FREE RADICAL INJURY, TYPES OF NECROSIS AND APOPTOSIS Dr. Mamlook Elmagraby

Objectives of the lecture: Upon completion of this lecture, students should be able to: Be aware of the concept of hypoxic cell injury and its major causes Understands the definitions and mechanisms of free radical injury Knows the definition of apoptosis, tissue necrosis and its various types with clinical examples Able to differentiate between necrosis and apoptosis

Consequences of cellular Injury

Causes of Cell and Tissue Injury: Hypoxia and Ischemia Physical agents Chemicals and drugs Infectious pathogens Immunologic reactions Genetic Defects Nutritional imbalances

Hypoxic Cell Injury Causes: Ischemia Decreased perfusion of tissues by oxygen-carrying blood (cardiac failure, hypotension, and shock) Anemia Carbon monoxide poisoning Poor oxygenation of blood secondary to pulmonary disease The susceptibility of cells to hypoxic injury varies with the tissue or cell type Intracellular enzymes and other proteins are released from necrotic cells into the circulation due to the loss of integrity of cell membranes (Indicators of necrosis) Hypoxia is a condition in which the body or a region of the body is deprived of adequate oxygen  supply at the tissue level. 

The functional and morphologic consequences of hypoxia and ischemia The functional and morphologic consequences of hypoxia and ischemia. ER, Endoplasmic reticulum.

Free Radical Injury Free radicals are unstable, highly reactive atoms or molecules that have an single unpaired electron in their outer orbit Free radicals initiate autocatalytic reactions; molecules that react with free radicals are in turn converted into free radicals The best-known free radicals are derived from oxygen and include the following: Superoxide (O2-) Hydrogen peroxide (H2O2) Hydroxyl radical (OH) Autocatalysis an increase in the rate of a chemical reaction in which a product of the reaction itself increase the rate.

The generation, removal, and role of reactive oxygen species (ROS) in cell injury. The production of ROS is increased by many injurious stimuli. These free radicals are removed by spontaneous decay and by specialized enzymatic systems. Excessive production or inadequate removal leads to accumulation of free radicals in cells, which may damage lipids (by peroxidation), proteins, and DNA, resulting in cell injury. SOD, Superoxide dismutase.

NECROSIS

Overview: Necrosis is one of two contrasting morphologic patterns of tissue death Necrosis is a form of cell death in which cellular membranes fall apart, and cellular enzymes leak out and digest the cell Necrosis is the summation of the degradative and inflammatory reactions occurring after tissue death Necrosis occurs within living organisms

Pathological changes in a dead cell Increased cytoplasmic eosinophilia occurs because of protein denaturation and loss of cytoplasmic RNA Nuclear changes (the morphologic hallmark of necrosis) include: Pyknosis, chromatin clumping and shrinking with increased basophilia Karyorrhexis, fragmentation of chromatin Karyolysis, fading of chromatin material Disappearance of stainable nuclei Dead cells may be replaced by large, phospholipid masses, (myelin figures) Which can be phagocytosed or degraded later

Morphological Patterns of Tissue Necrosis Coagulative Necrosis A form of tissue necrosis in which the component cells are dead but the basic tissue architecture is preserved In the end, the necrotic cells are removed Coagulative necrosis is characteristic of infarcts in all solid organs except the brain of infarcts in all solid organs except the brain

Coagulative necrosis.  A, A wedge-shaped kidney infarct (yellow).  B, Microscopic view of the edge of the infarct, with normal kidney (N) and necrotic cells in the infarct (I) showing preserved cellular outlines with loss of nuclei and an inflammatory infiltrate (seen as nuclei of inflammatory cells in between necrotic tubules).

Morphological Patterns of Tissue Necrosis Liquefactive Necrosis After the death of cells, liquefaction is caused by autolysis Digestion, softening, and liquefaction of tissue are characteristics, resulting in transformation of the tissue into a liquid viscous mass Hypoxic death of cells within the CNS induces liquefactive necrosis Suppurative infections characterized by the formation of pus (liquefied tissue debris and neutrophils)

Liquefactive necrosis Liquefactive necrosis. An infarct in the brain, showing dissolution of the tissue

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.

Morphological Patterns of Tissue Necrosis Caseous necrosis (cheesy) The term "caseous" (cheese-like) is derived from the friable yellow-white appearance of the area of necrosis The necrotic focus appears as a collection of fragmented cells with an amorphous granular appearance Unlike coagulative necrosis the tissue architecture is completely obliterated cellular outlines cannot be detected It is seen most often in foci of tuberculous infection

A tuberculous lung with a large area of caseous necrosis A tuberculous lung with a large area of caseous necrosis. The caseous debris is yellow-white and cheesy Typical tuberculous granuloma showing an area of central necrosis, epithelioid cells, multiple Langhans-type giant cells, and lymphocytes

Morphological Patterns of Tissue Necrosis Fat Necrosis It occurs in two forms. Traumatic fat necrosis, which occurs after a severe injury to tissue with high fat content (breast) Enzymatic fat necrosis, which is a complication of acute hemorrhagic pancreatitis The foci of necrosis contain unclear outlines of necrotic fat cells basophilic calcium deposits An inflammatory reaction

Omentum, fat necrosis in a case of pancreatitis - Gross  The chalky white areas in the omentum represent calcium combined with free fatty acids that were released upon digestion of intracellular lipids by lipase released from the injured pancreas Peripancreatic adipose tissue, fat necrosis in a case of acute pancreatitis - Low power  The pinkish-blue, somewhat granular material in the cytoplasm of some of the necrotic fat cells represents calcium deposition.

Morphological Patterns of Tissue Necrosis Fibrinoid (fibrin-like) necrosis It is usually seen in immune reactions involving blood vessels This pattern of necrosis is prominent when complexes of antigens and antibodies are deposited in the walls of arteries Deposits of these "immune complexes," together with fibrin result in a bright pink and amorphous appearance

APOPTOSIS

Apoptosis Apoptosis is a second morphologic pattern of tissue death Apoptosis is a pathway of cell death in which cells activate enzymes that degrade the cells’ own nuclear DNA and nuclear and cytoplasmic proteins Apoptosis is a programmed cell death that can result from endogenous or exogenous activation of cell suicide genetic pathways When the cell's DNA or proteins are damaged beyond repair, the cell kills itself by apoptosis Apoptosis is active, energy-dependent, regulated cell death type

Apoptosis initiation The two most important pathways are: Extrinsic pathway: This pathway is initiated by activation of the death receptors on the surface of the cell membrane Ligands for these receptors are proteins such as Fas ligand Intrinsic pathway: This pathway is initiated by an increased permeability of mitochondria, which release proapoptotic molecules, such as cytochrome “c” Cytochrome act on the initiator caspases The intrinsic, or mitochondrial, pathway, which is initiated by the loss of stimulation by growth factors and other adverse stimuli, results in the inactivation and loss of bcl-2 and other antiapoptotic proteins from the inner mitochondrial membrane. This loss results in increased mitochondrial permeability, the release of cytochrome c. Cytochrome c interacts with Apaf-1 causing self-cleavage and activation of caspase-9. a ligand is a substance that forms a complex with a biomlecule  to serve a biological purpose

Mechanisms of apoptosis The initial signal on the cell membrane or from the mitochondria activates the initiator caspases, which act on execution caspases Activated execution caspases act on enzymes, nucleic acids, and the cytoskeletal proteins the fragmentation of the nucleus and cytoplasm into membrane-bound apoptotic bodies Apoptotic bodies are phagocytized by neighboring cells or macrophages Caspases are aspartate-specific cysteine proteases The initial activating caspases are caspase-8 and caspase-9 The terminal caspases (executioners) include caspase-3 and caspase-6 Downstream caspases are activated by upstream proteases and act themselves to cleave cellular targets

Mechanisms of apoptosis. The two pathways of apoptosis differ in their induction and regulation, and both end in the activation of “executioner” caspases. The induction of apoptosis by the mitochondrial pathway involves the action of sensors and effectors of the Bcl-2 family, which induce leakage of mitochondrial proteins. Also shown are some of the anti-apoptotic proteins (“regulators”) that inhibit mitochondrial leakiness and cytochrome c–dependent caspase activation in the mitochondrial pathway. n the death receptor pathway engagement of death receptors leads directly to caspase activation. ER, endoplasmic reticulum; TNF, tumor necrosis factor

Feature Necrosis Apoptosis Cell size Enlarged (swelling) Reduced (shrinkage) Nucleus Pyknosis karyorrhexis karyolysis Fragmentation Plasma membrane Disrupted Intact; altered structure Cellular contents Enzymatic digestion; may leak out of cell Intact; may be released in apoptotic bodies Adjacent inflammation Frequent No Physiologic or pathologic role Always pathologic Often physiologic, means of eliminating unwanted cells; may be pathologic