DPT - Cell injury and Cell death

Pathology and Microbiology

1. Areesha Ahmad B.Sc (H), M.Sc , M.Phil Ph.D scholar ( Microbiology)

2. Cell injury and death Definitions: Cell injury:  Sequence of events that occurs when stresses exceed ability of cells to adapt. Responses are initially reversible, but may progress to irreversible injury and cell death . Cell death: Results when continuing injury becomes irreversible, at which time the cell cannot recover.

3. General Pathology Cell injury and death Causes of cell injury Cell death Necrosis Apoptosis

4. "embolus" is  a blood clot 

5. Anaphylaxis: is  a severe, potentially life-threatening allergic reaction .  something that has or can have both favorable and unfavorable consequences . D ouble edged sword:

6. An iatrogenic condition is a state of ill health or adverse effect caused by medical treatment; it  usually results from a mistake made in treatment, and can also be the fault of a nurse, therapist or pharmacist . HT:  Hormone therapy .

7. Cell death There are TWO principle types of cell death: 1. Necrosis  2. Apoptosis

8. 1. Necrosis Death of cells in living tissues characterized by the breakdown of cell membranes . These changes occur because of denaturation of cellular proteins, largely by release of hydrolytic enzymes from damaged lysosomes . In necrosis, excess fluid enters the cell, swells it, & ruptures its membrane which kills it. After the cell has died, intracellular degradative reactions occur within a living organism. Necrosis does not occur in dead organisms.

9. Necrosis occurs by the following mechanisms: Hypoxia Free radical-induced cell injury Cell membrane damage Increased intracellular calcium level

10. Hypoxia Hypoxia is decreased oxygen supply to tissues. It can be caused by: Ischemia: Ischemia is decreased blood flow to or from an organ. Ischemia can be caused by obstruction of arterial blood flow – the most common cause, or by decreased perfusion of tissues by oxygen -carrying blood as occurs in cardiac failure, hypotension, & shock. Anemia: Anemia is a reduction in the number of oxygen-carrying red blood cells. Carbon monoxide poisoning: CO decreases the oxygen-capacity of red blood cells by chemical alteration of hemoglobin. Poor oxygenation of blood due to pulmonary disease.

11. Free radical-induced injury Free radical is any molecule with a single unpaired electron in the outer orbital. Examples include superoxide & the hydroxyl radicals. When the production of free radicals exceeds their degradation, the excess free radicals cause membrane pump damage, ATP depletion, & DNA damage. These can cause cell injury & cell death.

12. Cell membrane damage Increased intracellular calcium level Direct cell membrane damage as in extremes of temprature , toxins, or viruses, or indirect cell membrane damage as in the case of hypoxia can lead to cell death by disrupting the homeostasis of the cell. Increased intracellular calcium level It is a common pathway via which different causes of cell injury operate. For example, the cell membrane damage leads to increased intracellular calcium level. The increased cytosolic calcium, in turn, activates enzymes in the presence of low pH. The activated enzymes will degrade the cellular organelles.

13. 2. Apoptosis Apoptosis  – Defined as programmed cell death characterized by nuclear dissolution, fragmentation of the cell without complete loss of membrane integrity, and rapid removal of the cellular debris.  Apoptosis can be physiological or pathological Apoptosis is the death of single cells within clusters of other cells. (Note that necrosis causes the death of clusters of cells.) In apoptosis, the cell shows shrinkage & increased acidophilic staining of the cell. This is followed by fragmentation of the cells. These fragments are called apoptotic bodies. Apoptosis usually occurs as a physiologic process for removal of cells during embryogenesis, menstruation, etc… It can also be seen in pathological conditions caused by mild injurious agents.

14. Reversible Cell injury I njurious stimulus applied in normal cell to injured cell If stimulus removed from cell so cell can perform normal function again and can reverse their normal morphology. Morphological and functional changes can be reverse. This stage is called as Reversible cell injury.

15. Morphology The two main morphologic correlates of reversible cell injury are C ellular swelling F atty change

16. Cellular swelling Mitochondria make ATP through oxidative phosphorylation that is imp for many cell function Due to schemia oxygen reduces due to which mitochondria stops oxidative phyosphorylation process so ATP production also stops. ATP is required for transferring sodium potassium through sodium potassium ATPs pump that transfer 3 Na to cell outside and 2 K to inside. Due to no production of ATP, pumps will inhibit and Na will not go outside. Due to high conc of Na water will also go inside so cell become swelling called as cellular swelling

17. In reversible cell injury first cellular swelling causes All cellular organelles will swell specially Mitochondria Endoplasmic reticulum will also dialted Due to swelling cell will heavy, cell weight and size will increase Due to swelling capillaries congestion will be occurred. In Rough endoplasm , R ibosomes synthesizes protein. primary, secondary and tertiary protein synthesizes. ATP also required for Proteins synthesis so due to no ATP , protein synthesis stops and protein become denatured. Ribosomes will also detached from R.E Ribosomes convert into monosomes from polysomes and get fragmented

18. In N ucleus chromatin material got clotted. In Cell membrane blubbing appear due to water influx bubbles appear. Phospholipids also found in cytoplasm called as myelin figure . ( Mylein figures are the component of phospholipids) 2. Fatty Change Fat vacuoles will be accumulated specially triglyceralaldehydes in those cells where fat metabolism occur specially in liver.

19. The cytoplasm of injured cells also may become redder ( eosinophilic ), a change that becomes much more pronounced with progression to necrosis. Other intracellular changes associated with cell injury include P lasma membrane alterations such as blebbing , dis t ortion of microvilli, and loosening of intercellular attachments. Mitochondrial changes such as swelling and the appearance of phospholipid-rich amorphous densities D ilation of the ER

20. Cell death (Morphology) Necrosis is characterized by changes in the cytoplasm and nuclei of the injured cells Cytoplasmic changes Necrotic cells show increased eosinophilia (i.e., they are stained red by the dye eosin—the E in the hematoxylin and eosin [H&E] stain), attributable partly to increased binding of eosin to denatured cytoplasmic proteins. Compared with viable cells, the cell may have a glassy , homogeneous appearance , mostly because of the loss of lighter staining glycogen particles. Myelin figures are more prominent in necrotic cells than in cells with reversible injury. When enzymes have digested cytoplasmic organelles , the cytoplasm becomes vacuolated and appears “ moth-eaten.” By electron microscopy , necrotic cells are char a cterized by discontinuities in plasma and organelle membranes, marked dilation of mitochondria associated with the appearance of large amorphous intramitrochondrial densities, disruption of lysosomes , and intracytoplasmic myelin figures . a breakdown of DNA and chromatin

21. Pyknosis is characterized by nuclear shrinkage and increased basophilia ; the DNA condenses into a dark shrunken mass. The pyknotic nucleus can undergo fragmentation ; this change is called karyorrhexis . Ultimately, the nucleus may undergo karyolysis , in which the basophilia fades because of digestion of DNA by deoxyribonuclease ( DNase ) activity. In 1 to 2 days, the nucleus in a dead cell may completely disappear. Fates of necrotic cells . Necrotic cells may persist for some time or may be digested by enzymes and disappear which are either phagocytosed by other cells Further Dead cells may be replaced by myelin figures degraded into fatty acids. These fatty acids bind calcium salts , which may result in the dead cells ultimately becoming calcified. Nuclear Changes

22. Other pathways of cell death In addition to necrosis and apoptosis, two other patterns of cell death have been described that have unusual features. Although the importance of these pathways in disease remains to be established, they are the subjects of considerable current research, and it is useful to be aware of the basic concepts. Necroptosis Pyroptosis

23. 1. Necroptosis The name necroptosis implies that there are features of both necrosis and apoptosis. Some infections are believed to kill cells by this pathway, and it has been hypothesized to play a role in ischemic injury and other pathologic situations, especially those associated with inflammatory reactions in which the cytokine TNF is produced. However, when and why it occurs and how significant it is in human diseases is not well understood.

24. 2. Pyroptosis. This form of cell death is associated with activation of a cytosolic danger-sensing protein complex called the inflammasome . The result of inflammasome activation is the activation of caspases , some of which induce the production of cytokines that induce inflammation , often manifested by fever , and others trigger apoptosis. Thus, apoptosis and inflammation coexist . The name pyroptosis stems from the association of apoptosis with fever (Greek, pyro = fire). It is thought to be one mechanism by which some infectious microbes cause the death of infected cells. Its role in other pathologic situations is unknown

25. Autophagy Autophagy (“self-eating”) refers to lysosomal digestion of the cell’s own components. It is a survival mechanism in times of nutrient deprivation , so that the starved cell can live by eating its own contents and recycling these contents to provide nutrients and energy. In this process, intracellular organelles and portions of cytosol are first sequestered within an ER-derived autophagic vacuole, whose formation is initiated by cytosolic proteins that sense nutrient deprivation. The vacuole fuses with lysosomes to form an autophagolysosome , in which lysosomal enzymes digest the cellular components. In some circumstances, autophagy may represent an adaptation that helps cells survive lean times. If, however, the starved cell can no longer cope by devouring its contents, autophagy may eventually lead to apoptotic cell death.

26. Mechanism of cell injury and cell death G eneral principles The cellular response to injurious stimuli depends on the type of injury, its duration , and its severity. Thus, low doses of toxins or a brief period of ischemia may lead to reversible cell injury , whereas larger toxin doses or longer ischemic times may result in irreversible injury and cell death. The consequences of an injurious stimulus also depend on the type, status, adaptability, and genetic makeup of the injured cell. The same injury has vastly different outcomes depending on the cell type. For instance, striated skeletal muscle in the leg tolerates complete ischemia for 2 to 3 hours without irreversible injury, whereas cardiac muscle dies after only 20 to 30 minutes of ischemia. The nutritional (or hormonal) status also can be important Genetically determined diversity in metabolic pathways can contribute to differences in responses to injurious stimuli.

27. Cell injury usually results from functional and biochemical abnormalities in one or more of a limited number of essential cellular components. D ifferent external insults typically affect different cellular organelles and biochemical pathways . However, it should be emphasized that the very same injurious agent may trigger multiple and overlapping biochemical pathways . T herefore, it has proved difficult to prevent cell injury by targeting an individual pathway.

28. Hypoxia and ischemia Deficiency of oxygen leads to failure of many energy dependent metabolic pathways , and ultimately to death of cells by necrosis. Most cellular ATP is produced from adenosine diphosphate (ADP) by oxidative phosphorylation during reduction of oxygen in the electron transport system of mitochondria . High-energy phosphate in the form of ATP is required for membrane transport , protein synthesis and lipogenesis , It is estimated that in total, the cells of a healthy human burn 50 to 75 kg of ATP every day! Oxygen deprivation is one of the most frequent causes of cell injury and necrosis in clinical medicine.

29. Cells subjected to the stress of hypoxia that do not immediately die activate compensatory mechanisms that are induced by transcription factors of the hypoxia inducible factor 1 (HIF-1) family. HIF-1 simulates the synthesis of several proteins that help the cell to survive in the face of low oxygen. Some of these proteins , such as vascular endothelial growth factor (VEGF), stimulate the growth of new vessels and thus attempt to increase blood flow and the supply of oxygen . Other proteins induced by HIF-1 cause adaptive changes in cellular metabolism by stimulating the uptake of glucose and glycolysis Anaerobic glycolysis can generate ATP in the absence of oxygen using glucose derived either from the circulation or from the hydrolysis of intracellular glycogen. N ormal tissues with a greater glycolytic capacity because of the presence of glycogen (e.g., the liver and striated muscle) are more likely to survive hypoxia and decreased oxidative phosphorylation than tissues with limited glucose stores (e.g., the brain ).

30. Persistent or severe hypoxia and ischemia ultimately lead to failure of ATP generation and depletion of ATP in cells. Loss of this critical energy store has deleterious effects on many cellular systems . Reduced activity of plasma membrane ATP-dependent sodium pumps , resulting in intracellular accumulation of sodium and efflux of potassium. The net gain of solute is accompanied by isoosmotic gain of water, causing cell swelling and dilation of the ER. The compensatory increase in anaerobic glycolysis leads to lactic acid accumulation , decreased intracellular pH, and decreased activity of many cellular enzymes. Prolonged or worsening depletion of ATP causes structural disruption of the manifested as detachment of ribosomes from the rough ER (RER) and dissociation of polysomes into monosomes , with a consequent reduction in protein synthesis protein synthetic apparatus. Ultimately, there is irreversible damage to mitochondrial and lysosomal membranes, and the cell undergoes necrosis.

31. Ischemia reperfusion injury T he restoration of blood flow to ischemic but viable tissues results, paradoxically, in increased cell injury . This is the reverse of the expected outcome of the restoration of blood flow , which normally results in the recovery of reversibly injured cells. This so-called “ischemia-reperfusion injury” is a clinically important process that may contribute significantly to tissue damage , New damage may be initiated during reoxygenation by increased generation of Reactive oxygen species (ROS). The inflammation that is induced by ischemic injury may increase with reperfusion because it enhances the influx of leukocytes and plasma proteins . The products of activated leukocytes may cause additional tissue injury. Activation of the complement system also may contribute to ischemia-reperfusion injury. Complement proteins may bind to the injured tissues , or to antibodies that are deposited in the tissues, and subsequent complement activation generates byproducts that exacerbate the cell injury and inflammation .

32. Oxidative stress Oxidative stress refers to cellular abnormalities that are induced by ROS , which belong to a group of molecules known as free radicals . Free radical-mediated cell injury is seen in many circumstances, including chemical and radiation injury , hypoxia , cellular aging , tissue injury caused by inflammatory cells, and ischemia-reperfusion injury . In all these cases, cell death may be by necrosis , apoptosis , or the mixed pattern of necroptosis . Free radicals are chemical species with a single unpaired electron in an outer orbit. Such chemical states are extremely unstable, and free radicals readily react with inorganic and organic molecules ; when generated in cells, they attack nucleic acids as well as a variety of cellular proteins and lipids . In addition, free radicals initiate reactions in which molecules that react with the free radicals are them selves converted into other types of free radicals , thereby propagating the chain of damage .

33. Generation and Removal of Reactive Oxygen Species The accumulation of ROS is determined by their rates of production and removal. ROS are produced by two major pathways. • ROS are produced normally in small amounts in all cells during the reduction-oxidation ( redox ) reactions that occur during mitochondrial respiration and energy generation. In this process, molecular oxygen is reduced in mitochondria to generate water by the sequential addition of four electrons. • ROS are produced in phagocytic leukocytes , mainly neutrophils and macrophages , as a weapon for destroying ingested microbes and other substances during inflammation and host defense . • Nitric oxide (NO) is another reactive free radical produced in macrophages and other leukocytes . It can react with O2 − to form a highly reactive compound , peroxynitrite , which also participates in cell injury

34. The generation of free radicals is increased under several circumstances: The absorption of radiant energy (e.g., ultraviolet (UV) light, x-rays). The enzymatic metabolism of exogenous chemicals (e.g., carbon tetrachloride etc. Inflammation , in which free radicals are produced by leukocytes

35. Cell Injury Caused by Reactive Oxygen Species ROS causes cell injury by damaging multiple components of cells Lipid peroxidation of membranes. Double bonds in membrane polyunsaturated lipids are vulnerable to attack by oxygen-derived free radicals. The lipid–radical interactions yield peroxides, which are themselves unstable and reactive, and an autocatalytic chain reaction ensues. Damage to plasma membranes as well as mitochondrial can have devastating consequences. Crosslinking and other changes in proteins . Free radicals promote sulfhydryl-mediated protein crosslinking, resulting in enhanced degradation or loss of enzymatic activity . Free radical reactions also may directly cause polypeptide fragmentation . Damaged proteins may fail to fold properly , triggering the unfolded protein response. DNA damage . Free radical reactions with thymine residues in nuclear and mitochondrial DNA produce single strand breaks. Such DNA damage has been implicated in apoptotic cell death, aging, and malignant transformation of cells.

36. In addition to the role of ROS in cell injury and the killing of microbes , low concentrations of ROS are involved in numerous signaling pathways in cells and thus in many physiologic reactions. Therefore, these molecules are produced normally but , to avoid their harmful effects , their intracellular concentrations are t ightly regulated in healthy cells

37. Cell injury caused by toxins Toxins , including environmental chemicals and substances produced by infectious pathogens, induce cell injury that culminates primarily in necrotic cell death. Different types of toxins induce cell injury by two general mechanism: Direct-acting toxins . Some toxins act directly by combining with a critical molecular component or cellular organelle. For example, in mercuric chloride poisoning (as may occur from ingestion of contaminated seafood), mercury binds to the sulfhydryl groups of various cell membrane proteins, causing inhibition of ATP-dependent transport and increased membrane permeability. Many anti-neoplastic chemotherapeutic agents also induce cell damage by direct cytotoxic effects. Also included in this class are toxins made by microorganisms. These often cause damage by targeting host cell molecules that are needed for essential functions, such as protein synthesis and ion transport. Latent toxins . Many toxic chemicals are not intrinsically active but must first be converted to reactive metabolites , which then act on target cells. Understandably, such toxins typically affect the cells in which they are activated. This is usually accomplished by cytochrome

38. Endoplasmic reticulum stress The accumulation of misfolded proteins in a cell can stress compensatory pathways in the ER and lead to cell death by apoptosis. During normal protein synthesis, chaperones in the ER control the proper folding of newly synthesized proteins, and misfolded polypeptides are ubiquitinated and targeted for proteolysis. If unfolded or misfolded proteins accumulate in the ER, they first induce a protective cellular response that is called the unfolded protein response . This adaptive response activates signaling pathways that increase the production of chaperones and decrease protein translation, thus reducing the levels of misfolded proteins in the cell. When a large amount of misfolded protein accumulates and cannot be handled by the adaptive response , the signals that are generated result in activation of proapoptotic sensors of the BH3 -only family as well as direct activation of caspases , leading to apoptosis by the mitochondrial ( intrinsic ) pathway.

39. Intracellular accumulation of misfolded proteins may be caused by abnormalities that increase the production of misfolded proteins or reduce the ability to eliminate them . Resason : This may result from gene mutations that lead to the production of proteins that cannot fold properly; aging , which is associated with a decreased capacity to correct misfolding ; infections, especially viral infections , when large amounts of microbial proteins are synthesized within cells , more than the cell can handle ; increased demand for secretory proteins such as insulin in insulin-resistant states; and changes in intracellular pH and redox state. Abnormality: Protein misfolding is thought to be the fundamental cellular abnormality in several neurodegenerative diseases . Deprivation of glucose and oxygen , as in ischemia and hypoxia , also may increase the burden of misfolded proteins

40. Protein misfolding within cells may cause disease by creating a deficiency of an essential protein or by inducing apoptosis . Misfolded proteins often lose their activity and are rapidly degraded, both of which can contribute to a loss of function. If this function is essential, cellular injury ensues. Diseases One important disease in which this occurs is cystic fibrosis, which is caused by inherited mutations in a membrane transport protein that prevent its normal folding. Cell death as a result of protein misfolding is recognized as a feature of a number of diseases, including the neurodegenerative disorders Alzheimer disease , Huntington disease , and Parkinson disease , and may underlie type 2 diabetes as well.

41. DNA Damage Exposure of cells to radiation or chemotherapeutic agents, intracellular generation of ROS , and acquisition of mutations may all induce DNA damage, which if severe may trigger apoptotic death . Damage to DNA is sensed by intracellular sentinel proteins, which transmit signals that lead to the accumulation of p53 protein . p53 first arrests the cell cycle (at the G1 phase) to allow the DNA to be repaired before it is replicated. However , if the damage is too great to be repaired successfully, p53 triggers apoptosis , mainly by stimulating BH3- only sensor proteins that ultimately activate Bax and Bak , proapoptotic members of the Bcl-2 family . When p53 is mutated or absent (as it is in certain cancers), cells with damaged DNA that would otherwise undergo apoptosis survive . In such cells, the DNA damage may result in mutations or DNA rearrangements (e.g., translocations ) that lead to neoplastic transformation.

42. Inflammation A common cause of injury to cells and tissues is the inflammatory reaction that is elicited by pathogens , necrotic cells, and dysregulated immune responses , as in autoimmune diseases and allergies . In all these situations, inflammatory cells , including neutrophils , macrophages, lymphocytes, and other leukocytes, secrete products that evolved to destroy microbes but also may damage host tissues . These injurious immune reactions are classified under hypersensitivity . Their mechanisms and significance.

43. Common events in cell injury from diverse causes Mitochondrial Dysfunction Mitochondria may be viewed as “mini-factories” that produce life-sustaining energy in the form of ATP . Not surprisingly, therefore, they also are critical players in cell injury and death. Mitochondria are sensitive to many types of injurious stimuli , including hypoxia , chemical toxins , and radiation . Mitochondrial changes occur in necrosis and apoptosis. They may result in several biochemical abnormalities: • Failure of oxidative phosphorylation leads to progressive depletion of ATP. • Abnormal oxidative phosphorylation also leads to the formation of ROS • Damage to mitochondria is often associated with the formation of a high-conductance channel in the mitochondrial membrane, called the mitochondria permeability transition pore . The opening of this channel leads to the loss of mitochondrial membrane potential and pH changes, further compromising oxidative phosphorylation. • Mitochondria also contain proteins such as cytochrome c that, when released into the cytoplasm , tell the cell there is internal injury and activate a pathway of apoptosis.

44. Defects in Membrane Permeability Increased membrane permeability leading ultimately to overt membrane damage is a feature of most forms of cell injury that culminate in necrosis. The most important sites of membrane damage during cell injury are the mitochondrial membrane, the plasma membrane, and membranes of lysosomes. Plasma membrane damage. Plasma membrane damage leads to loss of osmotic balance and influx of fluids and ions, as well as loss of cellular contents. The cells may also leak metabolites that are vital for the reconstitution of ATP, thus further depleting energy stores . Injury to lysosomal membranes results in leakage of their enzymes into the cytoplasm and activation of the acid hydrolases in the acidic intracellular pH of the injured (e.g., ischemic) cell. Activation of these enzymes leads to enzymatic digestion of cell components, and the cells die by necrosis .

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