INTRODUCTION
Rudolph Virchow, the father of modern pathology rooted his concepts of pathology from the changes he observed at cellular level. To appreciate the mechanism of injury to an organ it is must that we understand the changes that occur at the cellular level.
The cells form the basic structural and functional unit of a tissue/organ.
Every cell has a narrow range of structure and function. This is referred to as homeostasis. When the cells are subjected to physiologic stress they undergo certain structural and functional modifications which are called adaptations. When the stress is very severe then the cells unable to adapt to the stress gets injured and may end up as cell injury/death.
CELLULAR ADAPTATIONS
There are various ways by which a cell responds to a stressful stimuli.
They include:
- Hyperplasia
- Hypertrophy
- Atrophy
- Metaplasia.
These cellular adaptations are potentially reversible and the cells will return to normalcy when the stress is removed.4
Hyperplasia
It is defined as increase in the number of cells in an organ/tissue which in turn leads to increased volume of the organ. Histopathological picture of normal prostatic tissue is shown in Figure 1.
There are two types of hyperplasia—Physiological and pathological.
Physiologic hyperplasia may be induced by hormones, e. g. (i) increase in the size of female breast during puberty/pregnancy, (ii) increase in the size of uterus during pregnancy or may occur as a compensatory hyperplasia, e. g. regeneration of liver following hepatectomy.
Pathologic hyperplasia usually occurs due to excessive hormonal stimulation, e. g. (i) estrogen induced hyperplasia of the endometrial tissue, (ii) hormone induced hyperplasia of prostate (Figure 2). These hormone induced pathologic hyperplasia constitute a fertile soil for cancerous growth.
The other examples include, hyperplasia of the connective tissue as a part of healing process and hyperplasia of the surface epithelium induced by viruses producing warty lesions (viral warts caused by human papilloma virus).
Hypertrophy
It is defined as increase in the size of the cell and thereby increase in the size of the organ.
In contrast to hyperplasia, there are no new cells but the existing cells becomes larger.
Hypertrophy usually occurs in non-dividing cells.
There are two forms—Physiologic and Pathologic
Physiologic Hypertrophy
- Hypertrophy of the muscles due to exercise
- Hypertrophy of uterus in pregnancy.
Pathologic Hypertrophy
- Hypertrophy of the cardiac chambers due to hemodynamic overload as in hypertension and valvular heart diseases as in Figure 3.
The basic molecular mechanism for hypertrophy is due to the synthesis of newer cellular proteins or excessive secretion of growth factors.
Atrophy
It is defined as reduction in the size of the cell and thereby the size of the organ. An atrophic cell has diminished function but it is not dead.
Atrophy can be physiological or pathological.
Physiologic atrophy usually occurs during embryogenesis:
- Atrophy of the thyroglossal duct or notochord.
Pathologic atrophy are due to:
- Decreased work load (Disuse atrophy), e. g. limbs imobilised in plaster cast.
- Loss of nerve supply (Denervation atrophy), e. g. muscle wasting.
- Loss of blood supply—changes in brain due to ischemia.
- Inadequate nutrition—protein, energy malnutrition, cancer cachexia.
- Loss of endocrine stimuli—atrophy of uterus, ovaries, testis (Figure 4).
- Ageing (senile).
- Pressure—tissue compressed for a longer duration.
The major molecular mechanism of atrophy is due to excessive degradation of structural proteins or an imbalance between the protein synthesis and degradation.
Metaplasia
It is defined as a reversible change of one adult type epithelium into another. There are two forms of metaplasia—epithelial and mesenchymal.
Epithelial Metaplasia
- Change of columnar epithelium to squamous epithelium:
- Respiratory tract—chronic irritation, smoking
- Ducts of salivary gland, pancreas—calculi
- Urinary bladder—deficiency of vitamin A.
- Change of squamous to columnar epithelium:
- Lower end of esophagus (Barrett's esophagus)–—reflux of gastric acid (Figure 5).
Mesenchymal Metaplasia
Formation of bony tissue within a muscle—myositis ossifiicans—induced by trauma.
The major underlying molecular mechanism is genetic reprogramming of the stem cells. This occurs to withstand the stress to which the cell is exposed.
CELL INJURY
It usually occurs when the cells are subjected to severe stress, that they are no longer able to adapt.
There are two phases in cell injury—initial reversible phase and final irreversible phase (cell death).
The irreversibly injured cell undergoes various morphological alterations. The two most important ones are necrosis and apoptosis (Figure 6).
Causes for Cell Injury
- Hypoxia—due to lack of oxygen—most common cause of cell injury
- Ischemia—loss of blood supply
- Physical agents—trauma, pressure, radiation, extremes of temperature
- Chemicals—drugs, insecticides, environmental pollutants
- Biological—viruses, bacteria, fungi, parasites
- Immunologic—derangements in the immune mechanisms
- Genetic derangements
- Nutritional imbalances.
Mechanism of Cell Injury
The mechanism of cellular responses to various injurious stimuli depends on various factors, which include:
- Type of injurious stimuli
- Duration of injurious stimuli
- Severity of injurious stimuli
- Type of the cell
- Adaptability status of the cell.
In any form of cell injury there are few main cellular structures that are usually targeted they include:
- Aerobic respiratory mechanism
- Integrity of the cell membrane
- Protein synthesis
- Cytoskeleton
- Genetic apparatus.
The major biochemical mechanisms that are involved in cellular injury includes:
- Depletion of ATP
- Alterations in energy metabolism of cell
- Damage to cell membrane
- Failure of calcium homeostasis
- Mitochondrial damage.
Depletion of ATP
Occurs in hypoxic and chemical injury. Normally ATP is required for the energy dependent functions like membrane transport, protein synthesis and lipogenesis. Depletion of ATP leads to defective functioning of the Na/K dependent energy pump which causes excessive loss of potassium and high influx of sodium and water within the cell producing cellular swelling.
Alterations in Energy Metabolism of Cell
Switch from aerobic respiration to anaerobic pathway with prominent glycolysis. There will be rapid depletion of glycogen stores producing moth eaten appearance of the cytoplasm with accumulation of lactic acid and pyruvic acid. This causes intracellular acidosis which interferes with the functioning of various cellular enzymes.
Damage to Cell Membrane
Damage to the cell membrane is a major event in the cellular injury. The damage is induced by various mediators of cell injury like free oxygen radicals, rise in the cytosolic calcium levels and others. Cell membrane damage leads to the following:
- Loss of osmotic balance of the cell
- Influx of water and ions into the cell
- Loss of proteins, enzymes, coenzymes and metabolites
- Depletion of high energy phosphates
- Damage to the mitochondrial cell membrane leads to severe mitochondrial dysfunction
- Damage to the lysosomal membrane leads to leak of the lysosomal enzymes and activation which causes digestion of the cellular components.
Mitochondrial Damage
Important target for all types of injurious stimuli. Damage to the mitochondria occurs due to increased cytosolic calcium, oxidative stress, increased breakdown of phospholipids and breakdown of lipids. This leads to alteration in the mitochondrial membrane permeability and thereby defective oxidative phosphory–lation. There is release of cytochrome C into the cytoplasm which triggers the death of the cell.
Failure of Calcium Homeostasis
Normally calcium is kept in a very low level within the cytosol. Due to ischemia and certain toxins the intracellular calcium level is elevated which leads to activation of various enzyme systems 11like ATPase, phospholipase, protease and endonuclease. The elevated calcium levels also increases the membrane permeability of mitochondria.
Mediators of Cell Injury
The common mediators include the free oxygen radicals and cytosolic calcium.
The role of cytosolic calcium has already been highlighted.
Free Oxygen Radicals
These are reactive oxygen molecules which have an unpaired electron in its outermost orbit. They are extremely reactive and capable of undergoing chain reactions with the generation of more free oxygen radicals. They are usually represented as O2˙.
They mediate various forms of cell injury mostly due to radiation, chemical and ischemia—reperfusion. They also play a vital role in mediating the cellular damage due to ageing and aid in microbial killing by the phagocytes.
The common free radicals include—O2˙, H2˙O2˙, OH˙, NO˙
The free radicals causes:
- Lipid peroxidation of the cell membranes—cell membrane damage.
- Oxidative modification of proteins—affects protein synthesis
- Damage to DNA.
The action of the free oxygen radicals is usually balanced by substances called antioxidants. They inactivate the free radicals and terminate the damage induced by the radicals.
Common antioxidants include catalase, superoxide dismutase, glutathione peroxidase, transferrin, ferritin, lactoferrin, vitamins E and A.
Usually there is a well maintained balance between the functioning of the free radicals and antioxidants.
When does the cell actually die?
When the cell is unable to reverse the mitochondrial dysfunction as well as the cell membrane damage, the reversible phase of cellular damage transforms into an irreversible phase (cell death).
Morphological Patterns of Reversible Cell Injury
They include:
- Cellular swelling—hydropic/vacuolar degeneration
- Fatty change
- Hyaline and mucoid change.
Cellular Swelling
Most common morphological form of cell injury. This occurs due to the loss of maintainence of the fluid and ionic balance as a result of the defective functioning of the Na/K dependent pump.
This is manifested well in renal tissue and the change is called Cloudy Swelling of the renal tubules. The organ looks paler with increased turgor and weight. On light microscopy, the cells are swollen with small vacuoles within the cytoplasm and increased granularity of the cytoplasm. This leads to irregularity of the tubular lumina which is referred to as starry lumina (Figure 7). Electron microscopy shows widespread alterations in the plasma membrane, mitochondria, endoplasmic reticulum and nuclear chromatin.
Fatty Change
This is another form of reversible cell injury where the cell accumulates an intracellular substance in an excessive manner.
Lipid accumulation is the most common form and the cell accumulates triglycerides, cholesterol, phospholipids and esters of cholesterol. Fatty change occurs in organs like Liver, Heart, Muscle and Kidneys. Most common site is Liver, as it is the seat of lipid metabolism.
Causes of Fatty Change Liver:
- Chronic alcoholism (most common)
- Toxins—carbon tetrachloride
- Protein calorie malnurtrition
- Diabetes mellitus
- Obesity
- Anoxia
- Infections—hepatitis C virus.
- Late pregnancy
- Reye's syndrome
- Drugs—estrogen, corticosteroids, tetracycline.
Gross: The liver is enlarged, yellowish and greasy. The edges of the liver are blunt and it collapses on the table.
Microscopy (Figure 8): The hepatocytes are swollen with accumu–lation of microvesicles of fat which gradually fill the entire cell.
14The fat usually appears as clear spaces within the cytoplasm, because the fat is dissolved in the chemicals used for tissue processing in the laboratory. This pattern of staining is called Negative staining. Fat can be demonstrated using special stains like Sudan Black B, Sudan III, Sudan IV and oil red O in a frozen section.
Fatty change of the heart is less common and it presents as two morphological forms depending on the cause. In cases of moderate hypoxia as in anemia, the change is called as Tigered Effect– fatty yellow myocardium alternates with normal brown myocardium and in cases of severe hypoxia as in Diphtheria there is uniform fatty change within the myocardium.
Morphology of Irreversible Cell Injury (Cell Death)
The two major morphological forms of cell death includes Necrosis and Apoptosis.
Necrosis
Definition: The spectrum of morphological changes that follow cell death in a living tissue. This is mostly induced by the proteolytic degradative action of the enzymes on the injured cell. The enzymes may be released from the same cell (autolysis) of from the adjacent cells and inflammatory cells (heterolysis).
The two major events that occur in necrosis are:
- Denaturation of the cellular proteins
- Enzymatic digestion of cells.
Morphology of a Necrotic Cell:
- Cytoplasmic changes:
- Increased eosinophilia of the cytoplasm due to denaturation of proteins
- Glassy and moth eaten appearance of cytoplasm due to loss of glycogen
- Presence of calcification.
- Nuclear changes:
- Ultrastructural changes:
- Cell membrane and plasma membrane damage
- Mitochondrial alterations
- Presence of amorphous densities in the mitochondria
- Lysosomal membrane alterations
- Aggregation of denatured proteins.
Types of Necrosis
- Coagulative necrosis
- Liquefactive necrosis
- Caseation necrosis
- Fat necrosis
- Gangrenous necrosis
- Fibrinoid necrosis
- Osteonecrosis
- Zenker's degeneration.
Coagulative Necrosis (structured necrosis):
Causes | : Ischemia and hypoxia |
Organs involved | : Heart, kidney, spleen (solid organs with endareterial blood supply) (Figure 9) |
Major event | : Denaturation of proteins |
Features | : The basic structure of the cell is retained, so the cell type can be recognized. |
Causes | : Ischemia, bacterial infections (pyogenic) |
Organs involved | : Brain (Infarct brain), abscess |
Major event | : Enzymatic digestion |
Features | : The necrotic area is converted into a liquid viscous mass which undergoes cystic change later (Figure 10). |
Caseation Necrosis:
Causes | : Mycobacterium tuberculosis, Mycobacterium leprae |
Organs involed | : Lung, lymph node, skin and other tissues |
Major event | : A combination denaturation of proteins and liquefaction necrosis—This is due to an immune mediated delayed hypersensi–tivity reaction to mycolic acid present in the cell membrane of the bacteria. |
Features | : The necrotic area appears firm, dry and cheesy amorphous granular debri as shown in the Figure 11. The structure of the tissue is lost. |
Fat Necrosis:
Causes | : Acute pancreatitis, trauma (breast, Figure 12) |
Organs | : Pancreas, abdominal fat, breast |
Major event | : Enzymatic digestion with release of lipases |
Features | : Formation of chalky white necrotic areas due to abnormal release of lipases and subsequent calcification. |
Fibrinoid Necrosis:
Causes | : Immune mediated (Antigen-antibody reaction) |
Organs | : Blood capillaries, glomeruli |
Major event | : Immune mediated protein denaturation |
Features | : Deposition of powdery pinkish amor–phous granular substance in the blood vessels and capillaries as in Figure 13. |
Osteonecrosis:
Causes | : Ischemia, radiation, traumatic |
Organs | : Head of femur, navicular bone |
Major event | : Protein denaturation. |
Zenker's Degeneration:
Causes | : Enteric fever |
Organ | : Rectus abdominus, diaphragm |
Major event | : Protein denaturation. |
Features | : The muscle fibers loose the striations and appear as eosinophilic hyaline masses. |
Gangrene
Definition: It is a special type of necrosis with superadded putrefaction.
Cause: Due to ischemia with superadded severe bacterial infection.
Types: Three major types
- Dry gangrene
- Wet gangrene
- Gas gangrene
Dry gangrene: Ususally occurs in the distal part of extremity mostly due to ischemia (arterial occlusion). Gradual in onset. The affected part appears dry, shrunken and dark brown to black in color. The color change is due to the oxidation of hemoglobin and myoglobin into ferric sulphide. The line of demarcation is clearly made out. Amputated toe with dry gangrene is shown in Figure 14.
20Wet gangrene: Occurs in moist organs like small bowel, oral cavity, vulva, etc. The affected part appears swollen and dark. Mostly due to obstruction to the venous outflow. Rapid in onset. The line of demarcation is ill defined.
Contrasting features of dry and wet gangrene are summarized in the table below.
Features | Dry gangrene | Wet gangrene |
|---|---|---|
Cause | Arterial Occlusion | Venous outflow obstruction |
Sites | Extremities | Bowel loops, moist tissue |
Onset | Gradual | Rapid |
Macroscopy | Dry, shrunken and brownish black | Wet, congested, brownish black |
Putrefaction | Moderate | Marked |
Line of demarcation | Well defined | Ill defined |
Gas gangrene: Special type of wet gangrene due to infection with spore forming anaerobic bacteria of the Clostiridia family—Clostridium perfringens, C. novyi, C. septicum.
The most common predisposing factor is lacerated wounds of the extremities as in road traffic accidents contaminated with spores of the bacteria in the soil. The bacteria grows well in the dead and devitalized tissues and secretes powerful exotoxins which causes myonecrosis. The muscle carbohydrate is fermented into lactic acid, hydrogen and carbondioxide which accumulates in the area of injury. The affected organ appears swollen, tense, greenish black, creptitant (due to gas accumulation) and foul smelling (due to formation of hydrogen sulphide).
This can be prevented by proper wound toileting, wound debridement, use of hyperbaric oxygen and antigas gangrene serum.
Apoptosis
Definition: It is a type of coordinated and internally programmed cell death in which the cells destined to die, activate enzymes that degrade the cell's own DNA and cytoplasmic proteins (Figure 15).
This was described in 1972. The term is derived from a Greek word of “Falling off”(as the leaves fall from the tree during autumn).
Figure 15: Photomicrograph showing apoptotic cell death of hepatocytes, the arrows pointing apoptotic cells
This is a process to eliminate the unwanted and potentially harmful cells.
Apoptosis can be physiological or pathological.
Physiological apoptosis:
- During embryogenesis, e.g. implantation, organogenesis, and developmental involution, e.g. dispappearance of inter digital webs, involution of notochord, thyroglossal duct.
- Hormone dependent involution, e.g. endometrial breakdown in menstruation, regression of lactating breast.
- Maintainence of cell balance, e.g. deletion of cells in intestinal crypts.
- Death of cells after they have served the function, e.g. neutro–phils in acute inflammation, lymphocytes in immune reaction
- Elimination of potential harmful self-reactive lymphocytes.
- Cell death by cytotoxic T lymphocytes.
Pathological apoptosis:
- Cell death by various injurious stimuli—radiation, hypoxia, anticancer drugs.
- Viral induced cell injury—Viral hepatitis (Councilman bodies).
- Cell death in tumors.
- Induction of malignancy due to loss of apoptosis.
Morphological features:
- Cell shrinkage: The cell becomes small with dense cytoplasm and tightly packed cytoplasmic organelles. It becomes rounded and looses its specialized structures like the microvilli.
- Condensation of chromatin: It is the most characteristic feature with the aggregation of the chromatin under the nuclear membrane.
- Formation of cytoplasmic blebs: Extensive surface blebing with fragmentation.
- Phagocytosis of the apoptotic bodies by the macrophages due to surface expression of vitronectin, Beta 3 integrin, thrombospondin by the apoptotic cells.
- Absence of inflammatory response.
The major biochemical events in apoptosis include cleavage of proteins, breakdown of DNA and recognition by the phagocytes.
Mechanism: It occurs in two phases initiation and execution.
Initiation: It can be due the activation of the cell surface death receptors, e.g. TNF R1 and Fas or due to the release of pro-apoptotic signals from the mitochondria, e.g. Bak, Bax, Bim, Cytochrome C. The mitochondria normally contains anti-apoptotic molecules like bcl-2, bcl-x proteins. Due to stress, these molecules are lost and are replaced by the above mentioned pro-apoptotic proteins which initiates the process of apoptosis.
Execution: It is mediated by enzymes of the caspase family namely, transglutaminase and endonuclease. Transglutaminase causes cleavage of proteins and endonuclease causes DNA fragmentation. Contrasting features of necrosis and apoptosis are summarized in table on the next page.
CALCIFICATION
Definition: Depostion of calium salts in tissues other than teeth and enamel.
S.no | Features in apoptosis | Features in necrosis |
|---|---|---|
1 | Involves single cells | Involves sheets of cell |
2 | Intact tissue structure | Tissue structure lost |
3 | Shrunken cell | Swollen cells |
4 | Intact plasma membrane | Disrupted plasma membrane |
5 | Compact organelles | Disorganised organelles |
6 | Intact mitochondria | Amorphous densities in mitochondria |
7 | Pyknotic nuclei | Pyknotic nuclei |
8 | Active and energy dependent | Passive process |
9 | No inflammatory response | Inflammation present |
10 | Physiological/Pathological | Pathological |
Types: Dystrophic and metastatic calcification.
Morphology: Calcium is seen as granular deeply basophilic encrusted debri within the tissues. The special stains to demonstrate calcium are von Kossa and Alizarin red S.
Dystrophic: Calcification in dead and degenerate tissues. Plasma calcium levels are normal. Examples—Calcification of:
- Caseous necrotic material
- Dead parasites
- Fat necrosis
- Thrombi and infarct, hematoma
- Scar tissue
- Atherosclerosis
- Monckeberg's sclerosis: Deposition of calcium in the tunica media of the blood vessels, mostly uterine vessels seen in senility (as shown in Figure 16)
- Psammoma bodies: Circumscribed spherules of calcium seen in tumors like papillary carcinoma of thyroid, meningioma and serous papillary cystadenocarcinoma of ovary (Figure 17)
Metastatic: Calcium deposition in normal tissues. Plasma calcium levels are elevated. Examples:
- In the renal tubules—Nephrocalcinosis or renal calculi formation
- In the alveolar walls of the lung
- Wall of the blood vessels and cornea.
Figure 16: Photomicrograph showing dystrophic calcification (reddish brown) areas in the tunica media of the blood vessel (Monckeberg's sclerosis)
Common predisposing factors for hypercalcemia includes:
- Hyperparathyroidism
- Hypervitaminosis D
- Milk-Alkali syndrome
- Hypophosphatemia
- Destructive bone lesions.