Cell Injury & Cell Death (Nelson Mandela University)

Faculty of Health Sciences Medical School GENERAL PATHOLOGY Cell Injury & Cell Death Integrated Pathology &Microbiology YIHS300 Topic Overview Cell injury and cell death is the basis of all diseases. Reversible injuries result in cell adaptation or repair and h...

Faculty of Health Sciences Medical School GENERAL PATHOLOGY Cell Injury & Cell Death Integrated Pathology &Microbiology YIHS300 Topic Overview Cell injury and cell death is the basis of all diseases. Reversible injuries result in cell adaptation or repair and healing. Irreversible injuries result in cell death. In this series of lectures the pathology associated with various injuries, the pathogenesis and physiology of cell death are discussed.  Topic outline I. General Features of Cell Injury a. Causes of cell injury b. Progression of cell injury and death II. Reversible Cell Injury vs Cell Death a. Reversible cell injury b. Cell death: Necrosis vs Apoptosis, Other mechanisms, Autophagy III. Mechanisms and Selected Clinicopathologic Examples of Cell Injury a. Cellular targets of injurious stimuli: Mitochondrial, Membrane damage, DNA damage b. Biochemical alterations in pathways involved: Oxidative stress, Calcium homeostasis disturbances, ER stress. c. Clinicopathologic examples of cell injury and death: Hypoxia and ischemia, Ischemic‐reperfusion injury, Chemical (toxic) injury  Learning Outcomes At the end of this lesson you will be able to: Discuss the aetiopathogenesis of cell injury and cell death Differentiate apoptosis and necrosis Compare the different types of necrosis with respect to aetiology site, macroscopic and microscopic features Define sublethal cell injury, list the causes and sequelae Describe the Subcellular alterations that occur in the lysosomes, endoplasmic reticulum, mitochondria, and cytoskeleton as a result of cell injury Explain the significance of reperfusion injuries, free radical-induced injury (Underwood pgs. 78-80). Distinguish between disease and non-disease states (Underwood pgs. 12-14) Glossary Term Definition Aetiology Refers to cause or inciting agent. Pathogenesis Describes the mechanisms(biochemical and molecular) by which the disease develops, progresses, and either persists or is resolved. Morphologic refer to the structural alterations in cells or tissues that are characteristic of a disease and changes hence diagnostic of an etiologic process Hyaline A descriptive histologic term for glassy, homogeneous, eosinophilic appearance of material in haematoxylin and eosin-stained sections and does not refer to any specific substance. Hydropic Change Cell and organelle swelling= intracellular oedema Result of failure of energy-dependent ionic exchange/ membrane injury Adaptations Alterations that enable cells to cope with stresses without damage, such as increased muscle mass in response to increased workload. Reversible injury refers to structural and functional abnormalities that can be corrected if the injurious agent is removed. Cell death Irreversible degeneration of vital cellular functions culminating in the loss of cellular integrity. Http://www.public-library.uk/dailyebook/Stedman's%20Medical%20Dictionary.pdf Cell injury mind map Source :Dervan, P.A. and Harrison M. (2002).  Categories of Cell Injury Reversible cell injury Acute & self-limited (complete resolution) Adaptive (functional & morphological) changes. (incomplete resolution) Mild chronic injury -Subcellular alterations in various organelles Progressive & severe Irreversible Injury –cell death by necrosis or apoptosis Aetiology ,Forms and Sites of Damage of Cell Injury SOURCE: Cell Injury, Cell Death, and Adaptations Oakes, Scott A., Robbins & Cotran Pathologic Basis of Disease, Chapter 2, 33-69 Copyright © 2021 Copyright © 2021 by Elsevier, Inc. All rights reserved. Pathogenesis of Cell Injury SOURCE: Cell Injury, Cell Death, and Adaptations Oakes, Scott A., Robbins & Cotran Pathologic Basis of Disease, Chapter 2, 33-69 Copyright © 2021 Copyright © 2021 by Elsevier, Inc. All rights reserved.  Reversible Injury The cellular response to injury DETERMINED BY Type of injury, Duration (including pattern) of injury, Severity and intensity of injury, Type of cell injured, The cell’s metabolic state, and the Cell’s ability to adapt. Changes (Microscopy& Electron Microscopy) Cell swelling and impaired cellular regulation Mitochondria and endoplasmic reticulum swelling with detachment of ribosomes from the rough endoplasmic reticulum Surface blebs due to increased calcium which leads to changes in the microfilaments of the cytoskeleton Loss of microvilli structure  MildChronic Mild Chronic Injury Injury Subcellular Morphologic alterations in various organelles ▪Cellular swelling (hydrophobic- droplets of water in cell or vacuolar change) ▪Increased influx of water into the cytoplasm ▪Membrane bound vacuoles formed from the invaginations of the plasma membrane and ER ▪Plasma membrane – blebbing, blunting and loss of microvilli ▪Cytoplasm – myelin figures, lipid vacuoles in the cytoplasm (fatty change) ▪Mitochondrial changes – swelling and the appearance of small amorphous deposits ▪Dilation of ER – detachment of ribosomes and disassociation of the ribosomes ▪Nuclear alteration – clumping of the chromosomes  Irreversible Cell Injury Hallmarks of irreversible injury Plasma membrane damage results in Cytosolic enzymes leaking into the serum (e.g., cardiac troponin) Additional calcium entering into the cell Mitochondrial membrane damage results in Loss of the electron transport chain (inner mitochondrial membrane) Cytochrome c leaking into cytosol (activates apoptosis) Lysosome membrane damage results in hydrolytic enzymes leaking into the cytosol, which, in turn, are activated by the high intracellular calcium. In irreversible cell injury the extensive membrane damage and an increase in the concentration of calcium ions which mediate deleterious enzymatic processes results in cell death.  Biochemical Alterations In Cell Injury 1. Oxidative stress due to accumulation of oxygen‐derived free radicals Free radicals are chemical species with an unpaired electron in their outer orbit. Excess free radicals, results in a state of oxidative stress RESULTS IN Lipid peroxidation leading to membrane damage Protein modifications leading to mis-folding DNA damage leading to mutation implicated in aging,oncogenesis and reperfusion injury 2. Endoplasmic reticulum (ER) stress (unfolded protein response) ER stress leads to accumulation in the ER- activates adaptive mechanisms (unfolded protein response) i.e. signaling pathways that increase the production of chaperones, enhance proteasomal degradation of abnormal proteins via ubiquination and slow protein translation. 3. Calcium Changes Source: Underwood's Pathology Bury, Jonathan. © 2019. ER insults cause excessive increase in cytosolic Ca2+ acculumation in the mitochondrialeads to opening of the mitochondrial permeability transition pore and ATP depletion ( Ischaemic Ischaemiainjury and Cell Injury Source: Underwood's Pathology Bury, Jonathan. © 2019.  Clinicopathologic examples of cell injury and death ∙Ischaemic cell injury Hypoxia = lack of oxygen but energy production by anaerobic glycolysis can continue as blood flow is still maintained Ischemia = hypoxia +compromised delivery of substrates for glycolysis / accumulation of toxic metabolites due to reduced blood flow (due to arterial obstruction and/or reduced venous drainage), resulting in more rapid and severe cell and tissue injury compared to hypoxia ∙ Mechanism of ischemic cell injury: Decreased intracellular oxygen causes failure of oxidative phosphorylation and ATP depletion Ischemic‐reperfusion Injury Paradoxical exacerbation of cell injury / cell death when blood flow is restored to ischemic tissues Mechanisms of reperfusion injury: o Oxidative stress: Reoxygenation may cause increased generation of reactive oxygen and nitrogen species due to incomplete reduction of oxygen in leukocytes, and in damaged endothelial and parenchymal cells. Tissues may be more sensitive to free radical damage due to the preceding ischemia compromising antioxidant defence mechanisms o Intracellular calcium overload: Initiated during acute ischemia and exacerbated during reperfusion due to cell membrane damage and ROS‐mediated injury to sarcoplasmic reticulum o Inflammation: Presence of dead cells as well as cytokines from macrophages and increased expression of adhesion molecules by hypoxic parenchymal and endothelial cells recruit circulating neutrophils during reperfusion, causing additional tissue injury o Activation of complement system: During ischemia, IgM antibodies tend to deposit in ischemic tissue; during reperfusion, complement proteins can bind to these deposited antibodies, are activated and exacerbate cell injury and inflammation Chemical (toxic) injury ∙ Target :liver: metabolizes many drugs, organs of absorption or excretion. 2 main mechanisms: o Direct toxicity: chemicals combine with critical molecular components (e.g. cyanide binds to and inhibits mitochondrial cytochrome oxidase, preventing oxidative phosphorylation) o Conversion to toxic metabolites: causes membrane damage and cell injury via formation of free radicals, lipid peroxidation or direct binding to membrane proteins and lipids e.g. ∙CCl3 from CCl4  Cell Death :Point Of No Return https://www.clinicalkey.com/student/content/book/3-s2.0 B9780323531139000029#hl0000887 Two phenomena consistently characterize irreversibility, leading to cell death: 1. Inability to reverse mitochondrial dysfunction (lack of oxidative phosphorylation and ATP generation) even after resolution of the original injury 2. Profound disturbances in membrane function Source: Dervan, P. and Harrison, M., 2002. Mind maps in pathology. 1st ed. Edinburgh: Churchill Livingstone. Irreversible Cell Injury= Cell Death Feature Necrosis Apoptosis Inducing stimulus Pathological conditions Pathological or physiological conditions Number of cells Groups of cells Single cells Plasma membrane Loss of membrane integrity Membrane remains intact Morphology Cell swelling and lysis Cell shrinkage and nuclear fragmentation Eosinophilia Pyknotic nuclei Nuclear disintegration Chromatin condensation Blebbing Formation of characteristic apoptotic bodies Loss of contact with neighbouring cells/rounding up(shrink) Inflammation Inflammatory response due to No inflammatory response spillage of dead cell content Fate of cells Phagocytosed by neutrophils and Phagocytosed by neighbouring cells and macrophages macrophages Biochemical Energy-independent Energy-dependent Endonuclease activity mechanism Loss of ion homeostasis Protein denaturation Lysosomal degradation  Apoptosis Source https://www.clinicalkey.com/student/content/book/3-s2.0-B9780323531139000029#hl0001000 Dying cell shrinks, leading cytoplasm to become more eosinophilic ( Nucleus condenses (pyknosis) and fragments (karyorrhexis). Apoptotic bodies fall from the cell and are removed by macrophages; apoptosis is not followed by inflammation SOURCE: Cell Injury, Cell Death, and Adaptations Oakes, Scott A., Robbins & Cotran Pathologic Basis of Disease, Chapter 2, 33-69 Copyright © 2021 Copyright © 2021 by Elsevier, Inc. All rights reserved. Morphologic features of apoptosis. (A) Apoptosis of an epidermal cell in an immune reaction. The cell is reduced in size and contains brightly eosinophilic cytoplasm and a condensed nucleus with fragments of dense nuclear chromatin (B) This electron micrograph of cultured cells undergoing apoptosis shows some nuclei with peripheral crescents of compacted chromatin, and others that are uniformly dense or fragmented Apoptosis Activation of enzymes called caspases is central. Initiation phase, some caspases become catalytically active and unleash a cascade of other caspases; In the execution phase, terminal caspases then trigger cellular fragmentation. This entire process is regulated by a balance between pro‐apoptotic and anti‐apoptotic proteins. The initiation phase has two pathways, which are distinct but can intersect: 2 pathways DISC: Death inducing signaling domain (Extrinsic) :receptor mediated cell signaling whereby a trigger initiates cascades leading to the controlled death of a celI. Mitochondrial Intrinsic pathway Responsible for apoptosis in most physiologic and pathologic situations. A change in membrane potential or an altered membrane permeability results in mitochondria damage Damaged mitochondria secrete factors : apoptosis inducing factor, Bak and Bax. Bak and Bax dimerize, insert into the mitochondrial membrane, form channels through which cytochrome c and other mitochondrial proteins escape into the cytosol. After cytochrome c enters the cytosol, it, together with certain cofactors, activates caspase-9. Apoptosis SOURCE: Cell Injury, Cell Death, and Adaptations Oakes, Scott A., Robbins & Cotran Pathologic Basis of Disease, Chapter 2, 33-69 Copyright © 2021 Copyright © 2021 by Elsevier, Inc. All rights reserved. Mechanisms of apoptosis.. Apoptosis  Changes Leading to Necrosis https://www.clinicalkey.com/student/content/book/3-s2.0 B9780323531139000029#hl0000893 SOURCE: Cell Injury, Cell Death, and Adaptations Oakes, Scott A., Robbins & Cotran Pathologic Basis of Disease, Chapter 2, 33-69 Copyright © 2021 Copyright © 2021 by Elsevier, Inc. All rights reserved. Types Of Necrosis Source: https://www.clinicalkey.com/student/content/book/3-s2.0-B9780323531139000029#hl0000928  Liquefactive Necrosis-proteolytic Enzymes Proteolytic enzymes from microglial cells cause lysis of cells. Necrotic tissue becomes liquefied. The necrosed area on right side of the field shows a cystic space containing cell debris, while the surrounding zone shows granulation tissue and gliosis. SOURCE: Cell Injury, Cell Death, and Adaptations Oakes, Scott A., Robbins & Cotran Pathologic Basis of Disease, Chapter 2, 33-69 Copyright © 2021 Copyright © 2021 by Elsevier, Inc. All rights reserved. Coagulative Necrosis SOURCE: Cell Injury, Cell Death, and Adaptations Oakes, Scott A., Robbins & Cotran Pathologic Basis of Disease, Chapter 2, 33-69 Copyright © 2021 Copyright © 2021 by Elsevier, Inc. All rights reserved. Coagulative necrosis:kidney Initial injury results in cellular acidosis- denatures: structural proteins, + lysosomal enzymes ,therefore proteolytic disintegration does not occur Dead tissue is initially swollen and firm,later soft due to digestion by macrophages. Microscopically, the area of necrosis is demarcated adjacent to viable tissue. Tissue architecture is retained with the outlines of dead cells still visible but lacking nuclei (‘ghost cells’). Coagulative necrosis is usually caused by ischaemia and is seen in the distribution of the affected vessel Caseous Necrosis SOURCE: Cell Injury, Cell Death, and Adaptations Oakes, Scott A., Robbins & Cotran Pathologic Basis of Disease, Chapter 2, 33-69 Copyright © 2021 Copyright © 2021 by Elsevier, Inc. All rights reserved. Caseous (‘cheese‐like’) necrosis: Friable white gross appearance; microscopically amorphous granular debris surrounded by inflammation, often in granulomas.  Fat Necrosis Necrotic adipose tissue with chalky-white appearance due to deposition of calcium. Characteristic of trauma to fat and pancreatitis-mediated damage of peripancreatic fat Fatty acids released by trauma or lipase (e.g., pancreatitis) join with calcium via a process called saponification. Saponification is an example of dystrophic calcification in which calcium deposits on dead tissues. Microscopically: the necrotic fat cells have ‘ghost’ outlines, loss of nuclei +/ ‐ associated basophilic calcium deposits and inflammation The areas of white chalky deposits represent foci of fat necrosis with calcium soap formation (saponification) at sites of lipid breakdown in the mesentery SOURCE: Cell Injury, Cell Death, and Adaptations Oakes, Scott A., Robbins & Cotran Pathologic Basis of Disease, Chapter 2, 33-69 Copyright © 2021 Copyright © 2021 by Elsevier, Inc. All rights reserved.  Fibrinoid Necrosis Necrosis to blood vessel wall when immune complexes+ exuding plasma proteins are deposited in the walls of arteries. Manifest as a bright pink amorphous appearance on H&E Cause: Immunologically mediated vasculitis syndromes eg of malignant hypertension and vasculitis Characteristic of malignant hypertension and vasculitis (Source :Mckinney and Woodman, 2018)  Dry Gangrene Extensive coagulative necrosis +/ ‐ liquefactive necrosis due to superimposed bacterial infection usually in the context of ischemia of the lower limb. In picture :Second and third toes show gangrenous necrosis: coagulative necrosis of multiple tissue layers. SOURCE: Cell Injury, Cell Death, and Adaptations Oakes, Scott A., Robbins & Cotran Pathologic Basis of Disease, Chapter 2, 33-69 Copyright © 2021 Copyright © 2021 by Elsevier, Inc. All rights reserved.  Relationship Of Cell Injury To Disease CHRONIC REVERSIBLE ADAPTATIONS Disease Syndrome SOURCE: Cell Injury, Cell Death, and Adaptations Oakes, Scott A., Robbins & Cotran Pathologic Basis of Disease, Chapter 2, 33-69 Copyright © 2021 Copyright © 2021 by Elsevier, Inc. All rights reserved.  References Cross, S.S. and Underwood, J.C.E. (2019). Underwood’s pathology : a clinical approach. 7th ed. Edinburgh: Churchill Livingstone/Elsevier. Dervan, P.A. and HarrisonM. (2002). Mind maps in pathology. [online] Edinburgh: Churchill Livingstone. Available at: https://www.pdfdrive.com/pathology-mind-maps-e34399814.html [Accessed 15 Oct. 2022]. Dixon, M.F. and Quirke, P. (1993). Aids to Pathology. 4TH ed. Singapore: Churchill Livingstone, p.351. Kumar, V., Abbas, A.K. and Aster, J.C. (2021). Robbins & Cotran Pathologic Basis Of Disease. 10th ed. [online] S.L.: Elsevier - Health Science. Available at: https://www.clinicalkey.com/student/content/toc/3-s2.0C20160040871 Mckinney, O. and Woodman, I. (2018). Crash Course Pathology. 5th ed. Elsevier. Stedmans medical dictionary: Available http://www.publiclibrary.uk/dailyebook/Stedman's%20Medical%20Dictionary.pdf