• PA 2.

4
• Describe and discuss Cell
COMPETENCY death- types, mechanisms,
ADDRESSED necrosis, apoptosis (basic as
contrasted with necrosis),
autolysis.

Dr Anshu gupta
Specific Learning Objectives
• By the end of this lecture students must be able to define necrosis.
• Students must be able to describe necrosis and its subtypes with
suitable examples.
• Students should be able to define apoptosis
• Students must be able to describe the mechanism of apoptosis in
detail
• Students must be able to illustrate the role of apoptosis in both
physiological and pathological states
• Students must be able to differentiate between necrosis and
apoptosis

Dr Anshu gupta
 IRREVERSIBLE
CELL INJURY
• Living tissue – local/focal
change – necrosis/ apoptosis.

• Somatic death – autolysis –

self digestion by hydrolytic
enzymes of lysosomes. No
inflammatory reaction in
surrounding tissue

Dr Anshu gupta
Dr Anshu gupta

NECROSIS

• Sum total of morphologic changes that follow cell death in a living

tissue.
• Involves group of cells
• Loss of membrane integrity
• Liberation of lysosomal enzymes – enzymatic digestion of
cell(necrotic cell+ inflammatory cell)
• Denaturation of intracellular protein
• Accompanied by inflammatory reaction
• Takes hours to become detectable grossly
• Causes – hypoxia, infections etc
 CLINICAL
SIGNIFICANCE

• Liberated enzymes leak into

serum – detection serves as
a clinical parameter to detect
tissue specific injury
• Myocardial damage - CK-
MB/ cardiac troponin
• Hepatocyte damage -
SGPT,ALKP

Dr Anshu gupta
DAMPs – damage
associated molecular
pattern (ATP,uric acid)

Dr Anshu gupta
NECROSIS
• Morphology – H&E
• Cytoplasmic changes – glassy
homogenous cytoplasm, increased
eosinophilia (denatured proteins)
• Vacuolated(moth-eaten) cytoplasm-
(enzymatic digestion of cellular
organelles)
• Nuclear changes – Karyolysis,
pyknosis, karyorrhexis (due to break
down of DNA)
• Fate of necrotic cell – phagocytosed
by macrophages, later calcification
Digestion of DNA by
endonucleases
Dr Anshu gupta
 • Coagulative including
gangrenous necrosis
• Liquefactive
Morphological
• Caseous
Patterns Of • Fat
Tissue Necrosis • Fibrinoid

• Give clue about the cause

 COAGULATIVE ➢ Characteristic of ischaemic
necrosis (infarct) in solid
NECROSIS organs except brain.
➢ Denaturation of proteins and
enzymes, no proteolysis
➢ Gross – Firm & pale (infarct)
➢ M/E - Basic tissue
architecture & cell outlines
preserved for some days
(Tombstone appearance).
➢ Eosinophilic, anucleate
dead cells - removed by
leukocytes.

Dr Anshu gupta
LIQUEFACTIVE
NECROSIS
• Seen in focal bacterial or fungal
infections
• Microbes stimulate accumulation of
leukocytes which digest (liquify) the
cells.
• Transformation of tissue into a liquid
viscous mass - hydrolytic enzymes
• Necrotic material – creamy yellow- pus
• Hypoxic cell death of neurons in CNS-
liquefactive necrosis.
• Removal of pus – abscess cavity

Dr Anshu gupta
 Coagulative
• Mostly occurs d/t sudden cessation of
blood flow- Ischemia
• Gross – affected area pale, firm.
• M/E – tissue architecture preserved
• Denaturation of proteins
Liquefactive
• Due to bacterial/ fungal infections
• Gross – affected area soft with
liquefied centre
• M/E – tissue architecture completely
lost
• Hydrolytic enzymes cause tissue
degradation

Dr Anshu gupta
GANGRENOUS NECROSIS
• Coagulative necrosis with putrefaction of tissues
– two types:
• Wet gangrene :
• Blockage of both arterial and venous flow.
Ischemic necrosis + superadded virulent
infection.
• Bowel, lungs, diabetic foot, bed sores
• No clear cut line of demarcation
• Gross – soft, swollen, dark & putrid
• M/E – coagulative necrosis with intense acute
inflammation
• Gas gangrene
• Wet gangrene formed by gas forming anaerobic
bacteria clostridia – affected part swollen,
edematous, crepitant d/t accumulationDr Anshu
of CO2
gupta
GANGRENE
• Dry gangrene:
• Gradual small vessel obstruction -
atherosclerosis ,Burgers’ds
(thromboangiitis obliterans)
• Gangrene of foot/toes
• Line of demarcation present b/w viable
and gangrenous part
• Gross – dry, shrunken, black
(mummified)
• M/E – coagulative necrosis with minimal
inflammation

Dr Anshu gupta
CASEOUS NECROSIS
• Cheese like ( friable, yellow-white appearance)
• Tuberculous infection
• Tissue architecture not preserved
• Necrotic area - structureless, eosinophilic
granular material with nuclear debris
surrounded by lymphocytes & epitheloid cells
(granuloma)

Dr Anshu gupta
 FAT NECROSIS
• Focal areas of destruction of lipid deposits in the body by the
action of lipases.
• Seen in
i) acute pancreatitis (lipases)
ii) trauma to adipose tissue (breast /buttocks)

Dr Anshu gupta
 Neutral fat released – free
fatty acids – saponification

Dr Anshu gupta
 • Acute pancreatitis - Release of lipases -
lysis of fat cells, split triglycerides
• Released fatty acids combine with
FAT NECROSIS calcium form chalky white areas( fat
saponification)
• M/E – shadowy outlines of fat cells with
basophilic calcium deposits

Dr Anshu gupta
 TRAUMATIC FAT
NECROSIS
• Seen in tissues rich in fat – Breast,
gluteal region
• Cause – trauma, radiation
• Lipid laden foamy histiocytes and
multinucleated foreign body type
giant cells are due to phagocytosis of
necrotic adipocytes

Dr Anshu gupta
FIBRINOID
NECROSIS
• Seen in immune
reactions involving
blood vessels
• Deposition of immune
complexes + fibrin in
walls of arteries
• H&E –bright pink
amorphous
appearance

Dr Anshu gupta
Dr Anshu gupta

• Pathway of cell death in which cell

activate intrinsic enzymes that
degrade the cells own DNA and
cytoplasmic protein
• Programmed cell death / cell suicide
• Fragments of cell break off - “ Falling
APOPTOSIS Off”
• Plasma membrane intact but altered –
cell fragments phagocytosed.
• Dead cells cleared immediately
• No inflammation.
Dr Anshu gupta

Causes of Apoptosis

PHYSIOLOGICAL PATHOLOGICAL
Dr Anshu gupta

PHYSIOLOGICAL

• Normal phenomenon - eliminate cells no longer needed/ maintain constant number

• During embryogenesis – programmed destruction of cells
➢ During implantation, organogenesis, involution and metamorphosis for e.g. interdigital
cell death responsible for separating fingers
• Involution of hormone dependant tissue on hormonal withdrawal.
➢ Regression of lactating breast after weaning.
➢ Atresia of ovarian follicles in menopause
➢ Endometrial cell breakdown during menstrual cycle
• Cell loss in proliferating cell population – immature lymphocytes in bone marrow &
thymus & self reactive-lymphocytes
• Death of host cells after they have served purpose - neutrophils in acute
inflammation.
Dr Anshu gupta

PATHOLOGICAL

➢ Eliminates cells that are damaged beyond repair.

• DNA damage - radiation, anticancer drugs, ROS – triggers apoptosis if
beyond repair to prevent mutation
• Accumulation of misfolded proteins -mutations/ free radical injury. ER
stress--- Apoptosis- In Alzheimer’s disease, Parkinson’s
• Cell death in certain infections eg viral infections- viral hepatitis
• Pathologic atrophy in organs after duct obstruction – parotid gland,
kidney
Dr Anshu gupta

Cytoplasmic - cell shrinkage –

▪ MORPHOLOGIC cytoplasm dense, organelles more
CHANGES- tightly packed.
*Electron
microscope
*Light Nuclear changes - chromatin
microscope – condensation- chromatin aggregates
H&E sections peripherally under nuclear membrane
into dense masses of various shapes /
sizes – fragmented nucleus
• Formation of cytoplasmic blebs/ apoptotic bodies – extensive surface blebbing
→ fragmentation into apoptotic bodies.
• Phagocytosis of apoptotic bodies – ingested by macrophages
• No inflammation

Dr Anshu gupta
 Light Microscopy – H & E

•Cell shrunken
•Brightly eosinophilic cytoplasm
•Condensed nucleus
Dr Anshu gupta
 MECHANISM OF
APOPTOSIS
➢ Entire process two phases – initiation and
execution
➢ Critical event - activation of enzyme
caspases
• Exist in inactive forms-( proenzymes).
• Need to be activated.
• Activation - balance between pro-apoptotic
and anti-apoptotic proteins by two distinct
pathways –
• 1) Mitochondrial (intrinsic)
• 2) Death receptor pathway (extrinsic).

Dr Anshu gupta
Dr Anshu gupta

MITOCHONDRIAL/INTRINSIC
• Major mechanism
• Increased permeability of mitochondrial outer
membrane with release of death inducing pro-
apoptotic molecule (cytochrome c) into cytoplasm.
• In healthy cells, release of pro-apoptotic protein is
tightly regulated by BCL2 family of proteins.
• Three groups of BCL2 proteins
1. Anti-apoptotic – BCL2,BCL-XL – prevent
leakage of cytochrome-c
2. Pro-apoptotic – BAX, BAK – promote
mitochondrial outer membrane permeability
3. Sensors – BAD, BIM ,Puma (BH3- only
protein)- act as sensors of cellular stress and
damage. Regulate the balance b/w above two.
Dr Anshu gupta

• When cells deprived of survival signals/ DNA

damage/ER stress

• Sensor -BH-3only proteins –activated

• Activate pro apoptotic proteins BAX and BAK

• Block function of anti-apoptotic BCL2

• Create channels in mitochondrial membrane

• Cause cytochrome c to leak into cytosol

• Activate caspases
 INTRINSIC –
INITIATION
PHASE
• Cytochrome c binds to Apaf-1
in cytosol to form an
apoptosome.

• Apoptosome binds and

activates caspase 9 (initiator)
–auto amplification process

• Activates executioner
caspase -3.
Dr Anshu gupta
 DEATH
RECEPTOR/EXTRINSIC
• Initiated by engagement of cell surface death
receptors ,members of TNF receptor family –
TNFR1/Fas.
• Contain a cytoplasmic domain (death domain) –
triggers apoptosis – binds Fas ligand expressed on
T-cells.
• Cytoplasmic death domains forms a binding site
for FADD.
• FADD binds Pro caspase 8 to generate active
caspase -8/10
• Elimination of self- reactive T lymphocytes and
killing of target cells by cytotoxic T lymphocytes

Dr Anshu gupta
Dr Anshu gupta

• Both pathways converge to cascade of

caspase activation
• Mitochondrial pathway – activation of
initiator caspase -9
EXECUTION • Death receptor – initiator caspases 8 & 10.
PHASE • Activation of executioner caspases 3 & 6.
• Activate Dnase, endonuclease –Degrade
structural components of nuclear matrix.→
breakdown of nucleus
• Disrupt cytoskeleton
 REMOVAL OF DEAD CELLS
Apoptotic bodies are cleared before they leak Phagocyte Scavenger
Receptors
their contents.
Apoptotic cells undergo changes in ? Oxidized LDL-like Site

membrane → promote phagocytosis. PS

Phosphatidyl- Apoptotic
Phosphatidylserine present on inner layer flips serine
Cell
Receptors C1q
out on outer layer of plasma membrane Binding
Site
Recognized by phagocytes
C1q
Bridge
RAC-1
Apoptotic cells secrete soluble factors → DOCK 180
recruit phagocytes C1q Receptor ELMO

CRKII
Apoptotic cells coated with natural antibodies Cytoskeletal
Reorganization for
- recognised by phagocytes. Engulfment

Dr Anshu gupta
Dr Anshu gupta
Dr Anshu gupta

• Growth Factor deprivation – hormone sensitive cells

deprived of hormone, unstimulated lymphocytes die
by mitochondrial pathway d/t activation of pro-
apoptotic BCL2 members.
• DNA Damage – DNA damage results in accumulation
EXAMPLES of p53 protein - arrest cell cycle in G1 phase for
repair. If irreparable triggers apoptosis. If p53
OF mutated/absent neoplastic transformation
• ER Stress – Accumulation of misfolded proteins
APOPTOSIS induces unfolded protein response (ER stress).
Activation of caspases and apoptosis e.g Alzheimer,
Parkinson
• Apoptosis of self-reactive lymphocyte by apoptosis &
Fas death receptor pathway. Failure of apoptosis,
autoimmune disease.
Dr Anshu gupta
 NECROSIS Vs APOPTOSIS
NECROSIS APOPTOSIS
• Due to irreversible injury (accidental) • Programmed cell death (suicide)
• Large area involved • Single cell/ small clusters
• Accompanied by inflammation • No inflammation
• Plasma membrane disruption • Plasma membrane intact
• Brought about by leakage of ions, enzymes • No leakage, chain reaction by caspases
and proteins • Cells shrunken,dense eosinophilic
• Cytoplasm – vacuolated & moth eaten cytoplasm
• Nucleus – pyknosis, karyorrhexis, • Nuclear chromatin fragments & condense
karyolysis • Apoptotic body formed
• No apoptotic body • Both physiological and pathological
• Always pathological

Dr Anshu gupta
 PYROPTOSIS
• Proinflammatory programmed cell death
• Results in FEVER d/t IL-1
• Infected cells recognise microbial product and activate
multiprotein complex – inflammasome – activates caspase-1 –
IL-1 active form – leucocyte recruitment and fever
• Cellular swelling & loss of plasma membrane integrity resulting
in cell death

Dr Anshu gupta
 NECROPTOSIS
• Programmed necrosis – cell swelling, release of enzymes but
genetically programmed
• Starts in manner similar to extrinsic form of apoptosis (TNFR1
ligation), role of RIP1,3
• Caspases are not activated
• Terminal event like necrosis – lysosomal membrane disruption,
mitochondrial damage, ROS generation
• Both physiologic & pathologic – bone growth plate, acute
pancreatitis, hepatitis etc

Dr Anshu gupta
 AUTOPHAGY
• “Self eating”– lysosomal digestion of cells own components.
• Survival mechanism in times of nutrient deprivation.
• If not able to cope - signals cell death by apoptosis
• Also involved in clearance of misfolded proteins,defective
autophagy – neurodegenerative diseases.

Dr Anshu gupta
 CELLULAR AGING
• Result of progressive decline in
the life span and functional
capacity of cells.
• DNA damage that accumulates
overtime
• Aging associated with replicative
senescence of cells b/o
shortening of telomeres resulting
in cell arrest
• Defective protein synthesis

Dr Anshu gupta
Specific Learning Objectives
• By the end of this lecture students must be able to define necrosis.
• Students must be able to describe necrosis and its subtypes with
suitable examples.
• Students should be able to define apoptosis
• Students must be able to describe the mechanism of apoptosis in
detail
• Students must be able to illustrate the role of apoptosis in both
physiological and pathological states
• Students must be able to differentiate between necrosis and
apoptosis

Dr Anshu gupta
• On day 28 of her menstrual cycle, a 23-year-old woman experiences
onset of menstrual bleeding that lasts for 6 days. She has had regular
cycles for many years. Which of the following processes is most likely
occurring in the endometrium just before the onset of bleeding?
(A) Apoptosis
(B) Caseous necrosis
(C) Heterophagocytosis
(D) Atrophy

Dr Anshu gupta
• A 50-year-old man experienced an episode of chest pain 6 hours before his
death. A histologic section of left ventricular myocardium taken at autopsy
showed a deeply eosinophilic-staining area with loss of nuclei and cross-
striations in myocardial fibers. There was no hemorrhage or inflammation.
Which of the following conditions most likely produced these myocardial
changes?
(A) Viral infection
(B) Coronary artery thrombosis
(C) Blunt chest trauma
(D) Antibodies directed against myocardium
(E) Protein-deficient diet

Dr Anshu gupta
• A 40-year-old man had undifferentiated carcinoma of the lung. Despite
chemotherapy, the man died of widespread metastases. At autopsy, tumors
were found in many organs. Histologic examination showed many foci in
which individual tumor cells appeared shrunken and deeply eosinophilic.
Their nuclei exhibited condensed aggregates of chromatin under the
nuclear membrane. The process affecting these shrunken tumor cells was
most likely triggered by the release of which of the following substances
into the cytosol?
(A) Lipofuscin
(B) Cytochrome c
(C) Catalase
(D) Phospholipase

Dr Anshu gupta
• A 38-year-old woman experienced severe abdominal pain with
hypotension and shock that led to her death within 36 hours after the
onset of the pain. From the gross appearance of the mesentery, seen
in the figure at the bottom of the previous column, which of the
following events has most likely occurred?
(A) Hepatitis B virus infection
(B) Small intestinal infarction
(C) Tuberculous lymphadenitis
(D) Gangrenous cholecystitis
(E) Acute pancreatitis

Dr Anshu gupta