1. Adriana Acurio, M.D. Pathology Department Mount Sinai Hospital Chicago

2. Stages of Cellular Response to Injury Normal cell (STEADY-STATE) REVERSIBLE CELL INJURY ADAPTATIONS CELL INJURY IRREVERSIBLE CELL INJURY NECROSIS APOPTOSIS STRESS/ INJURY INSUFFICIENT ADAPTATION MILD TO MODERATE STRESS SEVERE STRESS CELL DEATH

3. Cell Injury and Cell Death REVERSIBLE CELL INJURY CELL DEATH Reduced energy production via oxidative process Depletion of cellular energy stores (ATP) Osmotic imbalance leading to water influx and cell swelling NECROSIS: Cell death caused by membrane and lysosomal damage leading to digestion and leakage of cell contents APOPTOSIS (Programmed cell death): Cell kills itself maintaining membrane integrity and avoidingleakage of contents that can damage surrounding cells. Apoptosis can be: PATHOLOGIC or PHYSIOLOGIC

4. Types of Cell Injury 1. Physical: direct physical trauma, thermal injury, radiation, electric shock 2. Hypoxic: decreased oxygen state 3. Chemical: Wide range of agents. Environmental pollutants, drugs, aberrations in normal cell solutes leading to osmolar imbalances 4. Infectious: From Prions to Parasites 5. Immunologic: Immune reactions to external injury and autoimmune disease 6. Genetic: Genetic aberrations, abnormal protein expression causing direct cell injury or increased susceptibility to deleterious agents 7. Nutritional: Under and overnutrition may lead to cell injury and disease

5. Morphologic Changes of Cell Injury REVERSIBLE CHANGES 1. Cellular Swelling: FIRST response to injury Loss of energy disables ATP-dependent ion pumps and leads to loss of osmotic homeostasis Morphological manifestations: Plasma membrane blebbing Mitochodrial and ER swelling Nuclear changes

6. Cellular Swelling/Reversible Injury ER MITOCHONDRIA LYSOSOME BLEBS AGGREGATION OF INTRAMEMBRANOUS PARTICLES/ loss of microvilli MITOCHONDRIAL SWELLING ACCUMULATION OF DENSITIES LIPID VACUOLES SWELLING OF LYSOSOME NUCLEAR SWELLING AND CHROMATIN CLUMPING NORMAL CELL REVERSIBLE CELL INJURY

7. Morphologic Changes in Cellular Injury NECROSIS IRREVERSIBLE CHANGES 1. Nuclear changes  breakdown of DNA 2. Severe mitochondrial damage  ATP depletion 3. Lysosome damage  autophagy 4. Cytoplasmic changes  eosinophilia 5. Membrane damage  leakage of cellular contents Necrosis often leads to inflammation/injury of surrounding cells

8. Morphologic changes of Necrosis Nuclear Changes: Due to breakdown of DNA Karyolysis: decreased nuclear basophilia due to DNA degradation by nucleases Pyknosis: Shrinkage and increased basophilia due to chromatin condensation (often seen in apoptosis) Karyorrhexis: Fragmentation of pyknotic nuclei Loss of Nuclei: with time necrotic nuclei dissapear

9. NUCLEAR changes of Necrosis

10. KaryolysisPyknosisKaryorrhexis NUCLEAR changes of Necrosis

11. Morphologic changes of Necrosis Cytoplasmic Changes Cytoplasmic eosinophilia: due to a decline in cytosolic mRNA, which is basophilic, and an increase in cytosolic concentration of denatured proteins Glassy cytoplasm: Loss of glycogen particles Myelin figures: Formed by broken cell membranes.

12. Patterns of Tissue Necrosis 1. Coagulative Necrosis: Overall architecture is preserved, individual cells have a ghostly appearance. Destruction of proteolytic enzymes hinders proteolysis of necrotic cell Necrotic cells remain until phagocytosis by infiltrating leukocytes occurs Example: Ischemia  infarction

13. Patterns of Tissue Necrosis Coagulative necrosis

14. Patterns of Tissue Necrosis 2. Liquefactive Necrosis: Digestion of dead cells which gives tissue a liquid appearance Infectious process leads to recruitment of white cells which release proteolytic enzymes which digest the dead cells Grossly, it is often seen as a circumscribed lesion filled with pus and cellular debri It is generally associated with abcess formation (INFECTIOUS) and for unknown reasons in brain hypoxic injury

15. Patterns of Tissue Necrosis Liquefactive Necrosis

16. Patterns of Tissue Necrosis 3.Gangrenous Necrosis: More than a pattern of necrosis it is a clinical descriptor (often a combination of coagulative and liquefactive necrosis) Term often used to describe limb necrosis secondary to loss of blood supply (coagulative necrosis) with superimposed bacterial infection (liquefactive necrosis)  wet-gangrene

17. Patterns of Tissue Necrosis Gangrenous necrosis

18. Patterns of Tissue Necrosis 4. Caseous Necrosis: Most commonly seen in mycobacterial infections and refers to the cheese-like appearance of the necrotic tissue Histologically, there is amorphous granular debris enclosed within an inflammatory border known as a granulomatous reaction. The tissue architecture is completely obliterated, unlike coagulative necrosis

19. Patterns of Tissue Necrosis Caseuos Necrosis Case of tuberculosis

20. Patterns of Tissue Necrosis 5.Fat Necrosis: Not a real pattern of necrosis, it refers to fat necrosis of pancreas and peritoneal cavity in seen in pancreatitis In acute pancreatitis, pancreatic enzymes leak into the peritoneum and liquefy surrounding fat cells releasing fatty acids These fatty acids combine with calcium to form calcium salts (soap)

21. Patterns of Tissue Necrosis Fat necrosis

22. Patterns of Tissue Necrosis 6. Fibrinoid Necrosis: Necrosis caused by deposition of immune-complexes and fibrin within walls of blood vessels. Pa thW ebsMuseum Polyarteritis Nodosa

23. Mechanisms of Injury/Necrosis 1. ATP Depletion 2. Mitochondrial Damage 3. Loss of Calcium Homeostasis 4. Oxidative Stress 5. Membrane Permeability Defects 6. DNA and Protein Damage

24. Mechanisms of Injury/Necrosis

25. Mechanisms of Cell Injury 1.Energy Depletion ATP is synthesized by one of two pathwyas: ADP phosphorylation in mitochondria (aerobic) Glycolysis (anaerobic) Causes of ATP Depletion: decreased oxygen and nutrients, mitochondrial damage, toxins All synthetic and degenerative processes require ATP, its depletion has widespread defect on many critical systems

26. Mechanisms of Cell Injury Membrane-bound pump malfunction Na/K-ATPase  cell swelling/ER dilation Ca++pump  influx of Ca++=derangement of cellular proceses Stimulation of glycolytic pathway  decrease pH ER detachment of ribosomes  decreased protein synthesis Protein missfolding, mitochondrial, lysosomal, nuclear membrane damage= Necrosis

27. Mechanisms of Cell Injury 2. Mitochondrial Damage Decreased ATP production: Electric potential across mitocondrial membrane is necessary for ATP production. Injury leads to formation of a high- conductance channel: the mitochondrial permeability transition pore  decreased potential  decreased ATP Activation of Apoptosis: Within the mitochondrial membrane lie proteins that activate apoptotic pathways, such as Cytochrome c and Caspases. Membrane injury and leakage of these proteins activates these pathways

28. Abou-Sleiman et al. Nature Reviews Neuroscience 7, 207–219 (March 2006) | doi:10.1038/nrn1868 Mechanisms of Cell Injury 2. Mitochondrial Damage Mitochondrial permeability transition pore (mPTP)

29. Mechanisms of Cell Injury 3. Loss of Ca 2+ Homeostasis Intracellular Ca 2+ is maintained at very low levels ( ∼ 0.1 μmol) in mitochondria and the ER Extracellular Ca 2+ levels are high (1.3 mmol), therefore a loss of homeostasis can lead to massive Ca 2+ influx This leads to: Activation of the mitochondrial permeability transition pore (mPTP) Activation of phospholipases, proteases, endonucleases Incrased mitochondrial permeability and activation of caspases and porcaspases which directly induce apoptosis

30. Mechanisms of Cell Injury 4. Oxidative Stress O 2 derived free radicals are species with an unstable single electron on its outer orbit which easily reacts with many key components of the cell Free radicals initiate autocatalytic reactions, converting molecules that they react with into free radicals, propagating the cellular damage ROS are produced normally in cells but they are degraded and removed by cellular defense systems. Loss of this steady state (where ROS are transient and in low concentration) results in OXIDATIVE STRESS

31. Mechanisms of Cell Injury 4. Oxidative Stress: Generation of ROS During normal respiration, O 2 reduction  2H 2 O by the transfer of four electrons to H 2, oxidative enzymes in the cell catalyze this reaction and produce partially reduced intermediates (ROS) during the process Radiation can break down water into OH and hydrogen (H) free radicals Inflammation leads to bursts of ROS: Activated leukocytes generate ROS via a Redox reaction lead by NADPH oxidase in plasma membrane Enzymatic drug metabolism (CCL4) Nitric oxide (NO) generated by many cell types can be converted to highly reactive peroxynitrite anion (ONOO - ), NO 2 and NO 3 -

32. Mechanisms of Cell Injury Oxidative Stress: Generation of ROS

33. Mechanisms of Cell Injury Oxidative Stress: Effects of ROS

34. Mechanisms of Cell Injury Oxidative Stress: Removal of ROS Spontaneous decay: Superoxide (O-2)  O 2 and H 2 O 2 in the presence of water

35. Mechanisms of Cell Injury Oxidative Stress: Removal of ROS Enzymatic and nonenzymatic cellular removal of ROS 1. Antioxidants block initiation of free radical formation or inactivate free radicals. Lipid-soluble vitamins E and A, ascorbic acid and glutathione in the cytosol. 2. Transferrin, ferritin, lactoferrin, and ceruloplasmin are transport and storage proteins that chelate iron and copper which can catalyze the formation of ROS 3. Enzymatic degradation of H 2 O 2 and O - 2 Catalase, H 2 O 2 (2H 2 O 2 → O 2 + 2H 2 O). Superoxide dismutases (SODs), O - 2  H 2 O 2 (2O - 2 + 2H → H 2 O 2 + O 2 ) Glutathione peroxidase H 2 O 2 + 2GSH → GSSG [glutathione homodimer] + 2H 2 O, or 2OH + 2GSH → GSSG + 2H 2 O)

36. Mechanisms of Cell Injury 5. Membrane Disruption 1. ROS cause injury to cell membranes via lipid peroxidation 2. Decreased phospholipid synthesis. Low ATP leads to decreased production of phospholipids affecting all cellular membranes 3. Increased phospholipid breakdown. Cell injury leads to activation of endogenous phospholipases and membrane breakdown. Lipid breakdown products have a detergent effect on membranes resulting in changes in permeability 4. Protease activation. Increased intracellular calcium proteases that damage cytoskeleton

37. Mechanisms of Cell Injury Causes of Membrane Disruption Membrane damage is a consistent feature of most forms of cell injury (except apoptosis)

38. Mechanisms of Cell Injury Effects of Membrane Disruption Mitochondrial membrane damage  mitochondrial permeability transition pore  decreased ATP, release of apoptotic proteins Plasma membrane damage  loss of osmotic balance and influx of fluids and ions  further decease in ATP Injury to lysosomal membranes  leakage of hydrolases into cytoplasm (RNases, DNases, proteases, phosphatases, glucosidases) and degradation of targets. Low intracellular pH allows for activation of hydrolases

39. Mechanisms of Cell Injury 6. Damage to DNA/Proteins DNA damage can be caused by toxic drugs, radiation, oxidative stress, etc Normal DNA repair mechanisms correct damage and maintain the steady state If damage is too extensive for repair, the cell initiates a self destructive program called apoptosis

40. Mechanisms of Cell Injury 1. ATP Depletion 2. Mitochondrial Damage 3. Loss of Calcium Homeostasis 4. Oxidative Stress 5. Membrane Permeability Defects 6. DNA and Protein Damage

41. Stages of Cellular Response to Injury Normal cell (STEADY-STATE) REVERSIBLE CELL INJURY ADAPTATIONS CELL INJURY IRREVERSIBLE CELL INJURY NECROSIS APOPTOSIS STRESS/ INJURY INSUFFICIENT ADAPTATION MILD TO MODERATE STRESS SEVERE STRESS CELL DEATH

42. Irreversible Cell Injury-Necrosis Irreversible cell injury is characterized by 2 main events: mitochondrial dysfunction (lack of oxidative phosphorylation and ATP generation) even after resolution of the original injury, and disturbances in membrane function Lysosomal injury results in the enzymatic degradation of the cell that is characteristic of necrosis Leakage of intracellular proteins through the damaged cell membrane leads to recruitment of inflammatory response also typical of necrosis

43. Questions

44. Question 1 At autopsy, a patient with known severe arthrosclerosis and occlusion of middle cerebral artery is found to have a cystic lesion in the parietal lobe. This finding most likely represents: A. Atrophy B. Gangrenous necrosis C. Apoptosis D. Coagulative necrosis E. Liquefactive necrosis

45. Question 1 At autopsy, a patient with known severe arthrosclerosis and occlusion of middle cerebral artery is found to have a cystic lesion in the parietal lobe. This finding most likely represents: A. Atrophy B. Gangrenous necrosis C. Apoptosis D. Coagulative necrosis E. Liquefactive necrosis

46. Question 2 Which of the following is/are event/s of irreversible cell injury (necrosis) A. Plasma membrane blebbing B. Mitochodrial and ER swelling C. Nuclear changes D. Membrane Permeability Defects E. DNA degradation

47. Question 2 Which of the following is/are event/s of irreversible cell injury (necrosis) A. Plasma membrane blebbing B. Mitochodrial and ER swelling C. Nuclear changes D. Membrane Permeability Defects E. DNA degradation

48. Question 3 True or False. Hypoxia (reduced oxygen availability) leads to a more rapid and severe cellular injury compared to ischemia (reduced blood flow), why?

49. Question 3 True or False. Hypoxia (reduced oxygen availability) leads to a more rapid and severe cellular injury compared to ischemia (reduced blood flow), why? False Hypoxia, reduced oxygen availability  decreased aerobic energy production/ switch to anaerobic glycolysis Ischemia, restriction in blood supply due to factors in blood vessels  decrease in aerobic and anaerobic energy production Ischemia leads to more rapid and severe damage than hypoxia alone