1. Dr (Prof) Vishal Saxena
Cell Injury
Dr (Prof) Vishal Saxena
MBBS, MD (Pathology)

2. I COME TO SCHOOL AT EIGHT I ALWAYS COME FOR ‘’PATH’’ CLASS
I GET UP IN THE MORNING
I COME TO SCHOOL AT EIGHT
I ALWAYS COME FOR ‘’PATH’’ CLASS
AND NEVER COME IN LATE !!!

3. I’LL BE A DARN GOOD DOCTOR IF PATHOLOGY DON’T DRIVE ME NUTS !!!
I LOOK AT BLOOD AND TISSUE
I LOOK AT PUS AND GUTS
I’LL BE A DARN GOOD DOCTOR
IF PATHOLOGY DON’T DRIVE ME NUTS !!!

4. A DOCTOR’S TOOL !!! "The Doctor's Doctor"
PATH IS SO COOL
A DOCTOR’S TOOL !!!
"The Doctor's Doctor"

5. Cell injury Normal cell is in a dynamic state of “Homeostasis”.
If the cells fail to adapt under stress, they undergo certain changes called cell injury.
The affected cells may recover from the injury (reversible) or may die (irreversible).

6. The precise moment of transition from reversible
injury to irreversible injury is known as the
POINT OF NO RETURN.
Irreversible
Adaptation
Reversible
Cell death
Etiologic agent
Point of no return

7. Causes of cellular injury
Hypoxia: inadequate oxygenation of tissues
Physical agents: mechanical trauma, burns,frostbite, sudden changes in pressure, radiations,electric shock
Chemical agents: poisons (toxins), insecticides, CO, asbestos, alcohol, tobacco
Infectious agents: viruses, rickettsiae, bacteria, fungi, parasites
Immunologic reactions: anaphylaxis, autoimmune disease.
Nutritional imbalances: protein calorie deficiency, vitamin deficiencies, excess food intake (obesity,atherosclerosis)
Genetic derangements: congenital malformations, abnormal proteins (hemoglobinopathies), abnormal or absent enzymes (storage disorders).

8. Types of cell injury Reversible cell injury
The cell can revert to normal if the stress or injury is removed.
Irreversible cell injury
Examples of cell injury:
Hypoxic cell injury
Ischemia- reperfusion injury
Free radical induced cell injury
Chemical injury

9. Factors determining the cell injury
Response to injury depends on
type, duration and severity of the injurious agent.
The consequences depend on:
the adaptability of cell.
3-5 minutes for neurons.
1-2 hours for myocardium.
hours for skeletal muscles.

10. Intracellular systems that are susceptible to injury
Production of ATP via aerobic respiration.
Cell membranes
Protein synthesis and
Genetic apparatus (DNA)
Note:
Damage to one system effects the other.

11. Sites of damage in cell injury- Mechanism

12. Mechanism of cell injury :
Loss of Calcium homeostasis: Causing Influx of calcium
ATP depletion
Mitochondrial damage
Increased cell membrane Permeability
Generation of oxygen derived free Radicals:
Causing damage to DNA, proteins and lipid membrane
CAMPR

13. Examples of cell injury
Hypoxic cell injury

14. Hypoxia Most common cause of cell injury.
Definition: inadequate oxygenation of tissue.
MAJOR CAUSES OF HYPOXIA
Ischemia: decreased arterial blood flow to tissues.
Most common cause of hypoxia
Ex: Atherosclerosis in coronary arteries.
Hypoxemia: decrease in the amount of oxygen dissolved in plasma. Seen in
Atelectasis, pulmonary embolus and interstitial fibrosis of lung
Decreased O2 carrying capacity of blood:
Anemia
Carbon monoxide poisoning
Most common cause of hypoxia: coronary artery atherosclerosis.

15. Consequences of tissue hypoxia
Loss of oxygen decreases ATP production by shutting down oxidative phosphorylation.
ATP required for:
Membrane transport
Protein synthesis
Lipogenesis etc.
ATP production: two ways
Oxidative phosphorylation of ADP
Anerobic glycolysis
Generation of ATP in absence of O2

16. ↓ oxidative phosphorylation
Ischemia
↓↓ ATP
 Na pump
Glycolysis
Ribosomal Detachment
Influx of Na, H2O
& Ca++
Efflux of K
 Glycogen
 Lactic acid
 Protein
Synthesis
Functional and morphologic consequences of decreased intracellular ATP during cell injury.
 pH
Cell Swelling
ER swelling
Loss of microvilli
Membrane blebs
Nuclear chromatin
clumping

17. ATP depletion : Consequences
Impaired Na,K-ATPase pump
Diffusion of Na and water into cells  cellular swelling.
Cellular swelling: First light microscopic finding of hypoxic cell injury
Swelling of the endoplasmic reticulum and mitochondria: First electron microscopic finding of hypoxic cell injury.
Cellular swelling = cloudy swelling or hydropic change.

18. Consequences of ATP depletion
2. Switching over to Anaerobic glycolysis for ATP synthesis:
It is accompanied by
Depletion of glycogen stores
Accumulation of lactic acid and
Decrease in intracellular pH  denaturation of proteins  decreased activity of many enzymes.

19. Consequences of ATP depletion
3. Decreased protein synthesis
Due to Detachment of ribosomes from the rough endoplasmic reticulum
Dissociation of polysomes into monosomes.
↓protein synthesis.
Reflected as accumulation of lipid in the cell = fatty change. (how??)
↓ protein synthesis  ↓ synthesis of apolipoproteins (lipid carriers in blood)  accumulation of lipid in the cell.
4. Impaired calcium ATPase pump
Increased cytosolic calcium

20. If oxygen is restored, all the above changes except increased cytosolic calcium are reversible.
If ischemia persists, irreversible injury follows.

21. Irreversible cell injury
Persistent or severe injury (hypoxia) takes the cell to:
the "point of no return" where the injury becomes irreversible.
At this point no intervention can save the cell.
Two phenomenon characterize irreversible injury:
Irreversible changes in mitochondrial functions.
Damage to the structural integrity of plasma membrane.
Calcium plays a major role in irreversible injury

22. Irreversible cell injury
Mechanism

23. Mitochondrial damage Damage can occur by: Increased cytosolic Ca++
Oxidative stress (free radicals)
Mitochondrial damage results in:
Formation of high conductance channels
= Mitochondrial Permeability Transition (MPT) channels.
Release of cytochrome c into cytosol
A trigger for apoptosis

24. MITOCHONDRIAL DYSFUNCTION or INJURY
↓ATP production H+
Mitochondrial dysfunction in cell injury
Cytochrome C
Mitochondrial
Permeability
Transition (MPT)
Apoptosis

25. Damage to plasma membrane
Biochemical mechanisms contributing to membrane damage
Mitochondrial dysfunction:
decreased production of phospholipids
Loss of membrane phospholipids:
Action of phospholipases
Lipid breakdown products:
Damage membrane.
Damage to cytoskeleton
Defects in membrane permeability
Can occur due to:
ATP depletion
Calcium modulated activation of phospholipases.
Bacterial toxins, viral proteins, lytic complement components and physical and chemical agents.

26. ↓O 2 ↑Cytosolic Ca++ Phospholipase activation Protease Activation
Mitoch.
dysfunction
↓Phospholipid
Synthesis
↑Phospholipid
Degradation
Cytoskeletal
Damage
Phospholipid loss
Lipid breakdown
Products
MEMBRANE DAMAGE

27. Consequences of membrane damage
Mitochondrial membrane:
Release of Cytochrome c  activates apoptosis.
Formation of MPT.
Plasma membrane:
Loss of osmotic balance
Influx of fluids and ions
Loss of proteins, enzymes, RNA.
Lysosomal membrane:
Leakage of lysosomal enzymes and their activation
RNases, DNases, proteases, phosphatases, glucosidases.
Enzymatic digestion of cell components
Cell death by necrosis.

28. Role of calcium in irreversible cell injury
Increased cytosolic calcium: Leads to :
Enzyme activation:
ATPases: Hasten ATP depletion
Phospholipases : cause membrane damage  increased permeability.
Proteases  damage membrane and structural proteins
Endonucleases  damage nuclear chromatin and DNA , causing fragmentation (karyolysis).
Increased mitochondrial permeability : release of Cytochrome c (activates apoptosis).
Damage to
Cell membrane
By activating phospholipase
Nucleus
By activating endonucleases
Cytoskleton
By activating calpain (a protease)

29. INJURIOUS AGENT Ca 2+ Ca 2+ Ca 2+ ATPase Phospholipase Protease
Increased Cytosolic Ca2+
ATPase
Phospholipase
Protease
Endonuclease
Sources and consequences of increased cytosolic calcium in cell injury.
Disruption
Of membrane
& cytoskeletal
Proteins
ATP
 Phospholipids
Nuclear
Chromatin
damage

30. ↓ oxidative phosphorylation
Ischemia
↓ oxidative phosphorylation
↓ ATP
Membrane
damage
 Na pump
Glycolysis
Ribosomal
Detachment
Influx of Na, H2O
& Ca++
Efflux of K
 Glycogen
Lactic
acid
 Protein
Synthesis
Functional and morphologic consequences of decreased intracellular ATP during cell injury.
Cytoplasmic
Enzyme
Leak
Out of cell
CK-MB, LDH
Influx Of
Calcium
 pH
Cell Swelling
ER swelling
Loss of microvilli
Membrane blebs
Nuclear chromatin
clumping

31. Free radical injury
Free radicals : chemical species that have a single unpaired electron in the outer orbit.
Initiate autocatalytic reactions that convert other molecules into free radicals.
The free radicals are generated by different mechanisms:

32. Free radical generation
Ionizing radiation
Water  OH radical +H free radical
Chemicals or drugs
CCL4  CCL3 radical (liver)
Reduction- oxidation reactions
O2  O2- (superoxide) , OH. And H2O2
Granulocytes  O2-
Transition metals: Iron & Cu
Fenton reaction  OH.
Fe2+ + H2O2 ----> Fe3+ + OH. + OH-

33. Consequences of free radical injury Oxidative modification
Lipid peroxidation
of membranes
Oxidative modification
of proteins
Lesions in
the DNA
Protein
fragmentation
And degradation
Single
stranded breaks
Increased
Permeability

34. Important free radicals
Oxygen derived Free radicals
Super oxide anion (O2 -. ):
neutralized by superoxide dismutase.
Hydroxyl radical (OH.):
neutralized by glutathione peroxidase.
Hydrogen peroxide (H2O2):
neutralized by catalase and glutathione peroxidase.
Drugs and chemicals free radicals
Conversion to free radical occurs via the cytochrome P-450 system in the liver.
FR from acetaminophen
FR from CCL4

35. FR neutralization
Cells have defense systems to prevent injury caused by FR.
Enzymes:
Superoxide dismutase (SOD)
Converts superoxide to H2O2
Catalase in Peroxisomes
Degrades H2O2
Glutathione peroxidase
Neutralizes H2O2 into water.
2. Antioxidants: (e.g. vitamin A/E/C)

36. Clinical correlations
Normal aging process:
Lipofuscin accumulates in cells damaged by FRs
Gives tissue a brown appearance.
Oxygen dependent myeloperoxidase (MPO) system
Most lethal bactericidal system present in neutrophils.

37. Oxygen toxicity:
Superoxide free radical damage
Damage to retinal tissue blindness = retrolental fibroplasia.
Ionizing radiation:
radiolysis of water  hydroxyl FR
Damages DNA  potential for cancer. E.g. Leukemia.
Reperfusion injury in the heart after myocardial infarction:
Superoxide FR and Ca++ irreversibly damage previously damaged cells on restoration of blood flow.

38. Morphology of reversible cell Injury
LIGHT MICROSCOPIC CHANGES:
Cell Swelling
First manifestation of cell injury.
Occurs when cells fail to maintain ionic and fluid homeostasis.
Manifests as small clear vacuoles.
Also known as cloudy swelling or hydropic change or vacuolar degeneration
Fatty change
Manifested by appearance of lipid vacuoles in the cytoplasm
Seen in kidney, heart and liver.

39. Normal cell
Normal cell
Injury (hypoxia)
Recovery
Reversible injury
Cell swelling,
Swelling of
ER and
mitochondria
Chromatin
clumping

40. Cell swelling - Light Microscopy
Cellular Swelling
= hydropic change
Normal epithelium
Hydropic change in renal tubular cells.

41. Myocardium : cell swelling= hydropic change
1: normal myocardial cells 2: myocardial cells with hydropic change 3: dead myocardium

42. Normal liver histology Hepatocytes showing fatty change

43. Morphological changes in the reversible and Irreversible cell injury

44. Normal cell
Endoplasmic
reticulum
Lysosome
Nucleus
Mitochondria

45. Reversible injury Surface blebs Generalized ER swelling swelling
Clumping of
Nuclear
chromatin
Dispersion of
Ribosomes
Mitochondrial
Swelling
Reversible injury

46. Morphology of irreversible cell injury

47. Light microscopy NUCLEAR CHANGES
Pyknosis = Shrinkage and darkening of the nucleus.
Karyorrhexis = fragmentation and breakdown of the nucleus, (into "nuclear dust").
Karyolysis = dissolution of the nucleus (nothing of the nucleus is visible except a purple haze).

49. Irreversible injury Rupture of Lysosomes and autolysis Lysis of ER
Defects in
Cell Membrane
Nuclear :
Pyknosis or
Karyolysis or
Karyorrhexis
Large densities
Mitochondrial
swelling

50. Timing of biochemical and morphologic changes in cell injury
2-3 hrs
6-12 hrs
Timing of biochemical and morphologic changes in cell injury.
Loss of cell function occurs much before cell death.
Cell death occurs much before it is apparent grossly or microscopically.
Example: Myocardial cells suffering ischemic injury
1-2 minutes : loss of cell function ( contractility)
20-30 minutes: Cell death
2-3 hours: appearance of first electron microscopic change of cell death
6-12 hours: required for light microscopic changes to occur.

51. Ischemia - Reperfusion injury
Ischemia-reperfusion refers to:
Cessation of blood flow followed by its restoration.
Restoration of blood flow to ischemic tissue can result in:
Recovery (if reversible injury)
May not affect the outcome (if irreversible injury)
May cause death of some cells by necrosis and apoptosis = reperfusion injury
Important process in myocardial infarction, stroke & transplant rejections.

52. How does reperfusion injury occur?
Reperfusion sets in motion new damaging processes.
Mechanisms:
Increased generation of Free Radicles
From: neutrophils, Xanthine (break down product of ATP).
Influx of calcium
Thrombolytic agents: Streptokinase and tissue plasminogen activator.
Neutralization of FR:
SOD and allopurinol (xanthine oxidase inhibitor)

53. Areas of coagulative necrosis Preservation of cellular shape.
Area of
Necrosis
Preservation of cellular shape.
Increased
cytoplasmic
eosinophilia

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