PATHOLOGY LABORATORY I CELL INJURY

Questions and Answers

Slide 4 - Liver, apoptosis

1. What are the two fundamental types of cell death?

Apoptosis (program cell death) and necrosis (pathologic cell death)
2. What are the morphologic features of apoptosis?

By light microscopy, apoptosis is characterized by condensation and shrinkage of nucleus and cytoplasm of single parenchymal cells. A key feature is scattered involvement of single cells. By electron microscopy apoptosis is characterized by condensation and fragmentation of nuclear chromatin and subsequent formation of multiple fragments of condensed nuclear material and cytoplasm. These multiple fragments are known as apoptotic bodies. They are eventually engulfed by adjacent cells. Inflammation with infiltrates of leukocytes is typically absent.

3. What is the pathogenesis of apoptosis? How is it different from oncositic necrosis?

Apoptosis involves a primary lesion of nuclear chromatin. Activation or de novo synthesis of endonucleases results in cleavage of DNA at linker regions to form fragments of doublestranded DNA. Endogenously programmed or induced but regulated activation of various genes is involved. Repressors as well as inducers of the lethal genetic program exist. Oncosis is a common form of pathologic cell death which occurs as a response to a severe or sustained injury. The major mechanism of oncosis or oncositic necrosis is progressive membrane damage. Apoptosis occurs as a physiologic process, counter-balancing mitosis. It is thought to play an important role in normal organogenesis and cell turnover. Multifocal single cell death resembling apoptosis also is seen in certain pathologic conditions, including viral hepatitis, death of cancer cells, and graft-versus-host disease. Necrosis, strictly speaking, refers to the sum of the degradative changes that accompany and follow cell death regardless of the cause. However, necrosis is generally used synonymously with oncosis or oncositic necrosis to refer to the most common problem of pathologic cell death.

Oncosis, as the name implies, refers to cell death with cell swelling, in contrast to cell shrinkage with apoptotic cell death.

Slide 17: Liver,fatty metamorphosis

1. What does fatty metamorphosis or fatty change indicate?

Accumulation of neutral lipid, specifically triglyceride, in the cytoplasm of parenchymal cells.

2. How does it differ from fatty ingrowth or stromal infiltration of fat or adipose tissue invasion?

All of these terms are synonymous for replacement of normal parenchyma by adipose tissue composed of adipocytes. In this condition, there is no accumulation of neutral lipid in the parenchymal cells. Rather there is ingrowth of fatty tissue replacing the normal parenchyma. 3. Is fatty change (metamorphosis) a reversible change?

Yes, one can tell this because the change involves the cytoplasm only, and the nuclei of the parenchymal cells are preserved.

4. Why does fatty change (metamorphosis) occur?

Fatty change can have a number of causes. The liver is particular prone to fatty change because it is a major organ of fatty acid processing and metabolism in the body. Fatty change can occur as adaptive change in response to excess mobilization of fats with presentation of an increased load of fatty acids to the tissue. Fatty change also can occur as a result of injury of the tissue and subsequent derangement of fatty acid metabolism. Because of the key role of the liver in fatty metabolism, it can manifest fatty change either as adaptation or as a manifestation of sublethal injury (degeneration). In contrast, fatty change in other organs such as the heart and kidneys is usually manifestation of sublethal injury to these organs.

5. What is the term for increasing the fat in normal cells in response to an oversupply of fat?

This is an adaptive change often referred to as fatty infiltration of the parenchymal cells.

6. What is the term for fatty metamorphosis that occurs as a result of injury.

This form of reversible sublethal injury is known as fatty degeneration.

7. What EM change would be present in fatty change or degeneration?

A single large or multiple small nonmembrane-bound lipid droplets which often exhibit a moderate electron density after staining with osmium and the lead citrate and urinyl acetate stains. By light microscopy, the lipid drops appear as clear, well defined, punched-out, vacuoles in contrast to the more irregular and less well defined vacuolization of the cytoplasm due to water accumulation (hydropic change).

8. Which do you think this slide is an example of?

Given the history that the tissue was obtained from a chronic alcoholic and given the fact that acute and chronic alcohol ingestion is known to cause injury and derangement of hepatocytes, this probably represents fatty degeneration rather than fatty infiltration. However, this cannot be determined just from examination of the slide.

9. Have biochemical changes occurred in hepatocytes? How do you know?

The very presence of large amounts of triglyceride in hepatocytes indicates that either the metabolic machinery of the hepatocytes has been overwhelmed by presentation of a large fatty acid load to the liver and/or metabolic alterations of the hepatocytes have occurred. Alcohol is a hepatotoxin that alters mitochondrial and microsomal functions. Alcohol is known to increase free fatty acid synthesis, diminish triglyceride utilization, decrease fatty acid oxidation, a block lipoprotein excretion, enhance lipolysis, and increase delivery and uptake of free fatty acids. All these factors have been implicated in alcohol-induced fatty liver.

10. What is the term for any sublethal reversible light microscopic change?

Degeneration.

11. What is another common light microscopy alteration indicating reversible injury? What causes the change? What changes occur by EM?

The other common light microscopic change is known as cloudy swelling or hydropic degeneration. This is characterized by increase in size of the cells with a finely vacuolated cytoplasm due to increased water accumulation, without displacement or otherwise alteration to the nucleus. By electron microscopy, one sees separation of the organelles indicating intracellular edema. The process is caused by impaired oxidated metabolism leading to insufficient energy to maintain the membrane pumps, in particular the sodium potassium ATPase. This is followed by accumulation of sodium, chloride, and water in the cell, with subsequent swelling.

Slide 63: Kidney, Infarct 1. Why do you think the patient developed renal infarcts?

Infarction is the development of irreversible injury, with subsequent coagulation necrosis, resulting from marked impairment in blood supply. Infarcts are often focal lesions due to anatomic obstruction of a major blood vessel by a thrombus (blood clot formed in vivo). Other times infarct can be multifocal due to a more generalized decrease in perfusion. In this case, we know by history that the patient had a myocardial infarct with a mural thrombus superimposed on the infarct in the left ventricle. It is most likely that a portion of this thrombus in the left ventricle detached and was carried through the blood stream, that is embolized, to a segmental branch of the renal vasculature thereby leading to severe impairment in blood flow to an area of the kidney.

2. What is the primary site of attack in hypoxia? What does this lead to?

Hypoxia which is a major component of ischemia leads to rapid shut down of oxidative phosphorylation in the mitochondria. This leads to progressive reduction in ATP content of the cells because of greatly diminished production and continued energy needs of the cells. There is a transient stimulation of anaerobic glycolysis, but because of the accumulation of lactate and other waste products, this pathway also becomes inhibited. As a result of energy depletion, marked derangements in electrolytes occur. This is followed by activation of degradative enzymes leading to progressive membrane damage.

3. What is the most common form of injury occurring with ischemia and toxic or thermal injury?

Coagulation necrosis.

4. What does the term infarct indicate?

Irreversible injury of tissue with subsequent coagulation necrosis due to marked impairment in blood flow (severe ischemia) or severe reduction in oxygenation (severe hypoxia or anoxia) of the tissue.

5. What does an infarct look like grossly?

In most tissue, infarcts appear as wedge shaped, pale yellow to gray tissue often with a reddish border due to hyperemia at the border. Several factors contribute to the pale yellow gross color of necrotic tissue. There is decreased or absent perfusion. Residual red cells trapped in the infarct, break down releasing their hemoglobin which defuses out. There is so called unmasking of structural lipids giving rise to increased accumulation of free lipids which have a yellow color. Finally, bilirubin from the blood diffuses into the dead tissue and stains it.

6. Why can you recognize the type of tissue present in the area of infarct?

This is because the characteristic feature of coagulation necrosis is initial preservation of the basic structure, and at the cellular level, the outlines of the cells. In coagulation necrosis, coagulation and denaturation of protein predominates over hydrolysis. It is only after the inflammatory process occurs that tissue break down happens.

7. What factors determine whether coagulation or some other type of necrosis will occur?

Necrosis is the sum of degradative changes that follow irreversible cell injury. The phenomena that occur are denaturation and coagulation of protein, and activation of hydrolytic enzymes, either by the lysosomal enzymes of the cells resulting in autolysis and/or by the influence of hydrolytic enzymes released from inflammatory cells given rise to heterolysis. Thus, the two basic processes are coagulation and liquefaction. In toxic or ischemic injury of protein rich tissues, the coagulation and denaturation of protein predominates early on. Only later does the inflammatory response lead to a breakdown of the tissue. In contrast,in lesions initiated by infection with marked neutrophilic inflammation, hydrolysis leading to liquefaction will occur rapidly. Also in protein poor tissues such as the central nervous system liquefaction is the predominate factor. Therefore, brain infarcts exhibit liquefaction necrosis. In other situations, there is a mixture of the two giving rise to caseous necrosis.

8. Is the change in the slide reversible?

The changes are irreversible. Evidence for this is the presence of marked nuclear alterations, i.e. pyknosis, karyorrhexis, and karyolysis in addition to the cytoplasmic changes.

9. What nuclear changes would indicate an injury is irreversible i.e necrosis? What would these changes look like by EM?

At the EM level, early on in injury there is mild clumping of chromatin, but this does not reach sufficient magnitude to be manifested by light microscopy. After the onset of irreversible injury, there is marked clumping of the nuclear chromin and shrinkage of the nucleus to give rise to the change of pyknosis which is recognized by light microscopy as well. Thereafter, the nuclear chromin fragments giving rise to karyorrhexis and karyolysis.

Slide 70: Toe, gangrene 1. What is gangrene? What caused it in this case?

Gangrene is coagulation necrosis modified by some other factor. In this case, the gangrene of the toe and foot was caused by severe ischemia due to peripheral arterial disease. Following necrosis of the superficial exposed tissue, drying occurred, leading to a combination of coagulation necrosis with superimposed desiccation.

2. How are dry and wet gangrene different? Both in cause and histologically.

Dry gangrene is usually caused by ischemia. It tends to involve a superficial site. Histologically, one sees features of coagulation necrosis coupled with shrinkage and drying of the dead tissue.

Wet gangrene usually results from superimposition of infection and neutrophilic inflammation on a focus of necrosis. It usually involves internal organs. A good example would be a lung infarct that has become secondarily infected. The tissue grossly is edematous and weepy. Histologically, one sees a lot of neutrophilic inflammation in addition to necrosis.

3. What is gas gangrene?

Gas gangrene is rapidly progressive coagulation caused by the toxic effects of infection by gas producing organisms such as Claustridia. One sees necrotic tissue filled with multiple gas bubbles.

Slide 21. Lymph node, granuloma with caseous necrosis

1. Caseous necrosis is actually a combination of what two types of necrosis?

Coagulation necrosis and liquefaction necrosis.

2. What is the classic gross description of caseous necrosis?

Soft, semisolid, gray to pale yellow tissue.

3. What is the microscopic appearance of caseous necrosis. How is it different from pure liquefactive and pure coagulation necrosis?

The classic microscopic appearance of caseous necrosis is an amorphous mass of granular eosinophilic material with no cell outlines as well as no identifiable nuclei. Pure coagulation necrosis shows, early on, well preserved cell outlines, whereas liquefactive necrosis shows disillusion and liquefaction of the dead tissue.

4. What are examples of when caseous necrosis occurs?

Caseous necrosis occurs with certain types of infections. Those produced by tuberculosis organisms and fungi and caseous necrosis is also seen in the centers of rapidly growing neoplasms.

Slide 10: Pancreas, acute pancreatitis

1. What are some of the causes of enzymatic fat necrosis in addition to pancreatitis?

Rupture of a gastric ulcer. Another example would be trauma or infection of the subcutaneous tissue.

2. What do these processes have in common?

Release of large amounts of hydrolytic enzymes which attack adipose tissue.

3. What causes the gross chalky white and microscopic bluish granularity of the fat cells?

As a result of the attack by hydrolytic enzymes, there is a break down of the neutral triglyceride into free fatty acids. These fatty acids complex with proteins which coagulate giving rise to an amorphous pink staining to the previously clear cytoplasm of the adipocytes. This is followed by an influx of calcium which complexes with the fatty acids to form calcium soaps. Since calcium deposits have an affinity for hematoxylin, the areas of fat necrosis then take on a bluish coloration.

4. What type of necrosis is found in the parenchyma of the pancreas and why?

In the parenchyma, one finds liquefactive necrosis. This is because the massive attack by the hydrolytic enzymes leads to rapid lysis of the dead tissue.

5. What are other examples of the type of necrosis in Question 4.

Infections with extensive neutrophilic inflammation, leading to suppuration and abscess formation. Another example of liquefactive necrosis would be infarction of the brain, leading to rapid lysis of the protein poor brain tissue.

Slide 93: Heart, hypertrophy with normal control

1. Define what hypertrophy is in an organ.

Hypertrophy is an increase in the mass of an organ due to an increase in the size of the individual parenchymal cells without an increase in cell number.

2. What evidence is there that hypertrophy is present in the abnormal heart, grossly and microscopically?

Grossly the heart is increased in weight. It may also exhibit an increase in wall thickness. Microscopically, the individual myocytes are increased in size. The nuclei are enlarged and are hyperchromatic.

3. Why do you think this patient had hypertrophy?

This patient likely had hypertrophy of the left ventricle due to an increased pressure load placed on the ventricle by hypertension.

4. Why does the heart not undergo hyperplasia?

The heart does not undergo hyperplasia because the adult myocytes have lost the ability to undergo mitotic division.

5. Why do other organs undergo hypertrophy or hyperplasia?

The key factor is whether or not the cells have the ability to undergo mitosis. Generally, the heart will undergo hypertrophy only, whereas other organs such as liver, adrenal gland, or prostate have the ability to have hyperplasia occur. In some cases, there is a mixture of hypertrophy and hyperplasia.