Reaction of Cells to injury - Reversible injury ( Degeneration) - Cell functions impaired but cell can recover - Irreversible injury : - Cessation of all cell functions with cellular death - Necrosis - Sum of the degradative & inflammatory reactions occuring after tissue death. - Apoptosis - programmed cell death
Causes of cell injury ( injurious stimuli) : 1- Hypoxia - lack of oxygen to tissues ( i.e. infarct) 2- Genetic - Enzyme deficiency - Abnormality ( e.g. diabetes mellitus) 3- Nutritional - Effects on cells & growth 4- Physical - Trauma - Cold - Heat - Electrical 5- Chemical - Therapeutic ( e.g. aspirin) - Non-therapeutic ( e.g ethanol abuse) 6- Biological - Infectious agents … Bacteria – viruses , fungi 7- Immunological - Hyposensitivity or Hypersensitivity 8- Aging : - Life span of cells - Environment
Targets of injurious stimuli 1- Aerobic respiration - Loss of ATP - Sodium pump failure ……water enters cell ……. Cell swells 2- Membranes - Defect in permeability ……. Water enters cells ……cell swells & even death 3- Synthetic mechanisms - Enzymatic & structural proteins are not synthesized …….. Cell swells 4-Genetic apparatus - DNA & RNA changes - Inherited or acquired - If enzymes deficient ……. Substrate accumulates …… cell swells
Changes of injury 1 - Congestion - increase of blood flow within vessels - Vessels are dilated & packed with RBCs 2 - Edema - Increase in interstitial fluid with widened space between interstitial components - Causes swelling except in bone 3 - Hemorrhage - Accumulation of blood outside of vessels - Extravasation of RBCs into the tissues or external surfaces. 4 - Thrombosis - Clot within a blood vessel formed during life 5 - Embolus - Detached intravascular solid , liquid or gaseous mass that is carried in the blood to a site distant from it is point of origin. Ex. Fat , bubble of air or N2 , atherosclerosis plaque , tumour , bone marrow , foreign bodies
Changes of injury - Cellular infiltration : - influx of cells usually from inflammation - Neutrophils , macrophages , lymphocytes , plasma cells , fibroblasts , angioblast. - Fibrosis : - Presence of collagenous tissue - Fibroblasts , fibrocytes , collagen & ground substance. - Substance accumulation - Deposition of substances in cells or interstitium - Ex. – lipid accumulation within cells as in Gaucher,s disease - Ex. – Amyloid * Fibrillary protein produced abnormally due to longstanding inflammation or immune dysfunction. * Accumulates in the interstitium between cells & ultimately kills the cells causing major damage to organ
Hypoxic Cell Injury * Results from cellular anoxia or hypoxia due to : 1- Ischemic * Obstruction of arterial blood flow , most common cause 2- Anemia * Reduction in number of oxygen – carrying red blood cells. 3- Carbon monoxide poisoning * Diminution in the oxygen – carrying capacity RBCs by chemical alteration of hemoglobin. 4- Decreased perfusion of tissues by oxygen carrying blood * Cardiac failure ; hypertension ; & shock 5- Poor oxygenation of blood secondary to pulmonary disease.
Hypoxic Cell Injury • Early stage : - Affect mitochondria Decreased oxidative phosphorylation & ATP synthesis - Decreased ATP availability consequences * Failure of the cell membrane pump 1 - Increased intercellular Na+ & H2o & decreased intracellular K+ cellular swelling & swelling of organelles 2 - Cellular swelling ( hydropic change) has large vacuoles in the cytoplasm 3 - Swelling of the ER ( early reversible EM change) 4 - Swelling of the mitochondria - Progresses from reversible to irreversible with marked dilatation of the inner mitochondrial space.
Hypoxic Cell Injury - Early Stage * Disaggregation of ribosomes & failure of protein synthesis - Ribosomal disaggregation is also promoted by membrane damage * Stimulation of phospholipids activity - Results in increased glycolysis - Accumulation of lactate - Decreased intracellular PH - Acidification causes reversible clumping of nuclear chromatin
Hypoxic Cell Injury - Late stage - membrane damage to plasma & to lysosomal & other organelle membranes , with loss of membrane phospholipides - Reversible ( or irreversible ) morphologic signs of damage including formation of : * Myelin figures : - Whorl – like structures probably originating from damaged membrane * Cell blebs : - Cell surface deformity most likely caused by disorderly function of the cellular cytoskeleton
* Cell death – caused by severe or prolonged injury - Point of no return is marked by irreversible damage to cell membranes …….. Massive calcium influx , extensive calcifications of the mitochondria , & cell death. - Intracellular enzymes & various other proteins are released from necrotic cells into the circulation as a consequence of the loss of integrity of cell membranes . * The basis of a number of useful laboratory determinations as indicators of necrosis ……..
* Myocardial enzymes in serum - Enzymes useful in the diagnosis of myocardial infarction - Aspartate aminotransferase ( AST aka SGOT) - Lactate dehydrogenase ( LDH) - Creatinine kinase ( CK aka CPK) - Troponins - Myoglobin * Liver enzymes in serum - Transaminases - AST - Alanine aminotransferase ( ALT) - Alklaline phosphatase - Gamma – glutamyltransferase ( GGT)
* Vulnerability of cells to hypoxic injury varies with the tissue or cell type - Irreversibilty occurs after : - 3-5 minutes for neurons - 1-2 hours for myocardial cells & hepatocytes - Many hours for skeletal muscle cells
Free Radical injury * Free radicales ( reactive oxygen metabolites) - Molecules with a single unpaired electron in the outer orbital - Exemplified by activated products of oxygen reduction which includes : - superoxide ( O2-) - Hydroxyl radicales ( OH- , H2O2 )
* Generation of free radicals - Normal metabolism - Oxygen toxicity - Ex. – alveolar damage in adult respiratory distress syndrome - Ionizing radiation - Ultraviolet light - Drugs & chemicals - Many promot proliferation of SER & induction the p-450 system of mixed function of oxidases of the SER - Ex. – proliferation & hypertrophy of the SER of the hepatocytes in barbiturate intoxication - Reperfusion after ischemic injury
The role of reactive oxygen species in cell injury. O2 is converted to superoxide (O2-) by oxidative enzymes in the endoplasmic reticulum (ER), mitochondria, plasma membrane, peroxisomes, and cytosol. O2- is converted to H2O2 by dismutation and thence to OH by the Cu2+/Fe2+-catalyzed Fenton reaction. H2O2 is also derived directly from oxidases in peroxisomes. Not shown is another potentially injurious radical, singlet oxygen. Resultant free radical damage to lipid (peroxidation), proteins, and DNA leads to various forms of cell injury. Note that superoxide catalyzes the reduction of Fe3+ to Fe2+, thus enhancing OH generation by the Fenton reaction. The major antioxidant enzymes are superoxide dismutase (SOD), catalase, and glutathione peroxidase. GSH, reduced glutathione; GSSG, oxidized glutathione; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate.
Chemical cell injury - Model – liver cell membrane damage induced by carbon tetrachloride ( CCl4) - CCL4 processed by P-450 system of mixed function oxidases within SER …….. CCl3 ( highly reactive free radical) ……. Diffuses throughout the cell ….. Lipid peroxidation of intracellular membrane. -Disaggregation of ribosomes …….. Decreased protein synthesis - Plasma membrane damage …….. Cellular swelling & massive influx of calcium ………. Mitochondrial damage , denaturation of cell proteins , & cell death.
Apoptosis * Programmed physiological cell death that removes unwanted cells - Cell deletion without rupture of the cell that otherwise would elicit the inflammatory process ( aka ; necrosis > Helps to maintain homestasis & growth in tissue - Distinguished from necrosis > Greek term meaning “ falling away from “ - Involutional process similar to physiologic loss of leaves from a tree > Has subtle cellular damage - With enzymes causes nuclear codensation & fragmentation
> important mechanism for the removal of cells with irreparable - Free radicals , viruses , cytotoxic immune mechanism - If falls then can lead tom cancers , viral infections & autoimmune diseases. > Plays a role in wound healing > Also important mechanism for physiologic cell removal during embryogenesis & in programmed cell cycling ( e.g. endometrial cells during menstruation )
* Morphologic characteristics : - Tendency to involve single isolated cells or small clusters of cell s within a tissue. - Progression through a series of changes marked by lack of inflammatory response > Blebbing of plasma membrane , cytoplasmic shrinkage & increased pink staining , chromatin condensation & fragmentation. > Budding of cell & separation of membrane – bound apoptotic bodies. > Phagocytosis of apoptotic bodies by neighboring macrophages & adjacent normal cells. - Involution & shrinkage of affected cells & cell fragments …….. Small , round eosinophilic masses with chromatin remnants > e.g. Councilman bodies of viral hepatitis
* Biochemical events - active , gene expression , protein synthesis , energy consumption - Initiated by diverse injurious stimuli ( free radicals , radiation , toxic substances , withdrawal of growth factors or hormones) - Signaling by molecules ( e.g. FAS ligand , tumour necrosis factor ) & associated protein - Release of cytochrome –c & AIF ( apoptosis inducing factor) from mitochondria ……. Caspase activation ( cytosolic cytotoxic proteases ; “ major executioners) > Aspartate – speciific cysteine proteases. > Analogues of interleukin -1beta converting enzyme
- Degradation of DNA by endonucleases …….. Nucleosomal chromatin fragments ( 180-200 base pairs) ……… laddering appearance of DNA on electrophoresis - Activation of transglutaminases ( cross- link apoptotic cytoplasmic proteins) - No inflammatory reaction
Apoptosis - Regulation * Mediated by a number of genes & their products > bcl-2 , gene product inhibits apoptosis > bax , gene product facilitates apoptosis > p53 , gene product facilitates apoptosis by decreasing transcription of bcl-s & increasing transcription of bax.
The intrinsic (mitochondrial) pathway of apoptosis. Death agonists cause changes in the inner mitochondrial membrane, resulting in the mitochondrial permeability transition (MPT) and release of cytochrome c and other pro-apoptotic proteins into the cytosol, which activate caspases
Necrosis - One of the two morphologic patterns of cell death ( the other is apoptosis) - Gross irreversible cellular injury - Sum of the degradative & inflammatory reactions occuring after tissue death caused by injury -e.g. hypoxia , exposure to toxic chemicals - Passive process since does not require gene involvement or new protein synthesis - Triggers or elicits a marked inflammatory response - Liberation of lysosomal enzymes , digestion of cell membranes , disruption of cells ; influx of macrophages due to release of chemotactic factors. - Removal of debris by phagocytic macrophages.
Necrosis * general & cellular characteristics - Change appears after the cells die. - Occurs within living organisms ; many contiguous cells , fixed cells of pathologic specimens are dead but not necrotic - DNA fragmentation is haphazard with smudge pattern on electrophoresis > Autolysis : - Degradative reactions in cells caused by intracellular enzymes indigenous to the cells. - Postmortem autolysis occurs after death of the entire organism & ≠ necrosis
> Heterolysis : Cellular degradation by enzymes derived from sources extrinsic to the cell ( e.g. , bacteria , leukocytes) ……… increased pink cytoplasm. - Nuclear changes : - Morphological recognizable light microscope nuclear changes ………. Progressive nuclear condensation with eventual disappearance of stainble nuclei. - Pyknosis : the shrinkage of the nucleus into a small deeply basophilic or black clumps of chromatin. - Karyorrhexis : a fragmentation of the nucleus into multiple small black dots or pieces. - Karyolysis : the fading of the nucleus ; less & less basophilic until it disappears.
Shrinkage (pyknosis), increased nuclear basophilic staining (hyperchromasia), nuclear fragmentation (karyorrhexis, karryolysis), are classic features of apoptosis.