irreversible cell injury, necrosis, apoptosis, free radicles, reperfusion injury

1. Item-3
Irreversible Cell Injury
Dr. A K M Maruf Raza
Associate Professor of Pathology
Based on Robbins and Cotran
9th edition

2. Irreversible cell injury:
 If the injurious stimulus persists or is severe
enough, the cell suffers irreversible injury and
ultimately undergoes cell death. Two types:
i. Necrosis.
ii. Apoptosis.

3. Necrosis
 Necrosis is the result of denaturation of
intracellular proteins and enzymatic digestion
of the lethally injured cell.
 Necrotic cells lost membrane integrity and
their contents leak out.
 The enzymes that digest the necrotic cell
derived from the lysosomes of that cells.

4. Nuclear changes in Necrosis
 Necrotic cells show increased eosinophilia
(increased pink colour).
 Nuclear changes appears in three patterns due
to breakdown of DNA:
I. Karyolysis.
II. Pyknosis.
III. Karyorrhexis.

5.  Karyolysis is due to loss of DNA. The
basophilia (violate color) taken by
chromatin fade away.
 Pyknosis characterized by nuclear
shrinkage and increased basophilia (pink
colour).
 Karyorrhexis, the nuclear fragmentation in
pyknotic cells and ultimately disappearance
of nucleus.

6. Types of Necrosis:
 Coagulative necrosis is a form of necrosis in
which the architecture of dead tissues is
preserved. It is due to denaturation of structural
proteins and enzymes.
 Exm: Necrosis effecting heart, kidney, lungs.
 Liquefactive necrosis is characterized by
digestion of the dead cells, resulting formation
of a liquid viscous mass called pus.
 Exm: Abscess, Boil, necrosis of brain.

7. Types of Necrosis:
 Gangrenous necrosis is a clinical term
applicable generally for necrosis affecting the
limp, specially the lower leg that has lost its
blood supply and has undergone necrosis.
Wet gangrene: When bacterial infection is
superimposed there is there is more liquefactive
necrosis in the gangrenous limb.

8. Types of Necrosis:
 Caseous necrosis is a special type of
coagulative necrosis present in tuberculous
infection (Tuberculosis).
 The term “caseous” (cheeselike) is derived
from the friable white appearance of the area
of necrosis.

9. Types of Necrosis:
 Fat necrosis refers to focal areas of fat
destruction due to release of activated
pancreatic lipases into the pancreas and
peritoneal cavity. Fat necrosis is two types:
i. Enzymatic fat necrosis: occurs in pancreas.
ii. Traumatic fat necrosis: in breast, in
subcutaneous tissue.
 Fibrinoid necrosis is a form of necrosis seen
in immune reactions involving blood vessels.

10. Apoptosis
 Apoptosis is genetically regulated cell death
induced by regulated suicidal program by
injured cells.
 It is referred to as programmed cell death.
 Apoptosis is a pathway of cell death in which
cells die by activating enzymes that degrade
the cells own nuclear DNA and nuclear and
cytoplasmic proteins.

11. Causes/ example of Apoptosis
Apoptosis in Physiologic Situations:
 The destruction of cells during embryogenesis.
 Epithelial Cell loss in the intestinal epithelium,
skin.
 Death of cells that served their purpose such as
neutrophils
 Regression of the lactating breast after
lactational period.

12. Causes/ example of Apoptosis
Apoptosis in Pathologic Conditions:
 Radiation, Cytotoxic anticancer drugs and
hypoxia can damage DNA. If DNA is not
repaired ultimately cells undergo apoptosis.
 Degenerative diseases of the central nervous
system.
 Cell death in viral infections due to viral
cytopathic effect.

13. Morphologic changes in
Apoptosis
 Cell shrinkage.
 Chromatin condensation.
 Formation of apoptotic bodies.
 Phagocytosis of apoptotic cells or cell
bodies, usually by macrophages.

14. Mechanisms of Apoptosis
 Apoptosis results from the activation of
enzymes called caspases.
 Caspases exist as inactive proenzymes within
the cells.
 The process of apoptosis divided into:
1.initiation/activation phase: caspases
become catalytically active
2.execution phase: degradation of cellular
components occurs.

15. Mechanisms of Apoptosis
 The activation of caspases depends on
balance between pro-apoptotic (BAX, BAC)
and anti-apoptotic (BCL2, BCL-XL) proteins.
 Two pathways of caspase activation:
i. The mitochondrial pathway (Mainly)
ii. The death receptor pathway

16. Difference between Necrosis and
Apoptosis
Feature Necrosis Apoptosis
Cell size Enlarged (swelling) Reduced (shrinkage)
Nucleus Pyknosis
karyorrhexis
karyolysis
Fragmentation into
fragments
Plasma
membrane
Disrupted Intact
Cellular
contents
Enzymatic digestion; leak
out of cell
Intact; may be released in
apoptotic bodies
Adjacent
inflammation
Frequent No
Physiologic or
pathologic
role
pathologic physiologic and also
pathologic

17. Mechanisms of Cell Injury/
Biochemical alteration in cell injury
 Depletion of ATP.
 Mitochondrial Damage.
 Influx of Calcium and Loss of Calcium
Homeostasis
 Accumulation of Oxygen-Derived Free
Radicals.
 Defects in Membrane Permeability.
 Damage to DNA and Proteins.

18. Mechanisms of Cell
Injury

19. ATP depletion
 Reduction of ATP and decreased ATP
synthesis is the most important cause of cell
death associated with hypoxic and toxic injury.
 The major causes of ATP depletion are:
i. reduced supply of oxygen and nutrients.
ii. mitochondrial damage.
iii. actions of some toxins (e.g., cyanide).

20. Mitochondrial damage
 Mitochondria supply life sustaining energy
ATP, its damage causes cell death.
 Damage to mitochondrial membranes results
in opening of the mitochondrial permeability
transition pore.
 Which leads to decreased ATP generation and
release of proteins that trigger cell death.

21. Influx of Calcium and Loss of
Calcium Homeostasis
 Calcium ions are important mediators of cell
injury. Low level of intracellular calcium
protects cells from injury.
 Ischemia and certain toxins cause an increase
in cytosolic calcium concentration, initially
from intracellular stores and later influx across
the plasma membrane.

22. Influx of Calcium and Loss of Calcium
Homeostasis
 Increased intracellular Ca2+ causes cell injury
by several mechanisms:
i. The accumulation of Ca2+ in mitochondria
results in opening of the mitochondrial
permeability transition pore.
ii. Increased cytosolic Ca2+ activates a
number of lysosomal enzymes.
iii. Increased intracellular Ca2+ causes
activation of caspases.

23. Influx of Calcium and Loss of
Calcium Homeostasis

24. Accumulation of Oxygen-Derived
Free Radicals
 Cell injury induced by free radicals causes cell
damage in many pathologic conditions.
 Such as chemical and radiation injury,
ischemia-reperfusion injury, cellular aging, and
microbial killing by phagocytes.

25. Defects in Membrane
Permeability
 Membrane damage of cell causes cell injury
and cell death.
 The most important sites of membrane
damage in cell injury are:
i. Mitochondrial membrane.
ii. Plasma membrane.
iii. Membranes of lysosomes.

26. Necroptosis
 Necroptosis is the form of cell death that
shares both necrosis and apoptosis.
 Morphologically it resembles necrosis but by
mechanism it is programmed cell death that i
Apoptosis.

27. Necroptosis
It is associated with cell death in
i.Steatohepatitis (Alcoholic fatty liver
disease)
ii.Acute pancreatitis
iii.Reperfusion injury
iv.Neurodegenerative diseases such as
Parkinson disease.

28. Pyroptosis
 Pyro (Fever)+ Optosis (Apoptosis).
 Pyroptosis is the release of fever inducing
cytokine IL-1 which bears biochemical
similarities with apoptosis.
 Pyroptosis occurs in cells infected by
microbial organism. Which causes death of
the infected cell.

29. Autophagy
 Autophagy is a process in which a cell eats its
own contents.
 Caused by the delivery of cytoplasmic
materials to the lysosome for degradation.
 Autophagy plays a role in human diseases like
Cancer, Alzheimer disease and also in ageing.

30. Ischemia-Reperfusion Injury
 Ischemia +Reperfusion Injury.
 Restoration of blood flow causes recovery of
cells of reversibly injured cells.
 Restoration of blood flow to ischemic tissues
can also increases the injury and cause cell
death, it is called Ischemia-Reperfusion
Injury.
 Exmp: Rapid restoration of blood flow in MI can
cause more myocardial injury.

31. Ischemia-Reperfusion
Injury
Why new tissue injury after restoration of
blood?:
i. Increased generation of reactive oxygen
and nitrogen species.
ii. Intracellular calcium overload.
iii. Repurfusion recruit circulating
neutrophils and causes tissue injury.
iv. Activation of the complement system.

32. Free Radicals; Accumulation of
Oxygen-Derived Free Radicals
(Oxidative Stress)

33. Free radicals
 Free radicals are chemical species that have a
single unpaired electron in an outer orbit.
 Unpaired electrons are highly reactive and
attack proteins, lipids, carbohydrates and
nucleic acids of cells causing cell damage and
cellular ageing.
 Superoxide anion. hydrogen peroxide, nitric
oxide; hydroxyl radical are example of free
radicals.

34. Free radicals
 Reactive oxygen species (ROS) are free radical
produced normally in cells during
mitochondrial respiration and energy
generation.
 Increased production or decreased
scavenging of ROS may lead to an excess of
these free radicals, a condition called oxidative
stress.

35. Generation of Free Radicals
 The reduction-oxidation reactions that occur
during normal metabolic processes.
 Absorption of radiant energy (e.g., ultraviolet
light, x-rays).
 Produced in activated leukocytes during
inflammation.
 Enzymatic metabolism of exogenous chemicals
or drugs generate free radicals (e.g., CCl4)
 Nitric oxide (NO) generated by endothelial cells,
macrophages act as free radical.

36. Effects of Free Radicals on cell
The effects of ROS and other free radicals are cell
injury and cellular ageing by:
i. Lipid peroxidation in membranes.
ii. Oxidative modification of proteins may
damage the active sites of enzymes, generate
misfolded protein.
iii. Free radicals causes DNA damage (single-
and double-strand breaks in DNA).

37. Removal of Free Radicals
1. Free radicals are unstable and decay
spontaneously.
2. Antioxidants either block free radical formation or
inactivate free radicals (Vit E, Vit A, Vit C).
3. A series of enzymes that breaks down free
radicals:
i.Catalase inactivates H2O2
ii.Superoxidase dismutases converts
superoxide anion
iii.Glutathione peroxidase

38. Free radicals
 Free radicals are chemical species that
have a single unpaired electron in an outer
orbit.
 Unpaired electrons are highly reactive and
attack proteins, lipids, carbohydrates and
nucleic acids of cells.

39. Generation of Free Radicals
 The reduction-oxidation reactions that occur
during normal metabolic processes
 Absorption of radiant energy (e.g., ultraviolet
light, x-rays).
 Produced in activated leukocytes during
inflammation.
 Enzymatic metabolism of exogenous chemicals
or drugs generate free radicals (e.g., CCl4)

40. Removal of Free Radicals
 Free radicals are inherently unstable and
decay spontaneously.
 Antioxidants
 A series of enzymes that breaks down free
radicals:
i.Catalase
ii.Superoxidase dismutases
iii.Glutathione peroxidase

41. Effects of Free Radicals on cell
 Lipid peroxidation in membranes.
 Oxidative modification of proteins.
 Lesions in DNA (single- and double-strand
breaks in DNA, cross-linking of DNA;
ultimately causing DNA damage)