GENERAL PATHOLOGY

CELLULAR INJURY & DEATH

INTRODUCTION
Q. What is PATHOLOGY? Ans. It is the study of nature & cause of disease which involves changes in structure and function The 4 aspects of a disease process that form the core of pathology are: 1. Its cause 2. The pathogenesis (sequence of events that give rise to the manifestations of the disease) 3. The morphologic changes (structural alterations induced in cells & tissues by the disease) 4. The functional consequences of the morphologic changes (Clinical features)

How does a cell react to excessive physiologic stresses or adverse pathologic stimuli ?
Adapting Sustaining reversible injury Suffering irreversible injury Dying

CELLULAR ADAPTATION
It occurs when excessive physiologic stresses, or some pathologic stimuli, result in a new but altered state that preserves the viability of the cell Examples - Hypertrophy ( in mass/size of a cell) - Atrophy ( in mass/size of a cell) - Hyperplasia ( in number of cells) - Metaplasia (Reversible change of one adult cell type by another) (Dealt in detail in the NEXT CHAPTER)

CELL INJURY
REVERSIBLE The pathologic changes can be reversed on removal of the stimulus or when the cause of injury is mild IRREVERSIBLE The pathologic changes gets permanent and cause CELL DEATH

PATTERNS OF CELL DEATH

NECROSIS
More common type of cell death after external stimuli.

APOPTOSIS
Cell death after activation of an internal suicidal program.

CAUSES OF CELL INJURY

1. HYPOXIA (Oxygen deprivation) as a result of
a. ISCHEMIA (Loss of blood supply) b. INADEQUATE OXYGENATION (Ex. Cardiorespiratory failure) c. LOSS OF OXYGEN CARRYING CAPACITY OF THE BLOOD (Ex. Anemia, CO poisoning) PHYSICAL AGENTS (TRAUMA, HEAT, COLD, RADIATION & ELECTRIC SHOCK) (Therapeutic ASPIRIN) (Non-therapeutic LEAD, ALCOHOL)

2.

3. CHEMICAL AGENTS & DRUGS 4. 5. 6. 7.

INFECTITOUS AGENTS IMMUNOLOGIC REACTIONS GENETIC DERRANGEMENTS NUTRITIONAL IMBALANCE (Lack or Excess)

MECHANISMS OF CELL INJURY

1. 2. 3. 4. 5. Decreased ATP synthesis & ATP depletion Oxygen derived free radicals Increased intracellular calcium Defects in membrane permeability Irreversible mitochondrial damage

Decreased ATP synthesis & ATP depletion

Increased intracellular calcium

Normal intracellular calcium < 0.1mmol Normal extracellular calcium 1.3 mmol Stored in mitochondria & ER Gradient maintained by Ca-Mg ATPase pump Ischemia & Toxins causes increase in intracellular calcium Increased intracellular calcium 1. activates many enzymes 2. Induce MPT 3. Induce apoptosis

Defects in membrane permeability

Mitochondrial damage causes decreased phospholipids synthesis that in turn leads to the formation of MPT Plasma membrane damage leads to loss of osmotic balance Loss of lysosomal membrane leads to leakage of its enzymes into the cytoplasm

Irreversible mitochondrial damage

Increased cytoplasmic calcium, Oxidative stress, Lipid breakdown products (Free fatty acids), Phospholipid breakdown

Loss of proton gradient ETC stops

(High conductance channels in the inner mitochondrial membrane)

OXIDATIVE STRESS
Oxygen derived free radicals are produced as a byproduct of mitochondrial respiration (O2 H2O) These may cause injury by 1. Lipid peroxidation of membranes (Double bonds of UFA attacked by free radicals formation of peroxides in turn these are also reactive) 2. Oxidative modification of proteins (Oxidation of amino acids protein-protein cross linking protein fragmentation) 3. DNA damage (Reacts with thymine single stranded breaks) (Theory of ageing) Cells have good counter mechanisms that prevent such damage. These include 1. Catalase (2 H2O2 O2 + 2 H2O) 2. Superoxide dismutase (In mitochondria & cytoplasm) (O2 + H H2O) 3. Glutathione peroxidase 4. Vitamins 5. Iron & Copper binders (Transition metals like Cu & Fe can generate free radicals) An imbalance between free radical generating & radical scavenging systems results in OXIDATIVE STRESS

Oxygen derived free radicals

MORPHOLOGY OF REVERSIBLE CELL INJURY

Light Microscope 1. Cellular swelling (Hydropic changes or Vacuolar degeneration) 2. Fatty changes (appearance of lipid vacuoles in the cytoplasm) (Secondary to hypoxia & toxic injury) (seen mostly in cells involved in and dependent on fat metabolism Hepatocytes & Myocardial cells) Electron microscope 1. Plasma membrane Blebbing, Loss of microvilli, Myelin figures (whorled phospholipid masses) 2. Mitochondrial swelling 3. Swelling of Endoplasmic reticulum 4. Clumping of chromatin

FATTY CHANGE

HYDROPIC CHANGE

MORPHOLOGY OF NECROSIS
1. 2. 3. 4. 5. 6. 7. Increased eosinophilia (d/t loss of RNA and increased eosin binding to denatured proteins) Cells become Glassy & Vacuolated Myelin figures Calcification of dead cells (Dystrophic calcification) Discontinuous plasma & organelle membranes Large amorphous mitochondrial densities Lysosomal swelling (Autolysis digestion of dead cells by lysosmal enzymes) (Heterolysis digestion of immigrant WBCs by lysosomal enzymes) Nuclear changes (can be one out of the following) Pyknosis Small, dense nucleus (increased basophilia) Karyolysis Faint (basophilia fades), dissolved nucleus Karyorrhexis Pyknotic nucleus broken up into many clumps

8.

Increased eosinophilia

Mallory bodies (the red globular material) composed of cytoskeletal filaments in liver cells chronically damaged from alcoholism. These are a type of "intermediate" filament between the size of actin (thin) and myosin (thick).

Two processes cause the basic morphologic changes of necrosis Denaturation of proteins Enzymatic digestion of organelles
Types of necrosis 1. Coagulative necrosis When denaturation of proteins is the primary pattern. Ex Ischemia of organs 2. Liquefactive necrosis When enzymatic digestion is the primary pattern. Ex. Infections 3. Caseous necrosis A form of coagulative necrosis. Ex. Tuberculosis 4. Fat necrosis

COAGULATIVE NECROSIS Here, the general tissue architecture is preserved for quite sometime (as the injury denatures not only structural proteins but also the lysosomal enzymes Proteolysis of the cell is blocked for weeks) Ultimately, the necrotic cells are removed by phagocytosis of the cell debris by WBCs & by the action of proteolytic lysosomal enzymes brought in by the immigrant WBCs LIQUEFACTIVE NECROSIS It is mostly due to bacterial infection Here, the end result is transformation of the tissue into a thick liquid mass (PUS) Wet gangrene Liquefactive necrosis over Coagulative necrosis

A, Normal myocardium. B, Myocardium with coagulation necrosis (upper

two thirds of figure), showing strongly eosinophilic anucleate myocardial fibers.

CASEOUS NECROSIS Cheesy white GROSS appearance of the area of necrosis Granuloma Fragmented coagulated cells enclosed within a distinctive inflammatory border Unlike coagulative necrosis, the tissue architecture is completely obliterated FAT NECROSIS Seen in acute pancreatitis Pancreatic lipases released into the substance of the pancreas & peritoneal cavity digests the TGs into free fatty acids FFA combines with Calcium to produce chalky white areas

Pulmonary TB, tissue necrosis

REPERFUSION INJURY
Sometimes, the restoration of blood flow to ischemic tissues may result in tissue death Mechanisms 1. Free radical induced injury (MOST ACCEPTED THEORY) Reoxygenation leads to increased generation of oxygen free radicals. This leads to tissue death as the cellular antioxidant defense mechanisms are compromised by ischemia. More so, the oxidases secreted by the immigrant WBCs also leads to free radical generation 2. The reactive oxygen species can further promote the MPT 3. Activation of complement pathway (Some IgM antibodies have an affinity to deposit in ischemic tissues restoration of blood flow complement proteins bind to the antibodies complement are activated cell injury & inflammation) 4. Increased recruitment of the circulating PMN to the reperfused site. (Hypoxic tissue produces cytokines & adhesion molecules)

CHEMICAL INJURY
Chemicals induce cell injury by one of the two general mechanisms 1. Mercury binds to the sulphydryl groups of the cell membrane increased membrane permeability & inhibition of NaK ATPase pump. Greatest damage occurs to the tissue that use, absorb, excrete or concentrate the chemical. In case of mercuric chloride, the cells of GIT & Kidney 2. Cyanide blocks mitochondrial oxidative phosphorylation 3. Some chemicals are not biologically active but must be converted to reactive toxic metabolites which then act on target cell

APOPTOSIS
Programmed cell death Activation of an internal suicide program Idea is to eliminate unwanted cells selectively, with minimal disturbance to surrounding cells and the host Examples 1. Programmed destruction of cells during embryogenesis 2. Hormone dependent involution of tissues (endometrium) 3. Cell death in tumors 4. Cell death by Cytotoxic T cells (CD8) 5. Death of B & T cells after cytokine depletion 6. Death of neutrophils after during acute inflammation 7. Cell death in certain viral diseases 8. Cell death produced by a variety of injurious stimuli that are capable of producing necrosis but when given in low doses induce apoptosis (Radiation, Mild thermal injury, anticancer drugs) 9. Cell deletion in proliferating cell population (Intestinal epithelium, bone marrow) 10. Pathologic atrophy in organs after duct obstruction

STEPS IN APOPTOSIS
1. SIGNALLING PATHWAYS (Death triggering signals) 2. CONTROL or INTEGRATION 3. EXECUTION PHASE 4. REMOVAL OF DEAD CELLS

SIGNALING & CONTROL

1. Lack of growth factor or a hormone 2. Receptor-ligand binding 3. Specific cell injury
SIGNALING BY TNF FAMILY of RECEPTORS

Tumor necrosis factor family of receptors (TNF-R) Some members initiate apoptosis (Fas, TNFRI) Some initiate cell proliferation And others can initiate both Fas & TNFRI subfamily is also called as Death receptors

Fas-mediated and Fas ligand (FasL)mediated apoptosis FasL or CD95L is a cytokine produced by the cells of the immune system that binds to Fas & activates a death program in lymphocytes FasL binds with Fas Activation of adaptor protein, FADD Activation of initiator CASPASE (procaspase 10) Cell death Certain viruses produce a protein called FLIP. It binds to procaspase 8 and prevents its activation viral infected cells are not killed

TNF-induced apoptosis TNF (a cytokine) binds to TNFRI Activation of adapter protein TRADD TRADD in turn binds to FADD Caspases activation Apoptosis Under certain conditions TRADD after activation does not bind to FADD. Instead it binds & activates TRAF 2 & RIP. This binding leads to activation of Nuclear Factor B (NF B), a transcriptional factor that promotes cell survival.

Cytotoxic T lymphocyte stimulated Apoptosis CTLs recognizes foreign antigen presented to them CTLs releases PERFORINS & cytotoxic granules in to the target cell These granules contains GRANYZYME B Activation of caspases directly DNA damage mediated apoptosis Radiation DNA damage accumulation of p53 gene p53 arrests the cell cycle at the G1 phase Tries to repair the damage If repair cannot be done trigger apoptosis by activating caspases Absence or mutation of p53 Cell survival

Intrinsic pathway of activation of apoptosis Injury to the cell Insertion of MPT release of cytochrome c apoptosis Growth factors stimulate the production of bcl-2 & bcl-x (anti-apoptotic proteins). These proteins are situated in the outer mitochondrial membrane. These binds to Apaf-1 (apoptosis activating factor) present in the cytoplasm Lack of growth factors bcl-2 & bcl-x are lost and are replaced by pro-apoptotic factors (bak, bax, bim) these induces MPT release of cytochrome c Cyt c in the cytoplasm binds to Apaf-1 Complex activates caspase 9 initiation of casapse cascade Apoptosis

EXECUTION
This final phase of apoptosis is mediated by cysteine proteases called CASPASES. Initiator caspases are caspase 8 & 9 Executioner caspases are caspase 3 & 6 Caspases exists as zymogens that must be cleaved for their activation Action of caspases 1. Protein Cleavage: Activated caspases cleave important cellular proteins like laminin thus break up nuclear scaffolds & cytoskeleton 2. DNA breakdown: Caspases activate DNAses (endonucleases) initiates internucleosomal DNA cleavage DNA is broken down into smaller fragments (200 bp) These pieces when visualized after electrophoresis gives a typical LADDER pattern

REMOVAL OF DEAD CELLS

Apoptotic cells & their fragments are phagocytosed by MACROPHAGES Apoptotic cells have marker molecules on their surfaces that facilitates *EARLY recognition by phagocytes [*If these cells are not removed in time secondary necrosis release of cellular contents inflammation] Marker molecules include 1. Phosphatidyl serine on the plasma membrane 2. Thrombospondin Adjacent viable cells prevent their own engulfment by macrophages by expressing CD31 on their surfaces

MORPHOLOGY (E/M)
1. Cell shrinkage Dense cytoplasm with tightly packed organelles (Seen as intensely eosinophillic cytoplasm) Chromatin condensation (Most characteristic) chromatin aggregates peripherally under the nuclear membrane Cytoplasmic blebs & apoptotic bodies Phagocytosis of apoptotic cells by macrophages

2.

3. 4.

CELLULAR ADAPTATIONS

DR SANDEEP SINGH

How does a cell react to excessive physiologic stresses or adverse pathologic stimuli ?
Adapting Sustaining reversible injury Suffering irreversible injury Dying

Cellular Adaptations

Size

Number

Type

Atrophy

Hypertrophy

Hyperplasia

Metaplasia

Dysplasia

Intracellular Accumulations

Calcifications

Dystrophic

Metastatic

Cells respond to increased demand & external stimulation by HYPERPLASIA (increased number) or HYPERTROPHY (increased size) Cells respond to reduced supply of nutrients and growth factors by ATROPHY (decrease or shrinkage in cell size) METAPLASIA - Cells change from one type to another DYSPLASIA - loss of uniformity of the individual cells, as well as a loss of their architectural orientation

HYPERPLASIA
Can be PHYSIOLOGIC or PATHOLOGIC PHYSIOLOGIC HYPERPLASIA 1. Hormonal hyperplasia Endometrial proliferation after estrogen stimulation 2. Compensatory hyperplasia Hyperplasia of liver after partial resection 3. Antigenic stimulation lymphoid hyperplasia Partial hepatectomy Secretion of growth factors (TGF , EGF) & cytokines (TNF , IL 6) Hyperplasia of the remaining cells Rebuilding of the lost hepatic tissue Secretion of TGF (growth inhibitor) Growth stops PATHOLOGIC HYPERPLASIA Excessive hormonal stimulation Hyperestrinism & atypical endometrial hyperplasia; Benign prostatic hyperplasia Locally produced growth factors on target cells Proliferation of connective tissue cells in wound healing Removal of stimulus hyperplasia disappears; cells respond to regular growth control (Diff from neoplasia) Pathologic hyperplasia is a fertile soil in which cancerous proliferation may sometime arise (Ex. Endometrial & cervical hyperplasia)

HYPERTROPHY
Can be PHYSIOLOGIC or PATHOLOGIC Mostly seen in cells that cannot divide (skeletal & cardiac muscles) It is caused by 1. Increased functional/mechanical demand Physiologic Hypertrophy of striated muscles in muscle builders Pathologic Hypertrophy in cardiac muscle in HTN 2. Specific hormonal stimulation Physiologic Uterine hypertrophy or breast enlargement during pregnancy Hyperplasia is often accompanied by hypertrophy Hyperplasia can occur only with cells capable of synthesizing DNA (such as epithelial, hematopoetic, connective tissue cells) Nerve, cardiac & skeletal muscle cells have little or no capacity for hyperplastic growth, and so muscle cells undergo almost pure hypertrophy

Heart hypertrophy in hypertension:

Left Ventricle

Tissue

Epithelial

Connective

Nerve

Muscle

Connective Tissue Proper

Cartilage

Bone

Blood Skeletal Cardiac Smooth

Loose Connective Tissues

Dense Connective Tissues

Regenerating Capability Good Moderate

Areolar

Adipose

Reticular

Dense regular

Dense Irregular

Elastic

Poor None

ATROPHY
Causes of atrophy are: - Decreased work load (Disuse atrophy Prolonged plastering) - Loss of inervation (Denervation atrophy) - Diminished blood supply - Inadequate nutrition (Malnutrition) - Loss of endocrine stimulation - Ageing (Senile atrophy) - Pressure Mechanisms: Endocytic mechanisms Autophagy (reduction in number of cell organelles). Those components that resists digestion persists as membrane bound residual bodies LIPOFUSCHIN granules (brown in color) (BROWN ATROPHY) Ubiquitin-Proteasome pathway leads to excessive proteolysis (proteins to be degraded are conjugated to ubiquitin and then degraded within a large proteolytic organelle called proteosome (Ex cancer cachexia) Thyroxine & steroids promotes this process while insulin opposes it

Cerebral atrophy - Alzheimers:

METAPLASIA
- Columnar to squamous (Most common metaplasia) Ex. Lungs of a smoker (mucus secretion is lost but) Stones in salivary, pancreatic, bile ducts Vitamin A Deficiency Squamous epithelium is able to withstand (survive) the adverse environment - Squamous to columnar. Ex Barrets esophagitis Mechanism: Genetic reprogramming of the epithelial stem cells (reserve cells) secondary to the influence of the growth factors and cytokines When the underlying stimulus abates, the metaplastic changes REVERSE

DYSPLASIA
Cells of varying sizes and shapes, lying topsyturvy (i.e., the cells have forgotten how to be good neighbors) Large, dark-staining nuclei with irregular surfaces (i.e., there's too many chromosomes, and the nucleus doesn't know how to pack them all) Increased nuclear-cytoplasmic ratio ( N/C ratio; i.e., there's a surplus of chromosomes) Basement membrane intact Potentially reversible Often a precurssor for cancer

INTRACELLULAR ACCUMULATIONS
It can occur when: 1. A normal endogenous substance is produced at a normal or increased rate, but the rate of metabolism is less (e.g., the accumulation of fat in liver cells). 2. A normal or abnormal endogenous substance accumulates due to defects in the packaging, transport, or secretion of these substances. Examples: a. Genetic defects leads to synthesis of abnormal proteins that fold improperly & accumulate, such as in: 1-antitrypsin disease (1-antitrypsin accumulates in the endoplasmic reticulum of liver cells that produce it) Sickle cell disease (hemoglobin S aggregates into polymers resulting in distortion of the RBC) b. Lysosomal storage diseases (lack of enzymes leads to accumulation of various types of complex lipids and carbohydrates) c. Cytoskeletal abnormalities (accumulation of pre-keratin intermediate filaments as they resist degradation Mallory bodies [alcoholic hyalin] in alcoholic liver disease) 3. Deposition of abnormal exogenous substances (e.g., macrophages laden with carbon dust from the air)

LIPIDS
1. STEATOSIS (FATTY CHANGE)
Triglycerides accumulate intracellularly formation of intracellular fat vacuoles It occurs occasionally in almost all organs but is most common in the LIVER Fatty change in the liver is reversible, but when excessive CIRRHOSIS
Pathogenesis of Fatty liver Causes of fatty liver include Alcohol abuse, Protein malnutrition, Diabetes mellitus, Obesity, Hepatotoxins, and Drugs Macroscopically Fatty livers are enlarged, yellow, and greasy Microscopically Fat is seen as small, cytoplasmic droplets or as large vacuoles The condition is caused by one of the following mechanisms: - Excessive entry of free fatty acids into the liver (e.g., starvation, corticosteroid therapy) - Enhanced fatty acid synthesis - Decreased fatty acid oxidation - Increased esterification of fatty acids to triglycerides as a result of an increase in -glycerophosphate (alcohol) - Decreased apoprotein synthesis (CCL4 poisoning, Protein malnutrition) - Impaired lipoprotein secretion from the liver (alcohol, orotic acid administration)

2. CHOLESTEROL AND CHOLESTEROL ESTERS Cholesterol is used for the synthesis of cell membranes. Accumulations intracellular vacuoles Seen in several pathologic processes, as follows: 1. In atherosclerosis, these lipids accumulate in smooth muscle cells and macrophages in the walls of arteries. Intracellular cholesterol accumulates in the form of small cytoplasmic vacuoles. Extracellular cholesterol gives characteristic rhomboid cavities formed by the dissolved cholesterol crystals 2. In acquired and hereditary hyperlipidemia, lipid accumulates in macrophages and mesenchymal cells, forming xanthomas 3. In inflammation and necrosis, lipid laden macrophages result from phagocytosis of membrane lipids derived from injured cells (foamy macrophages)

PROTEINS
Excess proteins within cells appear as round EOSINOPHILIC droplets in the cytoplasm They occur because of excessive synthesis, absorption, or defects in cellular transport. 1. Plasma cells - Filled with immunoglobulin within distended cisternae of the endoplasmic reticulum the protein creates - Russell bodies 2. Prolonged proteinuria reabsorption of protein forms droplets in PCT 3. Defects in protein folding After protein synthesis, partially folded intermediate arise, which can form intracellular aggregates among themselves or by entangling other proteins. Under normal conditions, these intermediates, are stabilized by CHAPERONES Transported across the ER, Golgi complex, & out of the cell A. Defective intracellular transport and secretion of critical proteins Examples include 1 antitrypsin deficiency - Mutations slow protein folding accumulation of partially folded intermediates in the ER of hepatocytes. Cystic fibrosis - Mutation delays dissociation of the protein from one of its chaperones, resulting in abnormal folding and loss of function. B. Toxicity of aggregated, abnormally folded proteins Aggregation of abnormally folded proteins, caused by genetic mutations, aging, is a recognized feature of neurodegenerative disorders, including the Alzheimer, Huntington and Parkinson diseases

GLYCOGEN
Glycogen is a readily available energy store that is present in the cytoplasm. Excessive intracellular, deposits are seen in abnormalities of glycogen and glucose metabolism (Ex Storage diseases)

PIGMENTS
Exogenous pigments: Anthracosis (accumulation of carbon in the macrophages of the lungs & lymph nodes from air pollution). Tattooing (injected pigment is taken up by macrophages and persists forever in the cells and extracellularly)

Endogenous pigments: Lipofuscin (the wear-and-tear pigment, seen microscopically as yellowbrown, fine, intracytoplasmic granules, usually associated with atrophy (brown atrophy). Melanin (an endogenous, brownblack pigment formed when the enzyme tyrosinase catalyzes the oxidation of tyrosine to dihydroxyphenylalanine in melanocytes). Hemosiderin (a hemoglobin-derived, golden yellow-to-brown pigment composed of aggregates of ferritin micelles). Intracellular accumulation occurs as a localized process or a systemic derangement. - Local hemosiderosis results from gross hemorrhage or rupture of small vessels. Macrophages take up hemoglobin, and lysosomal enzymes convert it to hemosiderin. - Systemic hemosiderosis occurs with Increased absorption of dietary iron (primary hemosiderosis) Impaired utilization of iron (e.g., thalessemia) Hemolytic anemias, causing excessive breakdown of red cells Transfusions, increasing the exogenous load of iron

PATHOLOGIC CALCIFICATION
Implies the abnormal deposition of calcium salts in soft tissues. DYSTROPHIC CALCIFICATION Occurs in arteries in atherosclerosis, in damaged heart valves, and in areas of necrosis (coagulative, casseous, and liquefactive). Calcium can be intracellular, extracellular or in both the locations.
METASTATIC CALCIFICATION Results from hypercalcemia, which has four principal causes: 1. Increased secretion of parathyroid hormone, as occurs in hyperparathyroidism resulting from parathyroid tumors and, in ectopic secretion of parathyroid hormone by malignant tumors (e.g., certain forms of lung carcinoma). 2. Destruction of bone tissue, as occurs with primary tumors of bone marrow components (e.g, multiple myeloma) or by diffuse skeletal metastasis (e.g., breast cancer) 3. Vitamin D-related causes, including vitamin D intoxication and systemic sarcoidosis (release of a Vitamin D precursor) 4. Associated with renal failure, which causes secondary hyperparathyroidism Calcium deposits are seen as amorphous BASOPHILLIC densities