CELL INJURY, ADAPTATION,

SECTION B

UERMMMC Class 2014

Dr. Teresita P. Bailon

June 14 and16, 2011

PATHOLOGY

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CELL INJURY, ADAPTATION,

ACCUMULATION AND CELL DEATH

 Due to excessive physiologic stress and some pathologic

stimuli resulting in a new altered state to preserve cell

viability

 Adaptations are reversible responses to physiologic stress and pathologic stimuli; takes several form

 Decrease in cell size and number  Decrease in tissue/organ size

 Shrinkage in the size of the cell by loss of cell integrity and substance (mitochondria, myofilaments, ER)

 Can be physiologic or pathologic in Etiology

 Example: Shrinkage in cell size due to loss of cell integrity and substance

Mechanism:

Decrease in protein synthesis due to decrease in metabolic activity and increase in protein degradation (via ubuquitin-proteosome pathway)

- Ubuquitin-proteosome pathway: Nutrient deficiency  Activation of ubuquitin ligases which target degradation of proteins in proteosomes

- Thought to be responsible for the accelerated proteolysis seen in a variety of catabolic conditions, including cancer cachexia (Robbins)

- Glucocorticoids and Thyroid hormones  Stimulate proteosome-mediated protein degradation

- Insulin  Suppressor

Notes:

Atrophy may also be accompanied by AUTOPHAGY (wherein in starved cells eat its own components to survive) marked by increase of autophagic vacuoles. These are vacuoles within the cell that contain fragments of cell components (e.g., mitochondria, endoplasmic reticulum) destined for destruction and into which the lysosomes discharge their hydrolytic contents, where the cellular components are then digested.

1. Disuse Atrophy

− Decrease in workload

− Immobilization of a fractured bone or complete bed rest  Skeletal muscle atrophy (reversible once activity is resumed).

− Skeletal muscle fibers decrease in cell size and

number  Increase bone resorption then to osteroperosis

2. Denervation Atrophy

− Nerves continuously give tone to muscles even at rest − Lesions may therefore lead to loss of tissue innervation − Example: Paralyzed patients

3. Decrease blood supply

− Ischemia – Arterial occlusive disease

− Example: Atherosclerotic cerebrovascular disease

− Atherosclerosis  Senile atrophy of the brain  Narrowing of gyri and widening of sulci

− May also affect heart.

4. Inadequate nutrition

− Marasmic patients (protein-calorie malnutrition) use skeletal muscle as energy source  Muscle wasting (Cachexia)

5. Loss of Endocrine Stimulation

− Loss of hormonal stimulation

− Examples:

a. Postmenopausal women: Loss of estrogen  Physiologic atrophy of the uterus (become thin and shiny), endometrium/vaginal epithelium and breasts.

b. Senile atrophy due to aging: Testicular atrophy; thickening of basement membrane

6. Pressure Atrophy

 Tissue compression for any given time

Ex. Benign tumours compressing surrounding tissues  atrophy

Notes:

Physiologic Atrophy: occurs during normal development. Ex. Atrophy of the notochord during normal development; Atrophy of the Thymus as the child grows older.

 Increase in the size of cells  Increase in tissue/organ size  New cells are NOT present

 Can be accompanied by hyperplasia among normally dividing cells

 Non-dividing cells do NOT undergo hypertrophy under stress  Most common stimulus: Increase in workload

Mechanism:

Increase production of cellular proteins (structural components). Occurs in non-dividing cells such as myocardial and skeletal muscle fibers.

Notes:

Most studies in the mechanism of hyperthrophy is

through the cardiac muscle, where it involves many signal

transduction pathways, leading to the induction of a number of genes, which stimulate synthesis of numerous cellular proteins. Genes induced include encoding transcription factors (such as c-fos, c-jun), growth factors (TGF-β, insulin-like growth factor-1

[IGF-1], fibroblast growth factor), and vasoactive agents

(α-adrenergic agonists, endothelin-1, and angiotensin II).

Mechanical triggers, such as stretch, and trophic trigers, such as polypeptide growth factors (IGF-1) and vasoactive agents (angiotensin II, α-adrenergic agonists) may trigger the changes in gene expression which may lead to hypertrophy of the cardiac muscle (Robbins).

CELLULAR ADAPTATIONS OF GROWTH AND DIFFERENTIATION

Atrophy

Causes of Atrophy

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UERMMMC Class 2014

Physiologic Hypertrophy

1. Hormonal Stimulation

Example: Estrogen  Increase in uterine size during pregnancy and increase in breast and sex organs during puberty

2. Increase functional demand Example: Resistance training

Pathologic Hypertrophy

1. Abnormal Hormonal levels.

Example: Hyperthyroidism  Increase T3 and T4  Feedback inhibition to TSH

2. Increase functional demand.

Example: Chronic Hemodynamic overload in left ventricular hypertrophy as seen in hypertension and valvular disease (Normal Left Ventricular wall thickness is at 1-1.5 cm, LVH greater than 2.0 cm is pathologic)

 Increase in number in cells  Increase in tissue/organ mass  Occurs in dividing cells

Mechanism:

Growth factor-driven proliferation of mature cells and sometimes, increased output of new cells from tissue stem cells.

− Caused by increased local production of growth factors, increased levels of growth factor receptors on the responding cells, or activation of particular intracellular signaling pathways. All these changes lead to production of transcription factors that turn on many cellular genes, including genes encoding growth factors, receptors for growth factors, and cell cycle regulators, and the net result is cellular proliferation (Robbins)

Physiologic Hyperplasia

1. Hormonal Stimulation.

Example: Pregnancy/ Puberty  Proliferation of glandular epithelium of breasts (usually accompanied by hypertrophy) 2. Compensatory.

Example: Liver regeneration via intrahepatic stem cells after partial hepatectomy. GF alpha  Activation of liver regeneration (counteracted by transforming GF beta)

− In massive destruction of liver cells (Example: Viral hepatitis  No regeneration due to destruction of CT framework necessary for regeneration

Pathologic Hyperplasia

1. Excessive Hormonal Stimulation.

Example: Hyperestrinism  Endometrial hyperplasia  May lead to CA if there are atypical changes)

2. Growth factors on target organs.

Examples: Mitogenic factors  Hyperplasia in wound healing. Papilloma virus  Skin warts)

3. Imbalance of Hormones. Examples:

a. Disturbance in the balance of estrogen and progesterone. Increase in Estrogen  Hyperplasia of endometrial glands  Abnormal Menstrual Bleeding b. Benign Prostatic Hyperplasia in response to androgens. NOTE: this remains controlled because it causes no mutation in genes that regulate cell division.

 Reversible change wherein one adult cell type is replaced by another adult cell type.

 Usually involves epithelial and mesenchymal cells.

 Columnar to squamous type is most common metaplasia.  Adaptive Substitution of cells better able to withstand the

adverse environment.

Mechanism:

Signals generated from Cytokines, GF’s and extracellular

matrix components  Reprogramming of stem cells normally present in tissues or undifferentiated mesenchymal cells present in connective tissue

1. Squamous metaplasia

− Chronic respiratory tract irritation (cigarette smokers); normal ciliated and bronchi replaced by stratified squamous columnar epithelium of trachea epithelium − Stones in excretory ducts, pancreas and bile ducts,

vitamin A deficiency; secretory columnar to stratified squamous; mucus secretion and ciliary function are lost

Squamous Metaplasia, Lungs , HPO

2. Osteoblastic or Chondroblastic metaplasia of fibroblasts 3. Apocrine metaplasia

− Seen in specimens of breast mass with fibrocystic change − Most likely are with apocrine metaplasia is benign − Persistent stimuli  Dysplasia/cancer may occur

Notes:

1. Barrett’s esophagus: esophageal squamous to intestinal-like columnar cells due to acid reflux.

2. Endocervix: Normal Columnar to Stratified Squamous

 Epithelial or mesenchymal cells undergo proliferation and atypical cytological changes involving size, shape and organization (Loss of polarity, increased mitosis, increased nuclear membrane etc.)

 Example: Cervical cancer. Desquamation is observed at the

squamocolumnar junction.

 Sometimes, cancer precursors (cervix and resp. tract)  Appearances:

o Variation in size and shape

o Nuclear enlargement; irregular hyperchromatism o Disarray arrangement of cells within epithelium

Hyperplasia

Metaplasia

Dysplasia

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UERMMMC Class 2014

 When cell’s exposure to injurious agents or stress exceeds

its capability for adaptive response  Can be reversible or irreversible

o Reversible cell injury initially is manifested as functional and morphologic changes that are reversible if the damaging stimulus is removed. Hallmarks are reduced oxidative phosphorylation, adenosine triphosphate (ATP) depletion, and cellular swelling caused by changes in ion concentrations and water influx.

o Irreversible injury and cell death continuing damage 

Injury becomes irreversible, at which time the cell cannot recover. Certain structural changes (e.g., amorphous densities in mitochondria, indicative of severe mitochondrial damage) and functional changes (e.g., loss of membrane permeability) are indicative of cells that have suffered irreversible injury (Robbins)  Irreversible  cell death

 Example:

In systemic hypertension, increase BP  Heart has to work against increase in peripheral resistance Cardiac muscle

adapts by increasing in cell size = Hypertrophy  When there is persistent hypertension (uncontrolled),

myocardial cell exceeds its limit of adaptation  Will decompensate and undergo changes (e.g. lysis of cells) = Irreversible cell injury (usually in the form of coagulative necrosis in the heart  Myocardial Infarction)

 Usually apoptosis is not seen in cardiac muscle

 4 common causes: Hypoxia, Reactive Oxygen Species, Chemicals, Viruses

1. Hypoxia

− Deficiency in oxygen which causes cell injury by decreasing aerobic oxidative respiration

− Causes are Ischemia, Cardiorespiratory failure

(Inadequate oxygenation of blood), Anemia

(decreased oxygen carrying capacity), Carbon Monoxide poisoning (block oxygen carriage), severe blood loss

Notes:

Duration of hypoxia needed to produce irreversible cellular injury varies according to cell type, nutritional state and hormonal status

Table. Susceptibility of cells to Ischemic Necrosis according to cell type

Neurons High 3-5 min

Myocardium, hepatocyte, renal epithelium Intermediate 30 min- 2h Fibroblasts, epidermis, skeletal muscle

Low Many hours

2. Physical agents

− Mechanical trauma, extremes of temperature, sudden changes in atmospheric pressure, radiation, electric shock

3. Chemical agents and drugs

− Hypertonic conc. of glucose/ salt, high oxygen concentrations, poisons, environmental and air pollutants, insecticides/ herbicides, CO and asbestos, alcohol, narcotics, etc.

4. Infectious agents

− Viruses, bacteria, rickettsiae, fungi, parasites

5. Immunologic reactions

− Anaphylaxis, autoimmune diseases

6. Genetic derangements

− Genetic injury (Down's syndromecaused by

chromosomal anomaly), sickle cell anemia inborn errors of metabolism, accumulation of damaged DNA or misfolded proteins

7. Nutritional imbalances

− Protein-calorie deficiency, vitamin def.,

atherosclerosis

A. Depletion of ATP production and synthesis

− Associated with hypoxic and toxic injury

− Major causes are reduction of oxygen supply, mitochondrial damage and actions of toxins

− Effects of depletion of ATP to 5% to 10% of normal levels are:

a. Decreased plasma membrane energy-dependent sodium pump; Sodium enters and accumulates inside cells and potassium diffuses out, causing cell swelling and dilation of the ER

b. Altered cellular energy metabolism  If supply of oxygen to cells is reduced

 Resulting to decreased cellular ATP and increased AMP

 Increased rate of anaerobic glycolysis will cause accumulation of lactic acid and inorganic phosphates, reducing intracellular pH and decreased activity of many cellular enzymes c. Influx of calcium due to pump failure: leads to influx

of Ca2+

d. Decreased protein synthesis: due to prolonged or worsening depletion of ATP, structural disruption of the protein synthetic apparatus occurs

Causes of Cellular Injury

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UERMMMC Class 2014

B. Mitochondrial damage

− May be caused by increased cytosolic Ca2+

, reactive

oxygen species and O2 deprivation

− Two major consequences:

a. Formation of mitochondrial permeability transition

pore  Loss of mitochondrial membrane potential 

Failure of oxidative phosphorylation  Decrease in ATP  necrosis

b. Leakage of proteins (Ex. Cytochrome c) that activate apoptosis into the cytosol

C. Ca2+influx and loss of Ca2+ homeostasis

− Calcium- important mediator of biochemical and morphologic alteration leading to cell death

− Ischemia and certain toxins causes this intracellular calcium causes cell injury by several mechanisms:

a. Opening of mitochondrial permeability transition pore b. Activation of a number of enzymes (phospholipases,

proteases, endonucleases)

c. Induction of apoptosis by direct activation of

caspases and by increasing mitochondrial

permeability

D. Accumulation of oxygen-derived free radicals

− Reactive Oxygen Species (ROS) – Produced normally in cells during mitochondrial respiration and energy generation, but degraded and removed by cellular defense systems

− Also produced by leukocytes, particularly neutrophils and macrophages

− Free radicals are chemical species that have a single unpaired electron.

a. Superoxide (O2) – Inactivated by superoxide dismutase (SOD) or spontaneously

b. Hydrogen peroxide (H2O2) c. Hydroxyl radicals

− Oxidative stress – Condition that is a result of excessive free radicals when production of ROS increases or the scavenging systems are ineffective (implicated in cell injury, cancer, aging, some degenerative diseases like Alzheimer’s)

− Main effect of O2 species:

 Lipid peroxidation - Oxidative damage is initiated when the double bonds in unsaturated fatty acids of membrane lipids are attacked by oxygen-derived free radicals, particularly by OH by the free radicals in the presence of oxygen.

 Oxidative modification of proteins: Formation of sulfhydryl bonds of proteins causing abnormal protein folding

 Mutation in genetic code − Fate of Free Radicals:

a. Spontaneous decay

b. Termination of inactivation of free radicals

 Antioxidants – Block the initiation of free radical formation and terminate radical damage

 Vitamin E

 Sulfhydryl containing compounds such as cysteine, glutathione and D-penicillamine  Serum proteins such as ceruloplasmin,

ferritin and transferring which bind to free iron

 Enzymes

 Superoxide dismutase (SOD - H2O2 system) converts O2 to H2O2

 Catalase which decomposes H2O2 to O2 and H2O

 Glutathione peroxidase – Catalyzes free radical breakdown(OHH2O2O2 and H2O)

− They may be generated by:

 Redox reactions during normal metabolic processes  Absorption of radiant energy

 During inflammation via redox reactions which use NADPH oxidase

 Enzymatic metabolism of exogenous chemicals or drugs (Ex. CCl4)

 Transition metals such as Fe and Cu  Nitric Oxide (NO)

E. Membrane damage

− In ischemic cells, membrane defects may be the result of ATP depletion and calcium-mediated activation of phospholipases

− Direct damage may also be caused by bacterial toxins, viral proteins, lytic complement components, physical and chemical agents

− Biochemical mechanisms contributing to membrane damage:

 ROS – Lipid peroxidation

 Decreased phospholipids synthesis – Due to

defective mitochondrial function or hypoxia

 Increased phospholipid breakdown – Due to

activation of endogenous phospholipases by

increased cytosolic Ca2+, lipid breakdown products

may insert into the lipid bilayer of membrane or exchange with membrane phospholipids, causing changes in permeability and electrophysiologic alterations

 Cytoskeletal abnormalities – Activation of proteases

by increased cytosolic Ca2+ results in detachment of

the cell membrane from cytoskeleton, rendering it susceptible to stretching and rupture

− Consequences of membrane damage

 Mitochondrial membrane damage  Opening of mitochondrial permeability transition pore

 Plasma membrane damage  Loss of osmotic balance

 Injury to lysosomal membranes  Leakage of enzymes into the cytoplasm and activation of acidic hydrolases, resulting to enzymatic digestion of proteins, RNA, DNA, and glycogen, and cell dies by necrosis

F. Damage to DNA and proteins

− If damage is too severe, the cell initiates a suicide program that results in death by apoptosis

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UERMMMC Class 2014

Notes:

The biochemical mechanisms responsible for cell injury are complex but there are a number of principles that are relevant to most forms of cell injury:

 The cellular response to injurious stimuli depends on the

type of injury, its duration, and its severity. Small doses of a toxin may lead to a reversible injury but larger toxins may induce an irreversible injury

 The consequences of cell injury depend on the type, state,

and adaptability of the injured cell. Nutritional and metabolic needs of the cell are important in its response to injury

 Cell injury results from functional and biochemical

abnormalities in one or more of several essential cellular components. Most important targets of injurious stimuli are: (1) aerobic respiration involving mitochondrial oxidative phosphorylation and production of ATP; (2) the integrity of cell membranes, on which the ionic and osmotic homeostasis of the cell and its organelles depends; (3) protein synthesis; (4) the cytoskeleton; and (5) the integrity of the genetic apparatus of the cell (Robbins)

A. Hypertrophy of SER – Adaptive response to medications (Ex. Protracted use of barbituates)

B. Mitochondrial alterations – Changes in size, shape and number

C. Abnormalities of cytoskeleton, contractile proteins, membrane skeleton

 Chediak-Higashi syndrome – Defective microtubule polymerization

 Colchicine – Disrupts microtubules, blocks mitosis in metaphase

 Cytochalasin B – Inhibits microfilament action and

phagocytosis, prevents polymerization of actin filaments

 Immotile cilia syndrome – Microtubule defect in respiratory cilia

 Intermediate filament accumulations – Mallory body (alcoholic hyaline in liver) and neurofibrillary tangle (brain in Alzheimer's disease)

D. Membrane skeleton – Seen in hereditary spherocytosis

Lysosomal catabolism

− Primary Lysosomes

 Membrane-bound intracellular organelles containing a variety of hydrolytic enzymes which are synthesized by RER and packaged into vesicles by Golgi apparatus − Secondary Lysosomes

 Fusion of primary lysosomes and membrane-bound vacuoles containing material to be digested  Phagolysosomes

Ways to Breakdown Phagocytosed Material

− Heterophagy

 Materials from the external environment are taken up through endocytosis, phagocytosis and pinocytosis  Process commonly exhibited by neutrophils and

macrophages − Autophagy

 Removal of damaged organelles during cell injury and cellular differentiation

 Common in cells undergoing atrophy due to nutritional deprivation or hormonal involution

 Most common type of cell injury in clinical medicine

 Ischemia – Reduced blood flow, usually as a consequence of a mechanical obstruction in the arterial system but sometimes as a result of a catastrophic fall in blood pressure or loss of blood compromising the delivery of substrates for glycolysis  Hypoxia – Any state of reduced oxygen availability

 Mechanism of Ischemic Cell Injury:

 Features of Ischemic/Hypoxic Injury: 1. Increased membrane permeability 2. Decreased mitochondrial function

- Cell’s aerobic respiration affected first (decreased oxidative phosphorylation and decreased ATP generation)

 Critical events leading to irreversible hypoxic cell injury: 1. Inability to reverse mitochondrial dysfunction upon

reoxygenation

2. Cell membrane damage – Central factor in the pathogenesis of ICI to irreversible cellular injury

- Calcium is an important mediator of biochemical changes leading to cell death (calcium influx).

Subcellular Alteration

Types of Cellular Injury

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UERMMMC Class 2014

Notes:

 IRREVERSIBLE INJURY is associated morphologically

with

1. SEVERE SWELLING of MITOCHONDRIA 2. EXTENSIVE DAMAGE to PLASMA MEMBRANE 3. SWELLING of LYSOSOMES

Leakage of intracellular enzymes and other proteins across

the abnormally permeable plasma membrane and into the blood provide important clinical indicators of cell death

Elevated serum levels of CKMB and Troponin are early

signs of MI and may be seen before the infarct is detectable morphologically

Ischemia-Reperfusion Injury

− Under certain circumstances, when blood flow is restored to cells that have been ischemic but have not died, injury is paradoxically exacerbated and proceeds at an accelerated pace

− Reperfused tissues may sustain loss of cells in addition to the cells that are irreversibly damaged at the end of ischemia − New damaging processes during reperfusion:

a. New damage may be initiated by reoxygenation by ↑ oxygen free radicals from infiltrating leukocytes b. Ischemic injury is associated with inflammation, as a

result of production of cytokines and adhesion molecules by hypoxic parenchymal and endothelial cells, which recruit circulating neutrophils

c. Activation of the complement system

Mechanism of Cell Injury by Activated Oxygen Species

Lipid Peroxidation Initiated by Hydroxyl Radical

 This can be observed from a decrease in glucose leading to electrolyte imbalance

1. Directly – Combine with molecular components or cellular organelle. Example:

a. Mercuric chloride – Mercury binds to sulfhydryl groups of the cell membrane proteins, causing an increase in membrane permeability and inhibition of ATPase-dependent transport; The greatest damage is usually to the cells that use, absorb, excrete, or concentrate the chemicals (GI TRACT and KIDNEY)

b. Cyanide – Poisons mitochondrial cytochrome oxidase and this inhibits oxidative phosphorylation

Notes:

Many antineoplastic chemotherapeutic agents and antibiotic drugs also induce cell damage by direct cytotoxic effects

2. Indirectly – Chemicals not biologically active in their native form and has to be converted to toxic metabolites which act on target cells by:

a. Direct covalent binding to membrane protein and lipids (Cytochrome P-450 mixed function oxidases in the smooth ER of the liver and other organs)

b. Formation of reactive free radicals and subsequent lipid peroxidation (is the most important membrane injury result)

− Ex. CCl4-induced liver necrosis and fatty change occur because conversion of CCl4 by cytochrome P450 to CCl3 (a highly reactive free radical), which causes lipid peroxidation and damages many cellular structures.

− Acetaminophen, an analgesic drug, is converted to a toxic product during detoxification in the liver.  Large doses of acetaminophen/paracetamol diminish reduced glutathione levels, consequently decreasing free radical breakdown

Sequence of Events Leading to Fatty Change and Cell Necrosis in CCl4 Toxicity

1. Direct Cytopathic Effect – Replicating virus particles which interfere with the cell’s metabolism, leading to cell damage such as:

− Cell lysis

− Cytoskeletal alterations

− Syncytial or Multinucleated giant cells (Ex. measles and herpes virus)

− Inclusion bodies – Contain virions or viral proteins; intranuclear, intracytoplasmic or both

Interstitial infiltrate.intracytoplasmic inclusion inside the giant cells in respiratory syncytial virus (RSV)

Chemical Injury

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UERMMMC Class 2014

Intranuclear inclusion (CMV)

2. Immune – mediated ell injury – Immune system will eliminate virus infected cells by apoptosis or lysis using complement system. Because of it, it will injure the other normal cells

Reversible (non-lethal) Irreversible (lethal) Cellular swelling. First manifestation.

Occurs when (a) cell is incapable of maintaining fluid and ionic homeostasis and (b) energy-dependent ion pumps on plasma

membrane have failed.

Fatty change. Occurs in hypoxic, toxic or

metabolic injury. Manifested as lipid vacuoles.

Cell necrosis/death

Cellular swelling of renal tubular cells (kidney) Reversible Injury

Two patterns of reversible cell injury can be recognized: 1. Cell Swelling

− First manifestation of almost all forms of injury to cells − Appears whenever cells are incapable to maintaining

ionic and fluid homeostasis

− Result of loss of function of plasma membrane energy-dependent ion pumps

2. Fatty Change

− Occurs in hypoxic injury and various forms of toxic or metabolic injury

− Appearance of small or large lipid vacuoles in the cytoplasm and occurs in hypoxic and various forms of toxic injury

− Encountered in cells involved in and dependent on fat metabolism (hepatocyte, myocardial cell)

 The ultrastructural changes of reversible cell injury include: 1. Plasma membrane alterations, such as bledding,

blunting, and distortion of microvilli; creation of myelin figures; and loosening of intercellular attachments 2. Mitochondrial changes, including swelling, rarefaction,

and the appearance of small phospholipid-rich amorphous densities

3. Dilation of the endoplasmic reticulum, with detachment and disaggregation of polysomes

4. Nuclear alterations, with disaggregation of granular and fibrillar elements (Robbins)

 Sum of morphologic changes that follow cell death in a living

tissue or organ, most commonly due to hypoxia

 With inflammation

 A major morphologic manifestation of Irreversible Cell Injury (ICI)

 morphologic appearance of necrosis is the result of

denaturation of intracellular proteins and enzymatic digestion of the cell

 enzymes are derived either from the lysosomes of the dead cells themselves (autolysis), or from the lysosomes of immigrant leukocytes (Inflammatory reaction)

 most necrotic cells and their debris disappear by a combined process of enzymatic digestion and fragmentation, followed by phagocytosis of the particulate debris by leukocytes.  Calcification happens if such cellular debris are not promptly

destroyed or reabsorbed (Dystrophic Calcification)

Morphologic changes

Nuclear

1. Pyknosis- nuclear shrinkage and increased basophilia

2. Karyorrhexis- pyknotic nuclear fragments 3. Karyolysis- basophilia or chromatin fades

Cytoplasmic

Increased eosinophilia; vacuolated,

moth-eaten appearance; calcification. Homogenous and glassy appearance.

Myelin figures- phospholipid masses of dead

cells.

Morphology of Injured Cells

Two Mechanism of Cell Death

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Types of Necrosis

TYPE DESCRIPTION EXAMPLE

C OA GU LA TI ON N E C R OSI S

− Most common; due to protein denaturation.

− Cell outline is retained, nucleus is lost  acidophilia (opacity, “tombstone)

− Usually follows sudden severe ischemia by obstruction of blood vessel

− Enzymes are also denature  block of proteolysis of dead cells  Eosinophlic ,

anucleated cells which persist for days  eventually phagocytosed by leukocytes and digested by leukocyte lysosomes Myocardial infaction, Ishchemia of all organs (except brain) due to obstruction of vessel. LI QU E FA C TI V E N E C R OS IS

− Digestion of dead cells  formation of liquid viscous mass

− Bacterial or fungal infections stimulate leukocytes to realease hydrolytic enzymes in cells

− Autolysis and heterolysis prevails over protein denaturation

− Pus production (due to dead leukocytes) Bacterial and fungal infection, suppuration, amoebic liver abscess and brain infarct-reason for liquefaction is unknown FA T N E C R O S IS

− Not a specific pattern of necrosis

− Pancreatic enzymes leak out of acinar cells and liquefy the membranes of fat cells in the peritoneum

− The released lipases split the TG esters contained within fat cells

− The Fats, so derived, combine with Calcium to produce grossly visible chalky-white areas(fat saponification) due to combining of calcium with released fatty acid, enables the identification of the lesion − Histologic examination: Takes

form of shadowy outlines of necrotic fat cells with basophilic Ca deposits

surrounded by an inflammatory reaction.

Acute pancreatitis

Types of Necrosis (cont’d)

TYPE DESCRIPTION EXAMPLE

C A S E OU S N E C R O S IS − Collection of fragmented or lysed cells and amorphous granular debris within an inflammatory border − Coagulation and liquefactive

necrosis

− Derived from the friable white appearance of the area of necrosischaracteristic of a focus of inflammation known as a GRANULOMA

− Gross: soft, friable, whitish-gray (cheesy)

− Microscopic: amorphous, granular pinkish debris

− Most often in foci of tuberculous Infection; “cheese-like” Tuberculosis GANGRE N OUS NE CR OS IS

− Refer to lost body part (limb) − Not a pattern of cell death − Coagulation + Liquefactive

action of bacteria and WBC’s

Wet (with infection) and Dry (black and

patchy) gangrene FI B R IN OI D N E C R OS IS

− CT and arterial walls are infiltrated by eosinophilic hyaline material which shows some of characteristics of fibrin − Occurs when complexes of

ANTIGENS and ANTIBODIES are deposited in the walls of arteries − FIBRINOID appearance in H and E stain Immunologically mediated vasculitis syndromes

 Pathway of cell death that is induced by a tightly regulated suicide program

 There is activation of enzymes that degrade the cells' own nuclear DNA and nuclear and cytoplasmic proteins  Breaks into fragments called apoptotic bodies

 Plasma membrane remains intact though altered to induce phagocytosis of apoptotic bodies (as a result, no leaking

occurs)

 Chief morphologic features: Chromatin condensation and fragmentation

 No ATP depletion

 Does not elicit inflammation  Involved only in living tissue

 Serves to eliminate unwanted or potentially harmful cells and cells that have outlived their usefulness

 Pathologic event when cells are damaged beyond repair, especially when the damage affects the cell's DNA; in these situations, the irreparably damaged cell is eliminated

Apoptosis of skin

Apoptosis

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Physiologic Causes

− For elimination of cells that are no longer needed as well as to maintain steady population levels of cells in tissues − Embryogenesis involves programmed destruction of cells in

implantation, organogenesis, developmental involution, and metamorphosis

− Involution of hormone-dependent tissues upon hormone

withdrawal such as endometrial cell breakdown during the

menstrual cycle

− Cell loss in proliferating cell populations (i.e. immature lymphocytes in the bone marrow that do no express the right antigen receptors)

− Elimination of harmful self-reactive lymphocytes

Pathologic Causes

− For elimination of cells injured beyond repair

− DNA damage due to radiation, hypoxia, free radicals, etc. − Accumulation of misfolded proteins due to mutations in

genes leading to ER stress and then to apoptosis − Cell death due to infections like viral infections (HIV) or

immune responses (viral hepatitis) via cytotoxic T lymphocytes

− Pathologic atrophy in parenchymal organs due to duct

obstruction such as in the pancreas or kidney

Morphological Changes

− Cell Shrinkage

− Chromatin condensation – Most characteristic feature of apoptosis. Chromatin aggregates peripherally then breaks up into smaller parts under the nuclear membrane − Formation of cytoplasmic blebs and membrane-bound

apoptotic bodies - extensive surface blebbing 

Fragmentation to membrane-bound apoptotic bodies composed of cytoplasm and tightly packed organelles, with or without nuclear fragments

− Phagocytosis of apoptotic cells or cells bodies by macrophages (phagocytes).

Notes:

 Plasma membrane remain intact in apoptosis until the last

stages where they become permeable to normally retained solutes (Robbins)

 Histological appearance of apoptotic cells

 In hematoxylin and eosin stain, involves single cells or

small clusters of cells, round or oval mass of intensely eosinophilic cytoplasm with dense nuclear chromatin fragments. Since it not elicit inflammation, it is more difficult to detect histologically(Robbins)

Biochemical Features

− Activation of Caspases

 Caspases are a family of cysteine proteases that activate apoptosis.

 Caspase-8 and caspase-9 are initiators  Caspase-3 and caspase-6 are executioners.  Must undergo enzymatic cleavage to become active. − DNA and Protein Breakdown

 Calcium and magnesium dependent endonuclease activity breaks down DNA.

 Changes of movement of phospholipids on the membrane (i.e. phosphatidylserine) from inner to outer leaflet are recognized by phagocytes.

Mechanism Initiation Phase

− Caspases become catalytically active.

− Occurs via intrinsic (mitochondrial) and extrinsic (death receptor-initiated) pathways

 Intrinsic Pathway.

 Involves release of mitochondrial proteins (i.e.

cytochrome c) into cytoplasm intiating apoptosis.

 Anti-apoptotic Bcl family regulates release

 Sensor proteins Bim, Bid, and Bad detect stress or damage and activate pro-apoptotic effectors Bax and Bak which allows mitochondrial leakage.  Bcl-2/Bcl-x levels decrease can increase

permeability of the mitochondrial membrane and several proteins that can activate the caspase cascade leak out

 Cytochrome c binds to Apaf-1 forming an

apoptosome that binds to initiator caspase-9

producing an auto-amplification process of the pathway

 IAP (inhibitors of apoptosis) are inhibited  Extrinsic Pathway

 Involves death receptors (TNF receptor family) such as TNFR1 and Fas (CD95).

 Ligands attach to receptors forming cytoplasmic death domains forming binding sites. Adapter proteins bind to these sites and their death domains bind to and allows activation of caspase-8 (caspse-10 in humans) leading to apoptosis.

 Fas is cross-linked by its ligand

 Pathway can be inhibited by FLIP proteins

Execution Phase

− Executioner caspases (caspase-3 and -6) are activated by either initiation pathways and degrade cytoplasmic structures and DNA.

Dysregulated Apoptosis

− Defective apoptosis allows increased cell survival.

 May involve mutation in p53 which fails to activate cell death leading to an increase in potentially harmful cells like lymphocytes.

− Increased apoptosis leads to excessive cell death.

 Neurodegenerative diseases involving loss of neurons caused by mutations or misfolded proteins.

 Ischemic injury.  Virus-infected cells

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UERMMMC Class 2014

Necrosis Apoptosis

Stimuli Hypoxia, toxins Physiologic & Pathologic

Histology

Cellular swelling Coagulation necrosis

Disruption of organelles

Single cells shrinkage Chromatin condensation

Apoptic bodies

DNA

Breakdown Random, diffuse Internucleosomal

Mechanisms

ATP depletion Membrane injury Free radical damage

Gene activation Endonuclease Tissue Reaction Inflammation No inflammation Phagocytosis of apoptotic bodies Features

Cell size Enlarged (swelling) Reduced (shrinkage)

Nucleus Pyknosis → karyorrhexis →

karyolysis Fragmentation into nucleosome-size fragments Plasma membrane Disrupted Intact; altered structure, especially orientation of lipids Cellular contents

Enzymatic digestion; may leak out of cell

Intact; may be released in apoptotic bodies Adjacent inflammation Frequent No Physiologic or pathologic role Invariably pathologic (culmination of irreversible cell injury) Often physiologic, means of eliminating unwanted cells; may be pathologic after some forms of cell injury, especially DNA

damage

 Sign of metabolic derangement due to abnormal amounts of various substances

 Most accumulations are attributable to three types of abnormalities:

o A normal endogenous substance is produced at a normal

or increased rate, but the rate of metabolism is inadequate to remove it.

o A normal or abnormal endogenous substance

accumulates because of genetic or acquired defects in the metabolism, packaging, transport, or secretion of these substances.

o An abnormal exogenous substance is deposited and

accumulates because the cell has neither the enzymatic

machinery to degrade the substance nor the ability of transport it to other sites

 From the type of abnormalities, four major mechanisms are present:

o Abnormal metabolism – Metabolic rate inadequate to remove normal substance (ex. fatty change)

o Enzyme deficiency – One can't metabolize certain substances (ex. inborn errors of metabolism like glycogen storage diseases)

o Inability to degrade or transport abnormal exogenous

substances (ex. hemosiderin, carbon pigments, silica) to

other sites

o Defects in protein folding and an inability to degrade

the abnormal protein efficiently. (ex. Accumulation of

mutated α1-antitrypsin in liver cells, various mutated proteins in the degenerative disorders of the CNS)

Fatty Change

− Steatosis

 Abnormal accumulation of triglycerides within the parenchymal cell

 Often seen in the liver since it is the major organ involved in fat metabolism, but may also be seen in the heart, muscle and kidney.

 Fatty change appears as clear vacuoles

Fatty Change gross specimen

− Causes:

 Alcohol – Most common cause in adults  Protein malnutrition

 Diabetes Mellitus

 Pregnancy – Acute fatty change  Obesity

 Some chronic diseases  Hepatotoxins

 Anoxia − Mechanism:

 Excessive entry of FFA into the liver brought about by starvation and corticosteroids

 Increased esterification of FA to triglycerides due to:  Increased alpha-glycerophosphate (ex. alcohol

poisoning)

 Enhanced FA synthesis  Decrease in FA oxidation

 Reduced apoprotein synthesis leading to a decrease in

fat mobilization (ex. CCl4 and protein malnutrition)

 Impaired lipoprotein secretion from the liver (oretic acid)

Necrosis versus Apoptosis

INTRACELLULAR ACCUMULATIONS

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UERMMMC Class 2014

− Morphology

 In all organs, fatty change appears as clear vacuoles within parenchymal cells

 In the Liver

 Mild fatty change may not affect the gross appearance

 Progression – Organ enlarges and becomes increasingly yellow until

 Extreme condition – Transformed into a bright yellow, soft, greasy organ.

 Development of minute, membrane-bound inclusions (liposomes) closely applied to the endoplasmic reticulum

 First seen as small vacuoles in the cytoplasm around the nucleus  the vacuoles coalesce, creating cleared spaces that displace the nucleus to the periphery of the cell  contiguous cells rupture, and the enclosed fat globules coalesce, producing so-called fatty cysts

 In the Heart

 In the form of small droplets, occurring in two patterns

o Prolonged moderate hypoxia – Intracellular deposits of fat, grossly apparent bands of yellowed myocardium alternating with bands of darker, red-brown, uninvolved myocardium o More profound hypoxia or by some forms of

myocarditis – Shows more uniformly affected myocytes

Other lipid accumulations:

1. Atherosclerosis

− Smooth muscle cells and macrophages in the intima and large arteries are filled with lipid vacuoles. These may rupture and release lipid into the extracellular space. Extracellular esters may crystallize in the shape of long needles, producing distinctive clefts in tissue sections.

2. Xanthomas

− Clusters of foamy cells in the subepithelial connective tissue of the skin and in tendons which form tumorous masses

3. Cholesterolosis

− Focal accumulations of cholesterol-laden macrophages in the lamina propria of the gallbladder. Mechanism of accumulation is unknown

4. Niemann-Pick disease, type C − A lysosomal storage disease

− Caused by mutations affecting an enzyme involved in cholesterol trafficking

− Results in cholesterol accumulation in multiple organs  Protein accumulations appear as rounded eosinophillic

droplets in the cytoplasm.

 Abnormal proteins deposit primarily in extracellular spaces in some disorders such as amyloidosis

 Causes of morphologically visible protein accumulation: o Reabsorption droplets in the proximal renal tubules

 In disorders with heavy protein leakage across the glomerular filter, renal absorption of protein into vesicles is increased.

 Protein appears as pink hyaline droplets within the cytoplasm of the tubular cell.

 This process is reversible. Once the proteinuria (protein loss in the urine) diminishes, the protein droplets are metabolized and disappear.

o Defective intracellular transport and secretion of critical proteins

o Accumulation of cytoskeletal proteins o Aggregation of abnormal proteins

 Abnormal or misfolded proteins may deposit in tissues interfere with normal functioning

 Deposits may be intracellular, extracellular, or both, and may directly or indirectly be the cause of the pathologic changes

 Excessive deposits of glycogen are due to an abnormality in either glucose or glycogen metabolism

 Glycogen appears as clear vacuoles within the cytoplasm.  Causes:

1. Diabetes Mellitus

− Glycogen is found in renal epithelial cells of PCT, liver cells, beta cells of islets of Langerhans and heart muscle.

2. Glycogen storage diseases or Glycogenoses

− Enzymatic defects in the synthesis or breakdown of glycogen result to massive accumulation of glycogen and, eventually, cell injury and death. − Ex. Von Gierke's disease, McArdle's syndrome,

Pompe's disease

 Colored substances, some of whichare normal constituents of the cell (eg.melanin), wheras others are abnormal and accumulate in cells only under special circumstances

Exogenous Pigments a. Carbon

− Ubiquitous air pollutant

− Most common exogenous pigment

− Picked up by macrophages within the alveoli and transported to lymph nodes at the tracheobronchial region

− Accumulation causes anthracosis (anthracotic

pigment) in lungs and involved lymph nodes OR coal

worker’s pneumocosis

 Blackened lung tissue

b. Tattoo

− Localized pigmentation of the skin

− Inoculated pigments are phagocytosed by dermal macrophages which reside in the skin for the duration of the person’s life.

− The pigments do not usually evoke any inflammatory response

Endogenous pigments 1. Lipofuscin

− Insoluble pigment, also known as lipochrome or wear

and tear (aging) pigment

− Appears as a yellow brown, finely granular cytoplasmic, often perinuclear pigment (brown atrophy)

− Composed of polymers of lipids and phospholipids in complex with protein

− Important as a telltale sign of free radical injury and lipid peroxidation

− Seen in cells undergoing slow, regressive changes and is particularly prominent in the liver and heart of patients with severe malnutrition and cancer cachexia

2. Melanin

− Endogenous, non hemoglobin derived, brown black pigment formed when tyrosinase catalyzes the oxidation of tyrosine to dihydroxyphenylalanine in melanocytes − The only endogenous brown-black pigment

Accumulation of Proteins

Accumulation of Glycogens

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UERMMMC Class 2014

3. Hemosiderin

− Hemoglobin-derived, golden yellow to brown, granular or crystalline pigment that serves as one of the major

forms of iron

− If engulfed by alveolar macrophage  Heart failure cells

or Hemosiderin-laden macrophage (seen in CPC) − In cells, iron is stored in association with a protein,

apoferritin, to form ferritin micelles .When there is local

or systemic excess of iron, ferritin forms henosiderin granules which are easily seen in the light microscope

 Hemosiderosis – Systemic overload of iron,

wherein hemosiderin is deposited in many organs and tissues; impaired use of iron

 Does not damage the parenchynmal cells or impair organ functions

 Main causes are: Increased absorption of dietary iron, hemolytic anemia, and repeated blood transfusions

 Hemochromatosis – Most extreme accumulation

of iron associated with liver, hear and pancreatic damage resulting in liver fibrosis, heart failure and diabetes mellitus.

4. Bilirubin

− Normal major pigment found in bile.

− Derived from hemoglobin but contains no iron − Evident in liver and kidney

− Jaundice: Common clinical disorder caused by excesses of bilirubin within cells and tissues − Kernicterus : Damage to the brain centers of infants

caused by increased levels of unconjugated bilirubin.

5. Hematin (Hemozoin)

− Also called malaria pigment (color: black) − Found in the liver and spleen; sinusoidal walls − Digest by macrophage

− (-) Prussian Blue

6. Copper

− Toxic levels of copper accumulate in Wilson’s disease, causing hepatolenticular degeneration

PATHOLOGIC CALCIFICATION

− The abnormal tissue deposition of calcium salts together with smaller amounts of iron, magnesium and other mineral salts

 Deposition usually occurs in dying tissues. Encountered in areas of necrosis, whether they are coagulative, casous, or liquefactive type and in the foci of enzymatic necrosis of fat.  MACROSCOPIC deposition of Calcium salts in injured tissue  Occurs despite normal serum levels of calcium and in the

absence of derangements in calcium metabolism.

 Calcium salts appear macroscopically as fine, white granules or clumps and often felt as gritty deposits

 Always present in atheromas of advanced atherosclerosis  Histologically, with the usual hematoxylin and eosin stain, the

calcium salts have a basophilic, amorphous granular,

sometimes clumped, appearance, may be intra or extracellular locations

Pathogenesis:

Ca2+ is concentrated in membrane-bound vesicles through a

process initiated by membrane damage

a. Ca2+ binds to phospholipids present in the vesicle

membrane.

b. Phosphates associated with the membrane generate

phosphate groups which bind to Ca2+.

c. Calcium-phosphate binding repeats.

d. Structural changes occur in the arrangement of calcium and phosphate groups generating a microcrystal which then propagate and lead to MORE calcium deposition E.g. Psammoma bodies – Seen in mesothelioma

 May occur in normal tissues whenever there is

hypercalcemia. Four principal causes of such include:

a. Increased secretion of parathyroid hormone (PTH) with subsequent bone resorption

b. Destruction of bone tissue

c. Vitamin D-related disorders

d. Renal failure

 Can occur widely throughout the body but principally affects the interstitial tissues of the gastric mucosa, kidneys, lungs, systemic arteries and pulmonary veins.

o In all these sites calcium salts morphologically resemble those described in dystrophic calcification. They may occur as noncrystalline amorphous deposits or at times hydroxyapatite crystals

o May cause massive deposits in the kidney (nephrocalcinosis) – May cause renal damage

 An alteration within or in the extracellular space which gives a homogenous, glassy, pink appearance in routine H&E sections

a. Intracellular

− Can be observed in the proximal convoluted tubules, Russell bodies, viral inclusions and alcoholic hyaline

b. Extracellular

− Examples are scars, hyaline arteriosclerosis, hyalinized glomeruli (chronic renal disease) and amyloid (positive Congo red stain; bipolar refringence)

 In long term hypertension, the walls of arterioles, especially in

the kidney, become hyalinized.

 Is the result of a progressive decline in cellular function and viability caused by genetic abnormalities and the

accumulation of cellular and molecular damage due to effects of exogenous influences.

 The balance between cumulative metabolic damage and the response to that damage could determine the rate at which we age.

 The known changes that contribute to cellular aging include:

a. Decreased cellular replication

− After a fixed number of divisions, all somatic cells become arrested in a terminally non dividing state known as senescence

b. Structural and Biochemical Changes with Cellular Aging

− Reduction of oxidative phosporylation, synthesis of nucleic acids and structural and enzymatic proteins, cell receptros and transcription factors

− Morphologic alterations in aging cells include irregular and abnormally lobed nuclei, pleomorphic vacuolated mitochondria, decreased endoplasmic reticulum, and distorted Golgi apparatus

− Steady accumulation of the pigment lipofuscin

CALCIFICATION

Dystrophic Calcification

Metastatic Calcification

HYALINE CHANGE

SECTION B

UERMMMC Class 2014

1. Hyp ertro ph y 2. Squ am ou s M eta pla sia /Colu mn ar t o Sq ua mo us Me tap la sia 3. Mito ch on dri al p erm ea bili ty t ran siti on po re 4. Me mb ran e d am ag e 5. Isc he mia -Rep erfu sio n I nju ry 6. Fatt y c ha ng e 7. Cas eou s Ne cro sis 8. Carbo n 9. Path olo gic Calc ifi ca tio n 10. Chron ic Pas siv e Co ng es tio n o f th e L un gs

c. Accumulation of metabolic and genetic damage

− E.g . Reactive oxygen species (ROS), byproducts of oxidative phosphorylation , causes covalent modification of proteins lipids and nucleic acids . − Increased oxidative damage could result from

repeated environmental exposure to such influences as ionizing radiation, mitochondrial dysfunction or reduction of antioxidant defense mechanisms with age.

Processes of Cellular Aging

1. Robbins Pathologic Basis of Disease 8th ed. 2. Dr. Bailon’s Lecture: Cell injury etc.

3. 2014-B Trans: Cell injury etc.

1) Cellular adaptation that has the mechanism of increasing its cellular proteins or structural components.

2) The most common epithelial metaplasia

3) Damage in mitochondria will result into leakage of proteins that activate apoptosis and formation of _____.

4) Central factor in pathogenesis of irreversible cell injury. 5) This injury is paradoxically exacerbated when the blood flow

is restored to cells that have been ischemic due to increasing numbers of ROS

6) Reversible cell injury has 2 manifestations: Cell swelling and ______.

7) Type of necrosis that has characteristic feature known as granuloma

8) Most common exogenous pigment seen.

9) Abnormal tissue deposition of calcium salts together with smaller amounts of iron, magnesium and other mineral salts 10) Hemosiderin is engulfed by alveolar macrophage resulting

into appearance of Heart Failure Cells. This manifestation can be seen in what disorder. NO ABBREVIATION.

Hi guys! 1st trans in Patho! Yehey! Don’t forget to read the Morphology boxes in the book. It’s important to know the morphology of each different pathologic cell for easier time to study the slides. Mas marami na pages ng mga patho trans since most of the topics are 2-day lecture plus pictures pa =) God Bless!!

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