Cell Injury

Gul Fatima/ [Link]

Lecturer, SICP, Lodhran
 Homeostasis
“When a cell maintain its normal structure and function it is called as
homeostasis”.
When a cell encounter physiologic stresses or pathologic stimuli, it
undergo three processes.
• It maintain its hemostasis.
• If stimuli become excessive, then it undergo Adaptation. (Change
in structure but function is same).
For example.
- Atrophy (↓ in size of cell).
- Hypertrophy (↑ in size of cell).
- Hyperplasia (↑ in number of cells).
• If the adaptive capability is exceeded cell injury develops
Cellular Responses to Stress and Noxious Stimuli
 Cell Injury
Cell injury results when cells are stressed so severely that they are no
longer able to adapt or when cells are exposed to inherently damaging
agents or suffer from intrinsic abnormalities (e.g., in DNA or proteins).

Cell injury is of two types:

1. Reversible injury: the stage of cell injury at which the deranged

function and morphology of the injured cells can return to normal
if the damaging stimulus is removed

2. Irreversible injury: With continuing damage, the injury becomes

irreversible, at which time the cell cannot recover and it dies.
 Factors of cell injury
There are following factors are involved in cell injury;
- Oxygen Deprivation

- Physical Agents

- Chemical Agents and Drugs

- Infectious Agents

- Immunologic Reactions

- Genetic Derangements

- Nutritional Imbalances
 Oxygen Deprivation
• The commonest cause in clinical practice is hypoxia where the cell is
damaged because aerobic respiration is diminished.

Reasons of Hypoxia

• Reduction in the blood supply to a tissue, as occurs in myocardial

infarction.

• Impairment in blood supply, ischemia,

• Hypoxia can also occur when there is a reduction in the overall

oxygenation of the blood as occurs in cardiorespiratory failure and in
carbon monoxide poisoning.
 Physical Agents
• Physical agents which cause cell injury include;

- Extremes of temperature

- Atmospheric pressure

- Radiation

- Direct and Indirect mechanical trauma

- Electrical currents.
 Chemical agents and poisons
• Many chemical agent may cause cell injury

• If glucose, salts or even water absorbed or administered in excess, can

so derange the osmotic environment that cause cell injury or death.

• Agents commonly known as poisons cause severe damage at the

cellular level by altering membrane permeability, osmotic homeostasis,
or the integrity of an enzyme or cofactor, and exposure to such poisons
can culminate in the death of the whole organism.

- include air pollutants, insecticides, CO, ethanol etc.

• Therapeutic drugs can cause cell or tissue injury in a susceptible patient

or if used excessively or inappropriately
 Infectious agents
• Agents of infection range from submicroscopic viruses to meter-
long tapeworms; in between are the rickettsia, bacteria, fungi, and
protozoans.
 Immunological Infection
• Immunological reactions are also a cause of cell injury

- when there is an exaggerated response to a foreign body

(Anaphylaxis)

- When there is breakdown of the normal tolerance mechanisms to

self-antigens (Autoimmune Disease).
 Genetic abnormalities
• Genetic diseases can lead to cell injury by a variety of
mechanisms.
• Chromosomal abnormalities which lead to major congenital
malformations (e.g., trisomy 21)
• Single base mutations in a gene leading to a protein product which
is abnormally folded, and which cannot be exported from the cell
(e.g. α1-antitrypsin deficiency).
• Many inherited metabolic abnormalities are caused by there being
a genetically determined enzyme defect or deficiency; these may
have consequences for only one tissue or organ system but most
commonly have systemic effects.
• Inborn errors of metabolism – G6PD deficiency
 Nutritional imbalances
• Nutritional imbalances can also cause cell injury
Nutritional deficiencies:
• Protein deficiency kwashiorkor is a malnutrition characterized
by severe protein deficiency.
• Iron-deficiency anemia can cause red blood cells to appear
small, oval-shaped, and pale
Nutritional Excess
• Excess of nutrition cause Obesity and the associated metabolic
syndrome are a major cause of serious disease.
• Diets rich in animal fat are strongly implicated in the
development of atherosclerosis as well as in increased
vulnerability to many disorders, including cancer.
 Aging
• Cellular senescence leads to alterations in replicative and repair
abilities of individual cells and tissues.

• All of these changes result in a diminished ability to respond to

damage and, eventually, the death of cells and of the organism.
 Morphology of cell injury
• Reversible injury

• Cellular swelling

• Fatty change

• Irreversible injury

• Cytoplasmic change

• Nuclear change
 Reversible injury-Cellular Swelling
• It is first sign of injury
• Failure of energy dependent ion pumps in the plasma membrane
leading to an inability to maintain ionic and fluid homeostasis.
• Microscopically it will not appear at tissue level, but it can appear at
the level of organ level.
• Microscopic examination
• small, clear vacuoles within the cytoplasm;
• distended and pinched off segments of the endoplasmic reticulum
(ER).
• It is also called hydropic change or vacuolar degeneration.
• At organ level it cause
• Pallor- pale appearance
• Increased turgor
• Increased organ weight
 Reversible injury-Fatty Change
• It occurs in hypoxic injury and various forms of toxic or metabolic
injury

• It can be reversed if cause is removed or corrected, but if the injury

persists or becomes severe, it can progress to irreversible injury.

• It form by the appearance of small or large lipid vacuoles in the

cytoplasm.

• Found in cells that involved in fat metabolism like hepatocytes,

myocardial cells.

• Injured cells may also show increased eosinophilic staining, which

becomes much more pronounced with progression to necrosis.
 Intracellular Changes of Reversible
Injury

• Plasma membrane alterations blebbing, blunting, or distortion of

microvilli, and loosening of intercellular attachments

• Mitochondrial changes swelling and the appearance of

phospholipid-rich amorphous densities

• Dilation of the ER  detachment of ribosomes and dissociation of

polysomes

• Nuclear alterations clumping of chromatin

 Irreversible cell injury
• It includes;
- Necrosis  uncontrolled cell death
- Apoptosis  programmed cell death
 Morphology of Necrosis
• Denaturation intracellular proteins

• Enzymatic digestion

• Cell loss its membrane integrity leaking of cell content 

inflammation in the surrounding tissue.

• The enzymes that digest the necrotic cell are derived from the
lysosomes of the dying cells themselves and from the lysosomes
of leukocytes that are called in as part of the inflammatory
reaction.
 Morphology of Necrosis-
cytoplasm
• Increased eosinophilia

• Digestion of cytoplasmic organelles by enzymes causes the cytoplasm

becomes vacuolated and appears moth-eaten.

• Dead cells may be replaced by large, whorled phospholipid masses called

myelin figures that are derived from damaged cell membranes.

• Precipitation of phospholipid

• phagocytosed by other cells

• degraded into fatty acids calcification of such fatty acid residues

results in the generation of calcium soaps.
Morphology of Necrosis-nucleus
• Nuclear changes appear in one of three patterns
• Pyknosis  nuclear shrinkage and increased basophilia.
dense, darkly-staining mass

• Karyorrhexis  fragmentation of pyknotic nucleus

• Karyolysis  loss of DNA because of enzymatic degradation by

due to endonucleases so the basophilia of the chromatin fades
 Fates of necrotic cells

• Necrotic cells may persist for some time or may be digested by

enzymes and disappear.

• Dead cells may be replaced by myelin figures, which are either

phagocytosed by other cells or further degraded into fatty acids.

• These fatty acids bind calcium salts, which may result in the dead
cells ultimately becoming calcified.
 Patterns of Tissue Necrosis
• Coagulative necrosis
• Liquefactive necrosis
• Gangrenous necrosis
• Caseous necrosis
• Fat necrosis
• Fibrinoid necrosis
 Coagulative necrosis
• A type of necrosis that occurs when the structure of dead tissues is
preserved for a period of time
or
• A type of necrosis caused by irreversible focal injury, mostly from
sudden cessation of blood flow, and less often from bacterial and
chemical agents
• It is commonly seen in
- ischemic injury which results from a lack of blood flow to an
area of the body, such as in the case of a blood clot or arterial
occlusion. It is not found in brain.
- Lungs  pulmonary infarction The necrosis found in
brain is liquefactive
- Kidney renal infarction necrosis
 Pathogenesis
• Injury causes
- Denaturation of proteins and enzymes (accumulation of lactic acid
because of ischemia, heavy metals and ionizing radiation)
- blocking proteolysis of the dead cells;
- Eosinophilic and anucleate cells may persist for days or weeks.
• At site of necrosis
- Leukocytes are recruited
- dead cells are digested by the action of lysosomal enzymes
- cellular debris is then removed by phagocytosis.
 Morphologic Features
• Grossly:
- early stage pale, firm, and slightly swollen.
- With progression more yellowish, softer, and shrunken.
• Microscopically:
• Hallmark of coagulative necrosis is the conversion of normal cells
into their ‘tombstones or ghost-like outline’.
• Cell outline remain intact but lost it cytoplasmic and nuclear
detail
• Necrotic cell swollen and become more eosinophilic
• Nucleus changes  pyknosis, karyorrhexis and karyolysis
• Digestion and liquefaction of fail to occur
• Necrotic area is infiltrated by inflammatory cells and the dead
cells are phagocytosed leaving granular debris
 Examples

• Myocardial infarction

• Zenker’s degeneration necrosis (occurs in striated muscle fibers)

 Liquefactive necrosis
• Liquefaction or colliquative necrosis
occurs commonly due to ischemic
injury and bacterial or fungal
infections.
or
• A type of necrosis that occurs when
the affected tissue undergoes rapid
dissolution or liquefaction.
 Pathogenesis
• Neutrophils or macrophages release hydrolytic enzymes that causes
Transformation of the tissue into a liquid viscous mass.

• Examples: infarct brain and abscess cavity.

• The digested tissue is removed by phagocytes.

• The necrotic material is frequently creamy yellow because of the

presence of dead leukocytes and is called pus.
Pancreatic Liquefactive necrosis

• Liquefactive necrosis can also occur in other tissues of the body, such
as in the pancreas in cases of acute pancreatitis,

• the pancreatic enzymes break down the pancreatic tissue and

surrounding fat into a liquid or semi-liquid form
 Morphologic Features
Grossly

• the affected area is soft with liquefied center containing necrotic

debris. Later, a cyst wall is formed.

Microscopically,

• the cystic space contains necrotic cell debris and macrophages

filled with phagocytosed material.

• The cyst wall is formed by proliferating capillaries, inflammatory

cells, and gliosis (proliferating glial cells at site of injury) in the
case of brain and proliferating fibroblasts in the case of abscess
cavity
The liver shows a small Abscess here
filled with many neutrophils. This abscess
is an example of localized liquefactive
necrosis.  Grossly, such an abscess appears
yellow to tan because it is filled with pus

At high magnification, liquefactive

necrosis of the brain demonstrates many
Macrophages at the right which are
cleaning up the necrotic cellular debris
 Caseous necrosis
• Caseous” (cheese like) is derived from the friable white appearance of
the area of necrosis.

• A type of necrosis characterized by the formation of a distinctive soft

and white or yellowish-white tissue that resembles cheese.

• It combines features of both coagulative and liquefactive necrosis

• Caseous necrosis is often seen in

- tuberculosis,

- Hypersensitivity reaction:

- Histoplasmosis, syphilis, coccidioidomycosis

• Necrotic area appears as;
• collection of fragmented or lysed cells and amorphous
granular debris i.e. surrounded by a granulomatous
inflammatory response, which is composed of immune cells,
such as lymphocytes and giant cells that wall off the affected
tissue.
• this appearance is characteristic of a focus of inflammation
known as a granuloma.
 Morphologic Features
• Grossly

- dry cheese , soft, granular and yellowish

• Microscopically, the necrosed foci are;

- structureless,
- eosinophilic,
- contain granular debris.

• Surrounding tissue shows characteristic

- granulomatous inflammatory reaction consisting of:

- epithelioid cells (type of phagocyte) with interspersed giant cells
of Langhans ((aggregates of macrophages)
- foreign body type and peripheral mantle of lymphocytes
This is more extensive caseous
necrosis, with confluent cheesy tan
granulomas in the upper portion of
this lung in a patient with
tuberculosis.

Microscopically, caseous necrosis is

characterized by acellular pink
areas of necrosis, surrounded by a
granulomatous inflammatory process.
 Fat necrosis
• It occurs when there is damage or death of fatty tissue in the body.
• Not a specific pattern.
• Reasons: trauma, surgery, radiation therapy.
• This occurs in the terrible abdominal emergency known as acute
pancreatitis.
• Pancreatic enzymes leaked out of acinar cells  fat cell
membranes liquify in peritoneum
• Lipases split the triglyceride esters contained within fat cells.
• Free fatty acids combine with calcium  produce grossly visible
chalky white areas (fat saponification)  lesion identify by the
surgeon and the pathologist
Microscopically, the necrosed fat
cells have cloudy appearance and
are surrounded by an
inflammatory reaction. Formation
of calcium soaps is identified in
the tissue sections as amorphous,
granular and basophilic material

Microscopically, the fat necrosis consists

of steatocytes (adipocytes) that have lost
their nuclei and whose cytoplasm has a
granular pink appearance as seen on the
right. Some hemorrhage is seen at the
left in this case of acute pancreatitis.
 Fibrinoid necrosis
• deposition of fibrin-like material

• visible by light microscopy

• fibrinoid necrosis is identified by;

- brightly eosinophilic,

- hyaline-like deposition in the vessel wall.

- surrounded by nuclear debris of neutrophils (leucocytoclasis-

vascular damage caused by nuclear debris from infiltrating
neutrophils)
- Local hemorrhage may occur due to rupture of the blood
vessels
• Immune reactions;

- antigens and antibodies complexes forms

- Complexes deposited in the walls of arteries

- deposited immune complexes + fibrin (leaked out of vessels) 

produce a bright pink and amorphous appearance on H&E
preparations called fibrinoid (fibrin-like) by pathologists
 Gangrene
• Gangrene is a serious and potentially life-threatening condition
that arises when a considerable mass of body tissue undergo
necrosis.

• gangrenous or necrotizing inflammation is characterized by

primarily inflammation provoked by virulent bacteria resulting in
massive tissue necrosis.

• examples of necrotizing inflammation are:

- gangrenous appendicitis,

- gangrenous stomatitis
 Types of Gangrene
• There are 2 main forms of gangrene

- Dry Gangrene

- Wet Gangrene

• The variant form of wet gangrene called gas gangrene.

• In all types of gangrene, necrosis undergoes liquefaction by the

action of putrefactive bacteria.
 Dry Gangrene
• This form of gangrene begins in the distal part of a limb due to
ischemia.
• It is mainly due to arterial occlusion & it’s a type of Coagulative
necrosis
• Examples
- dry gangrene in the toes and feet of an old patient due to
arteriosclerosis.
- dry gangrene foot include
- thromboangiitis obliterans (Buerger’s disease)
- Raynaud’s disease
- trauma,
- ergot poisoning.
• It is usually initiated in one of the toes which is farthest from the
blood supply, containing so little blood that even the invading
bacteria find it hard to grow in the necrosed tissue.

• The gangrene spreads slowly upwards until it reaches a point

where the blood supply is adequate to keep the tissue viable.
• A line of separation is formed at this
point between the gangrenous part
and the viable part.
 Morphologic Features
• Grossly,

• the affected part is dry, shrunken and dark black, resembling

the foot of a mummy.
• It is black due to liberation of hemoglobin from hemolyzed
red blood cells which is acted upon by hydrogen disulfide
(H2S) produced by bacteria resulting in formation of black
iron sulfide.
• The line of separation also known demarcation line as
usually brings about complete separation with eventual falling
off of the gangrenous tissue if it is not removed surgically
• Histologically, there is
necrosis with smudging of
the tissue. The line of
separation consists of
inflammatory granulation
tissue
 Wet Gangrene
• Wet gangrene usually develops rapidly due to blockage of venous
(mainly) and/or arterial blood flow from thrombosis or embolism

• Wet gangrene is Coagulative necrosis progressing to Liquefactive

necrosis.

• Wet gangrene occurs in naturally moist tissues and organs such as

the mouth, bowel, lung, cervix, vulva etc.

• Diabetic foot is another example of wet gangrene due to high sugar

content in the necrosed tissue which favors growth of bacteria.
• Bed sores occurring in a bed-ridden patient due to pressure on
sites like the sacrum, buttocks and heels are the other important
clinical conditions included in wet gangrene

• The affected part is stuffed with blood which favors the rapid
growth of putrefactive bacteria. The toxic products formed by
bacteria are absorbed causing profound systemic manifestations of
septicemia, and finally death.

• The spreading wet gangrene generally lacks clear-cut line of

demarcation and may spread to peritoneal cavity causing
peritonitis
 Morphologic Features
• Grossly, the affected part is soft, swollen, putrid, rotten and dark.

• Example: bowel gangrene  commonly due to strangulated

hernia, volvulus or intussusception. The part is stained dark due to

the same mechanism as in dry gangrene.

Histologically, there is coagulative necrosis with stuffing of affected part

with blood. There is ulceration of the mucosa and intense inflammatory

infiltration. Lumen of the bowel contains mucus and blood. The line of

demarcation between gangrenous segment and viable bowel is generally not

clear-cut.
 Gas Gangrene
• It is a special form of wet gangrene

• It caused by gas-forming clostridia (gram-positive anaerobic

bacteria) which gain entry into the tissues through open
contaminated wounds, especially in the muscles, or as a
complication of operation on colon which normally contains
clostridia.

• Clostridia produce various toxins which produce necrosis and

oedema locally and are also absorbed producing profound
systemic manifestations.
 Morphologic Features
• Grossly, the affected area is swollen, edematous, painful and
crepitant due to accumulation of gas bubbles within the tissues. he
affected tissue becomes dark black and foul smelling
• Microscopically, the muscle fibers undergo coagulative necrosis
with liquefaction.

• Large number of gram-positive bacilli can be identified.

• At the periphery, a zone of leucocytic infiltration, oedema and

congestion are found.

• Capillary and venous thrombi are common.

Feature Dry Gangrene Wet Gangrene Gas Gangrene
Site Commonly limbs More common in limbs
bowel
Mechanism Arterial occlusion More commonly Gases produced by
s venous clostridium bacteria
obstruction, less
often arterial
occlusion
Macroscopy Organ dry, Part moist, soft, Organ: Red, cold,
shrunken and swollen, rottenpale, numb,
black and dark shriveled up and
auto-amputation
Putrefaction Limited due to Marked due to Marked due to
very little blood stuffing of organ bacteria and
supply with blood infiltration of gases
produced by them in
tissues
 Feature Dry Gangrene Wet Gangrene Gas Gangrene

Line of Present at the No clear line of No clear line of

demarcation junction between demarcation demarcation
healthy and
gangrenous part
Bacteria Bacteria fail to Numerous present Major cause
survive
Prognosis Generally better Generally poor Generally poor due
due to little due to profound to quick spread to
septicemia toxemia surrounding tissue
 Mechanisms of Cell Injury
(Necrosis)

• Several general principles are relevant to most forms of cell injury:

• The cellular response to injurious stimuli depends on the type

of injury, its duration, and its severity.
• The consequences of an injurious stimulus depend on the type,
status, adaptability, and genetic makeup of the injured cell
• Cell injury results from functional and biochemical abnormalities in
one or more of several essential cellular components
• Depletion of ATP

• Mitochondrial Damage

• Influx of Intracellular Calcium and Loss of Calcium Homeostasis

• Accumulation of Oxygen-Derived free radical (Oxidative stress)

• Defects in Membrane Permeability

• Damage to DNA and proteins

• Multiple biochemical alterations may be triggered by any one

injurious insult.
 1. Depletion of ATP
• The cause of ATP depletion are

• reduced supply of oxygen and nutrients,

• mitochondrial damage

• actions of some toxins (e.g., cyanide).

• The depletion of ATP has widespread effects on many critical cellular

systems.

i. Decrease in ATP  ATP-dependent sodium pumps is reduced

intracellular accumulation of sodium and efflux of potassium 
gain of solute is by iso-osmotic gain of water causing cell
swelling and dilation of the ER.
ii. increase in anaerobic glycolysis  intracellular glycogen stores
are rapidly depleted, and lactic acid accumulates, leading to
decreased intracellular pH and decreased activity of many
cellular enzymes.

iii. Failure of ATP-dependent Ca2+ pumps  increase influx of

Ca2+ damaging effects on numerous cellular components

iv. Prolonged or worsening depletion of ATP  causes structural

disruption of the protein synthetic apparatus, detachment of
ribosomes from the rough ER (RER) and dissociation of
polysomes into monosomes  reduction in protein synthesis.
there is irreversible damage to mitochondrial and lysosomal
membranes, and the cell undergoes necrosis.
 2. Mitochondrial Damage and Dysfunction

• There are following causes of Mitochondrial damage and

dysfunction;
• Hypoxia,

• Toxins

• Cytosolic Ca2+

• Oxidative stress

• Lipid breakdown product

• Mitochondrial damage may result in several biochemical
abnormalities:

• Failure of oxidative phosphorylation  depletion of ATP necrosis

of the cell.

• Abnormal oxidative phosphorylation formation of reactive oxygen

species  cause deleterious effects

• Damage to mitochondria mitochondrial permeability transition

pore (formation of a high-conductance channel in the mitochondrial
membrane)  channel open  loss of mitochondrial membrane, pH
changes and release of protein that will cause activation of apoptosis.
 3. Influx of calcium and loss of calcium
homeostasis
• Increased intracellular Ca2+ causes cell injury by several
mechanisms.
• The accumulation of Ca2+ in mitochondria results in opening of
the mitochondrial permeability transition pore.
• Increased cytosolic Ca2+ activates a number of enzymes
(phospholipases, proteases, endonucleases, and adenosine
triphosphatases (ATPases) with potentially deleterious cellular
effects
• Increased intracellular Ca2+ levels also result in the induction of
apoptosis.
 4. Accumulation of oxygen-derived free
radicals (oxidative stress)
Free Radicals
• Free radicals are chemical species with a single unpaired electron in
an outer orbital.
Properties
• Extremely unstable  Rapidly destroyed
• Autocatalytic reaction  when they react with other molecule, they
convert them into free radical
• readily react with inorganic and organic chemicals.
Types
• Oxygen derived free radical (ROS)- superoxide (O2¯)­­, H2O2, -OH
• Reactive nitrogen species/ NO derived free radicals-peroxynitrile
(ONOO¯)
• Free radicals from drug and chemicals (CCl3)
Oxidative Stress

• Increased production or decreased scavenging of ROS may lead to

an excess of free radicals, a condition called oxidative stress

• Free radical-mediated cell injury includes:

• chemical

• radiation injury,

• hypoxia,

• cellular aging,

• tissue injury caused by inflammatory cells

 Reactive oxygen species (ROS
• Reactive oxygen species (ROS) are a type of oxygen derived free
radical whose role in cell injury is well established.

• ROS have been identified as the likely cause of cell injury in

many diseases and other damaging events. These include:
• The inflammatory process

• Chemical toxicity

• Ionizing radiation

• Chemical carcinogenesis

• Aging
 Generation of ROS
• The reduction-oxidation reactions that occur during normal metabolic
processes

• Absorption of radiant energy (ionizing radiation)  form reactive

oxygen

• Rapid bursts of ROS are produced inactivated leukocytes during

inflammation

• Enzymatic metabolism of exogenous chemicals or drugs (CCl3)

• Transition metal such as iron and copper

• Cellular ageing
 Mechanism of Production ROS
Mitochondrial pathway
• When oxygen undergoes partial or incomplete reduction 
superoxide anion by oxidative enzymes found in endoplasmic
reticulum, mitochondria, plasma membrane, peroxisomes, and
cytosol (O2  O2¯)
• Superoxide anion (O2¯)  hydrogen peroxide (H2O2) by
dismutation (catalyzed by superoxide dismutase-SOD)
• hydrogen peroxide (H2O2)  hydroxyl radical(-OH) in the
presence Cu2+ and Fe2+ (Fenton reaction)
• H2O2 is also derived directly from oxidases in peroxisomes.
• Resultant free-radical damage to lipid (by peroxidation), proteins,
and deoxyribonucleic acid (DNA) leads to various forms of cell
injury.
• Leukocytes Pathway

• ROS also generated in leukocytes during inflammation. The process

is known as respiratory burst.

• ROS are produced in phagocytic leukocytes, mainly neutrophils and

macrophages, as a weapon for destroying ingested microbes and other
substances during inflammation and host defense.

• Oxygen (O2) – NADPH oxidase (nicotinamide adenine dinucleotide

phosphate oxidase)  Superoxide anion(O2¯)  H2O2–
Myeloperoxidase(MPO) HOCl (hypochlorite)  cause cell injury
 Removal of free Radical
• Antioxidant  inactivation/ block free radical formation
• Many enzymes act as free radical-scavenging system and break
down of H2O2, and O2¯
1. Catalase: present in peroxisomes, decomposes H2O2
2H2O2  O2 + 2H2O
2. Superoxidase dismutase (SOD’s): found in many cells convert
O2 radical to H2O2
2O2 + 2H  H2O2 +O2
3. Glutathione peroxidase: it can also protect against injury by
catalyzing free radical breakdown
H2O2 + 2GHS  GSSG (Glutathione homodimer) + 2H2O
 Summary of ROS
Free Radical Mechanisms of Mechanisms of Pathologic Effects
Production Removal

Superoxide Incomplete reduction of Conversion to Direct damaging

anion(O2¯) O2 during mitochondrial H2O2 and O2 by effects on lipids
oxidative phosphorylation; superoxide (peroxidation),
by phagocyte oxidase in dismutase proteins, and
leukocytes DNA

Hydrogen Mostly from superoxide by Conversion to Can be converted

peroxide action of superoxide H2O and O2 by to .OH and
(H2O2) dismutase catalase, OCl¯, which
glutathione destroy microbes
peroxidase and cells
Free Radical Mechanisms of Mechanisms of Pathologic Effects
Production Removal

Hydroxyl Produced from H2O, Conversion to H2O Direct damaging

radical (•OH) H2O2, and O2¯ by by glutathione effects on lipids,
various chemical peroxidase proteins, and DNA
reactions

Peroxynitrite Interaction of O2¯ Conversion to Direct damaging

(ONOO• ) and NO mediated nitrite by enzymes effects on lipids,
by NO synthase in mitochondria proteins, and DNA
and cytosol
 Defects in Membrane Permeability
• The plasma membrane can be damaged
• ischemia,
• microbial toxins,
• lytic complement components,
• physical and chemical agents.
• Several biochemical mechanisms may contribute to membrane damage.
• Decreased phospholipid synthesis
• Increased phospholipid breakdown
• ROS. Oxygen free radicals cause injury to cell membranes by lipid
peroxidation, discussed earlier.
• Cytoskeletal abnormalities
• Lipid breakdown products Important sites of membrane damage during
cell injury are
• Mitochondrial membrane damage
• Plasma membrane damage
• Injury to lysosomal membranes
 Damage to DNA and Proteins

• Cells have mechanisms that repair damage to DNA, but if this

damage is too severe to be corrected (e.g., after radiation injury or
oxidative stress), the cell initiates its suicide program and dies by
apoptosis.

• A similar reaction is triggered by the accumulation of improperly

folded proteins, which may result from inherited mutations or
external triggers such as free radicals.
 Apoptosis
• Programmed cell death

• A pathway of cell death that is induced by a tightly regulated suicide

program in which cells destined to die activate enzymes that degrade
the cells' own nuclear DNA and nuclear and cytoplasmic proteins.

• Apoptotic cells break up into fragments, called apoptotic bodies,

which contain portions of the cytoplasm and nucleus.

• The plasma membrane of the apoptotic cell and bodies remains

intact, but its structure is altered in such a way that these become
“tasty” targets for phagocytes.
 Causes of Apoptosis
Apoptosis in Physiologic Apoptosis in Pathologic
Situations Conditions
• Programmed destruction of • Apoptosis eliminates cells that
cells during embryogenesis. are genetically altered or
• hormone deprivation  injured beyond repair and does
shrinkage of hormone- - DNA damage
dependent tissues upon - Accumulation of misfolded
• Cell loss in proliferating cell proteins
populations - Cell injury in certain
• Elimination of cells that have infections
served their useful purpose - Pathologic atrophy in
• Elimination of harmful self- parenchymal organs after duct
reactive lymphocytes obstruction
• Cell death by cytotoxic T
lymphocytes
 Morphology of Apoptosis
• Cell shrinkage:
- cell is smaller in size;
- cytoplasm is dense
- organelles are more tightly packed.
• Chromatin condensation:
- The chromatin aggregates peripherally into dense masses of various
shapes and sizes.
• Formation of cytoplasmic blebs and apoptotic bodies:
- apoptotic cell surface blebbing
- fragmentation into membrane-bound apoptotic bodies (cytoplasm
and tightly packed organelles)
- Phagocytosis
- apoptotic cells or cell bodies by macrophages.
Morphology of Apoptosis
 Biochemical Features of Apoptosis

Apoptotic cells usually exhibit a distinctive biochemical

modifications that underlie the structural changes in cells

• Protein cleavage -

• Activation of endonucleases

• Phagocytic recognition
i. Protein cleavage-
• Occurs via activation of several members of cysteine protease
family (caspases)Activates several proteases which acts on
microfilaments like laminin, actin etc.  Break up nuclear
scaffold & cytoskeleton
ii. Activation of endonucleases-
• Apoptotic cells exhibit characteristic breakdown of DNA
• There is intra-nucleosomal cleavage into multiple fragments by
CA2+ & Mg2+ dependent endonucleases.
iii. Phagocytic recognition
• Apoptotic cells express phosphatidylserine in outer layer of the
membrane
• Allows for early recognition of apoptotic cells by macrophages
resulting in phagocytosis without the release of pro-inflammatory
cellular components
 Mechanism of Apoptosis
• Apoptosis reflects several different pathways that lead to similar
end points.
Initiation

Extrinsic pathway Intrinsic Pathway

Caspases activation

Execution

Phagocytosis
 Apoptosis: Pathways

“Extrinsic Pathway”

Death Death Initiator

Ligands Receptors Caspase 8

Effector
“Intrinsic Pathway”
Caspase 3 PCD

DNA da Initiator
mage Mitochondria/
& p53 Cytochrome C Caspase 9
 Extrinsic pathway apoptosis

• In the “extrinsic” pathway to apoptosis, a signal is received from

outside the cell instructing it to commit programmed cell death.
This may occur if the cell is no longer needed, or if it is diseased

• The extrinsic pathway consists of;

• TNF-Pathway

• FAS- Pathway
 Extrinsic Pathway

TNF Pathway FAS- Pathway

Ligand  TNF-alpha Ligand  FAS-L

Cytokines of macrophages Transmembrane protein

 FAS- Pathway
• The FAS receptor (First apoptosis signal)

• It is a transmembrane protein of the TNF family which binds the FAS

ligand (FASL).

• The FAS pathway consists of two types of cell

• Signaling cell Cytotoxic T-Lymphocytes which contain

signaling molecule i.e. FAS-L
• Target cell  These molecules may be excreted by neighboring
cells if a cell is damaged or no longer needed. It contain receptor
i.e. FASR

• When FASL bind with FASR the process of apoptosis begins which
contain cascade of reaction
 FAS Pathway

Cell type

Signaling cell Target Cell

(cytotoxic T- Lymphocytes) FAS-Receptor
i n d (FAS-R)
B
FAS- Ligand
FAS-L
Induce apoptosis
 Death-Inducing Signaling Complex
(DISC)
• The interaction between FASR and FASL results in the formation of the
Death-Inducing Signaling Complex (DISC) ,

• The complex contain

- FADD,
- caspase-8 and caspase-10.

• In some types of cells (type I), processed caspase-8 directly activates

other members of the caspase family and triggers the execution of
apoptosis of the cell.

• In other types of cells (type II), the FAS-DISC starts a feedback loop
that spirals into increasing release of proapoptotic factors from
mitochondria and the amplified activation of caspase-8.
 Mechanism of FAS
• The cytotoxic T-lymphocytes contain FAS-L while the target cell
contain FAS-R (also called death receptor)when FAS-L bind with FAS-
R. It will induce changes in death domain found in intracellular matrix
of cell attached with FAS-R. After changes in death domain causes the
recruitment of FADD and procaspases-8. The FADD attached with
death domain and causes the activation of Procaspases-8 to caspases-8
and caspases-10. The caspases-8 causes the activation of procaspases-3
to caspases 3 which act on inactive apoptotic substances causes the
activation of apoptotic substances which will cause cell death.
 Cytotoxic T-cell FAS-L

Binding of FAS-L & FAS-R

Target Cell FAS-R Death Receptor Activation

Death domain

FADD

Procaspases-8
caspases-8 Procaspases-8

Inactive apoptotic substances

Procapases-3 Capases-3
Activation

Active apoptotic substances

 TNF-Pathway
• TNF-alpha is a cytokine produced mainly by activated macrophages
and is the major extrinsic mediator of apoptosis.
• The TNFR is present in an inactivated form which contain death
domain in intracellular matrix.
• The death domain is attached with silence of death domain (SODD)
which keep the cell alive and keep death domain inactivated.
• The binding of TNF-alpha to TNFR has been shown to initiate the
pathway that leads to caspase activation via the intermediate
membrane proteins TNF receptor-associated death domain (TRADD)
and FAS-associated death domain protein (FADD).
• The FADD causes the cleave or cut off of procaspases-8 to activated
caspases which will induce apoptosis.
BID pathway
• Another inactive molecule called BID (BH3 Interacting Domain
Death Agonist) is transformed into tBID when the activated
caspases cleave off the part of BID that keeps the molecule
inactive.

• tBID moves to the mitochondria.  it induces permeabilization of

the outer mitochondrial membrane that is dependent on the pro-
apoptotic proteins BAX (Bcl-2-associated X protein) and/or BAK
(Bcl-2 antagonist killer 1)

• tBID also activates the molecules BAX and BAK.

• BID acts as a protease-mediated death signals.

 Intrinsic pathway apoptosis
(Mitochondrial pathway)
Step 1

• The intrinsic pathway to apoptosis is triggered by stress or damage to

the cell.

• Types of stress and damage that can lead the cell to apoptosis include
damage to its DNA, oxygen deprivation, and other stresses that
impair a cell’s ability to function.

• The DNA lesion causes the release of ATM (ataxia telangiectasia

mutated) Serine/ Threonine kinase which causes the release of p53
protein
Mechanism

Cause release of
Stress/ DNA Release of p53
ATM serine/
damage protein
threonine

Causes release of
Activate BAX and
CYT. C from Induction MOMP
BAK
mitochondria

Caspases 9
Apoptosome
CYT. C form activation i. Activation of
complex Caspases 3 and 7
Apoptosome Procaspases-9
caspases-9 ii. Induce
apoptosis
Step 2:
• BH3-only proteins are a class of proteins including several pro-
and anti-apoptosis proteins.

• Apoptosis can be encouraged or discouraged, depending on which

BH3-only proteins are activated or expressed.

• Pro-apoptotic BH3-only proteins activate BAX and BAK – the

same proteins that are activated by tBID after it is created through
the extrinsic pathway to apoptosis.
Step 3:
• Activated BAX and BAK cause a condition known as “MOMP.”
(MOMP stands for “mitochondrial outer membrane permeability.)

• MOMP is considered the “point of no return” for apoptosis. The

steps leading up to MOMP can be stopped in their tracks by
inhibitor molecules, but once MOMP has been achieved, the cell
will complete the death process.

• MOMP plays its key role in apoptosis by allowing the release of

cytochrome C into the cytoplasm.
Step 4:
• Under normal circumstances, cytochrome C plays a key role in the
mitochondrial electron transport chain.

• During MOMP, however, cytochrome C can escape the

mitochondria and act as a signaling molecule in the cell
cytoplasm.

• Cytochrome-C in the cell cytoplasm prompts the formation of the

complex “apoptosome” – a complex of proteins that performs the
final step to beginning cellular breakdown.
• Step 5:
• The apoptosome, once it is formed, turns pro-caspase-9 into
caspase-9 is able to trigger further changes throughout the cell.
• Step 6:
• Caspase-9 performs several functions to promote apoptosis.
Cause the activation of caspases-3 and -7.
• Step 7:
• Once activated, caspases-3 and -7 begin the breakdown of cellular
materials.
• Caspase-3 condenses and breaks down the cell’s DNA.
 Cell Adhesion Molecules (CAMs)
• These are chemicals which mediate the interaction between cells
(cell-cell interaction) as well as between cells and extracellular
matrix (cell-ECM interaction).
• CAMs participate in following function;
• fertilization,
• embryogenesis,
• Tissue repair,
• haemostasias,
• cell death by apoptosis and in
• inflammation.

• CAMs may be detected on the surface of cells as well as free in

circulation.
 Groups of CAMs
i. Integrins: principal receptors used by animal cells to bind to the
extracellular matrix. They have alpha (or CD11*) and beta
(CD18) subunits and have a role in cell-ECM interactions and in
leucocyte-endothelial cell interaction.

ii. Cadherins: These are calcium-dependent adhesion molecules

which bind adjacent cells together and prevent invasion of ECM
by cancer cells.

Various types of cadherins include:

- E-cadherin (epithelial cell),
- N-cadherin (nerve cell),
- M-cadherin (muscle cell),
- P-cadherin (placenta).
iii. Selectins: Also called as lectins, these CAMs contain lectins or
lectin-like protein molecules which bind to glycoproteins and
glycolipids on the cell surface. Their major role is in movement
of leucocytes and platelets and develop contact with
endothelial cells. Selectins are of 3 types:
• P-selectin (from platelets, also called CD62),

• E-selectin (from endothelial cells, also named ECAM),

• L-selectin (from leucocytes, also called LCAM).

iv. Immunoglobulin superfamily: This group consists of a variety of
immunoglobulin molecules present on most cells of the body. These
partake in cell-to-cell contact through various other CAMs and
cytokines. They have a major role in recognition and binding of
immunocompetent cells.
• This group includes

• ICAM-1,2 (intercellular adhesion molecule, also called CD54),

• VCAM (vascular cell adhesion molecule, also named CD106),

• NCAM (neural cell adhesion molecule).

v. CD44: The last group of adhesion molecules is a break away
from immunoglobulin superfamily. CD44 molecule binds to
hyaluronic acid and is expressed on leucocytes. It is involved in
leucocyte-endothelial interactions as well as in cell-ECM
interactions.
 Necrosis vs Apoptosis
Features Necrosis Apoptosis

Definition Uncontrolled cell death Programmed cell death

Cell size Enlarged( Swelling) Reduced(shrink)

Nucleus Pyknosis → karyorrhexis Fragmentation into

→ karyolysis nucleosome size fragments
Plasma Disrupted Intact
membrane
Cellular Enzymatic digestion; may Intact; may be released in
contents leak out of cell apoptotic bodies
Etiology Gross irreversible cellular Subtle cellular damage,
injury
Adjacent Frequent No
inflammation
 Features Necrosis Apoptosis
Physiologic or Invariably pathologic Physiologic  eliminating
pathologic role unwanted cells; pathologic
 cell injury, especially
DNA and protein damage
Morphologic Inc. cytoplasmic Cytoplasmic shrinking and
changes eosinophilia increased eosinophilic
Nuclear condensation and staining Chromatin
fragmentation Preservation condensation and
of tissue architecture in fragmentation
early stages of coagulative
necrosis
Biochemical Cell death Passive form Active form of cell death
changes of cell death Requiring gene
not requiring gene expression, protein
involvement or new protein synthesis, and energy
synthesis consumption DNA
DNA fragmentation is fragmentation
haphazard
Reference
• Kumar V, Cotran RS, Robbins SL. Robbin’s Basic Pathology. 8th
Ed. W. B. Saunders Publishers; 2007.

• Mohan, Harsh. "Textbook of pathology." (6th edition): 842-854.

 Past Paper
2013 1. Discuss necrosis and its types. (5)
2015 1. Differentiate b/w necrosis and apoptosis (1)
2. Reversible and irreversible injury (1)
2017 1. Differentiate b/w necrosis and apoptosis in tabular form (5)
2. Define free radical and ROS (2 +2)
2018 Define reactive oxygen specie (2)
2019 Write a note on following
a. Intrinsic Pathway (mitochondrial pathway) of apoptosis (6)
b. Caseous necrosis (4)
c. What is difference b/w selectins and integrins (2)
2020 1. Define apoptosis and explain biochemical changes occurs (5)
in apoptosis
2. Define necrosis and define cytoplasmic and nuclear (5)
changes occur in necrosis.
3. Write down the three major consequences of ATP depletion (5)
due to ischemia
2021 1. Differentiate b/w necrosis and apoptosis (5)