Chapter 3

CELLULAR INJURY AND DEATH

 

 

 

        

 3.1 AN OVERVIEW:

At this time, the student has gained an appreciation for ways in which the normal cell can adapt to abnormal environmental influences. The cellular adaptation responses (atrophy, hypertrophy, hyperplasia, metaplasia) allow the cell to survive by achieving an altered but steady state. The adapted cell is not injured, ill or dead. It may revert back to a normal state if the abnormal environmental influences are removed. On the other hand, the cell may become ill, or even die, if the intensity of the abnormal environmental influences is increased sufficiently. In this section, reversible and irreversible cellular injury are considered in detail. Additional suggested references sources are:

References

  • -- Veterinary Pathology: Jones and Hunt; 5th Edition, 1983. (Refer to the
  • appropriate topics in Chapters 1 and 2, pages 1 thru 62).
  • -- Pathologic Basis of Disease: Robbins and Cotran; 2nd Edition, 1979. (Refer to the appropriate topic in Chapter 2; pages 22 thru 53).
  • -- Cell Pathology: Cheville; 2nd Edition, 1983. (Refer to Chapter 3).
  • -- General Veterinary Pathology: Thompson; 1978. (Refer to Chapter 2).

 3.2 TERMINAL OBJECTIVES:

At the completion of this section, each student should be able to perform the following tasks.

 3.3 KEY WORDS:

Please attempt to define, splee and use the following terms, etc., before and after embarking on a study of the sick and dead cell.

  

3.4 REVERSIBLE CELLULAR INJURY (The Sick or Ill Cell) 

3.4.1 GENERAL CONSIDERATIONS:

If the limits of adaptive capabilities are exceeded, the affected cell may become ill (reversible cellular injury) or even die (irreversible cellular injury). Actually, cellular adaptation, reversible cellular injury, and irreversible cellular injury are three states along a continuum of progressive encroachment on the cell's homeostasis. However, the manner in which a cell reacts to a specific injurious agent depends on varying factors, including the type, duration and severity of the injury (i.e., a cell may die acutely without becoming ill or adapting to the adverse influence).

Even though reversible cellular injury is one of the most common responses in disease, large gaps still exist in our knowledge and understanding of the process. Regardless, cells react to injurious agents in a limited number of ways. As mentioned earlier, damage to one biochemical or metabolic function may affect one type of organelle first, but eventually, other functions and organelles are involved. Therefore, whatever the primary target, in time, all forms of injury, if sustained, will extend to involve the entire cell.

Remember, the ill or sick cell is not a dead cell; it can revert back to a normal state or to an adapted state (spontaneously or after treatment with appropriate drugs, etc.). 

 

3.4.2 ULTRASTRUCTURAL CHANGES IN THE SICK OR ILL CELL:

In the ill cell, prominent morphologic or functional changes are usually associated with the cell membrane, mitochondria, endoplasmic reticulum and lysosomes; nuclear changes are minimal. Significant ultrastructural alterations that occur in the ill or sick cell are summarized.

3.4.3 PLASMA MEMBRANE:

The plasma membrane is involved (primarily or secondarily) in virtually all forms of injury. Very early in the cell's response to injury, no ultrastructural alterations are observed; however, biochemical studies disclose increased cell membrane permeability which is reflected by increased intracellular accumulation of sodium, water and calcium ions, as well as by a leakage of potassium, enzymes and cofactors. Later in the cell's response to injury, distortion of microvilli, blebs, and vesicles occur in the membrane. Still later, breaks are seen in both the plasma membrane and the membranes enclosing the organelles.

3.4.4 MITOCHONDRIA (Figure 2.29 of your textbook):

The mitochondria are almost always altered when the cell is injured. Alterations occur rapidly after hypoxic injury but are delayed in many forms of chemical injury (the following refers to hypoxic injury). The earliest response to hypoxic injury is condensation of the mitochondrial matrical proteins (associated with a loss of ATP). However, this is quickly followed by swelling of mitochondria (related to increased cell membrane permeability). With progressive injury, the mitochondrial matrix becomes translucent.

Remember, flocculation of mitochondrial matrical proteins correlates with the onset of cell death; this is the earliest absolute sign of cell death.

3.4.5 ENDOPLASMIC RETICULUM (Figure 2.18 of your textbook):

The endoplasmic reticulum responds very early when the cell is injured; it swells due to an intake of water. This swelling is followed by detachment of ribosomes and desegregation of polysomes resulting in decreased synthesis of protein. Subsequently, progressive fragmentation of the endoplasmic reticulum occurs.

Remember, swelling of the endoplasmic reticulum is usually the first manifestation of cell injury. 

3.4.6 LIGHT MICROSCOPIC CHANGES IN THE SICK OR ILL CELL (Figures 2.13 and 2.35; See Figure 2.28 for an overview)

As viewed with the light microscope, the alterations that develop within a sick cell are found principally in the cytoplasm. The nucleus is unaffected except for some clumping of chromatin against the nuclear membrane. Two patterns of reversible cellular injury can be recognized, "cellular swelling" and "fatty change." Cellular swelling (also referred to as cloudy swelling) occurs whenever the cell is unable to maintain ionic and fluid homeostasis. It reflects an excessive accumulation of water in the cell's cytoplasm. Fatty change refers to an abnormal accumulation of fat within parenchymal cells. It occurs principally in those cells involved in and dependent on fat metabolism. Fat accumulates in the cell's cytoplasm because the sick cell is unable to utilize or export fat that it normally receives. The term hydropic change is commonly used to refer to advanced stages of cellular swelling. Cellular swelling is an early manifestation of cellular illness whereas fatty change represents a more severely ill cell. However, cellular swelling and fatty change are reversible; affected cells may revert to a normal or adapted state (with treatment, etc.) or they may progress to the point of cellular death.

3.4.7 CELLULAR SWELLING:

Cellular swelling refers to an increase in cell size due to increased intracellular accumulation of water. It is the earliest or first manifestation viewed with the light microscope in almost all sick or ill cells. In the early stages, water tends to accumulate in the endoplasmic reticulum; later, all cytoplasmic structures are involved. As viewed with the light microscope, the involved cell is swollen and its cytoplasm has a cloudy, indistinct, ground-glass appearance. If an extensive amount of water accumulates, small clear vacuoles appear in the cytoplasm. This pattern is oftentimes referred to as hydropic change. As viewed with the naked eye, the involved organ may appear enlarged and pale (cellular swelling is difficult to appreciate on gross inspection). The ultrastructural alterations are the same as those described for the sick or ill cell.

Remember, cellular swelling or hydropic change occurs when an injurious agent causes disruption of controls for ionic and osmotic homeostasis at the cell membrane level, resulting in increased permeability. The so-called sodium-potassium pump is deranged and sodium concentration tends to equalize on both sides of the cell membrane; water passively follows sodium into the cell resulting in cellular swelling. Significant functional defects may not develop in involved cells. Cellular swelling is an indicator of mild injury and it is of primary importance as a possible incindence to more severe cellular injury (fatty change or even cell death).

"Cellular swelling is a difficult morphologic change to appreciate with the light microscope since early postmortem autolysis results in a similar appearance or pattern. Actually, cellular swelling is more readily recognized ultrastructurally and paradoxically, on gross examination of the whole organ than under light microscopy."

3.4.8 FATTY CHANGE:

"Fatty change" refers to an abnormal accumulation of fat within parenchymal cells. It occurs most frequently in those cells involved in, or dependent on, fat metabolism (cells of the liver, heart and kidneys). Fat accumulates in the cytoplasm of a "sick" or "ill" cell because such a cell is unable to metabolize, utilize and/or export the normal levels of lipids it receives on a daily basis. Fatty change may be preceded by "cellular swelling" and is, therefore, a morphologic expression of reversible cellular injury.

"In addition to its accumulation in the sick or ill cell, fat may accumulate within cells which are initially normal. The normal cell may synthesize and store excessive quantities of fat when presented with excess substrate. This occurs primarily in hepatic cells of obese animals. In other organs and tissues (heart, pancreas, etc.) the fat accumulates within adipose cells in connective tissue stroma when presented with excess substrate."

As viewed with the light microscope (utilizing routine hematoxylin and eosin stain) fat appear as small or large empty vacuoles within the cell's cytoplasm. Grossly, the involved organ is enlarged, friable, pale and the surfaces bulge when incised. Ultrastructurally, fatty change begins with the development of minute liposomes or membrane-bound inclusions which are closely applied and probably derived from endoplasmic reticulum.

In the liver, fatty change is manifested first by the appearance of small vacuoles in the cytoplasm near the nucleus. As the process progresses, the vacuoles coalesce and the larger vacuoles displace the nuclei to the periphery (hepatic cells may resemble adult adipose cells). In severe cases, the liver will float in water (fat lowers the surface tension). In the kidneys, fatty change is most prominent in the proximal convoluted tubules and the ascending limb of Henle's loop; however, all renal epithelial cells may be involved. Under light microscopy, the cytoplasmic fat droplets are small and indistinct. They are usually over-looked until demonstrated with special stain. In cats, large amounts of fat are commonly observed in the renal tubular epithelial cells, and their kidneys are quite pale grossly. In the heart, fat appears as very fine droplets within the cytoplasm of myocytes. These fat droplets are not easily detected unless a suitable special stain is employed. Grossly, the fat within myocytes cannot be judged accurately.

"Remember, fat appears as clear cytoplasmic vacuoles within parenchymal cells when routine hematoxylin and eosin stain is used in preparing microscopic slides. Likewise, intracytoplasmic accumulations of water and glycogen appear as clear vacuoles when hematoxylin and eosin is employed. Therefore, it becomes necessary to resort to special stains to distinguish between these three types of clear vacuoles. To identify cytoplasmic fat, it is necessary to prepare frozen tissue sections on fresh or formalin fixed tissues in order to preserve the lipid content of the vacuoles. These sections are then stained with a suitable fat stain (Oil-Red-O, Sudan II, Sudan IV, etc.). To identify glocygen, Best Carmine stain is commonly employed. When neither fat nor glycogen can be demonstrated within a clear vacuole, it can be assumed that water or fluid with a low protein content is present (water does not stain)."

3.4.9 STROMAL FATTY INFILTRATION:

Stromal fatty infiltration refers to the deposition of lipids in the cytoplasm of adipose tissue cells found among interstitial connective tissue cells throughout the body. If the amount of stromal fat is excessive, "obesity" is used as a descriptive term. This is not considered to be cellular injury.

NOTE: At this time, the student is familiar with the alterations that develop in "sick cells" or in reversible cellular injury. Remember, sick cells may recover spontaneously or they may recover if the injurious agent is removed via the treatment with drugs, etc. Unfortunately, however, cell death may ensue and under such circumstances, lesions and/or disease may be reflected in the body as a whole. 

 

3.5 IRREVERSIBLE CELLULAR INJURY (The Dead Cell) 

3.5.1 GENERAL CONSIDERATIONS:

Cellular death is oftentimes an unfortunate extension of cell injury. A dead cell cannot revert to a normal, adapted or ill state; it is doomed to be degraded. Remember, a cell may or may not exhibit cellular swelling, fatty change, etc., prior to death (depending on severity of injury). The point at which cell death occurs is still largely undetermined. Neither biochemical nor morphological studies can predict with certainty which sick cells have passed the "point of no return" and are doomed to die. Ultrastructurally, flocculation of the mitochondrial matrical proteins correlates with the onset of cell death. This is considered to be the earliest absolute sign of cell death. Utilizing the light microscope, a cell can be recognized as dead only after it has undergone a sequence of changes referred to as necrosis. There is a time lapse of 6 to 12 hours between the onset of cell death and the non-controversial recognition that the cell is dead by light microscopy. On the other hand, necrosis may be apparent to the naked eye after 3 to 4 hours, or much sooner (tissues become abnormally opaque and pale).

"Necrosis may be defined as the morphological changes caused by the degradative action of lysosomal enzymes on the lethally injured cell. It is the sum of the morphological changes following cell death within the living body."

After a cell dies, lysosomes rupture and their hydrolytic enzymes are released into the cell. The release and activation of these lysosomal enzymes are largely responsible for cell necrosis.

Remember, necrotic cells are dead cells, but dead cells are not necessarily necrotic.

 

3.5.2 ULTRASTRUCTURAL CHANGES IN DEAD/NECROTIC CELLS:

Immediately after cell death, the ultrastructural changes are similar to and cannot be distinguished from those of reversible cellular injury (sick cell). As mentioned previously, flocculation of mitochondrial matrical proteins correlates with the onset of cell death as seen ultrastructurally. Later, lysosomes rupture and disappear as recognizable structures. Cellular components are progressively degraded and the dead cell may become replaced by myelin figures (masses composed of phospholipids). These phospholoipd myelin figures are either digested by phagocytic cells or degraded further into fatty acids.

3.5.3 LIGHT MICROSCOPIC AND GROSS CHANGES IN DEAD/NECROTIC CELLS:

As stated previously, cells can be recognized as dead with the light microscope only when the morphologic changes of necrosis develop. Nuclear changes are the "hallmark" of cellular necrosis (cell death). The nuclear changes appear in the form of one of three patterns (pyknosis, karyorrhexis and karyolysis). Pyknosis refers to a nucleus that progressively shrinks and becomes transformed into a small, dense, wrinkled mass of tightly packed chromatin. Karyorrhexis refers to a nucleus that breaks or fragments into many clumps or pieces. Karyolysis refers to a nucleus in which there is progressive dissolution of the chromatin and eventual disappearance of the nucleus. The cytoplasm of a necrotic cell becomes transformed into an acidophilic, granular opaque mass. After a cell dies and undergoes the early changes of cell death, immediate dissolution may not occur. Instead, one of three distinctive patterns may ensue, depending on the balance between progressive proteolysis, coagulation of proteins and calcification. Thus, the necrotic cell may undergo coagulative necrosis, liquefactive necrosis or, in special circumstances, caseous, gangrenous or fat necrosis.

3.5.4 COAGULATIVE NECROSIS:

Coagulative necrosis refers to an area of necrosis in which the gross and microscopic architecture of the tissue and some of the cells are preserved. Presumably, the cell's structural and enzymatic proteins are denatured and rendered insoluble soon after cell death; thus, autolysis or self-digestion is hindered. As a result, the dead cells remain in a "state of coagulation" (cooked appearance), at least for a few days. Eventually, the coagulated cells become liquified slowly through heterolysis and/or phagocytized by phagocytes (neutrophils and macrophages). Microscopically, tissue structures and cellular outlines are recognizable; nuclei are pyknotic or absent; the cytoplasm is strongly acidophilic and opaque. (e.g., an entire renal tubule may undergo coagulative necrosis but still be recognized as a "tubule" due to preservation of its cylindrical shape and the outlines of the tubular epithelial cells.) Grossly, necrotic tissue is grey to white (unless filled with blood), firm, dense, and often depressed compared to surrounding normal tissue. Coagulative necrosis is most commonly the result of:

The term "Zenker's necrosis" refers to coagulation of proteins of sarcoplasm. The condition occurs only in striated muscle. Microscopically, individual fibers are swollen, homogeneous and hyaline in texture. The sarcoplasm is usually eosinophilic, the myofibrils are indistinct and the nuclei are pyknotic. Grossly, involved muscle fibers are pale, rather shiny, and swollen. (The term "hyaline degeneration" has been used to refer to this muscle change.)

3.5.5 LIQUEFACTIVE NECROSIS:

Liquefactive necrosis refers to an area of necrosis which disintegrates very rapidly into a liquid mass, resulting in a loss of cellular and architectural outlines. The very rapid liquefaction is due to autolysis (release of enzymes from the cell's own lysosomes) and to heterolysis (lysosomal enzymes from invading neutrophils). In liquefactive necrosis, the dead cells are digested, creating a defect which is filled usually by invading neutrophils. A tissue defect of this nature frequently occurs in nervous tissue soon after death due to the high content of lipid and small amounts of coagulable protein. Thus, there are two principal situations in which liquefactive necrosis occurs - abscesses found in any body site and in the central nervous system. Pyogenic bacteria (staphylococci, streptococci, etc.) are usually the cause of abscesses. Microscopically, the necrotic area may appear as empty spaces with frayed and irregular edges (commonly observed in the central nervous system); or it may be represented by a dehydrated residue of neutrophils, tissue debris and fibrin.

Remember, the fluid in liquified areas is removed in the process of tissue dehydration prior to sectioning. Grossly, the necrotic area is represented by a cavity containing clear fluid or by an abscess (pus).

"Remember, all necrotic tissue tends to disappear ultimately by a slow process of liquefaction. However, liquefactive necrosis refers to an area of necrosis that disintegrates very rapidly into a liquid mass."

3.5.6 CASEOUS NECROSIS:

Caseous necrosis refers to a distinctive pattern of necrosis which is a combination of coagulative and liquefactive necrosis. The gross and microscopic architecture of the cells/tissue is lost, but the necrotic tissue is not completely liquified. Caseous necrosis is associated with diseases in which granulomatous lesions occur (tuberculosis, mycotic infections, etc.). The caseous material usually remains in place for prolonged periods of time and is prone to undergo calcification. Liquefaction and disappearance seldom occur. Microscopically, the necrotic cells are not totally liquified nor are their outlines preserved, creating a distinctive amorphous granular debris. The necrotic material is usually enclosed by a connective tissue capsule. Grossly, the necrotic tissue is soft to firm, dry, friable, grayish-white to yellow, and resembles "milk curds" or cottage cheese. The term "caseous" is derived from the gross appearance of the necrotic tissue (white and cheesy).

3.5.7 FAT NECROSIS:

Fat necrosis is a distinctive type involving adipose tissue. I occurs in the body cavities (especially the abdomen) and beneath the skin.

Enzymatic fat necrosis occurs subsequent to pancreatic damage and the release of activated pancreatic enzymes into the abdominal cavity. The activated lipases split the triglyceride esters of adipose tissue into fatty acids and glycerin. The fatty acids combine with metallic ions (calcium, potassium sodium, etc.) to form a soap within what was once a fat cell. Microscopically, the fat within adipose tissue cells is replaced by a soap which is solid, opaque and nearly homogeneous. The necrotic fat cell takes a bluish to pinkish tinge, depending on the presence of sodium or potassium, respectively. It is purple if calcium is deposited. Cholesterol clefts are often present. Remember, the soap formed within necrotic fat cells is not dissolved out (as is fat) by fat solvents used in sectioning techniques. Grossly, necrotic fat is opaque, whitish, firm, chalky and somewhat granular. Enzymatic fat necrosis is not a specific form of necrosis. The cellular changes are essentially liquefactive.

Traumatic fat necrosis occurs primarily in subcutaneous adipose tissue. It is associated with mechanical trauma and pressure. However, the exact etiologic mechanism has not been clearly elucidated. Apparently, there is local damage to fat cells due to trauma with the release of fatty acids.

3.5.8 GANGRENOUS NECROSIS (GANGRENE):

Gangrenous necrosis or gangrene refers to an area of necrosis (usually coagulative) which is invaded by saprophytic and/or putrefactive bacteria.

"Initially, the tissues undergo coagulative necrosis; subsequently, the coagulated tissues are invaded by saprophytic and/or putrefactive bacteria which attract neutrophils to the area; the liquefactive action of the bacteria and the lysosomal enzymes released by the invading neutrophils modify the coagulated tissue."

Thus, gangrenous necrosis does not represent a distinctive pattern. It is, in reality, a combination of coagulative and liquefactive necrosis. If the coagulative pattern is dominant, the process is called dry gangrene. However, if the liquefactive pattern is more pronounced, it is designated as wet gangrene. Dry gangrene occurs primarily in the extremities (limbs, ears, etc.), whereas, wet gangrene occurs chiefly in visceral organs. Microscopically, the changes described for coagulative and/or liquefactive necrosis along with bacteria are observed. Grossly, in dry gangrene, the affected tissue is cool, dry, pale, shriveled and leather-like. There is a sharp line of demarcation between normal and gangrenous tissue. In moist gangrene, affected tissue is swollen, soft, pulpy, foul smelling and usually dark or black in color.

Gas gangrene is the term commonly used when necrotic tissue is invaded by bacteria that produce large amounts of gas from constituents of the dead tissue. Several species of genus Clostridium are capable of producing gas gangrene. These anaerobic, spore-forming bacteria can live in dead as well as in living tissue. For example, Clostridium septicum and Clostridium chauvoei are able to kill animal tissue and then continue to multiply in it, producing large amounts of gas.

NOTE: Necrosis of cells/tissues is one of the most common and important lesions observed in disease. If a sufficient number of cells die in a given organ or tissue, the animal as a whole may exhibit clinical signs of illness, or somatic death may ensue. Death of cells (necrosis) in one organ may lead to secondary effects in other organs and tissues (e.g., death and necrosis of heart muscle cells may lead to a weakened heart which is unable to pump blood at its normal rate; subsequently, blood accumulates in many organs and tissues; liver necrosis occurs, and edematous fluid collects in body cavities, etc.).

3.5.9 CALCIUM SALT DEPOSITION IN NECROTIC TISSUE:

The term dystrophic calcification is used when calcium salts are deposited in dead or dying tissues. This condition occurs in the presence of normal serum levels of calcium and in the absence of derangement in calcium metabolism. It is not related to calcium content of the blood which normally is around 10 mg/100 ml. Dystrophic calcification may be especially prominent in necrotic tissue that persists in the body for long periods of time. It occurs in areas of coagulative, liquefactive, caseous and fat necrosis. The precise pathogenesis is poorly understood and may involve several different pathways.

"The term metastatic calcification is used to refer to the deposition of calcium salts in living tissues. It occurs subsequent to some derangement in calcium metabolism that results in hypercalcemia."

Grossly, calcium salts appear as fine, white granules or clumps, which give a gritty feeling when incised. Microscopically, calcium salts have a basophilic, amorphorous appearance with H and E stain. However, it can be confirmed with special staining procedures such as Von Kossa and Alzarin-Red-S techniques.

3.5.10 CHOLESTEROL CLEFTS IN NECROTIC TISSUE:

Cholesterol clefts are empty spaces left by crystals of cholesterol dissolved out by solvent used in the preparation of microscopic sections. The cholesterol crystals are derived from the protoplasm of dead and/or dying cells. Thus, cholesterol clefts may be quite prominent in regions where there has been considerable necrosis of cells relatively rich in cholesterol. Microscopically, cleft-like empty spaces persist. They occur in picket fence-like groups. In frozen sections, cholesterol crystals may be observed. These crystals are anisotropic or birefringent. Grossly, cholesterol crystals are not observed unless deposited in large amounts. If visible, they appear as shiny, yellowish, granular or flaky material. Cholesterol clefts have no significance other than to indicate the presence of tissue damage or necrosi

3.5.11 INTRACELLULAR ENZYMES RELEASED BY DEAD OR DYING CELLS:

Injured or dead cells tend to leak intracellular enzymes across their abnormally permeable plasma membrane. The enzymes diffuse into the intercellular fluid and subsequently into the bloodstream. In the bloodstream, these enzymes can be assayed by relatively simple laboratory techniques. Thus, elevated blood levels of enzymes released from dead cells is an important diagnostic aid for the recognition of dead cells/tissues within the living body. Elevated blood levels of such intracellular enzymes as glutamic oxaloacetic acid transminase, lactic dehydrogenase and creatine phosphokinase suggest the presence of severely damaged or dead cells (necrosis) within the body (the diagnostic importance of the enzymes is discussed in your clinical pathology course).

3.5.12 THE OUTCOME OF NECROTIC TISSUE:

Overview: What do the outcomes mean to the host in terms of continued health?

Necrotic tissue tends to incite an inflammatory reaction in surrounding viable tissue since it acts as an irritant. Therefore, invading leukocytes surround the necrotic tissue and assist in its liquefaction. Eventually, the liquified tissue is removed via the bloodstream and/or lymphatic system. This is the usual outcome of necrotic tissue when the number of dead cells are few, and/or when the central nervous system is involved. Large necrotic masses of tissue are liquified and removed very slowly. In addition to liquefaction and removal by the bloodstream and/or lymphatic system, necrotic tissue may be handled by the body in the following ways.

NOTE: At this point, the student has been exposed to the ways in which a normal cell responds when it encounters stress or an injurious agent. Cellular adaptation, cellular illness and cellular death are the common responses to injury. Remember, the adapted cell achieves an "altered but steady state." Its health is preserved, but its structure is altered and its functions are performed at a different level than that of a normal cell. The reversibly injured or ill cell is actually damaged but not killed by the injurious agent. It may revert to an adapted or normal state. The irreversibly injured or dead cell is doomed to be completely degraded. In the next segment, an attempt is made to solidify aspects of cellular injury by discussing possible mechanisms of reversible and irreversible cellular injury.

 

3.6 MECHANISMS OF REVERSIBLE AND IRREVERSIBLE CELLULAR INJURY

Reference: Chapter 2, pages 53-75, of your textbook.

3.6.1 GENERAL CONSIDERATIONS:

The student is reminded that immensely complex problems are encountered in attempts to determine the precise molecular or biochemical biochemical event or events that initiate cellular injury or cellular death. It is apparent from the previous discussions that:

Despite the difficulties encountered, the mechanism and the site of primary attack for a few forms of injury have been fairly accurately elucidated:

3.6.2 HYPOXIA AS A MECHANISM OF CELLULAR INJURY/DEATH (Check Table 3.2, page 89, in Cell Pathology):

Since hypoxia may be the final pathway of action of many injurious agents, it is used as a "model" in the following discussion to assist the student in correlating and understanding aspects of reversible and irreversible cellular injury.

"A six year old Coonhound was submitted to the Small Animal Clinic with a history of difficult breathing and cyanotic or bluish mucous membranes. Somatic death occurred before treatment was instituted and a necropsy examination was performed immediately. On gross inspection, several branches of the blood vessel that supplies the heart musculature (coronary artery) were occluded by blood clots that formed prior to death (thrombi). The affected heart musculature was pale and firm (necrosis). On light microscopic examination, the cytoplasm of some heart muscle cells had a cloudy, indistinct, ground-glass appearance. The cytoplasm of other cells contained numerous small lipid vacuoles. Also, there were heart muscle cells in which the nuclei were dark and shrunken or fragmented into many tiny pieces."

At this point, it should be apparent to the student that the thrombi within the coronary artery hindered the flow of blood to the heart muscle cells, resulting in a decreased supply of oxygen. Thus, a hypoxic state occurred and some of the heart muscle cells become sick or ill as reflected by the cytoplasmic changes. Other cells died as reflected by the nuclear changes, etc.

Based on previous class discussions, we would be in a position to reconstruct the sequence of events (pathogenesis) that occurred in the Coonhound at the subcellular level prior to the development of the light microscopic and gross alteration described above. (Review Figures 2.28 and 2.33 in your textbook)

"Initially, the decreased oxygen supply to the Coonhound's heart altered the normal aerobic respiratory system of muscle cells. Oxidative phosphorylation by mitochondria was decreased and the generation of ATP slowed down or stopped -> The decreased cellular ATP and associated increase of cellular [ADP] stimulated the production of phosphofructokinase -> The phosphofructokinase caused an increase rate of anaerobic glycolysis (to maintain the cell's energy sources by generating ATP from glycogen) -> glycogen was rapidly depleted -> The increased anaerobic glycolysis resulted in the intracellular accumulation of lactic acid and inorganic phosphates from the hydrolysis of phosphate esters -> The accumulated lactic acid and inorganic phosphates reduced the intracellular pH (the reduced pH accounted for the early clumping of nuclear chromatin) -> At this point the reduced ATP concentration began to interfere with the sodium-potassium pump at the cell membrane -> Sodium and water began to accumulate within the cell as reflected by early dilatation of the endoplasmic reticulum, whereas potassium diffused out of the cell resulting in increased extracellular K+ (Cellular swelling or cloudy swelling became evident at this time) -> Subsequently, detachment of ribosomes from the rough endoplasmic reticulum and disassociation of polysomes into monosomes occurred -> The continued hypoxic state caused other alterations and these were due to decreased mitochondrial function and increased cell membrane permeability (such as formation of blebs and vesicles at cell surface, increasing concentration of sodium and water within the cell, marked swelling of the entire cell, loss os coenzymes, protein, and ribonucleic acid via the hyperpermeable cell membrane, etc.) -> At this time, the transition across the "point of no return" or cell death began -> Mitochondria exhibited high amplitude swelling and flocculation of the matrical proteins, lysosomes became swollen but did not rupture at this time, nuclear chromatin began to dissolve, denaturation of cellular proteins occurred -> Lysosomal membranes were damaged resulting in leakage and activation of hydrolytic enzymes initiated progressive degradation of all cellular components -> Widespread leakage of cellular enzymes into the extracellular spaces occurred -> Remember, at this point, immediate dissolution of dead cells may or may not occur; regardless, one of the patterns of necrosis described earlier (coagulative, liquefactive, caseous) will become apparent or gross and/or microscopic inspection -> Finally, the dead cells are completely degraded or ingested and removed by phagocytic cells -> If the Coonhound had lived, the defects in the heart musculature would have been repaired by connective tissue replacement. However, this Coonhound died subsequent to the heart lesions that developed as a result of coronary thrombosis and the associated hypoxia."

 

3.7 POSTMORTEM CHANGES

3.7.1 GENERAL CONSIDERATIONS:

Postmortem changes refer to cell death which accompanies death of the body as a whole (somatic death). The term "antemortem changes" refer to those alterations that occur in the living body (prior to somatic death). The student must learn to differentiate postmortem changes from antemortem changes in order to correctly interpret those lesions encountered at necropsy.

Somatic death refers to death of the entire body. The absence of heart beat, pulse, respiration, or brain waves have been used to define somatic death. In other words, somatic death is characterized by cessation of all organ function. It is quite difficult, however, to determine the precise moment at which somatic death occurs. In man, this difficulty assumes considerable medical, ethical and legal importance.

Remember, somatic death is clearly different from cellular death.

Following somatic death, cells become ischemic and survive for varying periods of time, depending on cell type, decreasing body temperature and other factors. Thus, it is possible to remove organs for transplantation from individuals who have been pronounced dead (e.g., fibroblasts may be successfully cultured many hours after somatic death). Regardless of the precise moment of death, once all vital body functions have ceased, a sequence of postmortem changes appears.

"The student should be able to distinguish between somatic death, necrosis and necrobiosis. Necrosis predominantly refers to the morphologic changes caused by lysosomal enzymes on the lethally injured cell within the living body. Necrobiosis refers to the death of cells at the end of their normal life-span. It occurs as a part of normal cell turnover. Cell death occurs without harm to the host since cell function has been fulfilled."

Postmortem autolysis refers to self-digestion by enzymes that are present within, or released into, the cytoplasm of cells after death.

Remember, all of the cytopathic changes described for necrosis occur in postmortem autolysis.

However, in postmortem autolysis, the changes are usually uniformly distributed throughout an organ and there is no inflammatory reaction. Postmortem autolysis is due to total diffuse anoxia. Organelles degenerate according to their oxygen requirement. Some tissue undergo autolysis very quickly after death and must be fixed (formalin, etc.) rapidly in order to preserve a lesion present at the time of death. The most sensitive are the retina (which becomes separated from the choroid), the seminiferous tubules (in which vacuoles appear within and between cells) and the intestine (in which the epithelium over the villi sloughs off. The rate at which postmortem autolysis occurs in an animal depends on a number of factors, including:

3.7.2 SPECIFIC TYPES OF POSTMORTEM CHANGES:

A description of some of the more commonly encountered postmortem changes are included below. Remember, it is important to distinguish postmortem changes from those changes that occur prior to death.

  

3.8 POST-INSTRUCTIONAL SELF-EXAMINATION 

After completing this section, each student should be in a position to provide appropriate answers for the following questions.

 

 

Questions

  • 1. Distinguish between reversible and irreversible cell injury. 
  • 2. Define and/or describe the following terms: hypoxia, anoxia, ischemia, necrosis.
  •  3. Discuss the differences and similarities between the injurious agents responsible for reversible cellular injury and those responsible for irreversible cellular injury.
  •  4. What is an injurious agent? 
  • 5. What is the interrelationship between biochemical, morphological and functional changes in an injured cell?
  •  6. What organelle is initially affected within a cell subsequent to hypoxic injury?
  •  7. What biochemical event is initially affected within a cell subsequent to hypoxic injury?
  •  8. Based on morphologic alteration, how would you distinguish a "sick" cell from one that recently died?
  •  9. What is formalin? Why is this chemical employed in veterinary medicine?
  •  10. How would you define the so-called "point of no return?"
  •  11. Approximately how many hours lapse occurs between the onset of cell death (point of no return) and incontroversial recognition that the cell is dead by light microscopy?
  •  12. How would you distinguish between necrosis, necrobiosis, cell death and somatic death?
  •  13. What factors are important in determining whether a cell will undergo adaptation, be reversibly injured or be irreversibly injured?
  •  14. Briefly discuss the ultrastructural changes of reversible cell injury as they occur in the lysosomes, mitochondria, Golgi apparatus and endoplasmic reticulum.
  •  15. What cellular organelle is the most sensitive indicator of altered fluid volume?
  •  16. What ultrastructural change is considered to be an early absolute sign of cell death?
  •  17. In what portion of the cell would you expect to observe morphologic changes of reversible cell injury when employing the light microscope?
  •  18. Under light microscopy, what portion of the cell would you expect to observe morphological changes of cellular death?
  •  19. How would you distinguish between "cellular degenerations" and reversible cellular injuries?
  •  20. Name and briefly describe the patterns of reversible cellular injuries recognized grossly and/or with the light microscope. 
  • 21. Distinguish between "cellular swelling," "cloudy swelling" and "parenchymatous degeneration."
  •  22. Briefly discuss the gross and light microscopic and the ultrastructural changes associated with cellular swelling. 
  • 23. Give a likely pathogenesis for a cell that was deprived of oxygen, beginning with the biochemical events.
  •  24. What is the interrelationship between "cellular swelling" and "hydropic change?"
  •  25. Under what circumstances would you expect cellular swelling to occur?
  •  26. What procedures and/or techniques would you employ in order to distinguish cytoplasmic lipid vacuoles from cytoplasmic vacuoles associates with cellular swelling?
  •  27. Discuss the significance of cellular swelling in the liver of a 6- year-old coonhound.
  •  28. What is "fatty change?" What is the interrelationship between fatty change, fatty degeneration and fatty infiltration? 
  • 29. What is the significance of fat that accumulates in the cytoplasm of injured cells in comparison to accumulated cytoplasmic fat in normal cells?
  •  30. Explain how injured cells accumulate cytoplasmic fat.
  •  31. Explain the mechanism(s) by which normal cells accumulate cytoplasmic fat.
  •  32, Why would you expect fatty changes to occur most commonly in the liver?
  •  33. Briefly outline the manner in which normal fat is metabolized.
  •  34. What is cyclic AMP? What is the importance of cyclic AMP in lipid metabolism? 
  • 35. What are ketones? Under what circumstances would you expect ketones to accumulate in the body?
  •  36. List and describe the factors that may result in fatty change. 
  • 37. Briefly discuss the gross and microscopic alterations associated with fatty change in the liver, heart and kidneys.
  •  38. Distinguish stromal fatty infiltration from fatty change.
  •  39. What are fatty cysts? 
  • 40. Discuss the significance of fatty changes in the kidneys, heart and liver of a 12-year-old Coonhound.
  •  41. How would you distinguish steatosis from steatitis and obesity?
  •  42. Please describe the differences and similarities between necrosis and postmortem autolysis. 
  • 43. What is the possible interrelationship between lysosomal hydrolytic enzymes, cell death and necrosis?
  •  44. Define and/or describe the following terms: pyknosis, karyolysis, chromatolysis, cytoplasmolysis, acidophilic cytoplasm, karyorrhexis, loss of differential staining, hemolysis, antemortem change, postmortem change.
  •  45. What type necrosis would you expect to observe in cells in which denaturation of protein is the dominant feature? 
  • 46. What is Zenker's necrosis? What is the possible interrelationship between Zenker's necrosis and coagulative necrosis? 
  • 47. What type necrosis is characterized by rapid disintegration of dead cells?
  •  48. What type(s) of necrosis would you expect to observe in the brain of a dog that suffered from prolonged hypoxia?
  •  49. What type necrosis would you expect to be associated with a large abscess in the liver of a 10-year-old Coonhound?
  •  50. What type necrosis is characterized by a combination of coagulative and liquefactive necrosis?
  •  51. What type necrosis is characterized by the presence of "soap" within affected cells?
  •  52. What type necrosis is usually associated with each of the following:

 

  • -- Infarcts
  • -- Tuberculosis
  • -- Granulomas
  • -- Activated lipases
  • -- Saprophytic and putrefactive bacteria
  • -- Ischemia
  • -- Pyogenic bacteria

 

  • 53. Discuss the differences and similarities between coagulative, liquefactive and caseous necrosis with emphasis on etiologic mechanisms, light microscopic findings, gross findings, prognosis and outcome. 
  • 54. Give a likely pathogenesis for massive fat necrosis that occurred in the abdominal cavity of an 8-year-old Coonhound that was hit by a car. How would you define pathogenesis?
  •  55. What is the possible interrelationship between gangrenous necrosis, coagulative necrosis and liquefactive necrosis?
  •  56. Under what circumstances would you expect dry and/or moist gangrenous necrosis to occur in an organ or tissue?
  •  57. Distinguish between dry and moist gangrene with emphasis on causative mechanisms, organs and/or tissues involved, microscopic changes and gross alterations.
  •  58. What is gas gangrene?
  •  59. Briefly discuss the ways in which an area of coagulative necrosis is handled by the body.
  •  60. Discuss the ways in which caseous necrosis is usually handled by the body.
  •  61. Discuss the ways in which an area of liquefactive necrosis is handled by the body.
  • 62.  ways in which an ulcerative skin lesion is handled by the body.
  •  63, Distinguish between dystrophic and metastatic calcification.
  •  64. Discuss the circumstances in which calcium salts are deposited in necrotic tissues.
  •  65. What are cholesterol crystals? How would you distinguish cholesterol crystals from cholesterol clefts?
  •  66. Under what circumstances would you expect cholesterol cleft and/or crystals to occur in tissues? What special procedures, techniques, etc., must be employed in order to demonstrate cholesterol crystals?
  •  67. Discuss the clinical significance of intracellular enzymes released by severely injured or dead cells.
  •  68. How would you define somatic death?
  •  69. Discuss the differences and similarities between cell death and somatic death.
  •  70. What factors are most likely responsible for rigor mortis?
  •  71. What is the possible relationship between postmortem autolysis and anoxia?
  •  72. Briefly discuss the onset and disappearance of rigor with emphasis on the muscles involved and the time of onset.
  •  73. What is putrefaction?
  •  74. How would you differentiate between the following?
  •  
    • -- Pseudomelanosis and melanin deposits
    • -- Antemortem and agonal changes
    • -- Postmortem rupture and an antemortem rupture
    • -- Postmortem emphysema and bloat
    • -- Imbibition of bile and pseudomelanosis
    • -- Postmortem clots and thrombi
    • -- Postmortem autolysis and autolysis associated with necrosis
    • -- Hypostatic congestion and hemorrhage
    • -- Algor mortis and livor mortis
  •  
  • 75. What is the importance of postmortem changes to the pathologist?
  •  76. Define and use the following terms:
  • (1) bacteremia, (2) toxemia, (3) thrombus, (4) septicemia, (5) infarct, (7) polarizing microscope, (8) anisotropic, (9) birefringent and (10) isotropic. 
  • 77. Which one of the following is most likely to occur first when a cell is damaged by an injurious agent?
    •  a. Pyknosis and karyorrhexis of the nucleus
    • b. Rupture of lysosomes
    • c. Lipid accumulation within cytoplasmic liposomes
    • d. Reversion of the cell to a more primitive type
    • e. Disruption of control mechanisms for ionic and osmotic homeostasis at the level of the plasma membrane
    • f. Flocculation of mitochondrial matrical proteins

 

  • 78. A 5-year-old Coonhound was kicked by a horse and rendered unconscious for a prolonged period of time. Normal blood circulation was altered and the brain received a decreased amount of oxygen. Which one of the following most likely occurred first in affected brain cells?
    •  a. Protein synthesis ceased due to damaged endoplasmic reticulum
    • b. Cell division ceased due to failure of DNA synthesis
    • c. Control mechanisms for ionic and osmotic hoemeostasis were disrupted at the plasma membrane level
    • d. Hydrolytic enzymes were released and activated subsequent to rupture of lysosomes
    • e. Flocculation of mitochondrial matrical proteins occurred.
    • f. Oxidatvie phosphorylation decreased subsequent to malfunction of the mitochondria