Que 1: Differentiate Reversible and Irreversible Cell Injury with Ultrastructural Changes

Reversible Cell Injury

• Cellular swelling (earliest manifestation) - caused by failure of the ATP-dependent Na+/K+ pump, leading to influx of water
• Hydropic change / vacuolar degeneration - small clear vacuoles representing distended segments of ER
• Cytoplasm appears more eosinophilic (loss of RNA which normally binds hematoxylin)
• Fatty change - in organs like the liver involved in lipid metabolism

1. Plasma membrane alterations - blebbing, blunting, and loss of microvilli
2. Mitochondrial changes - swelling and appearance of small amorphous densities
3. Accumulation of "myelin figures" in the cytosol (phospholipids from damaged membranes)
4. Dilation of the ER with detachment of polysomes (ribosomes)
5. Nuclear alterations - disaggregation of granular and fibrillar elements

Irreversible Cell Injury (Necrosis)

• Nuclear changes: Pyknosis (nuclear shrinkage and dark staining), Karyorrhexis (nuclear fragmentation), Karyolysis (nuclear dissolution by DNases)
• Cytoplasm becomes deeply eosinophilic
• Loss of cellular outlines in advanced necrosis

1. Membrane disruption - severe plasma membrane damage with leakage of cellular contents
2. Markedly swollen mitochondria containing electron-dense deposits (precipitated calcium and proteins) - this is the hallmark of irreversibility
3. Myelin figures - derived from damaged membranes
4. Flocculent amorphous densities in mitochondria
5. Nuclear fragmentation and dissolution
6. Lysosomal rupture with release of digestive enzymes into cytoplasm

Source: Robbins, Cotran & Kumar - Pathologic Basis of Disease, Fig. 2.9

Que 2: Mechanism of Ischemic Reperfusion Injury

1. Oxidative Stress (Reactive Oxygen Species - ROS)

• When blood flow is restored, the sudden burst of oxygen in previously ischemic tissue leads to massive generation of ROS (superoxide, hydrogen peroxide, hydroxyl radicals)
• Ischemic cells have reduced antioxidant defenses (depleted glutathione, SOD)
• ROS attack lipid membranes (lipid peroxidation), proteins, and DNA, causing further membrane damage
• Activated neutrophils (recruited during reperfusion) generate additional ROS via respiratory burst

2. Calcium Overload

• Ischemia depletes ATP, causing failure of the Na+/Ca2+ exchanger and Ca2+ ATPase
• During reperfusion, Ca2+ floods into cells
• Excess intracellular Ca2+ activates:
  • Phospholipases (membrane damage)
  • Proteases (cytoskeletal disruption)
  • ATPases (further ATP depletion)
  • Endonucleases (DNA damage)
• Ca2+ also accumulates in mitochondria, inducing the mitochondrial permeability transition (MPT)

3. Mitochondrial Permeability Transition (MPT)

• During reperfusion, the sudden influx of Ca2+ and ROS triggers opening of the MPT pore in the inner mitochondrial membrane
• This allows passage of molecules <1.5 kDa into the mitochondrial matrix
• Mitochondrial membrane potential collapses, ATP synthesis stops
• Cytochrome c is released - triggering apoptosis
• Mitochondria swell and rupture - causing necrosis

4. Neutrophil Activation and Inflammation

• Ischemic tissue produces cytokines (IL-1, TNF) and complement fragments that attract neutrophils during reperfusion
• Neutrophils adhere to activated endothelium (which upregulates selectins and ICAM-1 during ischemia)
• Activated neutrophils release:
  • ROS
  • Proteases (elastase, collagenase)
  • Myeloperoxidase
• This amplifies cell injury far beyond the original ischemic zone

5. Complement Activation

• Ischemic tissue activates complement via the alternative and lectin pathways
• Complement fragments (C3a, C5a) attract more neutrophils and cause direct membrane damage via the membrane attack complex (MAC)

Summary Diagram

Que 3: Compare Apoptosis and Necrosis with Examples

Definition

• Necrosis: Pathologic cell death due to severe injury, characterized by enzymatic digestion and protein denaturation of the cell
• Apoptosis: Regulated (programmed) cell death mediated by specific molecular pathways, executed with "surgical precision"

Comparison Table

Mechanisms of Apoptosis

• Triggered by DNA damage, oxidative stress, growth factor withdrawal
• Pro-apoptotic proteins (BAX, BAK) are activated and insert into mitochondrial membrane
• Anti-apoptotic proteins BCL-2 and BCL-XL are inhibited
• Cytochrome c is released from mitochondria
• Cytochrome c + APAF-1 → Apoptosome → Activates Caspase-9 → Caspase-3 → Cell death

• FasL binds to Fas (CD95) receptor on target cell
• FADD adaptor protein recruited to Fas
• Pro-caspase-8 binds FADD → Auto-cleaved → Active Caspase-8 → Caspase-3 cascade

Examples

Que 4: Classify Amyloidosis and Pathogenesis of AL and AA

What is Amyloidosis?

Classification (Table 6.17 - Robbins Pathology)

Systemic (Generalized) Amyloidosis

Localized Amyloidosis

• Deposits limited to a single organ
• Sites: lung, larynx, skin, bladder, tongue, eye
• Also: Endocrine amyloid in medullary thyroid carcinoma, type 2 DM islets (IAPP)

Other Forms

• Hemodialysis-associated amyloidosis - β2-microglobulin deposits in joints/carpal tunnel
• Familial Mediterranean Fever (FMF) - AA type; autosomal recessive, pyrin mutation → excess IL-1 → excess SAA production

Pathogenesis of AL Amyloidosis

1. A clonal plasma cell proliferation (in multiple myeloma or monoclonal gammopathy) synthesizes abnormal amounts of a single immunoglobulin (Ig)
2. Malignant plasma cells secrete free, unpaired κ or λ light chains (Bence-Jones proteins) into serum and urine
3. These light chains have specific amino acid sequences that confer amyloidogenic potential - they are intrinsically prone to misfolding
4. Misfolded light chains aggregate into beta-pleated sheet fibrils that are resistant to degradation
5. Fibrils deposit extracellularly in tissues (kidney, heart, liver, nerves) causing organ dysfunction
6. Most patients with AL amyloid have underlying monoclonal gammopathy even without overt myeloma

Pathogenesis of AA Amyloidosis

1. Triggered by chronic inflammatory conditions (rheumatoid arthritis, ankylosing spondylitis, IBD, chronic infections like TB, osteomyelitis)
2. Chronic inflammation causes prolonged production of cytokines IL-6 and IL-1 by macrophages
3. These cytokines stimulate the liver to synthesize large amounts of SAA (Serum Amyloid A), an acute phase protein
4. Normally, SAA is degraded by macrophage-derived enzymes to soluble end products
5. In amyloidosis patients, incomplete degradation of SAA occurs - likely due to:
   • Enzyme defects in macrophages
   • Structurally abnormal SAA molecules resistant to degradation
6. Insoluble AA protein fragments accumulate and deposit systemically (particularly in kidney, spleen, liver, adrenal glands)
7. Most common associated condition: Rheumatoid arthritis (amyloidosis in ~3% of RA patients)

Que 5: Mechanism of Intracellular Accumulation

Four Mechanisms of Intracellular Accumulation

1. Inadequate Removal of Normal Substance

• The cell produces a normal substance at normal rate but the metabolic pathway is defective and the substance cannot be removed
• Examples:
  • Fatty liver (hepatic steatosis): Normal triglycerides cannot be exported as VLDL (due to protein deficiency, toxins like alcohol, malnutrition). Lipid vacuoles accumulate in hepatocytes
  • Cholesterol accumulation in foam cells: In atherosclerosis, macrophages engulf oxidized LDL but cannot export cholesterol efficiently

2. Normal or Abnormal Endogenous Substance - Defective Metabolism (Enzyme Defect)

• A genetic defect in an enzyme causes failure to metabolize a normal substrate
• Substrate accumulates within cells (lysosomes)
• Examples:
  • Lysosomal storage diseases (e.g., Gaucher disease - failure to degrade glucocerebroside; Niemann-Pick disease - sphingomyelin accumulation; Tay-Sachs - GM2 ganglioside accumulation)
  • α1-antitrypsin deficiency - abnormal protein accumulates in hepatocyte ER (not in lysosomes)

3. Abnormal Endogenous Substance - Cannot Be Degraded

• Mutant proteins that are abnormally folded cannot be degraded by proteasomes and accumulate intracellularly
• Examples:
  • Mallory bodies (alcoholic hyalin) - cytokeratin intermediate filaments in hepatocytes of alcoholics
  • Neurofibrillary tangles in Alzheimer disease (hyperphosphorylated tau protein)
  • Lewy bodies in Parkinson disease (α-synuclein accumulation)

4. Exogenous Substance Deposited and Cannot Be Degraded

• A substance is ingested but the cell lacks machinery to degrade it
• Examples:
  • Carbon particles in macrophages (coal workers' pneumoconiosis / anthracosis) - carbon ingested by alveolar macrophages, accumulates as blackened deposits in lung
  • Silica particles in silicosis
  • Hemosiderin - after repeated red cell breakdown, iron-laden macrophages in tissues

Summary Table

All answers sourced from: Robbins, Cotran & Kumar - Pathologic Basis of Disease (Robbins Pathology)