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what is CELL INJURY ?


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INTRODUCTION

  • The normal cell is confined to a fairly narrow range of functions and structure by;
    • Its state of metabolism, differentiation and specialization
    • Constraints of neighboring cells and
    • The availability of metabolic substrates
  • It is nevertheless able to handle physiologic demands, maintaining a steady state called homeostasis

Stages of Impairment

  • The stages of progressive impairment following different types of insults are;
    • Adaptations
    • Reversible Injury
    • Cell death

Adaptation

  • Application of more excessive physiologic demands or some pathologic stimuli will lead to a new, but altered steady-state cellular adaptations
  • Adaptations are reversible functional and structural responses to changes in physiologic states (e.g. pregnancy) and some pathologic stimuli, allowing the cell to survive and continue to function

Reversible Injury

 If the limits of adaptive responses are exceeded or if cells are exposed to injurious agents or stress, deprived of essential nutrients, or become compromised by mutations that affect essential cellular constituents, a sequence of events follows that is termed cell injury  This can be reversible up to a certain point

Cell Death

  • It becomes irreversible when the stimulus persists or is severe enough from the beginning
  • Ultimately, such cells die
  • There are 2 principal pathways of cell death
    • Necrosis and
    • Apoptosis

Other cellular responses to injury include;

  • Subcellular alteration in sublethal and chronic injury
  • Intracellular accumulations
  • Pathologic calcification

Cellular adaptations

  • Cell aging

Stages of Cellular Response to Stress & Injurious Stimuli

  • This is oxygen deprivation which affects the aerobic oxidative respiration
  • In hypoxia, the PO2 in blood is reduced
  • In ischaemia, there is loss of blood supply from impaired arterial flow or reduced venous drainage
  • In hypoxia, anaerobic glycolysis can take place, but in ischaemia, metabolic substrates cannot be delivered to the tissues
  • Ischaemia, thus, injure tissues faster than hypoxia
  • Causes of hypoxia include;
    • Loss of O2-carrying capacity of the blood as in CO poisoning or anaemia
    • Inadequate oxygenation due to cardiorespiratory failure Physical agents
  • Mechanical trauma
  • Extremes of temperature (burns and deep cold)
  • Sudden changes in atmospheric pressure
  • Radiation
  • Electric shock

Chemical agents and drugs

Simple chemicals like glucose or salt in hypertonic concentration

  • O2 in high concentration
  • Poisons such as arsenic, cyanide or mercuric salts
  • Environmental and air pollutants, insecticides and herbicide
  • Industrial and occupational hazards e.g. CO, asbestos
  • Social stimuli – alcohol, narcotic drugs
  • Various therapeutic drugs
  • Some attack specific cells, tissues and organs
  • Others destroy sites of contact, absorption, concentration, metabolism and excretion of original chemicals and/or metabolites and activated forms Infectious agents
  • They range from prion, to virus, rickettsiae, bacteria, fungi, higher forms of parasite, to tapeworm
  • The infectious agents, that cause diseases in man, are referred to as PATHOGENIC

ORGANISMS

  • They cause disease in different ways Immunological reactions
  • They mostly serve in the defense against biological agents
  • They may however, cause cell injury and death
  • g. anaphylactic reaction to a foreign protein or drug; autoimmune diseases

Genetic derangements

  • There are 3 categories;
    • Single gene
    • Chromosomal
    • Multifactorial
  • They are inherited either as single and specific diseases such as SCD[1], Trisomy 21

(Down’s syndrome) or as increased susceptibility to specific disease Nutritional imbalances

  • Deficiency
  • Excesses

PRINCIPLES OF CELL INJURY

Important principles relating to most forms of cell injury include;

Cellular Response

  • Cellular response to injurious stimuli depends on;
    • The type of injury
    • Its duration; and
    • Its severity
  • The consequences of an injurious stimulus depend on the type, status, adaptability and genetic makeup of the injured cell
  • Genetically determined diversity in metabolic pathways can also be important
  • When exposed to the same dose of a toxin, individuals with polymorphisms in enzyme genes may catabolize it with different efficacies, leading to different outcomes

Systems Affected

  • 4 intracellular systems are particularly vulnerable to cell injury;
    • Maintenance of the integrity of cell membranes on which the ionic and osmotic homeostasis of the cell and its organelles depends
    • Aerobic respiration involving mitochondrial oxidative phosphorylation and production of ATP
    • Protein synthesis; and
    • The integrity of the genetic apparatus

Effects

  • The structural and biochemical components of a cell are so integrally connected that regardless of the initial locus of injury, multiple secondary effects rapidly occur
  • For example, impairment of ATP production disrupts the sodium pump that maintains the ionic and fluid balance of the cell
  • Cellular function is lost far before cell death occurs and the morphological changes of cell injury (or death) lag far behind both
  • Specific activities of a cell typically rely on all systems being intact, so cells lose functional activity relatively quickly even if they are not dead
  • Moreover, changes in the appearance of the cell are evident only after some critical biochemical system has been deranged and sufficient time has passed to manifest the change
  • For example, myocytes that died after 30 minutes of ischaemia do not appear dead by ultrastructural evaluation for 2-3 hours and by light microscopy for 6-12 hours

Sequential Development of Biochemical & Morphologic Changes in Cell Injury

 

 

BIOLOGICAL THEMES IMPORTANT IN MEDIATION OF CELL INJURY

  • ATP is required for membrane transport, protein synthesis, lipogenesis, etc.
  • The production of ATP can be through;
    • Oxidative phosphorylation of ADP in the inner leaflets of mitochondria ü Glycolytic pathway
  • A loss of ATP synthesis results in rapid shutdown of most critical homeostatic pathways  Mostly seen in both ischaemic and toxic injuries

Oxygen Deprivation or Generation of Reactive Oxygen Species

  • A lack of oxygen underlies the pathogenesis of cell injury in ischaemia
  • Small amount of partially reduced reactive oxygen forms are produced as an unavoidable byproduct of mitochondrial respiration
  • These free radicals can damage lipids, proteins and nucleic acids

Loss of Calcium Homeostasis

  • Intracellular calcium is low and sequestered in mitochondria and endoplasmic reticulum
  • Ischaemia or toxins allow a net influx of extracellular calcium across the plasma membrane, followed by release of calcium from the intracellular stores
  • The increased calcium in turn activates a variety of phospholipases, proteases, ATPases and endonucleases

Defects in Plasma Membrane Permeability

  • A consistent feature in most forms of cell injury (exception being apoptosis)
  • A loss of membrane barriers leads to a breakdown of the concentration gradients of metabolites necessary to maintain normal metabolic activities
  • May be a consequence of ATP depletion or calcium-modulated activation of phospholipases
  • Affects any cellular and organellar membrane
  • In addition, the plasma membrane can be damaged directly by;
    • Bacterial toxins
    • Viral proteins’ lytic complement components
    • Perforin from cytolytic lymphocytes; and
    • A number of physical and chemical agents

Mitochondrial damage

  • Since all mammalian cells are ultimately dependent on oxidative metabolism, mitochondrial integrity is critical for cell survival
  • A cell with irreparable damage to the mitochondria will ultimately die, irrespective of the glycolytic ability of the cell
  • Increases in cytosolic calcium, intracellular oxidative stress and lipid breakdown products can all damage the mitochondria
  • Damage to mitochondria is expressed as formation of a high-conductance channelmitochondrial permeability transition
  • These non-selective pores allow the proton gradient across the mitochondrial membrane to dissipate, thereby preventing ATP generation
  • Cytochrome C, which is an important soluble protein in the electron transport chain, also leaks out into the cytosol, where it activates apoptotic death pathways

Principal Biochemical Mechanisms & Sites of Damage in Cell Injury

 

ISCHAEMIC AND HYPOXIC INJURY

  • Ischaemia is the most common type of cell injury in clinical medicine
  • It results from hypoxia induced by reduced blood flow, most commonly due to a mechanical arterial obstruction
  • It can also be caused by reduced venous drainage
  • In hypoxia, energy production by anaerobic glycolysis can continue
  • But ischaemia compromises the delivery of substrates for glycolysis
  • Thus, in ischaemic tissues, not only is aerobic metabolism compromised, but anaerobic energy generation also stops after glycolytic substrates are exhausted
  • Or glycolysis is inhibited by the accumulation of metabolites that would otherwise be washed out by flowing blood
  • For this reason, ischaemia tends to cause more rapid and severe cell and tissue injury than does hypoxia in the absence of ischaemia

REVERSIBLE ISCHAEMIC AND HYPOXIC INJURY

  • As the blood and oxygen supplies become impaired, the first point of attack is the cell aerobic respiration
  • Leading to loss of phosphorylation and decreased generation of ATP
  • The depletion of ATP has widespread effects on many systems within the cell Features of Decreased ATP
  • Decreased activity of Na+-K+-ATPase pump
    • Consequently, Na+ accumulates intracellularly and K+ moves out
    • This net gain of solute is accompanied by isosmotic gain of water producing acute cellular swelling (cell or hydropic swelling)
  • Also, there is accumulation of catabolites
    • Such as inorganic phosphates, lactate and purine nucleosides and these also cause increased water load
  • Cellular energy metabolism is altered
    • Glycolysis, the sole source of energy as oxidative phosphorylation, ceases ü There is associated increase in cAMP
    • This coupled with the decrease in ATP stimulate phosphorylase and phosphofructokinase activities
    • This pathway was evolutionarily designed to maintain the cell’s energy by generating ATP from glycogen
    • These act on glycogen to produce ATP
    • Glycogen stores are thus rapidly depleted and are apparent histologically by reduced staining for carbohydrates
    • The increased glycolysis also results in the accumulation of lactic acid and inorganic phosphates from the hydrolysis of phosphate esters
    • Leads to a decrease in intracellular pH
  • Detachment of ribosomes from the rough ER and dissociation of polysomes into monosomes with consequent reduction in protein synthesis
    • This is a result of the loss of the energy relationship between them
  • Loss of function
  • Mitochondrial swelling
    • Owing to loss of volume control
    • Amorphous densities rich in phospholipid may appear
  • Plasma membrane
    • Blebs (focal extrusions of the cytoplasm) are occasionally noted

 They can be pinched off and released while the cell remains viable

  • The cytoskeleton disperses, resulting in the loss of microvilli
  • Myelin figures (derived from plasma as well as organellar membranes) may be seen within the cytoplasm or extracellularly
  • Nucleolus
    • In the nucleus, reversible injury is reflected principally in nucleolar change
    • The fibrillar and granular components of the nucleolus may segregate
    • Alternatively, the granular component may be diminished, leaving only a fibrillar core
    • These morphological changes are accompanied by reduced synthesis of ribosomal RNA species
  • It is important to recognize that after withdrawal of an acute stress, that has led to reversible cell injury, by definition, the cell returns to its normal state

Protective Responses

  • Mammalian cells have developed protective responses to deal with hypoxic stress
  • Best defined of these is induction of a transcription factor called hypoxia-inducible factor-1 (HIF-1)
  • This promotes new blood vessel formation, stimulates cell survival pathways and enhances glycolysis
  • Several promising investigational compounds that promote HIF-1 signaling are being developed

 

But there are still no reliable therapeutic approaches for reducing the injurious consequences of ischaemia in clinical situations

  • The strategy perhaps most useful in ischaemic and traumatic brain and spinal cord injury is the transient induction of hypothermia (lowering the core body temperature to 90°F)
  • This reduces the metabolic demands of the stressed cells, decreases cell swelling, suppresses the formation of free radicals and inhibits the host inflammatory response
  • All of these may contribute to decreased cell and tissue injury

Functional & Morphological Consequences of Decreased ATP

IRREVERSIBLE ISCHAEMIC AND HYPOXIC INJURY

  • If the acute stress to which a cell must react is too great, irreversible injury ensues  This will lead to the death of the cell Features
  • Mitochondria
    • Severe swelling
    • Appearances of large, flocculent, amorphous densities in the matrix
  • Massive Ca++ influx into the cell, particularly if the ischaemic zone is reperfused

Loss of protein, enzymes, coenzymes and RNA from the hyperpermeable membrane

  • Lysosomal membrane damage followed by leaks of their enzymes into the cytoplasm
    • These include RNAses, DNAases, Proteases, Glucosidases and Cathepsins
    • When these are activated, there is enzymatic digestion of cell components
  • When the cells die, the components are degraded and there is leakage of cellular enzymes into the extracellular space and entry of extracellular macromolecules from the interstitial space into the cells
  • The dead cell finally becomes replaced by large masses of phospholipid in the form of myelin figures
  • These are either phagocytozed or degraded into fatty acids
  • Leakage of intracellular enzymes and other proteins into the plasma provides important clinical parameters of cell death
  • g. elevated serum levels of ck- MB and Troponin, which are found in high concentration in cardiac muscle, are invaluable clinical criteria of myocardial infarction

Mechanisms

  • 2 phenomena illustrate the difference between irreversibly and reversibly injured ischaemic cells
    • An inability to reverse mitochondrial dysfunction on reperfusion or reoxygenation correlates with a similar inability to reverse cell injury in general
    • A disturbance in membrane functions, in general and in the plasma membrane in particular
  • Morphologic, functional and biochemical studies clearly suggest that defects in cell membranes is a central factor in the pathogenesis of irreversible cell injury

Mechanisms of Membrane Damage

  • Mitochondrial dysfunction:
    • Increased cytosolic Ca++, in addition to ATP depletion, results in uptake of Ca++ by mitochondria
    • This activates mitochondrial phospholipases and free fatty acids accumulate
    • There is thus, the loss of mitochondrial proton motive force
  • Loss of membrane phospholipids:
    • This is either due to activation of endogenous phospholipases by Ca++
    • Or decreased reacylation
    • Or diminished de novo synthesis of phospholipids
  • Cytoskeletal abnormalities:
    • Activation of proteases by increased intracellular calcium may result in damage to the cytoskeleton
    • In the setting of cell swelling, such injury may cause detachment of the cell membrane from the cytoskeleton, rendering the membrane susceptible to stretching and rupture

Reactive oxygen species:

  • They cause lipid peroxidation of membrane thereby damaging them
  • Lipid breakdown products
    • Acyl carnithine, unesterified FFA[2], lysophospholipid
    • They accumulate in the injured cells as a result of phospholipid degeneration
    • In the cells, they have a detergent effect and they insert into the lipid bilayer of the membrane or exchange with membrane phospholipid
  • Loss of intracellular amino acids

Effects of Membrane Damage

Damage to different cellular membranes has diverse effects of cells;

  • Mitochondrial membrane damage
    • Results in opening of the mitochondrial permeability transition pore
    • This leads to decreased ATP generation and release of proteins that trigger apoptotic death
  • Plasma membrane damage
    • Results in loss of osmotic balance and influx of fluids and ions as well as loss of cellular contents
    • Cells may also leak metabolites (e.g. glycolytic intermediates) that are vital for the reconstitution of ATP
    • Further depleting energy stores
  • Injury to lysosomal membranes
    • Results in leakage of their enzymes into the cytoplasm and activation of acid hydrolases in the acidic intracellular pH of the injured cell
    • The hydrolases include RNAses, DNAases, proteases, phosphatases and glucosidases
    • These degrade RNA, DNA, proteins, phosphoproteins and glycogen respectively and push cells into necrosis

ISCHAEMIA REPERFUSION INJURY

  • Restoration of blood flow to ischaemic tissues can result in recovery of cells if they are reversibly injured or not affect the outcome if irreversible cell damage has occurred
  • However, depending on the intensity and duration of the ischaemic insult, variable numbers of cells may proceed to die after blood flow resumes, by necrosis as well as by apoptosis
  • The affected tissues often show neutrophilic infiltrates
  • This ischaemia reperfusion injury is a clinically important process in such conditions as myocardial infarction and stroke and may be amenable to therapeutic interventions How does the injury occur?

A possibility is that a number of ischaemic cells are structurally intact

  • Meaning that while they are not necrotic yet, they are biochemically compromised nonetheless and they lose integrity during perfusion
  • Another possibility is that new damaging processes are set in motion during reperfusion  Causing death of cells that might have recovered otherwise

Proposed Mechanisms

  • Oxidative stress
    • New damage may be initiated during reoxygenation by increased generation of reactive oxygen and nitrogen species
    • These free radicals may be produced in reperfused tissue as a result of incomplete reduction of oxygen by damaged mitochondria
    • Or because of the action of oxidases in leukocytes, endothelial cells or parenchymal cells
    • Cellular antioxidant defense mechanisms may be compromised by ischaemia, favouring the accumulation of free radicals
  • Intracellular calcium overload
    • As mentioned earlier, intracellular and mitochondrial calcium overload begins during acute ischaemia
    • It is exacerbated during reperfusion due to influx of calcium (resulting from cell membrane damage) and ROS[3] mediated injury to sarcoplasmic reticulum
    • Calcium overload favours opening of the mitochondrial permeability transition pore with resultant depletion of ATP
    • This in turn causes further cell injury  Inflammation
    • Ischaemic injury is associated with inflammation as a result of “dangers signals” released from dead cells, cytokines secreted by resident immune cells such as macrophages, and increased expression of adhesion molecules by hypoxic parenchymal and endothelial cells, all of which act to recruit circulating neutrophils to reperfused tissue
    • The inflammation causes additional tissue injury
    • The importance of neutrophil influx in reperfusion injury has been demonstrated experimentally by the salutary effects of treatment with antibodies that block cytokines or adhesion molecules and thereby reduce neutrophil extravation
  • Activation of the complement system
    • May contribute to ischaemia-reperfusion injury
    • Some IgM antibodies have a propensity to deposit in ischaemic tissues for unknown reasons
    • And when blood flow is resumed, complement proteins bind to the deposited antibodies, are activated and cause more cell injury and inflammation

 

MORPHOLOGY OF REVERSIBLE CELL INJURY

  • Cellular swelling
    • First manifestation of all forms of injury to cells
    • Difficult to appreciate with light microscope
    • Crossly, the organ is pale with increased weight
    • Histologically, small clear cytoplasmic vacuoles are seen
    • Ultrastructurally, the number of organelles is unchanged though they appear dispersed in a larger volume
    • The excess fluid accumulates preferentially in the cisternae of the ER
  • Fatty change
    • Seen in hypoxic and various forms of toxic or metabolic injury
    • Small or large lipid vacuoles are seen within the cytoplasm
    • Mainly seen in cells involved in and dependent on fat metabolism e.g. hepatocytes and myocardial cells
  • Other changes
    • Plasma membrane changes – blebs, blunting, loss of microvilli, myelin figures
    • Mitochondrial changes – swelling, rarefaction, amorphous densities
    • Dilation of ER with detachment and disaggregations of polysomes
    • Nuclear alternatives with disaggregation of granular and fibrillar elements

CHEMICAL INJURY

  • Chemicals cause cell injury by either of 2 general mechanisms
    • Direct covalent binding
    • By conversion to reactive toxic metabolites

Direct Covalent Bonding

  • The chemicals act directly by combining with critical molecular components of cellular organelles
  • In mercuric chloride poisoning, mercury binds to the sulfhydryl groups of various cell membrane proteins
  • This will cause inhibition of ATPase dependent transport and increased permeability
  • Cyanide poisons mitochondrial cytochrome oxidase and thus inhibits oxidative phosphorylation
  • Other examples are chemotherapeutic agents and antibiotics that induce cell damage by similar direct cytotoxic effects
  • In these instances, the greatest damage is sustained by the cells that use, absorb, excrete or concentrate the compounds

Conversion to Reactive Metabolites

  • The chemicals in this group are not intrinsically biologically active
  • They must be first converted to reactive toxic metabolites, which then act on target cells
  • This conversion is by the P-450 mixed function oxidases in the SER[4] of the liver and other organs
  • The metabolites might cause direct membrane damage and cell injury, but most important mechanism of cell injury involves the formation of reactive free radicals
  • Examples of chemicals in this category include CCL4 and acetaminophen
  • [5]CCl4 is converted to CCl3, principally in the liver
  • [6]CCl3 causes autocatalytic membrane phospholipids peroxidation
  • Acetaminophen is a commonly used analgesic
  • It is detoxified in the liver through sulfation and glucoronidation and small amounts are converted by a cytochrome P-450-catalyzed oxidation to an electrophilic, highly toxic metabolite
  • This metabolite itself is detoxified by interaction with GSH[7]
  • When large doses of the drug are ingested, GSH is depleted and thus the toxic metabolites accumulate in the cells, destroy nucleophilic macromolecules and covalently bind proteins and nucleic acids

FREE RADICAL-INDUCED CELL INJURY (OXIDATIVE STRESS)

  • Free radicals are chemical species that have a single unpaired electron in an outer orbit
  • In this state, they are unstable and they can enter into chemical reactions with adjacent molecules–Proteins, Lipid, Carbohydrate, Nucleic acids–which are key components of cell membranes and nuclei
  • They have been identified as the likely cause of cell injury in an increasing number of diseases;
    • Chemical and radiation injury
    • Toxicity from oxygen and other gases
    • Cellular aging
    • Microbial killing by phagocytes
    • Inflammatory damage
    • Tumour destruction by macrophages
    • And other injurious processes
  • When generated in cells, they avidly attack and degrade nucleic acids as well as a variety of membrane molecules
  • They can be Oxygen, Nitrogen or Carbon-based

OXIDATIVE STRESS

  • Excess of free radicals
  • From increased production or decreased scavenging of ROS
  • Implicated in a wide variety of pathologic processes
    • Cell injury
    • Cancer
    • Aging
    • Some degenerative diseases such as Alzheimer disease

Initiation (Generation)

  • Absorption of radiant energy e.g. ionizing radiation can hydrolyze water into hydroxyl (OH-) and Hydrogen free radicals
  • Enzymatic metabolism of exogenous chemicals or drugs e.g. CCl4 can generate CCl3
  • Redox reactions that occur during normal metabolic processes;
    • As a part of normal respiration, molecular O2 is reduced by the transfer of 4 electrons to H2 to generate 2 H2O molecules
    • The conversion is catalyzed by oxidative enzymes in the ER, cytosol, mitochondria, peroxisomes and lysosomes
    • During this process, small amounts of partially reduced intermediates are produced in which different numbers electrons have been transferred from O2
    • These include superoxide anion (1 electron), hydrogen peroxide (2 electrons) and hydroxyl ions (3 electrons)
  • Transition metals e.g. iron (in Fenton reaction) and copper can donate or accept free electrons during intracellular reactions and therefore catalyze free radical formation
  • NO[8] can act as free radical and can also be converted to the highly reactive peroxynitrite anion
  • Rapid burst of ROS are produced in activated leukocytes during inflammation
    • This occurs in a precisely controlled reaction carried out by a plasma membrane multiprotein complex that uses NADPH oxidase for the redox reaction

Propagation

  • Free radicals initiate autocatalytic reactions whereby molecules with which they react are themselves converted into free radicals thus propagating the chain of damage

Termination (Removal)

  • They are inherently unstable and generally decay spontaneously
  • g. O2- is unstable and spontaneously dismutates into O2 and peroxide in the presence of water

Other Mechanisms

  • Antioxidants – they either block initiation or inactivate formed free radicals;
    • Vitamin A
    • Vitamin E
    • Ascorbic acid and
    • Glutathione
  • Storage and transport proteins that can bind the transitional metals and thereby render them unavailable;
    • Transferrin
    • Ferritin
    • Lactoferrin
    • Cerulloplasmin
  • Enzymatic degradation
    • Catalase which decomposes H2O2
    • Superoxide dismutase converts O2- to H2O2
    • Glutathione peroxidase

PATHOLOGIC EFFECTS OF THE FREE RADICALS

  • Lipid peroxidation of membrane
    • Double bonds in membrane phospholipids are vulnerable to attacks by oxygenderived free radicals
  • Cross-linking of proteins
    • They promote sulfhydryl-mediated protein cross-linking
    • Resulting in enhanced rate of degradation or loss of enzymatic activity
    • They may also directly cause polypeptide fragmentation
  • Lesions in DNA
    • Free radicals react with thymine in nuclear and mitochondrial DNA to produce single-stranded breaks
    • This is important in cell killing and in cancers
  • The final effects of free radicals depend on the net balance between free radical formation and termination

 

[1] Sickle Cell Disease

[2] Free Fatty Acids

[3] Reactive Oxygen Species

[4] Smooth ER

[5] Tetrachlorocarbon

[6] Trichlorocarbon

[7] Glutathione

[8] Nitric Oxide