HISTOLOGY

Course 1
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

Introduction Epithelial Tissues

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INTRODUCTION
The term histology - ancient Greek language, where histos - tissue and logos means science; thus we may define histology - the science which is studing normal h l h l ) human ti tissues ( (normal morphology). 4 main types of tissues are present in the human organism: p p g g y glandular 1. Epithelial tissues: epithelia of lining and covering, secretory or g epithelia and sensory epithelia. 2. Connective tissues - tissues of structure and support of the human body; here are also included hard varieties of connective tissue, the cartilage and the , g bone. 3. Musc. tissues: striated skeletal muscle, forming the large muscles of the tissues: body; the cardiac muscle or the myocardium - muscle of the heart and the y; y smooth muscle or visceral muscle - the walls of the hollow organs. 4. Nervous tissue, most specialized and highly differentiated tissue - neurons and glial cells. In a general definition, a tissue is a mass of cells - just one or several cell types, which may show or not morphological similarities, which have the same embryonic origin; the tissue is behaving as a unit to fulfill its functions in the homeostazis of the organism.
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These 4 tissue types - in all 3 embryonic sheaths – ectoderm, endoderm and mesoderm. The genes of the DNA of the nucleus are programming the mitosis of the cells and their differentiation into different tissue types. In the cytoplasm of the cells were identified regulatory proteins, capable to modulate gene expression. A special type of genes, were called ancestor genes; these genes are inducing the process of growth and differentiation of the cells. They promote the synthesis of growth factors. They are also called protooncogenes, because under certain circumstances, such as exposure to risk factors: ionizing radiations, toxic factors, viruses ( i l i f ti i (viral infections) - abnormal proliferations – malignant t ) b l lif ti li t tumors (cancers). ( ) The time period for the development of such proliferations, under the action of the risk factors, is very long (decades, usually). Via protooncogenes and growth factors, factors a normal function of the organism may become lethal in certain organism, lethal, conditions. In the human organism are present highly differentiated cells - neurons, which have l t th i capacity of cellular di i i h lost their it f ll l division, as well as undifferentiated cells, which ll diff ti t d ll hi h are keeping their capacity of cellular division, along with the adult life of the individual. They show an aspect very similar to that of the embryonic cells. These cells are responsible for the cellular regeneration and tissues renewal renewal. The natural cellular death is a genetically programmed phenomenon, called apoptosis. In human specie, in cultured cells, was established that the cells are capable of cellular di i i bl f ll l division, up t an age of 120 years; thi should b eventually, to f this h ld be, t ll the real limit of the human life.
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EPITHELIAL TISSUES
EpiEpi-, in ancient Greek - over or above, illustrating the main function of the epith above epith. tissues, which is that of covering and lining. The morphol. asp. of the epith. tissues is very much alike that of the embryonic layers. According to their function, epith. tissues may be divided into: epith. of covering and lining, gland. or secretory epith. and sensory epith. Epith. tissues – originate in all 3 embryonic layers, as follows: epidermis of the skin, epith. lining the natural cavit. of the body ( g y (oral cavity, anus, vagina, vestibule y g of the nose) and salivary glands, have all their origin in the ectoderm. Epith. lining the digestive and the respiratory tracts, as well as the epith. tissues of the liver and the pancreas, have their origin in the endoderm, while the epith. lining the vessels, mesothelia (pleura, pericardium and peritoneum) and the epith. of the nephrons are all deriving from the mesoderm.

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General characteristics of the epithelial tissues
1. Epith. tissues - a large no. of cells; in light microscopy, one may obs. a large no. of nuclei. Shape of the nuclei of the cells forming a tissue g p g gives us a clue, to the , shape of the cells forming the tissue, because intercell. limits are difficult to obs. with the light microscope; cuboidal epith. - round nuclei, columnar epith. - ovoidal nuclei and squamous epith. - flattened nuclei. 2. Epith. ll 2 E ith cells are closelly packed t l ll k d together and b t th d between th them - a very small amount ll t of intercell. cementary subst. When compared with other tissues, epith. cells establish one with another numerous junctions; crowded together, they show polyhedral faces faces. 3. Epith. tissues are not vascularized, in exchange they are very well inervated; they show numerous nerve endings – e.g. the skin, which is thus providing its tactile, sensory function. y 4. Epith. cells are alllways situated on a basement membrane, a complex, acellular struct. p p y g p y 5. Epith. cells show mitosis - the capacity of regeneration. This capacity is more obvious in the covering and lining epith., than in the glandular or sensory types. 6. Epith. cells show polarity, the orientation of the organels towards one pole of the cells; they also show specialzations of their apical and / or basal pole - directly connected with th f t d ith the functions of the epithelia. ti f th ith li
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The basement membrane
Beneath the epithelial tissue, we allways find connective tissue, which is the main tissue of support. Epithelial tissue does not come into direct contact with the connective tissue, but through an acellular membrane, with complex structure - the basement membrane. In light microscopy, in simple epithelia, the basement membrane is linear (straight), while in the stratified epithelia (made of several, superposed layers of cells), the basement membrane looks folded. With the usual staining techniques, the basement membrane is not visible; it becomes visible when special staining methods are used. In the structure of the basement membrane - compounds of both epithelial and connective tissues. Silver salts impregnation - the basement membrane is black, revealing connective tissue elements of the basement membrane. PAS method (periodic acid Schiff) stains it in red, revealing epithelial tissue compounds of the basement membrane (PAS method - the stain of the GP). The real structure of the b.m. may be studied only with the electron microscope. Depending on its localization, the basement membrane shows a thickness of 20 up to 100 nm. It has in its structure, a more electron dense layer - lamina densa, which shows inside a fine fibrilar network. Toward the epithelium - a more lucent layer (less dense) - lamina lucida.
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In the human org. are present also peculiar b.m. - that of the Malpighi`s copuscles of the nephrones, in the kidneys, which appears as the result of the fusion of 2 ordinary b. m. In this case - a central dense lamina, surrounded on each side by one lamina lucida. Lamina densa and lamina lucida form together lamina basalis. Usually, towards the connective tissue, it is present a third layer - lamina reticularis - a fine reticular fibers network. Biochemically, the compounds of the b.m. are: type IV collagen, inside lamina densa, GP (laminin, fibronectin) and PG, especially heparan-sulfate proteoglycan called heparanperlecan, in the lamina lucida; has a jelly-like conssistence. The attachment of the b. jellyp , ; j y m. to the connective tissue - anchoring fibrils made of collagen VII and elastic microfibrils - components of the connective tissue. Epithelial cells show receptors for GP, such as laminin, fibronectin, also known as adhesion molec., because they y function as attachment struct. of the epithelial tissue to the connective tissue of support. Through the b.m. - all the exchanges between the connective tissue and the g g epithelial tissue (water, macromolec., ions). The b.m. - a selective barrier, between the two tissues. Since epithelial tissues are not vascularized, the nutrition of the epithelia is made through the b.m., by diffusion processes.

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Figure 1. Two types of basement membranes. A: The thickness of this type of membrane results from fusion of 2 basal laminae produced by an epithelial and an endothelial cell layer, as found in the kidney glomerulus (shown here) and in the alveoli of the lung It consists of a thick central lamina lung. densa with a lamina lucida (lamina rara) on either side. B: The more common type of basement membrane that separates and binds epithelia to connective tissue is formed by association of the basal and reticular laminae. Note the presence of the anchoring fibrils formed by type VII collagen, p g y yp g which binds the basal lamina to the subjacent collagen.
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Intercellular junctions
Intercellular junctions between epithelial cells – of cellular adhesion or of cellular communication. communication Inside epithelia specialized in cellular transport the junctions transport, intraepithelial compartments. 1. Desmosomes - small, localized areas of membr. contact between cells, attaching pointcells one to another Being point-like junct they are also called maculae another. junct., adherentes. In a stratified epith., one may find hundreds of desmosomes on the surface of a single epith. cell. In light microscopy, desmosomes look like buttonbuttonlike points of contact, or projections between neighbor cells. The size of the desmosomes is at the limit of the resolution of the light microscope. They become better visible when the cells are contracted in the process of fixation (a step in obtaining histologic p p g g preparations) and the spaces between the cells are enlarged ) p g layer spinosum of the epidermis. Main comp. of the desmosomes are attachment plaques, interm. filam. and ta s e b transmembr. linker p ote s Attachment p aques a e intracell. pa ts o e proteins. ttac e t plaques are t ace parts of desmosomes; one desmosome has two attachment plaques, one in each cell. The plaques are made of filaments. Major part of the attachment plaque - cytoplasmic p p g proteins - desmoplakins and p plakoglobin. The p q contains as well the plaque cytoplasmic domains of the cadherin molec.
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Interm. filam. of the desmosomes - keratin filaments of the cytoskeleton, which attach to the plaque; they l h l h loop i and out of the plaques. Lik this, the plaques h in d f h l Like hi h l have a role of l f support for the cytoskeleton. Transmembr. linker proteins are in fact, GP, desmoglein and desmocollin - the adhesive parts of the desmosomes. Desmogleins and democollins are members of the cadherin family of calcium-dependent adhesion molec. These molec. show intracell. calciumand extracell. domains. The intracell. domain is part of the attachment plaques, while the t th extracell. d ll domain of th molec. extends i t th i t i f the l t d into the intercell. space and bi d with ll d binds ith similar molec. of the neighbor cellls, connecting cells together. Transmembr. linker proteins bind calcium, necessary for the normal funct. of the desmosomes as adhesion sites. sites Hemidesmosomes are desmosomal junct. of the basal pole of the epith. cells to the b.m. They show the same struct. as the desmosomes, being made of attachment plaques, i t l interm. fil filam. and t d transmembr. li k proteins. I contrast t th d b linker t i In t t to the desmosomes, the transmembr. linker proteins of the hemidesmosomes are part of the integrin family of adhesion molec., but they have the same role as that of the cadherins of desmosomes. desmosomes Hemidesmosomes are points of attachment of epith cells to the b m and epith. b.m. to the extracell. matrix. The laminin of the b.m. is concentrated in the areas of the hemidesmosomes. In conclusion, hemidesmosomes - similar in struct. to desmosomes, bioch. composition. but they are different as bioch composition
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2. Tight junctions are surrounding the cells j g j g joining them to the neighbor cells. This g g junct. is also called zonula occludens, because it is a belt-like junct., occluding beltvery tight the intercell. spaces. Tight junct. is a comp. of the terminal bar. Tight junctions - usual for the absorptive and secretory epith. They are situated close to the apical pole, on the lat. surfaces of the cells, especially in simple column. and simple cuboid. epith. One epith. cell has usually, just one tight junct., in contrast to numerous desmosomes and gap junct. In the electron microscopy, the tight junct. appears as an area of membr. contact by local connections. Each site of contact is called a membr. kiss. Studies by freezefreeze-fracture of the tissue, demonstr. that the tight junct. is made of ridges and grooves, forming a network, with a perfect correspondence on the neighbor cell. The depht and the width of the junct. may vary a lot, from a few ridges and grooves, to large complex arrangements of grooves and ridges. The strands or ridges are made of rows of particles. Th occlusion of th i t id d f f ti l The l i f the intercell. space i as ll is tight as the tight junct. is larger. In epith. specialized in transport, the tight junct. is forcing the watery materials to pass through the epith. The tight junct. are establishing limits between the two domains of the epith cells – the apical domain epith. and the basolat. domain, thus being important in the cell polarity and functioning like a molec. fence. The chemical comp. of the two domains is different (proteins funct. epith. and phospholipids) - important in the normal funct of these epith
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3. Adhesion junct. (adhesion belt) is appearing in the junct. complex beneath the tight junct. It is also called zonula adherens; it is an encircling adhering j g g junct. made of cadherin transmembr. linker proteins and a belt or a band of circumferential placed actin filam., as well as other contr. proteins, such as myosin, tropomyosin, vinculin, α-actinin. This is the site for the attachment of the actin filam. forming the terminal web. The junct. complex i a group of j Th j t l is f junct. b t t between 2 epith. cells. S h j ith ll Such junctional complexes are ti l l situated to the apical pole of the epith. cells and at the same time, on the lat. faces of the cells. Junct. complexes - present in the simple cuboidal and in the simple columnar epith., absorptive and secretory epith. (in the intestinal epith., in the acinar cells of the exocrine pancreas). The junct. complex is made of a tight junct. (zonula occludens), a zonula adherens and a desmosome. Some specialists include also a gap junct. in the junct. complex. Zonula occludens and zonula adherens are visible in the light microscopy as the terminal bar. In cross section it appears as a dot on the lat. surf. of the epith. cell. In sect. parallel to its plane it appears as a polyhedral frame encircling the cell. The terminal web is anchored on zonula adherens and is made of filam of actin and spectrin filam. spectrin, as well as interm. filam. The terminal web provides an attachment site for the bundles of actin filam. forming the core of the microvilli. Since it is achored on zonula adherens, the terminal web it is also placed towards the apical p p p pole of the cells. Zonula adherens and the terminal web form a contr. struct. to the apical pole of the epith. cells. This is usefull during embryonic develop. of the epith., in the arrangement of the epith. layers and also during adult life, when it ensures the deplacement and closure of the epith., for the expulsion of senescent, apoptotic cells. cells
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Figure 2. The main structures that participate in cohesion among epithelial ti i t i h i ith li l cells. The drawing shows 3 cells from the intestinal epithelium. The cell in the middle was emptied of its contents to p show the inner surface of its membrane. The zonula occludens and zonula adherens form a continuous ribbon around aro nd the cell ape whereas the apex, hereas desmosomes and gap junctions make spotlike plaques. Multiple ridges form the zonula occludens, where the outer , laminae of apposed membranes fuse. (Redrawn and reproduced, with permission, from Krstíc RV: Ultrastructure of the Mammalian Cell Cell. Springer-Verlag, 1979.)

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The terminal web is anchored on zonula adherens and is made of filam. of actin and spectrin, as well as interm. filam. The terminal web provides an attachment site for the bundles of actin filam. forming the core of the microvilli. Since it is achored on zonula adherens, the terminal web it is also placed towards the apical pole of the cells. Zonula adherens and the terminal web form a contr. struct. to the apical pole of the epith. cells. This is usefull during embryonic develop. of the epith., in the p g y p p , arrangement of the epith. layers and also during adult life, when it ensures the deplacement and closure of the epith., for the expulsion of senescent, apoptotic cells. The gap junct. is a communication junct.; such junct. are situated on the lat. faces of the epith. cells but they are also numerous in the liver myocardium and in the epith cells, liver, smooth muscle, as well. At the level of a gap junct., the cell membr. appear linear and very close one to another, with a space of 2 nm sepparating them. A gap junct. is made of small units called connexons.
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Connexons - a protein of approx. 26 000 – 30 000 Da, called connexin. 6 molec. of connexin form a connexon, which is a hexameric struct. with a hidroph. pore of 2 nm in diam. Each connexon has a perfect correspondant on the neighbor cell, forming an aqueous channel which i li ki th cytopl. of th cells i f i h l hi h is linking the t l f the ll involved i th l d in the junct.; this channel is made of 12 molec. of connexin. Gap junct. provide the metab. and electric coupling of the cells inside tissues, establishing continuity between the cytopl of adjacent cells Through the gap junct are passing water cytopl. cells. junct. water, ions and small molec., such as cAMP or cGMP. In this way, informations or stimuli are travelling along the tissue. Potentials are spreaded into the tissues by the electric coupling of the cells through gap junct since ions move freely through it cells, junct., it. Gap junct. - numerous in the myocardium and in the smooth muscle, where the funtion of the tissues is depending upon its electric coupling. The coupling of the cells through gap junct is influenced by the cytopl pH and by intracell calcium junct. cytopl. intracell. concentration. The increase of the intracell. calcium concentration and the decrease of the pH are uncoupling the cells. When a tissue is injured, the cells loose different materials through the connexons; if we administrate calcium to the tissue, we will provide the closure of the connexons and thus we prevent the extension of the lesion to the coupled uninjured cells.

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Figure 3. Electron micrograph of the apical region of an intestinal epithelial cell. Note the terminal web composed of a horizontal network that contains mainly actin microfilaments. The vertical microfilaments that constitute the core of the microvilli are clearly seen An extracellular cell coat seen. (glycocalyx) is bound to the plasmalemma of the microvilli. x45,000.
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Thank you for your attention!
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HISTOLOGY
Course 2
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

Epithelial Tissues The Glandular Epithelium

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Classification and characteristics of covering and lining epithelia d li i ith li
These epith. are forming large cellular membr., which provide resistance by opposing mechanical forces and other stress factors They are classified according to 2 main criteria: factors. according to the no. of layers of cells forming an epith. and according to the shape of the epith. cells. 1. according to the no. of layers of cells, we differentiate: cells, Simple epith. are made of just one layer of epith. cells, all into contact with the b.m., by their basal pole. Stratified epith. - made of 2 or several layers of epith. cells, superposed one on another and from which only the cells of the basal layer come into contact with the b m by their basal b.m. pole. Pseudostratified epith. (for ex. the respiratory epith.) are in fact simple epith. (pseudo false ). In these epith. all the cells have their basal pole into contact with the b.m. but they have different heights so not all of them are reaching with their apical pole the surface of heights, pole, the epith. In light microscopy, the nuclei situated at different levels give a false impression of stratif. Simple epith. come into contact with the int.l environment of the org., being more fragile, such epith. are lining cavitary organs. Stratif. epith. come into contact, by their superficial layer, with the ext. environment, how it is the case with the epidermis of the skin and the epith. lining the natural cavit. of the body, such as oral cavity, anus, vagina etc. These epith. are more resistant y p p , and they show an important p protective function. In the case of the epidermis, the keratinization process is making it even more resistant.
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2. according to the shape of the cells, we differentiate: cells, Squamous epith., which may be simple or stratif. epith., stratif. Cuboidal epith., which may be simple or stratif. epith., stratif. Columnar epith., which may be simple or stratif. epith., stratif. Simple squamous epith. is made of flattened cells; one cell is covering a large space epith. in surface and shows irreg. borders. The nuclei of the cells are flattened and centrally located; in cross section the cells show a lens asp. In simple epith. all the cells have the epith. same shape, while in the stratify. epith., they show different shape in different layers. In epith., stratify. epith., the name of the epith. is given according to the shape of the cells in its epith., epith. superficial layer. Simple squamous epith. looks like a very fine membr., which has selective epith. membr., permeability. The cells have pores, which are preferential channels fot the crossing of certain materials. In the e.m., the simple squamous epith. shows numerous pinocytotic e.m., epith. vesicles. Si l squamous epith. i li i th bl d and th l i l Simple epith. is lining the blood d the lymph vessels and i called ith h l d is ll d edothelium. edothelium. Simple squamous epith. is lining pleura, pericardium and peritoneum, the epith. serosal linings of the org. or mesothelia, since they develop from the mesoderm. mesothelia, Mesothelia - th movement of the visceral organs b sliding. Th l M th li the t f th i l by lidi The loop of H l i th f Henle in the nephron, nephron, is also lined by simple squamous epith., which is involved in the concentration epith., of the urine. Simple squamous epith. is specialized in the transport through diffusion of epith. molec. macromolec., pinocytosis. small molec. and in the active transport of macromolec., by pinocytosis.
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Simple cuboidal epith. is made of approx. cuboidal cells. In section, the cells epith. show almost all their diam. equal one with another. The nuclei - round and centrally placed Because in the light micr the intercell limits are not very clear placed. micr. intercell. clear, the epith. appears like a band of cytopl., with round nuclei in its midle. This is the criterion for its identif. in the light micr., since the asp. of the nuclei is giving us the clue for the type of epith obs Simple cuboidal epith is lining the small excretory epith. obs. epith. ducts of the liver (billiary ducts), pancreas, salivary gl. The simple cuboidal epith. is lining the thyroid follicles, in the thyroid; this is a gl. or secretory epith. Simple cuboidal epith. is covering the surface of the adult ovary and distal convoluted tubules of the nephron are as well, lined by simple cuboidal epith. Simple columnar epith. is made of cells which are more tall than large The epith cells, large. epith. cells are situated with their long. axis perpend. on the b.m. Columnar epith. cells show ovoidal nuclei, usually located towards their basal pole. These epith. are lining most of the digestive tract – the stomach, the intestine; it is also lining the stomach female genital tract, part of the male genital tract and the small bronchi of the lung. Usually, these cells show at their apical pole specializations involved in the function of these epith.
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Figure 4. Diagrams of stratified and pseudostratified epithelial tissue. A: Stratified squamous epithelium. epithelium B: Transitional epithelium. C: Ciliated pseudostratified epithelium The goblet cells secrete epithelium epithelium. mucus, which forms a continuous mucous layer over the ciliary layer.
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Figure 5. Simple squamous epithelium covering the peritoneum (mesothelium). Some blood capillaries are indicated by arrows. PT stain. stain Medium magnification magnification.

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Figure 6. Simple cuboidal epithelium from kidney collecting tubules. Cells of these tubules are responsive to the antidiuretic hormone and control the resorption of water from the glomerular filtrate, thus affecting urine density and helping retain the water content of y p g the body. PT stain. Low magnification.
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Figure 7. Simple columnar epithelium that covers the inner cavity of the uterus. Note that the epithelium rests on the loose connective tissue of the lamina propria. The epithelium and the lamina propria constitute the mucosa. H&E stain. Medium magnification.
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Figure 8. Diagrams of simple epithelial tissue. A: Simple squamous epithelium. B: Simple cuboidal epithelium. C: Simple ciliated columnar epithelium. All are separated from the subjacent connective tissue by a basement membrane. In C, note the terminal bars that correspond in light microscopy to the zonula occludens and the zonula adherens of the junctional complex.
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Specializations of the epithelial cells
The cell. polarity presumes specializations of the apical and of the basal pole of the epith. cells, correlated with the function of the epith. Microvilli - finger-lik projections of th cytopl. of th apical pole of th cells, Mi illi fi finger-like j ti f the t l f the i l l f the ll covered by the cell membr. They measure 0.5 – 1 μm in length and 0.08 - 1μm in thickness. Microvilli are involved in the enlargement of the surface of contact between the epith and the int environment, an adaptive mech for the absorption epith. int. environment mech. process. Their name suggests the resemblance with hair tafts (vilus-vili – hair tafts (vilusin latin lang.). Each microvillus has as a core a bundle of 20-30 actin filam. anast. 20one with another and with the cell membr through different other proteins At their membr., proteins. base the filam. are anchored on the terminal web, which is situated under the microvilli, towards the apical pole of the cells. The terminal web is an anast. network of interm. filam and actin filam To the outside the microvilli are covered interm filam. filam. outside, by a GP layer - glycocalyx, which is stained by PAS technique ( PAS positive). Microvilli are present in the columnar epith. of the small intestine, where they form the brush border of the enterocytes, the absorbtive cells of the intestine. The microvilli of the enterocytes are tall, and uniform in height. The brush border is visible in the light micr. like very fine striations of the apical pole of the cells. The p p p simple cuboidal epith. of the distal convoluted tubule of the nephron also shows a brush border, but here the microvilli are shorter and relativelly unequal in height.
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Microvilli - piston-like movements through the contr. of the actin filam.; the pistonmovements are correlated with the absorption process and in the intestine the rhythmical movements are li k d with th di h th i l t linked ith the digestive periods. ti i d Motile cilia are long, motile struct. of about 2-5 μm long and 0.3-0.5 μm in 20.3thickness. Cilia are much longer than microvilli and they have a different struct. They are situated at the apical pole of certain epith. cells. Each cilium has in its midle a struct. made of microtubules - axoneme. Microtubules are organelles of intracell. motion. The axoneme or the core of microtubules of each cilium is made of 9 pairs of microtubules surrounding a central pair. Th wall of th microtubules f i f i t b l di t l i The ll f the i t b l is made of heterodimers of α+ β tubulin. Each pair of the nine periph. pairs shares a common wall made of 2-3 heterodimers. In the central pair, the two tubules are 2separated one from another and are encircled by a central sheath Neighbor pairs sheath. are connected one to another by protein bridges of nexin and are linked to the central sheath by radial spokes. Each doublet - a subfiber A and a subfiber B. Subfiber A - a complete wall of 13 heterodimers while subfiber B - a wall of only heterodimers, 10 or 11 heterodimers. From the subfiber A of each pair are extending to the sufiber B of the neighbor pair, arms of dynein, which ATPase activ. Dynein arms are able to bind to the subfiber B of the next pair and to slide along it ATP - energy it. for the process, by ATPase hydrolysis. The sliding of the periph. pairs one from another is leading to the curving of the whole cilium and thus to the final lat. y g movement of the cilia. The movement is limited by bridges of nexin between the periph. pairs and by the radial spokes to the central sheath.
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The cuticle is a thickening of the apical pole of the epth. cells, found in the urothelium or transitional epith., which i li i the urinary passages. Fi h li ii l ih hi h is lining h i First histologists called it cuticle. In the e.m., it appears made of thicker membr. plates, separated by portions of usual cellular membr., which are falling between the l t inside the t l f the ll thus forming i t i intracytopl. vesicles, i f t f ld t l i l plates i id th cytopl. of th cells, th f in fact folds of cellular membr. As the urinary bladder is filling with urine, the cells of the superf. layer of the urothelium are stretched, their shape changes from dome-like cells into domesquamous cells and the folds of the apical cell membr are redressed The plates membr. redressed. are lipido-proteic pentameres, the lipid fraction containing surprisingly, lipidocerebrosides. In the empty bladder, the epith. shows 5-6 layers of cells, when the 5bladder is filled with urine the cells are rearranged in only 2-3 layers since this urine, 2- layers, epith. is pseudostratified and all the cells have contacts with the b.m. The basal pole of numerous epith. cells show folds or plications and processes of the cell membr which are increasing the surf of contact between the epith and membr., surf. epith. the connective tissue of support, through the b.m. This specialization is important in uptaking materials and/or secretion of different products, through the basal pole. Most often these cells also present microvilli at their apical pole The irreg folds often, microvilli, pole. irreg. of the basal pole, of the epith. cells were also called basal labirynth. We find this specialization in the distal convoluted tubule of the nephron, in the enterocytes of the intestine and in some of the gl.
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Specializations of the epithelial cells
The polarity of the epith. cells presumes the orientation of the organelles toward one pole of the cells; for example, mitochondria are usually found at the basal pole, in the irregular folds of the membr being involved in furnishing energy for diverse processes while the membr., processes, Golgi complex is situated toward the apical pole, being involved in synth. processes. The apical pole is the secretory pole of the epith. cells. One type of organelles is usually predominating (in secretory epithelia best developed organelles are those involved in the epithelia, synth. of the secretory product). Pseudostratif. epith. - a false impression of stratif. in light micr., with its nuclei at different levels, levels being in fact simple epith since all the cells have contact with the b m epith., b.m. Respiratory epith. is a pseudostratif. epith., typical being that of the trachea.It is a pseudostratif. columnar ciliated epith. Most numerous are the ciliated tall columnar cells, showing ovoidal nuclei located to the base of the cells A second type of cells - goblet nuclei, cells. cells, with their characteristic asp. of a champagne cup. Goblet cells - mucus- secreting mucuscells; 2/3 of their apical part is dilated, filled with mucus and they show a narrow, basal nucleus. nucleus A third categ of cells - regenerative basal cells which may show different categ. regenerative, cells, heights; generally, these are low cells, which are growing to become columnar. Between lining cells of the epithelium are spreaded neuroendocrine cells, cells with endocrine function, part of the diffuse neuroendocrine system (DNES). Neuroendocrine cells are usually situated at the base of the epith., in contact with the b.m.
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The urothelium or the transitional epith. is also a pseudostratif. epith.; it is lining the pelvis, the ureter, the urinary bladder and the first portion of the urethra. It is considered by some authors a stratified epithelium, since it is showing a stratif. asp., in light micr. With th electron microscope, it was revealed th t i f t all th cells h the l t i l d that in fact, ll the ll have contacts t t with the b.m., through anchoring bridges of cytopl. The basal layer of cells of the urothelium is made of columnar cells, perpend. disposed on the b.m. Interm. layers polyhedral or rocket-like cells while superficial cells are umbrella-like cells rather rocketcells, umbrellacells, flattened. When the urinary bladder is empty, the epith. is made of 5-6 layers of cells. 5When the urinary bladder is filled with urine (distended), the cells are rearranged in only 3-4 layers and the superf layer becomes squamous The superf layer of epith 3superf. squamous. superf. epith. cells shows plates of thicker membr. at their apical pole, providing the enlargement of the epith. cells, when the urinary bladder is filling with urine, the so-called cuticle, which sohave been described. described The stratif. epith. - at the level of contact between org. and the environment, such as epidermis of the skin; these are protective epith., the stratif. is providing a barrier function. function Being in contact with the atmosphere, epidermis is keratinized, a stratif. squamous keratinized epith. Keratin produced in the cells of the epidermis is a fibrous protein, providing resistance and impermeability to the epith epith.
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Figure 1. Stratified transitional epithelium of the urethra The basement membrane between the epithelium 1 urethra. and the underlying loose connective tissue is indicated by arrows. PSH stain. Medium magnification
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Epidermis shows the following layers: 1. 1 Layer germinativum or layer basale - the regenerative layer situated on a folded b m ; it layer, b.m.; consists of a single row of columnar cells, or low columnar cells, deep basophilic, showing numerous mitoses and it is most important in the renewal of the epidermis. 2. Layer spinosum is the thickest layer, made of 5-6 rows of cells, in the thin epidermis, up to 510 or more rows of cells in the thick epidermis. With the light micr., in this layer are visible the desmosomes, on which are anchored tonofilaments of keratin precursor; they are responsible for the characteristic thorny aspect of this layer and they are giving the name of the layer. 3. Layer granulosum is characterized by the presence in the cytopl. of the keratinocytes of basophilic gr. of keratohyalin, a keratin precursor. In layer spinosum and granulosum, cells are polyhedral in shape and they become gradually flattened as we go to the surface of the epith. This layer is continuous in the thick epidermis, covering the palms and the soles and shows discontinuity in the thin epidermis, which is covering the rest of the body. 4. Layer lucidum i present only i th thi k epidermis; it l k refringent, l 4 L l id is t l in the thick id i looks f i t lucent, i th li ht t in the light micr. (luce means light, in latin lang.). This asp. - by eleidin, a keratin precursor. 5. Layer corneum or the cornified layer is very thick in the thick epidermis and much more thiner in the thin epidermis Beginning with layer granulosum organelles are gradually epidermis. granulosum, dissapearing from the cells; in layer lucidum, the cells are loosing their nuclei and in the cornified layer, we can not differentiate individual cells any more. Cells are fusing together in sheath. sheaths. a keratin sheath The expelling or detachment of dead cells is done as keratin sheaths
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Figure 2. Stratified squamous nonkeratinized (moist) epithelium of the esophagus. PT stain. Medium magnification
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Stratif. squamous nonkeratinized epithelium is lining the nat. cavit. of the body, such as: oral cavity, anus, esophagus, vestibule of the nasal cavity, vagina. These are wet cavit., permanently moisturized b l t it tl i t i d by local secretions, preventing k ti f l ti ti keratin form. Th The superf. layer of this type of epith. is made of squamous cells with clear limits between one another, and with preserved nuclei. The expelling of this layer is done by detachment of isolated, individual cells, from which one may obtain smears, for the study of cytology of these epith. In this epith., we obs. 3 layers: 1. a regenerative, basal layer, made of a single row of columnar or cuboidal layer, basophilic cells. 52. An interm. layer, also called layer spinosum, made of 5-6 rows of cells, which layer, y tend to become flattened, as they become more superf. 3. A superf. layer made of squamous, flattened cells. The cells of the stratif. squamous nonkeratinized epith. contain only keratin precursors and under normal circumstances, complete keratinization is unusual. However, it is a thick, resistant epith., presenting numerous adherent j ith ti dh t junct., b t t between it cells. its ll In some cases, such as the stomach and the intestine, the protection of the simple epith. lining these cavit. org. is achieved through mucus secretion (mucus secreted by the simple columnar epith lining the stomach and by the goblet cells of the intestinal epith. lining epith.) - the epith. is at the same time, a lining, as well as a secretory epith. Neuroepith. cells are modified epith. cells, which are specialized in receiving sensory stimuli; such cells are found in the taste buds.
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The glandular epithelium
The gl. epith. is made of cells capable to synthesize and to secrete a material g p p y different in comp. from plasma and intercell. fluid. This presumes synthesis of macromolec., which are stored in the cytopl. of the secretory cells as secretory gr. The gl. epith. is organized as : unicellular g , as the g g p g gl., gobletgoblet-cells; groups of ;g p secretory cells, as the mucus secreting cells in the urethra, in both sexes; proper gl., as the pancreas, the salivary gl. In fact, many cells of the org. show secretory propert. (neurons - neuromed., plasma cells - antibodies etc.) but only gl. - specialized for secretion. Gl. are deriving from all 3 embryonic layers; they develop from the surface epith., which is growing inside the connective tissue; if the connection with the surf epith surf. epith. of origin is kept and the epith. cord is tunneled to form an excretory duct, an exocrine gl. will appear, while if the epith. cord is abs., an endocrine gl. will appear, which will establish close connections with the blood vessels (the horm. ( discharged directly into the blood).

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Figure 3. Formation of glands from covering epithelia. Epithelial cells proliferate and penetrate connective tissue. They may–or may not–maintain contact with the surface. When contact is maintained, exocrine glands are formed; without contact, endocrine glands are formed. The cells of endocrine glands can be arranged in cords or in follicles. The lumens of the follicles accumulate large quantities of secretions; cells of the cords store only small quantities of secretions in their cytoplasm. (Redrawn and reproduced with reproduced, permission, from Ham AW: Histology, 6th ed. Lippincott, 1969.)

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Thank you for your attention!
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HISTOLOGY
Course 3
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

Exocrine Glands Endocrine Glands

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Exocrine glands
Exocrine gl. are deriving from all 3 embryonic layers. Every exocrine gl. has 2 portions – a secretory portion and an excretory duct. In the secretory portion the secret. product is synthesized and through the excretory duct the secretion is discharged always onto an epith. duct, epith. covered surf. According to the chem.. nature of the secret. product - different types of secret. cells, in the exocrine gl.: 1. Proteins –secreting (enzymes-secreting) cells (enzymesSuch cells - in the pancreas, the parotid gl., in the mixed saliv. gl. Enzymes–secreting cells saliv. Enzymes– form gl. acini, they are called serous cells and they form serous acini (to differentiate them acini, from the mucus-secreting cells, which form mucus acini). In light micr. they are pyramidal mucusacini). micr. cells, with round nuclei at their base, with obvious nucleolus, intense basophilia of the basal pole and well-stained secret. gr. at the apical pole. In e.m., serous cells show well devel. RER welle.m., devel. and Golgi complex and numerous mitochondria Serous cells forming serous acini are also mitochondria. called zymogen cells, containing zymogenic gr. The stages of protein synthesis are : the uptake of aa from the blood; protein synthesis in the RER; the transfer of the proteins to the Golgi complex, where they are packed in membr. to membr. form secret. gr.; the accum. of the secret. gr. in the apical pole of th cells, where water i f t th accum. f th t i th i l l f the ll h t is removed from the gr., the secret. product is thus concentrated and the gr. are matured; the excretion from the cell is done by the fusing of the gr. with the membr. of the apical pole, the membr. g g y exocytosis. y gy y content of the gr. is discharged by exocytosis. The energy for the p process is furnished by mitochondria through oxidative phosphorylation. phosphorylation.
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2. Mucus –secreting cells are secreting GP. These cells have also well developed RER and Golgi complex. After protein synthesis in the RER, the coupling between proteins and carbohydrates is done in the Golgi complex by specific enzymes called glycosyl –transferases (these enzymes are localized in the membr. of the RER and of the Golgi complex). When are secreted sulfated GP, the sulfation g p ) , takes place in the Golgi complex. Mucus is discharged from the cells by exocytosis and forms a jelly-like, viscous, jellyelastic and hydrated film, which is protecting and lubricating the epith. MucusMucussecreting cells are the goblet cells (intestine, respiratory epith.), mucus-secreting mucuscells of the saliv. gl. (acinary cells), mucus-secreting cells are found in the genital mucustract, in both sexes. Mucus-secreting acinary cells show flattened nuclei at their Mucus, g y base, 2/3 of the apical cytopl. is very pale stained in light micr., because mucus is not stained with usual techniques. Mucus may be stained with special methods – alcian blue, carmine method, P.A.S. method. 3. The sebaceous cell is secreting triglycerides, uptaking fatty acids from the blood. The secret. product is called sebum. The droplets of lipid are gradually accum. in the cytopl., p y p providing a spongy, vacuolar, unstained asp., since the lipids are g p gy p p solved during the technique. The accum. of the secret. product is leading gradually to the degrad. of the organelles and of the nucleus, so that the cell dies and is discharged together with the secret. product. The cell has well develop. SER; the Golgi complex is absent (not needed in the synthesis).
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Figure 4. Section of large intestine g g showing goblet cells secreting mucus to the extracellular space. The mucus precursor stored in the cytoplasm of the goblet cells is also stained in a dark color color. PAS-PT stain. Medium magnification.

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3. The ions-transporting cells ionsThe ions t Th i transport may t k place f t take l from the base t the apex of the cells, or the th b to th f th ll th reverse. It may be simple diffusion (pasive) or an active transport (against the concentration gradient). The sodium ion - outside the cell a conc. of approximate 140 nmol / liter while inside the cell has a conc of only 5-15 nmol / liter In this situation liter, conc. 5liter. situation, the sodium has the tendency to enter into the cell. The cell is using sodium pumps to keep intracell. low sodium conc. The cells of the tubules of the nephrone, as well as the cells of the sweat gl are using this mech for transcell sodium transport Basal gl. mech. transcell. transport. folds of such cells show numerous mitochondria. Between the cells are present intercell. folds, which show activity of Na/K, Mg ATP-ase. In the cells of the tubules of ATPthe nephrone in the enterocytes (absorptive intestinal cells) and in the striated ducts nephrone, of the salivary gl., the transport takes place from the apex to the base. The reverse, from the base to the apex, the ions transport is present in the hydrochloric acid secret cells of the fundus gl in the stomach The cells are strongly secret. gl. stomach. acidophilic in the light micr., showing a richness of mitochondria in the e.m., because the synth. - with great energy consumption. The cells show between them intercell. canaliculi (hydrochloric acid is discharged outside the cell being harmfull by its very cell, low pH) and well developed SER, where the chemical dissociation takes place (from the blood are abs. NaCl and H2CO3 – sodium chloride and carbonic acid, y , Clp g ) dissociated they form Na+, Cl-, H+ and HCO3-; the coupling → HCl and NaHCO3.). HCO3Histology - 3 6

Figure 5. Ion and fluid transport can occur in different directions, depending on which tissue is g p p g involved. A: The direction of transport is from the lumen to the blood vessel, as in the gallbladder and intestine. This process is called absorption. B: Transport is in the opposite direction, as in the choroid plexus, ciliary body, and sweat gland. This process is called secretion. Note that the presence of occluding junctions is necessary to maintain compartmentalization and consequent control over ion distribution.
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Classification and description of the exocrine glands
According to the shape of the secret. portion, according to the number of the excretory ducts and according to the way of excretion of the secret. product. 1. According to the shape of the secret. portion, exocrine gl. - tubular or acinar gl. gl. Tubular gl. - simple tubular, branched tubular or coiled tubular gl. Simple tubular gl. may show or not an excretory duct. A straight simple tubular gl. - Lieberkuhn gl. of the intestine. The Lieberkuhn gl. - a simple invagination of the intestinal epith. into the lamina propria; that is why some authors are calling it intestinal crypt. Sinuous simple tubular gl. - the gl. of the fundus of the stomach and gl. of the endometrium, in the uterus. These gl. show no excretory ducts. An ex. of branched tubular gl. is the mucus-secreting gl. of the pylorus, in mucusthe stomach. This gl. - several tubular secret. units fusing to a single excretory duct. A simple coiled tubular gl. is the sweat gl.; its secret. portion is a highly tortuous tubule, while the excretory duct is linear and is opening to the pores of the skin The secret portion of the gl skin. secret. gl. is lined by simple cuboidal epith. and the excretory duct is lined by bistratif. cuboidal epith., darker stained than that of the secret. portion. Acinar gl. have a sphere-shaped secret. p g gl. spherep p portion - acinus. Secret. acini - serous, mucous and , mixed acini. Serous acinus - only of serous, enzymes-secreting cells. Serous cells are enzymespyramidal cells, with round nuclei at their base and a small, star-shaped, hardly visible starlumen. In light micr., serous cells are basophilic, filled 2/3 of their apical parts with well secret. gr. gl. gl., exclusiv. stained secret gr Exocrine pancreas and parotid gl - pure serous gl made exclusiv of serous acini.
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Mucous acinus is larger than the serous acinus, ovoidal or irregular-shaped. g irregularg p Mucous cells are rather cuboidal in shape, and the mucous acinus shows a larger lumen than the serous acinus. Mucous cells - flattened nuclei at their base; the cells are paler stained, apical 2/3 of the cells have a pale, foamy asp., being filled with mucus gr. Mucous acini are making part of the mixed saliv. gl. (submandib. and subling. gl.) and they form the palatine gl., which is a pure mucous gl. Mixed acinus is resembl. to the mucous acinus; 4/5 of its cells are mucous cells, while toward one pole of the acinus are crowded together some serous cells - 1/5 of the cells and forming a demilune of serous cells, called Gianuzzi`s demilune. Mixed acini - in the mixed saliv. gl. and in the lamina propria of the trachea. Sebaceous gl. is also an acinar gl., secreting triglycerides - sebum. The gl. shows in fact the asp. of a bag or of a sack, since it has no excretory duct; we may speek about a neck of the gland or a very short excretory duct. The sebaceous gl. is made of 3 types of cells – basal, well-stained, regener. cells, showing numerous wellmitoses; the big majority of cells are sebaceous, secret. cells, polyhedral-shaped, polyhedralwith central located nuclei and spongy, unstained asp. of the cytopl., which were already described; a third type of cells - keratinized cells of support. The cells of support are well-stained cells, with processes - a skeleton of support for the secret. wellcells, maintaining the gl. permanently opened.

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A peculiar type of gl. - the tubuloalveolar gl.; these gl. are similar to the acinar gl. gl. The alveolus (secret. porton) is an elongated acinus, with an ellipsoid shape. Secret. cells of the alveolus - in a single layer; each alveolus is continued with its own excretory duct. Such gl. - the mammary gl. and the prostate gl. 2. 2 According to the no. of the excretory ducts, the gl with a single unbranched no ducts, gl. duct - simple gl., while the gl. - several excretory ducts, that branch repeatedly compound gl. Compound gl. are tubular or acinar. Usually, large excretory gl. are compound tubuloacinar gl - the asp of a grape gl. asp. grape.

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3. 3 According to the way the secret products are discharged from the cells, secret. cells, exocrine gl. are classified as: merocrine, holocrine (holos in Greek - whole) and apocrine gl. Exocrine pancreas and saliv. gl. – ex. of merocrine gl.; the secret. p product is discharged from the cells through exocytosis, the integrity of the secret. g g y , g y cells being kept. Sebaceous gl. is a holocrine gl.; as the synth. of the secret. product occurs, the secret. cells are gradually degenerating, being expelled to the outside, together with the secretion. A typical apocrine g is the mammary g g yp p gl. y gland, milk is excreted this way. Simple molec., such as water and ions are discharged from the cells by simple diffusion; glucids and proteins are discharged by exocytosis, while lipids are fusing with the cell. membr. of the apical pole of the cells, being discharged as membrane -coated vesicles (not a single organelle is lost from the cell, during the process). The secret. cycle - the time period between the uptake of the precursors to the discharge of the final secret. product outside of the cell. The first stage of the secret. cycle is the selective uptake of the precursors, secret. through the cell. membr. of the basal p g pole of the secret. cells, which show , receptors for the specific precursors. The energy is furnished by mitochondria.

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Figure 6. Serous secretory cells of the pancreas disposed in acini. Note the basophilic cell basal region rich in RNA and the apex with the lightstained secretory vesicles PT stain Medium vesicles. stain. magnification.
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Figure 7. Esophageal mucous secretory gland with characteristic t l d ith h t i ti irregular, clear cytoplasm and basal nuclei. Loose connective tissue surrounds a secretory duct y

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Figure 8. Submandibular salivary gland 8 showing 2 types of secretory epithelial cells in a compound tubuloacinar gland. The light cells are mucous and the dark cells are serous. PT stain. Medium magnification.
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The sec. step of the secret. cycle - the stage of synth. and mobilization. The synth. begins when the cell is filed with precursors afterwards the secret gr are precursors, secret. gr. mobilized toward the apical pole (mobilization takes place by contr. of intracell. microtubules and contr. filam. of actin and myosin). Gl. deriving from the ectoderm (saliv. g , sweat g , lacrimal and mammary gl.) show at their base, myoepith. cells, gl., gl., ( yg ) , y p , which are cells with processes, containing interm. filam. and contr. filam. The processes of the myoepith. cells are embracing the secret. cells; by their contr. the mobil. of the secretion to the apical p p pole. The third, ultimate stage of the secret. cycle is that of excretion from the cells of secret. the secret product in the 3 ways already mentioned (merocrine holocrine or secret. product, (merocrine, apocrine). The cellular cycle is the time period from the cell. div. to the death of the cell. During a cell. cycle, the cells of the exocrine pancreas are accomplishing numerous sec et cyc es, while t e gob et ce s o y 1-2 secret. cyc es a d t e u e ous secret. cycles, e the goblet cells only 1- sec et cycles and the sebac. cells just one secretory cycle.

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The control of the gl activ - by the nerv system and by the endocrine syst but gl. activ. nerv. syst., usually one of the two syst. is predominant. For ex., the secretion of the exocrine pancreas is predominantly achieved by local horm. – cholecystokinin and secretin, while the secretion of the saliv gl is achieved especially by the autonomous veget saliv. gl. veget. nerv. syst. The sympathy. nerv. syst. acting especially on the secret. acini - an enzymes rich, reduced secretion (that is why the stress is inducing the dry mouth sensation) while the parasympath nerv syst acting on the striated excretory sensation), parasympath. nerv. syst. ducts of the saliv. gl. is provid. an abundant, watery secretion. In the human org. are present gl. in which the same cell shows both exocrine and endocrine activity; in the liver the hepatocyte (liver cell) is synth and secret synth. secret. exocrine the bile, but the same cell is secret. many products directly into the blood (even if the liver is not secreting horm., it is secreting many products on an endocrine pattern). The pancreas shows a double struct. – the serous acini, exocrine part, are secret. the pancreatic juice and the Langerhans islets (endocrine component) which are secret. horm. directly into the blood (insulin, glucagon, g gastrin). ) As histol. organiz., the exocrine gl. have an outer dense connective capsule from which are arising septa, dividing the parench. into lobes and/or lobules. They contain blood vessels and nerves.
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Endocrine glands
Endocrine gl. are also deriving from all 3 embryonic layers. Endocrine secret. cells - isolated or in small groups – cells of the diffuse neuroendocrine syst. ( g y (DNES) and ) endocrine cells of the ovary and of the testis, Langerhans islets of the pancreas, or may form proper gl. (pituitary, thyroid, adrenal gl. etc.) Most often the horm. are acting at some distance from the p g place of p production – the endocrine pattern; sometimes they act in vicinity, locally – the paracrine pattern; some of the neurotransmitters are also horm. (serotonin, epinephrine etc.) – the neurocrine pattern. According to their chemical nat. horm. are: proteins and glycoproteins; biologic active amines; hormonal steroids ( lipids). The endocrine cells which are secreting horm. which are proteins and GP show RER, Golgi complex and mitochondria. Because horm. are secreted in very small amounts, the organelles are not so well developed like in the exocrine cells, in conseq. the basophilia of the cells in light micr. is not so intense. For ex. in the q p g pituitary gl., the cells which are secreting TSH (thyroid stimulating horm.) basophilic, while the cells which are secreting GH (growth horm.) and prolactin acidophilic. The endocrine cells do not show polarity, they uptake precursors and they secrete all over their surf.
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Cells of the diffuse neuroendocrine syst. (DNES) are spread between the lining epith. cells of the digestive, respiratory tract etc., as well as in the parench. of the parench. org. First they were called A.P.U.D. cells (amine precursors uptake and decarboxylation), now they are called DNES cells. They contain small amount of RER and they secrete biol. active amines and horm. p yp p polypeptids. Many such horm. are secreted by neurons in the central nerv. syst. y y y and by cells of the DNES, in the periphery. The horm. of the DNES are usually acting locally, in a paracrine way. Cells are identified in the e.m. according to the asp. of the secret. gr. In light micr., cells of the DNES are stained with silver salts (argentaffin cells). Usually such a cell is secreting more than one horm. Endocrine cells secreting steroids with horm. activity - in the ovary, the testes and in the adrenal gl. In light micr. they appear acidophilic, round / polyhedral, with lipid clear vacuoles in the cytopl. and centrally located nucleus. In the e.m. - well developed SER and mitochondria, which contain the enzymes needed in the p y synth.; mitochondria are also furnishing the energy.

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The synth is the result of close cooperation between mitochondria and SER synth. SER, situated in proximity, in the cytopl. These cells do not store in their cytopl. the horm., they store only the precursor, the acetate. Acetate is transf. in cholesterol and cholesterol is turned into pregnenolone from which are deriving androgens pregnenolone, androgens, estrogens and progesterone. Acetate is solved in droplets of triglycerides, also synth. by the cells. The receptors for horm. proteins and GP are found on the cell membr. of the target cells. The horm. is the fist messenger, the cAMP is the sec. messenger. The activation of proteinkinases in the cell will ultimately lead to the activation of a synth.s or the blockage of a synth., representing the response of the target cell to the horm. message. The receptors for the steroid horm. - inside the cytopl. of the target cell. The hormonehormone-receptor couple is accepted by an acceptor. The three-sequence threecomplex i crossing the nuclear membr. acting directly on th genes of th DNA l is i th l b ti di tl the f the DNA, involved in the response of the target cells to the horm. message.

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The protein and GP horm. show already present enzymes in the target cells, which need only to be activated so they act rapidly but for a short period of time while activated, rapidly, time, the steroid hormones act a longer time, but they have a delayed action. Histol. organiz. of endocrine gl. is most often represented by cord-like arrangement cordof the endocrine secret. cells (pituitary, adrenal gl.). Thyroid is an exception, being made of follicles, lined by simple cuboidal epith. (thyroid cells); inside the follicles is present the thyroid colloid, th t th th id ll id thyroglobuline, a peculiar way of extracell. storing of th l b li li f t ll t i f the horm. precursor. The control of the activity of the endocrine gl. is very complex, involving the hypothalamus and the pituitary gl., by feed-back loops. The loops of control may feedbe long (e.g. the cortisol is acting directly on the hypothalamus) or short (thyroid horm. are acting first of all, on the pituitary gl.).

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Thank you for your attention! attention!
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HISTOLOGY
Course 4
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

Connective Tissues Collagen Synthesis

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CONNECTIVE TISSUE
The term connec. is origin. in Lt. lang., where nexus means connection. This is the main tissue of supp. of the org., linking together different struct. of the human body. Generally connec. body Generally, connec tissue does not come into contact with other struct but struct., epith.; however, some exceptions exist – the artic. cart. and the ant. chamber of the eye. Being well vasc., the connec. tissue is the place for intense diffusion processes, processes by this making possible the nutrition of the epith as well as immune epith., defense processes. The embryonic origin of the connec. tissue is the mesoderm. The primitive connec. tissue is the mesenchyme which has a viscous consistency jelly-like asp.; in this consistency, jellyasp ; ground subst. are found star-shaped cells – the mesench. cells, primitive connec. startissue cells. The Wharton gelatine of the umbilical cord of the new-borns and the newpulp of the young teeth are containing a connec tissue variety very much like the connec. variety, mesench. tissue. The mesench. cells - ovoidal nuclei with obvious nucleoli and fine dispersed chromatine. It has a very small amount of cytopl. and numerous processes. From the mesench. cell are deriving different connec. tissue cell types: the fibroblast, the white adipose cell, as well as cells of other types and tissues, such as endoth. cells, smooth muscle fibers.
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Figure 1. Simplified representation of the connective tissue cell lineage derived from the multipotential embryonic mesenchyme cell. Dotted arrows indicate that intermediate cell types exist between the examples illustrated. Note that the cells are not d ll t drawn i proportion t actual in ti to t l sizes, eg, adipocyte, megakaryocyte, and osteoclast cells are significantly larger than the other cells illustrated.

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The connec. tissue has 3 main comp.: cells, fb. and ground subst. (amorphous intercell. subst.). In contrast to the epith. and muscle, made predominantly of cells, cells the connec tissue main component is the intercell matrix made of fb and connec. intercell. matrix, fb. ground subst. The variations in the composition of the matrix, the predominance of one category of fb., or of one type of cells, or the different struct. of the ground subst., subst explains the large variety of connec tissue types connec. types. Between the cells of the connec. tissue it is found the ground subst.; that is why the ll th cells are usually situated at some di t one f ll it t d t dist. from another, so th d not show th they do t h intercell. junct. There are some exceptions to that rule: dentine secreting cells, the odontoblasts, adult cells of the bone (osteocytes) which may show intercell. junct. The intercell matrix contains a small amount of fluid - the main substrate for the intercell. diffusion processes in the whole organism (a solving environment for the small molec.).

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The ground substance (intercell. amorphous subst.)
The ground subst. of the connec. tissue is a complex, polymerized mixture of PG and GP, linking together the cells and the fb. of the connec. tissue. The Th ground subst. i t d b t is transparent, h t homogenous and colorless, i filli th sp. d l l is filling the between the cells and fb. of connec. tissue and forms a barrier against noxious particles, such as microorganisms. It does not stain in usual techniques in the light micr. micr and it has a fine granular asp in the e m Proteoglycans are made of asp. e.m. glycosaminoglycans (GAG) covalently bind to a protein core. GAG are linear polysaccharides made of repeating characteristics units of disaccharides which contain an uronic acid and a hexosamine The hexosamine is glucosamine or hexosamine. galactosamine, while the uronic acid is glucuronic acid or iduronic acid. The attach. of numerous chains of GAG to a protein core is forming the PG (with the exception of hyaluronic acid) acid). These molec. have the asp. of a hairbrush. The stem wire is the protein core, while the bristles, are the GAG. These molec. are rich in electronegative groups, such as carboxyl, carboxyl hydroxyl and sulfate which make them intensely hydrophilic and to act sulfate, as polyanions. With the exception of the hyaluronic acid, all the other GAG are sulfated to a certain degree. In the PG molec., the carbohydrate portion is best macromolec. s represented – 80-90% of macromolec.’s weight. 80Histology - 4 6

Figure 2. The molecular structure of proteoglycans and 2 glycoproteins. A: Proteoglycans contain a core of protein (vertical rod in drawing) to which molecules of glycosaminoglycans ( (GAGs) are covalently bound. A GAG is an unbranched ) y polysaccharide made up of repeating disaccharides; one component is an amino sugar, and the other is uronic acid. Proteoglycans contain a greater amount of carbohydrate than do glycoproteins. glycoproteins B: Glycoproteins are globular protein molecules to which branched chains of monosaccharides are covalently attached. (Reproduced, with permission, from Junqueira LCU, Carneiro J: Biologia Celular e Molecular, 7a ed. Editora g Guanabara Koogan. Rio de Janeiro, 2000.)
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Hyaluronic acid, the sole unsulfated GAG is a large highly polymer acid, large, polymer. molec., with a molec. weight of about 1000 kDa. In the cartilage matrix, the binding of the PG molec. to a chain of hyaluronic acid is forming larger molec. molec of proteoglycan aggregates These molec - electronegative aggregates. molec. (polyanions) and they bind a lot of cations (especially sodium) through electrostatic bonds. They are intensely hydrated molec.; although their hydration degree may vary, when th are f ll h d t d th i vol. h d ti d h they fully hydrated their l increases considerably. The hyaluronic acid, as well as other GAG are depolym. by hyaluronidase, an enzyme secreted by some bacteria (e.g. some types of streptoccocus). The invasive power of these bacteria is enhanced, since they reduce the normal viscosity of the ground subst., which is acting as a barrier against the p g g penetration force of bacteria. 4 sulfated GAG, together with a protein core are forming PG: dermatan , sulfate, chondroitin sulfate, keratan sulfate and heparad sulfate. The sulfated GAG are not so hydrophilic as hyaluronic acid but they form acid, bonds between hyaluronic acid chains as it is the case with the cartilage matrix, increasing the consistency of the connec. tissue.

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1. Dermatan sulfate - in the dermis of the skin, tendons, ligaments and fibrous cartilage, in all struct. that contain collagen type I collagen fb. 2. Chondroitin sulfate - in the hyaline and elastic cartilage, rich in collagen II fibrils. 3. Heparan sulfate - in tissues rich in retic. fibers, made of type III collagen and in the b.m. 4. Keratin sulfate is found in the cornea and in the intervertebral disks GAG are binding to collagen by electrostatic forces, established between acidic groups in the GAG molec and basic amino acid residues in the collagen molec molec. molec. The synth. of the PG begins in the RER, where the protein core is synthetized; the l l ti t t in the d is l t d in the Golgi l h glycosylation starts i th RER and i completed i th G l i complex, where th the sulfation of the sulfated molec., also occurs.

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Structural glycoproteins of the ground subst.
Structural GP are made of a protein to which are attached carbohydrates. In contrast to PG, in the case of GP, the protein usually represents the largest part of the molec. and they do not contain linear polysaccharides with hexosamines, instead the carbohydrate of the GP is a branched struct. f th i b h d t t Fibronectin (Latin lg. fibra-fiber, nexus-connection) is a GP sinth. by fibroblasts and fibranexussome epith. cells. Its molec. weight in of about 220000-240000 Da, it has binding sites 220000for cells, collagen and GAG, being a V-shaped molec. Fibronectin is an adhesion molec. Vstabilizing the entire struct. of the connec. tissue, made of cells, fb. and ground subst. It is involved, like others adhesion molec. in the migration of tumor cells, in cancers. Laminin is a large GP molec., found especially in the b.m.; it is responsible for the adhesion of the epith. cells to the b.m. The cell receptors for the adhesion molec. integrins. Tumor cells do not show receptors for the adhesion molec., which explains th i capacity of invasion b metastasis. Oth GP are: entactin, chondronectin it f i i by t t i Other t ti h d ti their (mediating the adhesion of condrocytes to type II collagen fibrils), osteonectin (in the bone) etc.

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Figure 3. Integrin cell-surface matrix receptor. By binding to a matrix protein and to the actin cytoskeleton (via alpha-actinin) inside the cell, the integrin serves as a transmembrane link. The molecule is a heterodimer, with alpha and beta chains. The head portion may protrude some 20 nm from the surface of the cell membrane into the extracellular matrix matrix.
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In the ground subst. - a small amount of fluid, called tissular fluid with a similar composition of the plasma (water, ions, small molec.s – aa and proteins with small molec. weight). molec weight) Under normal conditions, the amount of this fluid is very small. In edema, the fluid becomes greatly increased; main causes involved in the develop. of the edema are: venous obstruction (impairing the return of the blood from the periphery) heart periphery), failure (the pump function of the heart is insufficient), prolonged starving condition (the severe decrease of the plasma proteins will lead to an unbalanced difference between colloid osmotic pressure and hydrostatic blood pressure – starving edema), local inflammatory processes or release of histamine in alergic conditions and others. In different types of connec tissue the matrix may show different staining connec. tissue, affinities, for example the cartilage matrix is stain differently, according to its content in sulfated GAG. In young individuals, the higher content of polymer. GAG the matrix is basophilic methachromatic and P A S positive while in advanced basophilic, P.A.S. positive, ages, it becomes acidophilic, as the depolymer. of the sulfated GAG occurs.

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Fibers of the connective tissue
The fb. of the connec. tissue - long chains of p y g polymer. p proteins. There are 3 types yp of fb. in the connec. tissue: collagen, reticular and elastic. The 3 categ. of fb. - in different proportions in different connec. tissue varieties. Collagen and reticular fb. are made of collagen, while elastic fb. are made of elastin. Usually, the predominant fb. are the collagen fb. Generally, the predominance of one category of fb. is conferring specific properties to that connec. tissue type.

Collagen fibers
During evolutionary process, a group of proteins gradually developed, according to the environmental influences and required by the mechanical and functional needs of the org.; these proteins developed varying degrees of resistance and strength, being generically called collagen. The collagen is present in variable proportions in bone, cartilage, but also in the dermis of the skin, smooth muscle layers and b.m. Collagen is the best represented protein of the human body, it represents 30% of its dry weight.

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Collagen fb. are usually the most abundant fb. of the connec. tissue. They are almost transparent, when they are numerous they confer a whitish, shining asp. to the ti th tissue (like i th h li cartilage, t d (lik in the hyaline til tendons). Th orientation of th ) The i t ti f the tropocollagen molec. inside the fibrils makes collagen birefringent, when obs. with polarized light. Collagen fibers are not elastic, but they are flexible, very resistant. The resistance to tension forces is greater than that of the steel. A collagen fb. is made of closely packed fibrils of about 75 nm in diam. Collagen type I fb., best represented in the org., are f forming collagen b dl i ll bundles. C ll Collagen fb d not anast. one with another, fb. do t t ith th instead they clive the bundles and may pass from one bundle to another. Their thickness varies from 1 to 20 μm. Collagen fb. stain pink with eosin, blue Mallory s trichrome, Mallory’s trichrome green - Masson’s trichrome, blue - toluidine blue Masson s trichrome blue. If boiled, they form gelatine (jelly). If treated with weak bases or acids they hydrating and become swelled; they are degraded if treated with strong acids / basis; they b i th are di digested b specific enzymes called collagenases (f ex. th t of t d by ifi ll d ll (for that f the pancreatic juice) which cut the collagen molec. and afterwards the common proteases will lyse it.

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Figure 4. Schematic drawing of an aggregate of collagen molecules (tropocollagen), fibrils, fibers, and bundles. There is a stepwise overlapping arrangement of rodlike tropocollagen subunits, each measuring 280 nm (1). This arrangement results in the production of alternating lacunar and overlapping regions (2) that cause the crossstriations characteristic of collagen fibrils and confer a 64-nm periodicity of dark and light bands when the fibril is 64 nm observed in the electron microscope (3). Fibrils aggregate to form fibers (4), which aggregate to form bundles (5) routinely called collagen fibers. Collagen type III usually does not form bundles.
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More than 17 types of collagen are identified, more important are the first 5 identified, types: Collagen type I is best represented in the human org.; if forms collagen fb., aggregating into bundles Local. - bone tendons, dermis of the skin, dentine bundles. Local bone, tendons skin dentine, capsules of the parench. organs. Collagen type II is made of separate fibrils, which do not form fb. or bundles. Local. - hyaline cartilage, elastic cartilage, cornea. Collagen type III is the main component of the reticular fibers, usually associated with type I. It is found in the regenerative p yp g processes of the connec. tissue, is the first type of collagen synthetised, it may polymerize with other collagen types. Collagen type IV is local in the b m ; does not form neither fibrils nor fb local. b.m.; fb. Collagen type V appears in fetal membr., blood vessels, it is surrounding cells in different tissue types, for ex. musc. fb.; does not form fibrils.

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Figure 5. Electron micrograph of human collagen fibrils in cross and longitudinal sections. Each fibril consists of regular alternating dark and light bands that are further divided by cross-striations. cross striations. Ground substance completely surrounds the fibrils. x100,000.
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Collagen synthesis
First, it was believed that only connec. tissue cells are capable to synth. collagen – fibroblasts, osteoblasts. Later, was obs. that in fact, many other cell types may synth. synth collagen in certain conditions: smooth muscle cells Schwann cells liver cells, cells, cells, endoth. cells, some epith. cells etc. Main aa that form the collagen molec. are – glycine (33%), proline (12%) and hydroxyproline (10%) Two hydroxylated aa - characteristic for collagen – (10%). hydroxyproline and hydroxylysine. The hydroxylation of these aa takes place during collagen synth. Collagen fib il - polymerization of an elongated molec., th t C ll fibrils l i ti f l t d l the tropocollagen molec., ll l of about 280 nm long and 1.5 nm thick. Tropocollagen - 3 spiral polypeptide chains, assembled in a triple helix; it is an α-polypeptide, with 2 varieties – α 1 and αα 2. Glycine has an angular shape forming the turns of the spiral polypeptide 2 shape, chain. In collagen types I, II and III, tropocollagen molec. aggregate to form fibrils. Tropocollagen molec are united to form fibrils by hydrogen bonds hydrophobic molec. bonds, interactions and covalent cross-links between hydroxyl groups, present in the crosstropocollagen molec. (by oxidation of hydroxyl groups, in the presence of an enzyme, oxidase). enzyme the lysyl oxidase)
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Collagen fibrils show a transv. striation, with a characteristic periodicity of about 64 nm. nm The striation is the result of the characteristic overlapping arrangement of the tropocollagen molec., into fibrils. In e.m., the regular striation is visible like a succession of dark and light bands; the dark bands retain more of the lead solution used in e m technique than the light bands because the zones of the molec. e.m. bands, molec corresponding to the dark bands are richer in free chemical groups, reacting more intensely with the lead solution, than do the light bands. Type I II and III collagen form fibrils. Type I and III collagen - fibers and only fibrils I, type I collagen - bundles. These collagen types are also called interstitial collagen, to differentiate them from other collagen types, which do not form fibrils.

Steps of collagen synth.:
1. 1 The assembly of the aa in RER → polypeptide α chains formation; this is the aa, preprocollagen molec. As the signal peptide for stop of the synth. is cut off, results procollagen. 2. 2 Hydroxylation of proline and lysine takes place when the peptide chain has reached a certain length and is still bound to the ribosomes, under the influence of 2 enzymes – peptidyl proline hydroxylase and peptidyl lysine hydroxylase.

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3. Glycosylation of hydroxylysine with galactose, or glycosylgalactose. At each bottom of the α chains (both amino –NH2 and carboxyl – COOH ends) is appearing a supplementary peptide sequence, called registration peptide. R i t ti peptides are ensuring th tid ll d i t ti tid Registration tid i the correct α 1 and α 2 polypeptide chains assembly and the form. of the triple helix. This is the procollagen molec.; the registration peptides are also involved in the solubility of the molec., preventing the premature, intracell. fibril form. Procollagen is discharged from the cell by exocytosis. 4. Outside the cell, procollagen peptidases (specific proteases) are cutting off the registration p p peptides; thus are formed the tropocollagen molec., which are p y p g polymerizing into collagen g g fibrils. Hydroxyproline residues are establishing hydrogen bonds between the polypeptide chains of the tropocollagen molec., offering stability to the triple helix. 5. In collagen type I and type III, fibrils aggregate and form fb. The PG and the struct. GP of the th ground subst. are i d b t important in th polymer. of th t t t i the l f the tropocollagen i t fib il and i ll into fibrils d in aggregation of the fibrils to form collagen fibers. The fibrilar struct. is stabilized by covalent crosscross-links between hydroxyl groups on the tropocollagen molec. The oxidation of the hydroxyl groups is catalyzed by lysyl oxidase an enzyme acting in the extracell sp oxidase, extracell. sp. In the collagen synth. are encountered genetic defects, as it is the case with the EhlersEhlersDanlos syndr. (type VI and VII) which may lead to rupture of the eyeball, respectively to abnormal increased articular mobility, with frequent luxation. In scurvy, the nutritional lack y q y or deficiency of C vitamin (ascorbic acid – cofactor for proline hydroxylation) is developing ulcers and hemorrhages of the gums, as a conseq. of the defect of collagen synth. The
overaccumulation of collagen in the tissues is developing progressive systemic sclerosis, with fibrosis (high rigidity) of every org affecting especially the skin the digestive tract muscles and kidney org., skin, tract, kidney.

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Thank you for your attention! attention!
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HISTOLOGY
Course 5
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

Connective tissue
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Reticular fibers
Reticular fb. are very thin, about 0.5-2 μm thick. They do not form bundles, 0.5instead they are forming extensive networks representing a skeleton of support for networks, different tissues and org. Retic. fb. are not stained in usual techniques, but they are stained in black with silver salts (method of silver impregnation); they are also P.A.S. positive ( y have a higher content of carbohydrate in the molec., than the (they p g y , collagen fb.). Retic. fb. are made of collagen type III. They are made of loosely packed, very fine fibrils, hold together by PG and GP. Being so thin, they show only a weak birefringence, when obs. in p y g polarized light. g Retic. fb. - networks surrounding blood vessels, nerves (endoneurium), white adipose cells, smooth muscle fibers and acini of the acinary gl. They form the framework of hematopoietic organs – bone marrow, lymph node, spleen and the p g , y p , p stroma of parench. org., such as liver, kidney and endocrine gl. Retic. fb. are numerous during embryonic development, wound healing and during inflammatory processes, after a while being replaced by collagen fb. Because they form very flexible networks, they are present, together with the elastic fb., in org. which are changing form and/or vol., such as arteries, spleen, liver, uterus and smooth muscle layers of the intestine. Genetic defects of the retic. fb. lead to aortic and/or intestinal rupture, in type IV Ehlers-Danlos syndr. EhlersHistology - 5 3

Figure 6. Section of an adrenal cortex, silver stained to show reticular fibers. This is a thick section made to emphasize the networks formed by these fibers which consist of collagen type III Nuclei fibers, III. are black, and cytoplasm is unstained. Medium magnification.
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Elastic fibers
During their development, elastic fb. form the syst. of the elastic fb., which consists of 3 succesive types of elastic fb present in the embryonic as well as in the adult fb., embryonic, life. First stage is that of oxytalan fb., made of thin GP microfibrils. Oxytalan fb. - in the zonule of the eye and in the dermis of the skin. In the next stage, a small amount of elastin will link the microfibrils together; these are called elaunin fb and fb. are found around the sweat gl., in the dermis. In the third stage, elastin is accumulating in the center, surrounded by a thin layer of microfibrils. These are the elastic fibers best represented in the org fibers, org. While oxytalan fb. are tension resistant, elastic fb. due to elastin content, are truly elastic (5 times more elastic than the typical rubber band). Elastin is secreted as proelastin, proelastin a globular protein of 70 000 molec weight which polymerizes to form molec. weight, an amorphous GP, called elastin. It is secreted by fibroblasts in the dermis of the skin and by smooth muscle cells, in the wall of the large arteries. Elastin is resistant to boiling, acids and bases and to digestion by usual proteases, but is digested by pancreatic elastase. This peculiar resistance is determined by tertiary and quaternary struct. of the molec., with covalent bonds, between the peptide chains.

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Figure 7. Skin dermis, selectively stained for elastic fibers. Dark elastic fibers are interspersed with pale red collagen fibers. The elastic fibers are responsible for skin’s elasticity. Medium magnification.
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Elastin contains proline and glycine, like the collagen molec., but does not contain hydroxylated aa. Elastin contains 2 unusual aa – desmosine and isodesmosine, formed by covalent react. between 4 lysine residues. These react. are forming cross-links in the elastin molec. and is considered the key crossof the elastic property of this protein, ensuring the sliding of the peptide chains one from another and ulterior regain of the same shape and dimensions. Sole elastin, without microfibrils, forms the elastic fenestrated membr. (elastic laminae) present in the i th walls of l ll f large arteries, especially aorta. t i i ll t Elastic fb. - numerous in the org. which are changing a lot their vol., during fb. different funct. estates, such as: lung, uterus, vagina, urinary bladder, dermis of the skin, aorta. Elastic fb. are not stained in usual techniques; they are stained in dark brown with orcein and violet-blue, with Weigert method. violet-

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Figure 8. Elastin molecules are joined by covalent bonds to generate an extensive cross-linked network. Because each elastin molecule in the network can expand and contract like a random coil, the entire network can ti t k stretch and recoil like a rubber band. (Reproduced, with permission, from Alberts B et al: Molecular Biology of the Cell. Garland, 1983.) G l d 1983 )
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Connective tissue cells
According to their origin connec tissue cells - proper cells of the connective tissue origin, connec. and migrated cells, originating from the blood. Undifferentiated mesench. cells, fibroblasts, adipose cells and mast cells are proper cells of the connec. tissue, while macrophage, lymphocyte, plasma cell and polymorphonuclear cells (neutrophils, eosinophils and basophils) are cells migrated from the blood, with immune functions. The mesench. cells are similar to young fibroblasts for that being difficult to fibroblasts, recognize in adult life. They are situated usually along blood capillaries, called adventitial cells. They are smaller than fibroblasts, with elongated nuclei and coarse chromatin. These undifferentiated cells have the capacity to g p y give birth to proper cells of the connec. tissue, being involved in processes of restoring of damaged tissues. They can differentiate also in smooth muscle fb. In light micr. it has a basophilic cytopl., with p p y p processes which can come into contact with those of the neighboring cells and an obvious nucleolus.

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Fibroblasts are well represented in most varieties of connec. tissue; there are 2 cell types – the young fibroblast and the adult fibroblast, also called fibrocyte, which in fact are 2 functional stages of the same cell. The young fibroblast is responsible f th synth. of the fb. and of the amorphous i t ibl for the th f th fb d f th h intercell. subst. of th ll b t f the connec. tissue. In light micr., young fibroblasts are polyhedral, slightly fusiform or starstar-shaped, with numerous cytopl. processes, which are irreg. disposed. They are mediummedium-sized cells of about 20 μm with ovoid large pale nuclei and visible cells, m, ovoid, large, nucleolus and with basophilic cytopl. With the e.m., they have a well developed RER, as well as an abundant Golgi complex, involved in the synth. of fb. and intercell. intercell ground subst Mature fibroblasts are smaller than young ones with subst. ones, condensed chromatin in the nuclei and acidophilic cytopl., in light micr. It has also fewer and shorter cytopl. processes, than the young fibroblast. In e.m., mature fibroblasts have less developed RER and Golgi complex because the synth complex, synth. activity is finished. Even though, under adequate stimuli, it can regain its capacity of synth. This is the case of wound healing. Myofibroblast is a cell which has interm characteristics between fibroblast and cell, interm. characteristics, smooth muscle cell; in its cytopl. are present contr. filam. of actin and myosin. By its contr., this cell contributes to the retraction and the closure of the wounds, in the process of wound healing healing.
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Figure 1. Section of rat skin. A connective tissue layer (dermis) shows several fibroblasts (F), which are the elongated cells. H&E stain. Medium magnification.
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Fibroblasts - the capacity to synth. β interferons, with antitumoral and antiviral immunity; they express rec. for insulin, for LDL (low density lipopropteins), being able to decrease the levels of plasma cholest and lipids They also express rec cholest. lipids. rec. for the epidermic growth factor, EGF. By gradual storage of triglycerides in its cytopl., fibroblast becomes white adipose cell; in certain circumstances, fibroblast is able to change into chondroblat or into osteoblast osteoblast. Growth horm. and sexual horm. stimulate the synth. activity and mitoses of fibroblasts, while cortisol, ACTH and thyroid horm. inhibit the same processes. There are adipose cells or adipocytes of 2 diff Th di ll di t f different t t types: white and brown. I hit db . In adult life the white adipose cell is very well represented, while the brown adipose cell tends to almost disappear. The brown adipose cells are well represented in hibernating animals and in humans in the intrauterine life and in the newborn children, having an important role in the regulation of the body temperature, for its constant maintenance.

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The white adipose cell is also called unilocular, because it contains only one large droplet of lipids, in its cytopl., which is giving a characteristic asp. to this cell. White adipose cell is large, of about 100 μm in diameter, frequently disposed in groups, thus forming the white adipose tissue. The cell is accum. lipids gradually, each small amount of lipids which is stocked in the cytopl., is fusing with the already p y p , g y existent droplet, so that the unique droplet will become larger and larger, pushing the remaining cytopl. and the nucleus in the periphery. The usual techniques are using organic solvents, such as alcohol, xylol, which are solving the lipids from the cells, leaving an empty vacuole behind. So the image in light micr., of an white adipose cell will be of a thin layer of cytopl., containing a flattened nucleus, surrounding the vacuole left by the removed lipid g , g y p droplet, the so-called signet ring cell. If special techniques for lipids are used, the socells can be stained; this means a special fixation for lipids and staining methods, for ex. Sudan technique for lipids. With the e.m., the cells have the usual organelles, and small lipid droplets become visible; also, one can obs. that the droplets of lipids do not have membr., fact that makes possible the fusion of the droplets into one large vacuole. The white adipose cells are developing from lipoblasts or adipoblasts, cells very similar to fibroblasts, by gradual accum. of lipids into the cytopl. Under determined conditions, such as certain horm. stimuli, or after severe diets, white adipose cells regressed in young lipoblasts can regain their th i capacity of cell. di it f ll div.
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Unilocular adipose cells - the white adipose tissue or the unilocular adipose tissue. Isolated cells are rounded or ovoidal, but in the white adipose tissue they look polyhedral because they are closely packed together White adipose tissue is polyhedral, together. parted in incomplete lobules by small quantity of loose connec. tissue, containing blood vessels and nerves. Each cell is surrounded by retic. fb. and a b.m., forming a network of support for the white adipose cells Even if they are not always cells. apparent, blood vessels are well represented, in the tissue, white adipose tissue being richly vasc. Main functions of the unilocular adipose tissue are storing energy for the organism and offering temperature and mechanical isolation. White adipose cells store triglycerides; the source of fatty acids is the ingested food, chylomicrons, which are lipoproteins, synthetized in the liver – VLDL (very low density lipoproteins); white adipocytes are capable as well to synth. fatty acids and y p p ); p y p y y glycerol, from glucose. The coupling between fatty acids and glycerol phosphate (an interm. product of the glucose metab.) has as result triglycerides form. White adipose cells present insulin rec.; insulin is stimulating uptake of glucose into the cell, fatty acids synth. from glucose and synth. of a lipoprotein lipase, involved in the segregation of the fatty acids from the lipoprotein complexes.

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The lysis of triglycerides into fatty acids and glycerol is under the influence of the triglyceride lipase, a hormone-dependent lipase, which is activated by adenylate gy p hormone- p p y y cyclase, under the stimulation of norepinephrine. Norepinephrine is discharged at the level of sympath. nerve endings, present in the walls of the blood vessels of the white adipose tissue; unlike brown adipose cells, white adipose cells do not receive directly veget. sympath inerv veget sympath. inerv. Fatty acids are then transported by plasma albumin to other albumin, tissues and glycerol is metab. in the liver. The increase in the no. of unilocular adipose cells is leading to hyperplastic obesity, while an overaccum. of fat in the unilocular adipose cells, that become larger, is resulting in hypertrophic obesity. p , g , g yp p y Brown adipose tissue is also called multilocular, because the brown adipose cell has numerous lipid droplets in its cytopl., droplets are not fusing together. The appearance of this cell, in light micr. is that of a smaller cell than the unilocular di ll l l in h i ih ll located nucleus, which i d l hi h is adipose cell, polygonal i shape, sometimes with a centrally l not pushed by the lipids, visible like multiple empty unstained small vacuoles in an acidophilic cytopl. Brown adipose tissue is very rich in blood vessels, its arrangement looking very much like that of an endocrine gl otherwise responds to stimuli send by gl., sympathy. nerves, rapidly liberating the energy obtained by fat consumption. For this reason, it is well developed in hibernating species, being called improperly, hibernating gl., and in the human newborns. It is not developing during adult life, from any of the types of connec. tissue cells. In the e.m., brown fat cells have an abundance of mitochondria in their cytopl., involved in the oxidative mech. Mitochondria are rich in colored cytochromes, responsible for the brown color of the tissue. tissue
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Figure 2. Section of rat tongue. Several mast cells in the connective tissue surround muscle cells and blood vessels. PT stain. Medium magnification
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The exposure to low temperature is liberating norepinephrine into the brown adipose tissue, resulting in the activation of the horm. dependent lipase and hydrolysis of triglycerides into fatty acids and glycerol In contrast to the white glycerol. adipose (unilocular) adipose cell, in the brown adipose cell, fatty acids are metab. farther on, with an increase in oxygen consumption and heat prod. The heat is increasing the temperature of the tissue and of the blood which is passing through blood, it. Heat production is even more important, because the inner membr. of the mitochondria contain a protein called thermogenin, which impair the stockage of energy as ATP synth., instead the energy being dissipated as heat. Thermogenin may be increased in individuals which do not become obese, even when are overeating, while it may be reduced in obese individuals. The mast cells are proper cells of the connec tissue They orig from the bone connec. tissue. orig. marrow, their migration taking place during embryonic life, after that they become permanent residents of the connec. tissue. Even though they are very similar to basophil polymorphonuclear cells, they have different stem cells in the bone marrow. Mast cells are ovoid cells, of 20-30 μm in diam., but difficult to recognize 20in H.E. preparations; they become easily recognizable with toluidine blue, when y g p they have a reddish violet granulated asp.

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The abundance of gr. is covering the centrally situated nucleus, which often cannot be seen. The gr. are basophilic, of 0.3-0.5 μm in diam. and they contain a scroll-like 0.3scrollcore. The property that certain cells have, of changing the color of a dye they are stained with, is called metachromasia. Gr. of the mast cell are containing heparin, histamine, proteases, ECF-A (eosinophil chemotactic factor of anaphylaxis). These ,p , ECF- ( p p y ) subst. - pre-formed mediators, because the mast cell is liberating during allergic prereactions, newly formed mediators (leukotrienes –C4, D4, E4, also called slow reacting substance of anaphylaxis or SRS-A, prostaglandines etc.) which are synth. SRSand immediately released. Heparin is a well-known anticoagulant, while histamine is wellan inflammatory mediator. Anyway it has been well established that these subst. are synth. by the mast cells, being stored in its membr. packed gr. and released especially in the immediate hypersensitivity reactions (allergic reactions), such as the anaphylactic shock. This may occur when the org. is coming into contact with an antigen, called allergen, to which it was previously sensitized. The first contact with the antigen is inducing in the organism the synthesis of E immunoglobulins, a peculiar type of Ab, which will be fixed by specific rec. on the membr. of the mast cells. The sec. contact between the antigen and its corresponding antibody will determ. the form. of the antigen – antibody complexes, on the surface of the mast cells and thus will trigger the release of their gr. Histamine is contr. the smooth muscle fb. (for ex., in the small bronchi) and is increasing the permeability of the blood th bl d capillaries, producing b thi a d ill i d i by this dramatic f il ti failure of th bl d pressure. f the blood
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Figure 3. Mast-cell secretion. 1: IgE molecules are bound to the surface receptors. 2: After a second exposure to an antigen (eg, bee venom), IgE molecules bound to surface receptors are cross-linked by the antigen. This activates adenylate cyclase and results in the phosphorylation of certain proteins. 3: At the same time, Ca2+ enters the cell. 4: These events lead to intracellular fusion of specific granules and exocytosis of their contents 5: In addition phospholipases act on contents. addition, membrane phospholipids to produce leukotrienes. The process of extrusion does not damage the cell, which remains viable and synthesizes new granules. ECF-A, eosinophil chemotactic factor of anaphylaxis.
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Thank you for your attention!
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HISTOLOGY
Course 6
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

Connective tissue
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Newly formed mediators are enhancing the effects of the histamine. These reactions were first developed by the org as benefic immune defense reactions but in time at org. reactions, time, atopic individuals (approx. 10-15% of the whole population), they degenerated into 10pathologic conditions. Mast cells play an important role in the anaphylactic shock, which is sometimes a fatal condition, if it is not rapidly treated. , p y The molec. released by the mast cells are acting on a paracrine pattern, locally. There molec. are at least 2 populations of mast cells in the connec. tissue – one is called connec. connec. connec. tissue mast cell, found in the dermis of the skin and in the peritoneal cavity and the other is called mucosal mast cell, being present especially, in the lamina propria of the intestine and of the lung. The 2 mast cell populations have a different content of their gr. and they react differently to p y y pharmacologic ag. g ag. g Macrophages are the most important phagocytic cells of the org. In loose connec. connec. tissue is almost as well represented as the fibroblast and can be mistaken for it. They measure bet ee 10 a d 30 μm. Its nucleus ca have a pa e asp a d is usua y o a o easu e between 0 and ts uc eus can a e pale asp. and s usually oval or kidneykidney-shaped, eccentrically placed; in its cytopl. there are vacuoles or gr. of the cytopl. phagocytosed materials.

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The wandering macroph. are more active in phagocytosis than the fixed macroph. In e.m., the membr. of the macroph. has protrusions and identations, and the surf. is irreg. and waved. In the cytopl. of the macroph. lysosomes are abundant; they are fusing with the vacuoles of phagocytosed materials, forming so-called sec. lysosomes or sophagosomes, in which the digestion of the engulfed materials occurs. Fixed and wandering macroph. are different phases of the same cell and they can transform from one into another. The source of macroph. are blood monocytes, which are circ. cells in the blood for about 3 days, after that they are passing in the connec. tissue where they become macroph., li i and phagocytosing f 75 up t 100 d b h living d h t i for to days, t f to form th the mononuclear phagocytic syst. They are long-living phagocytic cells. Macrophages have longdifferent names, in different tissues, they are known as Kupffer cells in the liver, osteoclasts in the bone microglial cells in the nerv tissue Langerhans cells in the skin bone, nerv. tissue, skin. If a vital dye, such as trypan blue or India ink is inj. into a living org., macroph. are engulfing the dye in their cytopl. as phagocytic vacuoles, visible with the light micr. The transformation of the monocytes into macroph is illustrated by an increase in the cell macroph. size, in the protein synth., in the Golgi complex, in no. of lysossomes, microtubules and microfilam.

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Macroph. participate in cell-mediated immunity against bacteria, viruses, fungi, cellprotozoars, metazoars, as well as in the antitumoral and anti-graft immunity. They antiare involved in the extrahepatic bile production in iron and fat metab in production, metab., decreasing plasma lipids and in the destruction of aged circ. blood cells. In pathol. cond., such as chronic specific inflammations, macroph. may increase in vol. vol and arrange in clusters to form epithelioid cells or several cells may fuse cells, together, to form giant multinucleated cells. The lymphocytes found in the connec. tissue are usually small lymphocytes, of 668 µm i di in diam. Th appear with condensed, dark violet, rounded nuclei surrounded They ith d d d k i l t d d l i d d by a thin rim of slightly basophilic cytopl. This small amount of cytopl. is hardly visible, the cells being recognizable under their nuclei. Generally in org. exist 2 types of lymphocytes T and B lymphocytes They can be differentiated only using lymphocytes, lymphocytes. immunological techniques or special e.m., but not in the light micr. T lymphocytes are of several types, generally initiators of the cell-mediated immune response and cellin fact involved in both types of immune responses (humoral and cellular) B cellular). lymphocytes, after antigenic stimulus, suffer several cell. div. and cell. differentiation, generating plasma cells, which will secrete Ab or Ig specific to the Ag.
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Macroph. are very important in the immune defense of the org., they phagocytose remains of dead cells, waist products, microorganisms and, generally, foreign particles, penetrated in the org. In the same context, another important function of the th macroph. i th t of antigen - presenting cells, b i th most i h is that f ti ti ll being the t important antigen t t ti – presenting cells in the org., an obligatory stage in the develop. of the immune response. Plasma cells - usually few in the connec. tissue, an exception is the intestinal lamina propria where they are numerous because of the permanent propria, numerous, antigenic contact in this area. In light micr. plasma cells are ovoid, with slightly peripheral nucleus and basophilic cytopl. The h Th chromatin i th nucleus i di ti in the l is disposed i b l d in bulges i it periphery, l in its i h leaving i between the condensed zones, lighter areas, this arrangement giving to the nucleus the asp. of the spokes of a wheel or of a clock-face appearance, by which clockthe cell is recognizable in the light micr Ultramicroscopic the plasma cell has well micr. developed RER and Golgi complex, which is visible with the light micr. as a lighter zone, next to the nucleus. These organelles are involved in the synth of the Ab As biochemical nature synth. Ab. antibodies are GP, each Ab secreted by a plasma cell is corresponding to the antigen, which determined its production and reacts with it.

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The form. of the antigen-antibody complex, neutralize the harmful effect of the antigenantigen; for ex. of a bacterial toxin. This is the humoral immune response of the org. org The synth of the Ab in a plasma cell usually requires the intervention of the synth. macroph., as antigen -presenting cell, the coop. between different types of immune cells, being fundamental for the develop. of the immune response. Plasma cells live around 20 days while they secrete Ab and after that they die days, that, die. Granulocytes are also found in the connec. tissue as migrated cells; they are also known as leukocytes (leukos in Greek - white) - white blood el. From these PMN cells, cells one can more often see eosinophils which have acidophilic refractive gr eosinophils, gr., spreaded in all the cytopl. and nuclei with 2 lobes. In the e.m., the gr. show a denser ccrystalline core, called internum, containing the major basic protein, which is involved in the antiparasitic defense The perypheral part of the gr is less dense defense. gr. and contains enzymes, such as histaminase, involved in neutralization of histamine, released during allergic reactions These cells have also the capacity to engulf Ag-Ab complexes. They increase in no. in allergic and parasitic diseases, Agbeing involved in the neutralization of parasites and of the histamine.

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Neutrophils are not so common in the connec. tissue, with the exception of the intestinal mucosa, where they exert their antibacterial activity. They are easily recognizable by their nucleus with 3-5 lobes clearly visible The specific nucleus, 3- lobes, visible. specific, neutrophilic gr. of the neutrophils are hardly visible with the light micr. They are lysosomes and they contain enzymes, such as lysozyme, myeloperoxidase, phosphatases. phosphatases They are specialized in phagocytosis of bacteria penetrated in the bacteria, org.; dead neutrophils, with debris and bacteria form the pus. Unlike macroph., neutrophils are short –life phagocytic cells; they live about 7 hours in the blood, they pass through diapedesis in the connec. tissue, in different org., where their phagocytic activity is exerted once or twice (usually once) and after that they die. They live in the connec. tissue 1-3 days. 1Basophils are as rare in the connec tissue as they are in the blood They have connec. tissue, blood. an S-shaped or horse-shoe nucleus, which is masked by large, basophilic and Shorsemetachromatic gr., with the same content like those of the mast cell. They also exhibit rec. for E Ig on their cell. membr.; they release histamine into the blood, during allergic reactions.

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Figure 4. Photomicrograph of unilocular adipose tissue of a young mammal. Arrows show nuclei of adipocytes (fat cells) compressed against the cell membrane. Note that, although most cells are unilocular, there are several cells (asterisks) with small lipid droplets in their cytoplasm an indication that cytoplasm, their differentiation is not yet complete. Pararosaniline—toluidine blue (PT) stain. Medium magnification.

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Figure 5. Development of fat cells. Undifferentiated mesenchymal cells are transformed into lipoblasts that accumulate fat and thus give rise to mature fat cells. When a large amount of lipid is mobilized by the body, body mature unilocular fat cells return to the lipoblast stage. Undifferentiated mesenchymal cells also give rise to a variety of other cell types, including fibroblasts. The mature fat cell is larger than that shown here in relation to the other cell types types.

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Figure 6. Section of pancreas from a rat injected with the vital dye trypan blue. Note that 3 macrophages (arrows) have engulfed and accumulated the dye in the form of granules. H&E stain Low magnification stain. magnification.
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Figure 7. Portion of a chronically inflamed intestinal villus. The plasma cells are characterized by their size and abundant basophilic cytoplasm (rough endoplasmic reticulum) and are involved in the synthesis of antibodies. A large Golgi complex (arrows) is where the terminal glycosylation of the antibodies (glycoproteins) occurs. Plasma cells produce antibodies of importance in immune reactions. PT stain. Medium magnification.
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Types of connective tissue
According to the consistency of the amorphous intercell. subst. or to the predominance of one of the el. of the connec. tissue – cells, one category of fb., several types of connec. tissue are distinguished. Generally there exist soft connec distinguished. Generally, connec. tissues and hard varieties of connec. tissue – the cartilage and the bone. As varieties of proper connec. tissue one can distinguish loose connec. tissue and dense connec. tissue. connec tissue. Loose connec. tissue, which is also called areolar is much more well represented tissue, than the dense connec. tissue type. In loose connec. tissue all the comp. of the connec. connec tissue are in almost equal proportions - cells fibers and ground subst In are, cells, subst. light micr. it appears dispersed, with a small amount of fb., the most frequent cells being the fibroblasts and macroph., although any of the types of cells of the connec. tissue can be found Loose connec tissue is filling the sp between the muscle fb found. connec. sp. fb. and striated muscle bundles, free sp. between the org. and most important, it encircles the lymphatic and the blood vessels and the nerves, all over the org., thus forming a sp. of diffusion between different types of tissues. It is also found in the papillary dermis or superficial dermis of the skin, in the hypodermis, in the serous membr. or mesothelia like pleura, peritoneum and pericardium, and it is forming the pp p g g tissue of support for epith.l linings of mucous membr., which are lining natural cavit. or hollow organs – this is called the lamina propria. propria.
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Figure 8. Dense irregular connective tissue from human dermis contains thick bundles of collagen g g g fibers, fibroblast nuclei (arrowheads), and a few small blood vessels (bv). H&E stain. Medium magnification.
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In org. - several types of lamina propria. A so-called common type, which is found in sothe oral cavity and in the urinary bladder considered by some authors “of resistance bladder, of resistance” because of its higher content of fb. We distinguish 2 peculiar types of lamina propria, in which the main characteristic is the richness of cells situated in a loose connec tissue: the intestinal lamina propria and the cells, connec. lamina propria of the endometrium, the mucous membrane of the uterus. The intestinal lamina propria is the connec. tissue of support of the intestinal epith., it t d in the f the intestinal illi d between th i t ti l Li b k h gl., situated i th axes of th i t ti l villi and b t the intestinal Lieberkuhn l characterized by numerous migrated cells of the connec. tissue, which are cells of immune defense. Thus one will obs. many lymphocytes, macroph., plasma cells, eosinophils etc At a certain degree this is also true for the lamina propria of the etc. respiratory tract. The lamina propria of the uterus (endometrium) is rich in proper cells of the connec. tissue, tissue more precisely fibroblasts but which have rec for the female sexual horm rec. horm., estrogens and progesterone. Under the influence of the horm., the cells suffer cyclic changes, according to the ovarian cycle, for ex. they charge themselves with embryo trophic subst mainly glycogen but also protein and lipids This storing process subst., glycogen, lipids. enlarges the cells, which become rounded and even arranged epithelium-like, very epitheliumclose one to another, in the superf. part of the lamina propria, next to the epith.; these cells. are called decidual cells
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Figure 9. Longitudinal section of dense 9 regular connective tissue from a tendon. A: Thick bundles of parallel collagen fibers fill the intercellular spaces between fibroblasts. Low magnification. B: Higher magnification view of a tendon of a young animal. Note active fibroblasts with prominent Golgi regions and dark cytoplasm rich in RNA. PT stain
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Figure 10. Longitudinal section of dense 10 regular connective tissue (tendon). Bundles of collagen fibers fill the spaces between the elongated fibroblasts. H&E stain. Medium magnification
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The mucous membr. of the uterus, in this stage is called decidua, being capable to nourish the egg, if the fertilization of the ovum did occur. This is a cellular lamina propria, propria also called cytogenic being capable to regenerate after the expelling of cytogenic, the mucous membr., each month. The dense connec. tissue is characterized by a clear predominance of the collagen fb., fb it has fewer cells than the loose connec tissue fibroblasts being the most connec. tissue, common cells. This type of tissue is not so flexible like the loose type and is much more resistant to stress. There are 2 varieties of dense connec. tissue: dense irreg. connec. irreg connec tissue and dense regular connec tissue In the dense irreg connec connec. tissue. irreg. connec. tissue, collagen fb. are forming bundles, which do not have a definite orientation, thus giving to the tissue a certain degree of resistance to mechanical stress exerted from all directions Dense irreg connec tissue is characteristic to the directions. irreg. connec. proper dermis of the skin, while papillary dermis and hypodermis are made of rather loose connec. tissue. The standard preparation shows us bundles of collagen fb. of variable thickness, which seem to be totally disoriented, in fact these bundles are following in a certain degree the surf. of the skin. The fibroblasts, as well as the blood vessels are quite poorly represented. The capsule p g g of the parench. org. is also made of dense irreg. connec. tissue.

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In the dense regular connec. tissue, the collagen bundles have a definite direction, tissue, following a clear regular pattern. Collagen fb. - parallel one to another, at the same level, by this arrangement offering to the tissue great resistance to different mechanical forces. Most common ex. of regular dense connec. tissues are the aponeurosis, the tendon and the proper tissue of the cornea. The aponeurosis - superposed sheaths of collagen fb.; in each sheath or lamella the fb. are parallel to one another, but perpend. situated on the direction of the fb. of the neighboring plan. Between the fb. flattened fibroblasts are obs. The tendons are cylinders, which have a whitish appearance and are not extensible, cylinders, because of their collagen richness, their main funct. being that of attaching striated muscles to bones. They are made of parallel, closely packed bundles of collagen fb., with a small quantity of amorphous intercell. subst. between the fb. and flattened fibroblasts, which tend to envelop the collagen fb. with their cytop. With the light micr., only the nuclei of the fibroblasts are visible, elongated and parallel to the collagen bundles, th cytopl. b i di b dl the t l being dispersed b t d between th fb Th collagen b dl are the fb. The ll bundles enveloped in a fine layer of loose connec. tissue - endotendineum. These primary bundles are aggregating into larger bundles, sec. bundles, enveloped in their turn, in a layer of loose connec tissue - peritendineum containing blood vessels and nerves The connec. peritendineum, nerves. whole tendon is enveloped by a dense connec. tissue layer - epitendineum. An important asp. is that of the continuity between the connec. tissue sheaths of the tendon with the correspondent sheaths of connec tissue which are enveloping the striated connec. tissue, muscle bundles.
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The proper tissue of the cornea, or stroma of the cornea is situated between the cornea, ant. and the post. epith. of the cornea, which are squamous stratif. nonkeratinized and respectively, simple squamous epith. This tissue consists of approx. 200 lamellas of parallel collagen bundles, which cross to an angle to one another. The collagen fibrils are p g parallel between one another and they are occupying the whole y py g length of the cornea. Few fibroblasts can be seen between the lamellas, they are flattened in a manner, which suggests the wings of a butterfly. Lamellas are immersed in a small amount of amorphous intercellular subst., rich in chondroitin sulfate. Normally, cornea is an avascular tissue. Retic. tissue is containing abundant retic. fb. on which primitive retic. cells are anchored, considered by some authors to have the same developing p , y p g potential as the mesench. primitive cells. Retic. tissue is forming the framework of the bloodbloodlymph forming org., supporting the free cells of these org. Retic. cells - expansions, which sometimes are establishing contacts with those of the neighboring cells and large nuclei with dispersed chromatin and one or two obvious nucleoli. Even if they have all the same star-shape, their funct. are different; they can be phagocytic starcells, known as reticular-macroph., or of synth. of the retic. fb., forming the 3reticular3dimensional network, which are fibroblasts, or retic. cells funct. as ag-presenting agcells, in the developing of the immune response. Retic. tissue is stained by silver impregnation, the special method, which stains the retic. fb. in black.

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The predom. of the elastic fb. is consid. by some authors as a variety of connec. tissue, the elastic tissue. The abundance of elastic fb. confers to this tissue a yellowish asp Elastic tissue or the predom of elastic fb is characteristic for certain asp. predom. fb. ligaments, such as the yellow ligaments of the vertebral column. A regular disp. of the elastic fb. has been already discussed: in the walls of the large arteries, as the aorta, aorta where the elastic fb are forming fenestrated lamellas or membr with a fb. membr., concentric disp. Besides the lamellas, there exists here a small amount of collagen fb., intercell. subst. and few fibroblasts. Mucous connec tissue is abundant in amorph Intercell subst has a gelatin connec. amorph. Intercell. subst., consistency, few fb. and as predominating cells, fibroblasts. It is found in the umbilical cord, called Wharton`s jelly and in the pulp of young teeth. White and brown adipose tissues have been discussed with white and brown adipose cells cells.

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Thank you for your attention!
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HISTOLOGY
Course 7
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

Connective tissue The bone
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The cartilage
The cart. is a specialized type of connec. tissue, with a more consistent intercell. cart. matrix, even if this matrix is not calcified, how it is the case with the bone matrix. Cart. hibit th C t exhibits the property of resilience, which represents a li it d elasticity, t f ili hi h t limited l ti it comparable to that of a sponge (the capacity to suffer mech. stresses, without permanent distortion). Cart. is resilient and has a smooth surf., it is acting as a shock absorber and is a sliding area for joints supporting the movements of the joints, skeleton. The epiphyseal plates (growth cart.) are made of hyaline cart., providing the growth in length of the long bones, by endochondral ossification. The Th cart. is made of i t t i d f intercell. matrix and chondrocytes, cart. cells. Th i t ll ti d h d t t ll The intercell. ll matrix is made of fb., included in the ground subst. Chondrocytes - in cavit. or lacunae of the matrix, as a conseq. of the high consistency of the cart. matrix. Chondrocytes are not so numerous they represent only approx 1 up to 10% of numerous, approx. 1, the total vol. of the cart. In the human org., are present 3 types of cart.: hyaline cart. is the type, which is best represented and which contains type II collagen fibrils, in its matrix; elastic fibrils cart., besides type II collagen fibrils, contains in its matrix numerous elastic fb.; fibrous cartilage, most resistant to mechanical stress, contains type I collagen fb.

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Cart. - avascular, its nutrition - by diffusion from perichondrium (connec. tissue) or from the synovial fluid, in joint cavit. Cart. does not contain lymph. vessels or nerves. nerves Perichondrium is a dense connec. tissue layer, which is always covering the cart., with the exception of the artic. cart., which is devoid of perichondrium. Hyaline cart., b t represented in th org., h macroscopically a white, slightly H li cart. best t t d i the has i ll hit li htl bluish asp. and is almost translucent. It is localized in the skeleton of the embryo, till it is gradually replaced by bone, it is forming the epiphyseal plates, which are ensuring th growth i l i the th in length of th l th f the long b bones, until puberty, when growth i til b t h th is ceased. In adults, the hyaline cart. is form. the artic. cart. of the mobile joints; it is found in the respiratory tract – in the nose, larynx, trachea and large bronchi; it is covering the ventral ends of the ribs where they articulate with the sternum ribs, sternum. Cart. matrix contains approx. 80% water and electrolytes; 40% of its dry weight is represented by the collagen fibrils, embedded in the ground subst. (amorph. intercell. subst.). intercell subst ) Collagen fibrils of the cart matrix are not distinguishable in light cart. micr., because they are too small for the resolution power of the light micr. and because the refractive index of the fibrils is almost the same with that of the ground subst., included. fb. subst in which they are included Collagen type II fibrils do not form fb
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Figure 1. Photomicrograph of hyaline cartilage. The extracellular matrix was previously digested with papain to enhance the oriented aggregates of variously disposed collagen type II fibrils. These aggregates appear as black areas. Picrosirius—polarized pp p light stain. Medium magnification.
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Figure 2. Photomicrograph of hyaline cartilage. Chondrocytes are located in matrix lacunae, and most belong to isogenous groups The upper and lower groups. parts of the figure show the perichondrium stained pink. Note the gradual differentiation of cells from the perichondrium into chondrocytes. H&E stain. Low magnification
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The ground subst. of the cart. matrix is made of PG with sulfated GAG, especially subst. chondroitin 4-sulfate, chondroitin 6-sulfate and keratan sulfate. As many as 200 46PG molec. may bind with long hyaluronic acid molec., thus are appearing PG aggregates, which are binding to collagen. These complex molec. may reach up to 4 μm long, which explains the high consistency of the cart. matrix. PG molec. - the g, p g y asp. of a brush (a bottle brush), where the stem of the brush is the protein core and the bristles are the radiated GAG. Being electronegative molec. (polyanions), GAG are binding water and ions, which are acting as shock absorber; aspect important especially in the case of the artic. cart. The specific GP of the cart. matrix - chondronectin, an adhesion molec. which ensures the adherence of the chondrocytes to the matrix. The cart. matrix bordering the lacunae - a higher content of GAG and a lower content of collagen - capsular or territorial area, being intensely basophilic, metachromatic and PAS positive, while the cart. matrix between the lacunae, called interterritorial area, has a lower stain affinity. In living tissue, chondrocytes appear as round cells, of 10-30 μm in diam.; they tissue, 10are filing completely the lacunae, forming g p of 2-3, up to 8 cells in one cavity, g p y g groups 2p y result. from the mitotic div. of a single initial chondrocyte. Chondrocytes may form parallel rows -isogenous groups, like in the epiphyseal plates of the long bones. Chondrocytes shrink during histol. technique, so they appear in light micr. as starstarshaped cells, with irreg. shape, by their retraction from the periphery.
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In the e.m., especially young chondrocytes show fine processes, which penetrate into the matrix; by these processes, the chondrocytes are nourishing, since cart. is avascular. Through these processes, chondrocytes are also abs. and secreting small amounts of cart. matrix. They have abundant RER, Golgi complex and lysosomes. Chondrocytes secrete collagen and ground subst. and they keep the cap. of cell. div. Perichondrium is a layer of dense connec. tissue, which is always covering the hyaline cart., with the exception of the artic. cart. Perichondrium is vital for nutrition and growth of the cart. Perichondrium – 2 layers: an outer layer, rich in collagen type I fb., fibroblasts and blood vessels, being involved especially in the nourishment of the cart. and an inner layer, rich in cells resembling fibroblasts chondroblasts, secreting cart. matrix; as the matrix is secreted, the cells become chondrocytes and are trapped inside the lacunae. Young chondrocytes are elliptic cells, their log axis is parallel with the surf. of the cart. The growth of the cart. is accomplished by 2 processes – appositional growth and interst. growth. Appositional growth takes place by differentiation of the cells of the perichondrium, which are becoming chondroblasts and are secreting cart. matrix, deposited l d it d layer after l ft layer, i th periphery, b apposition. I t t growth t k in the i h by iti Interst. th takes place by cell. div. of the chondrocytes, inside the lacunae; this will increase the vol. of the lacunae and conseq., the vol.of the whole cart. Interst. growth is usually less important than real growth through apposition. i t t th l th th h iti
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Interst. growth occurs in artic. cart., which do not show perichondrium and in the case of the epiphyseal plates of the long bones, being involved in the process of the endochondral ossification The nutrition of the cart is achieved by diffusion ossification.The cart. through the solute water of the cart. matrix. Cart. has a low metab. rate, main being the anaerobic glycolysis. The synth. of the GAG of the cart. matrix is stimulated by GH (growth horm ) thyroid horm and testosterone being inhibited horm.), horm. testosterone, by estradiol and cortisol. Cart. growth is under the major influence of the pituitary GH or somatotropin. GH does not act directly on the cart., its action being mediated by somatomedin C synth. in the liver, under the influence of GH. Cart. is deriving from the embryonic mesench., which is forming the mesench. condensations. In the mass of these condensations will differentiate chondroblasts, which will secrete cart. matrix. Hyaline cart. may suffer degener., with age. Most common degener. process is the calcification of the matrix, leading to the death of the chondrocytes. Asbestiform degener. is characterized by the dehydration of the cart. and form. of thick, aggregated, abnormal, collagen fibrils; thus the cart. becomes fragile and may suffer ruptures when submitted to stresses. The regener. of the cart. is difficult g y and limited; for this reason, sometimes a graft of cart. may be needed.

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Elastic cart. is found in the auricle of the ear, in the wings of the nasal pyramid, in the external auditory canals, in the eustachian tubes, in the epiglottis, in the small bronchi and in the cuneiform cart. of the larynx. Elastic cart. is poorly represented in the org., compared to the hyaline cart. Besides collagen type II fibrils, the elastic cart. – has an abundant network of elastic fb., conferring it a macroscopically yellowish asp. Elastic cart. contains fewer chondrocytes in the lacunae, than the hyaline cart. and it may gradually continue with th h li cart. (f ex. i some of th cart. of th l ti ith the hyaline t (for in f the t f the larynx). Th ) The elastic fb. in the matrix are surrounding in arches the lacunae (nest-like). (nestElastic cart. has perichondrium, which ensures its nutrition and its growth. Elastic fb. of the elastic cart. matrix are making it more resistant to degener. processes, than the hyaline cart.

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Figure 3. Photomicrograph of elastic cartilage, stained for elastic fibers. Cells are not stained. This flexible cartilage is present, for example, in the auricle of the ear and in the epiglottis. Resorcin stain Medium magnification stain. magnification.

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Fibrous cart. - the most rare variety of all. It has interm. characteristics, between dense connec. tissue and hyaline cart. Fibrous cart. is usually assoc. with dense y y connec. tissue, when we obs. a gradual transition between the 2 types of tissues. It is found in the intervertebral disks, in the attachments of some tendons and ligaments to bones and in symphysis pubis. Chondrocytes of the fibrous cart. are usually disp. in parallel rows, separated by parallel collagen bundles. The matrix of the fibrous cartilage has an acidophilic asp. in light micr., because of the collagen type I bundles, the g g g yp ground subst. being less g abundant. Chondrocytes show a flattened asp., due to the compression exerted on them by the collagen bundles. Fibrous cart. is devoid of perichondrium; its nutrition is tributary to small regions of vasc. loose connec. tissue, interspersed between the collagen bundles. Intervertebral disks are made of an outer fibrous ring of dense connec. tissue and fibrous cart. (annulus fibrosus) and a centrally located pulpous nucleus (nucleus pulposus), made of a mucous or viscous consistent connec. tissue. When the disk is submitted to overcharge, its rupture is usually located in its post. region, where it contains fewer collagen bundles. The conseq. of the rupture, is the expulsion of the nucleus pulposus, which slips between the vertebrae, compressing the spinal nerves or the spinal cord. This condition is called herniation of the intervertebral disk and is resulting in severe pain, usually located in the lower lumbar region and in neurologic disturbances. di t b
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Figure 4. Photomicrograph of fibrocartilage. Note the rows of chondrocytes separated by y p y collagen fibers. Fibrocartilage is frequently found in the insertion of tendons on the epiphyseal hyaline cartilage. Picrosirius-hematoxylin stain. Medium magnification. M di ifi ti
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The bone
Bone tissue is one of the hardest tissues of the human org., with a calcified (mineralized) matrix. Bones are form. the skeleton, which supports the soft parts of the body, protects y vital org. situated in the thoracic and cranial cavit. and provides shelter for the bone marrow, where the blood cells are formed. It is the main organism’s reserve of calcium, phosphates and other ions; body movements are based on the lever syst., made by the human skeleton. Bone tissue is made of intercell. calcified matrix and 3 types of cells: osteoblasts, osteocytes and osteoclasts. Osteobl. are synth. the organic components of the matrix and are mineralizing it. Osteocytes are situated in lacunae (cavit.) inside the matrix. Osteocl. giant, multinucleated cells, responsible for the resorption and remodeling of the bone. Inside the matrix are present canaliculi, which are involved in the exchanges between p g blood and osteocytes. Osteocytes - very fine cytopl. processes, by which they establish connections with the neighbor cells, with the ext. and the int. bone surf. and with the blood capillaries. The microscopic studies of the bone, on ground sect. (grinding slices of bone with abrasives, until they become very thin and translucent, with no staining) make possible the obs. of the lacunae and canaliculi, which appear black, empty, filled with air. Another technique, used for obs. of the bone tissue, in light micr., is using the decalcification of the bone with acids; the decalcified bone is then sectioned and stained.
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Figure 5. Photomicrograph of dried bone ground very thin. The lacunae and canaliculi filled with air deflect the light and appear dark, showing th communication b t h i the i ti between th these structures through which nutrients derived from blood vessels flow. Medium magnification.

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Bone matrix contains inorganic matter, which represents approx. 50% of its dry weight. Best represented are calcium and phosphorus, but bicarbonate, citrate, potassium, sodium and magnesium, are also present. Studies using X-ray Xdiffraction, have demonstr. that calcium and phosphorus form hydroxyapatite p p y y p crystals (Ca10 (PO4)6 (OH)2). A significant amount of noncrystalline calcium phosphate is also present in the bone matrix. With the e.m., the hydroxyapatite crystals appear as parallelogram thin plates of 40 x 25 x 3 nm. They lie along collagen fibrils, which represents 90% of the matrix, being surrounded by the ground subst. Around the crystals forms a layer of water and ions, called the hydration layer or the solvation layer (hydration shell or ionic hydrated layer) which permits the exchange of ions between the crystals and the int. environment of the org. The org. matter, besides collagen type I, contains the ground subst., made of PG and bone specific GP, such as bone sialoprotein and osteocalcin. These GP contain numerous carboxy-glutamic acid residues, which bind calcium avidly, carboxyfacilit. the calcif. of the bone matrix. GAG found in the bone matrix are: chondroitin 4-sulfate, chondroitin 6-sulfate and k t sulfate. The association between the lf t h d iti 6- lf t d keratan lf t Th i ti b t th collagen type I fb. and the hydroxyapatite crystals, explains the characteristic hardness of the bone.

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Osteobl. are secreting the org. comp. of the bone matrix (collagen, PG and GP) and they are as well involved, in the calcification of the matrix. They appear always situated on the surf. of the bone lamellae, side by side, resembling to a simple epith. (epithelium-like). (epitheliumDuring synth. of bone matrix, osteobl. appear cuboidal to columnar, with basophilic cytopl. and high alkaline p g phosphatase activity, while when their synth. activity decreases, they p y, y y , y appear flattened, poorly basophilic and with low alkaline phosphatase activity. Osteobl. show processes by which they come into contact with the neighbor cells. As they secrete bone maytrix, they are trapped in cavit. or lacunae of the matrix. Lacunae and canaliculi are formed as a conseq. of the deposition of the matrix around a cell and its processes. Osteobl. are polarized cells, their apical pole is oriented toward the just synth. bone lamella. Osteobl. have well developed RER and Golgi complex, in e.m. Each newly synth. bone matrix layer, is lied down between osteobl. and older bone matrix, providing bone growth by apposition. After the synth. of the org. comp., takes place the calcification, by deposition of calcium salts in the org. matrix. Tetracycline is fluorescent, when penetrates in the bone matrix. Tetracycline is administered twice, in 2 inj., at an interval of 5 days and after that a bone biopsy is obtained. The secti. are studied with a fluorescent microscope; the dist. between the two fluorescent layers is proportional to the rate of the bone apposition. This method may diagnose diseases as osteomalacia, in which calcif. of the bone matrix is insufficient or osteitis fibrosa cystica, in which appears increased bone lysis by osteocl. and conseq. cystic and fib bone d degeneration. ti d fibrous b ti
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Figure 6. Events that occur during intramembranous ossification. Osteoblasts are synthesizing collagen, which forms a strand of matrix that traps cells. As this occurs, the osteoblasts gradually differentiate to become osteocytes. The lower part of the drawing shows an osteoblast being trapped in newly formed bone matrix
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Osteocytes derive from osteobl. and are situated in lacunae, disposed between bone lamellae. A single osteocyte is found in each lacuna The narrow canaliculi lamellae lacuna. canaliculi, between the lacunae are hosting processes of osteocytes, by which they establish gap junct., one with another. Chains of up to 15 cells may pass nutrients one from another. another A small amount of ground subst is lining the lacunae and the canaliculi subst. canaliculi, inside the bone matrix; the exchanges between osteocytes and the blood, take place through it. Osteocytes are almond-shaped, flat cells; in the e.m. - the decrease of RER and almondGolgi complex and a condensed nucleus when compared to osteobl. Under the influence of the horm. stimuli (e g parathyroid horm ) osteocytes are able to horm (e.g. horm.), secrete and to absorb small amounts of the surrounding matrix, being responsible for the viability of the bone matrix. Death of osteocytes is triggering the destruction of the bone matrix, by resorption matrix resorption.

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Osteocl. are giant, motile cells, which show numerous expansions and have a dilated dil d portion, where they contain 5 up to 50 nuclei, or more. Th i cell. i h h i l i Their ll expansions are branched and irreg. These cells are responsible for bone resorption; where they are engaged in bone lysis, they appear situated in ti ll d d lacunae on th b t i (lamellae) called H ll ) ll d Howship’s hi ’ enzymatically produced l the bone matrix (l lacunae. Osteocl. originate from the fusion of the blood monocytes, they are part of the mononuclear phagocyte syst. In light micr., th usually show acidophilic cytopl. I l h t t I li ht i they ll h id hili t l In active osteocl., engaged in bone resorption, the face of the cells facing bone lamellae is folded and irreg., form. the ruffled border. This struct. increases the surf. abs. surf of abs and provides a tight attach to the bone; here small particles of the attach. bone matrix may be easily submitted to enzymatic action. The ruffled border contains actin filam. Between the folds of the ruffled b d as well as i cytopl. vacuoles were B t th f ld f th ffl d border, ll in t l l identifed Ca crystals and partially digested collagen fb. In e.m., osteocl. contain RER, well developed Golgi complex, mitochondria and lysosomes. Osteocl. secrete collagenase and several enzymes; they are abs Ca and phosphate ions abs. from the bone matrix, liberating them into the blood and they are digesting the org. comp. of the bone matrix.

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Figure 7. Section showing 3 osteoclasts (arrows) digesting bone tissue. The osteoclast is a large cell with several nuclei and a ruffled border close to the bone matrix Note the clear matrix. compartment where the process of bone erosion occurs. This compartment is acidified by a proton pump localized in the osteoclast membrane. It is the place of decalcification and matrix digestion and can be compared to a giant extracellular lysosome. Chondroclasts found in eroded regions of epiphyseal calcified cartilage are similar in shape to osteoclasts.
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Periosteum and endosteum - layers of connec. tissue, containing bone-forming bonecells, which are covering the outer, respectively, the inner bone surf. Periosteum is made of an ext. layer, rich in collagen fb. and fibroblasts; bundles of these collagen fb. p g penetrate into the bone matrix, binding p g periosteum to the bone – Sharpey’s fb. The int. layer of the periosteum is richer in cells; it is containing flattened cells, which are dividing and differentiating into osteobl., called osteoprogenitor cells. They contain poorly developed RER and Golgi complex. Autoradiographic st. show that these cells incorporate tritium-labeled thymidine, tritiumwhich is afterwards detected in osteob. They are important in bone growth and bone repair. Endosteum is lining the int. of the bone cavit., found inside the bone. It is thinner than the periosteum, showing a single layer of osteoprogenitor cells. Periosteum and endosteum are involved in nutrition, growth and repair of the bone tissue; they represent a permanent source of osteobl. Periosteum and endosteum must be protected, during bone surgery, to ensure the complete restore of the bone.

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Thank you for your attention!
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HISTOLOGY
Course 8
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

The Bone
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Bone form.; calcif. of the bone matrix
A protein inducing the prolif. and differentiation of the osteoprogenitor cells – bone morphogenetic protein, was identified in the bone matrix. The decrease with age, of this protein, protein is involved in the develop of the senile osteoporosis The prod of autoAb develop. osteoporosis. prod. against bone morphogenetic protein, is incriminated in the develop. of the juvenile osteoporosis. The calcif of the bone matrix begins with local gradual accum of Ca and phosphate calcif. accum. ions, which reaching the adequate concentration, precipitates in calcium phosphate crystals. This process is stimulated by alkaline phosphatase, secreted by osteobl. Osteobl. Osteobl are able to concentrate calcium and phosphate in intracytopl vesicles and to intracytopl. release this content, when needed, outside the cell. Local concentration of Ca, phosphate and alkaline phosphatase will reach a critical threshold (mineralization threshold), threshold) resulting in bone spicules containing hydroxyapatite crystals; this is a calcif spicules, calcif. center or nucleus. Alkaline phosphatase is hydrolysing org. phosphates, thus liberating phosphate ions, which become free to combine with Ca to form inorg Ca phosphate Sulfated GAG are Ca, inorg. phosphate. promoting the calcif. of the bone matrix, by form. first, of Ca sulfate. High phosphates concentration will lead to the replace in the Ca sulfate molec., of sulfates, with exceeding phosphates; like this is formed Ca phosphate and the sulfated GAG molec. are restored.
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Bone involvement in the calcium and phosphate metabolism; metabolism; hormonal control of blood calcium
The skeleton contains 99% of the whole calcium in the organism, being body’s g g y calcium reserve. Continuous interchange takes place, between blood Ca and bone Ca. To maintain a stable blood Ca conc., Ca abs. from food is either rapidly deposited in bones or is excreted in stool or urine. Bone Ca is mobilized, when the blood Ca conc. decreases. A simple mech. of transfer of Ca ions from the hydroxyapatite crystals to interst. fluid, wherefrom Ca passes into the blood, is the first and simpler solution. This mech. involves especially the spongy bone; newly formed, slightly calcif. bone lamellae are those to furnish Ca, in the first place. Most important in the control of Ca metab. – horm. regulation. Parathyroid horm. is acting on all 3 types of bone cells, which show rec. for it. Main action of parathyroid horm. is that of extracting Ca from the bone matrix, thus increasing the blood level of Ca. It promotes the removal of osteobl. from the surf. of the bone lamellae, leaving them free, for the action of the osteocl. When a slight, transitory decrease of the blood Ca occurs, the parathyroid horm. will stimulate a limited bone lysis of the bone matrix bordering the lacunae, accomplished by osteocytes.
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In the case of a severe and prolonged drop of the blood Ca, parathyroid horm. will stimulate the form. of new osteocl. from the blood monocyyes and will induce in osteocl., the develop. of the ruffled border and the enzyme synth. The increase of the no. of osteocl. and their activation, will result in increased bone lysis and liberation of Ca ions, which will reach into the blood and will reestablish normal blood Ca level level. Hyperparathyroidism (increased secretion of parathyroid horm., in hyperplasia or adenoma of parathyroid gl.) results in bone decalcif., fragile bones, which are easily subject of fractures and because of the high blood Ca level abnormal level, deposits of Ca, affecting especially the kidneys (stone diseases) and the arterial wall. Hypoparathyroidism (insufficient secretion of parathyroid horm.) leads to a low blood Ca level, disturbing normal musc. contr., in its severe form develop. tetany. Tetany is caused by the excessive excitability of the nerv. syst., due to the lack of the Ca ions in the blood, being characterized by spastic contr. of the skeletal muscles and generalized convulsions. Intravenous administration of Ca is g the correct, rapid therapy.

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Calcitonin is synth by the parafollicular cells of the thyroid gl and by cells of the synth. gl. DNES and has the opposite effect on the bone, than that of the parathyroid horm. Calcitonin inhibits bone matrix resorption, inactivates osteocl. (inhibiting the form. of the ruffled border) and decreases the no. of osteocl. It is used in the treat. of osteoporosis, pathol. condition defined as a decrease in bone rate form., accompanied by loss of the bone mass. Osteoporosis affects especially p postmenopausal women and immobilized p p patients. Other horm., are influencing as well bone metab.; corticoids and thyroid horm. are stimulating bone lysis, while sexual horm., estrogens and androgens, are stimulating bone matrix synth. and are inhibiting bone mass destruction. g y g

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Types of bone tissue
There are 2 main types of bone – the spongy bone and the compact (haversian) bone. bone. Spongy bone - irregular-shaped bone lamellae, separating numerous irregularinterconnecting cavit. (an irreg. syst. of bone lamellae and bone cavit.). The compact bone - of concentrically, regular disp bone lamellae parallel one to concentrically disp. lamellae, another, surrounding a centrally located canal, the canal of Havers. In light micr., on decalcified and stained bone prep., both compact, as well as spongy b bone h have th same hi t l asp. ( t bl and osteocl. on th surf. of th the histol. (osteobl. d t l the f f the acidophilic bone lamellae and osteocytes, inside the lacunae). The epiphyses of the long bones - spongy bone, covered only by a thin layer of compact bone. The diaphysis of th l tb Th di h i f the long b bones, are made almost t t ll of d l t totally f compact bone, with a small portion of spongy bone, situated near the bone marrow canal. Short bones have a core of spongy bone, covered by compact bone. The flat bones of the skull - 2 layers of compact bone, called plates or tables, the int. containing spongy bone, called diploe.

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The cavit. of the spongy bone and the bone marrow canal of the long bones, contain bone marrow We differentiate 2 types of bone marrow: red active bone marrow. red, active, marrow and yellow, inactive bone marrow. The red, active bone marrow is filling bone cavit. in small children; in teenagers, it becomes restricted at the sternum, coxal bone, vertebrae and part of the femur, being gradually replaced by y , p , gg y p y yellow, , inactive bone marrow (made of lipid –stocking cells and connec. tissue). In certain cond., for ex. after severe haemorrhages, even in aged individuals, bone marrow may be react. to produce blood cells. y p First bone tissue to be formed is the primary bone tissue. This type of bone is temporary, being replaced in adults, by sec. bone tissues, with few exceptions: in teeth sockets, near the sutures of the flat bone of the skull and in the insertion on , the bone of some tendons. Differences between primary and sec. bone tissues consists in a lower mineral content in the primary bone tissue, an irreg. disp. of the collagen fb. and in a higher no. of osteocytes, in the primary, than in the sec. bone tissue.

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Figure 1. Lamellar (secondary) bone in which the collagen fibers can be parallel to each other (at left) or organized concentrically around neurovascular channels, to constitute the haversian systems, or osteons (in most of the figure). Among the numerous haversian systems are some interstitial lamellae. PSP stain. Low magnification.
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The compact (haversian) bone - 3-7 μm thick bone lamellae, concentrically disp. disp. around a central canal, which contains blood vessels, nerves and loose connec. connec. tissue – the canal of Havers. Such a complex, made of bone lamellae, parallel one to Havers. another and surrounding a central canal, is called haversian syst. or osteon. Lacunae syst. osteon. in which are situated osteocytes, are present usually between the lamellae and much y , p y more rare, inside the lamellae. In each lamella, collagen fb. are parallel one to lamellae. fb. another. another. Between the bone lamellae, it is found a cementing subst., with fewer collagen fb., subst. fb. made predom. of calcif. bone matrix. In the diaphysis of the long bones, bone predom. calcif. matrix. lamellae show a typical disp.: in the thickness of the diaphysis are present numerous disp.: haversian syst.; surrounding the whole diaphysis is the outer circumferential syst. syst.; syst. y ; g p y y (concentrically disposed bone lamellae); surrounding the medullar canal (bone lamellae); marrow cavity) is the inner circumferential syst.; between the haversian syst., in the syst.; syst. diaphysis, are appearing interst. syst., which are remainders of haversian syst., interst. syst. syst. destroyed during bone growth and remodeling. In the outer circumferential syst. remodeling. syst. more numerous lamellae than in the inner circumferential syst. syst. These asp. are easy identifiable in cross sect. of a diaphysis, in light micr. Main asp. p y sect. p y g micr. function of the haversian syst. is to nourish the compact bone and is easy to syst. understand why they are missing in the fine bone spicules of the spongy bone. In the bone. spongy bone, the nutrient subst. are diffusing easily from the blood capillaries found subst. in the cavit. of the bone. cavit. bone.
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Figure 2. Section of a haversian system, or osteon. Note the alternation of clear and dark circles resulting from the alternation in the direction of the collagen fibers The collagen fibers. fibers appear bright when cut longitudinally and dark when cross-sectioned. In the center of the osteon is a channel. PSP stain. Medium magnification.
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Each haversian canal is looking as a long, usually bifurcated cylinder, parallel to the long axis of the bone. In each haversian syst., the central canal is surrounded g y by 4-20 conc. bone lamellae. The haversian canal is lined by endosteum and cont. 4blood vessels, nerves and loose connec. tissue. Haversian canals are communicating one with another, with the marrow cavity and with periosteum, through transv. or oblique canals, the Volkmann’s canals. Volkmann’s canals are perforating the bone lamellae. Examination of the haversian syst. with polarized light shows bright layers alternating with dark layers. This asp. is due to the fact that collagen fb. are birefringent, when polarized light is at right angle (perpend.) to their length. The alternating bright and dark layers, are the conseq. of the orientation of the collagen fb. in the bone lamellae; in each lamella, the fb. are parallel one to another and follow a helical arrang.

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The pitch of the helix is different in different lamellae, so at a given point, fb. from neighbor lamellae intersect at right angles. Obs. of haversian syst. in cross section, in light micr., is showing cross section of the collagen fb. in one lamella (looking granular) and long. section of the collagen fb. in the next lamella (appearing as elongated struct.). l t d t t) Haversian syst. may vary a lot in their diamr. Each haversian syst. appears by deposition of bone lamellae around preexisting blood vessels, starting from periphery; so, younger syst., h i h t have l larger canals. I older syst., th newest l l In ld t the t lamella ll is that one closest to the central canal (most inner lamella). Even in adult life and not only during bone growth, there is continuous destruction and rebuilding of haversian syst., f thi reason, we may often obs. new syst., with few lamellae h i t for this ft b t ith f l ll and central large canals.

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Figure 3 Photomicrograph of the 3. epiphyseal plate, showing its 5 zones, the changes that take place in the cartilage, and the formation of bone. PT stain. Low magnification.
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Figure 4. Section of endochondral ossification. The osseous matrix, rich in collagen type I, is specifically stained with I picrosirius-hematoxylin. The cartilaginous matrix, containing collagen type II, stains blue with hematoxylin because of its high content of y g chondroitin sulfate. Medium magnification.
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Bone fracture repair
When a bone is fractured, the local rupture of the blood vessels will produce a fractured, hemorrhage and subsequently a blood clot and tissues debris. During the repair process, first the tissues debris and the blood clot are removed by macroph. The periosteum and endosteum are intensely prolif., providing osteoprogenitor cells. This cell. tissue will gradually surround the fracture and will penetrate between the edges of the fractured bone. Besides intramembranous ossif., in the connec. tissue surrounding the fracture, will appear small fragments of cart., which will be subjects of endochondral ossif. Areas of cart., endochondral and intramembranous ossify. appear side by side, at the place of the fractured bone. Irreg. bone lamellae of primary bone, will unite the bottoms of the fracture, forming a bone callus. The gradually acting stresses are remodeling the bone callus. Since these stresses are the same as those which acted during the growth of the bone, the original shape of the bone is restored. The primary bone tissue is gradually replaced by sec. bone tissue, thus restoring the initial struct. of the bone.

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Figure 5. Repair of a fractured bone by formation of new bone tissue through p g p y g periosteal and endosteal cell proliferation
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Thank you for your attention!
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HISTOLOGY
Course 9
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

Muscle Tissue

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MUSCLE TISSUE
Muscle tissue is made of highly differentiated cells, containing contr. proteins. Generally, muscle cells are originating in the mesoderm; their differentiation is done by gradual growth in length and accum of myofibrillar proteins Based upon accum. proteins. morph. and funct. characteristics, we differentiate 3 types of muscle tissue: striated skeletal muscle, cardiac muscle or myocardium and smooth muscle. Striated skeletal muscle is made of bundles of very long cylindrical long, cylindrical, multinucleated cells, with cross-striations. Their contr. are rapid, strong and crossvoluntarily controlled. Contr. mech. is based on the interaction of thick myosin filam. filam with the thin actin filam with a molec device which permits them to slide filam., molec. device, one upon another (bridges between actin and myosin filam.). Cardiac muscle shows cross-striations and is made of columns of junctioned crosscells, cells parallel one to another End-to-end junct. are visible in light micr as another. End-tojunct micr. intercalated disks, struct. proper to the cardiac muscle. Its contr. - vigorous, rhythmic and involuntary. Smooth muscle is made of fusiform (spindle-shaped) cells, which do not show (spindlecells striations, in light micr. They are capable of slow, involuntary contr. The cytopl. of the muscle cells (without myofibrils) - sarcoplasm; SER sarcoplasmic reticulum and th cell. membr. ( l l i ti l d the ll b (plasmalemma) - sarcolemma. l ) l
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Figure 6. Structure of the 3 muscle types. The drawings at right show these muscles in cross section. Skeletal muscle is composed of large elongated multinucleated fibers Cardiac muscle is composed of irregular branched cells bound large, elongated, fibers. together longitudinally by intercalated disks. Smooth muscle is an agglomerate of fusiform cells. The density of the packing between the cells depends on the amount of extracellular connective tissue present.
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Striated skeletal muscle - bundles of very long (up to 30 cm) cylindrical, multinucleated, muscle fb., with a diam. of 10-100 μm. Multinucleated cells are 10resulting from the fusion of mononucleated myoblasts, during embryonic develop. The oval nuclei are situated i periphery, under th sarcolemma; thi asp. i h l f l i l l i it t d in i h d the l this is helpful in differentiating the skeletal muscle from the cardiac muscle and the smooth muscle, which both show unique, centrally located nuclei. The variation in diam. of skeletal muscle fb is depending upon age sex nutrition estate type of muscle and physical fb. age, sex, estate, training. The increase of the vol. of the skeletal muscle fb., by accum. of myofibrils hypertrophy. The increase of the no. of the muscle cells - hyperplasia. This does not occur neither in the skeletal nor in the cardiac muscle but is common in the case of skeletal, muscle, the smooth muscle fb., which are keeping the capacity of cell. div. (in the pregnant uterus, both hypertrophy and hyperplasia of the smooth muscle fb. occur). The whole muscle is surrounded by a layer of dense connec tissue - epimysium; connec. epimysium; each bundle in a muscle is surrounded by less dense connec. tissue layer, derived from the epimysium, called perimysium. Each striated muscle fb. is surrounded by a thin layer of connec tissue consisting of retic fb and a b m -like struct connec. tissue, retic. fb. b.m.b.m. struct. A rich capillary network is surrounding the striated skeletal fb.; the capillaries are of the continuous, common type.

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Figure 7 Cross section of striated muscle stained to show collagens type I and III and cell nuclei The 7. nuclei. endomysium is indicated by arrowheads and the perimysium by arrows. At left is a piece of epimysium. Picrosirius-hematoxylin stain. High magnification.
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In light micr., the long. sect. skeletal fb., show cross-striations, made of alternating crosslight and dark bands. The dark bands are called A bands, being anisotropic, or birefringent in polarized light while the light bands are called I bands being light, bands, isotropic, which do not affect the polarized light. They appear as parallelogram, deep acidophilic and multinucleated cells, in long. sect. and as rounded or polyhedral multinucleated struct in cross section struct., section. In the e.m., each light, I band is bisected by a dark transv. line, the Z line. The smallest repetitive unit of the contr. device of the skeletal muscle is the sarcomere, which extends from one Z line to the next one It measures approx 2 5 μm in one. approx. 2.5 length, in relaxed muscle. The sarcomere is made of a central dark, A band and of 2 halves of light I bands, on each side of the A band. The sarcopl. contains bundles of contr filam called myofibrils. contr. filam., myofibrils. The myofibrils are parallel to the long axis of the muscle fb. and are made of endendtoto-end arrangement of sarcomeres. The e.m., revealed that the sarcomere appearance is due to the arrangement of 2 types of contr filam – thin actin and contr. filam. thick myosin filam., which are arranged in a parallel manner, to the long axis of the fb. and in a symmetric pattern.

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Figure 8. Striated skeletal muscle in longitudinal section (lower) and in cross section (upper). The nuclei can be seen in the periphery of the cell just under the cell, cell membrane, particularly in the cross sections of these striated fibers. H&E stain. Medium magnification.

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Figure 9. Striated skeletal muscle in longitudinal section. In the left side of the photomicrograph the insertion of collagen fibers with the muscle is clearly seen. Picrosirius—polarized light (PSP) stain. Medium magnification.
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Figure 10. L Fi 10 Longitudinal section of skeletal muscle fib it di l ti f k l t l l fibers. N t th d k t i d A b d and th Note the dark-stained bands d the light-stained I bands, which are crossed by Z lines. Giemsa stain. High magnification.
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Figure 11. Electron micrograph of skeletal muscle of a tadpole. Note the sarcomere with its A, I, and H bands and Z line. The position of the thick and thin filaments in the sarcomere is shown schematically in the lower part of the figure. As illustrated here, triads in amphibian muscle are aligned with the Z line i each ith th li in h sarcomere. In mammalian muscle, however, each sarcomere exhibits 2 triads, one at each A—I band interface (see Figure 10—16). x35,000. (Courtesy of KR Porter.) P t )
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Thick myosin filam. are 1.6 μm long and 15 nm thick; they occupy the A band, the central part of the sarcomere. Thin actin filam. are 1μm long and 8 nm thick; they 1μ are situated between and parallel to the thick filam and are anchored on the Z line filam. line. The I band consists of the portion of thin filam. which do not overlap the thick filam. (is made exclusively of thin filam.). The outer parts of the A band, are made of both thick myosin and thin actin filam ; the portion in which the actin filam are filam.; filam. overlapping the myosin filam. In the center of the dark A band is present a lighter zone, called H band, made only of thick myosin filam. (the portion of thick filam. which is not overlaped by thin filam.). In the middle of the H band is appearing the M line, where thick filam. are linked together by lat. connections (anchoring zone for the thick filam.). Because thick and thin filam overlap for some dist in the A band a cross section filam. dist. band, of this part of the sarcomere, is showing each thick filam. surrounded by 6 thin filam., in a hexagonal pattern. Striated muscle filam contain at least 4 proteins which are important in the musc filam. proteins, musc. contr. – actin, myosin, tropomyosin and troponin. Thin filam. are composed of actin, tropomyosin and troponin, while thick filam. are composed exclusively of myosin.
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Actin is present as long filaments (F-actin) made of 2 chains of glob monomers (G(Fglob. (Gactin), twisted to form a double helix. The glob. monomers of G-actin are asymmetric, Gshowing a binding site, for myosin. Actin filam. which anchor perpend. on the Z lines, exhibit opposite polarity on each side of the Z line At the level of the Z lines are found polarity, line. two proteins, α-actinin and desmin, which are linking actin filam. to the Z line and are holding the sarcomeres together, keeping the myofibrils in register. Tropomyosin is a 40 nm long molec made of 2 polypeptide chains; tropomyosin molec., filam. are situated over the actin glob. subunits, in the groove made by the 2 twisted actin chains. Each tropomyosin molec. covers 7 G-actin monomers and has a troponin Gcomplex bound to its upper part part. Troponin is a complex, with 3 subunits: TnT – which is attached to tropomyosin; TnC – which binds Ca ions and TnI – which inhibits the binding of myosin to actin. A troponin complex is attached to a specific site on each tropomyosin molec site, molec. Myosin is a large molec., with 500 000 Da molec. weight; it is made of 2 heavy chains and 2 pairs of light chains. Heavy chains of the myosin molec. have a rod-like (linear) rodasp., asp while the 4 light chains form the glob ends of the myosin molec and only a small glob. molec. part of its rod-like portion. The glob. ends of the myosin molec. show ATP-ase activity rodATPand binding sites for ATP. These glob. ends have the capacity to bind to actin.

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Using proteolysis on myosin heavy chains were obtained two fragments – light and heavy meromyosin. Light meromyosin forms most of the rod-like part of the molec., rodwhile heavy meromyosin forms the glob. end and only a small part of the rod. In a myosin thick filam. are found approx. 300 myosin molec., which have all their glob. ends directed to the outside, toward the thin actin filam., in the zone of the A band of the , , sarcomere where the actin and myosin filam. are overlapping each other. Because of this arrangement of the actin and myosin filam., their binding during musc. contr., becomes possible. During musc. contr., for the uniform depolarization of the musc. fb., release of Ca ions is necessary; for that, the striated musc. fb. shows a transv. tubular syst., assoc. to the SR. This syst. consists of finger-like invaginations of the sarcolemma, form. an anast. fingery g g , network, which surrounds especially the limits between the A and the I band, in every sarcomere. On each side of the T tubule, are present dilated cisternae of the SR. Thus are appearing complexes of 3 elements, SR-T tubule-SR, called triads. At this level, the SR- tubuledepolarization of the sarcolemma is transmitted through the T tubule, to the SR. SR is stocking Ca ions, which are needed for rapid contraction-relaxation cycles of the contractionmuscle. With the e.m., one may obs. that each myofibril is surrounded by a rich network y y y of SR. During depolarization, Ca ions are released from the SR and they are binding to troponin, allowing bridges form. between actin and myosin filam. and thus the musc. contr. When the depolarization ends, Ca is actively transp. into the SR, providing the cessation of the musc. contr.
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Figure 12. Electron micrograph of a transverse section of fish muscle, showing the surface of 2 cells limiting an intercellular space. Note the invaginations of the sarcolemma, forming the tubules of the T system (arrows). The dark, dark coarse granules in the cytoplasm (lower left) are glycogen particles The section passes through the A band particles. (upper right), showing thick and thin filaments. The I band is sectioned (lower left), showing only thin filaments. x60,000. (Courtesy of KR Porter.)
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Mechanism of contraction
During musc. contr., both actin and myosin filam. are keeping their length, but the sarcomere becomes shorter. A band - the same length, but is loosing H zone, g g while the 2 halves of I band are shortening. Energy release - ATP hydrolysis (ATP on the glob. ends of myosin filam.) is possible only when the binding between actin and myosin filam. occurs. Myosin binding to actin is not possible in the relaxed muscle, because the binding sites for myosin on actin molec. are masked by the troponin-tropomyosin troponincomplexes on the actin filam. During depolarize., the release of Ca ions from the SR occurs; Ca is fixing on the TnC subunit of the troponin. The spatial config. of the troponin-tropomyosin complex changes, leading the tropomyosin molec. troponindeeper into the groove of the actin helix. This modification discovers the myosinmyosinbinding site on the glob. molec. of actin, so that actin and myosin filam. are free to interact with each others. The binding between actin and myosin filam. → the hydrolysis of the ATP molec., fixed on the glob. heads of myosin; thus energy is released and ADP i prod. Th b di of th myosin glob. ends t l d d is d The bending f the i l b d toward th actin d the ti filam. is due to their hinge-like property. When connected to the actin filam., the hingemyosin filam. will pull actin filam. more to the inner part of the sarcomeres, inside A filam., int. band; the result will be a larger overlap between actin and myosin filam to the int of the sarcomeres.
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Figure 1. Schematic representation of the thin filament, showing the spatial configuration of 3 major protein components–actin, tropomyosin, and troponin. The individual components in the upper part of the drawing are shown in polymerized form in the lower part. The globular actin molecules are polarized and polymerize in one direction. Note that each tropomyosin molecule extends over 7 actin molecules. TnI, TnC, and TnT are troponin subunits.
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Figure 2. Muscle contraction, initiated by the binding of Ca2+ to the TnC unit of troponin, which exposes the myosin binding site on actin (cross-hatched area). In a second step, the myosin head binds to actin and the ATP breaks down into ADP, yielding energy, which produces a movement of the myosin head As a consequence of this head. change in myosin, the bound thin filaments slide over the thick filaments. This process, which repeats itself many times during a single contraction, leads to a complete overlapping of the actin and myosin and a resultant shortening of the whole muscle fiber. I, T, C are troponin subunits. (Reproduced, with permission, from Ganong WF: Review of Medical Physiology 20th ed. McGraw Physiology, ed McGrawHill, 2001.)
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Not all myosin g y glob. ends are reacting with actin filam., at the same moment. During g g musc. relax., Ca is transp. into SR and ATP is restored and is fixing again on the glob. ends of myosin. If ATP molec. cannot be restored, the actin–myosin complex remains actin– stable; this explains the extreme musc. rigidity which occurs after death (rigor mortis). During relax., the troponin-tropomyosin complex is covering again the myosin-binding troponinmyosinsite on the actin molec. In conclusion, the whole muscle, as well as the sarcomeres are shortening during musc. contr. Morph., histochemically and funct., we differentiate 3 types of striated skeletal muscle fb.: red fb. - a higher content of myoglobin and mitochondrial cytochromes (p g g y g y (pigments) ) conferring it a deep red color. They show a delayed contr., but are capable of prolonged contr. Energy is provided mainly from oxidative phosphorylation. The long muscles of the back, posture-maintaining muscles, are of this type. postureWhite fb. - lower content of myoglobin and mitochondria. These are short muscles, but with thicker fb. Extraocular muscles of the eye are of this type. They contr. rapidly, but for short time periods. En. for contr. - mainly from anaerobic g y y glycolysis. y Interm. fb. show characteristics situated between the two types, previously described. The differentiation of the muscle in one of the 3 categ. of fb. is also controlled by its , g inerv. In human, most of the skeletal muscles are a mixture of the 3 categ. of fb.

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Thank you for your attention!
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HISTOLOGY
Course 10
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

Muscle Tissue Circulatory system

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Cardiac muscle
Cardiac muscle cells have approx. 100 μm in length and 15 μm in diam. and they show cross-striations, like the striated skeletal muscle. In contrast to the striated crossskeletal muscle they have a single pale centrally located nucleus; surrounded by muscle, single, pale, a delicate layer of loose connec. tissue, which contains a very rich capillary network (one of the best vasc. tissue of the human org.). The most striking characteristic of the cardiac muscle, in the light micr., is the muscle, micr presence at irreg. interval of dark-stained lines, that cross the columns of end-todarkend-toend junct. cardiac cells. These are the intercalated disks, which are in fact, junct. complexes, complexes between neighbor cardiac muscle cells They may look as simple lines cells. lines, or they may have a step-like pattern. In the step-like junct., we differentiate a stepsteptransv. portion, perpend. situated on the myofilam. (crossing the filam.) and a lat. portion, portion which is parallel to the myofilam In the intercalated disks we find 3 types myofilam. disks, of junct.; in the transv. portion are found fasciae adherentes, which represent anchoring sites for the actin filam., halves of Z lines, binding the sarcomeres together and desmosomes, which bind the cardiac cells together, holding them during cardiac muscle contr. In the lat. portion of the junct. complex (intercalated disks) we find gap junct., which provide ionic coupling between the cells; because p g y g of this ionic coupling between the cardiac cells, the myocardium is acting like a syncytium, allowing the contr. signal to pass as a wave, from one cell to another.
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Figure 3. Drawing of a section of heart muscle, showing central nuclei, cross-striation, and intercalated disks
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Even if the struct. and the function of the contr. proteins is very much alike that in the striated skeletal muscle, some differences do exist. The T tubule syst. and the SR are not so regularly org like in the skeletal muscle T tubules are more org., muscle. numerous and larger in the ventricular cardiac muscle, than in the skeletal muscle. T tubules are situated at the level of the Z line and not at the level of the junction between A and I bands, like in the skeletal muscle bands muscle. The SR is not so well developed and appears irregul. disp.; in conseq. in the cardiac muscle, bundles of contr. filam., which form myofibrils, do not exist. Triads are as well absent in the cardiac muscle because the T tubules are assoc with well, muscle, assoc. only one lat. cisterna of SR, forming diads, instead. The cardiac cells are so rich in mitochondria, that they occupy about 40 % of the cell. vol., a fact explained by the extreme need of oxygen of the cardiac muscle (only 2 % in the striated skeletal muscle). In the cardiac muscle cells are present numerous lipid gr. and a small amount of glycogen, which broken into glucose is used in intense stress periods. The brown lipofuscin pigment (aging pigment) appears with age, accum. near the nucleus of the cardiac cells.

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Figure 4. Photomicrograph of cardiac muscle. Note the crossstriation and the intercalated disks (arrowheads). Pararosaniline— toluidine blue (PT) stain. High magnification. g

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Figure 5. Longitudinal section of portions of 2 cardiac muscle cells. The transversely oriented parts of the intercalated disk consist of a fascia adherens and numerous desmosomes. desmosomes The longitudinal parts (arrows) contain gap junctions. Mitochondria (M) are numerous. Fibrils of reticular fibers are seen between the two cells. x18,000. (Reproduced, with permission, from Junqueira LCU, Salles LMM: Ultra Estrutura Ultra-Estrutura e Função Celular Celular. Edgard Blücher, 1975.)
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Some differences in struct., appear between the atrial and the ventr. cardiac muscle. Atrial cardiac muscle cells are smaller than the ventr. ones and they contain drastically fewer T tubules than the ventr. cells. Membrane-coated gr. are Membranepresent near the nucleus, close to the Golgi complex, especially in the cytopl. of the cardiac cells of the right atrium, even if they appear as well, in the left atrium, ventricles and DNES. The gr. measure 0.2-0.3 μm in diam. and they are about g 0.2y 600/cell, in the right atrium. They contain the precursor of a horm., known as atrial natriuretic factor, also known as auriculin or atriopeptin. The stimulus for its release is represented by the p p p y increase of the blood circ. vol., which returns to the auricles. It is acting on the kidney, stimulating water and sodium excretion, an increase of the diuresis and natriuresis, thus reestablishing the normal circ. vol. ( has the opposite action of g (it pp the aldosterone, which conserves water and sodium). Another horm. subst. identified in these gr. is cardiodilatin, which main effect is that of decrease of the blood pressure, by decrease of the periph. resistance ( p , y p p (diastolic blood pressure) and by inhibition of the aldosterone secretion from the adrenal cortex, which leads to a decrease of the circ. vol. and conseq. of the blood pressure.

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Figure 6. Electron micrograph of 6 an atrial muscle cell showing the presence of natriuretic granules aggregated at the nuclear pole. (Courtesy of JC Nogueira.)
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The impulse-generating and conducting system of the heart is made of groups impulseof cells that form the sinoatrial node (node of Keith-Flack) and the atroventricular Keithnode (A h ff-T d (Aschoff Tawara), the bundle of Hi with it l ft and right b (Aschoff) th b dl f His, ith its left d i ht branches, made of h d f Purkinje cells. The main pacemaker of the heart is the sinoatrial node, which has a rhythmic activity of about 70 / minute, while the atrioventricular node has half of the activity of the sinoatrial node, with only 35 beats / minute. The nodes are made of nodal cells, which are modified cardiac muscle cells, smaller than atrial cells and with fewer myofibrils. f fib il The nodes are rich in blood vessels, they contain nerve endings of the autonomic veget. nerv. syst. and connec. tissue. Between the 2 nodes, the impulse is conducted through the thickness of the myocardium; there i no special anatomical d d h h h hi k f h di h is i l i l link, between the 2 nodes. Nevertheless, preferential passages in the myocardium, for the contr. impulse, were identified; passages consisting in a very large no. of gap j junct. between cardiac muscle cells. t b t di l ll

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Purkinje cells form the bundle of His its branches and the subendocardial network His, (also made of Purkinje cells). Purkinje cells are relatively large, of about 50 μm in diam. and linked one to another by numerous gap junct. In light micr., Purkinje cells show a typical asp with central nucleus surrounded by a large amount of glycogen conferring asp., nucleus, glycogen, them a pale asp. of the center of the cell and visible contr. filam., in periphery, beneath the cell. membr. They have large, pale nuclei, with obvious nucleoli. Purkinje cells also establish gap junct with ventr cardiac muscle cells thus ensuring the rapid conduction junct. ventr. cells, of the contr. impulse, all over the cardiac muscle. Transitional cells, were also described in the myocardium, with interm. characteristics, cells, between Purkinje cells and cardiac muscle cells cells. Both sympathy. and parasympath. autonomic nerv. syst. form large plexuses at the syst. base of the heart. Neurons and nerve fb. are present especially close to the nodes, but also in the thickness of the myocardium Even if the nerves do not influence the myocardium. generation of the impulses (process developed by the sinoatrial, pacemaker node), they affect the rhythm of the heartbeat and the force of contr. of the heart. Stimulation of the sympathy. sympathy nerves increases the force of contr of the heart and accelerates the rhythm contr. of the heartbeat, while stimulation of the parasympath. nerv. syst. (vagus nerve) induces a slowing of the heartbeat and decreases the contr. force of the heart.

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Figure 7. Purkinje cells of the impulse conducting system. They are characterized by a reduced number of 7 impulse-conducting system myofibrils that are present mainly in the periphery of the muscle cell. The light area around the nuclei of the conducting cells is caused by a local accumulation of glycogen. High magnification. H&E stain
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The heart - a musc. org., which by rhythmic contr. is pumping blood into the circ. syst. Its wall is made of 3 concentric tunics: endocardium, the inner tunic; myocardium, myocardium the middle tunic and epicardium the outer tunic epicardium, tunic. Endocardium corresponds to the intima (int. tunic) of the blood vessels. It is made of a simple squamous epith. (endothelium) and a thin subendothelial layer, consisting of loose connec tissue which may contain some smooth muscle cells connec. tissue, cells. Deeper, appears the subendocardial layer, a layer of connec. tissue, containing blood vessels, nerves, the branches of the bundle of His and the network of Purkinje cells cells. Myocardium is the thickest tunic of all, made of columns of cardiac muscle cells anast. in various directions, which are inserted on the fibrous cardiac skeleton. The myocardium contains also the impulse generating and conducting syst of the syst. heart. Epicardium is the serous covering of the heart, representing the visceral layer of the pericardium It is made of a simple squamous epith (mesoth ) situated on a pericardium. epith. (mesoth.) thin layer of loose connec. tissue. The subepicardial layer contains loose connec. tissue, veins, nerv. ggl. and white adip. tissue.

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The fibr. skeleton of the heart is made of dense connec. tissue and islets of fibr. cart. Its main comp. are: septum membranaceum, trigona fibrosa and annuli fibrosi; the septae are separating auricles from the ventricles and the left heart ventricles, heart, from the right heart, while the fibr. rings of the heart (annuli fibrosi) are limiting atrioventricular openings and the openings of the aorta and of the pulmonary artery. artery The cardiac valves are made of a core of dense, fibr. connec. tissue and a zone of mucous connec. tissue (viscous connec. tissue, rich in ground subst., similar to the mesench ) They are lined by endocardium on both sides Their base is mesench.). endocardium, sides. attached to the annuli fibrosi of the fibr. skeleton of the heart. Cardiac valves have blood vessels only at their base, that is why they cannot be regenerated, if they are injured; in this case a prosthesis of cardiac valve should be performed case, performed.

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Figure 8. Diagram of the heart, showing the impulse-generating impulse generating and -conducting system
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Smooth muscle
Smooth muscle is made of spindle-shaped elong cells which show not spindle-shaped, elong. cells, striations, in light micr. Smooth muscle cells are surrounded by a basal lamina and a network of retic. fb., which are important to concentrate the force generated by each smooth muscle fb. in a generalized action, how it is the case with the peristalsis of the intestine. In light micr., smooth muscle fb. are fusiform, thicker in their middle and thinner toward their ends In length they measure from 20 μm in the wall of small blood ends. length, vessels, up to 500 μm in the pregnant uterus. The cells - central located, unique nuclei. For the largest unity between the muscle cells, the thinner end of a cell comes into contact with the thicker middle of the neighbor cell, they are arranged g , y g like bricks in the wall. Due to this arrang., in cross section we obs. different diam. of sect. cells and only the thickest cells show nuclei in their middle. During muscle contr., the cell becomes folded and the nucleus may appear as a corkscrew.

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In the el.m., near the nucleus are present: ribosomes, RER, Golgi complex and mitochondria. The SR is poorly developed and the T tubules are absent. y Smooth muscle contr. is achieved also by the coupling between actin and myosin filam., but in the smooth muscle contr. filam. do not form sarcomeres. Instead, myofilam. bundles cross in zigzag the cytopl. of the muscle cells, in a oblique y g g y p , q direction, forming a lace-like network. Bundles of filam. (myofibrils) are made of lacethin filam. of actin and tropomyosin, of 5-7 nm in diam. and thick filam. of myosin of 51212-16 nm in diam. In contrast to the myosin filam. of the striated skeletal muscle, which show a rod-like portion and glob. heads only to their ends, myosin filam. of rodthe smooth muscle show glob. heads all their length and only a small bare region at the end of the filam. This molec. org. of the myosin filam. allows greater overlap, between actin and myosin filam. and conseq. a higher degree of contr. than in the striated skeletal muscle. Contr. mech. of the smooth muscle is based upon the same sliding filam. phenom., like in the striated muscle.

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Figure 9. Photomicrographs of smooth muscle cells in cross section (upper) and in longitudinal section (lower) Note the centrally located nuclei In many cells the nuclei were not included in the (lower). nuclei. section. PT stain. Medium magnification
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Figure 10. Transverse section of smooth muscle impregnated with silver to stain the reticular fibers. These fibers form a network that surrounds the muscle cells that are not stained by this method. At the right is an arteriole surrounded by thicker collagen fibers. x300.
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Initiation of smooth muscle contr. is achieved, here also, by Ca ions. However, the myosin of the smooth muscle is interact. with actin, only when the light chain of the myosin is phosphorylated. Troponin is absent in the smooth muscle, being replaced b another C -bi di protein, called calmodulin (i l d by th Ca binding Cat i ll d l d li (involved also i contr. l d l in t of nonmuscle cells - myoepith. cells). The Ca-calmodulin complex act. the myosin Calightlight-chain kinase, enzyme which phosphorylates the light chain of the myosin. Other factors but Ca are involved in the act of this myosin-kinase thus influencing act. myosin-kinase, the contr. act. of the smooth muscle. Contr. and relax. are controlled by horm., which are acting through cAMP. The increase of the cAMP will i d i f th AMP ill induce th activ. of th myosin-ki the ti f the myosin-kinase, th li ht chain of i the light h i f the myosin will be phosphorylated and the smooth muscle cell will contract. The decrease of the cAMP will have the opposite effect, with a decrease of the contr. The most illustrative ex is that of the uterine smooth muscle; estrogens are ex. increasing the cAMP and induce the phosphorylation of the myosin and contr. of the uterus, while progesterone is decreasing cAMP and induces the realx. of the uterus. uterus

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In the cytopl of the smooth muscle fb appear numerous interm filam of approx cytopl. fb. interm. filam. approx. 10 nm thick, which are waving in their cytopl. Desmin (skeletin) is the major protein comp. of the interm. filam. of the smooth muscle cells, vimentin is another one, p present especially in the vasc. smooth muscle. In the smooth muscle cells are p y present 2 types of dense bodies – membr. assoc. and cytopl. dense bodies; both of these contain α-actinin, being somewhat similar to the Z lines of the sarcomeres of the striated muscle. Both thin, actin filam. and interm. filam. are inserting into the g dense bodies; this is important in the transmission of the contr. force from one cell to another. Smooth muscles are innerv. by sympath. and p parasympath. autonomic nerv. y y p y p syst.; often, the axons of the veget. neurons and their terminal dilatations are found in the loose connec. tissue, surrounding the smooth muscle fb. Usually, smooth muscle forms large sheets, like those in the wall of the hollow org. (intestine, uterus, etc.) these smooth muscle cells show numerous gap junct., instead they are poorly innerv. Their contr. is achieved in a syncytial manner, being called visceral smooth muscle. The highly innerv. smooth muscle of the iris of the eye, achieves precise and graded contr.; this is the multiunit smooth muscle.

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The contr. of the smooth muscle is usually spont.; therefore the nerv. stimuli have more a modulating role, than that of initiating the contr.. Smooth muscle is innerv. by both adrenergic and cholinergic nerve end., which have antagonistic activ., stimulating/depressing it contr. I some org. th cholinergic nerve fb stim. ti ti l ti /d i its t In the h li i fb. ti contr. and the adrenergic nerve fb. inhibit it, while in others, the reverse is true. Smooth muscle cells show synth. activ., they synth. collagen, elstin, PG; for that, they have well dev. RER and Golgi complex

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Thank you for your attention!
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HISTOLOGY
Course 11
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

Circulatory system
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Regeneration of the musc. tissue

Cardiac muscle has no capacity of regeneration, after early childhood; the injury of the cardiac muscle (infarcts) is replaced by connec. tissue, forming myocardial scars. In the case of the striated skeletal muscle, although the multiple nuclei of the muscle fb. are not capable of mitosis, the regen. does occur, from the so-called sosatellite cells. This is a small population of mononucleated, spindle-shaped cells, spindlep p p p situated in the basal lamina, surrounding each mature muscular fb. They are identifiable only with the e.m. They are believed to be primitive myoblasts, that persist during adult life. After injury, the inactive satellite cells become active, they prolif. and they fuse together, to form new skeletal fb. In musc. hypertrophy, these cells are fusing with the adjacent mature fb.; thus is increasing the muscle mass, after intense physical exercise. Smooth muscle cells are keeping a limited cap. of regeneration; following injury, undamaged smooth muscle cells are dividing, replacing the damaged fb.

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General organization of the blood vessels (tunics)
The wall of the blood vessels is composed of 3 tunics or sheaths (in Lt. lg., tunica means coat) from the int. to the outside: 1. Tunica i ti 1 T i intima (intima) - of a simple squam. epith., th endoth., li i th vessel i (i ti ) f i l ith the d th lining the l in its int. Beneath the b.m. of the endoth. is found a thin subendoth. layer, made of loose connec. tissue, which may contain smooth muscle fb. Connec. tissue fb. and smooth muscle fb are arranged parallel with the long axis of the vessel fb. vessel. 2. Tunica media (middle tunic or media) - helically and circularly disp., concentric layers of smooth muscle fb. Interspersed between the smooth muscle fb., are found variable amounts of elastic and retic. fb and PG I arteries, th i ti f d i bl t f l ti d ti fb. d PG. In t i the intima i is separated from the media, by an elastic membr., called int. elastic lamina; this is made of some fenestrated elastic lamellae, with folded asp. In large arteries, between the media and the ext tunica advent is present a thinner ext elastic ext. advent., ext. lamina. In capillaries and postcapillary venules, media is made of connec. tissue cells, resembling the primitive mesench. cells - pericytes. 3. 3 Tunica advent. (outer tunic or advent.) is made of connec tissue especially advent advent ) connec. tissue, collagen and elastic fb., with long. orientation. The advet. connec. tissue becomes gradually continuous with the connec. tissue of the org. through which the vessel is passing. passing
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Capillaries
The capillaries - a simple squam. epith. (one layer of squam. cells) called endoth., situated on a b.m., not visible in the usual staining. Between the endoth. and the b m is a very thin space the under –endoth sp. The capillaries are very b.m. space, endoth. sp thin tubules with rich anast., in cross sect. they have 7-9 μm in diam.. The 7appearance of the simple squam. cells forming the endoth. is one with nuclei prominent to the lumen of the capillary In cross sect they are lined of only 2 or 3 capillary. sect. cells. Outside the capillaries are situated on their own b.m. the advent. cells or pericytes, undifferentiated cells, very similar to the primitive mesench cells which have a cells mesench. cells, great potentiality of transformation into diverse cell types. Capillaries are responsible for the level of exchanges between blood and tissues; total length of the human capillaries was estimated at approx 96 000 km approx. km. With the e.m., poor dev. RER and Golgi complex, as well as mitochondria are visible in the endoth. cells. They also contain interm. filam. and contr. filam., ensuring the contr property of the endoth Between the cells are present intercell contr. endoth. intercell. junct. – zonula occludens, rare desmosomes and gap junct. Numerous pinocytotic vesicles are also visible, since the endoth. achieves macromolec. transport, in both senses.
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These capillaries, just described are of the so-called common type or continuous socapillaries. Two other types of capillaries are the fenestrated or perforated capillaries and th sinusoidal capillaries. d the i id l ill i The fenestrated capillaries are characterized by the presence of pores into the endoth. cells, of 60-80 nm in diam.; in some org. the pores are closed by diaphragms 60with a complex molec. struct. The b.m. is continuous. This type of capillaries is found in org. where a rapid transport of subst. is needed between the blood and the tissues, as is the case with the kidney, some endocrine gl. and the intestine. Macromolec. cross these pores, thi mean of transport being much more rapid th th t realized b th this ft tb i h id than that li d by pinocytosis. A third type of capillaries, the sinusoidal type is characterized by a sinuous direction, portions of dil t d l ti f dilated lumen of about 30-40 μm, with unequal calib. and very rich anast. A f b t 30ith l lib d i h t An important characteristic is also the discontinuity of the walls of the sinusoidal capillaries, by abs. of a continuous endoth. and interrupted b.m., by this the circ. blood coming i close contact with th i t titi l cells and ti i in l t t ith the interstitial ll d tissues. C ll can pass th Cells through h these discontinuous walls, from the lumen to the interst. tissues or the reverse. Frequently one can find phagocytic cells in the walls of the sinusoidal capillaries. This struct., struct besides facilitating the interchanges between the vessels and the tissues is tissues, also capable to slow a lot the speed of the circ. blood, by this making easier a prolonged contact, between different cells, an asp. important in the develop. of immune processes. processes The sinusoidal capillaries are found in the liver and blood-lymph-forming blood-lymphorgans, as the bone marrow and the spleen.
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Figure 1. Small blood vessels from the microvasculature (arterioles and venules) surrounded by components of connective tissue. The arrowheads point to fibroblasts. H&E stain. Low magnification
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Figure 2. Cross section through a small g g artery and its accompanying muscular vein. Because of vasodilatation, the arteriole is unusually filled with blood. At this stage the internal elastic lamina is not distinguished distinguished. Many other small arterial branches and capillaries can be seen in the surrounding connective tissue. Pararosaniline—toluidine blue (PT) stain. Medium magnification.
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Figure 3. Types of microcirculation formed by small blood vessels. (1) The usual sequence of arteriole –> metarteriole –> capillary –> venule and vein. (2) An arteriovenous anastomosis. (3) An arterial portal system as is present in system, the kidney glomerulus. (4) A venous portal system, as is present in the liver. (Reproduced, with permission, from Krstíc RV: Illustrated Encyclopedia of Human Histology. Springer-Verlag, Histology Springer-Verlag 1984.)
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Arteries
The arteries are classif according to their size in small arteries or arterioles classif. arterioles, arteries of medium size or musc. arteries and large or elastic arteries. The arterioles are usually small, with a narrow lumen, they show less than 0.5 mm in diam In the edoth cells of the small arterioles the next larger in calib then diam. edoth. arterioles, calib. capillaries are present gr. of 3 μm long and 0.1 μm thick, Weibel-Palade gr.; gr. Weibelcontain Willebrand factor, factor VIII of the blood clotting process; deficiency in this factor is causing prolonged bleeding by affecting platelets adhesion which is one bleeding, adhesion, cause of the hemophilia. Arterioles have a tunica intima (int. tunic) with a reduced under-endoth. layer and undera thin or absent int l elastic limiting membr The middle layer is usually made of 2int.l membr. 23, up to 5-6 layers of smooth muscle fb. The advent. tunic is made of connec. 5tissue, with fibroblasts and all types of fb., but with a small quantity of elastic fb. This tunic seems generally to be poorly dev., because it shows no limits with the surrounding connec. tissue, but in reality, it can be as thick as the middle musc. tunic. The arterioles do not contain an ext. elastic limiting membr.

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With the light micr., the long. sect. through the arterioles, shows flattened nuclei of the endoth., a fine, refringent, sinuous line – the int. elastic limiting membr., which when does exist separates the int tunic from the middle one The middle tunic int. one. appears to be made of ovoid or rounded cytopl. masses of different sizes, the larger ones, with centrally located nuclei, these being cross-sectioned smooth crossmuscle fb In the transv sect through an arteriole we can obs rounded nuclei of fb. transv. sect. arteriole, obs. the endoth., prominent into the lumen and a waved int. elastic limiting membr., like a tortuous, shining line. The middle tunic appears formed by long.l sect. smooth muscle fb., the smooth muscle layers being circularly disp., thus giving to the arterioles the funct. Cap. to regulate the blood pressure, by their contr. In comp. with the small arteries, medium-sized arteries have a better repress. mediumunder-endoth. layer under-endoth layer, with a larger amount of fb and amorph subst a more clear fb. amorph. subst., int. elastic limiting membr.; the medium musc. tunic represents at least 50% of the whole thickness of the arterial wall, consisting of up to 30 -40 layers of smooth muscle cells. The smooth muscle fb. are united by connec. tissue. The advent. tunic is always smaller than the middle tunic, representing the most 40% of the total thickness of the wall. Here, one can obs. the ext. elastic limiting membr., p g separating the middle tunic from the advent.

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The ext elastic limiting membr is always less represented than the int elastic ext. membr. represented, int. limiting membr. The advent. tunic is made of denser connec. tissue, its inner portion, while the outer part of it is made of loose connec. tissue, gradually continued with the connec tissue around the vessels Here are situated small blood connec. vessels. vessels, which are nourishing the arterial wall, beginning from the outside, the advent. and only a small part of the middle tunic, called vasa vasorum and nerv. fb., also also. The large arteries are the elastic arteries including the aorta and its branches. The int. tunic is better dev. than that of the musc. arteries. The endoth. cells are bulging into the lumen of the aorta. In the e m the endoth cells have microvilli, pinocytotic aorta e.m. endoth. microvilli vesicles and even lysosomes and RER, some of the cells have the cap. of phagocytosis. The under-endothelial tissue is better represented, containing elastic underand collagen fb., fibroblasts, smooth muscle fb. and amorph. intercell. subst. The int. and ext. elastic limiting membr. are not visible, being confused with the middle elastic tunic. This is the thickest of all the tunics, representing up to 80% of the wall of the aorta; it is made of elastic fenestrated membr., concentrically disp. The no. of ; , y p these elastic lamellas is growing with age, from 30-40 in the newborn, up to 70 in 30the adult life.

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Between the lamellas are smooth muscle cells, fibroblasts and amorph. subst., with the same comp. like that of the connec. tissue. The advent. tunic is made of connec. connec tissue and is usually thin only 10% of the whole thickness of the wall thin, wall, containing vasa vasorum and nerves. The wall of the aorta can be better stained, using special dyes for the elastic fb., like orcein. The atherosclerosis process is up to a certain degree a normal process of is, degree, degeneration, affecting the arterial wall, beginning at the age of the newborn. Coronary arteries, irrigating the cardiac muscle are usually, precociously affected. ATS lesions appear as a local thickening of the intima accompanied by prolif of intima, prolif. the smooth muscle cells and of the connec. tissue fb. The deposits of cholest. in macroph. may lead to the asp. of foam cells, which form the macroscopically visible fatty steaks and plaques of the ATS process The gradual thickening may process. lead to the occlusion of the vessel. When arteries irrigate limited areas of an org., the occlusion of the vessel leads to necrosis (death of a tissue), which are infarcts, most common in the heart, brain and in the kidney. In some org., such as skin or striated skeletal muscle, the large no. of arterial anast. is ensuring the normal blood irrigation of the org., even if an artery is obliterated by an infarct.

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Figure 4. Drawing of a medium-sized muscular artery, showing its layers. Although the usual histologic preparations cause the layers to appear thicker than those shown here, the drawing is actually similar to the in vivo architecture of the vessel. At the moment of death, the artery experiences an i t i intense contraction; consequently, th l t ti tl the lumen i reduced, th i t is d d the internal elastic l l ti membrane undulates, and the muscular tunica thickens.
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Figure 5. Diagrams of a muscular artery prepared by H&E staining (left) and an elastic artery stained by Weigert’s method (right). The tunica media of a muscular artery contains predominantly smooth muscle, whereas the tunica media of an elastic artery is formed by layers of smooth muscle intercalated by elastic laminas. The adventitia and the outer part of the media have small blood vessels (vasa vasorum) and elastic and collagenous fibers.
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Figure 6. Oblique section of a small artery from the mesentery. Note the transverse section of âthe smooth muscle cells of the media and the endothelial layer covering the lumen of the vessel (arrowheads). PT stain. Medium magnification.
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Figure 7. Cross sections of small arteries. A: The elastic lamina is not stained and is seen as a pallid lamina of scalloped appearance just below the endothelium (arrowhead). Medium magnification. B: A small artery with a distinctly stained internal elastic lamina (arrowhead). From a preparation of the late George Gomori Gomori. Low magnification.
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Veins and lymphatic vessels
The veins have the same 3 tunics in their struct., but with different characteristics, they also have a larger lumen and a thinner wall than the arteries Veins can be wall, arteries. classified according to their size in venules, small, medium and large veins. Veins are considered stockage vessels, since they contain in any moment, at least 70 % of the total blood vol vol. The post-capillary venules have a peculiar struct. and a diam. slightly larger than postthat of the capillaries from which they are deriving. Besides the similar. with the capillaries, capillaries they have an unusual tall endoth and on the endoth cells are situated endoth. endoth. rec. cap. to recog. cells involved in the immune defense, mainly lymphocytes. By this recognition, the cells may be allowed to pass between the endoth. cells in the tissues; the wall of these vessels is thin with a simple struct for this purpose The thin, struct., purpose. common venules are small, they measure 0.2-1 mm in diam.; they have an int. 0.2endoth. tunic. Outside the endoth - pericytes; the larger venules and the small veins may contain endoth. a thin middle tunic made of a few smooth muscle cells and an advent. tunic, which is the thickest of all, made of connec. tissue, rich in collagen fb.

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Figure 8. Section showing part of a large vein. The vein has a very thin muscular tunica media that contrasts with the thick adventitia composed of dense connective tissue. Note the presence a valve. PT stain. Medium magnification
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The medium-sized veins have a thin under-edoth. layer, which may be inconstant, in mediumundery y the int. tunic. The middle tunic is made of thin layers of smooth muscle cells, interposed with long. disp. collagen bundles, few fibroblasts and a thick advent., as the outer tunic. These veins may be fibrous-muscular or muscular-fibrous, according fibrousmuscularto the predom. of the smooth muscle fb. or of the connec. tissue in their walls. Large veins have a better -developed int. tunic, but a very poor represented int. elastic limiting membr., invisible with the light micr. The middle tunic is very thin, consisting predominantly of connec. tissue and few smooth muscle cells. The advent. tunic is the thickest layer and contains smooth muscle fb. in long. and circ. disp. bundles, embedded in a connec. tissue atmosphere, which also contains vasa vasorum and nerves. The medium sized veins, especially those of the limbs have valves in their lumens. The valves are made of elastic connec. tissue lined by endoth., on both sides. They appear like folds of the int. tunic of the veins, prominent in the lumen and are automatically opened when the blood has the tendency to flow down, thus fragmenting the blood column and helping by this the return of the blood to the heart. The lymph. vessels have a struct. quite similar to that of the veins, with some differences: the lymph. vessels have even thinner walls and more numerous valves in the lumen; between the valves the lymph. vessels have a dilated asp.

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Thank you for your attention! attention!
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HISTOLOGY
Course 12
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

The Nerve Tissue

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THE NERVE TISSUE
Nerve tissue dev from the ectoderm being the most evolved on the phylogenetic dev. ectoderm, evolved, scale. Main funct. of the nerv. syst. are to coordinate and to integrate the org. into the environment. Human nerv. syst. contains at least 10 billion neurons. As struct the nerve tissue is made of 2 cell types: nerve cells or neurons and glial struct., cells or neuroglia, which have funct. of support, nutrition, protection and immune defense, for the neurons. Main properties of the nerv.. syst. are excitability, conduction and intercell trans of the nerve impulses through excit or inhib intercell. trans. impulses, excit. inhib. interconnec. The nerv. syst. has the property to detect, analyze, integrate, stock and transmit, inform. received from the environment or from the org. Although the p p properties of the nerve tissue are g generated by the neurons, they cannot function y , y in the abs. of the glial cells. The central nerv. syst. - the brain and the spinal cord; the periph. nerv. syst. nerve fb. (nerves) and small aggreg. of nerons, which are forming the nerve ggl.

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Neurons

Morph. neurons have the cell. organelles present in any cell. The cytopl. surrounding the neuronal nucleus - the cell body of the neuron or the perikaryon (in anc. Greek, karyon - nucleus, peri - around). Neuron has 2 types of processes – the dendrites and the axon. Dendrites are multiple and extensively branched, afferent (receiving stimuli) processes (dendron in anc. Greek - tree), while the axon is a single, long and efferent (transmitting stimuli) process. The distal end of the axon is branched, called the terminal arborization, which presents small dil ll d h i l b i i hi h ll dilatations, called end b lb i ll d d bulbs, involved in the synapse. Neurons may be classif. according to multiple criteria:

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Types of neurons neurons
Neurons may be classif. according to multiple criteria: 1. According to the no. and the size of their processes: unipolar neurons have just one type of processes; they are numerous during embryonic life; in adults, they are present in the retina of the eye – the amacrine cells, which show only dendrites, but some of them are functioning as axons. pseudounipolar neurons have one process, which after a short passage is dividing in two in T-letter shape one becomes dendrite and one becomes two, Tshape, axon. They are present in the spinal ggl. and cranial ggl.; they are sensory neurons, which are transmitting the inform. rapidly, since it is travelling directly to the a o , by-passing the ce bod es o the neurons. ( ey a e de by the o e axon, by-pass g e cell bodies of e eu o s (they are dev. e fusion of the two types of processes, during embryonic dev.) bipolar neurons have one dendrite and one axon, each at one pole of the cell, showing an elong. shape; they are present in the retina and in the olfactory mucosa. multipolar neurons have one axon and many dendrites; they are the most numerous of all all.
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Figure 9. Diagrams of several types of neurons. The morphologic characteristics of neurons are very complex. All neurons shown here, except for the bipolar and pseudounipolar neurons, which are not very numerous in nerve tissue are of the tissue, common multipolar variety.
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2. According to the length of the axon: Golgi type I neurons have a long, myelinated axon, which may reach 1 m or more, length. more in length Golgi type II have short, unmyelinated axons; these are interneurons or local circuit neurons (they provide inform. to other neurons, in their immediate vicinity). vicinity) 3. According to their funct.: motor (efferent) neurons, which transmit to effector org., such as muscle or gl. sensory (afferent) neurons, involved in receiving stimuli, from the environment or from the body, itself. y interneurons, which are establishing interconnect. between other neurons, thus forming complex chains or circuits. secretory neurons with a well-dev neurosecretory cap (they secrete neurons, well-dev. cap. neurotransmitters and neurohorm.).

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Figure 10. Motor neuron. The myelin sheath is produced by oligodendrocytes in the central nervous system and by Schwann y y cells in the peripheral nervous system. The neuronal cell body has an unusually large, euchromatic nucleus with a well-developed nucleolus. Th perikaryon contains Ni l l l The ik t i Nissl bodies, which are also found in large dendrites. An axon from another neuron is shown at upper right. It has 3 end bulbs, pp g , one of which forms a synapse with the neuron. Note also the 3 motor end-plates, which transmit the nerve impulse to striated skeletal m scle fibers Arro s sho the muscle fibers. Arrows show direction of the nerve impulse.
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4. According to the dimension of the neuronal cell body, which may vary a lot. g body, y y y Some of the neurons are among the smallest cells of the human body, like the granule cells of the cerebellum, of approx. 3-4 μm in diam., while other are 3between the largest cells of the body, like Purkinje neurons of the cerebellum and Betz neurons of the motor cortex (150 μm in diam., they are visible even with the naked eye); most of the neurons are medium-sized, with a cell body of approx. 20medium2040 μm in diam.). 5. According to the shape of the cell body; on one hand, the shape depends upon body; the no. of processes, thus the p p pseudounipolar neurons are round, the bipolar p p neurons are fusiform, while the multipolar neurons are star-shaped. On the other starhand, they may present a variety of shapes; the perikaryon may be ovoid, pyramidal or pear-shaped, like Purkinje neurons of the cerebellum (from the thicker pearextremity arises the axon and from the thinner one - the dendrites). 6. According to the local., the neurons may be part of the auton. veget. nerv. syst. (veget. neurons) or somatic neurons (involved in the voluntarily controlled funct.).

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The nucleus of the neuron is usually unique, centrally located, large, pale (contains finely dispersed chromatin) round and has a large well-visible nucleolus It is wellnucleolus. classically compared with the eye of an owl. Its asp. is reflecting the intense synth. act. of the neuron. H.E. H E technique and trichrome methods are not so good for neuron staining for that staining, were dev. special techniques to stain nerve tissue, such as: Golgi, Ramon J Cajal, Del Rio Cortega. Neuron h all th organelles, f N has ll the ll found i every cell, b t fi t hi t l i t when obs. d in ll but first histologists h b it, believed that some of the organelles are specific for the neuron. With the devel. of the e.m., was noticed that these so-called specific organelles are in fact socommon for any cell; yet the denomination of specific organelles was kept yet, kept. Mitochondria are numerous in the neuron, especially in the axon terminals. They are common, spherical or filamentous in shape. SER and the lysosomes are of common t type. In the cytopl. of the neuron are present moderate amounts of I th t l f th t d t t f glycogen and lipid gr.

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Figure 1 Ultrastructure 1. of a neuron. The neuronal surface is completely covered p y either by synaptic endings of other neurons or by processes of glial cells cells. At synapses, the neuronal membrane is thicker and is called the postsynaptic membrane. The neuronal process devoid of ribosomes (lower part of figure) is the axon hillock. The other processes of this cell are dendrites.
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Being highly differentiated cells, usually neurons do not show cell. div. cap., anymore. However, there are exceptions from this gen. rule; the neurons of the olfactory mucosa show mit. cap. Experimental studies demonstr. that in some cases neurons d di id with a very l do divide ith low mit. rate. it t The so-called specific organelles of the neuron are: soThe Golgi complex was first obs. by Golgi (an Italian researcher), in the vicinity of the nucleus. It is present exclusively in the perikaryon. It was stained with osmic acid or by silver impregnation and it appeared as a small irreg. network, close to y p g pp g the nucleus (first was considered specific for the neuron). Nissl bodies were obs. by the German researcher Nissl, who used basic aniline dyes, such as toluidine blue or metilen blue, which stained RER and free y , , ribosomes. Nissl bodies appeared as irreg. basophilic masses, in the perikaryon and in the dendrites, they are missing in the axon. The asp. of the nucleus, the presence of the Golgi complex and of the RER is demonstr. the protein synth. cap. of the neuron (e.g. neurotransmitters).

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Figure 2. Photomicrograph of a motor neuron, a very large cell, from the spinal cord. The cytoplasm contains a great number of Nissl bodies. The large cell process is a dendrite. Note the large, round, stained nucleus, with a central dark-stained nucleolus. Pararosaniline— toluidine bl (PT) stain. M di l idi blue i Medium magnification. ifi i
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Neurofilam. (neurofibrils) and microtubules (neurotubules) are stained with silver impregnation; they appear as a dense filamentous network, present in the cell body and also in the dendrites and in the axon of the neuron. In light micr., they were called neurofibrils and they stain black with silver salts, being 0.5-0.6 μm 0.5thick. With the e.m., they were identif. as interm. filam. and contr. filam. Contr. filam. are double chains of heavy meromyosin of approx. 10 nm thick and simple chains of actin of about 4-6 nm thick (called neurofilam., being found in the 4neuron). Microtubules have a diam. of 24 nm and have the common struct. of the microtubules (pairs of α + β tubulin); they are also called neurotubules. The network of microfilam. and microtubules are responsible for the mobil. of different materials, in the dendrites and in the axon; the axon’s and dendrite’s flux. Axon’s flux may be rapid or slow, afferent or efferent. Dendrites are usually numerous, extensively branched and they become thinner in their distal branches (while axon is keeping a constant diam.). They divide like the branches of a tree; with the e.m., on their surf. were identif. a large no. of small project., like fine spines, which are sites of synaptic contact (on a dendrite of the human cortex were encountered 20 000 of such project.). Dendrites contain Nissl bodies and neurofibrils.

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The axon is a unique, long p q g process, which may reach up to 1 m in length. Axon y p g origin. from a pyramid-shaped region of the cell body of the neuron, called the pyramidhillock of the axon. Unlike dendrites, the axon shows a constant caliber from one end to the other, of dendrites, , approx. 0.2 μm. It contains a thick network of neurofilam. and neurotubules, aggreg. into neurofibrils. It does not contain Nissl bodies. Its plasma membr. axolemma and its content - axoplasm. Axolemma and axoplasm are involved in the trans. of the nerve impulse. Sometimes, after a short passage, the axon gives rise to a branch that returns to the cell body, called collat. branch. The axon contains mitochondria, SER and y, , vesicles with content. The axon ending is branched, each branch is finished with small dilatations (bulbous expansions) – the end bulbs, which contain vesicles with neurotransmitters, part of the synapses.

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Thank you for your attention!
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HISTOLOGY
Course 13
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

The Nerve Tissue

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Nerve fibers (nerves)
Nerve fb. may be myelinated or unmyelinated. Myelinated nerve fb. show a myelin sheath and they have the cap to trans very rapidly the nerve impulse cap. trans. impulse. Unmyelinated nerve fb. do not have a myelin sheath, but both types of fb. are enveloped by Schwann cells. Schwann cells are glial cells of the periph. nerv. syst. In the myelinated fb the Schwann cells are forming the myelin sheath In the case fb., sheath. of the unmyelinated fb., the axons are situated in simple clefts of the Schwann cells. Nude nerve fb. are present in the gray matter of the central nerv. syst., which show no glial cells In the white matter of the central nerv syst the myelin sheath cells. nerv. syst., is prod. by a glial cell called oligodendrocyte. Usually, nerve fb. are represented by axons, but there are cases when dendrites are functioning like axons – the dendrites of the neurons of the spinal ggl ggl. The myelin form. begins with the penetration of the axon in an existing groove of the Schwann cell cytopl. The edges of the groove fuse together to form a mesaxon; afterwards the mesaxon wraps itself around the axon several times the times, no. of turns resulting in the final thickness of the myelin sheath. At the end of the process, an int.l and an ext. mesaxon can be differentiated. Myelin has a comp. cell. membr., content. similar to that of the cell membr but with a higher lipid content
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Figure 3. Upper: The most frequent type of unmyelinated nerve fiber, in which isolated axons are surrounded by a Schwann cell and each axon has its own mesaxon. Lower: Many very thin axons are sometimes found together, surrounded by the Schwann cell. In such cases, there is one mesaxon for several axons.
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The myelin sheath shows gaps along it, called nodes of Ranvier; they represent sp. b between 2 neighbor S h i hb Schwann cells. Th di b ll The dist. between 2 R Ranvier nodes i i d is called an internode and consists of one Schwann cell. Depending on the diam. of the axon, the length of an internode is situated between 1 and 2 mm. In the light micr., the myelin sheath also shows cone-shaped clefts, called coneincisures of Schmidt-Lanterman; these are areas in which the cytopl. of the Schmidt-Lanterman; Schwann cell was left behind, inside the myelin sheath (during its wrapping, around th axon, th S h d the the Schwann cell did not expelled it entire cytopl., b t ll t ll d its ti t l between th the layers of cell. membr. The cytopl. may also contain 2 or 3 microtubules; they appear as oblique incisures in the myelin sheath. The chemical comp. of the myelin sheath is close to that of the cell. membr., containing phospholipids, cholest., cerebrosides. Specific proteins identif. in the myelin sheath are : the myelin basic protein and proteolipid protein. t i Several demyelinating diseases are appearing as a result of autoantibodies against myelin sheath proteins. Close ex. of the myelin sheath reveals dark lines alternating with li ht li lt ti ith lighter lines. Th d k li The darker lines are called major d ll d j dense li lines and d represent lines of fusion of the cytopl. surf. of Schwann cell membr. The more diffuse lines are known as intraperiod lines and are sites of contact without fusion, between membr. bet een the extracell. s rf of Sch ann cell membr e tracell surf. Schwann
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Figure 4. Cross section of a thick nerve showing the epineurium, perineurium, and endoneurium. The myelin sheath that envelops each axon was partially removed by the histologic technique. PT stain. Medium magnification.
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Figure 5. C Fi 5 Cross sections of t ti f two small nerves with a thi covering l ll ith thin i layer. N t th S h Note the Schwann cell ll nuclei (arrowheads) and the axons (arrows). PT stain. Medium magnification.
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Synapses are highly specialized interconnect. between the neurons or between a neuron and another specialized cell ( d h i li d ll (e.g. muscle, gl., rec.). l l ) Synapses can be classif., according to their struct. in: electrical synapses, which y p g y p are in fact the gap junct., in which the trans. takes place rapidly, with no delay and chemical synapses, which are based on neurotransmitters, the trans. is achieved after a delay period, but they are more specific. According to their effects, synapses are: excit. synapses, like those which involve glycocol as neurotransmitter and inhib. synapses, like those which have gammagammaaminobutyric acid, as neurotransmitter. According to the types of cells involved synapses are: between neurons, involved, neurons between dendrites and receptors and between axons and effectors. Most effectors. specialized synapses are those between neurons. Most of these are between an axon and dendrite of another neuron (axodendritic) or between an axon and the (axodendritic) cell body of another neuron (axosomatic). More rare we find synapses between (axosomatic). the axons (axoaxonic) and between the dendrites (dendrodendritic). (axoaxonic) (dendrodendritic).

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Synapses are highly specialized interconnect between the neurons or between a interconnect. neuron and another specialized cell (e.g. muscle, gl., rec.). Synapses can be classif according to their struct in: electrical synapses which classif., struct. synapses, are in fact the gap junct., in which the trans. takes place rapidly, with no delay and chemical synapses, which are based on neurotransmitters, the trans. is achieved after a delay period but they are more specific period, According to their effects, synapses are: excit. synapses, like those which involve l l t itt d inhib. hi h have gammaglycocol as neurotransmitter and i hib synapses, lik th like those which h gammaaminobutyric acid, as neurotransmitter. According to the types of cells involved, synapses are: between neurons, between dendrites and receptors and between axons and effectors. Most specialized synapses are those between neurons. Most of these are between an axon and dendrite of another neuron (axodendritic) or between an axon and the cell body of another neuron (axosomatic). More rare we find synapses between the axons (axoaxonic) and between the dendrites (dendrodendritic).

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Presyn. membr. Presyn membr is capable to reincorporate the neurotransmitters which were not neurotransmitters, used and the membr. of the synaptic vesicles, which will be reused to form new synaptic vesicles. Neurotransmitters may induce an excitation (depol.) or an inhibition (hyperpol.) of the postsyn. membr. More frequently used synapses, will form chains of neurons, in the central nerv syst which are the basis of the memorization process nerv. syst., process. Nerve tissue has a high plasticity; chains of synapses are sometimes abandoned, especially d i fi t years of lif If an i j i ll during first f life. injury of th central nerv. syst. occurs, f the t l t these synapses, which were abandoned in the past, will be reactive., to perform the funct. recover of the individual.

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Figure 6. The main functional aspects of the two parts of the synapse: the presynaptic axon terminal and the postsynaptic region of the next neuron in the circuit. Numbers indicate the sequence of events during its activity SER smooth activity. SER, endoplasmic reticulum.
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Figure 7. Drawings of neuroglial cells as seen in slides stained by metallic impregnation. Note that only astrocytes exhibit vascular end-feet which end-feet, cover the walls of blood capillaries.
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Figure 8. Photomicrographs (prepared with Golgi stain) of glial cells from the cerebral cortex A: Fibrous astrocytes cortex. astrocytes, showing blood vessels (BV). x1000. B: Protoplasmic astrocyte showing brain surface (arrow). x1900. C: Microglial cell. ( ) g x1700. D: Oligodendrocytes. x1900. (Reproduced, with permission, from Jones E, Cowan WM: The nervous tissue. In: Histology: Cell and Tissue Biology 5th ed Biology, ed. Weiss L [editor]. Elsevier, 1983.)
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Neuroglia (glial cells) - in the nerve tissue, accompaning neurons. In usual techniques, only their nuclei are visible; special techniques used for their staining are using especially silver and gold salts. It h b i i ll il d ld lt has been estimated th t i th central ti t d that in the t l nerv. syst. are 10 glial cells for each neuron. Main funct. of neuroglia are to protect, to nourish neurons, to form myelin sheath and to perform phagocytosis. Astrocytes are the largest of all glial cells - numerous, long processes which are attaching to the walls of the blood capillaries, being called vascular feet. Vascular feet are isolating neuroglia from both, the blood vessels and the pia mater, thus forming the blood-b i b i Th h f i th blood-brain barrier. They have pale, spherical and centrally l bl d l h i l d t ll located t d nuclei. At the form. of the blood-brain barrier are also participating the blood bloodcapillaries, which are of the common continuous type, with continuous b.m. and without pores in the edoth cells edoth. cells. Protoplasmic astrocyte - in the gray matter of the central nerv. syst.; it has extensively branched cytopl. pocesses, which are shorter and thicker than those of the fib th fibrous astrocyte. It has abundant granular cytopl. Th i processes are t t h b d t l t l Their covering the synaptic areas, the cell bodies of the neurons and the blood vessels.

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Fibrous astrocyte - in the white matter of the central nerv. syst.; it has longer, not so branched processes, compared to the protoplasmic astrocyte. In silver-stained silverprep., it has numerous filam. (fibrils) which gave the name to this cell. Oligodendrocytes have short, few cytopl. processes, they are much smaller than astrocytes. Their nuclei are smaller and condensed, compared to the nuclei of the astrocytes. Oligodendrocytes form the myelin sheath in the white matter of the central nerv. syst., being the analogous of the Schwann cells, in the periph. nerv. syst. In contrast to the Schwann cells, oligodendrocytes may f t I t t t th S h ll li d d t form the myelin th li sheath for more than one axon. The no. of the oligodendrocytes increases together with the complexity of the nerv. syst., for that, in the human specie, they are most numerous. They also appear in the gray matter of the central nerv. syst., where they are in close contact with the cell bodies of the neurons. They appear surrounding neurons, in tissue cultures, h t ll t In the th h d t l d where th present cell. movements. I th e.m., they have a dense cytopl. and they well developed RER, Golgi complex, mitochondria and microtubules.

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Figure 9. B i section prepared with Ri H t Fi 9 Brain ti d ith Rio Hortega silver stain showing fib il t i h i fibrous astrocytes with t t ith their processes ending on the external surface of blood vessels. Medium magnification.
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Microglia are the phagocytic cells of the nerv. tissue, part of the phagocyte mononuclear syst. (the origin of the microglia is represented by monocytes in the blood). They have short processes, with numerous expansions, conffering them a thorny asp. They have ovoidal nuclei with condensed chromatin; they are present in both the gray matter and the white matter of the central nerv. syst., but they are not numerous. Ependymal cells derive from the int. lining of the neural tube. They are cuboidal or columnar cells and they line the cavit. of the brain and the spinal cord. They are bathed by the cerebrospinal fluid, showing motile cilia at their apical p pole. They are y p , g p y continuous with the epith. of the choroid plexus. In the e.m., they present RER, Golgi complex and mitochondria. Some of these cells are modified; they show a long basal pole, which is penetr. deep into the subjacent nerv. subst. – they are called tanycytes. They are found in the floor of the third ventricle and they are involved in the transfer of neurohorm., from the cerebrospinal fluid to the p p primary capillary p y p y plexus, of the hypothalamo, hypothalamoyp pituitary portal syst.

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Nerve ganglia - small aggreg. of nerve cell bodies, outside the central nerv. syst.; they also contain nerve fb. and are enveloped in a dense connec. tissue capsule. Dorsal root ggl. i a sensory ganglion, f D l t l is li found on th d d the dorsal ( l (post.) root of th t) t f the spinal nerves and in some of the cranial nerves. The sensory neurons are pseudounipolar, with only one T-shaped process. The cell bodies of the neurons Tare surrounded by dense nuclei of satellite cells which are specific glial cells (they cells, appear to border the neurons, in light micr.). In the spinal ggl., we obs. a regul. arrangement of the neurons, to the periphery, while the nerve fibers appear located toward the center of the ggl ggl. The auton. ggl. are larger ggl., of the sympathy. nerv. syst. and small intramural ggl., of the parasympath. nerv. syst. Sympath. ggl. present a capsule of dense connec. tissue, connec tissue to the outside They do not show the regul arrangement of outside. regul. neurons and nerve fb., like that of the spinal ggl., instead we obs. rows of neurons, alternating with the nerve fb. Neurons are surrounded by fewer satellite cells than it is the case with the spinal ggl ggl. Intramural ggl. are small parasympath. ggl., which contain only a few nerve cells; they are found in the walls of the cavit. org., especially of the digestive tract.

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Figure 10. Silver-impregnated sensory ganglion consisting of pseudounipolar neurons Medium neurons. magnification.
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Thank you for your attention!
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HISTOLOGY
Course 14
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

The Blood

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THE BLOOD

The blood is made of - a fluid, the plasma, or the serum and of the formed elements for the blood, or the blood cells. The plasma is made of p proteins p albumin, which is the most important, being responsible for the osmotic pressure of the blood and globulins -alfa, beta and gamma; gamma globulins are the alfa, antibodies, or the immunoglobulins; fibrinogen, which is involved in the blood immunoglobulins; coagulation. In the plasma are also found small molecule substances, ions and inorganic salts. To observe the blood elements or blood cells, are used the blood smears, a blood droplet being spread in a very thin layer, on a slide and air-dried. The most aircommon stain method is the Romanovsky technique and its variants, using a mixture of eosin and methylene blue, or derivatives, blended in different proportions.

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Figure 11. Scanning g g electron micrograph of normal human erythrocytes. Note their biconcave shape shape. x3300
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Erythrocytes (red blood cells)
Erythrocytes are the most numerous cells present on the blood smears, having a round, reddish appearance. The normal concentration of red blood cells in human, is of about 3.9 - 5 million/ ml in women, and of about 4.5 - 6 million/ ml, in men. The Th normal shape of th red bl d cell i th t of a bi l h f the d blood ll is that f biconcave di k resembling t a disk, bli to biconcave lens. This shape is increasing the surface to volume ratio, important in the gases exchanges, which is the main function of the erythrocytes. Some authors are even not calling them cells, because they do not have a nucleus, a major characteristic of a classical cell; they are nonnucleated elements, the nucleus being expelled from the future mature erythrocyte, at the moment of its liberation from the bone f th f t t th t t th t f it lib ti f th b marrow, into the circulation. The normal dimensions of the red blood cells are of about 7.5 micrometers, in di t d f i t l 2.5 i t i h d f l diameter, and of approximately 2 5 micrometers thi k i periphery, and of only thick, in 0.80.8-1 micrometers in the center, because of its shape.

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The content of the red blood cells is represented by 33 % hemoglobin (Hb), which is giving the acidophilia of the elements; hemoglobin is made of heme, containing iron and of a protein part the globine Hemoglobin is essential in the gases part, globine. transport, done by erythrocytes; it combines easily with oxygen, forming oxyhemoglobin, with carbon dioxide, forming carbaminohemoglobin, but with carbon monoxide is forming an irreversible combination carboxyhemoglobin combination, carboxyhemoglobin, which may lead to severe intoxication and even death (heavy smokers have a higher content of carboxyHb, in their blood). The cellular membrane of the red blood cells was very well studied because if studied, treated with hypertonic solutions, the content of the cells is expelled and the membrane keep its shape and composition (the so-called "ghost-cells"). The so"ghostcomposition of the membrane is of 50% proteins 40% lipids - cholesterol proteins, cholesterol, phospholipids etc. and 10% glucids. Glycophorines are fractions of the membrane, which when modified, are triggering a signal for the destruction of the cells by macrophages (especially in the spleen), as if is the principal sign of aging of the erythrocytes.

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Their normal life in the blood is of about 120 days. The membrane proteins of the erythrocytes are classified i i h l ifi d in: integral membrane proteins, which span the whole l b i hi h h h l lipid bilayer and peripheral proteins, associated with the inner face of the cellular membrane. These latter, are responsible for the maintenance of the unusual shape f th th t th t ib t t th t k l t t th ith th ll l of the erythrocytes, they contribute to the cytoskeleton, together with other cellular filaments, forming also a membrane skeleton (e.g. spectrin, stromatin). Besides the shape conservation, they are involved in the great flexibility of the red blood cells, cells during their passage through the narrower capillaries when they may capillaries, appear, packed, as cup-shaped. cupThe decrease in the number of erythrocytes are pathologic conditions, generically called anemias; are occurring by decreased production of red blood cells in the bone marrow, by loss through hemorrhages, by increased destruction (for example in the spleen), or by production of red blood cells with a low content of Hb, especially by iron deficiency; this is called hypochromic anemia The Hb anemia. reverse, consisting in an overproduction of red blood cells, is called polycythemia or erythrocytosis, and may be a physiological estate, in the case of the people living at high altitude where the pressure of the oxygen is low or a pathological altitude, low, condition, leading sometimes to thrombosis.

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There are also anemias produced by genetic diseases, as in the case of sickle cell disease; in this case, the mutation of one nucleotide in the DNA molecule, involving the gene for beta chain of Hb is resulting in the replacement of an Hb, aminoacid by another, in this case, glutamic acid is replaced by valine. It will appear an abnormal Hb, called Hb S, the red blood cells will be sickle-shaped (like sickledemilunes), demilunes) they are also very fragile with a shorter life leading to severe anemia fragile, life, anemia, and consequently, anoxia of the tissues. The red blood cells during their formation in the bone marrow, are gradually loosing their organelles and finally their nucleus; in some young erythrocytes erythrocytes, however, are persisting, for about 24-48 hours, remains of ribosomal RNA, like a 24few granules, or a small network, giving them a characteristic aspect; these young erythrocytes are called reticulocytes Normally they represent only 1% of the red reticulocytes. Normally, blood cells; they may be increased in the treatment of anemias, reflecting a good chosen therapy. Red blood cells are dying after 120 days, being removed by macrophages, especially from the spleen and bone marrow. By erythropoiesis, bone marrow is providing permanently new erythrocytes, to the circulating blood.

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Leukocytes
In the hematocrit tube, when the blood is centrifuged, the thick lower layer is red tube, and made of erythrocytes (hematocrit -Ht, being the volume of packed erythrocytes per unite volume of blood; normally of about 37%-45% in adult) while the thin 37%-45%, adult), layer above is looking white-grayish and is made of leukocytes (white blood cells), whitewhich are less dense than the red blood cells. Above, is a very fine layer of platelets, platelets not visible with the naked eye eye. Leukocytes are, according to the aspect of the nucleus -polymorphonuclear cells, with lobes in the nucleus and mononuclear cells, with no lobes in the nucleus, which is round with a compact aspect - lymphocytes and monocytes According to round, monocytes. the aspect of the cytoplasm, leukocytes are divided into granulocytes, with characteristic granules stained differently, in their cytoplasm -neutrophils, eosinophils and basophils which are also polymorphonuclear cells and basophils, cells, agranulocytes, with fewer azurophilic granules in the cytoplasm (which are binding azure dyes), these are mononuclear cells - monocytes and lymphocytes. The normal number of leukocytes per blood microliter, in adult, is of about 6000-8000. 6000Leukocytes are involved in the humoral and cellular immune defense of the organism, being capable to pass by diapedesis, through the wall of the capillaries g p y y in the organs and tissues, especially in the connective tissue, all over the body.

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Neutrophils - are representing 60-70% of the blood leukocytes. They have a 60diameter of about 12-15 micrometers, and 3-5 lobes in the nucleus, united by thin 123chromatin bridges not always visible with the light microscope and for that they bridges, microscope, that, received the denomination of polymorphonuclear cells. Old cells have 5 or even more lobes, while the young neutrophils may show a horseshoe-shaped horseshoenonsegmentated nucleus (in the so-called Arneth formula -the predominance of soyoung elements is called left deviation, and presumes a high reactivity of the bone marrow, while the right deviation, is found in the case of hyporeactivity of the bone marrow, meaning of course, predominance of old neutrophils in the blood). In female sex, the second X chromosome is appearing sometimes, like a drumstick appendage, on one of the lobes of the nucleus. It is called the sexual chromatin, and is important in the clinical diagnosis of some genetic diseases involving the diseases, sexual chromosomes (e.g. - Klinefelter syndrome, consisting in males with XXY; Turner syndrome -females with XO; in the first case we find an extra X chromosome, in the second, a missing X chromosome).

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In the cytoplasm of the neutrophils are found two types of g y p p yp granules, most numerous are the neutrophilic or the specific ones, or secondary granules, violet stained and generally smaller than the primary or azurophilic granules. The granules are primary lysosomes, containing different enzymes as: lysozyme, phosphatases, lactoferrin, etc. Neutrophils, Neutrophils, as well as basophils and eosinophils are terminal cells, they live for 1-3 days in the connective tissue and after that, they die. Their principal activity is y y y phagocytosis of small particles, in comparison with the macrophages, which may phagocyte larger particles, also. After the surrounding of the particle by pseudopodia, and the formation of a vacuole, or phagosome, by oxygen consumption, are formed free radicals, such as superoxide anions O2-, and O2hydrogen peroxide H2O2. Together with the halide ions, myeloperoxidase and acid ph, all these are functioning as cytotoxic for bacteria, fungi, viruses, killing them. Lactoferrin, b linking iron i l di t d th of b t i b L t f i by li ki i is leading to death f bacteria, because i iron i essential is ti l in the nutrition of bacteria. Dead neutrophils, with bacteria and digested materials are forming the pus.

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Eosinophils are only 2-4%, of the leukocytes in the blood. Characteristically, they have 2only two lobes in the nucleus (bilobed), few organelles and numerous, refractive, eosinophilic granules, the specific granules of this cell. The granules are large, about 200 granules per cell. The granules are containing a crystalline core, a denser part, in the electron microscopy, also called internum; the major component of the internum is the major basic protein, involved in the killing of the parasites, antiparasitic defense. The external part of the granules is lesser dense, called extemum, is containing different enzymes. The increase in number of the eosinophils, called eosinophilia is found in parasitic i f t ti iti infestations and i allergic di d in ll i diseases, since th granules are also containing an i the l l t i i enzyme called histaminase, capable to neutralize histamine, released during allergic diseases. Characteristically, corticoids, hormones of the adrenal cortex, also used in therapy, produce a rapid fall of the number of the eosinophils, especially by stopping them to pass from the bone marrow, into circulating blood. Basophils have the same size like the other granulocytes (12-15 micrometers, in (12diameter) and their nucleus is masked by large numerous basophlic granules, also metachromatic. The granules are containing heparin and histamine, like those of the mast cells, and th h t ll d they have th same receptors f E i the t for immunoglobulins, on th i cellular l b li their ll l membrane, being involved, as well, in the allergic reactions, of immediate hypersensitivity. Even if they are resembling a lot with the mast cells, they have different stem cells i th b t ll in the bone marrow.
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They are very hard to find on the blood smears representing only 0 6-1% of the smears, 0.60.6 leukocytes. A specific dermatologic inflammatory reaction is containing predominantly basophils, being called cutaneous basophilic hypersensitivity. Lymphocytes are spherical cells, most numerous i th circulating bl d b i th L h t h i l ll t in the i l ti blood being the small lymphocytes, mature cells, of about 6-8 micrometers (7 in average); a small 6percent of the lymphocytes in the blood are medium-sized and large lymphocytes, mediumwhich may reach up to 16 micrometers in diameter The large lymphocytes are micrometers, diameter. young cells, from the bone marrow, or resulted after the antigenic contact, of a mature, small lymphocyte, which suffered the process of blastic transformation (the cell is regressing in the young stage and is beginning to divide leading to the divide, formation of several lymphocytes capable to recognize the antigen which initiated the process). The small mature lymphocyte has a condensed nucleus deep violet and a small nucleus, violet, amount of cytoplasm, pale basophilic, surrounding like a rim the nucleus. In the cytoplasm are found a few azurophilic, enzymes-containing granules. The enzymeslymphocytes are divided into to big categories: B and T lymphocytes according to lymphocytes, markers on the cellular membrane of the lymphocytes, called CD markers (clusters of differentiation).

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B and T lymphocytes can be differentiated only using immunological techniques or scanning electron microscopy B lymphocytes are formed into the bone marrow microscopy. and are colonizing after that, the secondary lymphoid organs - lymph nodes, spleen, tonsils, etc., while T lymphocytes after being formed in the bone marrow are colonizing the thymus where they suffer a complex process of differentiation thymus, into different types of T lymphocytes -helper, cytotoxic and supressor, being the main types of T lymphocytes. After fully matured in the thymus, the T lymphocytes are colonizing as well, the secondary lymphoid organs. When they come in contact with an antigen (foreign or non-self particle for the nonorganism), both T and B lymphocytes are capable to produce, by division, effector cells and memory cells The effector cell of the B lymphocyte is the plasma cell cells. cell, which will secrete antibodies, while the effector cell of the T lymphocyte is the cytotoxic T lymphocyte, the main cell responsible for the cellular immunity of the organism (cellular immunity consists in the defense against tumor cells, virus infected cells, graft rejection, etc.). Memory cells are living long time in the organism (20,30 years) giving us the immunity against some diseases we had already, like a wide category of contagious diseases of the childhood, which y, g y g , normally are providing afterwards, a permanent immunity. Most of the blood lymphocytes are T, about 85%, the rest of10-15% being B lymphocytes. of10Lymphocytes represent approximately 25-28% of the leukocytes in the blood. 25-

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Figure 13. The 5 types of human leukocytes. Neutrophils, eosinophils, and basophils have granules that stain specifically with certain dyes and are called granulocytes. Lymphocytes and monocytes are agranulocytes; they may show azurophilic granules, granules which are also present in other leukocytes.
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Figure 14. Photomicrograph of a blood smear showing 3 neutrophils and several erythrocytes. Each neutrophil has only one nucleus with a variable number of lobes Giemsa stain High nucleus, lobes. stain. magnification.
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Figure 15. Photomicrograph of an eosinophil. Note its typical bilobed nucleus and coarse cytoplasmic granules. Giemsa stain. High magnification.
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Figure 16 E i Fi 16. Eosinophil with its bilobed nucleus and coarse cytoplasmic hil ith it bil b d l d t l i granules. Giemsa stain. High magnification
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Figure 17 T small l Fi 17. Two ll lymphocytes with th i round, d k t i d nuclei. h t ith their d dark-stained l i Giemsa stain. High magnification.
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Monocytes are larger cells, of about 15-20 micrometers in diameter, with an ovoid 15or kidney-shaped nucleus, lighter stained than that of the lymphocytes, because it kidneycontains more dispersed chromatin In the cytoplasm are found very small chromatin. azurophilic granules, giving a grayish aspect to the cytoplasm; these granules are lysosomes. Monocytes have also nucleoli in the nucleus and some common organelles, organelles in their cytoplasm cytoplasm. Monocytes are the main source of the macrophages of the mononuclear phagocyte system, present all over the organism. They represent approximately 446% of the leukocytes in the blood and after about 72 hours circulating in the blood blood, they pass into the tissues, between the endothelial cells of the capillaries walls and they become macrophages. They keep the capacity of cellular division, in the tissues, tissues and also they are capable to renew their enzymatic equipment being the equipment, main phagocyte cell of the organism, involved in the immune responses, together with the lymphocytes. Macrophages are also the main antigen presenting cells of the human organism.

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Figure 18. Photomicrograph of a monocyte. This cell type has a kidneyshaped nucleus with delicately stained chromatin. The cytoplasm is slightly basophilic. Giemsa stain. High magnification.
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Platelets
Platelets or thrombocytes are nonnucleated disk-like fragments of about 2-4 disk2micrometers, in diameter. They are fragments of a giant polyploid cell, the megakaryocyte, present in the bone marrow, which is the source of platelets. They are most important in blood clotting and in keeping the blood inside the vessels; their normal number is of about 200,000-400,000 per microliter of blood. Their life 200,000in the blood, is of about 10 days. In the blood smears, usually they appear in small clusters The platelet has a smears, clusters. peripheral lighter zone, called hyalomere and a central well stained, violet zone, called the granulomere. With the electron microscope the platelet has an open canalicular system, made of canaliculi which may be continuous with invaginations y , y g of the plasmalemma. These canaliculi are helpful in the release of different molecules during the process of blood clotting. In the periphery they contain bundles of microtubules, which are maintaining the disk-shape of the p g diskp platelet, and together with actin filaments are involved in the process of platelet adhesion and platelet aggregation, in the formation of the blood clot. The granulomere contains different types of granules, mitochondria and glycogen granules.

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The granules called delta granules or dense bodies, are granules of about 300 nm in diameter, containing especially calcium ions, ADP and ATP; these granules are accumulating serotonin, f l ti t i from th plasma. Al h granules may b l the l Alpha l be larger and d contain fibrinogen, platelet-growth factor. The smallest granules, are in fact plateletlysosomes, and are called lambda granules. The granules are azurophilic, most of them are alpha gran les granules. Platelets after adhesion to a damaged blood vessel, are initiating the platelet aggregation, also by the release of the alpha and delta granules. The real blood coagulation, i possible b cascade i t l ti is ibl by d interaction of approximately 13 plasma ti f i t l l proteins, leading to the formation of a fibrin polymer. In the network of the fibrin clot, are trapped the formed elements of the blood, thus resulting in the formation of a blood final coagulum coagulum. The platelets are also involved in the clot retraction, by the contractile filaments they do contain, and in the clot lysis, by the enzymes released from their lambda granules. l

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Figure 12. Electron micrograph of human platelets x40 740 platelets. x40,740. (Courtesy of M Harrison.)
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Thank you for your attention!
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1.HEMATOPOESIS 1 HEMATOPOESIS
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

HEMATOPOESIS
1. Th bone marrow The b 2. Erythropoiesis 3. Granulopoiesis 4. Megakaryocytopoiesis 5. Monocytopoiesis 6. Lymphocytopoiesis 7. Thymus

Hematopoiesis is the process by which, the blood cells are formed. The stem cell is a precursor cell, which give rise to all types of blood cells; stem cells are called colony-forming cells (CFCs) or colony-forming units (CFUs). Stem cells are undifferentiated elements, - a sufficient proliferative capacity. A CFC that is capable to develop in all blood-cell types is a pluripotential stem cell. As the restriction toward the development into the cells of three/two/one lineage of blood cells is increasing, the proliferative capacity is decreasing; → a multipotential stem cell → a committed progenitor cell. it ll Colony-stimulating factors (CSF) or growth factors or factors, hematopoietins are stimulating the divisions of the precursors, and are selecting the program of development of the precursors into one lineage of blood cells. The completely restricted precursor cells (blasts) are the sole, which are recognizable in the microscopy. light microscopy

Hematopoiesis is initiated at approximately two weeks of embryonic life, and has three stages: 1. The extra-embryonic stage - in the mesenchyme of the sac of Yolk, inside the blood vessels; are first developing islets of erythropoiesis, and thus are appearing nucleated erythrocytes, containing fetal hemoglobin. In the islets are also present CFUs. The hepato-spleno-thymic stage is initiated at 4-5 weeks of intrauterine life, and its development is maximum at 5-6 months of intrauterine life. In this stage islets of hematopoiesis - in the liver, spleen, and in the thymus. In the islets from the liver and the spleen are found precursors for al blood cell types, while in the thymus are developing exclusively T lymphocytes. Bone marrow hematopoiesis begins in the second month of intrauterine life, when first bone cavities are formed; bone marrow is developing and this will ; p g become the permanent site of hematopoiesis, after birth. The hematopoiesis in the liver and in the spleen is gradually decreasing; up to birth, a few islets of hematopoiesis are still present in the liver and in the spleen, but they are disappearing during the first and the second months of life.

2.

3.

Figura 1. Differentiation of pluripotential stem cells during hematopoiesis.

1. The bone marrow
All hematopoietic organs are made of a capsule, a stroma and a p p g p parenchyma. The y capsule, usually made of dense connective tissue, - in the case of the bone marrow the endosteum, lining the bone cavities. Stroma consists of a tridimensional network of reticular fibers, enveloped by reticular cells, with phagocytic and secretory properties. Red bone marrow is present in the childhood and in the young individuals, being the active bone marrow; with age, red bone marrow becomes restricted to the sternum, vertebrae and coxal bone. It is replaced by yellow bone marrow, with grayish appearance, containing - white adipose tissue and connective tissue. pp , g p Parenchyma of the red bone marrow - hematopoietic islets or cords, and sinusoidal capillaries. Sinusoids are thin-walled capillaries by which the mature blood cells may capillaries thincapillaries, which, enter into the blood circulation. In case of severe blood loss, yellow bone marrow may regress into red bone marrow.

Figure 3 Section of active bone marrow (red bone marrow) showing some of its components Five 3. components. blood sinusoid capillaries containing many erythrocytes are indicated by arrowheads. Note the thinness of the blood capillary wall. Giemsa stain. Medium magnification.

Figure 2. Drawing showing the passage of erythrocytes, leukocytes, and platelets across a sinusoid capillary in red bone marrow. Because erythrocytes (unlike leukocytes) do not have sufficient y y y pressure motility to cross the wall of the sinusoid, they are believed to enter the sinusoid by a p gradient that exists across its wall. Leukocytes, after the action of releasing substances, cross the wall of the sinusoid by their own activity. Megakaryocytes form thin processes that cross the wall of the sinusoid and fragment at their tips, liberating the platelets.

2. Erythropoiesis
CFUe is sensitive to erythropoietin, the main factor which stimulates red blood cells differentiation differentiation. Proerythroblast is the first stage in the red blood cells development, this cell is recognizable in the light microscopy. Most important gradual changes of the precursors till the mature erythrocyte are: progressive decrease in the cell size precursors, erythrocyte, size, also in the size of the nucleus, which becomes condensed, is loosing nucleoli; accumulation of hemoglobin, till the organelles are disappearing. Proerythroblast is a large cell of approximately 20-25 micrometers. The cytoplasm cell, micrometers is intense basophilic, nucleus is large, with dispersed chromatin and well defined nucleoli. Basophilic erythroblast is smaller than the proeythroblast Basophilia is intense proeythroblast. intense, due to large amounts of polyribosomes. Nucleus is more condensed, and the cell is beginning to synthesize protein part of the hemoglobin and edocytosis of iron. Polychromatophilic erythroblast starts to accumulate hemoglobin hemoglobin. Polychromatophilia means a combination of basophilia (from ribosomes) and acidophilia (from hemoglobin); this may result in a grayish aspect of the cytoplasm cytoplasm, or in separate acidophilic and basophilic regions of the cytoplasm in the light microscopy.

Figura 4. S Fi 4 Summary of erythrocyte maturation. The stippled part of the cytoplasm (on the left) shows the continuous f th t t ti Th ti l d t f th t l ( th l ft) h th ti increase in hemoglobin concentration from proerythroblast to erythrocyte. There is also a gradual decrease in nuclear volume and an increase in chromatin condensation, followed by extrusion of a pyknotic nucleus. The times are the average life span of each cell type. In the graph, 100% represents the highest recorded concentrations of hemoglobin and RNA.

Normoblast or the orthochromatic, or acidophilic erythroblast is the last stage in which a nucleus is identifiable. The normoblast is smaller than the polychromatophilic erythroblast; the nucleus first becomes pyknotic and finally is expelled from the cell, surrounded by a thin rim of cytoplasm and a membrane. The expelled nucleus will be p g y phagocytized by macrophages, resident in the bone marrow. The cytoplasm is y p g , y p acidophilic, consequence of large amount of hemoglobin. Reticulocytes are nearly mature red blood cells, released in the periphery. Reticulocytes have almost same size as the mature erythrocytes; they contain in the cytoplasm remains of polyribosomes, which become visible like reticulum; even if they are not yet fully matured erythrocytes, they are circulating cells, which are continuing hemoglobin synthesis for 24-48 hours. The normal percentage of reticulocytes in the g y p g y circulating blood is of about 1 %. The last dividing cell, of the red blood cell developmental stages is the p y polychromatophilic erythroblast. Normoblasts and reticulocytes are p p y y postmitotic cells, , they do not divide anymore. Besides erythropoietin, in the regulation of erythropoiesis are involved hormonal factors (thyroid hormones and corticoids are stimulating erythropoiesis, while the estrogens are inhibiting it). Spleen is the main site of destruction of aged erythrocytes and is triggering a feed-back on erythropoiesis. Nutritional factors, such as the amount , , g , , of iron, folic acid, found in the ingested food, secretion of intrinsic factor, in the stomach, are also involved in the process of erythropoiesis, being important in the synthesis of the hemoglobin.

Figure 5. Stages in the development of erythrocytes and granulocytes.

3. Granulopoiesis

Figure 6. Section of stimulated red bone marrow. Note 4 mitotic figures ( g g (arrows) and a p ) plasma cell (arrowhead). Regions of erythropoiesis and of granulopoiesis are also evident. Most immature granulocytes are in the myelocyte stage: their cytoplasm contains large, dark-stained azurophilic granules and small, less darkly stained specific granules. Giemsa stain. High magnification.

The development of g p granulocytes - neutrophils, eosinophils and basophils - is stimulated y p p p by more or less specific stimulating factors, such as G-CSF (colony-stimulating factor for G(colonygranulocytes); interleukin 3 (IL3) which stimulates the production of all blood cell types. Recently, was discovered that each granulocyte (neutrophilic, eosinophilic and basophilic) has its own precursor, in the bone marrow. Myeloblast is the first stage in the formation of the granulocytes. The myeloblast is a large cell of approx. 15-20 µm in diameter, with a large, euchromatic nucleus, with well15welldefined nucleoli. The cytoplasm is agranular and basophilic, because of the presence of RER. Myeloblast is hard to recognize in the light microscopy. Promyelocyte is the next stage in the development of the g y y g p granulocytes; it may be a little y y bit larger than a myeloblast, nucleus is not yet identated, and nucleoli are visible. In this stage are appearing azurophilic, primary, or nonspecific granules. Nonspecific granules are present, in this stage. Azurophilic granules are lysosomes. The azurophilic, primary granules, are first appearing in the periphery of the cell, they are coated with membranes. Myelocyte is the stage in which the specific g granules of the g granulocytes are first y y g y appearing. In the bone marrow are identified neutrophilic, eosinophilic and basophilic myelocytes. The nucleus became identated, condensed; nucleoli are no more visible. As the number of specific granules, are increasing, the azurophilic granules are gradually declining. The specific, or secondary granules are first appearing in the proximity of the nucleus, they are smaller than the primary ones; they gradually fill the cytoplasm.

Figure 7. Drawing illustrating the sequence of gene expression in the maturation of granulocytes. Azurophilic granules are blue; specific granules are pink pink.

Metamyelocyte, the next stage, has a deep identated nucleus, becoming kidney-shaped. The nucleus is more condensed than that of the previous stage. The specific granules are well developed, the number of specific granules is larger than that of the azurophlic granules. Both types of granules are fully developed in this stage. The band cell, or the nonsegmentated neutrophil is the almost mature neutrophil; its nucleus is like a peripheral band, or like a horseshoe. At this time starts the lobulation of the nucleus; only the cells with 3-5 lobes in the nucleus are considered completely matured polymorphonuclear leukocytes, or neutrophils. A small percent of the circulating neutrophils are band cells, approximately 3-5 %. In the bone marrow, approximately 50 % of the cell population, is represented by cells of the neutrophilic lineage. The number of mature neutrophils and band cells in the marrow, is 15 times more numerous than that of the circulating neutrophils, representing a storing compartment, which is used in special conditions (for example, in severe infections of the organism).

4. Megakaryocytopoiesis (platelets formation)

Megakaryocytes are the precursors of the platelets. The first cell derived from the CFUCFUMeg, is the megakaryoblast Megakaryoblast is a large cell of approximately 30-50 Meg megakaryoblast. 30micrometers in diameter, with a large, nonlobulated nucleus, with numerous nucleoli; the cell has 32 N, or 64 N chromosomes, but no cytokinesis is occuring, thus is beco becoming a po yp o d ce As the ce beco es larger a d po yp o d, it is ca ed a g polyploid cell. s e cell becomes a ge and polyploid, s called magakaryocyte. Some authors are distinguishing different types of megakaryocytes, during the platelet formation: basophilic megakaryocyte, the basophilia being generated by the appearance in the cell of the azurophilic granules, the granulomeres of the future platelets; the platelets generating megakaryocyte, is the stage of demarcation of each p platelet, by channels developed at the surface of the megakaryocyte. In the , y p g y y development of the platelets, when the surrounding channels of each platelet are fusing together, the platelets are released, all at the same time, a phenomenon known as release of the platelets.

Figure 8. Cells of the megakaryocyte series shown in a bone marrow smear. Note the formation of platelets at the lower end of the megakaryocyte

5. Monocytopoiesis

The precursor of the monocyte is the monoblast, which becomes a promonocyte, cell which is very similar to the monocyte, and hard to recognize in the light microscopy. microscopy After living a short time in the blood (36-72 hours) the monocytes pass through the (36wall of the capillaries in different tissues and organs becoming mononuclear organs, phagocytic cells, generically called macrophages, the main APC of the organism.

6. Lymphocytopoiesis
The process of development of the T and B lymphocytes is very complex and not restricted to the bone marrow. As previously signaled, lymphocytes have a separate precursor in the bone marrow from which are arising T and B lymphocytes During fetal marrow, B, lymphocytes. life, T lymphocytes will colonize the thymus, where they will further divide and mature. B lymphocytes developed in the bone marrow, will colonize the thymus-independent y y p g y p areas, in the secondary lymphoid organs. The thymus-independent areas are follicular or nodular zones, forming lymphoid nodules. T lymphocytes developed in the thymus will populate the thymus-dependent areas, of the secondary lymphoid organs; these areas do not show nodules of lymphocytes, instead, T lymphocytes have a diffuse, nonnodular di d l disposition. Mi iti Microenvironmental (h i ) f t i t l (homing) factors are most i t important i th t t in the colonization of different areas of the secondary lymphoid organs, with one of the two types of lymphocytes. The principal three stages in the development of the lymphocytes are – lymphoblast lymphoblast, prolymphocyte and lymphocyte. Lymphocytopoiesis may be either antigen-independent, which means the usual development of the lymphocytes, from the young stage of lymphoblast to that of a lymphocytes lymphoblast, mature small lymphocyte, and in this case we speak of a primary lymphocytopoiesis, or an antigen-dependent lymphocytopoiesis, also called secondary lymphocytopoiesis. y p y ( g ) gg The contact of a lymphocyte with a non-self (antigen) will trigger the blastic transformation of the mature lymphocyte → a young cell, capable of cellular division, a phenomenon, which is one of the most important steps of the immune response.

7. Thymus
The thymus is a lymphoepithelial organ, since it has a double origin; it is developing f d l i from both endoderm and mesoderm. Th th b th d d d d The thymus i l is located i th t d in the upper part of the mediastinum; it is divided into thymic lobules. Each thymic lobule has a peripheral cortical, and a central medullary zone. The stroma of the thymus is made of networks of epithelial reticular cells. Epithelial reticular cells establish desmosomal junctions, one with another; in the cytoplasm the cells contain keratin filaments. Lymphocytes are closely surrounded by epithelial reticular cells, which are an important part of the blood-thymus barrier. bloodThe thymus cortex is more efficient isolated by the blood-thymus barrier, than the bloodthymus medulla. The blood-thymus barrier is providing an antigen-free bloodantigenenvironment, in the cortex of the thymic lobules, suitable for the cellular division and differentiation of the T lymphocytes, here instructed to recognize the self, as well as different types of antigens, according to the immune memory of the specie.

Figure 9. Photomicrograph of a section of thymus showing the lobules. Two lobules show the dark cortical and the light medullary zones. At the upper left are blood vessels and the connective tissue capsule. Pararosaniline-toluidine blue (PT) stain. Low magnification.

The thymus is colonized with precursors of T lymphocytes, at approximately 10-12 weeks 10of intrauterine life. At birth, the weight of the thymus is around 15 grams; its maximum development is reached at puberty after that a gradual involution of the organ is noticed; puberty, the lobules of the thymus are replaced by connective tissue and white adipose tissue, but the T lymphocytes in the degenerated thymus, are still dividing and differentiating, here. Thymus is enveloped by a dense connective tissue capsule which is continuous and has capsule, no lymphatic vessels, being part of the blood-thymus barrier. bloodThe blood-thymus barrier becomes efficient only after the first two-three months of bloodtwoe b yo c e; during s embryonic life; du g this time, T lymphocytes a e immunologically instructed to e, y p ocy es are u o og ca y s uc ed o recognize the self. Trabeculae of dense connective tissue are parting the lobules only at the level of the cortical zones; the lobules are communicating one with another, by their medullary zones. ; g , y y Each thymic lobule shows two distinct zones: a deep basophilic cortex in the periphery, and a paler central zone, the medulla. In the central part of the medulla are found structures, which are unique to the medulla of the thymus, the Hassal or the thymic corpuscles. Th H l The Hassal corpuscle i made of concentrically di l l is d f t i ll disposed epithelial reticular d ith li l ti l cells; they may measure between 30 to 150 µm, in size. The centrally located squamous epithelial reticular cells show no nuclei, resembling to keratinized epidermal cells. It seems that the number and size of the thymic corpuscles is increasing with the age of the individual. In the cortex, as well as in the medulla of the thymus, are found the same cell types, only the proportion of distinct cell types is different in the two regions. Generally, T y p y , p g p , lymphocytes, macrophages and epithelial reticular cells are most numerous, while other cell types, such as antigen presenting cells (dendritic and interdigitating cells) are more rare.

T lymphocytes are clearly predominating in the cortex; in the medulla we find the reverse, T lymphocytes are rare and the epithelial reticular cells are predominating, together with macrophages. macrophages In the cortex the epithelial reticular cells are masked by numerous T cortex, lymphocytes. Epithelial reticular cells show long slender processes, in which are hosting dividing T lymphocytes, or T cells during cellular differentiation; this aspect, gave to epithelial reticular cells, the name of "nurse" cells. B h i h li l i l ll d i i ll i h i f Both epithelial reticular cells and antigen presenting cells, are expressing on their surface HLA antigens (antigens of the major system of histocompatibility) thus helping T lymphocytes, to learn the difference between self and non-self. During this complex process of nond e e a o , os of e y p ocy es are excluded differentiation, most o the T lymphocytes a e e c uded by apop os s, the result be g that apoptosis, e esu being a only approximately 8-10 %, of the T cells in the cortex of the thymic lobules are reaching the 8medulla. The rest are phagocytized by macrophages. Epithelial reticular cells are secreting thymic hormones, which are essential in the differentiation of the T lymphocytes, after mitosis, such as thymosin and th diff ti ti f th l h t ft it i h th i d thymopoietin. i ti The blood-thymus barrier is protecting T lymphocytes from the antigenic contact. The barrier bloodwas found effective in the thymic cortex, between blood vessels and parenchyma, and less effective or even absent, in the medulla. The components of the blood-thymus barrier are: the bloodcontinuous capsule of dense connective tissue, enveloping the organ; tight junctions between the endothelial cells of the blood capillaries. The absence of afferent lymphatic vessels in the thymus and the envelopment of the inner face of the capsule, as well as the enveloping of the blood capillaries, by epithelial reticular cells processes are important components of the capillaries processes, bloodblood-thymus barrier. Perivascular macrophages phagocytize any particle, which cross the capillary wall. y y p y , y p y The thymus is a source of T lymphocytes, even after its involution. Most of the T lymphocytes excluded in the thymus are self-reacting T cells (which were programmed to react against selfself).

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2. SECONDARY LYMPHATIC ORGANS. MUCOUS-LINING MUCOUSASSOCIATED LYMPHATIC TISSUE
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

SECONDARY LYMPHATIC ORGANS; MUCOUSLINING ASSOCIATED LYMPHATIC TISSUE 1. 1 The lymph node 2. The spleen 3. 3 Mucosal attached lymphatic tissue

The lymphatic tissue is divided into loose and dense lymphatic tissue. In the loose lymphatic tissue, lymphocytes - interspersed with the connective tissue elements, and with the blood vessels; lymphocytes are not so numerous here (the medulla of the lymph node and the red pulp of the spleen). Dense lymphatic tissue - a high density of the lymphocytes, which appear disposed in nodules, a characteristic of the B lymphocytes, or in a diffuse manner the way of disposition of the T lymphocytes in the so-called thymusmanner, lymphocytes, so called thymus dependent zones – in the secondary lymphatic organs. The lymphatic tissue may also be divided into transitory and permanent structures. Transitory structures are lymphatic nodules or lymphatic infiltrations (with a diffuse arrangement of the lymphocytes); they are often solitary, and they appear and disappear, according to the intensity of the antigenic stimulation, from the environment. Such transitory lymphatic tissue structures are found especially in the lamina propria of the digestive tract, and in a lesser degree, of the respiratory, genital and urinary tract. Permanent lymphatic tissue structures are both nodular and diffuse zones of lymphocytes, found in the secondary lymphoid organs such as: lymph nodes spleen Peyer patches organs, nodes, spleen, patches, tonsils, appendix. The lymphoid nodule has two different morphologic aspects, resulting from the presence of the antigenic stimulation or the reverse the lack of it In the absence of the antigenic stimulation, reverse, it. stimulus, the lymphoid nodules are called primary, or nonreactive nodules, and they look like nodular crowding of small (mature) B lymphocytes, rounded, or ovoidal, with a homogenous, aspect, microscopy. homogenous basophilic (violet) aspect in the light microscopy If the antigenic stimulation occurs, the lymphatic nodule becomes reactive, or secondary nodule, with a clear (germinal) center, in its middle.

The secondary nodules are the morphologic expression of the regression of the B lymphocytes into a younger stage - the lymphoblasts, which are capable of numerous mitosis, th enlarging th clone of l it i thus l i the l f lymphocytes capable t recognize th antigen, h t bl to i the ti which was initiating the whole process, in fact a immune response. The secondary nodule is an ovoid structure, with a paler (clear) center, or germinal center, and a crown of small mature deep basophilic lymphocytes in the periphery The crown of small, mature, lymphocytes, periphery. the nodule usually exhibits a cup-like thickening, at one pole. The germinal center is made of B lymphoblasts, or large lymphocytes, result of the regression of mature cells; lymphoblasts have a larger amount of cytoplasm nucleus with dispersed chromatin 1 cytoplasm, chromatin, 12 visible nucleoli, with such characteristics, they give the pale aspect of the germinal center. Macrophages and dendritic reticular cells, are antigen presenting cells, found in the clear centers of the secondary nodules, which cannot be differentiated in the light microscopy. I th germinal center are also present: h l i In the i l t l t helper T l lymphocytes (CD4+) h t important in the process of immune cooperation, plasma cells, resulting by transformation from B lymphocytes, effective cells of the humoral immune response, which are secreting antibodies (Ig) (Ig). In the dense crown of lymphocytes of a secondary nodule are found: small, mature, B lymphocytes, h l helper T l lymphocytes, suppressor T l lymphocytes and memory B l h h h d lymphocytes.

1. The lymph node
The lymph nodes are sites of immune responses to lymph-carried antigens, situated y p p lymphy p g on the lymphatic circulation. Lymph is filtered - through the lymph nodes. Usually the lymph nodes show a kidney-shape, or ovoid. They have a convex and a concave kidneymargin, with a hilum. They have the common histologic organization of the lymphatic organs - a capsule, a stroma and a parenchyma. The capsule is made of dense connective tissue; is penetrated by afferent lymphatic vessels on its convex site, and by efferent lymphatic vessel on its concave site, to the hilum, where are also found the blood vessels and the nerves of the organ. g From the capsule - septa of dense connective tissue, which are penetrating into the interior of the lymph node node.

Figura 2. Schematic representation of the structure of a lymph node. Note the outer and inner cortex, the g p y p medulla, and the blood and lymph circulation. Also note that the lymph enters through the convex side of the node and leaves through the hilum. The lymph percolates through the node, exposing its contents to the action of defensive cells (macrophages, lymphocytes, APCs).

The stroma of the lymph node is represented by a tridimensional network of reticular fibers, fibers anchored on the inner face of the capsule and on the septa limiting two types septa, of spaces: small and large spaces. Small spaces - the parenchyma of the lymph node (dense lymphatic tissue), while the large spaces - the lumen of the lymphatic sinusoid capillaries (loose lymphatic tissue) Finally this arrangement is describing two zones tissue). zones, inside the lymph node: the cortex, the outer zone and the medulla, the inner zone. O the reticular network forming the stroma, are anchored -fibroblasts, macrophages, ti l t kf i th t h d fib bl t h On th dendritic reticular cells (providing microenvironment for B lymphocytes) and interdigitating reticular cells (which are creating the suitable microenvironment for T lymphocytes). lymphocytes) The cortex of the lymph node is situated superficial to the medulla, beneath the capsule, and has two subdivisions - a superficial, outer or nodular cortex and an inner, non-nodular or paracortex. The nodular (outer) cortex is a thymus-independent area, colonized with B lymphocytes. Most of the lymphatic nodules in this region, are secondary nodules, with germinal centers. The plasma cells produced here, are migrating to the medulla (medullary cords); here they secrete antibodies for approximately 2 weeks, then they die.

Figure 3. Section of a lymph node showing the structure of the cortex and the medulla. The medullary cords and sinuses are clearly visible. PT stain. Low magnification.

The inner (nonnodular) cortex, or paracortex is situated between the outer cortex and the cortex, medulla. The lymphocytes in this area are numerous, with a diffuse disposition, being mainly T cells. This is a thymus-dependent area cells thymusarea. The medulla of the lymph node is situated between the cortex and the hilum. Medulla consists of large medullary sinuses; between the sinuses are situated medullary cords. The cords - lymphocytes, plasma cells, macrophages. The cords are ending up to the hilum. The lymphatic sinuses are beginning with the subcapsular sinus, which is visible with the light microscope, like a space between the capsule and the outer cortex. The subcapsular sinus has a continuous wall, to the capsule, while the wall to the cortex is discontinuous. , p , Cortical sinuses are narrow, being masked by the cortical parenchyma. The medullary sinuses - much larger, between them are situated the medullary cords; medullary sinuses can be seen in the light microscopy. The medullary sinuses are continuous with the efferent lymphatic vessels; the afferent lymphatic vessels are opening into the subcapsular sinus. The wall of the sinuses is discontinuous, endothelial cells, with their basement membrane are not continuous, part of th wall of th l ll f the lymphatic sinuses i made only of reticular fib h ti i d l f ti l fibers. Thi f t t t in the is This feature i i is important i the free circulation of the cells, from the parenchyma to the sinuses and the reverse. In the lumen of the lymphatic sinuses are situated reticular fibers, with anchored reticular cells (in fact macrophages and fibroblasts); thus is provided a prolonged antigenic contact contact, the lymph circulation inside the sinuses, being delayed. Postcapillary venules (high endothelial venules) are present in the inner cortex (paracortex) venules, of the lymph node; through postcapillary venules mature T and B lymphocytes pass from the blood stream, into the parenchyma (B lymphocytes are migrating afterwards to the nodular cortex).

Figure 4. Section of a lymph node showing the capsule, subcapsular sinuses, and a small portion of the outer cortex. Some adipose tissue is seen outside the capsule. PT stain. Medium magnification.

Figure 5. Photomicrograph of the medulla of a lymph node; the medullary sinuses are separated by medullary cords. PT stain. Medium magnification.

Functions of the lymph nodes:
They are functioning as a barrier for the lymph, and they represent a site of phagocytosis, for any foreign material. Lymphocytes may pass into the sinuses, from the parenchyma of the lymph node, travelling with the lymph to other sites of immune response of the organism; thus the chance to find the antigen for which the lymphocytes were programmed is increased. Resident and free macrophages of the lymph node show intense phagocytosis. The processes of the resident macrophages are projected into the lumen of the sinuses. sinuses Lymph nodes are sites of immune responses. The outer, or nodular cortex is a site of humoral immune response, here are produced plasma cells, and B memory fh li h d d l ll d cells. The deep or inner cortex is a site of cellular immune response; for example metastasis of most malignant tumors takes place in the lymph node, of the region adjacent to the tumor; here are produced cytotoxic killer T lymphocytes and T memory cells.

Figure 6. Photomicrograph of a high endothelial venule in a lymph node. Arrowheads indicate high endothelial cells. The venule is crossed by lymphocytes (arrows). PT stain. High magnification.

2. The spleen
Spleen is the largest lymphatic organ of the body. In contrast to the lymph nodes, it is situated on the blood circulation system and is filtrating, the blood. The spleen has an arched ovoid shape, with two faces, two margins and a hilum; its weight is of about 150-180 grams. During intrauterine life, the spleen has hematopoietic 150function, after birth exclusively lymphocytes are developing in the spleen. In leukemia, the so-called myeloid metaplasia of the spleen may occur (appearance of hematopoietic soislets in the spleen). It may be surgically removed (after severe trauma with rupture of the spleen, or in hyperfunction, with overdistruction of blood elements), but only during adult life; in children till 3, or 5 years of age, the removal of the spleen may cause severe infections, by impairing the normal immunity of the organism. The histologic organization of the spleen includes a capsule of dense connective tissue, with a few smooth muscle fibers, a stroma and a parenchyma. Septa of dense connective tissue are arising from the capsule, and are penetrating into the parenchyma of the spleen. No splenic lobules are visible, in humans. With the naked eye, in the spleen are b i ll yellowll i h l dispersed i a mass of b d in f brownobvious small yellow-grayish zones - th white pulp, di the hit brownreddish tissue - the red pulp of the spleen. While the white pulp is represented by crowding of lymphocytes (dense lymphatic tissue), surrounding arterial vessels, the red pulp is made of loose, or reticular lymphatic tissue (most of the red pulp is made of venous sinusoid capillaries of the spleen).

Figure 7 S ti of spleen showing it capsule sending t b Fi 7. Section f l h i its l di trabeculae t th i t i of th organ. N t th l to the interior f the Note the white pulp with its arterioles. The red pulp occupies most of the microscopic field. Picrosirius stain. Low magnification.

The white pulp
The splenic artery is branching gradually, first following the trabeculae of connective tissue, arising from the capsule, the so-called trabecular arteries. As the trabecular arteries are branching, branching their caliber is decreasing and at approx 0 2 mm in diameter they are leaving decreasing, approx. 0.2 mm, diameter, the trabeculae, and are penetrating into the parenchyma; here they loose their adventitial tunic, and the arteriole becomes closely surrounded by mature, small, T lymphocytes, forming a periarteriolar lymphatic sheath (PALS). PALS are thymus-dependent areas of the thymus dependent spleen, comparable to the deep cortex (paracortex) of the lymph node. To the outside of the PALS, are appearing lymphatic nodules, made of B lymphocytes, thymus-independent areas comparable to the superficial cortex of the lymph node thymus independent areas, node. The marginal zone is situated at the limit between the white pulp and the red pulp. This is an important site of antigenic contact; blood injected antigens are attracted to this zone. From the marginal zone of the spleen activated lymphocytes are leaving the white pulp to spleen, pulp, enter into the blood stream, or the reverse (this is part of the recirculation of lymphocytes, in the process of immune surveillance). As a result of nodule formation, the arteriole may be centrally located, in the dense lymphatic tissue of the white pulp, but more often, has an eccentric situation to the dense lymphatic tissue. The arterioles give straight branches in the red pulp, called penicillar arterioles; these vessel, arterioles may end with a small vessel surrounded by macrophages (periarteriolar macrophage sheath). Arterial capillaries may end in the cords of the red pulp, or more rarely, in the venous sinuses.

The red pulp
The two components of the red pulp are: the venous sinusoids, and the cords of Billroth, situated between the venous sinuses. The venous sinuses have a discontinuous wall and an irregular passage; the endothelial cells have between one another free spaces, through which, the blood elements may pass one way, or another. A network of reticular fibers and the discontinuous endothelium, are forming the wall of the venous sinuses. The stroma of the spleen is made of a 3-dimensional network of reticular fibers, 3anchored on the inner face of the capsule, and on the septa. On the reticular fibers network are found reticular cells, of different types, macrophages, fibroblasts and antigen presenting cells. Two theories were advanced, concerning the blood flow, in the venous sinusoids. The closed circulation theory is stating that the blood flows directly from the arterial capillaries into the venous sinuses. In contrast, the open circulation hypothesis, is suggesting that the blood first passes through the red pulp cords, and after that enters inside the venous sinusoids. The open circulation theory, seems to be more correct, since it would provide a better contact between blood and macrophages, in the red pulp cords. S d Some authors b li th believe th t if th amount of bl d i i ti th spleen i l that the t f blood irrigating the l is large, the blood circulation is open, while when the amount of blood reaching the spleen is smaller, the circulation becomes closed. Red l lymphocytes, R d pulp cords contain all bl d cell types, as well as macrophages, l d i ll blood ll ll h h plasma cells.

Figure 8. Structure of the red pulp of the spleen, showing splenic sinusoids and splenic cords with reticular p g (some macrophages contain ingested material). The disposition of the reticular p g g ) p cells and macrophages ( fibers in the red pulp is illustrated. In the splenic cords they form a 3-dimensional network; in the sinusoids they are mainly perpendicular to the long axis of the sinusoid. Both the open and closed theories of circulation are illustrated. Arrows indicate blood flow and options for movement of blood cells.

Functions of the spleen
White pulp of the spleen is a site of lymphocytes formation and immune responses. Red pulp is a site of blood filtration, destruction of aged blood elements takes place here. The iron from the hemoglobin is stored to be reused and the heme portion is transformed into stored, reused, bilirubin, which transported to the liver, will be excreted in the bile. By distension, the spleen is functioning as a blood reservoir, like in the case of cardiac failure.

3. Mucosal attached lymphatic tissue
The attached lymphatic tissue is more abundant in the digestive tract the "gut tract, gutassociated lymphoid tissue", or GALT. The lymphoid tissue associated to the respiratory tract is called "bronchial-associated lymphoid tissue", or BALT. Skinassociated lymphoid tissue - SALT SALT. The striking characteristic of the attached lymphatic tissue consists in the presence of aggregated lymphatic nodules, areas of B l f t dl h ti d l f lymphocytes, T l h t lymphocytes b i h t being the clear predominant type found in the skin-associated lymphatic tissue. Permanent lymphatic tissue structures associated to the digestive tract are - the tonsils, the Peyer patches and the appendix. All these structures are functioning as secondary lymphatic organs, and have common features, such as: the absence of a covering capsule, the absence of a proper stroma, the lymphatic elements are situated on the stroma of the organ, hosting the lymphatic tissue; they do not have their own blood vessels, being irrigated by the vessels of the organ.

The tonsils are part of the ring-like lymphatic tissue around the openings of the ringdigestive and respiratory tract. P l i tonsil i a pair organ, situated on b h sides of di i d i Palatine il is i i d both id f the pharynx; - stratified squamous epithelium, without keratin, oral type epithelium. The epithelium is invaginating and forms crypts; each palatine tonsil - 10-20 crypts. 10I id th crypts are usually f t ll found b t i d b i migrated l d bacteria, debris, i t d lymphocytes, expelled h t ll d Inside the epithelial cells. Most of the aggregated lymphatic nodules of the tonsils, show germinal centers. A layer of dense connective tissue, also called capsule of the tonsil, is separating the lymphatic tissue of the tonsil from the deeper structures of the pharynx tonsil, pharynx. If the palatine tonsils become subjects of chronic inflammation, they are surgically removed (amigdalectomia). Pharyngeal tonsil is a single mass of lymphatic tissue, in the posterior wall of the nasonaso-pharynx. This tonsil - pseudostratified ciliated columnar epithelium (respiratory type); the epithelium shows some folds. In its deepest region, it may also show a thin capsule of dense connective tissue. The enlargement of the pharyngeal tonsil is forming the adenoids. The lingual tonsil is situated at the posterior one third of the tongue. Usually, it has one crypt; excretory ducts of salivary glands are opening into the crypts, saliva is thus washing the crypts and therefore, this tonsil is rarely inflamed.

Figure 9. Section of Peyer’s p g y patch of the small intestine showing the epithelial covering of enterocytes and g p g y goblet cells (right), the intestinal lumen (center), and the covering of the patch with a row of M cells and groups of lymphocytes (left). The small dark nuclei belong to B and T lymphocytes, and the large palestained nuclei belong to M cells. PT stain. Medium magnification.

The Peyer patches are aggregations of lymphatic nodules, located in the lamina propria, and in the submucosa of the ileum Each Peyer patch is made of hundreds of lymphatic ileum. nodules; the total length of the ileum contains 20-30 such patches. The nodules seem to originate in the lamina propria, but they extent into the submucosa, through muscularis mucosae, distorting the villi and the Lieberkuhn glands. M cells (microfolded cells or membrane-like cells) are covering the lymphatic tissue of the digestive tract, at the level of the epithelium; they have the capacity to transport the ti h ti tissue, without changing it molecular structure. Th presence of ith t h i its l l t t f antigen t the lymphatic ti to th l The the M cell is suggested in the light microscopy, by the presence of 2, or 3 lymphocytes, inside the epithelium. Approximately 60 % of the Ig secreted in the digestive tract, is represented by A immunoglobulin, secretory type. This is made of two units of A immunoglobulins assembled by a so-called secretory piece secreted by epithelial cells; this structure is sopiece, resistant to the action of the digestive enzymes. Lymphocytes formed in these areas are migrating through the afferent lymphatic vessels to the lymph nodes, from here, the activated lymphocytes may reach the blood stream, some of the lymphocytes will populate the spleen, others will reach once again the lymphatic tissue attached to mucosal linings. During breast-feeding activated lymphocytes breastand antibodies, are reaching i th colostrum and i th maternal milk, and will ensure th the d tib di hi in the l t d in the t l ilk d ill immunity of the new-born. new-

Figure 10. General view of the mucosa immunity in the intestine. Luminal antigens are captured by dome-shaped M cells present in the covering of Peyer’s patches and transported to subjacent lymphocytes, macrophages, and dendritic cells. Macrophages and dendritic cells migrate to neighboring lymph nodes, where they stimulate B and T lymphocytes, which then enter the lymphatic circulation and later the blood circulation (lymph flows to the blood) The stimulated lymphocytes blood). home in other tissues, including the mucosa lamina propria, where plasma cells produce considerable amounts of IgA. The lymphoid cells of the lamina propria of intestinal mucosa are a major antibody producer, because of their extension and close contact with antigens introduced into the digestive tract.

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3. THE ENDOCRINE GLANDS (1)
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

THE ENDOCRINE GLANDS 1. The pituitary gland (hypophysis) 2. The pineal gland (epiphysis)

The endocrine system is a complex control system of the organism, which together with the nervous system is involved in the integration of different structures of the human body. The chemical messengers are called hormones and are acting a long distance from the place of their production , on so-called target cells. so-

The endocrine system is represented by proper endocrine glands - pituitary, thyroid, adrenal etc gro ps of endocrine cells disposed in the parench ma of other organs adrenal, etc.; groups parenchyma - the endocrine cells of ovary and testis, the endocrine islets of the pancreas, and a third possibility is the presence of isolated endocrine cells between lining epithelial cells, cells of different tracts especially digestive but also in the respiratory and urinary tracts, digestive, passages, forming the diffuse neuroendocrine system (DNES).

1. The pituitary gland (hypophysis)
Is a small gland of about 0.5 g, in adult, situated in a depression of the sphenoid bone, bone the sella turcica; it is connected with the hypothalamus by a nervous link link, called infundibulum, or the pituitary-hypothalamic stalk. It has a double origin, from pituitarythe ectoderm of the primitive oral cavity, its epithelial, endocrine part (the anterior lobe) and from the nervous system, its nervous lobe (neurohypophysis). ) y , ( yp p y ) In the development, the 2 components are fusing together; the ectodermal component growing in the superior part is forming the Rathke's pouch while the component, part, Rathke s pouch, nervous component is forming a prominence of the hypothalamus, at the level of the floor of the third ventricle, the mediane eminence. From the mediane eminence will arise a nervous expansion, the infundibulum, which is fusing with the Rathke's pouch, forming the nervous lobe of the pituitary gland. gland The Rathke's pouch is loosing gradually the link with the epithelium of origin - primitive oral cavity, giving birth to the anterior lobe of the pituitary, or the adenohypophysis, or pars distalis. The 2 other lobes - tuberalis and intermediary important, specie, rudimentary. lobe are not important in human specie being rudimentary

Figure 1. Development of the adenohypophysis and the neurohypophysis from the ectoderm of the g p yp p y yp p y roof of the mouth and from the floor of the diencephalon.

Supraoptic and paraventricular nuclei of the hypothalamus, have long axons, which are forming the stalk or the infundibulum, and after that they spread into the nervous lobe of the pituitary, representing most part of it. Other nuclei of the hypothalamus have shorter axons, which end at the level of the mediane eminence. These nuclei are synthesizing neurohormones or neurohormonal factors → the regulation of the endocrine secretion of the endocrine cells, in the anterior lobe of the g pituitary. Two categories of such factors are secreted by neurons of the hypothalamus - the releasing factors, which are stimulating the synthesis and secretion of the pituitary hormones (GH-RH -growth releasing hormone; CRF - corticotropine (GHreleasing factor; TRH - tireotropine releasing hormone etc.) and inhibiting factors -PIF - prolactine inhibiting factor, somatostatin, inhibiting the secretion of GH. The neurohormones of the hypothalamus are reaching in the anterior pituitary, especially by the hypothalamic-pituitary portal system; a portal system represents a hypothalamicplexus of capillaries united with another network of capillaries, by numerous p p p y anastomoses. The superior pituitary artery is penetrating in the mediane eminence, where is forming a network of capillaries, called primary capillaries, because they give birth to venules which are forming a secondary network of capillaries in the anterior lobe of the pituitary gland.

Figure 2. The hypothalamo-hypophyseal system, with its vascularization and sites of hormone production, storage, and release.

At the level of the mediane eminence the neurohormones or factors are eminence, exocytosed from the end bulbs of the axons, into the perivascular spaces; they enter into the blood stream, being transported to the secondary capillaries, in the anterior lobe of the hypophysis, like this they reach to influence the secretory capacity of the endocrine cells of the pituitary gland. Another way, by which the nervous regulatory factors of the hypothalamus are way reaching the primary capillary network, is by so-called tanycytes, glial ependimal socells, which have very long expansions, in the floor of the third ventricle; they are capable to uptake neurohormones from the cerebrospinal fluid and they discharge them at the level of the mediane eminence, in the perivascular spaces of the primary capillary network, of the hypothalamic-pituitary portal system - this second hypothalamicmode is less important, in this transport.

The anterior lobe of the pituitary, or pars distalis – 2 categories of cells: with no p y p g affinity for dyes – chromophobes, looking grayish, representing approx. 40 % of all the cells and another category which have affinity for different dyes - chromophils, representing approx. 60 % of all the cells. According to this affinity, the chromophils are - acidophilic cells, with affinity for acidic dyes and basophilic cells, with affinity for basic dyes. Acidophilic cells - polypeptidic hormones, the cells are P.A.S. negative (the secretory product has no sugar in the molecule) - growth hormone, GH or somatotrop (STH), another category of cells is secreting prolactine. The GH-secreting cells have affinity for an orange dye, they measure approx. 20 µ g y g y y pp µm, they are situated predominantly at the periphery of the anterior lobe; with the electron microscope - secretory granules of approx. 400 nm. The exact secretion of each pituitary endocrine cell, was first suspected, comparing the pathology, e.g. hypersecretion, with the type of cell overdeveloped, but the exact determination was done by electron microscopy and immunohistochemistry techniques. GH is acting on the g growth cartilages or epiphyseal disks, where they stimulate the g g y y ossification process, and by this, the normal growth of the long bones, and thus the final stature of the individual. GH is also involved in the glucidic, proteic and lipidic metabolism, of the organism.

Figure 1 - Some stains allow the recognition of cell t iti f ll types of th f the pars distalis: chromophils (acidophils and basophils) and chromophobes. Gomori’s trichrome stain. High magnification.

The lack of hormone is leading to the pituitary dwarfism, determining a short stature, with 20-30 cm, or even more, than the normal height of the age; nevertheless, this condition does neither affect the mental development, nor the proportion between different segments of the body, being a proportional dwarfism. The hypersecretion of GH, may lead during growth period to a giant stature, while if occuring after the growth period is finished, it will determine only the overgrowth of the extremities, tangue, lips, fingers - the so-called acromegaly. The regulation of the GH secretion is depending upon the. GH-RH, releasing factor of the hypothalamus, which is stimulating th production and lib ti of th GH f ti l ti the d ti d liberation f the GH, from th pituitary. It secretion i the it it Its ti is inhibited by somatostatin, secreted either by the hypothalamus and the DNES cells, especially in the pancreas and the intestine; GH is not acting directly on the target cells, cells but mediated by so called somatomedines or insulin like growth factors - IGF1 so-called somatomedines, insulin-like and IGF2; the 2 factors are secreted in the liver, under the influence of GH. The prolactin-secreting cells - pink-redish (carmine) and they are situated especially in the i th center of the anterior lobe of th pituitary. The secretory granules may reach t f th t i l b f the it it Th t l h during lactation period, up to 600 nm. Outside lactation period the prolactin secretion is normally inhibited by dopamine, dopamine being the prolactin inhibiting factor (PIF). The gradual growth of estrogens progesterone etc during pregnancy is leading to growth, estrogens, progesterone, etc., pregnancy, the overlap of the inhibitory power of the dopamine, like this prolactin secretion from the pituitary gland will act on the mamary glands, determining the milk synthesis and secretion. secretion When breast-feeding is stoped the dopamine is increasing once again stoped, again, and the prolactin secretion is dropping to basal values; the secretory granules are lysed inside the prolactin cells - autolysis.

The chromophobes cells - several categories they may be either reserve categories, elements, or degranulated chromophils, while an important number is represented by the adrenocorticotropin-secreting cells (ACTH). The ACTH-secreting cells have small secretory granules, of about 150 nm, in the e.m. and they become basophilic yg , , y p when the adrenal gland is damaged and the feed-back of the cortisol acts nomore on the pituitary ACTH-secreting cells. First it is secreted a larger molecule, called big ACTH, from which by cleavage are obtained: ACTH, MSH ( g y g (melanocytey stimulating hormone), endorphins and enkephalins. ACTH is stimulating zone fasciculata and zone reticularis of the adrenal gland and reticularis, the production of glucocorticoids (produced in zone fasciculata, mainly cortisol) and of androgens of adrenal origin (produced by zone reticularis). The regulatory feed back feed-back of the ACTH is done by cortisol, acting directly on the hypothalamic corticotropin-releasing factor (CRF) - a high or long loop of retrocontrol. The MSH is acting on the melanin synthesis stimulating it in the melanocytes → synthesis, it, skin pigmentation. Enkephalins and endorphins are modulating the reactions of the organism, when facing important stress.

The basophilic cells of the anterior lobe of the pituitary gland are secreting glycoproteic hormones, the cells are stained P.A.S. positive. The TSH-secreting cells are secreting the thyrotropin; they appear like triangular, deep basophilic cells, with granules of about 200 nm, in the e.m. The TSH is a large molecule, of approx. 200 000 Da, which is acting on the thyroid cell, stimulating either the growth, as well as the function of the thyroid gland. The feed-back feed back of control is represented by a short loop, the thyroid hormones -T3 and T4, loop T3 T4 acting directly on the hypophysis (TSH). In hypothyroidism, like for ex. in the case of iodine deficiency, which is leading to an overaccumulation of thyroglobuline in the thyroid follicles, which cannot be iodinated, the TSH will increase in the plasma, leading to a goiter enlargement of the thyroid gland. In contrast, when the thyroid is secreting more than normal -hyperthyroidism, the TSH will be inhibited at the point that it cannot be stimulated, not even with TRH (hypothalamic hormone) injection. The gonadotropin-secreting cells are larger, rounded cells, with a lower basophilia, disposed in small groups; they secrete the two gonadotropins, the follicle-stimulating hormone, FSH and the luteinizing hormone, or LH. Their secretion and release is stimulated by the g , y hypothalamic FSH-RH and LH-RH. The FSH → the estrogens secretion and of the maturation of the ovarian follicle, till the stage of mature ovarian follicle, in female sex, while in the male sex is determining the first stages of spermatogenesis (mitosis). The LH is triggering the ovulation in the female sex and is inducing the progesterone secretion, by the luteal body; in male sex, where is also known as ICSH - interstitial cells stimulating hormone, LH is stimulating the secretion of testosterone, by Leydig interstitial cells of the testicle, testicle the most important sexual androgen The secretion of FSH is inhibited especially by androgen. inhibine, secreted by both hypothalamus and ovary, respectively testicle, while the secretion of LH is inhibited by progesterone, in female, and by testosterone, in male.

The intermediary lobe, as well as the pars tuberalis are not so developed in human, the majority of secretory cells are migrating into the anterior lobe of the pituitary; in pars intermedialis are found some MSH-secreting cells and in pars tuberalis, only rare MSHgonadotropingonadotropin-secreting cells. The nervous lobe or the posterior lobe of the p p pituitary g y gland, is made of the axons of the neurons of the supraoptic and paraventricular nuclei, forming the infundibulum, or the pituitary stalk; finally the axons are spreading in the nervous lobe, forming most part of it. The end bulbs of the neurons are visible in the light microscopy, the Herring bodies. Here are deposited the neurohormones secreted by the hypothalamus, the two neuropeptides of nine aminoacids, vassopresin or antidiuretic hormone (ADH) and oxytocin. The two hormones are secreted together with transporting proteins, called neurophysins, one for each peptide. ADH is stimulating the facultative absorbtion of water, at the level of the distal and colecting tubules of the nephrone. The lesion of the infundibulum, or its experimental section, is leading to diabetes insipidus, characterized by the loss of the capacity of concentrating the urine; as a result the patient will excrete 10, up to 30 liters of urine per day (polyuria) and in compensation, will drink the same quantity of water. Oxytocin is stimulating the contraction of the uterus, initiating the expulsion of the fetus, and is stimulating the contraction of the myoepithelial cells of the ducts of the mammary gland, by this determining the milk ejection, from the mammary gland. The secretion of oxytocin is triggered by neurohormonal reflex, starting from the distended uterine muscle, and respectively, form the nipple, by suction, during breast-feeding. breast-

2. The pineal gland (epiphysis)
The pineal gland is a very small gland, of about 5-7 mm, with a weight of 100 mg, in relation 5with the roof of the third ventricle; the gland is covered by pia-mater forming a connective pia-mater, tissue capsule of the gland; from the capsule are arising trabeculae, which are dividing the gland into small cords, surrounded by sinusoidal blood capillaries. More than 90 % of the cells - pinealocytes, secreting melatonin and other related p p p y , g peptides, all derived from triptofan , p and serotonin. The gland is also containing specific astroglial cells, with abundant intermediate filaments in the cytoplasm. Melatonin is considered to be the main gonadotropin-inhibiting hormone, till the age of g gonadotropinp g g puberty. It is also inhibiting the development of the receptors of the pituitary cells, for the hypothalamic releasing factors, and the receptors of the ovary and testes, for the pituitary gonadotropins. The tumors arised from the pineal gland, which are secreting melatonin, are leading to delayed puberty, while th t l di t a d l d b t hil the tumors which are di t i th pineal, are l di t hi h distroying the i l leading to precoceous puberty. Recently, melatonin was discovered to be involved in the so-called jet syndrome, appearing soas a disorder of sleep and a ake periods when we fl b plain at very long distances and awake periods, hen e fly by plain, er distances, the hour is also changed. Some studies have shown the involvement of the melatonin in depressions, and its decrease during ovulatory period. One of the most important effects of radicals, organism, melatonin is its ability in neutralizing the free radicals keeping the youth of the organism and thus demonstrating antitumoral activity. The gland is light-sensitive, being inhibited by lightlight, via the optic nerves. In Orient is involved in spiritual practices, yoga, etc. being called "the third eye".

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4. THE ENDOCRINE GLANDS (2)
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

THE ENDOCRINE GLANDS 3. The th 3 Th thyroid gland id l d 4. The parathyroid g y glands 5. The adrenal (suprarenal) glands

3. The thyroid gland
Thyroid gland is the largest endocrine g y g g gland, situated at the base of the neck, anterior to the larynx and the first and second tracheal rings. In adult has approximately 25 g, being formed of two lobes united by an isthmus; sometimes from the isthmus is arising a third lobe, called the pyramidal lobe. The gland is made of a capsule of connective tissue, envelopping it at the exterior, a fine stroma of reticular fibers, the parenchyma being represented by follicles. The thyroid follicles are spherical structures, lined by a simple cuboidal epithelium, the thyroid epithelium, made of thyrocytes, which are secreting the 2 thyroid hormones - thyroxine or T4 and triiodothyronine, or T3. Inside the follicles is found y y , the thyroid colloid, with a gelatinous consistence, and acidophilic. This is the way of stockage of the thyroid precursor, the thyroglobulin, as if the gland is functioning on the endocrine pattern, by synthesis of hormones and also on an exocrine pattern, by the stockage of the precursor, inside the follicles. When the gland shows a hypersecretion, the epithelium of the follicles becomes columnar, while when the gland is in hypofunction, the colloid accumulated in the follicles, is gradually flattening the epithelium, which becomes squamous.

Figure 2. Photomicrograph of a section of thyroid and parathyroid parathyroid. The thyroid is formed by thousands of spheres called thyroid follicles. They are filled with a glycoprotein, the colloid which appears colloid, fragmented here because of an artifact. The parathyroid is separated from the thyroid by a thin connective tissue capsule. H&E stain. L i l i Low magnification.

The thyroid cells are basophilic; with the e.m. - well developed rough ER, Golgi complex and mitochondria, as well as microvilli at their apical pole and invaginations of the cell membrane t b toward th i b d their basal pole. B t l l Between th f lli l are present numerous bl d the follicles t blood capillaries, of the common type, and a fine, reduced network, of reticular fibers. Between the follicles are present a second category of cells, called parafollicular cells, the C cells, or clear cells, which are secreting a peptide hormone, called calcitonin; they contain small secretory garnules, in e.m., of about 100-150 nm. The principal action of 100calcitonin is that of decreasing the blood calcium, by inhibiting bone resorbtion, opposite effect than that of the parathyroid hormone (PTH). The secretion of calcitonin is triggered by an elevation of the blood calcium. The appearance in the light microscopy of the parafollicular cells, is that of pale or clear cytoplasm cells, slightly larger than the thyroid cells; they are situated like isolated cells, y p g y g y y between the follicles, or in small groups of 2-3 cells. They are also present in the DNES, 2of the lung, in the pituitary gland, in the intestine, etc.

The synthesis stages of the thyroid hormones: 1. First the thyroid cells, lining the follicles are uptaking from the blood stream aminoacids and glucids (most important - thyrosine, manose, galactose); which on the pattern of protein synthesis are used for the synthesis of a polypeptide, which glycosilated in the Golgi complex, will form the thyroglobulin. 2. The cells are trapping the circulating iodide; this is done by the iodide pumps, situated at the basal pole of the thyroid cells. These first two stages, as well as the next ones, p y g , , are stimulated by TSH, of the pituitary gland. Drugs as perchlorate and thiocyanate, may inhibit this stage, by competing with iodide. y y p , g y 3. Iodide is oxidized by the thyroid peroxidase, becoming ready to combine with the thyrosine residues of the thyroglobulin. 4. The thyroglobulin is endocytosed in the cells (the iodide is activated outside the cells, at the apical pole, inside the follicle) and by cleavage of the iodinated molecule are resulting monoiodotyronine (MIT) and diiodotyronine (DIT). MIT + DIT = T3; DIT + DIT = T4. 5. 5 Finally only T3 and T4 are released into the plasma active thyroid hormones while plasma, hormones, the restant MIT and DIT are deiodinated by a dehalogenase, the iodide being reused in the synthesis of active thyroid hormones. hormones, quantity, potent, From the two secreted hormones T3 is in a less quantity but is more potent rapidly metabolized, while T4 represents 80 % of the circulating thyroid hormones, has a milder action, but acts longer on the cells.

Figure 3. High magnification of a section of a thyroid. Calcitoninproducing parafollicular cells can be distinguished from the follicular epithelial cells because they are g g larger and their nuclei stain lighter. H&E stain. High magnification.

Almost any cell of the organism has receptors for the thyroid hormones, the hormones being involved in maintaining the normal metabolic rate, are ensuring the general catabolism of the cells and the normal oxygen consumption. The hyperthyroidism will increase the metabolic rate, with weight loss, tachycardia, etc., while the hypothyroidism will have the reverse effect, with a decrease of metabolic rate, increased weight, infiltration of the skin, slow cardiac rhythm, slow cerebral function, etc. Most common cause of hyperthyroidism is Grave-Basedow disease, an Graveimmunologic entity, in which are produced antibodies acting on the thyroid cells TSH receptors, stimulating the function of the g g gland ( (LATS - long acting thyroid g g y stimulator antibodies). Most common cause of hypothyroidism is the iodide deficiency, present in many regions of the world, because of the absence of the iodide in the drinking water; a common clinical sign, will be the goiter. The congenital myxedema, characterized by a lack of thyroid hormones from birth, usually by agenesia of the thyroid, is leading to thyroid cretinism, with severe y y g y , g y , mental retardation and a typical dwarfism. The thyroid hormones are important in the development of the central nervous system, during childhood, and are interintermediating the action of the g g growth hormone, at the level of the g growth cartilages; g the administration, of the thyroid hormones, immediately after birth, will prevent these severe handicaps.

4. The parathyroid glands
Parathyroid glands are small g y g glands, usually 4, situated at the 4 p y poles of the 2 thyroid lobes, of the thyroid gland, inside the thyroid capsule, sometimes they may be situated in the thyroid parenchyma; more rare they may be 6, or even 8, situated in the mediastinum, or in the pericardium. They derive from the third (inferior glands) and from the fourth (superior glands) pharyngeal pouches, having a common origin with the thymus. The parenchyma of the gland is made especially of chief or principal cells, secreting the parathyroid hormone. These cells look basophilic, having a lymphocyte-like aspect, p y lymphocytey p y p , polyhedral; in the e.m., they show secretory granules of ; , y yg about 200-400 nm. A second type of cells are the oxyphil cells, larger than the 200chief cells, with an intense acidophilia of the cytoplasm; these cells have abundant mitochondria and their role is not yet fully understood. It seems to be aged elements, since the number of the oxyphils is increasing with age. Adipose cells are accumulating in the gland, in older individuals, where they may reach more than 50 % of the cell population.

Figure 1 - Section of a parathyroid gland showing th th id l d h i the chief cells of the gland arranged as cords separated by blood capillaries. H&E stain. Medium magnification.

Parathyroid hormone (PTH) is acting on the bone, inducing bone resorbtion, and an icrease of the blood calcemia. A decrease of the blood calcemia, is stimulating the release of the PTH from the parathyroid glands, while an increase of the biood calcium has the opposite effect. PTH is also responsible for the decrease of the blood phosphate, by reducing its absorbtion in the kidney, in change with an p p , y g y, g increased calcium absorbtion. PTH is also responsible for the absobtion of the calcium, in the intestine, in the presence of the D vitamin. PTH is acting mainly on the bone osteoclasts, stimulating their formation, from the blood monocytes, and activating them to form the ruffled border and to uptake calcium from the bone matrix, demineralizing the bone tissue matrix. Calcitonin secreted in the thyroid parafollicular cells, has the opposite effect, stimulating osteogenesis, in stade of bone resorbtion. In hyperparathyroidism, bones become very fragile, by decalcification, leading to yp p y , y g , y , g spontaneous fractures, blood calcium is elevated, with deposit in different organs kidney stone disease, etc. This pathologic condition is known as osteitis fibrosa cystica. In contrast, in hypoparathyroidism, the blood calcium is decreased, leading y yp p y g to a hyperexcitability of the striated muscle and of the nervous system, called tetany - generalized spastic contractions of the skeletal muscle and convulsions, which are ceassing when calcium is administered to the patient. It seems that the main stimulus for the PTH secretion, is the level of the blood calcium.

5. The adrenal (suprarenal) glands
The adrenal glands are situated at the upper poles of the kidneys, having a shape of a half-moon They are in fact represented by two different glands which have fused half-moon. glands, together, during embryonic development, having different origin, different nature of the secreted hormones, as well as, different functional dependence. The adrenal cortex has a mesodermal origin is secreting steroid hormones and is origin, under the control of the pituitary ACTH, and of the renin-angiotensin-aldosterone renin-angiotensinsystem, while the adrenal medulla has a nervous origin, from the neural crests, is secreting catecholamines, and is depending upon the sympathetic nervous system. The adrenal medulla was first a sympathetic ganglion, in which had become endocrine secretory cells. The adrenal cortex is covered by a dense connective tissue capsule from which are capsule, arising thin septa, into the parenchyma of the gland; the stroma is represented by a network of reticular fibers offering support to the secretory cells. The endocrine cells a e arranged cords, according the different appearance of the cells and of the are a a ged in co ds, acco d g to t e d e e t appea a ce o t e ce s a d o t e different orientation of the cellular cords, we differentiate 3 zones, in the adrenal cortex. A thiner zone, called zona glomerulosa is situated just beneath the capsule 1010-15 % of the thickness of the cortex - made of small arched cords of cuboidal cells, looking more basophilic than the cells from the next zones. This zone is secreting the mineralocorticoids, tipically aldosterone, also corticosterone.

The next zone is made of parallel cords of cells, with a radiary disposition, face to p y p the surface of the gland. The cells are larger than that of the glomerulosa, polyhedral, with a vacuolated cytoplasm, because of the lipid droplets inside it. This is zona fasciculata, the best represented, occupying approximately 80 % of the cortex, which is synthesizing the glucocorticoids, most important being the cortisol. The last zone is zona reticularis, made of cords anastomosed in an irregular network; these cells are containing abundant lipofuscin pigment granules, like degenerated elements. This zone is reduced - only 8-10 % of the cortex. It 8secretes adrenal androgens, in significant amount, only the dehydroepiandrosterone (DHEA), which is marked less potent than the testosterone, from the testicle and has reduced physiological effects in organism. The adrenal cortex cells are not stocking the hormones, only their precursor, which is the acetate; from it is synthesized the cholesterol, and from the latter is synthesized the pregnenolone, etc. The best developed organelles involved in the synthesis, are the smooth endoplasmic reticulum and the mitochondria. The mineralocorticoids, most important being the aldosterone, are involved in the water and sodium homeostasis, stimulating the absorbtion of sodium, consequently of water, at the level of the distal tubule of the nephrone, of the gastric mucosa, and by sweat and salivary glands.

The glucocorticoids mainly cortisol are acting on all the metabolisms of the glucocorticoids, cortisol, metabolisms, organism, glucidic, lipidic and proteic. They promote the glycogen synthesis and stockage, in the liver, while in the periphery, they rather induce the glucids catabolism as well as the lipids and proteins catabolism They exert an effect of well, catabolism. immune suppression, acting on the immune system, for example on the B lymphocytes, they inhibit the antibody synthesis. Cortisol is one of the stress hormones of the organism inducing also the increase of blood presure of the organism, presure, blood glucose, etc. The androgens, mainly DHEA, have masculinizing and anabolic effects, but being secreted in small amounts, DHEA has a reduced importance in the body physiology. The control of the secretion is done by the ACTH, from the pituitary, but the feedfeedback of the cortisol is acting especially on the hypothalamic CRF The aldosterone CRF. secretion is under the control of the renin-angiotensin-aldosterone system; the renin-angiotensindecrease of the blood pressure in the afferent arteriole of the kidney corpuscle is leading to the release of renin from the juxtaglomerular apparatus renin is apparatus, triggering the formation of I and II angiotensin, with a constriction of the arterioles smooth muscle and increase of the blood pressure and the stimulation of secretion of aldosterone, from the glomerulosa zone.

In pathology, the hypersecretion of cortisol is called Cushing's syndrome, p p gy yp g y produced most often by a pituitary adenoma, ACTH-secreting, leading to a hypertrophy of the ACTHadrenal cortex and hypersecretion of glucocorticoids. The hypersecretion of aldosterone is called Conn's syndrome, characterized especially by hypertension. The androgens overproduction may produce virilization, in females. The vascularization of the gland is peculiar, the adrenal arteries are giving branches for the cortex and also for the medulla; the venules of the cortex are then drained to medulla, so the medulla is receiving both arterial and venous blood; the venous blood is bringing to the medulla, hormones of the adrenal cortex, involved in the regulation of the catecholamines production. Some studies revealed that this is important either in the synthesis i th medulla of th t th i in the d ll f the transferase, i f involved i th epinephrine synthesis, l d in the i hi th i from norepinephrine. The adrenal medulla is situated deep, in the middle portion of the adrenal gland. The cells - short cords, anastomosed in a network; these are large polyhedral cells, with granules of catecholamines, in the cytoplasm, visible in the light microscopy; granules of about 150-300 nm, in the e.m. Unlike the adrenal cortex, the medulla is stocking in 150the cells the hormones - epinephrine and norepinephrine. Different cells are secreting the epinephrine and the norepinephrine, dopamine, as a precursor is also present in the th granules. l

A hydroxylase is transforming the dopamine into norepinephrine and a transferase is i transforming the norepinephrine i f i h i h i into epinephrine. Th cells which are secreting i hi The ll hi h i the epinephrine have less dense granules, with homogenous content, while the norepinephrine secreting cells have denser granules, with a lucent, clear layer in th i h ti is t d by i h i (70- %). the periphery. M t of the secretion i represented b epinephrine (70-80 %) Most f th The catecholamines are the most important stress hormones of the organism, especially the epinephrine, determining, when released, the fright, fight and defense reactions of the individual. This is triggering the increase of the blood pressure, the increase of the power of the heart muscle contraction, an accelerated heart-beat, the increase of the blood glucose, etc.; all these reactions heartare offering a support of stress resistance. Other adrenal tissue is present beside the autonomic ganglia, called paraganglia, they usually secrete norepinephrine. The adrenal medulla is controlled by the sympathetic nervous system, sympathetic large neurons are present, between the secretory cells of the adrenal medulla. The proliferation of the cells of the adrenal medulla, is called pheochromocytoma, a tumor which is leading, especially, to crysis of paroxistic arterial hypertension.

Figure 2 - Structure and physiology of th adrenal h i l f the d l cortex

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5. THE DIGESTIVE SYSTEM (1)
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

THE DIGESTIVE SYSTEM
1. 1 The general structure

of the digestive tract 2. 2 Esophagus 3. Stomach

The digestive system - the digestive tract and the glands associated with the digestive tract (liver pancreas and salivary glands) (liver, glands). The oral cavity is the first portion of the digestive tract, important in the nourish of the organism, but also in articulate speech, facial expression, etc. The anterior opening of the oral cavity is limited by two muscular folds the lips which have an folds, lips, exterior face, covered by skin (stratified keratinized epithelium) and an inner face, covered by the mucous-lining of the oral cavity, with stratified nonkeratinized mucousepithelium epithelium. The tongue is a muscular organ present in the oral cavity - on its superior, or dorsal face, numerous small prominences, called papillae. In human are present 3 types of tongue papillae - filiform fungiform and circumvallate papillae These are filiform, papillae. expansions of the lamina propria, covered by the stratified nonkeratinized epithelium, of the oral cavity. The filiform papillae have a sharp edge, usually enclined toward the surface of the tongue they do not contain taste buds; they are tongue, numerous on the superior face of the tongue. The fungiform papillae are resembling to mushrooms, with a narrow foot and a dilated upper part they have a few taste buds The cicumvallate papilla is the part, buds. largest of all, is present especially at the level of the V of the tongue. They have a flattened edge, and they do not exit from the surface of the tongue epithelium, like types, prominent. the other two types which are more prominent On its lateral faces - most numerous taste buds, the receptors of the taste sensations.

Figure 1. Surface of the tongue on the region close to its V-shaped boundary, between the anterior and posterior portions. Note the lymphoid nodules (lingual tonsil), glands, and papillae.

The taste buds - in light microscopy, like small ovoid structures, situated in the thickness of the epithelium, l ki much paler, than the epithelium around. I hi k f h i h li looking h l h h i h li d In their structure - 3 types of cells -columnar curved cells of support, in the periphery, giving them a characteristic aspect of a barrel, these cells have small microvilli, t d their i l l by hi h they form th opening i th oral cavity of th i in the l it f the toward th i apical pole b which th f the taste buds - the gustative pore; receptor cells, with long microvilli at their apical pole, which are prominent in the oral cavity; at their basal pole they make synapse with the dendrites of cranial nerves transmitting to the nervous system the taste nerves, stimuli. A third category of cells is that of cuboidal basal cells of regeneration. At the base of the circumvallate papillae, are opening excretory ducts of pure serous glands, called Ebner's glands. In the lamina propria of the tongue are also present the acini of minor salivary glands. To the edge of the tongue, in the lamina propria, are f i found all t d ll types of acini, t f i i toward th middle part (th li d the iddl t (the lingual V) are f l found d only serous acini, of Ebner's gland, and to the base of the tongue are found exclusively mucous acini. The lamina propria of the tongue is penetrating between the striated muscular bundles of the tongue disposed in a tridimensional manner tongue, manner, thus providing a tight adherence of the tongue mucosa, with the striated muscle of the tongue.

The minor salivary glands, unlike the large salivary glands -parotid, submaxilary, glands, etc. etc. are secreting continuously providing the necessar humidity for the oral cavity continuously, humidity, mucosa. mucosa. In the gingiva and in the hard palate the lamina propria is adherent to the palate, periosteum of the bone, while on the inner face of the cheeks is continuous with the perimisium of the striated muscles, this adherence is preventing the folds formation, during mastication. Up to 70-80 % of the oral cavity epithelium has an incomplete mastication. 70keratinization, especially at the level of the gingiva and the hard palate. palate. The Th rest of th oral mucosa epithelium i nonkeratinized. Th k ti i ti occurs t f the l ith li is nonkeratinized. The keratinization k ti i d especially because of the mechanical forces, exerted on the epithelium during mastication, as well as a result of the chemical and termic agression, provided by the ingested aliments. aliments.

The teeth are hard structures prominent in the oral cavity 32 in number in the structures, cavity, number, adult. They are made of two hard materials - the dentin and the enamel, the latter covering the dentin at the level of the crown, while the cementum is covering the dentin, in the roots of the teeth. The part of the tooth visible in the oral cavity is , p y called crown, and the part located in a socket of the bone of the maxilla, is called root. These bone sockets are named alveoli, made of alveolar bone. Inside the crown is found the pulp cavity, hosting blood vessels and nerves of the tooth, in a p p y g loose connective tissue atmosphere. The pulp cavity is continuous with the root canal, inside the tooth's root, with an orifice at its apex, by which are penetrating vessels and nerves into the pulp cavity. Dentin is made of a calcified matrix, similar to that of the bone, but harder, because its hydroxyapatite crystals have a higher content of calcium salts, approximately 70 %. In the compositon of the dentin are present as well, collagen type I fibrils and glycosaminoglycans. The cells which are secreting the dentin are called odontoblasts, they are present all the life of the individual at the p p y of , y p periphery the pulp, in contact with the secreted dentin. The odontoblasts show long, slender processes, penetrating in canals, inside the dentin, the so-called dentinal tubules, sohosting odontoblasts expansions - Tomes' fibers.

Figure 2. Diagram of a sagittal section from an incisor tooth in position in the mandibular bone. (Redrawn and reproduced, with permission, from Leeson TS, Leeson CR: Histology, 2nd ed. Saunders, 1970.)

Enamel is the hardest structure of the human body, highly mineralized (95 %) containing b id the calcium salts, fl id salts, responsible f the i i i besides h l i l fluoride l ibl for h increased d hardness. It is made of perpendicularly disposed prisms, enamel prisms, occupying the whole thickness of the enamel layer. The enamel between the prisms is poorly mineralized and may be the subject of tooth decay. Besides the hydroxyapatite crystals, the tooth is containing specific proteins, called amelogenins. The cells which are secreting the enamel are called ameloblasts; they have an ectodermal origin, while odontoblasts are of mesenchymal origin. Cementum, covering the dentin, at the root of the tooth has a structure very similar to that of the bone, containing cells - cementocytes, disposed in lacunae of the , g y , p calcified matrix, communicating with one another, by expansions, like the osteocytes, in the bone. The development of the tooth is a complex process, starting from the ectoderm of the primitive oral cavity, giving birth first to a dental lamina, and further on to teeth buds, inside the beneath mesenchyme.

At the level of the tooth bud is differentiating first, from the mesenchyme, the dental papilla, from which will arise the dentin secreting odontoblasts and the tooth pulp. The odontoblats are inducing the differentiation of ameloblasts, from pulp. the so-called enamel organ, which will secrete the enamel. soenamel. The enamel organ is made of an internal epithelium - ameloblasts, an intermediary layer with star-shaped epithelial cells and an external squamous starepithelium. epithelium. In its development, the tooth with its enamel organ, has first a cap shape (the cap stage) and a later bell stage, in which is resembling to a bell. bell. Where the two epithelia (inner and outer) of the enamel organ are fusing together, is appearing the so-called Hertwig's root sheath, from which will arise the root of sothe tooth. As the teeth are erupting into the oral cavity, the enamel organ is tooth. p g y, g dissapearing; dissapearing; thus the enamel can not be replaced anymore, during adult life. life.

1. The general structure of the digestive tract
The general structure of the digestive tract is found all over the digestive tract, from the esophagus, esophagus to its terminal anal region 4 concentrically disposed layers or tunics from terminal, region. tunics, the interior to the exterior: a mucosa, a submucosa, a muscular layer and the peritoneal serosa or the adventitia. The mucosa - an epithelium and a lamina propria; the epithelium is simple columnar, all over the digestive tract, with the exception of the esophagus and the anal region, which h hi h have a squamous stratified nonkeratinized epithelium. L i propria i made of t tifi d k ti i d ith li Lamina i is d f loose connective tissue, beneath the epithelium, abundant in migrated cells of immune defense, especially in the intestine, the so-called lamina propria of immune defense. soIn the lamina propria of the intestine are present simple tubular glands, called Lieberkuhn glands, all over the small and large intestine. In the rest of the digestive tract we find glands in the lamina propria, in the upper and the lower region of the esophagus, in the stomach and in the duodenum (Brunner's glands). The limit between the mucosal layer and the next is considered to be a fine sheath of smooth muscle fibers, called muscularis mucosae - a fi i fine inner circular sheath and an outer l fib ll d l i i l h th d t layer, longitudinaly disposed.

The submucosa is consisting of loose connective tissue with numerous lymphoid tissue, structures, most abundant in the ileum and isolated, in the rest of the digestive tract. The submucosa contains glands, all over the esophagus, and in the duodenum; mucous secreting glands Besides the vascular elements are present glands. numerous nerve fibers and microganglia, forming the submucosal or Meissner's nerve plexus. The muscular layer is made of smooth muscle fibers with the exception of the fibers, upper 2 thirds of the esophagus, and the terminal part of the rectum, which have striated muscle fibers. The muscular layer, or tunica muscularis - two layers of smooth muscle fibers; the inner layer with a circular disposition and the outer layer with a longitudinal orientation; only the stomach has three layers of smooth muscle fibers, a third inner oblique layer, being present. Between the layers of smooth muscle, is found the myenteric nerve plexus of Auerbach, with numerous microganglia. The serosa is represented by the peritoneum, where it envelopes the digestive tract, tract with the structure of a mesothelium - a thin layer of loose connective tissue tissue, covered by a simple squamous epithelium. Esophagus and rectum, show an adventitia of loose connective tissue, instead of a serosal covering.

Figure 3. Organizarea generală a tradusului digestiv

2. Esophagus
Esophagus is a muscular tube, of approximately 25 cm long, making the link between the oral cavity and the stomach, transporting the food. It has some longitudinal folds, food. in its mucosa, in repause. In its structure, has the same 4 tunics, like all the digestive repause. tract. tract. The mucosa - a squamous stratified nonkeratinized epithelium, in the lamina propria we d not see so many migrated cells, lik i other regions, of th di i do t i t d ll like in th i f the digestive ti tract. tract. In the lamina propria, close to the pharynx, and next to cardia, are present, tubular branched glands, the so-called cardiac glands, which are mucus-secreting somucusglands. glands. The submucosa is made of a denser connective tissue, than the lamina propria and is containing on all its length, tubular-acinary glands, called proper esophageal glands, tubularalso mucus-secreting glands. The mucus is lubricating the mucosa. The muscular mucusglands. mucosa. layer of the esophagus is made in its upper part, exclusively of striated muscle fibers, i it middle portion, of a mixture of striated and smooth muscle fib ti f i t f ti t d d th l fibers and i it l d in its lower in its iddl region, only of smooth muscle fibers. At its outer part, esophagus has an adventitial fibers. layer, of loose connective tissue, with vessels, nerves and adipose tissue. tissue.

Figure 4. Photomicrograph of a section of the upper region of the esophagus. M h Mucous esophageal h l glands are in the submucosa; striated skeletal muscle is in the muscularis. PAS and PT stain. Low magnification.

3. Stomach
The stomach is the most dilated part of the digestive tract, where the ingested food is very well mixed with the gastrc juice transformed into a viscous mass - the gastric mixed, juice, chyme; pepsin is initiating the protein digestion. The stomach has 4 regions - cardia, body, fundus and pylorus. The lining epithelium of the stomach is a simple columnar epithelium, epithelium and the transition from the stratified squamous epithelium of the esophagus to the simple columnar epithelium of the stomach is abrupt - the so-called eso-gastric soesojunction. The epithelium of the gastric mucosa is invaginating into the lamina propria, forming the gastric pits The lining epithelium of the stomach is mucus-secreting with pits. mucus-secreting, tight junctions, both these features being involved in the protection of the gastric mucosa against the corosivity of the gastric juice. The epithelial lining cells are pale, with an ovoid nucleus, in the inferior part of the cells. , p The cardiac region is reduced -2-4 cm, at the opening of the esophagus and has short gastric pits; in the lamina propria are present tubular branched mucus-secreting glands mucusglands, opening at the bottom of the pits. The region of the fundus and the body is the most important functionally - the fundic glands secrete the gastric juice. At the bottom of each gastric pit are opening 2 or 3 fundic glands thus the lamina propria between the pit, glands, numerous glands is reduced.

Figure 5. Photomicrograph of the basal portion of the gastric gland in the fundus. This section shows parietal cells rich in mitochondria and their characteristic intracellular canaliculi ( (arrowheads). Chief cells show red ) secretory granules in their cytoplasm. Pararosaniline toluidine staining.

The fundic glands are simple tubular glands, with a neck, a body and a base. At the neck of the gland are present undifferentiated low columnar cells, which by mitosis are replacing the epithelial cells of the gastric pits, which have a rapid turnover, of 3-4 days (even if injured by the acidity of the gastric secretion, they are 3rapidly replaced). They also replace the cells of the fundic glands, with a much slower rate. In the neck of the fundic gland are also present mucus-secreting cells, secreting a mucusmucus different as chemical nature from that secreted by the lining epithelium The epithelium. cells show flattened nuclei, to their base (the cells are also called accesory mucus cells).

Figure 6. Photomicrograph of a section of the pyloric region of the stomach. Note the deep gastric pits with short pyloric glands in the lamina propria. H&E stain. Low magnification.

Figure 7. Photomicrograph of a section of the gastric glands in the fundus of the stomach. Note the superficial mucussecreting epithelium. Parietal cells ( g g p (lightstained) predominate in the mid and upper regions of the glands; chief (zymogenic) cells (dark-stained) predominate in the lower region of the gland. MM, muscularis mucosae. PT stain. Low magnification.

Thank you for your attention!

6. THE DIGESTIVE SYSTEM (2)
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

THE DIGESTIVE SYSTEM 4. The small intestine 5. Appendix 6. Large intestine (colon)

The parietal cells (oxyntic or acidophilic) - in the body of the fundic glands, to the periphery of the gland; they look polyhedral or rounded, with central placed nucleus and intense acidophilic cytoplasm. In the e.m. they have abundant mitochondria and a well developed system of intercellular canaliculi, in which is discharged the HCI acid, p g , produced by these cells. The HCI acid is obtained from y chlorides in the blood and from carbonic acid, with the help of the carbonic anhydrase (CO2 - carbonic acid - bicarbonate -H+, the cation with Cl- = HCI). The Clcell is accomplishing this synthesis with high oxygen consumption. The presence of canaliculi between two adhiacent cells, is important in the protection of the cells against HCI acid, here produced. The parietal cells are also producing the intrinsic factor, or the Castle's factor, which will combine with B12 vitamin, and only in this combination, this vitamin is absorbed in the intestine. The lack of B12 vitamin is leading to the impairment of erythropoieis, with pernicious anemia, resulting in production of abnormal large, red blood cells. The zymogenic or chief cells are secreting the pepsin and a gastric lipase. They have the characteristics of protein synthesizing and exporting cells; they show an intense basophlia of the cytoplasm and violet granules of secretion toward their apical pole. They are situated to the base and body of the fundic gland, forming most part of it. The pepsinogen, inactive, in the acidity of the gastric juice, is transformed into active pepsin; pepsin is however a weak proteolitic enzyme, with a reduced role i di d d l in digestion, while th h d hl i acid i responsible f th ti hil the hydrochloric id is ibl for the proteins degradation, in the stomach.

Another type of cells, in the fundic gland - the neuroendocrine cells, part of the DNES, here secreting serotonin, also histamine and gastrin, both stimulators of the hydrochloric acid secretion. Pylorus has deep gastric pits; at their base are opening the branched tubular mucusmucus-secreting glands; they also secrete lysozyme. The glands are the same with those of the cardiac region, but instead of short pits and long glands, here we find the reverse long pits and short tubular glands Cells of the DNES are reverse, glands. secreting gastrin and somatostatin, which is inhibiting, here, the release of the gastrin. The submucosa has no peculiar aspects, but that we find more migrated cells than in the esophagus (also in the lamina propria) -especially mast cells, lymphocytes, macrophages and eosinophils The muscularis mucosae is a continuos layer eosinophils. layer, because in the submucosa are not present glands, to interrupt it. Smooth muscle fibers of the muscularis mucosae are inserting on the basal lamina of the lining epithelium of the stomach; by their contraction is stimulated the discharge of the contraction, glands secretion. The muscular tunic has 3 layers, in the stomach - outer -longitudinal, middlemiddleoblique, fibers. common. circular and inner - oblique of smooth muscle fibers The serosa is common

4. The small intestine
The small intestine is the principal site of digestion and metabolite absorbtion. Small intestine is of approx. 5 m long; according to characteristics of its mucosa was divided into duodenum jejunum and ileum duodenum, ileum. The main features of the mucosa are the same for the three segments. Most important adaptive feature is the enlargement of the surface of the intestine, in vue of an increased absorbtion of the nutrients With the naked eye are observable nutrients. observable, large folds of the mucosa and submucosa, of circular, spiralated or demilunar aspect, called plicae circulares or Kerckring's valves, best developed in the jejunum. jejunum All the surface of the small intestine - small projections of the lamina propria, covered by the epithelium (mucosa), called intestinal villi, of about 0.5-1.5 mm 0.5long. long At the base of the villi - numerous simple tubular glands called Lieberkuhn glands, glands, or crypts. The epithelium of the villi is continuous with that of the glands; in the Lieberkuhn glands are found numerous undifferentiated cells, responsible for the renewal of the intestinal epithelium epithelium. These cells are located especially at the base of the glands, they migrate and they replace gradually, the epithelium, up to the top of the villi, capable to differentiate in 4- days. absorbtive cells and goblet cells; the turnover of the epithelium is of 4-6 days

Figure 1. Photomicrograph of the epithelium covering the small intestine. A: Columnar epithelial cells with th b h b d ( ith the brush border (arrowhead) i t h d) interspersed with mucus-secreting goblet cells. Th PAS d ith ti bl t ll The PAShematoxylin staining gives a positive reaction for the glycoproteins present in mucus and the brush border. Medium magnification. B: Numerous absorptive cells with their brush borders and the clearly visible intercellular limits. PT stain. High magnification

The majority of the cells of the small intestine epithelium are absorbtive cells, or cells, enterocytes. enterocytes. They are tall columnar cells with numerous microvilli at their apical pole - 3000, for each absorbtive cell. They show an ovoid nucleus to their basal pole, perpendicular on the basement membrane and basophilia of the cytoplasm, in the light microscopy In e m they have abundant RER supranuclear Golgi microscopy. e.m. RER, complex, mitochondria and lysosomes to their apical pole. Besides this polarization, the microvilli are forming the so-called striated border or brush border, sovisible with the light microscope, covered by a layer of glycoproteins, called glycocalix. Microvilli are increasing the absorbtion surface of the small intestine; the striated border is also the site of specific enzymes - disaccharidases and dipeptidases, able to hydrolyze the disaccharides into monosaccharides, and the p p , y y , dipeptides into amino acids. The goblet cells are interspersed between the enterocytes they produce mucus enterocytes, to lubricate and protect the intestinal lining and they become more numerous in the ileum, and in the large intestine. They have the appearance of a glass of champaigne. champaigne The Paneth cells are pyramidal cells, filled with shining acidophilic granules - at the base of the Lieberkuhn glands, especially in the duodenum. Paneth cells are intestine, colon. present in the small intestine but they dissapear in the colon They secrete lysozyme, which has antibacterial activity and is important in protecting the intestinal mucosa.

The M (membranous) cells are covering especially the Peyer's patches of the ileum; i the li h microscopy, they are suspected b the presence of i il in h light i h d by h f intraepithelial i h li l lymphocytes. These cells have long processes, forming a sort of crypts, in which they host activated lymphocytes; their important role is that of transporting ti (f i ti l ) to the lymphoid ti h id tissue of th i t ti ( f the intestine (e.g., P ' antigens (foreign particles) t th l Peyer's patches) and even farther on, to the local lymph nodes, being vital in the development of the immune response. The basement membrane may be discontinuous under these cells facilitating their passage cells, passage. Neuroendocrine cells are numerous in the small intestine. The lamina propria of the small intestine is a typical lamina propria of immune defense, with numerous migrated cells -lymphocytes, macrophages, p , g y p y , p g , plasma cells etc. In the long axes of the villi are present an arteriole, a venule and a lymphatic vessel, beginning with a blind end, in the villi. The arteriole is forming a capillary network just beneath the epithelium of the villi, important in the absorbtion of the nutrients. Another vascular plexus is found in the submucosa.

Figure 2. Structure of a microvillus. A cytoskeleton of actin filaments, associated with other proteins, keeps the shape of the microvillus. The actin microvillus filaments are continuous with the microfilaments of the terminal web (see Chapter 4), which also contains intermediate filaments. N t th t i thi i t di t fil t Note that in this location actin filaments have a structural role and are not related to movement, as is usually the case when these microfilaments are present. To fulfill its supportive role, actin is associated with other proteins that link the , microfilaments to one another to fibrin, and to the cell membrane and a specific protein–villin–in its tip.

Smooth muscle fibers from the muscularis mucosae climb to insert on the basal lamina, in the top of the villi; their contraction is changing the length of the villi, during absorbtion, as well, these movements are exerting a pump effect in emptying the lymphatic vessel, found in the middle of the villi. The products resulted from digestion are absorbed in the small intestine; the amino acids and the monosaccharides are absorbed by active transport, directly into the blood capillaries. The most powerfull protease is the pancreatic trypsin acting on the proteins; the lipids are digested by the action of pancreatic lipase and bile. The result is an emulsion of glycerol and fatty acids, which cross p gy y pasively the cellular y membrane of the microvilli, and in the smooth ER, they form once again triglycerides. Triglycerides are packed in the Golgi complex, by a thin layer of proteins, forming the so-called chylomicrons. By the lateral cellular faces, the chylomicrons flow in sothe direction of the vessels of the lamina propria. Most of the chylomicrons go to the lymphatic vessels. The muscular tunic and the serosal lining of the small intestine have nothing uncommon.

Figure 3. Photomicrograph of the basal portion of 2 glands (crypts) of the small intestine. Note the enteroendocrine cell, Paneth cell, goblet cell, and a cell in mitosis.

Figure 4. Photomicrograph of the duodenum, showing villi and duodenal glands in the submucosa. submucosa H&E stain Low stain. magnification

DUODENUM - first portion of the small intestine, with shorter and larger villi, resembling to leaves; the Lieberkuhn glands are either, shorter. In the lamina propria, as well as in the submucosa, are present numerous branched tubular-acinary mucus tubularsecreting glands, called Brunner's glands; they secrete a mucus with an alkalin ph of 778, containing bicarbonate; this is the principal factor of neutralization of the gastric acidity. Because the glands are extending from the lamina propria into the submucosa, the muscularis mucosae appear interrupted, hard to observe. In the duodenum the absorbtion i reduced; main processes of di b bti is d d i f digestion are t ki place h ti taking l here. JEJUNUM – the second portion of the small intestine, with no peculiar glands in the lamina propria, or submucosa, thus the muscularis mucosae appears continuous, well deffined. H d ffi d Here we fi d th l find the longest villi, of th small i t ti t illi f the ll intestine, and th l d the longest t Lieberkuhn glands, at the base of the villi; the villi have a characteristic aspect of fingers. In the lamina propria may appear isolated lymphoid nodules, or irregular crowdings of l di f lymphocytes, which are t h t hi h transitory structures. J j it t t Jejunum i th main site of is the i it f the absorbtion process. ILEUM - is the third, last portion of the small intestine, with fewer and shorter villi, as we approach th l h the large i t ti intestine. Th number of goblet cells i l The b f bl t ll is larger. Th Li b k h The Lieberkuhn glands are also shorter, fewer and deviated, by the lymphoid tissue in the lamina propria and submucosa, forming the Peyer's patches. The Peyer's patches are made of nodules, zones, hundreds of lymphoid nodules associated together; they also contain T cells zones with a diffuse aspect; they are sites of the immune response and immune defense, of the intestine. The mucosa is smoother in these zones, with numerous M cells.

Figure 5. Photomicrograph of a group of neurons (with large nuclei) and satellite cells (with small nuclei) constituting a component of the myenteric plexus between 2 smooth muscle layers. Note the red stained collagen fibers. Picrosirius-hematoxylin. Medium magnification.

5. Appendix

The appendix was first considered as a remaining of the species evolution; in fact evolution; it has the role of a secondary lymphoid organ. It's a glove finger-like organ, organ. fingerattached at the posterior and lateral part of the cecum. cecum. Being part of the large intestine, it has no villi, only short, deviated Lieberkuhn glands. glands. The glands are deviated by the lymphoid tissue, which is encircling the organ, in its lamina propria and submucosa, that we do not find anymore the muscularis mucosae. mucosae. It has no teniae coli in the longitudinal muscular layer. Because appendix is blindlayer. blindended, it is an usual site of inflammation, called appendicitis. appendicitis.

6. Large intestine (colon)
The large intestine has no more villi, since here the absorbtion process is much reduced. The mucosa - numerous long Lieberkuhn glands, crowded, so the lamina propria b t i between th them i reduced. Th epithelium of th mucosa h f is d d The ith li f the has fewer absorbtive b bti enterocytes, but much more numerous goblet cells, than in the small intestine. The microvilli of the absorbtive cells are less numerous, shorter and irregular. Water is absorbed, following pasively absorbed follo ing pasi el the sodi m gradient as well as vitamins, especiall of sodium gradient; ell itamins especially the group B and K vitamin. The mucus is important in the propelling of the fecal mass and in protection of the mucosa. In the lamina propria we find relativelly numerous lymphoid structures, which may penetrate into the submucosa. These are transitory lymphoid structures, but they are frequently found, because of the septic content of the colon. f l f d b f h i f h l The outer longitudinal layer of smooth muscle fibers is concentrated into 3 bands of g y muscle, called teniae coli. The peritoneum is forming protuberances of adipose tissue, called appendices epiploicae. In the rectum are present longitudinal folds of the mucosa - the rectal columns of Morgagni; a few cm before the anal opening, the simple columnar epithelium is replaced by stratified squamous nonkeratinized epithelium.

Thank you for your attention!

7. THE DIGESTIVE SYSTEM (3)
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

THE DIGESTIVE SYSTEM 1. 1 The Liver 2. Functions of the liver 3. 3 Biliary ducts system 4. The pancreas 5. Salivary Glands 5 S li Gl d

1. The liver
The liver is the largest gland of the body, of about 1.5 kg in weight, with both exocrine function, by the production of the bile, and endocrine function, by uptaking products from the blood intense synthesizing activity the discharge of blood, activity, the synthesized materials, being into the blood, on the endocrine pattern, even if these products are not hormones. The liver - two lobes and a hilum by which are penetrating the portal vein and the hilum, hepatic artery and are exiting the hepatic veins, that empty in the inferior vena cava. At its exterior, the liver is covered by a dense connective tissue capsule, Glisson's capsule thicker to the hilum surrounding in the parenchyma the vessels Glisson s capsule, hilum, parenchyma, and the bile ducts, with sheaths of connective tissue. Liver is typically a parenchymal organ, the liver cells, or hepatocytes, being the predominating component of it while the stroma is made of a very fine network of reticular fibers it, fibers, only for the support of the liver cells. In histologic section, one may observe, triangular spaces, called portal spaces (of Kiernan), Kiernan) containing the portal triads three elements - a venule branch of the triads, venule, portal vein, an arteriole, branch of the hepatic artery, a bile duct, part of the bile ducts system and lymphatic vessels, not visible in the light microscopy. The venule is usually the largest element of all; the bile duct is lined by simple cuboidal epithelium.

The liver cells or hepatocytes are situated in p p y plates of cells, oriented from the portal spaces to the center of the liver lobules. In light microscopy, the plates of hepatocytes are appearing as cords of liver cells, distributed radiary, to the center of the lobule, finishing to the central vein of the lobule. Between the cords of liver cells, are present sinusoidal capillaries of the liver, emptying into the central vein of the lobule. The so-called classic hepatic lobule is a hexagonal teritory of radiary disposed liver socells, with the central lobulary vein in its center; the plates of liver cells, visualized as cords, in light microscopy, are surrounded by the liver sinusoid capillaries, which sometimes are perforating the liver cells plates. In other species, the liver lobules are clearly limited by sheaths of connective tissue; in human, this does not occur, because of that, the liver lobules are hard to observe. Usually in three corners, from the six of the hexagon, which is the hepatic lobule, are present portal spaces, containing th d t i i the described elements. I f t i space, th li ib d l t In fact, in the liver l b l lobules appear as hexagonal prisms, with no clear limits, of connective tissue, in human. There were also described in the liver, other types of lobules, the so-called sofunctional lobules - the portal lobule is considering especially the exocrine function of the liver, the bile production; the portal lobule has in its center the portal triad, with the biliary duct, situated in the adjoining corners of three classic hepatic lobules. l b l

Figure 6. Photomicrograph of the liver. A: A central (centrolobular) vein. Note the liver plates that anastomose freely, limiting the space occupied by the sinusoids. PT stain. Medium magnification. B: A portal space with its characteristic small artery, vein, lymph vessel, and bile duct surrounded by connective tissue. PT stain. Medium magnification. C: Collagen III reticular fibers in the lobule, forming a scaffold for the hepatic tissue. Silver impregnation. Medium magnification.

The bile duct is colecting bile from three classic lobules, thus the portal lobule, will appear triangular, with a central vein of classic lobules at the tip of each of its angles, with the center in the portal space and will contain parts of the three adjacent lobules (see space, scheme). Another functional lobule is the hepatic acinus of Rappaport - an area of hepatocytes, situated in two adjacent classic lobules, receiving blood, from the same arteriole and venule (branches of each following the faces of the classic lobule) The lobule). hepatic acinus is romboidal, with the center at the level of the two adjacent classic lobules, and the two angles in two central lobular veins (see scheme). This is a primary acinus; the acini irigated by the same arteriole and venule of a portal space - a secondary acinus. In the liver sinusoids are mixed the arterial and the venous blood; most of the blood is from the portal vein - 70-80 %, the rest is arterial blood from the hepatic artery Liver cells 70- % blood, artery. in the hepatic acinus were divided in three zones - zone I is the first which receive blood with oxygen and nutrients (situated in the center of the hepatic acinus and in the periphery of the classic lobule) the detoxification capacity of zone I is the bigest, is the most resistant zone of the liver, to anoxya, or toxic substances, is the first zone stocking glycogen and the last one to loose it. Zone II, in the middle part of the classic lobule, is the second to receive blood with nutrients and oxygen, which were not used by zone I, yg , y , has a medium metabolic activity, while zone III, in the periphery of the hepatic acinus and in the center of the classic hepatic lobule, is the most vulnerable zone, the last one to capture the glucose and the first which is loosing it, when needed by the organism, the first which will suffer from the toxic products, in the blood, and when anoxya occurs. In different types of hepatic lesions, zone III will be affected, first.

Figure 7. Three-dimensional aspect of the normal liver. In the upper center is the central vein; in the lower center, the portal vein. Note the bile canaliculus, liver plates, Hering’s canal, Kupffer cells, sin

The hepatocytes, or liver cells, are polyhedral cells, of about 20-30 µm, in diameter; they 20show one or two centrally located nuclei, with visible nucleoli, many times, the nucleus is polyploid. In H.&E. stained microscopic sections, the cytoplasm is eosinophilic, especially because of the abundant mitochondria and the presence of smooth endoplasmic reticulum. The RER is well developed either, p p , present in aggregates, in the cytoplasm, as gg g , y p , well as the Golgi complexes, up to 50/cell. The hepatocytes contain, as well, dense granules of glycogen, lipid granules. Hepatocytes show usually six faces, at least two are facing the liver sinusoids. Between the liver cells and the endothelial cells of the sinusoids - a microscopic space, called the space of Disse, containing microvilli of the hepatocytes. In consequence, the blood may pass easy, from the sinusoid, through its pores, into the Disse's space; the vascular - permanent exchange of macromolecules, between hepatocytes and blood, being the substrate of the endocrine functions of the liver permanent processes of exocytosis and endocytosis. Where two adjacent hepatocytes come into contact, they form a tubular space between them - the bile canaliculus, improper name, since this tube does not have its own wall, being limited by two adjacent hepatocytes. Hepatocytes establish here tight junctions, thus impairing the bile to penetrate into the blood stream. Toward the billiary pole, are found numerous Golgi complexes and lysosomes, participating in the bile synthesis. The polarization of the hepatocytes is mainly represented by the presence of a vascular, or endocrine pole and a billiary, or exocrine pole, through which bile is excreted; the intercellular f be ith i t ll l faces of adjacent h f dj t hepatocytes may b either, simply j t t i l junctional adhesion f ti l dh i faces.

Figure 8. Liver section showing sinusoid capillaries with their endothelial cells close to the hepatocytes. hepatocytes The thin slit between the hepatocytes and the endothelium is the space of Disse. Kupffer cells can be seen inside the i i id th sinusoid. PT stain. id t i High magnification.

The liver sinusoid capillaries present between the plates of hepatocytes are capillaries, hepatocytes, vessels with an irregular caliber, a tortous passage, with a discontinuous wall, made of fenestrated endothelial cells and a discontinuous basal lamina. The lamina. fenestrae of the endothelial cells - 100 nm in diameter without diaphragms usually diameter, diaphragms, grouped in clusters, forming "sieve areas". areas". The sole continuous structure of the sinusoid capillary wall is represented by a delicate network of reticular fibers. Besides the endothelial cells in the liver fibers. cells, sinusoids are present macrophages of the liver, elements of the mononuclear phagocyte system, called Kupffer cells. Their main functions are to digest aged cells. erythrocytes and resulted Hb to secrete immunologic factors and to act as antigen Hb, presenting cells. They have luminal processes, as well as prominences which are cells. penetrating into the Disse's spaces. spaces. The fat-storing cells or Ito cells are found between hepatocytes outside the fatcells, cells, hepatocytes, capillary wall. They have the capacity to store vitamin A, in triglycerides droplets, in wall. their cytoplasm, but their exact role is still unknown. It was supposed they are unknown. mesenchymal cells capable to secrete reticular fibers and to store lipids similar to cells, lipids, fatfat-storing cells in the bone marrow, since the liver has hematopoietic function, during fetal life. life.

Figure 9. Ultrastructure of a hepatocyte. RER, hepatocyte RER rough endoplasmic reticulum; SER, smooth endoplasmic reticulum. x10,000.

2. Functions of the liver

1. Metabolic functions - in the glucids metabolism, the liver is the principal site of glucogenesis, with glycogen stockage, under the influence of insulin; under the influence of epinephrine and glucagon, glycolysis occurs, both systems being balanced by the level of the glucose, into the blood. A complex process, called gluconeogenesis, able to synthesize glucose, starting from lipids and amino acids, is also taking place in the liver. In the lipids metabolism, the liver is responsible of synthesis of lipoproteins, from faty acids and chylomicrons (LDL - low density lipoproteins; HDL - high density lipoproteins; VLDL - very low density lipoproteins; HDL have antiatherogenic properties). In the liver are synthesized also esters of cholesterol, an i h l t l important component, for example of the cellular membranes. t t t f l f th ll l b In the proteins metabolism, the liver cells are synthesizing various types of proteins, y g y found into the plasma of the blood: albumin, alfa and beta globulins, coagulation factors - fibrinogen, transferin, etc. In the liver are also taking place deaminations of amino acids, with production of urea, which will be excreted by the kidney.

2. 2 Detoxification function - many drugs and toxic substances of which many of them substances, which, used in therapy, are inactivated in the liver, especially by oxidation, conjugation and methylation. Steroid hormones are also metabolized and inactivated in the liver. Bilirubin, resulted from the break-down of Hb, is conjugated into the smooth breakendoplasmic reticulum of the hepatocytes, with glucuronic acid, forming from the insoluble bilirubin, a water-soluble bilirubin glucuronide, which is then excreted in waterthe bile. All these functions are reflecting the endocrine p g pattern of behavior of hepatocytes. 3. 3 Bile secretion is the exocrine function of the liver; main components of the bile are - water, electrolytes, bile acids, cholesterol and conjugated bilirubin. About 90 % of the bile acids content of the bile, are resulting from reabsorbtion of these substances from the intestine (enterohepatic recirculation) and only approx. 10 % are synthesized in the liver. Bile acids are important in emulsifying lipids, in the intestine, facilitating their digestion and absorbtion. They are also helpful in solubilization of cholesterol, facilitating its excretion. , g

3. Biliary ducts system
The bile produced by the liver cells flows to the bile canaliculi, between adjacent hepatocytes, at the periphery of the hepatic lobules is discharged into the bile ductules or Hering's H i ' canals, lined b flattened cuboidal cells. l li d by fl tt d b id l cells. ll The bile ducts are forming an anastomosing network, and they become gradually larger, g g y g y g being lined by simple columnar epithelium. The left and the right hepatic ducts are epithelium. forming, when uniting, the hepatic duct, which is fusing with the cystic duct - from the gallbladder; gallbladder; to the duodenum is appearing the common bile duct or ductus choledochus, which is opening into the duodenum with the sphincter of Oddi. Besides the mucosa, with Oddi. the simple columnar epithelium and a thin lamina propria, at the outside of the ducts is appearing a thin layer of smooth muscle fibers, which finally forms the sphincter of Oddi, i the d d in h duodenum wall. wall. ll Gallbladder appears as a p -shaped organ, attached to the inferior surface of the liver pp pearpear p g and united with the common hepatic duct, by the cystic duct. Is a reservoir for the bile, being capable to store approx. 30-50 ml of bile. In its structure has a mucosa, attached 30directly to the muscular layer and a connective tissue and serosal lining, to the outside.

The mucosa makes folds and is made of a simple columnar epithelium, responsible for the concentration of the bile; its epithelium is specialized in the ion and water transport. Water is following, pasively, the sodium gradient. To the neck of the gallbladder, close to the cystic duct are present a few tubuloacinar mucusmucussecreting glands which secrete most of the mucus found into the bile The glands, mucus, bile. muscular layer is disposed plexus-like. While the inferior surface of the gallbladder plexusis covered by the peritoneum, its superior face is attached by a thick layer of connective tissue to the inferior face of the liver tissue, liver. The vegetative autonomic nervous system is controling the discharge of the bile, from the gallbladder, t f th llbl dd together with th DNES th parasympathetic nervous system th ith the DNES; the th ti t is stimulating the contraction of the gallbladder and the bile discharge into the duodenum. Cholecystokinin, a local hormone, produced by the DNES cells in the intestine, intestine is also stimulating the contraction of the gallbladder with bile discharge; gallbladder, the release of cholecystokinin is stimulated by the presence of the ingested fats, in the small intestine. Secretin, another DNES hormone is promoting the secretion of bicarbonate into the bile conffering an alkalin ph Bile is also containing the bile, ph. secretory IgA, the hepatocytes having the capacity to produce the secretory piece, which by binding IgA monomers, from the serum, will form the dimer form of IgA, which is part of the mucosal local immune defense, of the organism; secretory IgA, in the bile, will protect the intestinal mucosa.

Figure 1 Photomicrograph 1. of a section of gallbladder. Note the lining of columnar epithelium and the smooth muscle layer (M) PT stain (M). stain. Low magnification.

4. The pancreas
Pancreas is a mixed exocrine and endocrine gland (Langerhans islets), which produces digestive enzymes and also hormones, most important, insulin. The exocrine pancreas is a g y , p , p pure serous gland, compound acinar gland, similar with the parotid gland. In contrast with the parotid gland, the pancreas has no striated ducts, but has the Langerhans islets, looking paler, in light microscopy than the exocrine acini, these being the main criteria for differentiating the two glands in light microscopy microscopy. The pancreas is situated outside peritoneum, has a thin capsule of connective tissue, from which are arising septa, which are dividing the parenchyma of the pancreas, into lobules. proteinThe pancreatic acini are typical serous acini containing granulated protein-secreting cells acini, cells, with a peculiar characteristic, also differentiating them from those of the salivary glands; the smalest excretory ducts, called intercalated ducts, lined by a simple cuboidal epithelium, are beginning, in the pancreas, inside the lumen of the acini, the so-called socentroacinar cells. The centroacinar cells are visible in some acini, like pale cells, with their nuclei, in the lumen of the serous acini, of the pancreas. The intercalated ducts are fusing and are forming larger, interlobular ducts - simple columnar epithelium; finally, these ducts are forming the principal excretory duct of the pancreas - Wirsung duct and sometimes sometimes, the accesory duct of Santorini. The principal ducts are lined also by simple columnar epithelium, which may contain some goblet cells. The pancreatic acini do not contain myoepithelial cells, like the acini of the salivary glands; pancreas has an endodermal origin, unlike the salivary glands originating from the ectoderm.

Figure 2. Schematic drawing of the structure of pancreatic acini. Acinar cells are pyramidal, with granules at their apex and rough endoplasmic reticulum at their base. The intercalated duct partly penetrates the acini. These duct cells are known as centroacinar cells. Note the absence of myoepithelial cells.

The pancreatic juice contains the most powerfull digestive enzymes - the protease trypsin, secreted as precursor -trypsinogen, the pancreatic lipase, amylase, and also ribonuclease, deoxyribonuclease, elastase, carboxypeptidase, etc., and of course, water and ions. , The pancreatic secretion is controlled by both vegetative nervous system - the parasympathetic nervous system (vagus nerve) is stimulating the secretion of the pancreas, as well as secretin and cholecystokinin - secreted by cells of the intestinal DNES. Secretin is providing a secretion rich in water and bicarbonate, able to neutralize the acidity of the gastric chyme while cholecystokinin is chyme, stimulating a less abundant secretion, but rich in enzymes. Islets of Langerhans - small clusters of polyhedral or round endocrine cells cells, disposed in anastomosed cords, in the mass of the exocrine pancreatic acini. Langerhans islets - paler than the serous acini of the pancreas; they do not have a capsule to isolate them from the acini only some reticular fibers are forming a fine acini, stroma to support the endocrine cells; between the cords of endocrine cells are present fenestrated blood capillaries. The islets are receiving nerve fibers from the system. autonomic nervous system

Figure 3. Photomicrograph of a pancreas showing the exocrine portion (acini) and the endocrine portion ( (islet of Langerhans). The g ) acini contain secretory cells with basophilic cytoplasm. Different types of endocrine cells are seen in the islet. PT stain. Medium magnification.

Immunocytochemical techniques have differentiated several cell types, secreting different hormones, inside the i l diff h i id h islets: – A cells, represent maximum 20 % of the islets cell p p population, are situated p mainly in the periphery of the islets, they have regular granules, with dense core, surrounded by a clear region, bounded with a membrane, are secreting glucagon, which is increasing the blood glucose, by glycogenolysis and lipolysis. – B cells, represent approx. 70-75 % of the cell population of the islets; they are 70situated mainly in the midle part of the islets, they contain irregular granules, with central crystal core; they secrete insulin, main hormone capable to decrease the glucose in the blood, by promoting the entrance of the g g , yp g glucose into the cells. – D cells represent maximum 5 % of the cells of the islets they contain granules cells, islets, with homogenous aspect, they secrete somatostatin, main action of somatostatin is that of inhibiting the release of other hormones, secreted by the Langerhans islets, through a local, paracrine mechanism.

F cells, are rare, they represent less than 1 % of the cells of the islets, they secrete the pancreatic polypeptide (PP), with local, not yet fully understood actions. Insulin is the most important of the secreted hormones, being fundamental in the homeostazis of glucose, in the organism. The lack of insulin is leading to a severe condition - diabetes melitus, type I; the clinical signs are the consequence of the inability of the cells to use the glucose, when insulin is missing are appearing: increase of the food and fluids ingestion, but weight loss, since insulin is the main anabolism providing hormone, polyuria and gradual accumulation of toxic catabolites, in the organism, which may finally lead to a diabetic coma, when central nervous system is affected by this toxicity. One of the causes of the diabetes is the abnormal production of autoantibodies against B cells of the pancreatic islets (autoimmune diabetes). Insulinoma are pathologic proliferations of the B cells leading to abnormal sudden decrease of the blood glucose, reflected by severe hypoglicemia. The sympathetic nervous system is decreasing the insulin secretion, while the parasympathetic nervous system is stimulating insulin secretion.

Thank you for your attention!

8. THE DIGESTIVE SYSTEM (4)
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

SISTEMUL DIGESTIV 4. 4 Salivary Glands

4. Salivary glands
Salivary glands are pair organs, which are producing saliva, with digestive, lubricating and immunologic functions 3 pairs of salivary glands - the parotid glands the functions. glands, submandibular (submaxillary) glands and the sublingual glands. The glands are branched acinar glands, surrounded with a capsule of connective tissue - septa which are dividing the g g glands into lobules. The acini of the salivary g y glands have the classical structure of the mucous, serous and mixed acini and they contain myoepithelial cells at the base of the secretory cells, salivary glands having an ectodermal origin. The parotid is a pure serous gland, and is containing exclusively serous acini, while the th th others are mixed glands, containing serous, mucous and mixed acini. Th i d l d t i i d i d i i The submaxillary glands show serous cell predominance, while the sublingual glands, the reverse. The submaxillary glands contain serous and mixed acini, and a few mucous acini; sublingual glands contain almost none serous acini having only mucous and acini, mixed acini. The excretory ducts are begining to a pole of the secretory acini, the centroacinar cells of the pancreas are absent. These intralobular ducts are called intercalated ducts of Boll, lined by simple cuboidal epithelium; from the joining of the i t l t d ducts lti the t i t d ducts f Pfl l intralobular ducts, intercalated d t are resulting th striated d t of Pfluger, also i t l b l d t lined by simple columnar epithelium. The striated ducts show in the electron microscopy, numerous mitochondria and folds of the basal cellular membrane, microvilli at their apical pole being ion-transport specialized cells These organelles pole, cells. ionare conffering the striated aspect, visible in light microscopy.

Figure 4. Photomicrograph of a submandibular gland. Note the presence of dense serous cells forming demilunes and pale-staining mucous cells grouped along the tubular portion of this tubuloacinar gland. Medium magnification.

The columnar epithelium of these ducts is responsible of secretion and absorbtion p processes, leading to the concentration or dilution of the secreted saliva. The g striated ducts are uniting and form larger ducts, present in the connective tissue septa, parting the glands into lobules - interlobular ducts. These ducts are first lined by bistratified cuboidal epithelium, which is becoming in the main excretory duct of each gland (e g Stennon canal of the parotid gland) columnar stratified (e.g. epithelium, with goblet cells. In the stroma of the glands are present vascular and nervous elements and small lymphoid structures, adipose cells. The striated ducts are more numerous in the submaxillary glands and less numerous in the p yg parotid, , and especially in the sublingual glands. Saliva is secreted in an amount of approx. 1 liter per day; 70 % of it is secreted by the submaxillary glands, 25 % by the parotids, and only 5 % is secreted by the bli l l d i l f h li is h i i d lubrication f h sublingual glands. A main role of the saliva i the moistening and l b i i of the oral cavity; by its amylase activity, saliva is providing the digestion of carbohydrates; the alkaline ph is neutralizing the acidity of the oral cavity; by the content of secretory IgA, lysozyme, lactoferrin saliva is also providing an IgA lysozyme lactoferrin, immunologic activity of defense of the oral cavity from noxious agents and is facilitating mastication. The secretion of saliva is regulated by the vegetative nervous system the g y g y parasympathetic nervous system is stimulating an abundant, watery secretion, acting predominantly on the striated ducts, while the sympathetic nervous system is acting mainly on the acini, providing a viscous, reduced secretion, rich in enzymes. system, condition, enzymes The stimulation of the sympathetic nervous system in stress condition is responsible for the dry mouth sensation.

Figure 5. Photomicrograph of a sublingual gland showing the predominance of mucous cells. H&E stain. Low magnification

THE RESPIRATORY SYSTEM (1)
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

RESPIRATORY SYSTEM
1. Nasal Cavity 2. The Larinx 3. The Trachea

The respiratory system is made of the lungs and a system of tubes, which are leading the air to the lungs - the air passages. In the respiratory system, it is usual, to divide the air passages into: a conducting portion - the nasal cavity, the nasopharynx, the larynx, the trachea, the bronchi and bronchioles, including the terminal b t i l bronchioles, and a respiratory portion, made of th respiratory hi l d i t ti d f the i t bronchioles, the alveolar ducts and the alveoli, the latter, being mainly the site of the gases exchanges, between blood and air. The respiratory passages are also considered according t th i situation t id d di to their it ti toward th l d the lung, as outside th l t id the lung (extrapulmonary) and inside the lung (intrapulmonary respiratory passages).

The nasal cavity
The two nasal fossae - external vestibule, the anterior and dilated portion of the nasal cavities. The outer integument, covering the nose is continuous with the g g internal one, gradually becoming nonkeratinized stratified squamous epithelium, containing numerous sebaceous and sweat glands, as well as numerous short hairs, called vibrissae, which are trapping the large particles, from the inspired air.

The nasal fossae are continuous in their posterior part with the nasopharynx continuous, nasopharynx, being sepparated one from another by the nasal septum. Most of the mucosa is containing a respiratory epithelium (pseudostratified columnar ciliated) with the exception of a small superior and posterior area with a yellowish colour the area, colour, olfactory mucosa. The movements of the cilia are directed to the nasopharynx (to the interior). The lamina propria of the mucosa, is attached directly to the bony and cartilage structures, forming the walls of the nasal cavity. In the lamina propria of the nasal cavity are present tubuloacinar mixed glands secreting both a mucus and serous glands, fluid, moistening the mucosa. Another characteristic of the lamina propria, is the presence of anastomozed large venous plexuses, known as the swell bodies. At every 20-30 minutes, the swell bodies on one side become filled with blood with a 20- minutes blood, distention of the mucosa, and a decrease of the air flow, thus, most of the air is directed through the other nasal fossa; this periodic decrease of the air flow, is preventing the desiccation (overdry) of the respiratory epithelium The abundant epithelium. vascularization of the nasal fossae is also responsible for the warming of the inspired air; in cases of blood coagulation impairment, the bleeding in this area is frequent, the so-called epistaxis. so-

The olfactory mucosa
The olfactory mucosa is a specialized area, in the roof of the nasal cavity. It consists of a pseudostratified columnar epithelium, with 3 types of cells - supporting, sensory and basal regenerative cells The supporting cells are narrower toward their bases cells. bases, with a nucleus closer to their apical pole, with microvilli at their apical pole, floating in the mucus and serous secretion, in the nasal cavity. They show junctions with the neighbor cells and they contain a yellow pigment vitamin A aldehyde very similar pigment, aldehyde, with the visual pigment, which expelled in the secretion, is helping the receptor cells in capturing the olfactory stimuli. The sensory olfactory cells are in fact bipolar olfactory neurons which are coming in sensory, neurons, contact with the environement, a peculiar situation. They have the nuclei, to their basal pole, below the level of the nuclei of the supporting cells. At their apical pole, they have dilated structures, olfactory vesicles from which are arising 6-20 nonmotile structures vesicles, 6cilia. These cilia have receptors, for the odoriferous molecules, which they are capable to recognize. The axons of the neurons are forming the olfactory nerve, directed to the central nervous system The basal cells are small round cells they system. cells, come into contact with the basement membrane of the epithelium, like all the others cells; they are regenerative cells. The receptors of the sensory cells are capable to recognize a huge variety of smell sensations, but only from substances which are g g y , y waterwater-soluble, they solve into the secretion covering the olfactory mucosa; it seems that the receptors are specialized to recognize each, a certain olfactory sensation.

Figure 6. Olfactory mucosa showing the 3 cell types (supporting, olfactory, olfactory and basal) and a Bowman’s gland.

The larynx
The larynx is like an irregular tube, connecting the pharynx to the trachea. The medium layer is containing several cartilage pieces, laryngeal cartilages; hyaline cartilages - the thyroid, cricoid and part of the arytenoids and elastic cartilages - the epiglottis, cuneiform, corniculate and the tips of the arytenoids. The cartilages are held together by membranes, capsules and ligaments and by the muscles of the larynx, which are striated muscles. They maintain an open airway, serving as support structures, they are important in phonation and they are acting as a valve, in preventing the penetration of the ingested food or fluids into the trachea. The mucosa of the larynx has two pairs of folds; the superior pair is made of the false vocal cords, being covered by a respiratory epithelium, like most of the larynx mucosa, in the lamina propria are present numerous serous glands. The inferior pair of folds is represented by the true vocal cords, covered by a nonkeratinized stratified squamous epithelium, with a lamina l i propria made of d i d f dense connective ti ti tissue. T True vocal cords are th main l d the i phonation structures, they are linked together by the vocal ligament and by striated muscles. As air is directed between the folds, the muscles contraction is regulating production. exterior, tunic, the sounds production At its exterior the larynx has an adventitial tunic of connective tissue, fixing it to the surrounding structures.

The trachea
The trachea is a tube - 10-12 cm long, with a low flexibility, that extends from the 10base of the larynx, to the point where it branches into the two principal or primary bronchi. The trachea and the primary bronchi have the same structure of the wall, made of a mucosa, a medium tunic, containing the cartilage rings and an adventitia, with th diff d titi ith the difference th t th primary b that the i bronchi contains complete cartilage hi t i l t til rings in their wall, while the trachea has incomplete C-shaped cartilage rings. The Ctracheal mucosa is made of a typical respiratory epithelium, pseudostratified, ciliated columnar epithelium The respiratory epithelium in the electron epithelium. epithelium, microscopy, contains 5 types of cells: 1. Ciliated columnar cells are the most numerous, with approx. 300 cilia, each cell, at its apical pole. 2. Mucous goblet cells, mucus secreting cells, are absent during embryonic life; they begin to develop rapidly after birth, according to the polution degree of the inspired air, the stimulus for their differentiation, being the particles in the air.

3. Brush cells, or brush border cells are so-called, because of the numerous microvilli soat their apical pole; they are columnar cells, and b h i i l l h l ll d between them were diff h differentiated i d two categories of cells. One type of brush cells has fewer organelles in the cytoplasm, but they present toward their basal pole, sensory nerve endings; these cells are acting as sensory receptors (i t ll ti t (intraepithelial chemoreceptors). A th ith li l h t ) Another category of brush cells has better represented organelles in the cytoplasm and basal bodies in the cytoplasm beneath the microvilli; they represent future ciliated cells. cells 4. Basal small cells are regenerative cells, which by mitosis are capable to differentiate into different cell types. They do not reach the surface of the epithelium, epithelium even if they lie on the basal lamina like all the others The situation of lamina, others. the nuclei at different levels is the reason for the false stratification appearance of this epithelium. 5. Small 5 S ll granular cells are very much lik th b l ll h like the basal regenerative cells, b t th h l ti ll but they have abundant granules of about 100-300 nm, in their cytoplasm, being cells of the 100DNES. These cells are secreting especially serotonin, calcitonin, bombesin (which has a stimulating effect on the gastrin production) and also enkephalins and endorphins. The enkephalins and endorphins secreting cells are especially abundant before birth, they are capable to induce a local vasodilatation, thus increasing the blood flow in the pulmonary circulation which is much reduced circulation, reduced, before birth.

Figure 7. Photomicrograph illustrating the main components of the respiratory epithelium. Pararosaniline— toluidine blue (PT) stain. High magnification.

The lamina propria of th t h Th l i i f the trachea i containing abundant elastic fib is t i i b d t l ti fibers, l lymphoid h id tissue, sometimes forming lymphoid nodules (part of the bronchial associated lymphoid tissue - BALT) also present in the larger bronchi, especially at the point of their bifurcation. In the lamina propria of the trachea are also present numerous mixed bifurcation. acinary glands, with both mucus and serous secretion. secretion. The secretion is produced and discharged as a reflex, consequence of the stimulation of the receptor brush cells in the respiratory epithelium; according to the type of the cells, epithelium; stimuli, the secretion may be predominantly mucous or predominantly serous, in order to cleanse, by the cilia beats, the air passages. The medium tunic of the trachea is passages. made of 16-20 C-shaped or horse-shoe hyaline cartilage rings united together by their 16horserings, perichondrium, and an elastic continuous membrane. The openings of the tracheal membrane. rings are oriented to the posterior part of the trachea, through where the esophagus is located. located. The cartilage rings are keeping the lumen of the trachea open, the missing portion of cartilage rings is closed by smooth muscle fibers - from one end of the cartilage to the other, other forming the tracheal muscle. In the lamina propria as well as in the medium muscle. propria, tunic of the trachea are present numerous vascular and nervous elements. The outer elements. tunic of the trachea is an adventitia, of loose connective tissue, with adipose tissue, structures, vascular and nervous structures continuous at some degree with that of the esophagus. esophagus.

Figure 8. Section of trachea showing the respiratory epithelium with goblet cells and columnar ciliated cells. Also shown are serous glands in the lamina propria and hyaline cartilage. The mucous fluid produced by the goblet cells and by the glands forms a layer that permits the y propel g particles out of ciliary movement to p p foreign p the respiratory system. PT stain. Medium magnification.

Thank you for your attention!

9. THE RESPIRATORY SYSTEM (2)
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

RESPIRATORY SYSTEM
• The Bronchi • The Alveoly

The Bronchi
As they enter into the lungs the two primary bronchi are dividing into lobar bronchi lungs, - 2 for the left and 3 for the right lung. From the lobar bronchi → the segmentary bronchi, distributing air to a pulmonary segment. As they penetrate into the lung lobules - intralobular bronchioles. From this level, the lobule, the small bronchi are called bronchioles. The intralobular bronchioles are dividing into 5-7 terminal 5bronchioles, each terminal bronchiole is branching into 2-3 or more, respiratory 2bronchioles, from which are opening the alveolar ducts and the alveoli. , p g The air passages are involved in the conditioning of the inspired air. The gradual branching of the bronchial tree is decreasing the speed of the air and is offering a larger surface of contact, between the inspired air and the mucosa. This contact is important for the warming, the moistening and the cleansing of the air. The air is uniformly distributed and is purified by the continuous cilliary beat, which is moving the secretion on the surface of the epithelium, toward the pharynx (outside). The extensive capillary network in the lamina propria is warming the inspired air.

The b Th bronchial associated l hi l i t d lymphoid ti h id tissue (BALT) i secreting I - secretory IgA, and is ti Ig t I A d from the serum are diffusing small amounts of IgM and IgG - the immune defense of the respiratory mucosa. The bronchi have a structure very similar to that of the trachea, with a mucosa, a medium tunic and an adventitia. In the initial portion of the bronchial tree, the mucosa is identical with that of the trachea, while gradually the number of the goblet cells is decreasing and in the intralobular bronchiole, with a diameter of maximum 1 mm, the epithelium change from respiratory type (pseudostratified ciliated columnar) to ciliated simple columnar epithelium, with no goblet cells. I th place of th goblet cells are i l l ith li ith bl t ll In the l f the bl t ll appearing the Clara cells, with some microvilli at their apical pole, they contain secretory granules and they secrete a material somewhat similar with the pulmonary surfactant. surfactant The lamina propria of the bronchi is first rich in mixed acinary glands, mucous and serous glands, secreting the fluid covering the surface of the epithelium; the glands are also decreasing in number, as the bronchi are becoming smaller and they dissapear at the level of the bronchioles. The lymphoid tissue becomes much reduced as th b the bronchi are b branching. d d hi hi

The medium tunic of the bronchi is made of hyaline cartilage, connective tissue and smooth muscle fibers; first the bronchi have a complete hyaline cartilage ring (cylinder) first, as they branch inside the lungs, the cartilage becomes fragmented into cartilage plates or islets. First the cartilage plates are larger, with small, few spaces between them, as the bronchi become smaller the cartilage plates are smaller (typical in the histologic preparations - the segmentary bronchus) The cartilage plates are completed by spirally bronchus). arranged smooth muscle; as the cartilage islets become smaller, the smooth muscle is forming a double helix, oriented in the two directions. In the intralobular bronchioles, the cartilage pieces are dissapearing and the medium tunic is represented by a continuous smooth muscle layer, with helical or circular orientation. The bronchial mucosa appear f ld d i th hi t l i preparations, b Th b hi l folded in the histologic ti because of th f the smooth muscle contraction after death; in bronchioles, where the medium tunic is made only of smooth muscle fibers, the mucosa appears with deep, characteristic folds. The smooth muscle ring of the bronchioles is submitted to large caliber variations variations. The adventitia of the bronchi is linking them with the surrounding structures - elastic fibers common with those in the alveoli walls, the tension exerted on the bronchioles wall, is keeping them opened.

Figure 1. Photomicrograph of a section from the wall of a terminal bronchiole. Note that no cartilage is present, but there is an incomplete ring of smooth muscle. PT stain. Low magnification.

From each intralobular bronchiole are arising 5-7 terminal bronchioles, lined by g 5y simple ciliated cuboidal epithelium, with Clara cells; the terminal bronchioles present as well, smooth muscle fibers in their walls. From each terminal bronchiole are arising 2 or more respiratory bronchioles, with a simple cuboidal epithelium, devoid of cilia, gradually flattened and continuous at the opening of the alveoli, with the simple squamous cells of the alveoli - type I alveolar cells. We find here too, some smooth muscle fibers in the wall. The respiratory bronchioles are continuous with the alveolar ducts, opening into the alveolar sacs, as well as isolated alveoli. The alveolar ducts have a discontinuous wall, with elastic fibers, and some smooth muscle fibers, at the opening of the alveolar sacs. They are lined by the same squamous epithelium, as th alveoli. ith li the l li The lobes of the lungs are made of pulmonary segments, each receiving air from a segmentary bronchus; the segments are divided, during fetal life, and poorly in the adult into pulmonary lobules, which are pyramidal territories, with the base to the periphery and the apex toward the pulmonary hilum. Each lobule is penetrated by an intralobular bronchiole; the terminal bronchiole is distributing air to the pulmonary acinus, generally considered the morphological and functional unit of the lung. Some authors are further considering the pulmonary unit, receiving air from a respiratory bronchiole, subdivision of th t f i t b hi l bdi i i f the terminal b i l bronchiole. hi l

Figure 2. Section of a terminal bronchiole with a small portion of a respiratory bronchiole continuous with an alveolar duct and many alveoli. PT stain. Low magnification

The Alveoli
The alveoli of the lung are appearing like small chambers, where the gases exchanges take place. They are responsible for the spongy structure of the lungs, g y gy g resembling to small pockets, open on one side, or like the honeycombs of a beehive, of about 200 µm in diameter. The walls are sepparating adjacent alveoli, for that they are called the alveolar septa. The alveolar septum is a very thin structure, permitting the gase changes; in its interior is lined by the alveolar cells, squamous alveolar cells, or type I cells and it contains - capillaries, fibroblasts, reticular and elastic, collagen fibers and macrophages. The air in the alveoli is seggregated from the blood, b at l t df th bl d by t least 3 components, which are k t t hi h known as th the bloodblood-air barrier - the cytoplasm of the alveolar cells, the fused basal laminae of the alveolar cells and of the blood capillaries, and the cytoplasm of the endothelial cells. ll Type I cells, squamous alveolar cells, also called membranous peumocytes, are very flattened cells, lining the alveoli - approx. 97 % of the alveolar surface; they d their t l h big f ll bl to spread th i cytoplasm on such a bi surface, th t 2/3 cells are capable t cover that the interior of one alveolus. The nucleus and the organelles are restricted to the midle part of the cells, the rest being free cytoplasm, by which the gases place. vesicles, exchanges take place In the cytoplasm - numerous pinocytotic vesicles involved in absorbtion of the surfactant.

Type II alveolar cells, or g yp granular p pnemocytes, are interspersed among type I y p g yp alveolar cells, they represent only approx. 3 % of the cells of the alveoli, they establish junctions with the membranous pneumocytes. They look like cuboidal cells, with a few microvilli, at their apical pole, found usually in the corners of the alveoli, often in groups of 2-3 cells. 2The granules seen in the light microscopy, are in the e.m., the lamellar bodies, of approx. 1-2 µm, with parallel lamellae, enveloped by membranes, like an 1alternance of dense and clear bands. They contain phospholipids, GAG and p proteins, being compared with the myelin sheath, as structure; when discharged, g p y g they form on the surface of the alveolar lining, the pulmonary surfactant, which is decreasing the alveolar surface tension. Alveolar lining is covered by a watery fluid; the surfactant is covering this fluid, to the surface, so is impairing the tendency of the alveoli to collapse, during expiration. Also, by reducing the surface tension, is decreased the respiratory effort. In premature births, the surfactant is not yet synthesized, leading to major breathing difficulty. The respiratory distress syndrome of the newborn, may be y , , treated by administration of g glucocorticoids, to the p g pregnant woman, or to the newborn.

Alveolar macrophages are present either in the alveolar septum, and inside the alveoli; they often contain dust and carbon particles, being also called dust cells. y g After a while, they are expelled with the secretion from the lung and they reach, by swallowing, into the digestive tract, being destroyed. In congestive heart failure, the blood is passing into the lung and the macrophages are phagocytosing red blood cells; because hemosiderin is accumulating inside the cytoplasm, these macrophages are called heart failure cells. y p , p g The gases exchanges are done by the differences between the partial pressures of the oxygen and carbon bioxide; the oxygen is diffusing through the blood-air bloodbarrier and is uptaken by the hemoglobin; the carbon bioxide is soluble in the plasma, the dissociation of the carbonic acid is catalyzed by the carbonic anhydrase present in the red blood cells and in the endothelial cells of the anhydrase, capillaries.

Figure 3 Th Fi 3. Threedimensional schematic diagram of pulmonary alveoli showing the structure of the interalveolar septum. Note the capillaries, connective tissue, and macrophages. These cells can also be seen in–or passing into– the alveolar lumen. Alveolar pores are numerous. Type II cells are identified by their abundant apical microvilli. The l Th alveoli are li d with a li lined ith continuous epithelial layer of type I cells.

Figure 4. Alveoli and interalveolar septum showing blood capillaries and epithelial cells type I and type II. PT stain. Medium magnification.

The lung has a double nutrient and functional circulation, as well as a rich network of lymphatic vessels, considered as a deep lymphatic network, carring the lymph to the l th lymph nodes of th pulmonary hil h d f the l hilum and a superficial network, of th visceral d fi i l t k f the i l pleura. Besides its important respiratory function, the lung is accomplishing some other functions, in the organism: 1. 2. 3. 4. 4 Catabolism of catecholamines - MAO (monoaminooxidase) here produced. Heparin is present, synthesized by the lung mast cells - reactions of immediate hypersenzitivity hypersenzitivity. Production of angiotensin II, from angiotensin I, by synthesis of the conversion enzyme. The l Th lung - i containing cells of th DNES secreting especially serotonin, is t i i ll f the DNES, ti i ll t i calcitonin, bombesin, enkephalins etc.

Thank you for your attention!

10. THE URINARY SYSTEM
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

THE URINARY SYSTEM
1. The Nephron 2. 2 The juxtaglomerular

apparatus 3. 3 The urinary passages

The urinary system - pair organs, the kidneys and the urinary bladder; by producing the urine, the excretory system i eliminating various waste products, d i h i h is li i i i d thus maintaining the homeostasis of the body. The kidneys are bean-shaped organs, with a hilum to their concave margin, of beanabout 150 g, each. From the hilum are exiting the ureters and veins and are entering the arteries; the dilated upper portion of the ureter is called the renal pelvis, divided in 2-3 major calyces, each with several minor calyces. When 2sectioned, the kidney has two zones, visible with the naked eye - a cortex, looking uniform granular, and a medulla, entirelly made of sectioned tubules. The cortex is situated in the periphery, beneath the connective tissue capsule, enveloping the organ. In human, the medulla is represented by the medullary p g g , p y y pyramids, 10-18 pyramidal structures, to the inner part of the kidney. From the 10base of each medullary pyramid, are arising into the cortex, the so-called somedullary rays of Ferrein, surrounded by cortical zones. Between the medullary pyramids are also found zones of cortical tissue, as columns of cortex, between medulla, called columns of Bertin. Each medullary pyramid, together with the surrounding cortex is forming a renal lobe, and each medullary ray, with the cortical tissue around it, is considered a renal lobule.

Figure 1. Bird’s-eye view of the renal cortex, which is composed mainly of proximal (P) and distal (D) convoluted tubules, and renal glomeruli (G). Pararosaniline—toluidine blue (PT) stain. Low magnification.

The Nephron
The nephron is the morphological and functional unit of the kidney, each kidney has 1, 1 up t 4 million nephrons. to illi h The nephron has a dilated portion, called the renal corpuscle, always situated in the cortex of the kidney; the tubules of the nephron are situated in both areas, cortex and medulla, while the collecting tubules are situated exclusively in the medullary zone. The renal corpuscle is continuous with the proximal convoluted tubule (PCT), followed by the loop of Henle and by the distal convoluted tubule(DCT); finally the y p y ( ) y uriniferous tubule is opening into the collecting tubules. The nephrons have the renal corpuscles situated on 3 levels in the cortex of the levels, kidney - superficial, medium and deep, next to the medullary zone; the loop of Henle of the deep situated corpuscles is descending on a long distance in the medullary pyramids; these are most important nephrons, in the process of formation and concentration of the urine.

In the structure of the renal corpuscle, one may differentiate 3 components: the p y p vascular component is made of a tuff of capillaries, resulted from the afferent arteriole, with 3-4 primary branches, further forming capillaries, they give rise to the 3efferent arteriole, which is leaving the corpuscle. The epithelial component is represented by the double layer of the Bowman's capsule, with its visceral and its parietal layer. The third component is represented by the mesangial cells, situated between the capillaries branches. Each renal corpuscle has a vascular pole, by which the afferent arteriole enters and the efferent arteriole leaves and a urinary pole, where the PCT yp begins. At its exterior, the renal corpuscle has the parietal layer of the Bowman's capsule, lined by a simple squamous epithelium, which is continuous to the urinary pole, with the PCT. In the interior of the renal corpuscle - nuclei, of different cell types, which cannot be differentiated with the light microscope. During embryonic development, the blood vessels are invaginating in the spherical ending of the future nephron, so that the inner visceral layer of the Bowman' capsule is continuous with the parietal, outer layer. The basement membrane of the visceral layer of the Bowman's capsule is fusing with the basement membrane of the y p g capillaries of the corpuscle, forming together a single common, thicker membrane.

The cells of the visceral layer of the Bowman's capsule are g y p greatly modified, the y soso-called podocytes. Podocytes show numerous processes, called primary processes, giving rise to secondary and tertiary, thiner processes, called pedicels, embracing the capillaries, over the common basal lamina, leaving small free spaces, which are called filtration slits, with small diaphragms, of 6 nm thick, comparable with the diaphragms of the fenestrated capillaries. The podocytes are covered with a glycocalyx (GAG layer) giving them an electronegative charge. The capillaries have numerous fenestrae, in their endothelial cells, of about 70-90 70nm in diameter, with no diaphragms. The thicker basement membrane, has a p g central lamina densa, a more electron-dense midle layer and on each side, two electronlaminae lucidae or rarae. On the lamina lucida interna is found the endothelium of the capillaries, while on the lamina lucida externa are found the podocytes. The filtration space is represented by the thick, common basal lamina and by the filtration slits of the podocytes, between the pedicels. Lamina densa of the basal lamina is containing collagen type IV and collagen type V, acting as a filter in the filtration process, while the 2 lamina lucidae, or rarae, contain large amounts of heparan sulfate, giving to the filtration membrane an electric negative charge, important in the filtration process. Large molecules being negative charged, are rejected b th same negative charge of th filt ti membrane. j t d by the ti h f the filtration b

The third cellular component of the renal corpuscle is represented by the mesangial cells; they are hard to recognize with the light microscope they resemble to microscope, macrophages. Being situated in the corners between the capillaries branches, they have a role of support for the elements of the renal corpuscle and they are responsible for the continuous phagocytosis of the common basal lamina or the lamina, filtration membrane, situated between endothelial cells and podocytes. The filtration membrane is continuously produced by the podocytes and the endothelial cells, but being damaged during filtration, must be continuously replaced. It seems that the mesangial cells are functioning as well, as antigen presenting cells. The filtration process is filtrating all the elements of the plasma except the blood cells plasma, and the molecules of approx. 68 kDa, the molecular weight of albumin, or more. By the filtration process are produced approx. 150-180 liters of primary urine, in 24 150hours, hours from which most is reabsorbed in the tubules of the nephron and only 1 5 liters 1.5 of urine are excreted, daily. The filtration is possible because of the big difference between the hydrostatic pressure and the oncotic pressure; important hydrostatic pressure is resulting in filtration Large molecules like albumin immunoglobulins filtration. molecules, albumin, immunoglobulins, fibrinogen are not passing through the filtration membrane. The molecular configuration is also important in the crossing of the filtration membrane; the straight p g y ; g g molecules are passing more easy than the g globular molecules; the negative charge of the membrane is important in the filtration process, the so-called electrical pores of sothe filtration membrane.

The PCT is beginning at the urinary pole of the renal corpuscle; being longer than the DCT most sections surrounding the renal corpuscles in the cortical zone of DCT, corpuscles, the kidneys, are sections through proximal convoluted tubules. The proximal convoluted tubules are lined by a simple low columnar epithelium, the diameter of the proximal tubules is of about 60-70 micrometers. The cells are polarized at the 60- micrometers polarized, apical pole they have microvilli, in light microscopy -they have a brush border. At the basal pole, the cells - numerous folds, with abundant mitochondria, giving them a more intense acidophilia, than the cells of the distal convoluted tubules. The cells have tight junctions, important for the reabsorbtion process. Because the cells have also lateral folds, we do not see the intercellular limits of the epithelial lining of the proximal tubule. p Most part of the reabsorbtion process is taking place in the PCT; here are absorbed the glucose sodium is absorbed together with the glucose In the glucose, absorbed, glucose. proximal convoluted tubules, are also absorbed water, the phosphate, calcium, peptides and amino acids. More than 90 % of the absorbtion process is done in the PCT, PCT peptides and the albumin which yet escaped through the filtration albumin, membrane, are absorbed by an active absorbtion process, by pinocytosis. The absorbed components are passing in the interstitium, through the basal pole of the p p y p epithelial cells and are uptaken by the blood capillaries.

Figure 2. Renal cortex section showing a proximal convoluted tubule (PCT) with its large cuboidal cells presenting a brush border formed by numerous microvilli. Distal convoluted tubules (DCT) are also present. PT stain. Medium magnification.

The loop of Henle is situated between the proximal and the distal convoluted tubule and is U-shaped, with a thin portion and a thick portion, which is similar to Uthe DCT. There are 2 types of Henle's loops - short and long; the short loops are of the cortical superficial situated nephrons, while the long loops are of the so-called soj t d ll h juxtamedullary nephrons. Juxtamedullary nephrons have a very long loop, which is descending deep in the medullary, these being most important in the process of concentration of the urine, in the formation of the hypertonic urine. In the short loops, the contumation is at the level of the thick segment of the loop, while in the long loops, the contumation is at the level of the thin segment of the loop. Thus, the long loops have a thick descending limb, a long thin descending and , g p g , g g ascending limb and a thick ascending limb. Cortical or superficial nephrons have a short thin descending limb and the thick segment, with the contumation, so they g show no thin ascending limb.

The thin segment of the loop has the structure of a blood capillary, with a simple squamous epithelium, but with no red blood cells in the lumen. In the e.m., the cells lining the thin segment have few irregular microvilli microvilli. The thick segment of the Henle's loop has a structure identical with that of the DCT. In the thin DCT I th thi segment of th l t f the loop of H l water and sodium are absorbed f Henle, t d di b b d pasively, according to the difference of gradient, between the lumen and the interstitium. In contrast, in the thick segment of the loop, sodium is transported actively, with i i pumps; thi segment i concentrating th urine against th ti l ith ionic this t is t ti the i i t the gradient, especially under the influence of the aldosterone. The distal convoluted tubules have a smaller total diameter than the proximal ones, of about 40 micrometers, the contour of the lumen is more clear, because they have fewer microvilli at the apical pole of the epithelial cells. Distal convoluted tubules are shorter than the proximal tubules, for that we observe in the histologic preparations fewer sections through distal, than through proximal convoluted tubules.

They also have mitochondria, but not so numerous like the cells lining the proximal tubules and they appear less acidophilic than the proximal, in light microscopy; because the epithelium is simple cuboidal, not so tall, like that of the proximal tubule, we observe a larger diameter of the lumen, in this tubule and because the lateral folds of the cells are less numerous, we may distinguish the intercellular , y g limits of the cuboidal cells. The main role of the DCT is to absorb actively sodium and water, by ionic pumps, under the influence of the mineralocorticoids, secreted in the glomerulosa zone of the adrenal cortex, especially aldosterone. In this mechanism an important role has the renin, secreted by the juxtaglomerular apparatus. The distal convoluted tubules are followed by the collecting tubules lined by tubules, simple cuboidal epithelium and as they become larger, they show simple columnar epithelium, reaching the tips of the medullary pyramids. They occupy most of the medullary zone of the kidneys, visible in light microscopy, both in longitudinal and y y , g py, g in cross sections. They appear pale stained, with clear limits between the lining cells; in the e.m. they have pale looking cells and darker cells, richer in organelles, considered to be involved in the secretion processes, by which the urine is formed. The collecting tubules are absorbing water, actively, under the influence of ADH, antidiuretic , p y g , hormone, of the posterior p pituitary. If the water ingestion is limited, the ADH will act on the collecting tubules, stimulating water absorbtion, from the urine.

2. The juxtaglomerular apparatus
The juxtaglomerular apparatus - three types of cells: the juxtaglomerular cells, modified smooth muscle cells, in the wall of the afferent arteriole, with secretory properties, the cells of th macula d ll f the l densa, situated i th wall of th DCT next t th afferent arteriole, it t d in the ll f the DCT, t to the ff t t i l and the lacis cells, also known as external mesangial cells, or polkissen cells (pole cushions). The juxtaglomerular apparatus is situated adjacent to the renal corpuscle, toward the vascular pole of the corpuscle. The juxtaglomerular cells become modified, rounded, with centrally located ellipsoid nuclei, in the cytoplasm are present secretory granules, stained with PAS technique. The granules are of approx. 10-40 nm; there are 2 10categories of granules - numerous, dense, and fewer, smaller and less dense. At the level of the j l l f h juxtaglomerular cells, the afferent arteriole h no i l l ll h ff i l has internal elastic l l i membrane, thus the cells are in direct contact with the endothelium. The macula densa is a small portion of the DCT, where the cells of the tubule are columnar, taller, the nuclei seem more numerous, better stained; because of that, the macula densa received this name (a coloured spot). The basement membrane is missing in the zone of the macula densa, the cells have a changed polarization, oriented to the juxtaglomerular cells, with the Golgi complex to the juxtaglomerular cells.

Figure 1. Photomicrograph of renal medulla with 2 collecting ducts consisting of cuboidal cells resting on a g g p g g g basement membrane. In this hypertonic region of the kidney, because of the action of the hypophyseal antidiuretic hormone, water is reabsorbed, controlling the water balance of the body. PT stain. Medium magnification.

Figure 2. R Fi 2 Renal cortex showing a di t l convoluted t b l with a macula d l t h i distal l t d tubule ith l densa f formed b closely packed d by l l k d epithelial cells (broken line). This structure is sensitive to the ionic concentration of the filtrate in the distal tubule and is believed to influence glomerular filtration. PT stain. Medium magnification.

The lacis cells are situated in the angle between the afferent and the efferent arterioles, at the vascular pole of the renal corpuscle. These cells have processes between the juxtagfomerular cells, they are continuous with the mesangial cells, inside the renal corpuscle ( p (internal mesangial cells), being also called external g ), g mesangial cells. The juxtaglomerular apparatus is an endocrine component of the kidney The kidney. juxtaglomerular cells secrete hormonal substances -renin, erythropoietin, and others, as prostaglandins, etc. Erythropoietin is stimulating erythropoiesis. Renin is acting on the plasma protein called angiotensinogen forming an inactive protein, angiotensinogen, decapeptide, called angiotensin I. On this is acting the converting enzyme, produced especially, by endothelial lung cells, and cutting 2 amino acids, is forming the active octapeptide, angiotensin II. The angiotensin II is constricting the arteriole, increasing the blood pressure and is also acting on the adrenal cortex, stimulating the secretion of aldosterone, which by increased absorbtion of sodium and water, especially at the level of the distal tubule, will also increase the blood , p y , pressure, restoring the filtration pressure. When the high blood pressure is acting on the wall of the afferent arteriole, the juxtaglomerular cells do not secrete renin, they are stimulated by the decrease of the blood pressure, and they release the renin.

The cells of the macula densa are stimulated by the changes in the concentration y g of the chloride and sodium ions, they stimulate in such cases the production of renin, by juxtaglomerular cells, acting like osmotic receptors; the aldosterone secretion being stimulated, will promote sodium, water absorbtion, with the same purpose, to establish the effective filtration pressure. The lacis or external mesangial cells are regenerating the internal mesangial cells and are in contact with sympathetic nerve endings, stimulating via nervous system, the renin secretion. The vascularization of the kidney is from the renal artery, which is entering through the hilum and gives branches, between the medullary py g y pyramids, the interlobar arteries; at the level of junction between cortex and medulla, these form the arcuate arteries, at the base of the pyramids. From the arcuate arteries are arising interlobular arteries, between the medullary rays, parting the renal lobules; from interlobular arteries are deriving the afferent arterioles of the renal corpuscles. From the efferent arterioles is resulting the peritubular capillary network, nourishing the proximal and distal tubules and uptaking the absorbed materials, from the filtered primary urine. The efferent arterioles associated with the juxtamedullary nephrons are forming straight capillaries, called vasa recta, descending deep in the medulla and looping back to the cortex; these provide vascularization for the medulla. V i are f ll i th same course, i th opposite di ti following the in the to form d ll Veins it direction, t f finally, the renal veins, exiting from the kidneys.

3. The urinary passages
The urinary passages are made of a dilated portion, the renal pelvis, with the calices, calices the ureter and the urinary bladder an organ of storage of the urine bladder, urine, continued with the urethra. All these have a common structure, made of a mucosa, a muscular layer and an adventitia, adventitia with the exception of the bladder covered in its upper part by the bladder, peritoneum. The mucosa is made of transitional epithelium, or urothelium, and an usual lamina propria, propria devoid of glands The transitional epithelium has 5-6 layers of cells giving a glands. 5cells, false impression of stratification, because the cells have contact with the basal lamina; the cells look rounded, the superficial layer is made of rounded umbrella-like umbrellacells. cells When the epithelium is stretched by dilation most obvious in the urinary stretched, dilation, bladder, filled with urine, the epithelium become made of 3-4, even 2 layers of cells. 3The superficial layer of cells are frequently polyploid or binucleate and they present an adaptive structure made of polygonal plates of thicker membrane; these are forming an osmotic barrier for the hypertonic urine and they also provide the folding of the normal portions of membrane, between the apical pole plates, like a membrane reservoir.

When the epithelium is distended, the folds will redress, permitting the enlargement of the epithelium surface, in the full urinary bladder. These aspects are most obvious in the bladder, the distension of the ureters and pelvis being minimum. L i propria i made of l i i Lamina i is d f loose t d to dense connective ti ti tissue, with f ith few migrated elements; it is thicker in the urinary bladder. The muscular layer is made in the pelvis of a single layer of helically arranged smooth muscle fibers; in the ureter we find two layers of smooth muscle fibers, in the upper part of the ureter, with the reverse arrangement than in the intestine outer circular and inner longitudinal smooth muscle layer. In the lower part of the ureter is appearing a third longitudinal disposed, outer, smooth muscle layer; here we find an outer longitudinal, a midle circular, and an inner longitudinal smooth muscle l l layer. The bladder has a thicker muscular tunic, with a plexus-like arrangement of the plexusmuscle fibers, oriented in every direction, with apreciable connective tissue between the smooth muscle fibers. In the adventitia and in the muscular tunic we find numerous vascular and nervous elements, as well as vegetative microganglia.

Figure 3. Compare the structure of the transitional epithelium when the urinary bladder is empty (A) or full (B). When the bladder is full, the capacity of epithelial cells to slide upon one another reduces the thickness of the epithelium. As a result, the interior surface of the bladder increases. In B, note the thin strands of collagen fibers separating bundles of smooth muscle cells PSH stain Medium cells. stain. magnification.

Urethra is short in female sex, of approx. 3-4 cm, lined by stratified or 3pseudostratified columnar epithelium and to its end, by stratified squamous epithelium. Male urethra is longer, made of a prostatic, membranous and pendulous urethra. d l th The prostatic urethra is lined by transitional epithelium, while the other segments are lined by pseudostratified or stratified columnar epithelium, becoming to the end part of the urethra squamous stratified epithelium. Both male and female urethrae have two sphincters - an inner involuntary one, and an outer sphincter under the voluntary control of the individual.

Thank you for your attention!

11. THE FEMALE REPRODUCTIVE SYSTEM (1)
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

THE FEMALE REPRODUCTIVE SYSTEM
1. The Ovaries 2. Ovulation 3. The uterine tubes (oviducts)

The female reproductive system is made of the ovaries, uterine tubes, uterus, vagina, and the mammary glands. The system is functional during the fertile life of the woman, which is situated between menarche, the time of the first menses, and menopause, when the menses dissapear. All this time period, of approx. 30-40 years, the female reproductive system p pp 30- y p y undergoes cyclic changes, controlled by neurohormonal mechanisms; the mammary glands, are as well submitted to similar control, and by their function, are associated to the female reproductive system.

The Ovaries
The ovaries are almond-shaped structures, ovoidal, of about 3 cm long, 1-2 cm almond1wide and 1 cm thick; they accomplish both exocrine function, by formation of the ova, ova and endocrine function consisting in the female sexual hormones secretion function, secretion. The histologic structure of the ovary consists of a compact zone in the periphery, the cortical region and a looser, central zone, the medullary region. In the cortex are found numerous ovarian follicles while in the medulla are present blood follicles, medulla, vessels in loose connective tissue. During fetal life, in the ovary are migrating, from the endodermal yolk sac, primordial germinal cells populating the genital ridges - oogonia Oogonia start to cells, oogonia. divide and at the finish of the fifth month of fetal life they enter into the prophase of the first meiosis, becoming primary oocytes (2n chromosomes). At birth, the ovaries do not contain anymore oogonia but approx 7 million primary oocytes oogonia, approx. oocytes. The stromal cells of the cortex, will surround the oocyte and will form a primordial ovarian follicle. The primordial follicles are most numerous in the ovaries but most exposed to a ovaries, degenerative phenomenon, called atresia. From birth to puberty, many of them are degenerating, so in the adult ovaries, we find approx. 400, 000, from these, in appox. 30-40 years of fertile life, only about 400 will reach the stage of mature 30follicles and will release ova.

The ovary is covered by a simple cuboidal epithelium, improper called, the germinal epithelium, since i part of the peritoneum; under the epithelium i f i l i h li i is f h i d h i h li is found d a dense connective tissue tunic - tunica albuginea, giving an whitish appearance to the ovary. The stroma of the cortical region is peculiar rich in spindle-shaped spindlefib bl t with h l t tl following helical t fibroblasts, ith hormonal receptors, f frequently f ll i a h li l arrangement. Till the puberty, all the follicles are in the primordial stage; beginning with the puberty they respond to the pituitary FSH stimulus, and they begin to mature, passing through the stages of primary and secondary follicles to the ultimate mature follicles, ultimate, stage. Primordial follicles are the smalest and most numerous of all, they are situated mainly in the periphery under the tunica albuginea The oocyte is of about 25 µm periphery, albuginea. µm, surrounded by one layer of flattened follicular cells; the nucleus is eccentrically placed, with dispersed chromatine and obvious nucleolus. The primordial follicles are surrounded by a membrane called membrane of Slavjanski sepparating them membrane, Slavjanski, from the stroma. The primary follicles are the first stage of maturation, under FSH influence; the primordial follicles selected by the pituitary hormone to undergo the cyclic maturation - privileged follicles. Several such follicles are selected, but only one will reach, each month, to the mature stage. The privileged follicles - to inhibit, way, follicles. paracrine way the development of the other follicles

The primary follicles are characterized by the growing of the follicular cells, p y y g g becoming cuboidal or low columnar, called from this stage granulosa cells; this is a unilaminar primary follicle. As the granulosa cells are p g proliferating, by mitosis, they form a stratified g g, y , y granulosa layer, disposed on several layers around the oocyte. The oocyte gradually become eccentrically placed inside the granulosa layer, is increasing in dimension, to about 40 µm. The granulosa cells and the oocyte are secreting an acellular glycoproteic material, called zona pellucida. This is looking like a stained membrane, surrounding the oocyte, eosinophilic, in H&E method and PAS positive. Oocyte has microvilli and the g p y granulosa cells show processes, both are penetrating inside the zona pellucida. In the stage of the primary follicles are appearing the follicular thecae - outside the follicle will appear the theca interna, outside it, the theca externa. Between the two, pp , , , is no clear limit. Theca interna is abundant in cells, of endocrine nature, which will secrete androgens. The theca externa is rich in connective tissue fibers and blood vessels and shows no secretory properties. From the stage of primary follicle, the ovarian follicles are beginning to secrete sexual steroid hormones, part of the endocrine y function of the ovary.

Figure 1. Cortical region of an ovary 1 ovary. Besides primordial follicles formed by an oocyte and flat follicular cells, a few follicles at the initial stage of growth (unilaminar primary f lli l ) are present. ( il i i follicles) t These are formed by an oocyte and one layer of cuboidal granulosa cells. Pararosaniline—toluidine blue (PT) stain. Low magnification.

The hormonal secretion is achieved on the so-called, principle of the two cells. The soendocrine cells of the techa interna have receptors for the pituitary LH which will LH, stimulate here, the production of androgens, while the cells of the granulosa layer, show enzyme activity, aromatase, capable to convert, the androgens into estrogens. estrogens Cells of the granulosa are linking pituitary FSH under this influence at FSH, this level, will be secreted estrogens, most important female sexual hormones. The Th secondary f lli l are representing a l t stage of th growing ovarian d follicles ti later t f the i i follicles; the granulosa cells are proliferating further on, between the cells are appearing first small cavities filled with so-called follicular fluid, secreted by the sogranulosa cells containing GAG proteins (also steroid-binding proteins) and cells, GAG, steroidsexual hormones -androgens, estrogens and small amounts of progesterone. As the liquid is gradually accumulating, will form a single larger cavity, called antrum. A crowding of granulosa cells will contain the large oocyte of approx 100 µm or oocyte, approx. µm, more, this zone is called cumulus oophorus, prominent in the antrum. These last characteristics are true for the late stage of the secondary follicles.

The mature or graafian follicles are the largest, they reach about 1-2 cm, before 1ovulation are visible on the surface of the ovary, like a prominent vesicle, the zone of stigma. The accumulation of liquid is so important, that the granulosa cells are situated in periphery, on 2 or 3 layers. Cumulus oophorus contains a large oocyte and the cells next to it become columnar perpendicularly on the thick zona pellucida - the columnar, corona radiata. Corona radiata is kept after ovulation, even during fertilization of the ovum is still present The first meiotic division is taking place before ovulation but all the present. ovulation, cytoplasm is kept by one of the daughter cells, the other cell is made almost only of nuclear material, being expelled, as the first polar body. About at the moment of the ovulation, the nucleus of the ovum begins the second meiotic division, which will stop in the metaphase and which will be completed only during fertilization of the ovum, if this occurs. The ovum is viable and may be fertilized by a spermatozoon for about 24 hours if spermatozoon, hours, not, suffers autolysis. During fertilization, if occurs, will be completed the second meiosis, with expelling of second polar body. Fertilization will restore the diploid cell, with the characteristic number of chromosomes of the specie.

Figure 2. Photomicrograph of an antral follicle showing the oocyte surrounded by the g g g p g y y granulosa cells of the corona radiata and supported by the cells of the cumulus oophorus. The remaining granulosa cells form the wall of the follicle and surround the large antrum. A theca surrounds the whole follicle. PT stain. Medium magnification.

Ovulation
Ovulation is consisting in the rupture of the mature follicle and liberation of the not yet mature ovum. It was speculated that the increase of the pressure of the follicular fluid and the contraction of some smooth muscle cells, in the ovarian stroma, should be important in this process. In fact this is not fully demonstrated. A role may play either, the proteases activity, in the follicular fluid, capable to lyse, the follicular wall. Indispensable for ovulation is, in any case, a sudden discharge of LH, from the pituitary, under the positive feed-back exerted by a high level of feedplasma estrogens. Besides estrogens, several hypothalamic neurohormones are released and seem to trigger LH ovulatory cascade, from the pituitary, such as, Y neuropeptide, prostaglandines, substance P, etc. After ovulation, the walls of the mature follicle collapse, become folded and they will form under the influence of LH, a temporary endocrine structure, called corpus luteum, or progestative body. The corpus luteum will secrete progesterone and estrogens; progesterone will prepare the endometrium for nidation. Corpus luteum is made most of it of granulosa cells, which increase in size, at approx. 25-30 µm, 25they will look clear, vacuolated, containing lipids, since they secrete steroid hormones. hormones.

Cells of the ex-theca interna are fewer, but also present in the p g ex, p progestative body, y, similar to the granulosa cells, but smaller and darker stained. The two categories are called granulosa lutein cells and theca lutein cells. In the central part of the corpus luteum is visible a small blood clot, gradually replaced by connective tissue, g y y remaining of the rupture of the mature follicle, during ovulation. If the fertilization of the ovum occurs the corpus luteum will further develop occurs, develop, secreting larger amounts of progesterone and estrogens, stimulating the development of the pregnancy; it may reach 4-5 cm and it will last till the sixth 4month of pregnancy, after that will decline. This is the corpus luteum, or y p g y, p , yellow body of pregnancy. Relaxin is a hormone produced by the corpus luteum of pregnancy, with the property to relax the symphysis pubica and to infiltrate the connective tissue, facilitating p g parturition. If the fertilization did not occur, the corpus luteum is lasting only approx. 10-14 10days, days the second period of the menstrual cycle The progesterone secretion is cycle. inhibiting LH, the lack of LH is leading to the involution of the corpus luteum, which will degenerate and will form a scar of dense connective tissue - collagen fibers, with whitish aspect, called corpus albicans. Corpus albicans is gradually absorbed, by macrophages, in weeks or months.

Estrogens are stimulating LH and are inhibiting FSH secretion, by the p g g g , y pituitary. Inhibin y is inhibiting also the FSH secretion. Progesterone is inhibiting LH secretion, while the progesterone secretion is stimulated by LH. The follicular atresia may occur in any stage of the ovarian follicles, even in the mature stage. The degeneration of the follicles takes place from birth, in any life period, period but is peculiar important in some periods of the woman's life when hormonal woman s life, changes are numerous - at puberty, during pregnancy and during menopause. Main events of atretic phenomenon are the cessing of proliferation of granulosa cells cells, detaching of the granulosa cells one from another and from the oocyte and the death of oocyte, which will be phagocytosed. The zone pellucida is lasting more time, being acellular, acellular looking waved and empty since the oocyte is dissapearing empty, dissapearing. Small parts of the theca interna of the atretic follicles and some granulosa cells, are persisting i th ovary and secrete continuously small amounts of steroid h i ti in the d t ti l ll t f t id hormones. These are the endocrine interstitial cells, forming the interstitial gland of the ovary, present from childhood, and after menopause and ensuring a basal sexual steroid secretion. secretion

Figure 2. Photomicrograph of a small portion of a corpus luteum. Most cells present in the figure are granulosa lutein cells. PT stain. High magnification.

The uterine tubes (oviducts)
The uterine tubes are tubular organs, of approx. 12 cm long, with a dilated portion opened to the surface of the ovaries, and a zone inside the uterine wall, opened in p , , p the uterine cavity. The wall of the oviduct is made of 3 concentrically disposed tunics - a mucosa, a muscular layer and the peritoneal serosa. The mucosa is made of a simple columnar epithelium, with 2 types of cells: cells with motile cilia, at the apical pole and secretory cells, without cilia, but with secretory granules accumulated in the apical pole. The two types of cells are in fact, different functional stages of the same cell; in the first period of the ovarian cycle and menstrual cycle, when estrogens are secreted predominantly, ciliated cells are the most numerous, while in the second half of the ovarian cycle, when , y , progesterone is secreted in the ovary, secretory cells, with no cilia, are clearly predominant. The beat of the cilia is directed to the uterine cavity, helping the fertilized ovum to reach the uterine cavity, allthough it is suspected that, of most importance, is the contraction of the muscular wall of the uterine tube. The mucosa of the oviduct has p j , fringesg , y , numerous projections, fringes-like, at the free extremity of the uterine tube, these are long, and are called fimbriae.

Figure 3 Photomicrograph of part of the 3. wall of an oviduct. The highly folded mucosa indicates that this region is close to the ovary. PT stain. Low magnification.

The numerous folds inside the lumen, are giving in cross section, the image of a labyrinth. The secretory cells are secreting a nourishing fluid, capable to activate the spermatozoa the so-called capacitation and to nourish the egg because the spermatozoa, socapacitation, egg, fertilization usually takes place in the lateral, external third of the oviduct. The lamina propria of the oviduct is made of loose connective tissue, with migrated cells and an abundant network of capillaries; the rich vascularization of the lamina propria is responsible for the edema, developed at the mid part of the cycle, when ovulation should occur. The fimbriae of the oviduct become enlarged, and they approach more the ovaries like this being capable to capture the expelled ovum ovaries, ovum. Cells of the epithelium and lamina propria of the mucosa have receptors for estrogens and progesterone, and they react to hormonal changes, during the ovarian cycle. cycle The muscular tunic is made of a circular, inner layer, and a more longitudinal arranged, outer layer, of smooth muscle fibers. In case of abnormal nidation, in the tube, tube it appears an ectopic pregnancy which in evolution by rupture of the oviduct pregnancy, evolution, oviduct, is resulting in severe hemorrhage, which may be fatal, if not surgically treated in emergency.

Thank you for your attention!

12. THE FEMALE REPRODUCTIVE SYSTEM (2)
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

THE FEMALE REPRODUCTIVE SYSTEM

• Th uterus The t • The mammary glands • The implantation and the placenta

The uterus
Uterus is a pear-shaped organ, continued with a cylindrical structure, the cervix, pearwhich is opening in the vagina. The uterus - a body, with a fundus in the upper part, part above the opening of the uterine tubes and a narrow lower region the tubes, narrow, region, internal os. The wall of the uterus is made of 3 tunics - mucosa or endometrium, muscular tunic, tunic or myometrium and the outer layer - the peritoneum or an adventitia, adventitia according to the zone of the uterus. The endometrium - a simple columnar epithelium, with simple tubular glands, resulted f lt d from th invagination of th surface epithelium. Th epithelium h th the i i ti f the f ith li The ith li has the same 2 types of cells, like in the uterine tubes - ciliated cells and secretory cells. The endometrium is strongly hormone-dependent mucosa, his aspect being hormonerelated to the hormonal secretion of the ovaries ovaries. The number of ciliated cells of the epithelium is more numerous in the first half of the cycle, but in the case of the uterus, in contrast with the oviduct, the secretory cells are usually more abundant abundant. The lamina propria is rich in fibroblasts, the so-called cytogenic lamina propria, rich soin proper cells of the connective tissue; the cells of the lamina propria show receptors for estrogens and progesterone, suffering cyclic changes, according t t f t d t ff i li h di to the phase of the ovarian cycle.

Conventionally, the first day of the menstrual cycle is considered the day when the menstrual bleeding is appearing. The thickness of the endometrium is divided in two zones - the deepest 1/4 or 1/3 of the endometrium is called layer basalis, representing a d ti deep portion of th l i propria, containing th b ti f the lamina i t i i the bases of th f the simple tubular endometrial glands; this is the portion of the mucosa which is not expelled with the menstruation and, from which, each month, the endometrium will be regenerated The 3/4 or 2/3 upper part of the thickness of the endometrium is regenerated. 3/4, the layer functionalis, which is growing after each menstruation, from the resting layer basalis. The Th menstrual cycle i superposed with th ovarian cycle; after 3-5 d t l l is d ith the i l ft 3- days of f menstrual bleeding, under the influence of the estrogens secreted by the ovary, from the discovered lamina propria, containing the base of the glands, the endometrium is growing the glands and the superficial epithelium as well as the growing, epithelium, lamina propria, are restored. This is the proliferative phase, or the follicular phase, or estrogenic phase, since is superposed with the development of the ovarian follicles and with the secretion of estrogens At the finish of this phase the estrogens. phase, endometrium is 2-3 mm thick, with almost straight simple tubular glands, and a 2lamina propria rich in cells. The duration of this phase is up to approx. the 14th day, day of a normal cycle lasting in average 28 days cycle, days.

The next phase is called the secretory p p y phase, or luteal, or p g progesteronic. A menstrual cycle is considered from the first day of a menstruation, to the first day of the next menstrual bleeding, coinciding with the 28th day of the previous menstrual cycle. In the 14th day, ovulation is usually taking place, and corpus luteum is beginning to secrete progesterone; the progesterone will lead to the accumulation in the epithelium of the glands and in the superficial epithelium, of the endometrium, of glycogen, lipids and proteins, with the purpose to nourish the egg, till the development of the placenta, in case nidation occurs. The simple columnar epithelium will have nuclei moved to the apical p p p p pole of the cells, after accumulating especially glycogen, the nucleus will regain its basal position. The cells in the lamina propria will also accumulate nutrient substances, till they become spherical and crowded next to the superficial epithelium of the endometrium, in an epithelium-like manner, forming the compact decidual layer of epitheliumthe endometrium, the decidua - part of the maternal placenta, in case of pregnancy. The glands of the endometrium become highly coiled, tortuous, like a corkscrew, or the teeth of a saw, due to the edema and secretion accumulated. The endometrium is reaching its maximum thickness - 5 mm, or more.

Figure 1. Photomicrograph of the superficial layer of the endometrium during the proliferative phase. The surface epithelium and the uterine glands are embedded in a g lamina propria made of very loose connective tissue. PT stain. Medium magnification.

If the fertilization of the ovum did not occur, the menstrual phase will follow, p determined by the drop of the sexual hormones in the plasma, and the sudden decrease at minimum levels of the pituitary LH and FSH. The layer basalis is irrigated by the straight arterioles, from the myometrium, while the layer functionalis is vascularized by the coiled arteries, with a helical passage. The consequence of the lack of progesterone, is the further coiling and spasm of the coiled arteries, followed by ischemia and necrosis of the functional layer of the endometrium, which will be expelled, with a moderate amount of blood, the f menstrual bleeding, and the cycle will restart. The myometrium is a thick muscular tunic, with 3 layers - an outer and an inner more longitudinal disposed layers and a midle, circular and plexus-like disposition plexusof the smooth muscle fibers, being thicker and also a vascular layer - numerous blood vessels. The arteries form in the midle vascular layer, the arcuate arteries, which will give b hi h ill i branches f th endometrium - straight arteries, f the basal l h for the d ti t i ht t i for th b l layer and the coiled arteries, for the functional layer of the endometrium. The blood vessels of the myometrium do not have adventitia, they are surrounded directly by smooth muscle fibers of the uterus forming by contraction a haemostatic device uterus, contraction, device, by obliterating the lumen of the vessels, after parturition, an important adaptive mechanism. During pregnancy, the myometrium undergoes both hyperplasia and fibers, dimensions, months, hypertrophy of its muscle fibers with a regain of dimensions in a few months after birth.

Figure 2. Photomicrograph of uterine glands. During the luteal phase, the uterine glands become t t b tortuous and their lumen i fill d with d th i l is filled ith secretions. Some edema is present in the connective tissue. H&E stain. Medium magnification. Inset: High magnification.

The uterine cervix is the cylindrical structure prominent in the vagina - the inner structure, channel is called the endocervix and the portion in the vagina, the exocervix. The epithelium lining the endocervix is mucus-secreting, simple columnar mucusepithelium, with tubular-branched mucus secreting cervical glands, which does not tubularundergo cyclic changes, like that of the endometrium; however, the quality of the t d t in different phases of th ovarian cycle. t h f the i l secreted mucus i diff is different, i diff Close to the 14th day, when ovulation occurs, the mucus is fluid, and may be easy penetrated by the sperm cells. During the luteal, or progesteronic phase, the mucus become more viscous, and in pregnancy, will form a viscous mucous plug, obliterating the uterine cavity. Sometimes the lumen of the glands is blocked, and they become dilated, filled with mucus, forming the nabothian cysts. The exocervix is lined by stratified squamous nonkeratinized epithelium, continued, y q p , , with the same type of epithelium, in the vagina. The midle tunic of the cervix is thinner than that of the uterus, with a single layer of circular smooth muscle fibers, but with abundant connective tissue, instead.

Figure 3. Photomicrograph of stratified squamous epithelium of the vagina supported by a d t db dense connective ti ti tissue. Th The cytoplasm of these epithelial cells is clear because of accumulated glycogen. PSH stain. Medium magnification.

The mammary glands
Mammary g y glands are compound tubuloalveolar g p glands, active only during y g lactation; they are made of 15-25 lobules, each with its own excretory duct, 15lactiferous ducts, opened separatelly, at the nipple. The alveoli, secretory portion of the glands, ovoidal in shape are developing, only during pregnacy, they are active after birth. The cells of the alveoli have different heights, according to the stage of the secretory cycle. The intralobular ducts have simple cuboidal epithelium; interlobular ducts are lined by stratified columnar or cuboidal epithelium Inside the columnar, epithelium. lobules is found loose connective tissue and adipose tissue, in which will develop the alveoli, during pregnancy; the interlobular connective tissue is denser. The interlobular ducts are continuous with the lactiferous ducts, lined in their terminal portion, by squamous stratified epithelium, opening at the level of the nipple. i l

The implantation and the placenta
Placenta is a transitory organ, present only during pregnancy; in its structure has both fetal and maternal components. The fetal and the maternal parts of the placenta are genetically different, pregnancy being partially a graft, yet it is not expelled. This fact is not well explained yet; it is suspected that it is an induced immune tolerance estate (e.g. placenta does not express HLA or histocompatibility antigens). Fertilization of the ovum takes place in the oviduct, immediately after that the egg undergoes numerous cellular divisions, and the morula will be formed. In the interior of the morula will develop a small cavity - the blastocyst stage. The blastocyst is formed of a cavity with liquid content, surrounded by a layer of cells (blastomeres) forming the trophoblast, and the inner cell mass, a crowding of cells, from which will arise the embryo. In the blastocyst stage, the uterus is reached, after 4-5 days, from the fertilization moment and after the migration, the nidation of the egg occurs. The nidation or implantation is done in the thickness of the endometrium; the endometrium changed by the progesterone secretion, during implantation of the egg, is called decidua. The implantation is possible through the lysis of the endometrium, by the trophoblast, which secretes enzymes.

The trophoblast, an epithelial structure, will differentiate into two layers syncytiotrophoblast and cytotrophoblast. The former, i th outer l Th f is the t layer of th t h bl t a continuous l f the trophoblast, ti layer of cytoplasm, f t l with numerous nuclei, forming a syncytium. The cytotrophoblast is the inner layer, a simple cuboidal epithelium, with individual cells (there are cellular limits between the cells). cells) The lysis of the trophoblast causes rupture of both arterial and venous maternal blood vessels and lacunary spaces, filled with maternal blood, will develop between trophoblast and decidua; f b t t h bl t d d id from th these, embryo will t k nutrients and b ill take t i t d oxygen. The endometrium in contact with the trophoblast is called decidua basalis. The endometrium which will grow and will cover th bl t d ti hi h ill d ill the blastocyst, after it nidation, i t ft its id ti is called decidua capsularis, while the resting endometrium is called decidua parietalis. During the development of the pregnancy, the decidua capsularis is gradually fusing with decidua parietalis, becoming as one. Decidua basalis, developing with the trophoblast forms the placenta.

Figure 5. D i pregnancy th endometrial connective cells t Fi 5 During the d ti l ti ll transform i t d id l cells. Th f into decidual ll The endometrium is then called decidua, and 3 regions of mucosa can be recognized: decidua basalis, capsularis, and

From the extraembryonic mesenchyme, will develop the connective tissue, which will form the chorionic plate or chorion of the placenta The trophoblast develops plate, placenta. numerous expansions, called chorionic villi, initially on all its surface, later, only the surface in contact with the maternal decidua basalis will further develop its villi, the rest becoming smooth surfaced In development first are appearing primary villi surfaced. development, villi, made only of trophoblast, with the two layers - syncytio- and cytotrophoblast. In syncytiotime, the connective tissue forming the chorial plate will penetrate the villi, forming secondary villi; with the growth of the blood vessels of the fetus, these will penetrate also the villi, forming finally, tertiary villi. The villi of the fetal placenta may be anchored or free: the anchored villi are attached to the maternal decidua basalis, instead, the free villi are floating freely, in the maternal blood. , , g y, The lysis of the endometrium leaves some undestroyed areas behind, which will form the septa of the maternal placenta intercalated with the chorionic villi of the placenta, villi, fetal placenta. Septa of maternal decidua have the same zones, as the endometrium, in the second half of the menstrual cycle, the secretory, luteal or progesteronic phase, with the same territories in the lamina propria - a superficial phase compact decidua, and a deeper spongy zone, where the cells of the lamina propria are nomore so numerous and crowded together, in an epithelial manner, like in the p , compact zone, or decidua.

In its development the trophoblast is proliferating inside the maternal blood vessels, the vascular trophoblast, as well as inside the lamina propria of the maternal decidua, the so-called interstitial trophoblast. soDuring first half of pregnancy, the trophoblast has two layers of cells, being thicker - the syncytiotrophoblast to the exterior and the cytotrophoblast, to its interior, bordering the villi. Cytotrophoblast undergoes mitosis, being a regenerative layer; when placenta has reached its full development, the cytotrophoblast is gradually dissapearing, so in the second half of pregnancy, the fetal villi are bordered only by the syncytiotrophoblast, this single layer being kept till p y y p , g y g p parturition. Both layers of the y trophoblast are rich in organelles, contain glycogen and inclusions of lipids. Syncytiotrophoblast contains to its apical region a brush border corresponding to border, the microvilli, in the e.m.; by microvilli, the embryo is uptaking nutrients and oxygen, from the maternal blood, and is discharging into it the waste products, of his own metabolism metabolism.

Syncytiotrophoblast has also endocrine function, secreting hormones, important in the viability and development of the pregnancy most important being - chorionic pregnancy, gonadotropin, an LH-like hormone, which is mantaining the corpus luteum in the ovary, LHforming the pregnancy corpus luteum, stimulating at this level the secretion of progesterone and estrogens; human placental lactogen which has lactogenic and lactogen, growthgrowth-stimulating properties, acting like a growth factor; estrogens, progesterone, chorionic thyrotropin, chorionic corticotropin. Syncytiotrophoblast has receptors for Fc fragment of G immunoglobulins which are immunoglobulins, passing all the pregnancy, to the fetus, and are ensuring the immunity of the fetus, as well as the immunity of the newborn, in the first 3-4 weeks of life, till he will synthesize 3his own antibodies antibodies. Even if the chorionic villi are floating in the maternal blood, the fetal blood is isolated inside the villi, forming the placental barrier, made of - syncytiotrophoblast and cytotrophoblast (only during first half of pregnancy); the basal lamina of the trophoblast trophoblast, the connective tissue in the axes of the villi, the basal lamina of the fetal capillaries and the endothelium of the capillaries. The placental barrier is effective in protection against bacteria, bacteria but is not effective against virus infections which may induce abortion genetic infections, abortion, mutations to the embryo, resulting in fetal malformations, especially if the infection occurs in the first 2-3 months of pregnancy, when organogenesis takes place. The 2placenta is also crossed by most of the drugs, alcohol and nicotine. In parturition, placenta is expelled, the delivery of the placenta; the rupture of it is taking place at approx. the same level, where endometrium should be expelled, during menstruation.

Thank you for your attention!

13. MALE REPRODUCTIVE SYSTEM
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

MALE REPRODUCTIVE SYSTEM
• • • • •

The seminiferous tubules Ductuli efferentes Epididymis The ductus (vas) deferens The prostate

The male reproductive system is made of the two testes, g p y genital or sperm ducts, p accesory glands and penis. The testes have double function, on one hand to produce mature sexual cells, spermatozoa, and on the other hand, to produce male sexual hormones, androgens, the main one being testosterone. The genital ducts are divided, according to their situation, in intratesticular and extratesticular, or excretory. The accesory exocrine glands are producing a fluid for the transport and nutrition of the spermatozoa. All connective tissue components of the testes are of mesenchymal origin - the outer tunica albuginea, the mediastinum testis, the trabeculae inside the testes, etc. The mesodermis forms the genital ridges; from the genital ridges are arising the genital or sexual cords, in which the differentiation of the supprting cells, called Sertoli cells, occurs. From the extraembryonic area, are generated the primordial germinal cells, spermatogonia, which will be placed, in the genital cords, on the basal lamina, between Sertoli cells, in the future seminiferous tubules. In the second semester of intrauterine life, the testicle begins to migrate, from the abdominal cavity, to the scrotum, passing along the inguinal traject. When the testicle remains in the abdomen, this is the socalled cryptorhidism. This condition must be surgically solved, because spermatogenesis takes place only at a temperature with 2-3 Celsius degrees bellow the temperature of the human body, ensured b th position of th t t t t f th h b d d by the iti f the testes, in th scrotum; if i the t not the individual will be sterile.

Figure 1. The male genital system. The testis and the epididymis are shown in different scales than the other parts of the reproductive system. Note the communication between the testicular lobules

The scrotum is pigmentated and folded skin, under it is found the cremasterian muscle, the inner layer covering the testis is the tunica vaginalis, part of the peritoneum, peritoneum transported with the testis into the scrotum testis, scrotum. The testis is covered by thick dense connective tissue, the same tunica albuginea, like the ovary, which is rich in collagen fibers, giving a whitish appearance to the organ To its superior and posterior inner surface tunica albuginea has a organ. surface, thickening, called mediastinum testis, pyramidal-shaped, in which is placed an pyramidalanastomotic network of channels, called rete testis, representing the intratesticular ducts. ducts From the mediastinum are arising trabeculae parting each testis in approx trabeculae, approx. 200200-300 lobules, which are triangular compartments. The septa are incomplete, so the testicular lobules are communicating with one another. another Each lobule contains 2-5 seminiferous tubules in loose connective tissue 2tubules, tissue, containing also vessels, nerves and interstitial Leydig cells. Leydig cells are the endocrine cells of the testis, found isolated, or in small groups, secreting androgens male sexual hormones the main one testosterone The androgens, hormones, one, testosterone. seminiferous tubules begin with a blind end inside the lobules, they are highly tortous and twisted, and they open inside the mediastinum, in the rete testis.

Figure 2. Part of the wall of a seminiferous tubule. Several cells of the spermatogenic lineage are present: a spermatogonium, primary spermatocytes, and young and late spermatids. The approximate limits of a Sertoli cell holding several spermatids are delineated. H&E stain. High magnification

The seminiferous tubules
Seminiferous tubules have an outer fibrous connective tissue tunic, with a basal lamina, lamina on which is situated a complex germinal or seminiferous, epithelium complex, germinal, seminiferous, epithelium. Besides connective tissue, the outer tunic of the seminiferous tubules contains, to its inner part, so-called myoid cells, with characteristics of smooth muscle cells, soimportant in the movement of still nonmotile spermatozoa along the length of the spermatozoa, tubules, to the seminiferous ducts. The germinal epithelium is made of 2 types of cells - cells of support, Sertoli cells, and cells of the spermatogenic lineage The cells of the spermatogenic lineage are lineage. situated on 4, up to 8 layers, occuping the space between the basal lamina and the lumen of the tubules. Till the puberty, in the tubules are present exclusively spermatogonia, spermatogonia, on the basal lamina, between Sertoli cells of support Beginning lamina cells, support. with the puberty, the discharge of gonadotropins, FSH and LH (ICSH), from the gonadotropins, anterior pituitary, is leading to the start of spermatogenesis process. Spermatogenesis can be divided into 2 stages - spermatocytogenesis, consisting spermatocytogenesis, in the mitotic divisions and the 2 meiosis, from which finally are formed the spermatids, spermatids, haploid cells (n chromosomes); the second stage is called p g p , g p spermiogenesis, by which are formed spermatozoa, through a complex cellular spermiogenesis, y differentiation, without any cellular division.

From the puberty, in the seminiferous tubules - type A spermatogonia and type B spermatogonia. While type A spermatogonia are undifferentiated germinal cells, like reservoir elements, elements type B spermatogonia, by mitosis, give rise to primary spermatocytes spermatogonia, mitosis spermatocytes. Soon after their formation, primary spermatocytes enter the prophase of the first meiotic division. During this division, the cell is reducing to half the number of the chromosomes, to 23 from 46, bu not the DNA quantity. The resulted cell, has 23 double h th i d during first chromosomes, so i still a di l id cell, b is till diploid ll because DNA i synthesized, d i fi t is meiosis, by the demiconservative replication (the chromatids duplicate themselves, without duplication of cetromeres). In this phase, crossing over genes exchanges takes place between the chromosomes place, chromosomes. Thus are resulting secondary spermatocytes, which have 23 double chromosomes (2 N); the secondary spermatocytes enter rapidly in the second meiotic division, resulting in spermatids, real haploid cells, with 23 simple chromosomes and half of the DNA quantity, also (1 N), since nomore DNA synthesis occurs, this time. With fertilization, th di l id characteristic number of chromosomes, f th specie, will the diploid h for the f tili ti t i ti b f h i ill be restored.

Spermatogonia are visible on the basal lamina of the seminiferous tubules with visible, tubules, small pale nuclei; primary spermatocytes have the largest nuclei, of the seminiferous lineage, situated approx. at the mid part of the seminiferous epithelium, epithelium with the chromatin condensed in large clumps clumps. Secondary spermatocytes are difficult to observe, in sections, in light microscopy, b h t d they t idl in the d i i because th are short-lif cells, and th enter rapidly i th second meiosis. they short-life ll Spermatids are small cells, 7-8 micrometers, with condensed nucleus, situated to 7the lumen of the tubules, intercalated with spermatozoa; they show eccentrically placed nucleus and a small amount of acidophilic cytoplasm cytoplasm. Because of the crossing over processes, the genetic material of the individual is changed, during spermatogenesis, and the cells must be isolated from the immune system of the organism, otherwise being considered as non-self and destroyed. nonSpermatozoa are sequestrated or segregated antigens, because they may function, in contact with th i f ti i t t ith the immune system, as autoantigens, d t t t ti determining th i i the production of autoantibodies. For this reason is developed a blood-testis barrier, bloodinvolving especially the Sertoli cells.

Spermiogenesis is a complex process of cellular differentiation, by which spermatids are transformed into spermatozoa without cell division The first stage of spermatozoa, division. spermiogenesis is called the Golgi phase -the well-developed Golgi complex is placed wellto a pole of the nucleus, accumulating enzymes, and will form, gradually, an acrosomal vesicle, vesicle and finally an acrosome (acrosomal phase) At the other pole of the nucleus finally, phase). nucleus, migrate the centrioles and will initiate microtubuli, with axoneme, forming the flagellum, or the tail of the spermatozoon. The maturation phase is consisting in the aggregation of the mitochondria around the initial portion of the flagellum following a helical flagellum, arrangement, forming the middle piece, a thickened region, where the movements of the spermatozoa are generated. During this maturation process, small fragments of cytoplasm are phagocytized by Sertoli cells. Spermatozoon - a small head, with an anterior acrosome, containing hyaluronidase, peptydases and acid phosphatase, for the penetration of the zona pellucida and the membrane of the ovum during fertlization; a middle piece - mitochondria involved in ovum, mitochondria, generating the movements of the tail, and a flagellum, or the tail, the longest portion of the spermatozoon, of about 40-50 µm, with the structure of a motile cillium. Dynein, the 40protein with ATPase activity essential for the movement of the tail, is activated in the epididymis, by a protein here secreted. Only the mature spermatozoa are liberated in the lumen of the seminiferous tubules; all the other cells of the sperm lineage are held together by small cytoplasmic bridges, thus being ensured the syncronism of the whole g y y p g , g y process.

Figure 3. Diagram showing the clonal nature of the germ cells. Only the initial spermatogonia divide and p produce separate daughter cells. Once committed to differentiation, the cells of all subsequent divisions p g , q stay connected by intercellular cytoplasmic bridges. Only after they are separated from the residual bodies can the spermatozoa be considered isolated cells. (Modified and reproduced, with permission, from Bloom W, Fawcett DW: A Textbook of Histology, 10th ed. Saunders, 1975.)

Sertoli cells, cells of support of the spermatogenic lineage, high columnar cells, occuping all the thickness of the germinal epithelium with the basal pole on the basal epithelium, lamina and the apical pole in the lumen of the tubules. Sertoli cells have in their cytoplasm places for the cells of the sperm lineage. Sertoli cells appear recognizable, in light microscopy, only by their nucleus, because of the crowding of cells, in the germinal epithelium. The nucleus - ovoidal or triangular (the l l ith this t because all th sperm cells show a round nucleus) with ll the ll h d l ) ith sole nucleus with thi aspect, b obvious nucleolus and 1-2 masses of chromatin, close to the nucleolus. 1To their basal part, the Sertoli cells establish one with another occluding junctions, isolating the spermatogonia from the other cells of the spermatogenic lineage, forming a basal compartment and an adluminal compartment. This is the blood-testis barrier, important in isolating the more advanced spermatogenic bloodcells, from blood, and like this, from the immune system. If this barrier is injured, the , , , y j , autoimmunity developed, is leading to infertility by antisperm autoantibodies, representing approx. 20 % of all causes of infertility, for both sexes. In the e.m., Sertoli cells - well-developed Golgi complex, rough and smooth ER, mitochondria, lysosomes welland contractile filaments toward their lateral and apical faces.

Besides hosting in lateral invaginations of the cytoplasm, different cells of the spermatogenic lineage, they also protect spermatozoa, till their liberation - with the head in invaginations of their apical pole and the tails, visible like tufts, to the lumen of the tubules. Sertoli cells have important roles: 1. Role of support for all the cells of the spermatogenic lineage; nutrition of the cells in the adluminal compartment (except spermatogonia, taking nutrients from the blood) and uptaking the metabolic products; providing the blood-testis barrier bloodbarrier. 2. Phagocytosis of cytoplasm excess, of spermatids, the so-called residual bodies; solib ti of mature spermatozoa, b phagocytosis of th cytoplasmic b id t t t i f the t l i bridges. liberation f by h 3. Secretion of a fluid for the transport and nutrition of the liberated spermatozoa, which are not yet motile; secretion of androgen-binding protein ABP, under the androgencontrol of FSH and testosterone, serves to concentrate the testosterone inside the seminiferous tubules, where is indispensable for the spermatogenesis; secretion of inhibin, which is inhibiting the FSH secretion, important in the regulatory feed-back feedcontrol of the hormonal secretion; conversion of small amounts of testosterone into estradiol, main estrogen, production of anti-Mullerian hormone, important in the antiembryonic d b i development, i hibiti th d l t inhibiting the development of th M ll i d t i l t f the Mullerian ducts, is directing the differentiation of the embryo toward the male sex.

Figure 1. The Sertoli cells form the blood-testis barrier. Neighbor Sertoli cells are attached by occluding junctions that divide the seminiferous tubules into 2 compartments and impede the passage of substances between both compartments. The basal compartment comprises th b l t t i the interstitial space and the spaces occupied by the spermatogonia. The adluminal compartment comprises the tubule lumen and the intercellular spaces down to the level of the occluding junctions (OJ). In this p p y , compartment are spermatocytes, spermatids, and spermatozoa. Cytoplasmic residual bodies from spermatids undergo phagocytosis by the Sertoli cells and are digested by lysosomal enzymes. The myoid cells surround the seminiferous epithelium.

The control of the spermatogenesis and of the endocrine function of the testis is dependent upon the pituitary FSH and LH also known in male as ICSH -interstitial LH, male, cells stimulating hormone. FSH is indispensable for the initiation of the spermatogenesis, stimulating the mitosis of the spermatogonia. FSH is also increasing the receptivity of the Leydig cells to the ICSH. ICSH, or LH is stimulating the meiosis and spermiogenesis, indirectly, by stimulating the t t t ti b L di ll testosterone secretion, by Leydig cells. Testosterone inhibits the ICSH secretion, while inhibin, secreted by Sertoli cells, inhibits the FSH secretion, by the pituitary. For the complete spermatogenesis process, in human are necessary approx. 70 days; from these, 10 days are needed for the spermiogenesis process, alone. In human, one ejaculate contains 2-5 ml of sperm, with an average of 60-120 million spermatozoa / ml. 60The Leydig, or interstitial cells of the testis - isolated, or in small groups, between y g, , g p , the seminiferous tubules. Leydig cells - rounded or polyhedral, with central located nuclei; they have 20 µm, in diameter and characteristics of steroid-secreting cells. steroidThey show acidophilic cytoplasm, containing lipid droplets, looking vacuolated, in light microscopy. In e.m. - well developed smooth ER, mitochondria.

During intrauterine life, the testis is secreting testosterone, under the influence of the placental gonadotropin, resulting in the differentiation of the male sex of the fetus. fetus After birth, the interstitial cells are silent till the puberty, when under the influence of the pituitary gonadotropins, they begin t secrete testosterone, which will f th it it d t i th b i to t t t t hi h ill determine the development of the secondary male sexual characteristics and the maturation and normal functioning of the male reproductive system. The intratesticular male ducts are the tubuli recti, or the straight tubules and the rete testis. The seminiferous tubules are opening into the rete testis by the tubuli recti. The tubuli recti are first lined only by Sertoli cells, and after that by a simple cuboidal epithelium. They are short and they empty into the rete testis, found inside the mediastinum, like an anastomotic network of channels, irregular in shape, lined by simple p p q p cuboidal, simple columnar, or simple sqamous epithelium.

Ductuli efferentes

From the rete testis are arising 15-20 ductuli efferentes, highly coiled, twisted 15tubules, forming together the head of the epididymis, representing first part of the extratesticular male ducts. The epithelium of the ductuli efferentes is made of an alternation of groups of higher, ciliated cells, with motile cilia, beating in the direction of the epididymis, and lower, cuboidal cells, which absorb the fluid in which the spermatozoa reach here, from the seminiferous tubules, creating a fluid flow, that atracts the spermatozoa to the epididymis. p y The difference in height of cells gives the waved aspect, of the lumen of these tubules. tubules At the outside they show a thin circular layer of smooth muscle fibers outside, fibers. The ductuli efferentes are opening into the epididymis.

Epididymis
Epididymis is a highly coiled duct, a single, long tube, packed in connective tissue, of about 3-5 meters long. It is li d b pseudostratified columnar epithelium, with f b t 3t l i lined by d t tifi d l ith li ith predominant columnar cells, which show very long microvilli, called stereocilia. These peculiar microvilli are fusing together, giving in light microscopy, an image of a candle flame flame. At the base are found small, round cells, regenerative cells, giving the false impression of stratification. The long microvilli are involved in water absorbtion, thus concentrating the seminal fluid, and exerting an aspyration force, which will move the sperm, further on, along the genital ducts. At the outside of the epididymis are found one or two layers of smooth muscle fibers; by peristaltic contractions, the muscle layer has also the role in moving the sperm along the ducts. In the epididymis is secreted a maturation protein, capable to act on the dynein of the flagellum, transforming the non-moving spermatozoa, noninto motile cells.

Figure 2. The highly coiled ductus epididymidis, sectioned several times. Its wall i made of a ll is d f pseudostratified columnar epithelium surrounded by connective tissue and smooth muscle. PSH stain. Medium magnification. Inset: Higher magnification of the epithelial cells with their long microvilli (stereocilia).

The ductus (vas) deferens
The vas deferens is continuing the epididymis, like a straight tube, with a thick muscular wall giving it a star-shaped folded lumen aspect in cross section It is wall, star-shaped, aspect, section. lined, most of it, by pseudostratified columnar epithelium, sometimes with zones of simple columnar, or stratified columnar epithelium. The muscular tunic is made of 3 layers of smooth muscle fibers - outer and inner longitudinal layers and a layers, medium circular layer. The Th contraction of thi duct is propelling th sperm t th ejaculatory d t which i t ti f this d t i lli the to the j l t duct, hi h is opening into the prostatic urethra. Ejaculatory duct is lined by simple columnar or pseudostratified columnar epithelium, which is continued with the urinary epithelium, epithelium lining the prostatic urethra in male a common urinary and genital duct urethra, male, duct. The final portion of the ductus deferens is dilated, forming the ampulla; from it originates the seminal vesicles. g p y The two glands associated with the male reproductive system are the p prostate and the seminal vesicles.

Figure 3. Section of the ductus deferens showing the mucosa formed by pseudostratified columnar p epithelium with stereocilia and a lamina propria. The thick outer wall is formed of smooth muscle (brown) and collagen fibers (blue) (blue). Trichrome stain. Low magnification.

The prostate
The prostate gland is made of 30-50 tubuloalveolar glands, the ducts of these 30glands are opening separatelly into the prostatic urethra The gland has three urethra. lobes, the medium one is containing the first portion of the urethra. The stroma and the capsule of the prostate are rich in smooth muscle fibers. Each dilated secretory portion, called alveolus, is lined by psedostratified columnar epithelium and has its p , , yp p own excretory duct, lined by simple columnar epithelium. The prostatic fluid is discharged during ejaculation. In the lumen of the prostatic glands and ducts are found spherical bodies of g y p p glycoproteic accumulatios, called p prostatic concretions or corpora amylacea. They become calcified, in time, like small stones; they have an acidophilic concentrical aspect, in light microscopy, in usual stain, being a criterion for the p , g py, , g recognition of prostate, as organ. The prostatic secretion has an alkaline ph, of about 6, contains citrate, lipids, prostaglandins, lecithin, enzymes, like acid phosphatase, which is also a marker for the prostate adenocarcinoma. Both prostate and seminal vesicles have hormone-dependent epithelia, which are hormoneregressing with age. g p Bulbourethral glands or Cowper's g glands are two small tubuloalveolar or tubuloacinar glands, in the lamina propria of the membranous urethra; they secrete a mucus which is lubricating the urethra.

Figure 4. Seminal vesicle. A section of this tortuous tubular gland with a muchfolded mucosa gives the impression that the gland consists of many tubules. PSH stain. Medium magnification.

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14. THE SKIN (INTEGUMENT)
Conf. Univ. Dr. Ioana Cristina AMIHĂESEI UMF “Gr. T. Popa” Iasi Facultatea de Medicina Disciplina Histologie Di i li Hi t l i

The skin is the heaviest organ of the body, representing approx. 15 % of the total body weight, and having a total suface of about 1-2.3 meters. 1The epidermis is stratified squamous keratinized epithelium with four layers in the thin epidermis - germinativum (basale), spinosum, granulosum and corneum and five layers of cells, i th thi k epidermis. fi l f ll in the thick id i Thick epidermis is p p present only on the p y palms of the hands and the soles of the feet and is glabrous or nonhairy skin, containing layer basale, spinosum, granulosum, lucidum and a much thicker layer corneum than the thin skin, made of comified, fully keratinized cells. The layers of the epidermis have two big regions - the malpighian region is made of living nonkeratinized cells; it consists of layer germinativum (the basal layer), layer spinosum and granulosum. The surface layer is made of comified, fullyfullykeratinized cells.

Figure 1. Section of the stratum spinosum showing the localized deposits of melanin covering the cell nuclei. Melanin protects the DNA from the UV radiation of the sun. This explains why people with light skin have a higher incidence of skin cancer than do people with dark skin. The highest concentration of melanin occurs in the cells that are more deeply localized; these cells divide more actively. actively (The DNA of cell populations that multiply more actively is particularly sensitive to harmful agents.)

The basal layer contains one or more, rows of cells (this layer may be thicker in the thick epidermis). The cells of this layer show permanent mitotic divisions, for the renewal of the superior layers. The cells contain tonofilaments, or keratin intermediate fil i t di t filaments of approximately 10 nm thi k t f i t l thick. The layer spinosum is made of up to 10 rows of cells, in the thick epidermis, while in the thin variety, covering the whole body, is made only of a few rows of cells. Thi l ll This layer h a thorny appearance, with small prominences connecting cells has h ih ll i i ll one with another; these are sites of the desmosomes, which are visible, in this layer, with the light microscope. Filaments of keratin are aggregating to form tonofibrils, in this layer, as well as in the superior, layer granulosum. In this layer are produced granules with lipid content, important in the function of water barrier, of the skin. In the layer granulosum are present keratohyalin granules, looking basophilic, in the light microscopy. The granules contain keratin precursor proteins, rich in cystine and histidine, with numerous phosphate groups. Filaggrin is a protein associated with the keratin filaments, which will be dispersed in the cells, and finally will englobe the keratin filaments into a compact material, filling the keratinized cells. Layer granulosum is discontinuous in the thin epidermis.

Figure 2. Photomicrograph of a section of thick skin. Note the blood vessels in the dermal papillae improving the nutrition of the thick epithelium. Picrosiriushematoxylin stain. Medium magnification.

The comified layer corneum is made of dead keratinized cells, 5-7 rows of cells, y 5forming keratin lamellae, in the thin epidermis, while in the thick type of skin, we may find a layer corneum, of up to 30 comified cells thick. Lipid material accumulated in granules, is discharged from the cells at the limit between layer granulosum and layer corneum, filling the intercellular spaces of the deep portion of the layer corneum, providing the water barrier function of the skin. A discontinuous layer lucidum is present only in the thick epidermis, at the limit between layer granulosum and layer corneum This is a transitional layer, between living granular cells of the granulosum layer, to the dead fully keratinized cells of the comified layer corneum. This layer is absent in the thin epidermis epidermis. The dermis is the connective tissue supporting the epidermis; the proper dermis is made of irregular dense connective tissue while the superficial and the deep tissue, dermis, or hypodermis, contain loose connective tissue. The skin of the palms and the feet contains ridges and grooves, arranged in individually genetical patterns, dermatoglyphics, called dermatoglyphics or fingerprints; they are of medical and forensic importance.

Figure 3. Several types of sensory skin nerve endings. (Modified and reproduced, with permission, from p , p , Ham AV: Histology, 6th ed. Lippincott, 1969.)

The hair is an elongated keratinized structure with a root from where it grows; at root, the level of the root, found in the dermis, an invagination of the epidermis forms the hair follicle, with a terminal dilatation, called the hair bulb. In the hair bulb enters connective tissue with capillaries forming the dermal papilla which is nourishing tissue, capillaries, papilla, the hair follicle. The hair has a midle region with fewer cells, slightly keratinized, called the medulla and a thicker outer layer the cortex with highly keratinized fusiform cells medulla, layer, cortex, cells, better stained in usual techniques. At the level of the hair follicle, or the root of the hair, we differentiate an external root sheath, continuous with the basal layers of the epidermis like an invagination of the epidermis containing the same cells of epidermis, epidermis, cells, the layer germinativum (basale) and spinosum. The internal root sheath has acidophilic cells, charged with keratohyalin, keratin precursor, precursor and is dissapearing at the level of the opening of the sebaceous gland gland. The layer intimately adherent to the hair cortex is called the hair cuticle, first columnar, the cells become flattened to the upper part of the root, forming a layer of shinglelike, keratinized cells covering the cortex shinglelike cells, cortex. The glassy membrane is a thickening of the basal lamina, separating the hair follicle from the connective tissue sheath, surrounding it to the exterior. The hair, in keratin. contrast to the skin produces a hard and compact variety of keratin

Main functions of the skin are - the protection of the body, like a barrier, by the stratified type of epithelium, by the keratinized layer corneum and by permanent descuamation of the skin. Besides mechanical and chemical protection, the keratin prevents the water loss. Sebum, produced b th sebaceous glands, i l t th t l S b d d by the b l d is lysed i t d into free fatty acids by the saprophite bacteria, and by the low, acid ph, thus created, is protecting the skin against microorganisms. The Th sweat glands, b their i transporting cells, are i glands, by h i ion l d i ll important i water and in d electrolytes homeostasis; together with the kidney, they are functioning also, as excretory organs. The temperature regulation of the body is dependent as well, on the th amount of secreted sweat, which by evaporation, i cooling th surface of th t f t d t hi h b ti is li the f f the skin, when the temperature of the environment is high. If the temperature is low, the decreased sweating and constriction of the blood vessels, will reduce the loss of heat heat. The skin contains sensory receptors for tactile sensations, the pacinian corpuscle, in the dermis, is the best studied; the central nerve ending is surrounded by several collagen fibers sheaths with fibroblasts resembling with a sliced onion in sheaths, fibroblasts, onion, histologic section. Other encapsulated nerve endings, as well as free nerve endings are found in the dermis and in the epidermis, forming networks, and receiving heat cold pain and tactile stimuli heat, cold, stimuli.

Figure 4. Sebaceous gland. This is a holocrine gland, because its product is secreted with the remnants of a dead cell. Stem cells (arrows) in the base of the gland p proliferate to replace the lost cells. Collagen p g fibers are stained in red. PSP stain. Medium magnification.

The skin associated lymphoid tissue - SALT - is important in the specific immune defense of the organism; skin associated lymphoid tissue is made especially of T g y y lymphocytes, important in the cell-mediated immunity, like antitumoral immunity, cellgraft rejection, etc. A peculiar cell type, the Langerhans cell, p of the p g y mononuclear p yp , g , part phagocyte system, is found mainly in the layer spinosum, being phagocytic and antigen presenting cell, of the skin. Besides Langerhans cells, in the basal layers of the epidermis are present melanocytes and Merkel's cells. Melanocytes are producing the skin pigment, melanin, starting from an amino acid tyrosine; melanin is protecting like a shield, the deeper layers of the epidermis, from UV radiations. Melanocytes are transffering the p g y g pigment in its p processes, to the epidermal cells. Merkel's cells are originating from the neural crests, as well as the melanocytes. They look similar to the epithelial cells, but they have dense g y p , y granules in their cytoplasm, resembling to the cells of the diffuse neuroendocrine system. They are located especially in the layer spinosum and are closely associated with nerve endings, forming the sensory Merkel complex.

Figure 5. Low-magnification photomicrograph of a section of sweat p g p gland. This is a simple coiled tubular gland. H&E stain.

Figure 6. Section of sweat gland. Note the duct lined by stratified cuboidal epithelium. The myoepithelial cells, whose contraction helps to discharge the glandular secretion, yp surround the secretory portion. H&E stain. Medium magnification.

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