Cell Death Dr (Prof) Vishal Saxena MBBS, MD(Path)

Cell Death Some important terms ! Autolysis: degradation of cell and its constituents caused by its own enzymes. Heterolysis: degradation of cell and its constituents by enzymes derived from sources extrinsic to the cell (e.g. neutrophils, macrophages). Putrefaction: lysis of dead tissue by bacterial enzymes. Irreversible injury inevitably leads to cell death. Types of cell death: Necrosis Apoptosis

Necrosis (Greek - Nekrosis = deadness) is defined as the spectrum of gross and light-microscopic appearances that indicate cell death in living tissue, Caused by action of hydrolytic enzymes on the injured cells. Is accompanied by inflammation. Cell expands and nucleus becomes pyknotic ( or is lost) Always pathological.

How do you tell a cell is dead?? Necrotic cells show certain changes: Cytoplasm becomes hypereosinophilic due to:. Loss of RNA Binding of eosin to coagulated proteins Loss of glycogen: Cell smaller, more pink. Nuclear changes: Karyorrhexis, Karyolysis and pyknosis. After these early changes, mass of necrotic cells may have different appearances. Appearance depends upon two processes: Denaturation of proteins Digestion by enzyme (Enzymatic digestion) If Denaturation is predominant  coagulation necrosis develops If enzymatic digestion is predominant  liquefactive necrosis develops.

Morphologic types of necrosis Coagulation necrosis Liquefactive necrosis Caseous necrosis Fat necrosis Traumatic Enzymatic Gangrenous necrosis Gummatous necrosis

Coagulative necrosis Is secondary to denaturation and coagulation of structural and enzymatic proteins due to intracellular acidosis. Denaturation of lysosomal enzymes by intracellular acidosis prevents autolysis. Preserving cell outlines and tissue architecture. Most common cause: ischemia (secondary to atherosclerosis thrombus formation). Also produced by denaturation of proteins in Heavy metal poisoning (lead) and Irradiation. Commonly seen in solid organs like: Heart, kidney, spleen

Infarction: Refers to a localized area of tissue necrosis resulting from loss or reduction in blood supply (= ischemic necrosis). The dead tissue is called an infarct. Coagulation necrosis is the type of necrosis associated with infarction except in infarction of brain. In Brain: Lack of good structural support and cells rich in lysosomal enzymes and lipid result in liquefactive necrosis.

Coagulation necrosis GROSS APPEARANCE: Dead tissue: appears dense and dry Cut surface: white-grey MICROSCOPY: Loss of the nucleus but preservation of cellular shape. Increased cytoplasmic eosinophilia.

Preservation of cellular shape. Area of necrosis Area of Necrosis Preservation of cellular shape. In this gross photograph of the heart , note the area of pale gray myocardial infarction (arrows) in the anterior portion of the left ventricle and extending into the anterior portion of the interventricular septum. This high-power photomicrograph contains normal myocytes (1), vacuolated myocytes (2), and infarcted myocytes (3). Increased cytoplasmic eosinophilia

Liquefaction necrosis Definition: necrosis with complete loss of cell and tissue structure. Primarily results due to Destruction of dead cells by hydrolytic enzymes. Enzymes derived from either : Cell’s own lysosomes (autolysis) or from neutrophils and macrophages (heterolysis).

Liquefaction necrosis Seen in Suppurative bacterial infections: characterized by formation of pus (liquefied necrotic debris and neutrophils). In ischemic injury to brain tissue and abscess of brain. Enzymatic destruction of brain tissue leaves behind a cystic cavity. Gross appearance Necrotic area: soft and filled with fluid. Microscopy: Structure-less tissue debris (no cellular outlines) Neutrophils NOTE: Liquefaction by leukocyte enzyme is called Suppuration and the resultant fluid is called pus

Lung Abscesses Necrotic material with loss of cellular architecture The two lung abscesses seen here are examples of liquefactive necrosis in which there is a liquid center in an area of tissue injury. One abscess appears in the upper lobe and one in the lower lobe. Liquefactive necrosis is typical of organs in which the tissues have a lot of lipid (such as brain) or when there is an abscess with lots of acute inflammatory cells whose release of proteolytic enzymes destroys the surrounding tissues. Necrotic material with loss of cellular architecture Lung Abscesses

Neutrophils Necrotic tissue

liquefactive necrosis Cerebral infarction: liquefactive necrosis Grossly, the cerebral infarction at the upper left here demonstrates liquefactive necrosis. Eventually, the removal of the dead tissue leaves behind a cavity. At high magnification, liquefactive necrosis of the brain demonstrates many macrophages at the right which are cleaning up the necrotic cellular debris. The job description of a macrophage includes janitorial services such as this, particularly when there is lipid.

Coagulative Vs liquefactive necrosis Coagulative and liquefactive necrosis. A, Kidney infarct exhibiting coagulative necrosis, with loss of nuclei and clumping of cytoplasm but with preservation of basic outlines of glomerular and tubular architecture. B, A focus of liquefactive necrosis in the kidney caused by fungal infection. The focus is filled with white cells and cellular debris, creating a renal abscess that obliterates the normal architecture. Coagulative necrosis Focus of liquefactive necrosis

Caseous necrosis Definition: a distinct form of necrosis without preservation of cellular outlines and tissue architecture, but firm in consistency. A combination of Coagulative and liquefactive necrosis. Most commonly associated with Formation of granulomas in tuberculosis and Systemic fungal infection like histoplasmosis.

Yellowish white, “cheese like” material. Cheese like appearance GROSS: Yellowish white, “cheese like” material. Cheese like appearance due to release of lipid from the cell walls of M.tuberculosis and systemic fungi (histoplasma). (Latin “caseous” – cheese) MICRO: Eosinophilic material surrounded by inflammatory cells, activated macrophages, multinucleated giant cells and helper T cells (= a granuloma). Granuloma: a lesion seen in conditions referred to as granulomatous disease. Examples of granulomatous diseases: tuberculosis systemic fungal infections like Histoplasmosis leprosy sarcoidosis berylliosis The main cell which forms the granuloma is modified macrophage (also known as epithelioid cell). In other words granuloma is a localized collection of modified macrophages or epithelioid cells. Granulomas may be associated with caseous necrosis (caseating granulomas) : e.g. tuberculosis, histoplasmosis or May no be associated with caseous necrosis (non caseating granulomas) e.g. sarcoidosis.

Caseous necrosis Lymphnode: histoplasmosis Lung: tuberculosis B B: Lymph node, mediastinal, histoplasmosis - Gross, cut surface  The caseous necrosis in this case of fungal infection has a white, cheese-like appearance. Caseous necrosis has a similar appearance, irrespective of the tissue involved. B Lymphnode: histoplasmosis Lung: tuberculosis

Caseous necrosis Langhan’s giant cells Lung, pulmonary tuberculosis - Low power : The area of caseous necrosis appears as amorphous, granular debris without preservation of the tissue architecture. At its edge, granulomatous inflammation with multinucleated giant cells is apparent. Lung, pulmonary tuberculosis - High power  Caseous nodules are pink and structureless, and unlike coagulation necrosis, architecture and cell outlines have been largely destroyed. Once again, Langhans giant cells are prominent Langhan’s giant cells

Granuloma Epithelioid cell Langhan’s giant cell

Enzymatic fat necrosis Definition: a focal areas of fat destruction. Occurs due to action of pancreatic enzyme on fatty tissue located around pancreas. Occurs as a complication of Acute pancreatitis. Acute pancreatitis: Release of lipases and amylase from pancreas. Lipases break triglycerides into fatty acids (FA). FA combine with Ca++ via the process of saponification to form chalky white calcified deposits in the pancreas as well as in the omental fat. Precipitating factors for Ac. Pancreatitis: Alcoholism.

Enzymatic fat necrosis Clinical significance: Deposits visible on abdominal X ray Marker of pancreatitis. Consequence of extensive enzymatic fat necrosis Tetany (due to lowering of the Ca levels) Gross appearance Chalky white calcium deposits (calcium soaps) Pancreas and adjacent tissues Microscopy: Necrotic fat cells – pale outlines. Cytoplasm filled with basophilic staining calcified material. Hematoxylin stains calcium blue.

Area of necrosis with blue discoloration White nodules on the surface of pancreas Foci of fat necrosis with saponification. The areas of white chalky deposits represent calcium soap formation at sites of lipid breakdown. Area of necrosis with blue discoloration

Normal Fat tissue Vs. Fat necrosis Left: control; right: fat necrosis: the cellular outlines vaguely remain, the fat cells have lost their peripheral nuclei and their cytoplasm has converted to a mass of basophilic amorphous necrotic material.

Traumatic fat necrosis Differs from enzymatic fat necrosis by being Secondary to trauma. Trauma to fatty tissue  acute inflammatory reaction (neutrophils)  healing by fibrous tissue and dystrophic calcification. Commonly seen in women with pendulous breasts. Clinical significance: Scar tissue feels firm, retracts the overlying skin and shows calcifications on mammography. “these findings also seen in breast cancer”. Traumatic fat necrosis of breast: differential diagnosis breast cancer.

A famous surgeon went on a safari in Africa A famous surgeon went on a safari in Africa. When he came back, his colleagues asked him how it had been. 'Oh, it was very disappointing,' he said. 'I didn't kill a thing. I’d have been better off staying here in the hospital.'

Gangrenous necrosis Most often occurs in lower limbs and bowel secondary to loss of blood supply. Two types: Dry gangrene: Is a form of infarction that results from ischemia. Characterized primarily by coagulative necrosis without liquefaction. Dead tissue has mummified appearance. Characteristic finding in diabetic foot. Dry gangrene If the blood flow is interrupted for a reason other than severe bacterial infection, the result is a dry gangrene. Persons with impaired peripheral blood flow, such as diabetics, are at greater risk for dry gangrene. The early signs of dry gangrene are a dull ache and sensation of coldness in the area, along with pallor of the flesh. If caught early, the process can sometimes be reversed by vascular surgery. However, if necrosis sets in, the affected tissue must be removed just as with wet gangrene.

Dry Gangrene A A: This is gangrene, or necrosis of foot. In this case, the toes were involved in a frostbite injury. This is an example of "dry" gangrene in which there is mainly coagulative necrosis from the anoxic injury. B: Foot, dry gangrene - Clinical presentation  Gangrenous necrosis usually implies coagulative necrosis and is commonly seen in a limb that has lost its blood supply. Wet gangrene is necrosis combined with bacterial infection.   B

Gangrenous necrosis Wet gangrene: Refers to necrosis with superimposed bacterial infection. Liquefactive necrosis is the primary type of necrosis in wet gangrene. Associated Acute inflammatory reaction leads to Ulceration Gas production and Oozing of purulent material.

This is gangrene of the lower extremity This is gangrene of the lower extremity. In this case the term "wet" gangrene is more applicable because of the liquefactive component from superimposed infection in addition to the coagulative necrosis from loss of blood supply. This patient had diabetes mellitus. Wet Gangrene

Gummatous necrosis Refers to a type of coagulative necrosis seen in tertiary syphilis. It is associated with formation of Gummas (soft rubbery masses composed of plasma cells, macrophages, lymphocytes etc.) Gummas are very destructive lesions and can be found in any organ, esp. in skin and bones. Necrosis in syphilis is due to endarteritis obliterans (the arterial lumen is obliterated leading to ischemic damage to tissue and necrosis).

Apoptosis From Gk. Meaning falling off Is genetically controlled programmed single cell death by activation of particular enzyme pathways. Helps eliminate unwanted cells. Plays role in embryogenesis, normal physiologic and pathologic cell death. Different from necrosis!! Absence of inflammation Cell shrinks and nucleus becomes pyknotic An active process and involves RNA and protein synthesis. Apoptosis: refers to individual cell death without an inflammatory response. Main function is to remove unwanted cell selectively with minimal disturbance to surrounding cells.

Remember ! IN APOPTOSIS: Cells actually expend energy in order to die. The cell membrane does not rupture, The cell contents are not released into the extracellular space, and Inflammation does not occur.

Physiologic examples of apoptosis Embryogenesis Disappearance of Mullerian and Wolffian duct structures. Development of lumen within hollow organs (e.g bowel and heart) Hormone-dependent involution in adults Endometrial breakdown in menstruation Post-lactational atrophy of breast Prostate atrophy following castration. Involution of Thymus in the adult. Cells that are programmed to die; for example, the cells of the outer layers of epidermis, cells in the gut epithelium. Destruction of inflammatory cells

Pathologic examples of apoptosis Toxin induced injury Diphtheria toxin  myocarditis Councilman bodies= dead hepatocytes in viral hepatitis. Psammoma bodies: apoptosis of neoplastic cell with subsequent calcification. Tumor cell death by cytotoxic T cells Neurons that are lost in Alzheimer's disease HIV-positive T-lymphocytes die by apoptosis. Psamomma bodies: seen in Papillary carcinoma of thyroid papillary cystadenocarcinoma of the ovary meningioma

Mechanism Divided into 3 phases Initiation phase: Execution phase: Removal of dead cell:

Initiation phase Characterized by activation of caspases. Activation of caspases occurs by a death signal from: Outside the cell (Extrinsic or receptor initiated pathway) Inside the cell (Intrinsic or mitochondrial pathway) Regulated by genes Death signal

Initiation phase A: Extrinsic pathway: Receptor-ligand interactions: Binding of Tumor necrosis factor (TNF) to its receptor(TNFR1) Fas binding to the Fas ligand. Result: activation of caspases B: Intrinsic pathway: Withdrawal of hormones or growth factors Damage to DNA: viruses, radiation, free radicals. Result: increased permeability of mitochondrial membrane  release of cytochrome c  activate caspases

Initiation phase Whatever the signal for apoptosis ; the ultimate result is activation of enzymes k/a caspases. Caspases Death Signal

Execution of apoptosis Mediated by caspases (group of proteases). Caspases: Activate enzymes endonuclease and protease Activation of proteases: leads to breakdown of the cytoskleton. Activation of endonucleases: DNA degradation (size- 180-200 base pairs)  laddering appearance of DNA on electrophoresis.

Execution phase Endonuclease Caspases Protease Death Signal

Execution phase Result is: DNA breakdown Cytoplasmic buds forming apoptotic bodies. Endonuclease DNA breakdown, Cytoplasm budding = apoptotic bodies Caspases Protease Death Signal

Lane A: Control Lane B: Apoptosis Lane C: Necrosis Ladder pattern of DNA DNA electrophoresis

Removal of dead cells Formation of cytoplasmic buds on the cell membrane containing Nuclear fragments, mitochondria and protein fragments. Breaking off of cytoplasmic buds  apoptotic bodies. Phagocytosis of apoptotic bodies by neighboring cells or macrophages.

Proapoptotic molecules Formation of apoptotic bodies Receptor ligand Interaction FAS TNF receptor Withdrawl of GF and Hormones Proapoptotic molecules Cytochrome c Adaptor proteins Initiator caspases Injury Rad,FR,Toxin (DNA damage) Execution caspases Activation of Endonuclease & proteases Cytoskeletal breakdown DNA fragmentation Formation of apoptotic bodies Phagocytosis

Apoptosis is regulated by genes Bcl-2 gene (inhibits apoptosis) Manufactures gene products that inhibit apoptosis by Preventing release of cytochrome c from mitochondria p53 : Tumor suppressor gene (Stimulates apoptosis). Elevated by DNA injury and arrests the cell cycle to repair DNA damage. If DNA repair is impossible p53 stimulates apoptosis by activating BAX apoptosis gene. Bax: stimulates apoptosis

Morphologic appearance Apoptotic cells: Have deeply pink staining cytoplasm. Have pyknotic nucleus which fragment. Are smaller in size Breakdown into fragments (apoptotic bodies)

Apoptosis of epidermal Apoptotic cell in liver A, Apoptosis of epidermal cells in an immune-mediated reaction. The apoptotic cells are visible in the epidermis with intensely eosinophilic cytoplasm and small, dense nuclei. H&E stain. B, High power of apoptotic cell in liver in immune-mediated hepatic cell injury. Apoptosis of epidermal cells Apoptotic cell in liver

Apoptotic cell with eosinophilic Cytoplasm

Councilman bodies Apoptosis is a more orderly process of cell death in which there is individual cell necrosis, not necrosis of large numbers of cells. In this example, liver cells are dying individually (arrows) from injury by viral hepatitis. The cells are pink and without nuclei.

What if apoptosis is too little or too much!! = dysregulated apoptosis Two groups of disorders: Decreased apoptosis with increased cell survival can give rise to: Cancers Autoimmune disorders. Increased apoptosis with decreased cell survival: Neurodegenerative diseases Death of virus infected cells Lymphocyte depletion as in AIDS

A pipe burst in a doctor's house. He called a plumber A pipe burst in a doctor's house. He called a plumber. The plumber arrived, unpacked his tools, did mysterious plumber-type things for a while, and handed the doctor a bill for $600. The doctor exclaimed, "This is ridiculous! I don't even make that much as a doctor!." The plumber waited for him to finish and quietly said, "Neither did I when I was a doctor."

Apoptosis Vs. Necrosis

The sequential ultrastructural changes seen in necrosis (left) and apoptosis (right). In apoptosis, the initial changes consist of nuclear chromatin condensation and fragmentation, followed by cytoplasmic budding and phagocytosis of the extruded apoptotic bodies.

Apoptosis Vs. Necrosis Feature Necrosis Apoptosis Cell size Enlarged (swelling) Reduced (shrinkage) Nucleus Pyknosis  karyorrhexiskaryolysis Fragmentation Cellular contents Enzymatic digestion ; may leak out of cell Intact, may be released in apoptotic bodies.

Feature Necrosis Apoptosis Adjacent inflammation Frequent No Physiologic or pathologic role Always pathologic Often physiologic, may be pathologic

Enzyme markers of cell death

Enzyme markers of cell death Diagnostic use Aspartate aminotransferase (AST) Marker of diffuse liver cell necrosis (e.g. viral hepatitis) Also increased in alcohol induced liver disease Alanine aminotransferase (ALT) More specific for liver cell necrosis than AST

Enzyme markers of cell death Diagnostic use Creatine kinase MB (CK-MB) Elevated in AMI or myocarditis Marker enzyme for acute pancreatitis. Lipase more specific than amylase for pancreatitis. Amylase also increased in salivary gland inflammation (e.g. mumps) Amylase and lipase

Self study : extra images

Gross appearance: Caseous necrosis Necrotic area

Microscopic appearance of caseous necrosis

Caseous Necrosis

Granulomas

Granuloma 3 1 2 Granuloma: 1: modified macrophage = epithelioid cell 2: Langhans giant cell 3: rim of lymphocytes

Kidney infarct showing coagulation necrosis Preservation of tissue architecture. Kidney infarct showing coagulation necrosis Kidney infarction showing coagulation necrosis with preservation of tissue architecture.

Splenic infarction: coagulation necrosis Splenic infarction showing coagulation necrosis

General Practitioners know nothing and do little General Practitioners know nothing and do little. Surgeons know little and do everything. Internists knows everything and do nothing. Pathologists know everything and can do everything, but it's usually too late.

She told me that she loved me She said her love was true But what is the use in loving When “Path” has got you blue?

Thank You !