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

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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).

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

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).

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

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.

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.

Sites of damage in cell injury- Mechanism

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

Examples of cell injury Hypoxic cell injury

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.

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

↓ 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

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.

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.

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

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

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

Irreversible cell injury Mechanism

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

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

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.

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

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.

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)

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

↓ 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

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:

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-

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

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

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)

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.

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.

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.

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

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

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

Normal liver histology Hepatocytes showing fatty change

Morphological changes in the reversible and Irreversible cell injury

Normal cell Endoplasmic reticulum Lysosome Nucleus Mitochondria

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

Morphology of irreversible cell injury

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).

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

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.

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.

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)

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

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