Cell injury and cell death| reversible-irreversible| cellular swelling| Reactive oxygen species| free radical| necrosis| type of necrosis|

CELL INJURY & CELL DEATH 

An Insightful Introduction to Pathology: -

Pathology serves as a crucial gateway to understanding the intricate mechanisms of disease. It is the study of the nature and causes of illness, bridging the gap between clinical practice and laboratory science. This field delves deep into the cellular and tissue-level changes that occur in the body due to various pathological processes, unveiling the mysteries behind symptoms and diagnoses. Through histological examinations, biomolecular analyses, and a range of cutting-edge techniques, pathology illuminates how diseases disrupt the harmony of normal function, fostering insights that are vital for effective treatment and patient care. Pathology (definition): Pathology is the study of the structural, biochemical & functional changes in cells, tissues, and organs that underlie disease. 

Divisions of pathology: 

Traditionally, the study of pathology is divided into two parts- 

1. General pathology: General pathology is concerned with the common reactions of cells and tissues to injurious stimuli. 
2. Systemic pathology: Systemic pathology examines the alterations & underlying mechanisms in diseases of particular organ systems. 

Functions of pathology: 

1. By the use of morphologic, microbiologic, immunologic, and molecular techniques, pathology attempts to explain the whys and wherefores of the signs and symptoms manifested by patients while providing a rational basis for clinical care and therapy.
2. It thus serves as the bridge between the basic sciences and clinical medicine and is the scientific foundation for all of medicine. 

Aspects of pathology: 

The four aspects of a disease process that form the core of pathology, they are as follows- 

1. The causation (etiology).
2. Biochemical & molecular mechanisms (pathogenesis). 
3. The associated structural (morphologic changes) & functional alterations in cells & organs. 
4. The resulting clinical consequences (clinical manifestations).

Father of modern pathology: 

Virtually all diseases start with molecular or structural alterations in cells. This concept of the cellular basis of disease was first put forth in the nineteenth century by Rudolf Virchow, known as the father of modern pathology. Virchow emphasized the idea that individuals are sick because their cells are sick. 

Cell injury 

Define cell injury: -

Cell injury: If the limits of adaptive response to a stimulus are exceeded or if cells are exposed to damaging insults, deprived of essential nutrients, or compromised by mutations that affect essential cellular functions, a sequence of events follows that is termed as cell injury.

Causes of cell injury: -

1. Oxygen deprivation: 

• Hypoxia: Hypoxia is a deficiency of O2 to the tissue. It occurs due to cardio-respiratory failure, anemia, CO poisoning etc. 
• Ischemia: Ischemia means loss of blood supply from impeded arterial flow or reduced venous drainage in a tissue. 

     2. Physical agents: 

• Mechanical trauma. 
• Extremes of temperature (burn and deep cold). 
• Sudden changes in atmospheric pressure.
• Radiation. 
• Electric shock. 

3. Chemical agents and drugs: 

• Glucose or salt in hypertonic concentrations.
• Oxygen at high concentrations is toxic.
• Poisons, such as arsenic, cyanide, or mercury. 
• Environmental pollutants, insecticides, and herbicides. 
• Industrial and occupational hazards, such as carbon monoxide and asbestos. 
• Recreational drugs such as alcohol. 
• Variety of therapeutic drugs. 

4) Infectious agents: Virus, bacteria, fungi, protozoa and high forms of parasites. 

5) Immunological reactions: Anaphylactic reaction to foreign body and autoimmune diseases.

6) Genetic abnormalities: Genetic injury may result in congenital malformations e.g. Down syndrome, sickle cell anemia, enzymatic abnormalities etc. 

7) Nutritional imbalance: such as protein-calorie deficiency, vitamin deficiency, nutritional excess (atherosclerosis, obesity) etc.

Types of cell injury: 

🕮According to severity: 

1. Reversible / mild cell injury. 
2. Irreversible / severe cell injury. 

🕮Morphological types: 

1. Reversible cell injury: 

• Cellular swelling. 
• Fatty change. 

      2. Irreversible cell injury: 

• Necrosis. 
• Apoptosis. 

Reversible cell injury: 

Reversible cell injury is characterized by functional and structural alterations in early stages or mild forms of injury, which are correctable (reversible) if the damaging stimulus is removed. 

Features of reversible cell injury: 

🕮Gross changes / features: Two features are consistently seen in reversibly injured cells. 

1) Early alterations in reversible injury include: 

• Generalized swelling of the cell and its organelles, 
• Blebbing of the plasma membrane, 
• Detachment of ribosomes from the endoplasmic reticulum (ER), and 
• Clumping of nuclear chromatin. 

2) Fatty change occurs in organs that are actively involved in lipid metabolism (e.g. liver). 

🕮The ultra-structural changes: (Visible by electron microscopy) 

1. Plasma membrane alterations, such as blebbing, blunting, and loss of microvilli. 
2. Mitochondrial changes, including swelling and the appearance of small amorphous densities.
3. Accumulation of 'myelin figures' in the cytosol composed of phospholipids derived from damaged cellular membranes.
4. Dilation of the ER, with detachment of polysomes.
5. Nuclear alterations, with disaggregation of granular and fibrillar elements. 

Irreversible cell injury: 

If the injurious stimulus persists or is severe enough from the beginning, the cells or tissues are damaged to such extent that it becomes unable to recover when injurious stimulus is withdrawn, is called irreversible cell injury. 

🕮Pathogenesis / features / criteria of irreversible cell injury:

Irreversible cell injury occurs due to irreversible damage of cell membrane, mitochondria & nucleus. It is characterized by - 

• Increasing swelling of the cell. 
• Swelling and disruption of lysosomes.
• Vacuolization of mitochondria with reduced capacity to generate ATP. 
• Disruption of cellular membranes.
• Profound nuclear changes. 

🕮Hallmarks of reversible cell injury: 

1. Reduced oxidative phosphorylation.
2. ATP depletion.
3. Cellular swelling that causes changes in ion concentration & water influx. 

🕮Hallmarks of irreversible cell injury: 

Irreversible damage of cell membrane, mitochondria & nucleus. 

🕮 Reversible cell injuries become irreversible

Transformation of reversible cell injury to irreversible cell injury; It is useful to consider the possible events that determine when reversible injury becomes irreversible and progresses to cell death. Two phenomena consistently characterize irreversibility - 

1. The inability to reverse mitochondrial dysfunction (lack of oxidative phosphorylation and ATP generation) even after resolution of the original injury.
2. Profound disturbances in membrane function (of cell and cellular organelles of those membrane- bound organelles like mitochondria and endoplasmic reticulum). 

🕮Morphological pattern of irreversible cell injury/cell death: 

Irreversibly injured cells invariably undergo morphologic changes that are recognized as cell death. There are two morphological patterns of cell death - 

• Necrosis. 
• apoptosis. 

Cellular swelling:

Cellular swelling is the earliest manifestation of almost all forms of injury to cells 

🕮Causes of cellular swelling: 

1. Hypoxia.
2. Radiation.
3. Toxins. 

🕮Pathogenesis of cellular swelling: 

Hypoxia, mitochondrial damage by radiation or toxins, 

 ↓ 

Depletion of ATP.

↓ 

Failure of ATP-dependent Na*-K pump in the plasma membrane. 

 ↓ 

Influx of water into the cell. 

 ↓ 

Cellular swelling. 

Morphology of cellular swelling: 

• It causes pallor, increased turgor, and increased weight of the affected organ. 
• On microscopic examination, small clear vacuoles may be seen within the cytoplasm; these represent distended and pinched-off segments of the ER. 
• This pattern of nonlethal injury is sometimes called hydropic change or vacuolar degeneration. 

Cellular responses to stresses & noxious stimuli: 

Cellular response to injury varies in its effects on cell structure and function according to the type of involved and the nature and severity of the agent responsible. They include - 

1) Cellular adaptations: 

• Atrophy. 
• Hypertrophy.
• Hyperplasia.
• Metaplasia. 

2) Cell injury: 

a) Acute reversible injury. 
b) Irreversible injury / cell death: 

✓ Necrosis. 

✓ Apoptosis. 

3) Intracellular accumulations. 

4) Pathological calcifications. 

5) Cellular aging. 

Figure: Stages of the cellular response to stress and injurious stimuli. 

Subcellular responses to cell injury 

 These conditions are associated with distinctive alterations in cell organelles or the cytoskeleton. The responses are - 

1) Lysosomal catabolism: Lysosomes are involved in the breakdown of phagocytosed materials in 

one of the following two ways - 

• ➤ Heterophagy: It is the process of lysosomal digestion of materials ingested from the extracellular environment, e.g. uptake and digestion of bacteria by neutrophils and removal of apoptotic cells by macrophages. 
• ➤ Autophagy: It refers to lysosomal digestion of the cell's own components, and involved in the removal of damaged organelles during cell injury. 

2) Induction (hypertrophy) of smooth endoplasmic reticulum. 

3) Mitochondrial alterations: 

• ➤ In cell hypertrophy: There is increase in number of mitochondria. 
• ➤ In cell atrophy: There is decrease in number of mitochondria. 
• ➤ Extremely large and abnormal shape mitochondria (mega-mitochondria) are seen in liver in alcoholic liver disease and in certain nutritional deficiencies. 

4) Cytoskeletal abnormalities: 

• ➤ Defects in the function such as cell locomotion and movements of intracellular organelles. 
• ➤ In some instances, there is intracellular accumulation of fibrillar material.  

Biochemical mechanisms of cell injury 

Mechanism/biochemical mechanism / intracellular targets of cell injury: 

1.  ATP depletion. 
2. Mitochondrial damage. 
3. Membrane damage & defects in membrane permeability.
4. Damage to DNA.
5. Oxidative stress: Accumulation of oxygen derived free radicals.
6. Disturbances in calcium homeostasis: Influx of intracellular calcium & loss of calcium homeostasis.
7. Endoplasmic reticulum stress: The unfolded protein response. 

Description of each mechanism of cell injury: 

1) Depletion of ATP: 

Causes of ATP depletion: 

✓ Ischemia & hypoxia: Reduced supply of oxygen (hypoxia) & nutrients.

✓ Mitochondrial damage: By radiation and toxins. 

Effects / consequences of ATP depletion: 

a) Failure of ATP dependent Na+-K+ pump & Ca2+ pump: 

• Na+-K+ pump failure → influx of Na* (and accumulate inside the cell) and K to diffuse out
• Failure of the Ca2+ pump → influx of calcium

↓  

Net gain of solute is accompanied by isosmotic gain of water 

↓ 

✓ Cellular swelling. 

✓ Dilatation of endoplasmic reticulum. 

✓ Loss of microvilli. 

✓ Cellular blebs. 

b) Cellular energy metabolism is altered: Oxidative phosphorylation, ATP & ↑AMP (adenosine monophosphate) → ↑Glycolysis & Glycogenolysis. 

c) Rapid depletion of glycogen store & increased anaerobic glycolysis: Lactic acid & 

inorganic phosphates →↓pH→↓activity of many cellular enzymes. 

d) Detachment of ribosome from ER: ↓Protein synthesis. 

e) Misfolding of proteins & their accumulation in the ER: Cell injury & cell death. 

f) Ultimately, there is irreversible damage to mitochondrial & lysosomal membranes, and 

the cell undergoes necrosis. 

2) Mitochondrial damage: 

Mitochondria have a critical role in all pathways leading to cell injury & cell death. 

Causes of mitochondrial damage: 

✓ ↑ Cytosolic calcium. 

✓ Reactive oxygen species (ROS). 

✓ Oxygen deprivation (make the mitochondria sensitive to virtually all types of injurious stimuli). 

✓ Mutation of mitochondrial gene (in some inherited diseases). 

Effects of mitochondrial damage: 

Major consequences of mitochondrial damage are- 

a) ATP depletion: (discussed earlier). 

b) Formation of a 'high-conductance channel (mitochondrial permeability transition pore)' in the mitochondrial membrane 

↓ 

Loss of mitochondrial membrane potential.

↓

• Failure of oxidative phosphorylation. 
• Progressive depletion of ATP. 
• Cell necrosis. 

c) Formation of reactive oxygen species & their effects (please see below). 

d) Leakage of apoptotic proteins, e.g. cytochrome c & caspases (situated in between outer & inner mitochondrial membrane) into the cytosol → apoptosis. 

3) Membrane damage & defects in membrane permeability: 

Causes/mechanism of membrane damage: 

Early loss of selective membrane permeability, leading ultimately to membrane damage. Causes are- 

✓Free radical (causes injury to the cell membrane).

✓Decreased phospholipid synthesis. 

✓Increased phospholipid breakdown. 

✓Cytoskeletal abnormalities. 

Consequences of membrane damage: 

Membrane damage is a consistent feature of most form of cell injury except apoptosis. 

a) Mitochondrial membrane damage: Discussed earlier. 

• ➤Failure of oxidative phosphorylation.
• ➤Progressive depletion of ATP.
• ➤Cell necrosis.
• ➤Apoptosis. 
• ➤ Formation of reactive oxygen species (ROS). 

b) Plasma membrane damage: 

• ➤Loss of osmotic balance → influx of fluid & ions. 
• ➤Loss of cellular contents.
• ➤"Leakage of metabolites, those are vital for the reconstitution of ATP → further depletion of energy stores. 

c) Injury to lysosomal membranes: Loss of lysosomal membrane

 ↓

Leakage of lysosomal enzymes into the cytoplasm; e.g. RNases, DNases, proteases, phosphatases & glucosidases

↓  

Enzymatic digestion of RNA, DNA, protein, phosphate & glycogen respectively

↓ 

Cells death & necrosis 

4) Damage to DNA: Damage to nuclear DNA activates sensors that trigger p53-dependent pathways. Cells have physiological mechanisms that repair damage to DNA, but, DNA damage persists. 

Causes of DNA damage: 

✓Exposure to radiation. 

✓Chemotherapeutic (anti-cancer) drugs. 

✓Oxidative stress (produces ROS). 

✓ Aging (may occur spontaneously as a part of aging). 

Effects of damage to DNA: 

a) Aging. 

b) Cell cycle arrest. 

c) If physiological repair mechanism fails → apoptosis (by activation of caspases). 

d) If DNA damage is too severe to be corrected or mutations in p53 → malignant transformation. 

5) Oxidative stress – accumulation of oxygen-derived free radicals: 

Causes of accumulation of O2-derived free radicals: 

✓The reduction-oxidation reactions that occur during normal metabolic processes. 

✓Absorption of radiant energy (e.g. UV light, X-rays etc.). 

✓Rapid bursts of reactive oxygen species (ROS). 

✓Enzymatic metabolism of exogenous chemicals or drugs (CCl, generates CCl3"). 

✓Transition metals (e.g. Fe & Cu) → donate/accept free electrons during intracellular reactions → catalyze free radical formation. 

✓Nitric oxide. 

Effects of accumulation of O2-derived free radicals: 

a) Lipid peroxidation in membranes. 

b) Oxidative modifications of proteins. 

c) Lesions in DNA (DNA damage) 

6) Disturbance in calcium homeostasis: 

Calcium ions normally serve as 2 2nd messengers in several signaling pathways. If released into the cytoplasm of cells in excessive amounts, are also important sources of cell injury. Most intracellular Ca2+ is sequestrated in mitochondria & ER. 

Causes of increased intracellular Ca2+ level: Initially Ca2+ releases from intracellular stores & later influxes across the cell membrane. 

✓Ischemia. 

✓Certain toxins. 

Effects of increased intracellular / cytosolic Ca2+ level: 

a) Opening of the 'mitochondrial permeability transition pore' → failure of ATP generation (discussed earlier). 

 b) Activation of potentially harmful enzymes: Increased cytosolic Ca** activates a number of enzymes, with potentially deleterious cellular effects, e.g. 

• ATPase→ Leads to ATP depletion.
• Phospholipase→ Membrane damage. 
• Protease → Breakdown of membrane and cytoskeletal proteins.
• Endonucleases → Fragmentation of DNA and chromatin. 

c) Direct activation of caspases & Mitochondrial permeability → apoptosis. 

7) Endoplasmic reticulum (ER) stress: 

The unfolded protein response Causes of intracellular accumulation of misfolded proteins: (Increase production or decreased ability to repair & elimination) 

✓Ischemia. 

✓Hypoxia. 

✓Radiation. 

✓Mutation. 

✓Viral infection. 

✓Insulin-resistant states. 

Effects of damage to DNA & proteins: 

a) Trigger apoptosis by activating pro-apoptotic proteins. 

b) Cellular aging. 

c) Neurodegenerative disease: 

• ➤ Alzheimer disease. 
• ➤ Familial hypercholesterolemia. 
• ➤ Cystic fibrosis. 
• ➤ⲁ-antitrypsin deficiency. 
• ➤Tay-Sachs disease. 
• ➤ Creutzfeldt-Jacob disease. 

ATP is produced in two ways: 

1) Oxidative phosphorylation: It is the major pathway in mammalian cells in non-dividing (e.g. brain and liver) cells by the mitochondrial electron transport system. 

2) Glycolysis. 

Roles of ATP in the body: 

✓High-energy phosphate in the form of ATP is required for virtually all synthetic and degradative processes within the cell. These include membrane transport, protein synthesis, lipogenesis, and the deacylation-reacylation reactions necessary for phospholipid turnover. 

✓Depletion of ATP to 5% to 10% of normal levels has widespread effects on many critical cellular systems.  

Oxidative stress: 

Increased production or decreased scavenging of reactive oxygen species (ROS) may lead to accumulation of an excess of free radicals, a condition called oxidative stress. 

Reactive oxygen species (ROS) / free radicals: 

❖ Free radicals are chemical species that have a single unpaired electron in an outer orbit.

❖ Unpaired electrons are highly reactive and 'attack' and modify adjacent molecules, such as inorganic or organic chemicals (carbohydrates, proteins, lipids, nucleic acids etc.), many of which are key components of cell membranes & nuclei. 

❖ Some of above reactions are autocatalytic, whereby molecules that react with free radicals are themselves converted into free radicals, thus propagating the chain of damage. 

Types of free radicals: 

1. Oxygen derived free radicals: Superoxide (O2), H2O2, OH 
2. Carbon derived free radicals: CCl2 Hoc 
3. Nitrogen derived free radicals: NO, NO2, NO3".
4. Transitional metal: Feat. 

Characteristics of free radical: 

1. Has unpaired electron (s) in the outer orbit.
2. Highly reactive chemical species.
3. Half-life is very short.
4. Interact with other substance to lose or gain electron.
5. Cause damage to the living cells, tissues and responsible for early aging process & different diseases (neurodegenerative diseases, atherosclerosis, IHD etc.). 

Sources/mechanism of formation of free radicals: 

1. The reduction-oxidation reactions that occur during normal metabolic processes: 

✓Small amount of free radicals produce spontaneously during mitochondrial respiration & energy generation by oxidative enzymes in the ER, cytosol, mitochondria, peroxisomes and lysosomes. 

✓Free radicals that produces normally: They are- 

• Superoxide anion (O2, one electron).
• Hydrogen peroxide (H2O2, two electrons), &
• Hydroxyl ions ("OH, three electrons). 

2. Absorption of radiant energy (e.g. UV light & X-rays): Ionizing radiation can hydrolyze water into 'OH & hydrogen (H) free radicals. 

3. Rapid bursts of reactive oxygen species (ROS): Produced in activated leukocytes during inflammation. 

4) Enzymatic metabolism of exogenous chemicals or drugs (e.g. CCL generates CC13'). 

5) Transition metals (e.g. Fe & Cu) → donate/accept free electrons during intracellular reactions → catalyze free radical formation. 

6) Nitric oxide (NO): It is an important chemical mediator generated by endothelial cells, macrophages, neurons & other cell types. It can act as a free radical and can also be converted to highly reactive peroxynitrite anion (ONOO) as well as NO, & NO. 

Cell injury caused by free radical / reactive oxygen species (ROS) / oxidative stress: 

Cell injury induced by free radicals (particularly by ROS), is an important mechanism of cell damage in many pathologic conditions, such as- 

1. Chemical and radiation injury.
2. Ischemia-reperfusion injury (induced by restoration of blood flow in ischemic tissue).
3. Cellular aging.
4. Cancer.
5. Microbial killing by phagocytes.
6. Some degenerative diseases (e.g. Alzheimer disease). 

Injuries caused by free radicals: 

1. Lipid peroxidation of membranes: 

O2-derived free radicals (particularly by 'OH) 

↓ 

Attack double bonds in unsaturated fatty acids of membrane lipids

↓ 

Initiation of oxidative damage 

↓ 

Peroxidation of lipids occurs within plasma and organellar membranes 

↓ 

The 'lipid-free radical interactions' yield peroxides, which are themselves unstable and reactive 

↓ 

An autocatalytic chain reaction ensues (called propagation) 

↓ 

Further extensive membrane damage occurs (viscous cycle continues) 

2) Oxidative modification of protein: 

Free radicals causes: 

• Oxidation of amino acid side chains. 
• Formation of covalent protein-protein cross-lins (e.g. disulfide bond), and 
• Oxidation of protein backbone resulting in protein fragmentation. 

The effects of oxidative modifications are- 

• Damage of active site of proteins. 
• Disruption of structural proteins. 
• Enhance proteasomal degradation of unfolded or misfolded proteins. 

3) Lesions in DNA: Free radicals are capable of causing- 

• Single- & double-strand breaks in DNA. 
• Cross linking of DNA strands. 
• Formation of adducts 

Inactivation / destruction of free radicals: Free radicals are destroyed by- 

1. Spontaneous destruction: Free radicals are inherently unstable and generally decay spontaneously.
2. Cellular mechanism: 

• ➤Antioxidants: They block synthesis of free radicals, inactivate them & terminate radical damage e.g. vitamin A, C & E (ACE) and glutathione etc. 
• ➤ Binding with storage and transport proteins: Iron and copper can catalyze the formation of reactive oxygen species. The levels of these reactive metals are minimized by binding of the ions to storage and transport proteins (e.g. transferrin, ferritin and caeruloplasmin), thereby minimizing formation of reactive oxygen species. 
• ➤Enzymes: 

✓Catalase: It breaks H2O2 (2 H2O2 → O2 + 2H2O). 

✓Superoxide dismutase: It converts superoxide (O2 ̄) to H2O2. 

202+ 2H⭑→ H2O2 + O2 

✓Glutathione peroxidase: 

H2O2+2GSH → GSSG (glutathione homodimer) + 2H2O 20H ̄ +2GSH → GSSG (glutathione homodimer) + 2H2O 

Example of cell injury: 

• ➤Ischemic &/or hypoxic cell injury. 
• ➤Reperfusion injury. 
• ➤Chemical (toxic) injury. 

Ischemic & Hypoxic cell injury 

Ischemia, the most common cause of cell injury in clinical medicine, results from hypoxia induced by reduced blood flow, most often due to a mechanical arterial obstruction. It can also occur due to reduced venous drainage. 

Mechanism: 

As intracellular oxygen tension falls.

↓ 

Oxidative phosphorylation fails and ATP generation decreases. 

↓ 

Loss of ATP results initially in reversible cell injury (cell and organelle swelling). 

↓ 

Later in cell death by necrosis 

Sequence of events / pathogenesis / mechanism of ischaemic &/or hypoxic cell injury: 

Reversible events/changes following ischaemic &/or hypoxic cell injury: 

1) Loss of oxidative phosphorylation & generation of ATP: 

a) Failure of ATP dependent Na' pump: 

• ↑ influx of Ca, H2O and Na*, Efflux of K → Cellular swelling, swelling of ER, loss of microvilli & cellular blebs. 

b) Increased anaerobic glycolysis: ↓ Glycogen, ↑ lactic acid, ↓ pH → Clumping of nuclear chromatin. 

c) Detachment of ribosomes: ↓ Protein synthesis. 

2) Mitochondrial membrane damage: 

• ➤ Failure of oxidative phosphorylation & progressive depletion of ATP. 
• ➤ Formation of reactive oxygen species (ROS). 
• ➤ Swelling and the appearance of small phospholipid-rich amorphous densities. 

3) Plasma membrane damage: 

• ➤ Loss of osmotic balance → influx of fluid & ions → cellular swelling, dilatation of ER, loss of microvilli & cellular blebs. 
• ➤ Leakage of metabolites, those are vital for the reconstitution of ATP→ further depletion of energy stores. 
• ➤ Loosening of intercellular attachments. 

4) Nuclear alterations: Clumping of chromatin. 

5) Gross changes / macroscopic changes: 

• ➤ Cellular swelling → pallor, ↑turgor & Weight of the organ. 
• ➤ Fatty changes. 
• ➤ Hydropic change or vacuolar degeneration. 

Irreversible changes following ischemic / hypoxic cell injury: 

1. Increased eosinophilia of cells. 
2. Loss of glycogen particles → more glassy homogeneous appearance than that of normal cells. 
3. Lysis of endoplasmic reticulum (ER) → damage of protein synthesis machinery. 
4. Cytoskeletal alteration.
5. Extensive damage of plasma membrane: 

• ➤ Loss of cellular contents.
• ➤ Giving rise to myelin figures. 

 6. Severe swelling of mitochondria & damage of mitochondrial cell wall: 

• ➤ Large, flocculent, amorphous densities develop in the mitochondrial matrix. 
• ➤Leakage of apoptic proteins, e.g. cytochrome c & caspases (situated in between outer & inner mitochondrial membrane) into the cytosol → apoptosis. 
• ➤Cell necrosis. 

7. Injury to the lysosomal membranes & swelling of the lysosomes: 

Loss of lysosomal membrane

 ↓ 

Leakage of lysosomal enzymes into the cytoplasm, e.g. RNases, DNases, proteases, phosphatases, lipase & glucosideases

↓ 

Enzymatic digestion of RNA, DNA, protein, phosphate, lipid & glycogen respectively

↓ 

Cell dies by necrosis 

8. Nuclear changes: 

• ➤ Pyknosis.
• ➤ Karyorrhexis. 
• ➤ Karyolysis. 

Ischaemia tends to cause more and severe cell and tissue injury than hypoxia: 

1. In hypoxia, there is only reduced oxygen availability to cells and tissues. On the other hand, in ischaemia, the supply of oxygen and nutrient is decreased.
2. In hypoxia, energy production by anaerobic glycolysis can continue. But in ischaemia, delivery of substances for glycolysis is compromised. So, in ischaemia, both aerobic and anaerobic glycolysis is inhibited.
3. Again, in ischaemia, there is accumulation of metabolites that would have been removed otherwise by blood flow. 

For these reasons, ischaemia tends to cause more rapid and severe injury than does hypoxia alone. 

Ischaemia-reperfusion injury / reperfusion injury: 

Paradoxical & progressive cell injury followed by cell death even after restoration of blood flow to ischaemic tissues in a reversibly injured cell (which was caused by ischaemia) is called ischaemia reperfusion injury. 

• ✓ Restoration of blood flow to ischemic tissues can promote recovery of cells if they are reversibly injured. 
• ✓But in case of ischaemia-reperfusion injury, reperfused tissues sustain & continue the cell injury and ultimately irreversible cell injury & cell death occurs. 

Importance: It is clinically important because it contributes to tissue damage during myocardial infarction, cerebral infarction following therapies to restore blood flow. 

Mechanism of reperfusion injury: 

1. 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 in leukocytes, and in damaged endothelial cells and parenchymal cells. Compromise of cellular antioxidant defense mechanisms during ischemia may sensitize cells to free radical damage.
2. Intracellular calcium overload. 

Intracellular & mitochondrial calcium overload begins during acute ischemia

 ↓ 

Ca2+ overload exacerbated during reperfusion due to influx of calcium resulting from- 

• Cell membrane damage and 
• ROS mediated injury to sarcoplasmic reticulum. 

 ↓ 

Oening of the mitochondrial permeability transition pore with resultant depletion of ATP.

 ↓ 

Further cell injury 

3) Inflammation: 

Ischemic injury is associated with inflammation caused by resident immune cells (e.g. macrophages) 

 ↓ 

Increased expression of adhesion molecules by hypoxic parenchymal & endothelial cells

 ↓

Reperfusion recruits more circulating neutrophils to the injured tissue

 ↓

Further tissue injury & cell death 

4) Activation of the complement system: 

Some IgM antibodies have a propensity to deposit in ischemic tissues 

 ↓

For unknown reasons & when blood flow is resumed, complement proteins bind to the antibodies, are activated

 ↓

Further cell injury and inflammation 

Chemical injury: 

Cell injury caused by chemicals (mostly environmental), drugs & toxins is called chemical injury. 

✓ Most of the drugs are metabolized in the liver & is a frequent target of drug toxicity. 

✓ In fact, toxic liver injury is perhaps the most frequent reason for terminating the therapeutic use or development of a drug. 

✓ Chemical injury remains a frequent problem in clinical medicine and is a major limitation to drug therapy. 

Mechanism of chemical toxicity: 

🕮Direct toxicity: Some chemicals can injure cells directly by combining with critical molecular components, e.g. 

• ➤In case of mercuric chloride poisoning, mercury binds to the sulfhydryl groups of cell membrane proteins → ↑membrane permeability & inhibition of ion transport → cell injury. 
• ➤Cyanide poison inhibits oxidative phosphorylation 

🕮Conversion to toxic metabolites: 

• Most toxic chemicals are not biologically active in their native form but must be converted to reactive toxic metabolites, which then act to target molecules.
• This modification is usually accomplished by the cytochrome P-450 mixed function oxidase (Cyt. P450 MFO) enzymes in the smooth ER of the liver and other organs.
• The toxic metabolites cause membrane damage and cell injury mainly by formation of free radicals and subsequent lipid peroxidation.
• Direct covalent binding to membrane proteins and lipids may also contribute. 

Example: 

✓ CCI, (widely used in the dry-cleaning industry), is converted by cytochrome P-450 to the highly reactive free radical ̊CCl3 → causes lipid peroxidation & damages many cellular structures. 

✓ Acetaminophen (Paracetamol) also converted to a toxic product during detoxification in the liver → cell injury. 

Necrosis: 

Necrosis is a pathologic process that is the consequences of severe injury, which is characterized by denaturation of cellular proteins, leakage of cellular contents through damaged membranes, local inflammation, and enzymatic digestion of the lethally injured cell. 

Necrosis refers to a spectrum of morphologic changes that follow cell death in living tissue, largely resulting from the progressive degradative action of enzymes on the lethally injured cell. 

Classification / types of necrosis with example: 

🕮Basic types of necrosis: 

1. Coagulative necrosis: e.g. Ischaemic necrosis of- 

• Heart (myocardial infarction),
• Kidney,
• Liver,
• Adrenal gland & 
• Other solid organs. 

2) Liquefactive / colliquative necrosis: [Mnemonic- BBA] 

• Ischaemic necrosis of brain tissue. 
• Boil.
• Abscess. 

🕮Special types of necrosis: 

1. Caseous necrosis: e.g. Granuloma of tuberculosis.
2. Fat necrosis: 

• Enzymatic fat necrosis: e.g. enzymatic fat necrosis of pancreas and omental tissue. 
• Traumatic fat necrosis: e.g. traumatic fat necrosis of breast. 

3) Gangrenous necrosis: Any necrosis with superadded putrefaction. 

4) Fibrinoid necrosis: Acute rheumatic fever, rheumatoid arthritis, systemic lupus erythematosus.

5) Necrosis of muscle: e.g. Zenker's degeneration. 

6) Osteonecrosis: Necrosis of bone can affect the medullary bone with its medullary cavity and trabecular bone or affect both medullary and cortical bone. 

Ischaemic necrosis: 

Ischaemic necrosis means necrosis of tissue that result from impaired arterial supply or venous drainage from that tissue. 

Morphology of ischaemic necrosis: 

🕮Ischaemic necrosis of all solid organs except brain 

🕮Ischaemic necrosis of brain 

Sequelae of necrosis/cell death: 

1. When small numbers of cells are involved, the cellular debris is removed by phagocytosis.
2. With larger numbers of dead cells, there is an inflmmatory response with organization and firous repair.
3. When the necrotic tissue cannot be completely removed or organised, deposition of calcium may be an additional feature, for example in tuberculous caseous necrosis. This feature is important in radiologic diagnosis. It is known as 'dystrophic calcifiation'.  

Autolysis:

When enzymatic digestion of a cell occurs by its own lysosomal enzyme, is called autolysis. 

Heterolysis:

When enzymatic digestion of a cell occurs by the lysosomal enzymes of immigrant leukocytes during inflammatory reaction, is called heterolysis. 

Morphological changes of necrosis 

🕮Cytological changes in necrosis: 

1. Staining: Increased cosinophilia in hematoxylin and eosin (H & E) stains.
2. Appearance: More glassy homogeneous appearance than do normal cells (due to loss of glycogen particles).
3. Cytoplasm: Enzymatic digestion of cytoplasmic organelles → cytoplasm becomes vacuolated and appears moth-eaten. 
4. Myelin figures: Replacement of dead cells by large, whorled phospholipid masses → called myelin figures (derived from damaged cell membranes). 

Fate of myelin figures are- 

🕮Mayelin figures may phagocytosed by other cells or 

🕮They may further degraded into fatty acids → calcification of fatty acid residues → generation of calcium soaps → thus, the dead cells may ultimately become calcified. 

Nuclear changes in necrosis (due to non-specific breakdown of DNA): 

1) Karyolysis: 

• The basophilia of the chromatin may fade, 
• This change presumably reflects loss of DNA because of enzymatic degradation by endonucleases. 

2) Pyknosis: 

• Pyknosis is also seen in apoptic cell death. 
• It is characterized by nuclear shrinkage and increased basophilia. 
• Here the chromatin condenses into a solid, shrunken basophilic mass. 

3) Karyorrhexis: 

• Here, the pyknotic nucleus undergoes fragmentation. 
• With the passage of time (a day or two), the nucleus in the necrotic cell totally disappears. 

Figure: -Nuclear and cytoplasmic changes in necrosis. 

Pathogenesis of inflammation in necrosis: 

Cellular contents leak through the damaged plasma membrane into the extracellular space, e.g. ATP (released from damaged mitochondria), Uric acid (a breakdown product of DNA) & other molecules that are released after severe cell injury.

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These molecules are recognized by receptors present in macrophages and most other cell types 

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Trigger phagocytosis of the debris and there is production of cytokines

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Cytokine induces inflammation 

Coagulative necrosis / structured necrosis: 

• ➤ Coagulative necrosis is a form of necrosis in which the basic cellular shape, outline & architecture of dead tissues is preserved for at least some days. 
• ➤The affected tissues exhibit a firm texture.
• ➤ A localized area of coagulative necrosis is called an infarct. 
• ➤Also called structured necrosis. 

Pathogenesis of coagulative necrosis: 

The injury denatures both structural & enzymatic proteins due to↑ Intracellular pH

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Proteolysis of the dead cells is blocked (due to lack of proteolytic enzymes)

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Eosinophilic, anucleate cells may persist for days or weeks 

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Ultimately the necrotic cells are removed by - 

• Phagocytosis of the cellular debris by infiltrating leukocytes &
• By digestion of the dead cells by the action of lysosomal enzymes of the leukocytes. 

Examples: Ischemic necrosis in all solid organs except the brain, such as - 

• ➤Heart (myocardial infarction).
• ➤Kidney.
• ➤Liver. 
• ➤Adrenal gland.
• ➤Gumma of tertiary syphilis. 

Morphology: 

🕮The affected tissues exhibit a firm, dry, opaque and homogenous architecture. 

🕮Microscopic: -

• Cellular outline: Basic cellular outline is preserved. 
• Cytoplasm: Becomes opaque and acidophilic.
• Nucleus: Nucleus shows pyknosis, karyorrhexis & karyolysis.  

Liquefactive / colliquative necrosis: 

• ➤Liquefactive necrosis is characterized by digestion of the dead cells, resulting in transformation of the tissue into a liquid viscous mass.
• ➤It is seen in focal bacterial & occasionally in fungal infections.
• ➤For unknown reasons, hypoxic death of cells within the CNS often manifests as liquefactive necrosis.
• ➤If the process had been initiated by acute inflammation → the material is frequently creamy yellow because of the presence of dead WBC & called 'pus'. 

Pathogenesis: 

Microorganisms (usually bacterial & occasionally fungal) stimulate the accumulation of leukocytes and the liberation of enzymes from these cells

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Enzymatic digestion of the dead cell completely

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Transformation of the tissue into a liquid viscous mass 

Example: 

• ➤CNS infarction: Ischaemic necrosis of brain tissue.
• ➤Suppurative inflammation: Abscess & boil. 

Morphology: 

🕮Macroscopic: 

• Softening of the necrosed area (liquid viscous mass).
• Colour: Creamy yellow (due to presence of dead WBC) if pus is formed. 

🕮Microscopic: Basic cellular outline is not preserved. 

Caseous necrosis: 

Caseous means 'cheese-like' 

• ➤ It is a distinctive form of coagulative necrosis.
• ➤It occurs most commonly in foci of tuberculous infection.
• ➤There is formation of soft, friable, amorphous granular debris resembling white & cheesy material (hence named as 'caseaous'). 

 Example:  

• ➤Granuloma of tuberculosis.
• ➤Cat scratch disease. 

Morphology: On microscopic examination-

➤The tissue architecture: Tissue architecture is lost & the cell outlines are not preserved. 

➤Formation of granuloma: The necrotic area appears as a structureless collection of fragmented or lysed cells and amorphous granular debris enclosed within a distinctive inflammatory border, this appearance is characteristic of a focus of inflammation known as a 'granuloma'. 

Gangrenous necrosis 

• ➤Gangrenous necrosis is not a specific pattern of cell death, but the term is commonly used in clinical practice.
• ➤It is usually applied to a limb, generally the lower leg.
• ➤Loss of blood supply in lower leg causes necrosis (typically coagulative necrosis) → dry gangrene.
• ➤When bacterial infection is superimposed → liquefactive necrosis occurs due to actions of degradative enzymes of bacteria and the attracted leukocytes → giving rise of wet gangrene. 

Fat necrosis:

It occurs in adipose tissue due to action of lipase. 

Classification of fat necrosis: 2 types. 

🕮Enzymatic fat necrosis: 

Site: Pancreas & omental tissue.

Pathogenesis: 

In milder form of acute interstitial pancreatitis 

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Pancreatic enzymes leak out of acinar cells 

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Release of activated pancreatic lipases into the pancreas, peripancreatic fat & peritoneal cavity 

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The released lipases split the triglycerides to free fatty acids 

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Free fatty acid combines with calcium to produce grossly visible chalky-white areas (fat saponification) 

Morphology: On histologic examination the necrosis takes the form of foci of shadowy outlines drop necrotic fat cells, with basophilic calcium deposits, surrounded by an inflammatory reaction. 

❖ Traumatic fat necrosis: 

Site: Breast, abdominal fat etc. 

Pathogenesis: Unlike pancreatic fat necrosis, it is not enzyme mediated. 

Surgery of blunt trauma of the breast tissues or abdominal fat 

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Acute lesions may be hemorrhagic and contain central areas of liquefactive fat necrosis with neutrophils and macrophages 

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Over the next few days proliferating fibroblasts and chronic inflammatory cells surround prio the injured area 

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Subsequently, giant cells, calcifications, and hemosiderin make their appearance 

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Eventually the focus is replaced by scar tissue or is encircled and walled off by fibrous tissue. 

Morphology: 

❖Macroscopically: Ill defined, firm, graywhite nodules containing small chalky-white foci are seen grossly. 

❖Microscopically: Nutrophil, lipid-laden macrophages, giant cells & fibroblast is seen macroscopically.  

Fibrinoid necrosis: 

❖Fibrinoid necrosis is a special form of necrosis usually seen in immune reactions involving blood vessels. 

Site: -

• ✓ The immune-complex mediated vasculitis syndromes, e.g. polyarteritis nodosa. 
• ✓ Acute rheumatic fever.
• ✓ Connective tissue disorder, e.g. Rheumatoid arthritis (RA), SLE etc.
• ✓ Arthus phenomenon.
• ✓ Malignant hypertension in arterioles. 

Pathogenesis: 

Antigens & antibody complexes are deposited in the walls of arteries

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Deposition of these "immune complexes", together with fibrin that has leaked out of vessels 

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Result in a bright pink and amorphous appearance in H&E stains, called 'fibrinoid' (fibrin-like) necrosis 

Reference: -

🖋Robbins 10th

🖋Pathology Tutorial 

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