General anatomical

pathology. Class 3
CELLULAR DEATH
Cell features

Vicente Hernández Rabaza, PhD

 Index

Brain Warm-up
Class 3
Exercises
Work/Game
Presentation made by Vicente Hernández Rabaza, PhD
 Brain

Warm-up

Presentation made by Vicente Hernández Rabaza, PhD

 Who was Galen?

Image by: CAROL YEPES (GETTY IMAGES) Presentation made by Vicente Hernández Rabaza, PhD
 Cambios celulares:
Rigor mortis, sangre y membranas

Giuseppe Sanmartino, 1753. Il Cristo velato nella cappella di San Severo Napoli

Presentation made by Vicente Hernández Rabaza, PhD

Index
A. Cell death overview.
B. Apoptosis
- Cell features
- Apoptosis regulation
C. Necrosis
- Cell features
- Necrosis types
D. Beyond classic deaths
- Necroptosis
- Pyroptosis

Presentation made by Vicente Hernández Rabaza, PhD

 Cell death.
Overview

Image from: Robbins & Cotran Pathologic Basis of Disease Presentation made by Vicente Hernández Rabaza, PhD
 APOPTOSIS
Why said bye?

• Cell Senescence.
• Physiological processes (regeneration,
development, hormone drive
processes, self –reactive lymphocytes)

Pathology conditions:

o Inviable cellular environment (high

inflammation reaction)
o Risk or unfunctional mutations
(replication)
o Metabolic derangements (proteins)
o Certain infection types.

Presentation made by Vicente Hernández Rabaza, PhD

 Apoptosis cellular features

http://www.cellimagelibrary.org/images/43705

http://www.cellimagelibrary.org/images/50503

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 Apoptosis cellular features

On histologic examination, in tissues stained
with hematoxylin and eosin, the apoptotic cell
appears as a round or oval mass of intensely
eosinophilic cytoplasm with fragments of
dense nuclear chromatin
This electron micrograph of cultured cells undergoing
apoptosis shows some nuclei with peripheral crescents of
compacted chromatin, and others that are uniformly dense or
fragmented

Which type of cell death do you thing would be easier to detect histologically?
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 Summary. Apoptosis cellular features
The following morphologic features, some best seen with the electron microscope, characterize cells
undergoing apoptosis

Cell shrinkage. The cell is smaller in size, the cytoplasm is dense, and the organelles, although relatively
normal, are more tightly packed. (Recall that in other forms of cell injury, an early feature is cell swelling, not
shrinkage.)

Chromatin condensation. This is the most characteristic feature of apoptosis. The chromatin aggregates
peripherally, under the nuclear membrane, into dense masses of various shapes and sizes. The nucleus itself
may break up, producing two or more fragments.

Formation of cytoplasmic blebs and apoptotic bodies. The apoptotic cell first shows extensive surface
blebbing, then undergoes fragmentation into membrane-bound apoptotic bodies composed of cytoplasm and
tightly packed organelles, with or without nuclear fragments

Phagocytosis of apoptotic cells or cell bodies, usually by macrophages. The apoptotic bodies are
rapidly ingested by phagocytes and degraded by the phagocyte’s lysosomal enzymes.

Plasma membranes are thought to remain intact during apoptosis, until the last stages, when they become
permeable to normally retained solutes.

Image from: Robbins & Cotran Pathologic Basis of Disease Presentation made by Vicente Hernández Rabaza, PhD
 Apoptosis regulation

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 Apoptosis regulation: caspases
Apoptosis results from the activation of enzymes called caspases (so named because they are
cysteine proteases that cleave proteins after aspartic residues).

Apoptotic bodies nuclear fragmentation Caspase 3

These images of cultured cells undergoing apoptosis show blebbing and formation of apoptotic bodies (left panel, phase contrast micrograph), a stain for DNA showing nuclear fragmentation (middle
panel), and activation of caspase-3 (right panel, immunofluorescence stain with an antibody specific for the active form of caspase-3, revealed as red color). (B, From Kerr JFR, Harmon BV: Definition
and incidence of apoptosis: a historical perspective. In Tomei LD, Cope FO (eds): Apoptosis: The Molecular Basis of Cell Death. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press, 1991, pp 5-
29; C, Courtesy Dr. Zheng Dong, Medical College of Georgia, Augusta, Ga.)

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 Apoptosis regulation:
Intrinsic mitochondria pathway

Activators:

• Lack of survival signals

• DNA damage (irradiation, novo
mutations, toxins)
• Protein unfolded (ER stress)

Immunofluorescence image of Human Embryonic

Stem cells stained with an antibody to the active
form of Bax (red) and Hoechst (blue). The
distribution of Bax, an apoptosis mediator
indicates that it resides in the Golgi apparatus
under these conditions.

Image from: Robbins & Cotran Pathologic Basis of Disease Presentation made by Vicente Hernández Rabaza, PhD
Apoptosis regulation

Presentation made by Vicente Hernández Rabaza, PhD

 Apoptosis regulation:
Extrinsic mitochondria pathway

Tumor necrosis factor receptor superfamily (TNFRSF)

• Tumor necrosis factors (TNF)

• More than apoptosis

Apoptosis pathways:

1. Death domain, ligand : TNf-alpha; such as TNFR1,

2. Fas R; Ligand FasL

Image from Wikipedia

Presentation made by Vicente Hernández Rabaza, PhD

 Death domain, ligand : TNf-alpha; The extrinsic (death receptor initiated) pathway of
such as TNFR1 apoptosis

Regulation of the immune

system
Macrophages T-Cells control
Lymphocytes control
• (in immune privileges
regions, such as cornea)
Crosslinking of TNFR1 by TNF causes
• Tumour hacker the
recruitment of RIP1 and RIP3 along vwith
lymphocytes
caspase 8.

Activation of the caspase leads to apoptosis.

Presentation made by Vicente Hernández Rabaza, PhD Image from: Robbins & Cotran Pathologic Basis of Disease
 APOPTOSIS. FILL IN THE BLANK
Regulated mechanism of cell death that serves to eliminate unwanted and irreparably damaged
cells, with the least possible host reaction

Characterized by enzymatic degradation of proteins and DNA, initiated by caspases; and by

recognition and removal of dead cells by phagocytes

Initiated by two major pathways:

1. Mitochondrial (intrinsic) pathway is triggered by loss of survival signals, DNA damage, and
accumulation of misfolded proteins (ER stress); associated with leakage of pro-apoptotic proteins
from mitochondrial membrane into the cytoplasm, where they activate caspases; inhibited by
anti-apoptotic members of the BCL2 family, which are induced by survival signals including
growth Factors

2. Death receptor (extrinsic) pathway is responsible for elimination of self-reactive lymphocytes and
damage by cytotoxic T lymphocytes; is initiated by engagement of death receptors (members of
the TNF receptor family) by ligands on adjacent cells.

Presentation made by Vicente Hernández Rabaza, PhD

 NECROSIS

Source: https://tenor.com/search/thanos-gifs

Presentation made by Vicente Hernández Rabaza, PhD Image from: Robbins & Cotran Pathologic Basis of Disease
Presentation made by Vicente Hernández Rabaza, PhD Image from: Robbins & Cotran Pathologic Basis of Disease
 The morphologic appearance of necrosis is the result of denaturation of intracellular proteins and
enzymatic digestion of the lethally injured cell

What do you see?

Morphologic changes in reversible cell injury and necrosis. A, Normal kidney tubules with viable epithelial cells. B, Early (reversible) ischemic injury showing surface blebs, increased
eosinophilia of cytoplasm, and swelling of occasional cells. C, Necrosis (irreversible injury) of epithelial cells, with loss of nuclei, fragmentation of cells, and leakage of contents. (Courtesy
Drs. Neal Pinckard and M. A. Venkatachalam, University of Texas Health Sciences Center, San Antonio, Texas.)

Presentation made by Vicente Hernández Rabaza, PhD Image from: Robbins & Cotran Pathologic Basis of Disease
MORPHOLOGICAL CHARACTERISTICS OF PYKNOSIS AND OTHER FORMS OF NUCLEAR
DESTRUCTION

Bile infarct with pyknotic nuclei (arrows)(400X).

The morphologic appearance of necrosis as well as necroptosis is the result of denaturation of intracellular
proteins and enzymatic digestion of the lethally injured cell
What do you see?

Ultrastructural features of reversible and irreversible cell injury (necrosis) in a rabbit kidney. A, Electron micrograph of a normal epithelial cell of the proximal kidney tubule. Note abundant microvilli (mv) lining the
luminal surface (L). B, Epithelial cell of the proximal tubule showing early cell injury resulting from reperfusion following ischemia. The microvilli are lost and have been incorporated in apical cytoplasm; blebs have
formed and are extruded in the lumen. Mitochondria would have been swollen during ischemia; with reperfusion, they rapidly undergo condensation and become electron-dense. C, Proximal tubular cell showing
late injury, expected to be irreversible. Note the markedly swollen mitochondria containing electron-dense deposits, expected to contain precipitated calcium and proteins. Higher magnification micrographs of the
cell would show disrupted plasma membrane and swelling and fragmentation of organelles. (A, Courtesy Dr. Brigitte Kaisslin, Institute of Anatomy, University of Zurich, Switzerland. B, C, Courtesy Dr. M. A.
Venkatachalam, University of Texas Health Sciences Center, San Antonio, Texas.)
Image from: Robbins & Cotran Pathologic Basis of Disease
 COAGULATIVE NECROSIS

Coagulative necrosis (IGM)

• Liquefactive necrosis (IGM)

• Caseous necrosis (IGM)
• Fat necrosis (IGM)
• Fibrinoid necrosis (MM)
• Gangrenous necrosis (EM)

25
 infarct in the brain

• Liquefactive necrosis
• Caseous necrosis

Microscopic appearance of caseous necrosis. A well-formed granuloma

containing epithelioid macrophages, with a rim of lymphocytes and several
Tuberculosis of the lung Langhans giant cells can be seen. Centrally, caseous necrosis is apparent as
amorphous pink material. This figure was uploaded by Juanita Bezuidenhout.
 • Fibrinoid necrosis

Fibrinoid Necrosis of Small Arteries, Edema Disease, Stomach, Submucosa, Pig.

Note the circumferential eosinophilic (arrows) material in the walls of the arterioles and the extensive
edema and mild hemorrhage in surrounding submucosa. H&E stain.
(Courtesy School of Veterinary Medicine, Purdue University.)

autoimmune diseases (e.g., systemic lupus erythematosus) or malignant hypertension

28
• Fat necrosis

Resulting from release of activated pancreatic lipases into the substance of the Breast lump showing an area of fat necrosis showing shadowy outlines of
pancreas and the peritoneal cavity. necrotic adipocytes surrounded by an inflammatory reaction with cholesterol
clefts [H&E stain 4X]
mesentery
29
• Gangrenous necrosis

http://www.ijpmonline.org/viewimage.asp?img=IndianJPatholMicrobiol_2011_54_4_847_91520_u1.jpg
Necrosis: Increased eosinophilia; nuclear shrinkage, fragmentation, and dissolution;
breakdown of plasma membrane and organelle membranes; abundant myelin figures;
leakage and enzymatic digestion of cellular contents

Patterns of tissue necrosis: Under different conditions, necrosis in tissues may assume
specific patterns: coagulative, liquefactive, gangrenous, caseous, fat, and fibrinoid

What do you see?

Image from: Robbins & Cotran Pathologic Basis of Disease

Beyond the classics
 This form of cell death is a hybrid that shares aspects of both necrosis and apoptosis

https://www.creative-diagnostics.com/necroptosis-signaling-pathway.htm

Image from: Robbins & Cotran Pathologic Basis of Disease

Molecular mechanism of TNF-mediated necroptosis.

Crosslinking of TNFR1 by TNF causes recruitment of RIP1 and RIP3 along

with caspase 8.

Activation of the caspase leads to apoptosis.

Inhibition of caspase 8, as may occur in some viral infections, allows

RIP1 and RIP3 to initiate signals that affect mitochondrial generation of
ATP and ROS.
This is followed by events typical of necrosis. (Adapted from Galluzi L, et
al: Programmed necrosis from molecules to health and disease. Int Rev
Cell Molec Biol 289:1, 2011.)
“Programmed necroptosis regulated by RIP3 (receptor interacting protein
kinase 3) and MLKL (mixed lineage kinase domain-like protein) is the most
well-researched necrotic pathway. Programmed necroptosis may involve a
series of defense processes against intracellular infections, and related
studies have revealed that programmed necroptosis plays an important role
in the pathology of many human diseases, such as myocardial infarction,
stroke, arteriosclerosis, and ischemia-reperfusion injury.”

https://www.creative-diagnostics.com/necroptosis-signaling-pathway.htm
 PYROPTOSIS fever inducing cytokine IL-1

Pyroptosis occurs in cells infected by microbes .It

https://www.the-scientist.com/daily-news/how-hiv-destroys-immune-cells-38230
involves activation of caspase-1 which cleaves the
precursor form of IL-1β to generate biologically active
IL-1β.
Caspase-1 along with closely related caspase-11 also
cause death of the infected cell.
35
TIME TO WORK
AND PLAY
• Necroptosis resembles necrosis morphologically and apoptosis mechanistically as a form of
programmed cell death.

• Necroptosis is triggered by ligation of TNFR1, and viral proteins of RNA and DNA viruses.

• Necroptosis is caspase-independent but dependent on signaling by the RIP1 and RIP3 complex.

• RIP1-RIP3 signalling reduces mitochondrial ATP generation, causes production of ROS, and
permeabilizes lysosomal membranes, thereby causing cellular swelling and membrane damage as
occurs in necrosis.

• Release of cellular contents evokes an inflammatory reaction as in necrosis.

37
38
Path:

1. Mechanical arterial obstruction

2. Reduced blood flow
3. Reduced oxidative phosphorylation
4. Reduced ATP generation
5. Failure of Na pump
6. Influx of sodium and water
7. Organelle and cellular swelling
8. Membrane Permeabilization
9. Release of cellular contents
10. Inflammation and deaths
1. What is fibrinoid necrosis? And in which type of disease did you expect find
it?
2. What type of necrosis did you expect to find in an acute pancreatitis?
3. Coagulative necrosis is a form of necrosis in which the architecture of dead
tissues is preserved for a span of at least some days; Which is the cell
architecture of dead?

Necrosis: Increased eosinophilia; nuclear shrinkage, fragmentation, and dissolution;

breakdown of plasma membrane and organelle membranes; abundant myelin figures;
leakage and enzymatic digestion of cellular contents

Patterns of tissue necrosis: Under different conditions, necrosis in tissues may assume
specific patterns: coagulative, liquefactive, gangrenous, caseous, fat, and fibrinoid
 1. Cellular swelling
1. Which is the first manifestation of almost all
2. Necrosis
forms of injury to cells?
3. Yes. Oesophagus, from stratified to simple columnar (goblet)
2. Which cellular death type is always linked with by acid reflux.
pathological conditions?
4. Necrotic cells show increased eosinophilia in hematoxylin and
eosin (H & E) stains, attributable in part to the loss of
3. Metaplasia can be physiological?; provided one cytoplasmic RNA (which binds the blue dye, hematoxylin) and
example in part to denatured cytoplasmic proteins (which bind the
red dye, eosin).

4. Why necrotic cells show increased eosinophilia? 5. The necrotic cell may have a more glassy homogeneous
appearance than do normal cells, mainly as a result of the loss
5. Why necrotic cells show a slight glassy of glycogen particles
appearance?
6. When enzymes have digested the cytoplasmic organelles, the
cytoplasm becomes vacuolated and appears moth-eaten.
6. During the early stages of injury and lately Dead cells may be replaced by large, whorled phospholipid
during the cellular death a phospholipid masses masses called myelin figures that are derived from damaged
cell membranes.
will appear in the cytoplasm. Which is the name
of these masses? 7. These phospholipid precipitates are then either phagocytosed
by other cells or further degraded into fatty acids; calcification
7. Why death cells calcified? of such fatty acid residues results in the generation of calcium
soaps. Thus, the dead cells may ultimately become calcified.
 REFERENCES
➢ Vicente writings.
➢ The Internet Pathology Laboratory for Medical Education. The University of Utah. Eccles Health Science Library
/ Pathology images and text for medical education - WebPath (utah.edu)
➢ UC San Diego, School of Medicine, MedPics / https://medpics.ucsd.edu/index.cfm
➢ Histology Guide. University of Leeds
➢ Robbins & Cotran Pathologic Basis of Disease

• Megías M, Molist P, Pombal MA. (2019). Atlas de histología vegetal y animal. mmegias.webs.uvigo.es/02-
english/index.html
• The University of Western Australia. Department of Anatomy and Human Biology.
http://www.lab.anhb.uwa.edu.au
Social networks:
• Diagnostic pathology (Instagram)
• #histoløgy - Búsqueda de Twitter / Twitter
• Pinterest: https://www.pinterest.es/vhrabaza/
Recommended books or blogs, for own interest:
- La vida en cuatro letras. Carlos López-Otín (Editorial Paidós Contextos)
- Blog – José Ramón Alonso (jralonso.es)
- https://culturacientifica.com/
- https://www.uchceu.es/media

Presentation and PDF created by Professor, Vicente Hernández Rabaza, vicente.hernandez@uchceu.es