1 Cell Injury

Introduction to pathology • Pathology is the study (logos) of suffering (pathos) • Pathology is devoted to the study of structural and functional changes in cells, tissues and organs that underlie diseases

Pathology General pathology Basic reactions of cells and tissues to abnormal stimuli, i.e. common features of various disease processes in various cells and tissues

Systematic pathology The descriptions of specific diseases as they affect given organs or organ systems

Pathology focuses on 5 aspects of disease 1) Occurrence in populations (epidemiology) 2) Its cause (etiology) environmental, genetic, multifactorial, etc.

3) The mechanism of its development (pathogenesis) 4) The structural alterations induced in organs, tissues and cells: macroscopical and microscopical (patho)morphology 5) The functional consequences: clinical significance

Pathology of cellular injury and death Cells react to adverse influences by

Reversible cell injury Changes that can be reversed when the stimulus is removed

Pathology of cellular injury and death Cells react to adverse influences by

Reversible cell injury

Irreversible cell injury

Changes that can be reversed when the stimulus is removed

Changes that cause cell death

Pathology of cellular injury and death Cells react to adverse influences by

Reversible cell injury

Irreversible cell injury

Cellular adaptation

Changes that can be reversed when the stimulus is removed

Changes that cause cell death

Stimuli result in new but altered state that maintaines the viability of the cell

Causes of cellular injury Hypoxia • Ischemia (loss of blood supply) • Inadequate oxygenation (cardioresp. failure) • Loss of oxygen carrying capacity of the blood (e.g., anemia, CO poisoning)

Causes of cellular injury Hypoxia (⇓ of O2)

• Ischemia (loss of blood supply) • Inadequate oxygenation (cardioresp. failure) • Loss of oxygen carrying capacity of the blood (e.g., anemia, CO poisoning)

Physical agents • Trauma • Heat • Cold • Radiation • Electric shock

Infectious agents • • • • •

Bacteria Viruses Fungi Rickettsiae Parazites

Causes of cellular injury Hypoxia • Ischemia (loss of blood supply) • Inadequate oxygenation (cardioresp. failure) • Loss of oxygen carrying capacity of the blood (e.g., anemia, CO poisoning)

Physical agents • Trauma • Heat • Cold • Radiation • Electric shock

Chemical agents and drugs

Infectious agents • • • • •

Bacteria Viruses Fungi Rickettsiae Parazites

Others • Immunologic reactions • Genetic derangements • Nutritional imbalances

Intracellular mechanisms vulnerable to cellular injury • Maintenance of membrane integrity Critical for cell and organellar ionic and osmotic homeostasis

• Aerobic respiration, involving mitochondrial oxidative phosphorilation and ATP production

• Synthesis of enzymes and structural proteins • Preservation of the integrity of the genetic apparatus

Features of Hypoxic injury

Injury induced by free radicals

Chemical injury

Hypoxic injury Main biochemical events • ATP depletion • Influx of intracellular Ca ++ ions and loss of Ca++ homeostasis Ca ++ ions activates phospholipases ⇒ degradation of membrane phospholipids proteases ⇒ membrane and cytoskeletal protein degradation ATPases ⇒ enhance ATP depletion endonucleases ⇒ chromatin fragmentation

Reversible hypoxic injury • Hypoxia prevents oxidative phosphorilation, thus reducing the capacity to generate ATP • ATP provides fuel for the Na+/K+ ATPase, which acts as a pump, keeping the high concentration of sodium in the intercellular fluid and the high concentration of potassium inside the cell

Reversible hypoxic injury • ATP provides fuel for the Na+/K+ ATPase, which acts as a pump, keeping the high concentration of sodium in the intercellular fluid and the high concentration of potassium inside the cell • Hypoxia prevents oxidative phosphorilation ⇒ ATP ⇓ • Hypoxia ⇒ malfunction of Na+/K+ ATPase ⇒ influx of sodium and water from the extracellular space ⇒ cellular swelling: hydropic change

Reversible hypoxic injury • ATP provides fuel for the Na+/K+ ATPase, which acts as a pump, keeping the high concentration of sodium in the intercellular fluid and the high concentration of potassium inside the cell • Hypoxia prevents oxidative phosphorilation ⇒ ATP ⇓ • Hypoxia ⇒ malfunction of Na+/K+ ATPase ⇒ influx of sodium and water from the extracellular space ⇒ cellular swelling: hydropic change • Anaerobic glycolysis starts ⇒ depletion of cytoplasmic glycogen ⇒ ⇑ of lactic acid in the cytoplasm ⇒ ⇓ of the intracellular pH, ⇓action of enzymes

Hydropic change in the proximal tubule: water ⇑ in the cytoplasm, in the invaginations of the surface plasma membrane (hydropic vacuoles), in the cisterns of the RER, and in the mitochondria. Loss of microvilli.

Hydropic change in the proximal tubule: water ⇑ in the cytoplasm, in the invaginations of the surface plasma membrane (hydropic vacuoles), in the cisterns of the RER, and in the mitochondria. Loss of microvilli.

The changes are reversible if oxygenation is restored

Irreversible hypoxic injury • The transition from reversible to irreversible state is gradual and occurs when adaptive mechanisms have been exhausted • Signs of irreversible injury - Amorphous densities in swollen mitochondria - Formation of myelin figures from whorls of mitochondrial membranes -

Irreversible hypoxic injury • The transition from reversible to irreversible state is gradual and occurs when adaptive mechanisms have been exhausted • Signs of irreversible injury - Amorphous densities in swollen mitochondria - Formation of myelin figures from whorls of mitochondrial membranes - Surface membrane blebs - Rupture of cell and plasma membranes - Leakage of lysosomal enzymes ⇒ digestion of cell and nuclear components

The mitochondria are swollen, their membranes are ruptured, and amorphous densities are in their matrix

Sublethal hypoxic injury

Lethal hypoxic injury: rupture of cell membranes and lysis of chromatin

LM features of lethal hypoxic injury: loss of nuclear staining, the cytoplasm is eosinophilic (pink)

Dead cells show typical nuclear changes • Pyknosis (pyknos, dense) - condensation of chromatin • Karyorrhexis - (rhexis, tearing apart) - fragmentation of nuclear material • Karyolysis - lysis of chromatin due to the action of endonucleases (loss of nuclear staining)

Laboratory markers of irreversible cell injury • Cytoplasmic enzymes are released through damaged cell membranes into the blood • Creatine kinase (CK) - cardiac or skeletal muscle injury • Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) - liver cell injury • Lactate dehydrogenase (LDH) is released from ruptured RBCs and many other cells

Can hypoxic cell injury be reversed or repaired by providing the cells with adequate oxygen? • Irreversibly damaged cells cannot be revived by O2 • The function of reversibly damaged cells can be improved by O2

Can hypoxic cell injury be reversed or repaired by providing the cells with adequate oxygen? • Irreversibly damaged cells cannot be revived by O2 • The function of reversibly damaged cells can be improved by O2 • Reestablished blood flow to a myocardium made hypoxic to coronary obstruction may cause reperfusion injury of still living myocardial cells at the marginal zone of a myocardial infarction

Can hypoxic cell injury be reversed or repaired by providing the cells with adequate oxygen? • Irreversibly damaged cells cannot be revived by O2 • The function of reversibly damaged cells can be improved by O2 • Reestablished blood flow to a myocardium made hypoxic to coronary obstruction may cause reperfusion injury of still living myocardial cells at the marginal zone of a myocardial infarction • Reperfusion injury is caused by oxygen-derived free radicals that may form under such conditions, and is an irreversible damage to cells injured previously by hypoxia

Oxygen free radicals

• Superoxide anion radical (O2.-), hydrogen peroxide (H2O2), hydroxyl radical (OH.) and nitric oxide (NO.) • Free radicals cause lipid peroxidation ⇒ membrane damage cross-link proteins ⇒ inactivation of enzymes cause DNA breaks ⇒ blockade of DNA transcription

Chemical injury

• Two mechanisms • Direct damage, by binding to some critical molecular component of cell membrane proteins, causing ⇑ permeability • Indirect damage, by conversion to reactive toxic metabolites, which cause cell injury by - direct binding to membrane proteins and lipids - formation of free radicals

Necrosis (morphology of irreversible injury)

• Necrosis (necros, dead) is death of cells, tissues, or organs in a living organism • Histological signs: same as those of irreversible hypoxic injury: cell membrane rupture, nuclear changes: pyknosis, karyorrhexis or karyolysis

Main types of necrosis • Coagulative necrosis • Liquefactive necrosis • Caseation • Fat necrosis • Gangrene • Fibrinoid necrosis

Main types of necrosis

Grossly visible

• Coagulative necrosis • Liquefactive necrosis • Caseation • Fat necrosis • Gangrene • Fibrinoid necrosis

Coagulative necrosis • Most common form of necrosis, predominated by protein denaturation with preservation of the cell and tissue framework • This pattern is characteristic of hypoxic death in all tissues except the brain

Coagulative necrosis Anaemic infarct • Cause: occlusion of an end artery • In the heart, spleen, kidney • Gross: pale tissue, with well-defined boundaries; later it becomes yellowish because the lysosomal enzymes of the necrotized cells autodigest the infarcted area • Healing: by connective tissue replacement (fibrosis)

Haemorrhagic infarct

Coagulative necrosis Anaemic infarct • In the heart, spleen, kidney • Cause: occlusion of an endartery • Gross: pale, firm tissue, with well-defined boundaries; later yellowish because the lysosomal enzymes of the necrotized cells autodigest the infarcted area • Healing: by connective tissue replacement (fibrosis)

Haemorrhagic infarct • In the lungs • Cause: occlusion of a segm. pulmonary artery • The necrotized area undergoes secondary haemorrhage via bronchial arteries • Firm, wedge-shaped, pleural-based, haemorrhagic, airless focus • Healing: by fibrosis

Anaemic infarction of the myocardium, the margins are hyperaemic

The infarcted myocardial fibers are eosinophilic, there is no nuclear staining; neutrophilic granulocytes accumulated at the margins of infarction (vital sign)

Liquefactive necrosis: softening of the necrotic tissue due to action of hydrolytic enzymes released from • Dead cells, as in brain infarct • Healing: with glial scar

Liquefactive necrosis: softening of the necrotic tissue due to action of hydrolytic enzymes released from • Dead cells, as in brain infarct •Healing: with glial scar

• Neutr. granulocytes invading the tissue, as in an abscess • Healing: with fibrosis

Caseous necrosis • Distinctive form of coag. necrosis in foci of tuberculous infection • Gross: caseous necrosis is white and cheesy

The necrotic area is eosinophilic, amorphous, and is surrounded by epitheloid cells and Langhans’ giant cells. Healing: by fibrosis + calcification

Fat necrosis • Refers to necrosis in adipose tissue, induced by the action of lipases derived from injured pancreatic cells or macrophages • Lipases catalyse decomposition of triglycerides to fatty acids, which complex with calcium to create calcium soaps

Fat necrosis • Refers to necrosis in adipose tissue, induced by the action of lipases derived from injured pancreatic cells or macrophages • Lipases catalyse decomposition of triglycerides to fatty acids, which complex with calcium to create calcium soaps • Observed in the course of pancreatitis, or in traumatic injury of subcutis or breast • Healing: by fibrosis

Yellowish foci of enzymatic fat necrosis in acute pancreatitis

Gangraene • Gangraene results when putrefactive bacteria invade necrotic tissue • Three types (detailed later) • Dry gangraene: in the leg of patients suffering from atherosclerosis-related occlusion of the tibial arteries

Gangraene • Gangraene results when putrefactive bacteria invade necrotic tissue • Three types (detailed later) • Dry gangraene: in the leg of patients suffering from atherosclerosis-related occlusion of the tibial arteries • The affected tissues appear black because of the deposition of iron sulphide from degraded haemoglobin

Gangraene of the great toe

Fibrinoid necrosis • Limited to medium-sized and small arteries • The wall of these vessels undergo necrosis and is impregnated with fibrinogen and other plasma proteins • It can be recognized only in histologic slides • Observed in malignant hypertension, arteritis, rejection

Fibrinoid necrosis of small arteries, the necrotized SMCs are eosinophilic

Apoptosis: programmed cell death • A form of energy-dependent process for deletion of unwanted individual cells • Cell death occurs by activation of the internal suicide program

Apoptosis: programmed cell death • A form of energy-dependent process for deletion of unwanted individual cells • Cell death occurs by activation of the internal suicide program • Prevented or induced by a variety of stimuli • ⇓ Apo contributes to cell accumulation, e.g. neoplasia • ⇑ Apo results in extensive loss, e.g. atrophy

Inhibitors • Growth factors • Sexual steroids (e.g., testosteron)

Inducers • Growth factor withdrawal • Glucocorticoids • Injuries: - Viruses (hepatitis virus, HIV) - Free radicals - Ionising radiation - DNA damage

Intrinsic (mitochondrial) pathway of apoptosis

Mitochondrion Bcl-2 inhibits Bax activates

Execution caspases

When cells are deprived of survival signals or subjected to stress, anti-apoptotic Bcl-2 protein is lost from the mitochondrial membrane, and is replaced by pro-apoptotic Bax protein

Extrinsic (death receptor) pathway of apoptosis Mitochondrion Bcl-2 , Bax

Execution caspases

Death receptors Cytotoxic T-cells

If death receptors on the cell surface (TNF-R, FAS-R) cross-link with the ligand, activation of execution caspases occurs. Cytotoxic molecules derived from CD8+ T-cells directly activate these caspases

Execution pathway of apoptosis

Bax

Death receptors Cytotoxic (TNF, FAS) T-cells

Execution caspases: cascade of proteolytic enzymes

• Breakdown of cytoskeleton • Cell shrinkage • Chromatin condensation and fragmentation • Formation of apoptotic bodies

Apoptosis: cell shrinkage, and condensation of nucleus induced by cytotoxic T- lymphocytes

Adaptations Changes that occur in cells and tissues in response to prolonged stimulation or chronic injury • Atrophy • Hypertrophy • Hyperplasia • Metaplasia • Dysplasia (to be lectured later) • Intracellular accumulation of various substances

Atrophy • Decreased cell mass: reduction in size of cells (nucleus and cytoplasm), tissue, or organs. • Atrophied organs are smaller than normal. • Normal weight (g) of parenchymal organs: - spleen 150 - kidneys 150-150 - heart 300 to 350 - lungs 400-400 - brain 1300 - liver 1500

Physiologic atrophy - Involution of the thymus in adolescence - Senile atrophy in aging - Atrophy of female genitalia in menopause

Pathologic atrophy • Disuse. Muscles atrophy in people who do not use them (prolonged bed rest, immobilization of an extremity for healing of fracture) • Loss of innervation of skeletal muscle • Lack of trophic hormones in pituitary disease •

Pathologic atrophy • Disuse. Muscles atrophy in people who do not use them (prolonged bed rest, immobilization of an extremity for healing of fracture) • Loss of innervation of skeletal muscle • Lack of trophic hormones in pituitary disease • Ischaemia. Reduced blood supply leads to renal atrophy or atrophy of the brain • Malnutrition. Protein-energy deficiency cause atrophy of skeletal muscles, parenchymal organs, and general wasting (marasmus) • Increased pressure, e.g., hydrocephalus or hydronephrosis

Obstruction of the CSF flow leads to pressure atrophy of the brain, with the enlargement of ventricles: hydrocephalus

Hydronephrosis: obstruction of the ureter leads to sac-like dilation of renal pelvis and calyces, and pressure atrophy of parenchyma

Hydronephrosis: dilated calices, atrophied papillae, thinned parenchyma

Hypertrophy • An increased cell mass leading to an increased size of organs • Physiologic: hypertrophy of uterus in pregnancy, compensatory hypertrophy of the remnant kidney after unilateral nephrectomy (in both conditions, cell division is also present)

Hypertrophy • An increased cell mass leading to an increased size of organs • Physiologic: hypertrophy of uterus in pregnancy, compensatory hypertrophy of the remnant kidney after unilateral nephrectomy (in both conditions, cell division is also present)

• Pathologic: occurs e.g. in the muscles • Muscles are not able to divide, therefore an increased demand for action can be met only by enlarging the size of cells

Increased exercise leads to hypertrophy of muscles

Hypertrophy of heart, triggered by action of mechanical stimuli (⇑ workload) and vasoactive substances (e.g., angiotensin II). Free wall thickness: above 15 mm

Hypertrophy of the muscles of urinary bladder due to urethra obstruction

Hyperplasia • An increase in the size of a tissue or organ due to an increased number of constituent cells. The cells may have an increased volume. • Physiologic: hormonal hyperplasia: - proliferation of the glandular epithelium of the breast during lactation; - compensatory hyperplasia of liver after partial hepatectomy

Hyperplasia • An increase in the size of a tissue or organ due to an increased number of constituent cells. The cells may have an increased volume. • Physiologic: hormonal hyperplasia: - proliferation of the glandular epithelium of the breast during lactation; - compensatory hyperplasia of liver after partial hepatectomy

• Pathologic: due to hormonal stimulation - endometrial hyperplasia, induced by oestrogens - adrenal cortex hyperplasia, induced by ACTH - hyperplasia of prostate, induced by dihydrotestosterone, oestrogens and peptide growth factors - hyperplasia of thyroid, induced by anti-TSH antibodies

Metaplasia • Replacement of one mature cell type by another type. • E.g.: - Squamous metaplasia of the bronchus: chronic irritation-induced replacement of bronchial stratified columnar epithelium by squamous epithelium in smokers

Metaplasia • Replacement of one adult cell type by another adult cell type; reversible. • E.g., - Squamous metaplasia of the bronchus: chronic irritation-induced replacement of bronchial stratified columnar epithelium by squamous epithelium in smokers - Gastric metaplasia of the oesophagus: chronic irritation induced by gastric juices in gastrooesophageal reflux leads to the replacement of squamous epithelium by gastric epithelium • If the adverse circumstances persist, metaplasia may progress to dysplasia

Squamous metaplasia of the bronchus

Intracellular accumulations • Lipids - triglycerides, cholesterol • Proteins • Pigments

Accumulation of triglycerides • Most common in the liver, but also occurs in the heart; reversible • Fatty change/steatosis of liver: due to - alcohol abuse - morbid obesity - diabetes - protein-energy malnutrition - hypoxia - hepatotoxins • Biochemical pathways of uptake and metabolism of fatty acids by the liver, formation of triglycerides, and secretions of lipoproteins: not detailed here

Steatosis: the liver is enlarged, yellow and greasy, resembles to goose liver

The lipid molecules accumulate in large vacuoles

Frozen section, Oil Red O

Accumulation of cholesterols and cholesterol esters • In the intima of aorta and large arteries in atherosclerosis. • In macrophages - in hyperlipidaemia: collections of foamy macrophage produce yellowish nodules in the palpebra (xanthomas) -

Accumulation of cholesterols and cholesterol esters • In the intima of aorta and large arteries in atherosclerosis. • In macrophages - in hyperlipidaemia: collections of foamy macrophage produce yellowish nodules in the palpebra (xanthomas) - in cholesterolosis: foamy macrophages accumulate in the lamina propria of gallbladder -

Accumulation of cholesterols and cholesterol esters • In the intima of aorta and large arteries in atherosclerosis. • In macrophages - in hyperlipidaemia: collections of foamy macrophage produce yellowish nodules in the palpebra (xanthomas) - in cholesterolosis: foamy macrophages accumulate in the lamina propria of gallbladder - in cerebral infarction: macrophages phagocytosemembrane lipids derived from dead oligodendrocytes

Atheromatous plaque: the lipids are dissolved during normal histologic processing

The dissolved cholesterol crystals appear as cleftlike cavities

Foamy macrophages scavenge necrotic debris rich in lipids

Accumulation of proteins • Hyaline change: any alteration within cells that imparts a homogeneous, glassy pink appearance in H&E-stained histologic sections •

Accumulation of proteins • Hyaline change: any alteration within cells that imparts a homogeneous, glassy pink appearance in H&E-stained histologic sections • Intracellular: - Hyaline droplets in proximal tubular cells in heavy proteinuria - Mallory-hyaline in hepatocytes in alcoholic liver injury

Hyaline droplets in proximal tubular epithelial cells

Mallory-hyaline in chronic alcohol abuse

Accumulation of pigments • Exogeneous - Inhaled coal dust (black) - leading to anthracosis of lungs; stored in pulmonary macrophages - Pigments of tattooing, taken up by macrophages

Accumulation of pigments • Exogeneous - Inhaled coal dust (black) - leading to anthracosis of lungs; stored in pulmonary macrophages - Pigments of tattooing, taken up by macrophages • Endogeneous - Lipofuscin (brown), associated with tissue atrophy, in the myocardium of elderly people

Accumulation of pigments • Exogeneous - Inhaled coal dust (black) - leading to anthracosis of lungs; stored in pulmonary macrophages - Pigments of tattooing, taken up by macrophages • Endogeneous - Lipofuscin (brown), associated with tissue atrophy, in the myocardium of elderly people - Haemosiderin (brown), haemoglobin-derived intracellular pigment composed of aggregated ferritin, indicates previous haemorrhage. Systemic accumulation: termed haemosiderosis - Melanin (brown): product of naevus cells - Jaundice (icterus): systemic bilirubin retention; yellow skin and sclera discoloration

Pathologic calcification Abnormal deposition of Ca-salts in soft tissues Dystrophic In nonviable or dying tissues; the serum Ca++ level is normal. •Arteries in atherosclerosis •Damaged heart valves •Areas of various necrosis Precipitation of a crystalline Ca-phosphate starts with nucleation (initiation) on membrane fragments, followed by propagation of crystal formation

Pathologic calcification Abnormal deposition of Ca- salts in soft tissues Dystrophic In nonviable or dying tissues; the serum Ca++ level is normal. •Arteries in atherosclerosis •Damaged heart valves •Areas of various necrosis Precipitation of a crystalline Ca-phosphate starts with nucleation on membrane fragments, followed by propagation of crystal formation

Metastatic Results from hypercalcaemia: • ⇑ secretion of parathormone in hyperparathyroidism • Destruction of bones by myeloma, metastases, accelerated bone turnover, or immobilization [space travel] • Vitamin D-intoxication • Systemic sarcoidosis • In chronic renal failure ⇒ sec. hyperparathyroidism due to phosphate retention Deposits in the arteries, kidneys, lungs, and stomach

Dystrophic calcification: calcifying aortic stenosis

Metastatic calcification of arteries in ESRD Radial art. Ulnar art.

Bereczki Csaba, SZTE Pediatrics

Metastatic calcific deposits of the lung

The calcific deposits fill the alveolar spaces