Chapter 1

CELL INJURY
Sr. No. TOPIC Page No.
1 Cellular adaptation 2

Hypertrophy 2
Hyperplasia 2
Atrophy 3
Metaplasia 4
Difference between metaplasia and dysplasia 5

2 Cell injury 6

Causes of cell injury 6

Mechanism (pathogenesis) of cell injury 7

3 Reversible cell injury 10

Cellular swelling (hydropic change, vacuolar degeneration) 10

Hyaline change 10
Fatty change 11

4 Irreversible cell injury 12

Necrosis 12
Apoptosis 13
Difference between necrosis and apoptosis 16

5 Changes after cell death 16

Gangrene 16
Pathologic calcification 17
1. Dystrophic calcification 17
2. Metastatic calcification 19

6 Important questions 20

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 CELLULAR ADAPTATION

HYPERTROPHY

Definition

Hypertrophy is defined as an increase in the size of cells, resulting in an increase in the size of the
organ, without any change in the number of cells. The increased size of the cells is due to the synthesis
of more structural components of the cells. Non-dividing or permanent cells (neurons, cardiac and
skeletal muscles) with little or no capacity to regenerate undergo hypertrophy.

Causes

Hypertrophy can be physiologic or pathologic.

Physiological hypertrophy Pathological hypertrophy

1. Pregnant uterus 1. Skeletal muscle hypertrophy: in body builders and athletes
2. Cardiac muscle hypertrophy: LVH
3. Smooth muscle hypertrophy: in pyloric stenosis, intestinal stricture
4. Compensatory hypertrophy: of other kidney in a patient having
undergone unilateral nephrectomy

Mechanism

Hypertrophy is caused by increased functional demand or stimulation by hormones and growth factors.
Any of the following factors may lead to hypertrophy:
1. mechanical sensors (physiological/pathological demand)
2. growth factors (e.g. TGF-β, insulin-like growth factor-1 [IGF-1], FGF)
3. vasoactive agents (e.g. α-adrenergic agonists, endothelin-1, and angiotensin II)

Biochemical pathways involved in muscle hypertrophy are:

1. phosphoinositide 3-kinase/Akt pathway
2. signaling of G protein
3. switch of contractile proteins from adult to fetal or neonatal forms; α-isoform of myosin heavy
chain is replaced by the β-isoform.
E.g. atrial natriuretic factor (ANF) gene is expressed in the embryonic heart, but down-regulated
after birth. Cardiac hypertrophy is associated with re-induction of ANF gene.

Morphology

Affected organ is enlarged and heavy; e.g. normal adult heart weighs 350 gm while in LVH, heart
weighs up to 700-800 gm
Ultrastructure: cells show increase in DNA, RNA, cell organelles and proteins

HYPERPLASIA

Definition
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Hyperplasia is an increase in the number of cells in an organ or tissue, usually resulting in increased
mass of the organ or tissue. Hyperplasia occurs in cells that are capable of dividing. Hyperplasia and
hypertrophy frequently occur together. Labile (continuously divide- squamous cells, cells of bone
marrow and lymph node) and stable cells (low rate of replication- cells of thyroid, liver and kidney) can
undergo hyperplasia, while permanent cells (do not divide- neurons) can undergo only hypertrophy.

Causes

Hyperplasia can be physiologic or pathologic.

Physiological Pathological
Hormonal Compensatory
1. Hyperplasia of female breast 1. Regeneration of liver after partial 1. Endometrial hyperplasia
at puberty and during hepatectomy 2. Benign prostatic
pregnancy and lactation 2. Regeneration of epidermis after hyperplasia
2. Hyperplasia of pregnant skin abrasion 3. Skin warts due to HPV
uterus 3. Hyperplasia of nephrons of other infection
3. Proliferation of endometrium kidney in a patient having
after menstruation undergone unilateral nephrectomy

Hyperplasia is distinct from cancer, but pathologic hyperplasia constitutes a fertile soil in which
cancerous proliferation may eventually arise.

Mechanism

Hyperplasia is growth factor–driven proliferation. In some cases tissue stem cells help.

Morphology

Affected organ is enlarged and heavy

Increase in number of cells

Increased DNA synthesis, hence increase in mitotic figures

ATROPHY

Definition

Atrophy is reduced size of an organ or tissue resulting from a decrease in cell size and number.

Causes

Atrophy can be physiologic or pathologic.

Physiological Pathological
1. Atrophy of notochord and 1. Starvation atrophy: E.g. protein malnutrition
thyroglossal duct during fetal (marasmus) is associated with the use of skeletal muscle
development as a source of energy after other reserves of energy are
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2. Atrophy of uterus post delivery depleted. This results in marked muscle wasting
3. Loss of estrogen stimulation after (cachexia).
menopause results in physiologic 2. Ischemic atrophy: slowly developing arterial occlusive
atrophy of the endometrium and results in ischemic atrophy of the tissue. E.g. atrophic
breast kidney due to atherosclerosis of renal artery
3. Neuropathic atrophy: e.g. poliomyelitis
4. Disuse atrophy: skeletal muscle atrophy in a patient on
complete bed rest or fractured bone immobilized in a
plaster cast
5. Endocrinal atrophy: hypopituitarism, hypothyroidism
6. Pressure atrophy: an enlarging benign tumor, cyst or
aneurysm can cause atrophy of the surrounding
uninvolved tissues.

Mechanism

Atrophy is due to decreased protein synthesis and increased protein degradation.

Decreased protein synthesis is due to reduced metabolic requirement.
Degradation of cellular proteins occurs through ubiquitin-proteasome pathway. Ubiquitin ligases attach
ubiquitin to cellular proteins, which are in turn destroyed by proteasomes.

Atrophy is also caused by increased autophagy (“self eating”). When the cell is starved, it eats its own
components for survival. Lysosomal enzymes digest autophagic vacuoles. Vacuoles, which are resistant
to digestion, persist as residual bodies. E.g. lipofuscin granules, when present in sufficient amounts,
impart brown discoloration to the tissue, called brown atrophy.

Morphology

Affected organ is small and shrunken.

Ultrastructure: cells show decrease in cell organelles. Autophagic vacuoles containing cellular debris
and residual bodies may be seen.

METAPLASIA

Definition

Metaplasia is a reversible change in which one differentiated cell type (epithelial or mesenchymal) is
replaced by another cell type. Cells that are sensitive to stress are replaced by cell types, which can
withstand the adverse environment.

The metaplastic epithelium is usually less specialized epithelium. For e.g. with squamous metaplasia in
respiratory epithelium, mucus secretion and ciliary action of the columnar epithelium is lost. Moreover,
in most circumstances, it represents an undesirable change. It may initiate malignant transformation in
metaplastic epithelium.

Types

Metaplasia can be epithelial or mesenchymal.

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Epithelial metaplasia can be squamous or columnar. Squamous metaplasia is most common.

Squamous metaplasia Columnar metaplasia

1. Pseudostratified columnar ciliated epithelium of the 1. Barrett esophagus: esophageal squamous
bronchus is replaced by squamous epithelium in epithelium is replaced by intestinal-like
chronic smokers columnar cells under the influence of
2. Columnar endocervical epithelium replaced by refluxed gastric acid. Adenocarcinomas
squamous epithelium with age and in prolapsed may arise in these regions.
uterus 2. Intestinal metaplasia in gastric ulcers
3. Stones in the excretory ducts of the salivary glands,
pancreas, or bile ducts may replace columnar
epithelium
4. Vitamin A (retinoic acid) deficiency induces
squamous metaplasia in the respiratory epithelium

Mesenchymal metaplasia can be osseous or cartilaginous.

Osseous metaplasia Cartilaginous metaplasia

1. Monckeberg’s medial calcific sclerosis: calcification 1. In healing of fractures, cartilaginous
in arterial wall in old age metaplasia may occur due to undue
2. Myositis ossificans- ossification in soft tissue mobility
3. Ossification in scar of chronic inflammation
4. Ossification in leiomyoma (benign smooth muscle
tumor of uterus)

Mechanism

Metaplasia does not result from a change in the cell that already exists but due to reprogramming of
stem cells that exist in normal tissues. Differentiation of stem cells to a particular lineage occurs under
the influence of cytokines, growth factors, and extracellular matrix components.

DIFFERENCE BETWEEN METAPLASIA & DYSPLASIA

Metaplasia Dysplasia
Metaplasia is a reversible change in which one Defined as disorderly non-neoplastic proliferation
differentiated cell type is replaced by another cell of the epithelium
type. Replacing cell type is more suited to a
change in environment.
Always found in association with tissue damage, It is a pre-malignant condition
repair, and regeneration. It may initiate malignant
transformation
Metaplasia can be epithelial (squamous or Characterized by pleomorphism, hyperchromasia,
columnar) or mesenchymal (osseous or high N/C ratio, loss of architecture, mitosis are
cartilaginous) increased and at abnormal locations
E.g. squamous metaplasia of endocervical cells, E.g. dysplasia of ectocervix
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Barrett esophagus

CELL INJURY

CAUSES OF CELL INJURY

Cells can be broadly injured by genetic or acquired causes.

Genetic causes

Congenital malformations associated with Down syndrome, sickle cell anemia, inborn errors of
metabolism are some of the examples of genetic abnormalities.

Acquired causes

Hypoxia:
It causes cell injury by reducing aerobic oxidative respiration. Causes of hypoxia: include
1. reduced blood supply (ischemia)
2. inadequate oxygenation of blood due to cardiorespiratory failure
3. decreased oxygen-carrying capacity of blood, as in anemia, carbon monoxide poisoning or after
severe blood loss

Physical agents: Mechanical trauma, extremes of temperature, sudden changes in atmospheric pressure,
radiation, and electric shock

Chemicals and drugs:

1. oxygen in high concentration
2. strong acid and alkali
3. hypertonic glucose and salt
4. poisons: arsenic, cyanide, mercury
5. environmental and air pollutants
6. insecticides and pesticides
7. industrial and occupational hazards: e.g. asbestos
8. alcohol and narcotics
9. therapeutic drugs

Infectious agents: Rickettsiae, viruses, bacteria, fungi, parasites

Immunologic reactions: Hypersensitivity reaction, anaphylaxis and autoimmune disorders are examples
of immunologic tissue injury.

Nutritional imbalances:
Deficiency or excess of nutrients may result in nutritional imbalance.
Nutritional deficiency: protein-calorie deficiencies (marasmus, kwashiorkar), vitamin deficiency,
anorexia nervosa (self-induced starvation)
Nutritional excess: cause atherosclerosis, obesity, heart disease, hypertension

Aging: Cellular aging leads to impaired ability of cells to undergo replication and repair leading to cell
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death.

Psychogenic diseases

Iatrogenic causes

Idiopathic causes

MECHANISM (PATHOGENESIS) OF CELL INJURY

The mechanisms responsible for cell injury are complex. Cellular response to injury depends on:
1. the nature of injury, its duration and severity
2. type, state and adaptability of the injured cell
3. cell's nutritional, hormonal status and its metabolic needs

Biochemical mechanisms that contribute to cell injury are:

Depletion of ATP

ATP is required by cell for synthetic and degradative processes.

ATP is produced by oxidative phosphorylation of ADP in mitochondria and anaerobic glycolytic

pathway. Hypoxia/toxins cause mitochondrial damage and reduce production of ATP by oxidative
pathway. Depletion of ATP to 5% to 10% of normal levels has widespread effects on cell:

Na +, K + -ATPase pump: Activity of the pump is reduced. Failure of this causes sodium to enter and
potassium to diffuse out of cell. The net gain of solute is accompanied by gain of water, causing cell
swelling, dilation of ER, loss of microvilli and bleb formation.

Anaerobic glycolysis: Hypoxia causes increased rate of anaerobic glycolysis, which results in the
accumulation of lactic acid. This reduces intracellular pH, causing clumping of nuclear chromatin.

Failure of Ca2+ pump: leads to influx of Ca2+ damaging cellular components.

Reduction of protein synthesis: prolonged ATP depletion detaches ribosomes from rough ER and
dissociation of polysomes. This results in reduced protein synthesis and increased lipid deposition.

Unfolded protein response: In cells deprived of oxygen or glucose, proteins become misfolded, which
culminate in cell injury and even death.

Mitochondrial damage

Increased cytosolic Ca2+, reactive oxygen species and lipid peroxidation can damage mitochondria. Two
major consequences of mitochondrial damage are:
1. Opening of mitochondrial permeability transition pore: leads to failure of oxidative
phosphorylation and hence ATP depletion.
2. Release of pro-apoptotic proteins: Mitochondria contain proteins capable of activating apoptotic
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 pathway (cytochrome c and caspases) between its inner and outer membrane. Damage to the
outer membrane releases these proteins to the cytosol causing death by apoptosis.

Influx of calcium and loss of calcium homeostasis

Cytosolic free calcium level is low (-0.1 μmol) as compared to extracellular levels (1.3 mmol).
Intracellular calcium is stored in mitochondria and ER. Ischemia and certain toxins increase cytosolic
calcium by:
1. releasing Ca2+ from intracellular stores
2. increasing influx across the plasma membrane

Increased cytosolic calcium causes cell injury by:

1. Opening mitochondrial permeability transition pore, leading to failure of ATP generation
2. Activating enzymes with deleterious effects. E.g.
a. phospholipases- cause membrane damage
b. proteases- break membrane and cytoskeletal proteins
c. endonucleases- fragment DNA and chromatin
d. ATPases- hasten ATP depletion
3. Result in induction of apoptosis by activation of caspases

Accumulation of oxygen-derived free radicals (oxidative stress)

Free radicals are chemical species that have a single unpaired electron in their outer orbit. Free radicals
also initiate autocatalytic reactions, whereby molecules with which they react are themselves converted
into free radicals. Free radical injury is particularly common with chemical and radiation injury,
ischemia-reperfusion injury, cellular aging, and microbial killing.

Reactive oxygen species (ROS) (a free radical) are produced normally in cells during mitochondrial
respiration and ATP production but they are degraded and removed by cellular defense systems. When
the production of ROS increases or degradation reduces, the cell is said to be in oxidative stress.

Mechanisms of generation of free radicals:

1. Reduction-oxidation (redox) reactions: It occurs during normal mitochondrial metabolism, where
molecular oxygen is reduced in mitochondria to produce water. The reaction liberates toxic
intermediates like superoxide radicals (O2Ÿ), H2O2 and hydroxyl radicals (ŸOH).
2. Transition metals: like iron and copper also accept and donate free radicals. E.g. Fenton reaction:
Fe2+ (Ferrous) + H2O2 à Fe3+ (Ferric) + OH- + OHŸ
3. Ionizing radiation: Ultraviolet light and X-rays can hydrolyze water into hydroxyl (ŸOH) and
hydrogen (HŸ) free radicals.
4. Enzymatic metabolism of exogenous chemicals or drugs: can generate free radicals that are not
ROS. E.g. CCl4 can generate CCl3 (free radical).
5. Nitric oxide (NO): produced by endothelial cells, macrophages, neurons, and other cells, can act
as a free radical and also get converted to highly reactive peroxynitrite anion (ONOO-), NO2 and
NO3-.

Mechanisms of removal of free radicals:

Free radicals are inherently unstable. They either decay spontaneously or are removed by
nonenzymatic/enzymatic mechanisms, thus minimizing cell injury.
1. Antioxidants: like lipid-soluble vitamins E and A, ascorbic acid and glutathione in cytosol either
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 block the initiation of free radical or degrade them.
2. Levels of these reactive metals like iron and copper are minimized by binding them to storage
and transport proteins like transferrin, ferritin, lactoferrin, and ceruloplasmin.
3. Enzymatic breakdown of free radicals:
a. Catalase: decomposes H2O2 (2H2O2 à O2 + 2H2O)
b. Superoxide dismutases (SODs): convert O2Ÿ to H2O2 (2 O2Ÿ + 2H à H2O2 + O2).
c. Glutathione peroxidase: catalyzes the breakdown of free radicals by
i. H2O2 + 2GSH à GSSG + 2H2O or
ii. 2OH + 2GSH à GSSG + 2H2O

Pathologic Effects of Free Radicals:

Free radical injury is caused by the following three reactions:
1. Lipid peroxidation in membranes: In the presence of O2, free radicals may cause peroxidation of
lipids within plasma and organellar membranes. The peroxides, initiate autocatalytic chain
reaction, resulting in extensive membrane damage.
2. Oxidative modification of proteins: Free radicals promote oxidation and cross-linkages of
proteins. This damages enzymes and structural proteins and enhances degradation of unfolded or
misfolded proteins.
3. Lesions in DNA: Free radicals are capable of causing single- and double-strand breaks in DNA,
cross-linking of DNA strands, and formation of adducts. Oxidative DNA damage has been
implicated in cell aging and in malignant transformation of cells.

Defects in membrane permeability

Mechanisms of Membrane Damage:

1. Reactive oxygen species: cause injury to cell membranes by lipid peroxidation.
2. Decreased phospholipid synthesis: ATP production is reduced due to defective mitochondrial
function or hypoxia. Decreased ATP synthesis affects all cellular membranes.
3. Increased phospholipid breakdown: Severe cell injury leads to increased degradation of
membrane phospholipids, due to activation of endogenous phospholipases by increased levels of
cytosolic and mitochondrial Ca2+. Phospholipid breakdown leads to the accumulation of lipid
breakdown products, including unesterified free fatty acids, acyl carnitine, and
lysophospholipids, which have a detergent effect on membranes.
4. Cytoskeletal abnormalities: Cytoskeletal filaments connect plasma membrane to cell interior.
Activation of proteases by increased cytosolic calcium damages them. This leads to detachment
of cell membrane from cytoskeleton, rendering it susceptible to stretching and rupture.

Consequences of Membrane Damage:

1. Mitochondrial membrane damage: results in opening of the mitochondrial permeability
transition pore leading to decreased ATP and release of proteins that trigger apoptotic death.
2. Plasma membrane damage: results in loss of osmotic balance and influx of fluids and ions.
3. Injury to lysosomal membranes: Lysosomes contain RNases, DNases, proteases, phosphatases,
glucosidases, and cathepsins. Activation of these enzymes leads to enzymatic digestion of
proteins, RNA, DNA, and glycogen, and the cells die by necrosis.

Damage to DNA and proteins

Cells have mechanisms that repair damage to DNA, but if this damage is too severe to be corrected, the
cell initiates a suicide program that results in death by apoptosis. A similar reaction is triggered by
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improperly folded proteins, which may be the result of inherited mutations or external triggers such as
free radicals.

REVERSIBLE CELL INJURY

Cellular swelling (hydropic change), hyaline change, mucoid change and fatty change are the common
forms of reversible cell injury.

Cellular swelling (hydropic change, vacuolar degeneration):

Cellular swelling is the most common and first manifestation of almost all forms of cell injury.

Etiology

It is the earliest change following cell injury by bacterial toxins, chemicals, poisons, burns, high fever,
etc.

Pathogenesis

There is failure of sodium and potassium pump at the plasma membrane level. This leads to
accumulation of sodium and loss of potassium. Sodium is followed by water in the cell to maintain the
iso-osmotic condition. In addition there is influx of calcium.

Morphological features

Grossly: The affected organ is enlarged and pale. The cut surface bulges outward.
Microscopically:
1. Light microscopic changes:
• Small clear vacuoles are seen within the cytoplasm, which represent distended ER; hence the
term vacuolar degeneration.
• Cells show increased eosinophilic staining
• Nucleus appears pale
2. Ultrastructural changes:
• Plasma membrane shows blebbing, blunting, and loss of microvilli
• Mitochondrial changes: swelling and small amorphous densities
• Dilation of ER, with detachment of polysomes
• Intracytoplasmic myelin figures may be present
• Nuclear alterations, with disaggregation of granular and fibrillar elements

Hyaline change:

Hyaline means glassy. It describes glassy, homogenous, eosinophilic appearance of proteinaceous

material on H&E section and does not refer to any particular substance. Hyaline change can be
intracellular or extracellular.

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Intracellular hyaline

1. Hyaline droplets in proximal tubular epithelium in proteinuria

2. Hyaline degeneration of rectus abdominalis muscle (Zenker’s degeneration) in typhoid fever
3. Mallory hyaline body are intermediate filaments in hepatocytes seen in alcoholic liver disease
4. Hyaline inclusions seen in viral infections
5. Russell’s bodies are excess Ig in rER of plasma cells

Extracellular hyaline

1. Hyaline degeneration in leiomyoma

2. Hyalinised old scar
3. Hyaline atherosclerosis of renal vessels in HT and DM
4. Hyalinised glomeruli in chronic glomerulonephritis
5. Corpora amylasea are rounded masses of concentric hyaline material in glandular lumen in
benign prostatic hyperplasia

Fatty change (steatosis)

Definition

Fatty change is defined as abnormal accumulations of triglycerides within parenchymal cells.

Sites

Most common: liver and heart

Others: muscle, kidney

Causes

1. Toxins (CCl4), protein malnutrition, diabetes mellitus, obesity, and anoxia.

2. Fatty liver could be due to alcohol abuse and nonalcoholic fatty liver disease, associated with
diabetes and obesity

Morphology

Fatty change appears as clear vacuoles within parenchymal cells. Intracellular accumulations of water or
glycogen also produce clear vacuoles. Fat can be identified by staining frozen tissue sections with Sudan
IV or Oil Red-O, both of which impart orange-red color to lipid. Alternatively osmic acid, which is a
fixative and a stain for fat can be used. Periodic acid-Schiff (PAS) stain is used to identify glycogen,
which stains PAS magenta. When neither fat nor polysaccharide can be demonstrated within a clear
vacuole, it is presumed to contain water.

Liver: Mild fatty change may not affect the gross appearance. With progressive accumulation, the organ
enlarges, the capsule is tensed and glistening, the margins are rounded and the organ becomes yellow,
soft and greasy.

• Microvesicles: Accumulation of fat is first seen by light microscopy as small vacuoles in the

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 cytoplasm around the nucleus.
• Macrovesicles: With progression, the vacuoles coalesce, displacing the nucleus to the periphery
of the cell.
• Fatty cysts: Occasionally contiguous cells rupture and the enclosed fat globules coalesce,
producing so-called fatty cysts.
• Lipogranulomas: may appear as a reaction to extravasated fat.

Heart: Lipid in cardiac muscle may occur in two patterns:

• Tigered effect: Is seen in cases of prolonged moderate hypoxia (e.g. profound anemia). Grossly,
affected myocytes appear like yellow bands, which alternate with darker, red-brown, uninvolved
myocardium, called tigered effect.
• Uniformly affected myocytes: The other pattern of fatty change is produced by more profound
hypoxia or by some forms of myocarditis (e.g., diphtheria infection) and shows more uniformly
affected myocytes.

IRREVERSIBLE CELL INJURY

NECROSIS
Necrosis is defined as a localized area of cell death followed by degradation of the dead tissue by
hydrolytic enzymes either secreted by the dying cell or leukocytes.

Necrotic cells shows increased eosinophilia is due to loss of cytoplasmic RNA, which binds blue dye,
hematoxylin and denaturation of cytoplasmic proteins, which bind the red dye, eosin. The nuclear
changes appear in one of the three patterns:

1. Karyolysis: basophilia of the chromatin fades due to loss of DNA

2. Pyknosis (also seen in apoptosis): characterized by nuclear shrinkage and increased basophilia
3. Karyorrhexis: the pyknotic nucleus undergoes fragmentation

Morphological types of necrosis:

1. Coagulative necrosis 2. Liquefactive (colliquative) necrosis
Cause: ischemia; less commonly bacterial and Cause: bacterial or, occasionally, fungal
chemical agents infection
In this form of necrosis architecture of dead tissues In this form of necrosis dead tissue is
is preserved for a span of at least some days. transformed into a liquid viscous mass.
This occurs because ischemia not only denatures This occurs because microbes stimulate the
structural proteins but also proteolytic enzymes, accumulation of leukocytes and the liberation of
which blocks cell destruction, initially. proteolytic enzymes from these cells leading to
cell destruction.
However, later leukocytes are recruited and the
necrotic cell is digested by lysosomal enzymes of the
leukocytes and removed by phagocytosis, leaving
granular debris.
Grossly, the tissue is pale, firm and slightly swollen. Grossly, the affected area is soft with liquefied
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Later yellowish, soft and shrunken
Microscopically, the hallmark is ‘tombstones’, i.e.
outline of cells are retained but cytoplasmic and
nuclear details are lost. The cells are swollen, more
eosinophilic and anucleate.
E.g. coagulative necrosis occurs in all organs except
brain. Most commonly affected organ(s): heart,
kidney, spleen
center containing necrotic debris. Later cyst wall
is formed.
Microscopically, center contains necrotic debris
with macrophages filled with phagocytosed
material. The cyst wall is formed by formed by
blood vessels, inflammatory cells and gliosis (in
brain) and fibroblasts (in abscess).
E.g. necrosis in brain and abscess (pus)

3. Caseous necrosis The term “caseous” (cheese-like) is derived from the friable white appearance of the area of
necrosis. It is seen in tuberculous infection. It combines features of both coagulative and liquefactive necrosis. It
is seen in tuberculosis, syphilis and fungal infections like histoplasmosis, cryptococcosis, coccidiodomycosis.

Grossly, the necrotic focus resembles cheese- soft, granular, white to yellowish.

Microscopically, the necrotic area comprises of structureless eosinophilic material with scattered
granular deposits of disintegrated nuclei. This is surrounded by chronic granulomatous inflammation
comprising of epithelioid cells (modified macrophages with slipper-shaped vesicular nuclei) interspersed
with Langhan’s and foreign body giant cells and rimmed with lymphocytes.

4. Fat necrosis occurs in fat-rich anatomic locations of the body, e.g. traumatic fat necrosis of breast and
mesenteric fat necrosis in acute pancreatitis.

Acute pancreatitis: In acute pancreatitis, activated pancreatic lipases are released from the acinar cells of
injured or inflamed pancreas into the substance of the pancreas and the peritoneal cavity. Lipases liquefy
the membranes of fat cells in the peritoneum and split triglyceride esters contained within fat cells. The
fatty acids, so derived, may combine with calcium, called fat saponification or calcium soap.

Grossly, the area is yellowish-white and firm. Formation of calcium soaps imparts chalky white color.

Microscopically, necrotic fat cells have shadowy outlines or cloudy appearance. They are surrounded by
inflammatory cells. Calcium soaps are identified as amorphous granular basophilic material.

5. Fibrinoid necrosis: is seen in immune reactions involving blood vessels. Deposits of these “immune
complexes,” together with fibrin that leaks out of vessels, result in a bright pink and amorphous
appearance in H&E stains, called “fibrinoid” (fibrin-like). E.g. immune complex vasculitis (polyarteritis
nodosa), malignant hypertension, Arthus reaction (type III hypersensitivity reaction), peptic ulcer.

Microscopically, it is characterized by brightly eosinophilic hyaline-like deposition in vessel wall.

Necrotic debris is surrounded by nuclear debris of neutrophils (leucocytoclasis). Local hemorrhage may
occur due to rupture of blood vessel.

APOPTOSIS

Definition

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Apoptosis is a form of coordinated and internally programmed cell death having significance in a variety
of physiologic and pathologic conditions. The process of apoptosis was discovered in 1972. Apoptosis
means, “falling off” in Greek.

Causes of apoptosis

Apoptosis occurs normally during development and throughout adulthood, and removes unwanted, aged
or potentially harmful cells.

Apoptosis in physiologic situations:

1. Apoptosis is often used interchangeably with “programmed cell death.” Programmed destruction
of cells occurs during embryogenesis, implantation, organogenesis, developmental involution,
and metamorphosis.
2. Involution of hormone-dependent tissues upon hormone withdrawal. E.g. endometrial cell
breakdown during menstrual cycle, regression of lactating breast after weaning, prostatic atrophy
after castration
3. Involution of thymus in early age
4. Death of neutrophils after acute inflammation and lymphocytes after immune response

Apoptosis in pathologic conditions:

Apoptosis eliminates cells that are injured beyond repair without eliciting a host reaction.
1. DNA damage: Radiation, cytotoxic anticancer drugs, and hypoxia can damage DNA. Apoptosis
eliminates these cells, thus preventing them from getting mutated and resulting in malignant
transformation.
2. Accumulation of misfolded proteins: Damage caused by free radicals leads to formation of
misfolded proteins. Excessive accumulation of misfolded proteins in the ER is called ‘ER stress’.
Apoptosis caused by accumulation of misfolded proteins is the basis of several degenerative
diseases of CNS like Alzheimer’s disease, Parkinson’s disease and chronic infective dementia.
3. Cell death by cytotoxic T cells in GVHD and transplant rejection
4. Progressive depletion of CD4+T cells in the pathogenesis of AIDS
5. Cell death in viral hepatitis e.g. Councilman bodies in viral hepatitis
6. Pathologic atrophy in parenchymal organs after duct obstruction, such as occurs in the pancreas,
parotid gland, and kidney.

Morphologic and biochemical changes in apoptosis

Cell shrinkage: Cell is smaller in size; cytoplasm is dense, organelles are tightly packed

Chromatin condensation (pyknosis): the most characteristic feature of apoptosis. The chromatin
aggregates peripherally, under the nuclear membrane. Nucleus may break up, producing two or more
fragments, called karyorrhexis.

In H&E section, apoptotic cell appears as a round or oval mass of intensely eosinophilic cytoplasm with
fragments of dense nuclear chromatin.

Cytoplasmic blebs and apoptotic bodies: apoptotic cell shows extensive surface blebbing, which
fragment into membrane-bound apoptotic bodies composed of cytoplasm and tightly packed organelles,
with or without nuclear fragments.

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Plasma membrane: remains intact until last stages

Phagocytosis: apoptotic cells and bodies are rapidly ingested by phagocytes and degraded by lysosomal
enzymes. Because of quick phagocytosis, apoptosis does not elicit any inflammatory response.

Mechanisms of apoptosis

The process of apoptosis is divided into an initiation phase, during which some caspases become
catalytically active, and an execution phase, during which other caspases trigger the degradation of
critical cellular components. The dead apoptotic cell develops membrane changes, which promotes their
phagocytosis.
1. Initiation phase: occurs by signals from two distinct pathways:
a. Intrinsic or mitochondrial pathway: It is the major mechanism of apoptosis. In health, anti-
apoptotic proteins Bcl-2, Bcl-x, and Mcl-1 are released thus preventing cell death.

When cells are deprived of survival signals or their DNA is damaged, or misfolded proteins
induced ER stress, pro-apoptotic proteins Bax and Bak are released. Sensors, “BH3-only
proteins”- Bim, Bid, and Bad are activated, which in turn activate pro-apoptotic proteins. Bax
and Bak create channels in the mitochondrial membrane that allow proteins to leak out into the
cytoplasm. One of the major proteins released in the cytoplasm is Cytochrome c, which binds to
Apaf-1 (apoptosis-activating factor-1) to form apoptosome. This complex binds to caspase-9.

Other mitochondrial protein released into the cytoplasm is Smac/DIABLO, which bind to
inhibitors of apoptosis (IAPs) and neutralize it. The normal function of the IAPs is to block the
activation of caspases. Thus, the neutralization of IAPs permits the initiation of a caspase
cascade.

b. Extrinsic or death receptor–initiated pathway: This pathway of cell death is initiated by

activation of death receptors on the cell membrane; e.g. Fas (CD95). The ligand for Fas, FasL is
expressed on T cells. When FasL binds with Fas on outer surface of cell membrane, 3 or more
molecules of Fas are brought together. The cytoplasmic death domain of Fas now forms a
binding site for FAS-associated death domain (FADD) protein. FADD causes activation of
caspase-8 and -10 leading to activation of executioner caspases.

2. Execution phase: Activation of caspase-9 by mitochondrial pathway and caspase-8 and -10 by death
receptor pathway leads to activation of executioner caspases, -3 and -6. They cleave inhibitor of
DNase, thus making DNase active; which in turn causes cleavage of DNA. They act on other cellular
components causing cytoskeletal damage, disruption of ER, and mitochondrial damage.

3. Phagocytosis: The apoptotic cells and their fragments undergo several changes in their membranes
that actively promote their phagocytosis so they are cleared before they can elicit inflammatory
response. In healthy cells phosphatidylserine is present on the inner leaflet of the plasma membrane,
but in apoptotic cells this phospholipid “flips” out and is expressed on the outer layer of the
membrane, where it is recognized by several macrophage receptors.

Apoptotic cells may secrete soluble factors, e.g. thrombospondin, which are recognized by
phagocytes.

Apoptotic bodies may become coated with C1q, which are recognized by phagocytes.
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 Macrophages themselves may produce proteins that bind to apoptotic cells.

DIFFERENCE BETWEEN APOPTOSIS AND NECROSIS

FEATURE APOPTOSIS NECROSIS

Definition Programmed and coordinated cell Cell death along with degradation of
death tissue by hydrolytic enzymes
Causative agent Physiological & pathological Hypoxia, toxins
processes
Morphology 1. No inflammatory reaction 1. Inflammatory reaction always
present
2. Death of single cells 2. Death of many adjacent cells
3. Cell shrinkage 3. Cell swelling initially
4. Cytoplasmic blebs on membrane 4. Membrane disruption
5. Apoptotic bodies 5. Damaged organelles
6. Pyknosis (chromatin 6. Karolysis (nuclear fading),
condensation) pyknosis (chromatin
condensation) and/or karyorrhexis
(nuclear fragmentation)
7. Phagocytosis of apoptotic bodies 7. Phagocytosis of cell debris
Molecular changes 1. Lysosomes and other organelles are 1. Lysosomal breakdown with
intact liberation of hydrolytic enzymes
2. Initiated by loss of signal for 2. Initiated by hypoxia, chemicals,
normal cell survival physical agents, microbes
3. Cell death occurs by mitochondrial 3. Cell death by ATP depletion,
and death receptor induced membrane damage and free radical
activation of caspases injury

CHANGES AFTER CELL DEATH

Two types of pathological changes may superimpose following cell injury- gangrene and pathologic
calcification.

GANGRENE
Gangrene is necrosis of tissue associated with superadded putrefaction.

DRY GANGRENE WET GANGRENE

Onset: insidious Onset: rapid
Usually seen in toes and foot Usually seen in moist tissues and organs like
bowel, lung, mouth, cervix, vulva etc.
Cause: Ischemia (blockage of arterial supply). Cause: blockage of both arterial supply and venous
Usually starts in one of the toes, which is farthest drainage. The affected part is stuffed with blood,
from blood supply, containing so little blood that which favors rapid growth of bacteria.
invading bacteria also find it difficult to survive.
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Grossly, the affected part is dry, shrunken, dark Grossly, the affected part is soft, swollen, putrid
black resembling foot of mummy. Black color is and dark (same mechanism as in dry gangrene).
due to formation of iron sulfide (hemoglobin
released from hemolysed RBC + H2S produced by
bacteria)

Clear line of demarcation between gangrenous and
normal tissue
Microscopically, necrosis with smudging of tissue.
Line of demarcation shows inflammatory
granulation tissue
Prognosis: leads to spontaneous amputation of the
affected part
E.g. gangrene is distal parts of toes and feet due to
severe atherosclerosis, thromboangitis obliterans
(Berger’s disease), Raynaud’s disease, trauma,
ergot poisoning
Lacks clear line of demarcation
Microscopically, coagulative necrosis with red
blood cells, intense acute inflammation and
thrombosed blood vessels
Prognosis: has profound systemic manifestations
like septicemia and finally death
Gangrene of bowel due to strangulation, volvulus
or intussusception, diabetic foot, bed sore
Gas gangrene is a special form of wet gangrene
caused by gas-forming gram-positive anaerobic
bacteria- clostridia. The organism gains entry
through contaminated wounds especially in
muscles or through operation on colon, which
normally contains clostridia. The area becomes
crepitant due to gas bubbles of carbon dioxide.

PATHOLOGIC CALCIFICATION

Definition

Abnormal deposition of calcium salts, together with smaller amounts of iron, magnesium, and other
minerals, in cells and tissues that are normally not mineralized, is called ‘pathological (ectopic)
calcification’.

Classification

Pathological calcification is classified into dystrophic and metastatic calcification.

Dystrophic calcification:

Definition

Calcium deposition occurring in dead, dying, or degenerating tissues, with normal calcium metabolism
and normal serum calcium level is called dystrophic calcification.

Pathogenesis

The process is likened to formation of normal hydroxyapatite of bone i.e. binding of phosphate ions with
calcium ions to form calcium phosphate. It involves two steps- ‘initiation’ and ‘propagation’
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Initiation begins with tissue degeneration or necrosis

Calcium gets concentrated either extracellularly (in membrane-bound vesicles of about 200 ηm size
derived from degenerating cells) OR intracellularly within the mitochondria of degenerating cells.

Membrane damage releases membrane phospholipids along with phosphatases, which liberate phosphate
(PO4-) ions

Calcium binds with PO4- ions

Structural change occurs in Ca2+ and PO4- groups, leading to microcrystallization

Microcrystals then ‘propagate’ and perforate the membrane, depending on the concentration of Ca2+
and PO4- and inhibitors in the extracellular space

Dystrophic calcification in dead and degenerating tissues

IN DEAD TISSUE IN DEGENERATING TISSUE

1. Caseous necrosis in tuberculosis 1. Dense old scar
2. Liquefactive necrosis in abscess 2. Atheromas in aorta and coronary arteries
3. Fat necrosis resulting in formation of calcium 3. Monckeberg’s calcific sclerosis in degenerated
soaps tunica media of muscular arteries in elderly
4. Gamna Gandy bodies in chronic venous people
congestion (CVC) of spleen 4. Goiter
5. Infarcts 5. Stroma of tumors like uterine fibroid, breast
6. Thrombi, especially in veins may produce cancer, thyroid adenoma
phleboliths 6. Dystrophic calcification may appear in a
7. Hematomas in the vicinity of bones characteristic lamellated concentric form,
8. Dead parasites like hydatid cysts, schistosoma called psammoma bodies (grains of sand) or
eggs, cystisercosis calcospherites, which has diagnostic
9. Congenital toxoplasmosis of brain implications for certain tumors:
10. Microcalcification in breast cancer detected by - papillary carcinoma of thyroid
mammography - serous cystadenoma of ovary
11. ‘Stone child’: dead fetus in ectopic abdominal - meningioma
pregnancy, may get completely calcified or - Metanephric adenoma of kidney
ossified, appearing as a fossil. 7. Epidermal and pilar cysts
8. Calcification in aging or damaged heart valve
cusps can lead to stenosis
9. Asbestosis of lung: beaded dumbell shaped
forms develop upon accumulation of calcium
and iron on long slender spicules of asbestos.

Morphology

Morphologically both dystrophic and metastatic calcification resemble normal mineralization of bone.

Grossly, seen as fine or large white granules with gritty or eggshell consistency
Microscopically, seen as amorphous, basophilic granular, clumped, or lamellated pattern on H&E stain.
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Special stain: von Kossa (stains calcium black) or alizarin red S (stains calcium red)

Metastatic Calcification

Definition

Calcium deposition occurring in normal tissue, secondary to hypercalcemia, is called metastatic

calcification. The deposition is reversible, when the metabolic disturbances are corrected.

Pathogenesis

Certain tissues are prone to calcium deposition secondary to hypercalcemia. These tissues are those,
which lose acid, therefore develop an internal alkaline compartment, which predisposes them for
calcium deposition.

Causes of hypercalcemia

Excess mobilization of calcium from bone Excess absorption of calcium from gut
Hyperparathyroidism: Hypervitaminosis D:
1. Parathyroid adenoma 1. Excess intake
2. Parathyroid hyperplasia 2. Sarcoidosis
3. Chronic renal failure
Hypercalcemia: Milk-alkali syndrome:
1. Paraneoplastic syndrome produced by 1. Excess oral intake of milk
parathyroid-like hormone in SCC of lung, 2. Calcium carbonate for treatment of peptic
breast/renal/ovarian carcinoma, Adult T-cell ulcer
leukemia/lymphoma
Destruction of bone Idiopathic hypercalcemia of infancy (William’s
1. Primary tumor of BM (MM, leukemia) syndrome)
2. Diffuse skeletal metastasis
3. Accelerated bone turnover (Paget’s disease)
4. Immobilization
Renal failure causes retention of phosphate

Site of deposition

Commonly seen in the interstitial tissue of:

1. Gastric mucosa
2. Kidney (nephrocalcinosis)
3. Lung parenchymal vasculature in alveolar septa
4. Vasculature
5. Synovium

Morphology

Similar to dystrophic calcification

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 IMPORTANT QUESTIONS

SHORT NOTES:
1. Hypertrophy and hyperplasia
2. Free radicals in cell injury
3. Necrosis
4. Apoptosis
5. Describe gangrene and it’s different types.
6. Pathological calcification

SHORT ANSWERS:
(Note: Answers only to those questions not addressed in the text or written scattered are given here.
In some answers are given in detail to cover relative topics.)

1. Name two pathological causes of localized atrophy.

Ans. Cervical spondylosis, spina bifida, spinal tumor, syringomyelia, peroneal muscular atrophy,
spastic hemiplagia
2. Name two causes of unilateral limb muscle atrophy.
Ans. Neuropathic: Poliomyelitis, motor neuron disease, nerve section
Disuse: wasting of muscles of limb in plaster cast, prolonged rest
3. Give two differences between metaplasia and dysplasia.

4. Mention four free radicals.

Ans. Superoxide (O2Ÿ), hydroxyl (ŸOH), hydrogen (HŸ), CCl3, peroxynitrite anion (ONOO-), NO2
and NO3-
5. Mention two antioxidants.
Ans. Endogenous: superoxide dismutase; catalase, glutathione peroxidase, ceruloplasmin,
transferrin
Exogenous: Lipid-soluble vitamins E and A, ascorbic acid
6. Types of hyaline change with examples.

7. Morphological features of fatty change in liver and heart.

8. Mention nuclear changes seen in necrosis.

1. Karolysis: nuclear fading
2. Pyknosis: chromatin condensation
3. Karyorrhexis: nuclear fragmentation
9. Mention two differences between coagulative and colliquative necrosis.

10. Two examples of liquefactive necrosis.

Ans. Brain infarct, abscess
11. Fat necrosis

12. Morphology of an apoptotic cell.

Ans. Cell shrinkage, pyknosis/karyorrhexis, cytoplasmic blebs, apoptotic bodies, phagocytosis
13. Name pro-apoptotic and anti-apoptotic proteins.
Ans. Pro-apoptotic proteins: Bax and Bak

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 Anti-apoptotic proteins: Bcl-2, Bcl-x, and Mcl-1
14. Mention two differences between necrosis and apoptosis.

15. Give two differences between dry and wet gangrene.

16. Mention any two differences between dystrophic and metastatic calcification.

17. What is dystrophic calcification? Give two causes of dystrophic calcification.

18. Two clinical conditions in which metastatic calcification occurs.

19. List exogenous and endogenous pigments.

Ans. Exogenous pigment: carbon (coal dust), tattoo
Endogenous pigment: lipofuscin, melanin, hemosiderin, ferritin

The End

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