Cellular Pathology (VPM 152) Lecture 4 (Web) Paul Hanna Jan 2018

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2 Cellular Pathology (VPM 152) Lecture 4 (Web) Paul Hanna Jan 2018

3 IRREVERSIBLE CELL INJURY 1) Necrosis describes the range of morphologic changes that follow cell death in living tissue the morphologic appearance is due to 2 concurrent processes: denaturation of proteins (nonproteolytic alteration 2 o or 3 o structure) Raw egg white (albumin protein) is a transparent / clear viscous liquid Adding heat, acid or alkali will denature (misfold) the proteins which results in a change of the color to white / opaque & the texture becomes solid enzymatic digestion autolysis* (self digestion) = endogenous enzymes derived from the lysosomes of the dead cells themselves heterolysis = lysosomes of immigrant leukocytes *autolysis also used for changes that occur in all cells after death, ie postmortem autolysis

4 1) Necrosis distinctive morphologic patterns depending on whether enzyme catabolism or protein denaturation predominates morphologic patterns of necrosis include: Coagulation Necrosis Liquefactive Necrosis Caseous Necrosis *Gangrenous Necrosis *Fat Necrosis

5 a) Coagulation Necrosis most common manifestation of cell death characteristic of hypoxic / ischemic death of cells in all tissues (except brain) on LM, basic outline of the coagulated cell persists at least a few days (protein denaturation predominates over enzymatic digestion) necrotic cells eventually removed by leukocyte proteolysis & phagocytosis

6 a) Coagulation Necrosis Gross Appearance architecture resembles normal tissue, but color and texture are different lighter in color- denatured cytoplasmic proteins and decreased blood flow usually firm necrotic tissue may be swollen or shrunken may see a local vascular / inflammatory reaction to necrotic tissue

7 Figure 01-18A (McGavin & Zachary). Coagulation necrosis, infarcts, kidney, cow. A, Note the pale regions of acute coagulation necrosis surrounded by a red rim of active hyperemia and inflammation (far left). Bovine, kidney, renal infarct, cut surface. Note pyramidal (wedge) shaped area of necrosis with hyperemic border.

8 Bovine, fetus, liver, multifocal hepatic necrosis. Bovine herpesvirus 1 (BoHV-1) causes IBR (infectious bovine rhinotracheitis) in adult cattle and can also cause abortion when a pregnant cow is infected. Often see multifocal hepatic necrosis (necrotizing hepatitis) in the fetus.

9 Bovine, liver: Multifocal hepatic necrosis (coagulative) due to infection with the bacterium Fusobacterium necrophorum. The majority of bacterial infections cause liquefactive necrosis due to the marked infiltration of neutrophils with associated heterolysis; however in this particular bacterial infection there are toxins which result in coagulative necrosis (at least initially).

10 Bovine, heart, myocardial necrosis (coagulative); note irregular area of pallor within myocardium.

11 a) Coagulation Necrosis Microscopic Appearance original cell shape & tissue architecture is preserved (ie eosinophilic "shadow ) cytoplasm: nucleus: increased eosinophilia (H&E stain) usually hyalinized (homogeneous / glassy) may be mineralized karyolysis pyknosis karyorrhexis

12 Figure 01-17B (McGavin & Zachary). Pyknosis and karyolysis, renal cortex, chloroform toxicosis, mouse. Some epithelial cells exhibit hydropic degeneration, whereas others are necrotic. Some necrotic cells exhibit pyknosis (arrow), whereas others have lost the nucleus or have a very pale nucleus, ie karyolysis (arrowheads)

13 Figure 01-17C (McGavin & Zachary) Karyorrhexis, lymphocytes, spleen, dog. Spleen of a dog with parvovirus infection. Lymphocyte nuclei have fragmented because of the infection (arrow). H&E stain.

14 White muscle disease, skeletal muscle, calf. Note coagulation necrosis of myofibers characterized by fragmentation and hyalinization; also note extensive mineralization (blue-purple staining)

15 Ultrastructural Appearance of Irreversible Injury Path Basis of Vet Disease, 8 th ed

16 Irreversible ischemic injury - renal tubular epithelium Disrupted cell membranes Marked mitochondrial swelling with amorphous densities Dense pyknotic nucleus Fig 2-10 (Robbins). C, Proximal tubular cell showing late injury, expected to be irreversible. Note the markedly swollen mitochondria containing electron-dense deposits, expected to contain precipitated calcium and proteins. Higher magnification micrographs of the cell would show disrupted plasma membrane and swelling and fragmentation of organelles.

17 Irreversible Injury Normal human epithelial cell (Scanning EM). Epithelial cell 24 hours after exposure to sulfur mustard (SEM). Note loss of microvilli and perforations / invaginations of the plasma membrane.

18 Irreversible Injury after cell death: - cell parts continue to be digested by lysosomal acid hydrolases - leakage of cellular enzymes into ECF (eg muscle CK, liver ALT) - influx of ions (eg Ca 2+ ) and macromolecules from the ECF - cells debris removed by phagocytosis or broken into fatty acid residues

19 b) Liquefactive Necrosis when enzymatic digestion of necrotic cells predominates many bacterial infections; neutrophils contain potent hydrolases in hypoxic damage of the CNS Gross appearance affected tissue is liquefied, ie soft to viscous to fluid mass in inflammation, the liquid is often mostly dead WBC s (pus) Microscopic appearance may see degenerate neutrophils and/or amorphous necrotic material

20 Porcine, abscess in ventral neck / shoulder area. Escape of purulent exudate (pus) when the abscess is lanced. Note, suppurative (= purulent) exudate is a common type of liquefactive necrosis.

21 Cytology of an aspirate from abscess / suppurative exudate. The predominate cell type is neutrophils and there are fewer scattered macrophages. Porcine, kidney, suppurative pyelonephritis, note purulent exudate (again an example of liquefactive necrosis).

22 Figure 01-20A (McGavin & Zachary). Liquefactive necrosis. Acute polioencephalomalacia, brain, goat. A thiamine deficiency has resulted in polioencephalomalacia (ie polio = grey, encephalo = brain and malacia = necrosis). This is liquefaction necrosis with varying degrees of tissue separation (arrows). Scale bar = 2 cm. Note: this same lesion can result from either ischemia, lead poisoning or salt poisoning.

23 c) Caseous Necrosis typical seen with specific bacterial diseases, eg TB, caseous lymphadenitis infections in birds, since heterophils lack myeloperoxidase Gross appearance grey-white, dry, friable to pasty (caseous = cheese like) Microscopic appearance dead cells persist as amorphous, coarsely granular, eosinophilic debris retain cellular outline (coagulative) caseous complete dissolution (liquefactive)

24 Ovine, submandibular lymph node, with caseous lymphadenitis an infectious disease caused by infection with the bacteria Corynebacterium pseudotuberculosis; note the type of necrosis in this exudate is caseous (not quite liquefactive, but more broken down than coagulation necrosis, ie it would be a thick pasty texture, if you could cut / feel it)

25 Equine, lung tissue, pyogranulomatous pneumonia due to Rhodococcus equi; note caseous exudate (arrows).

26 Figure Tuberculosis, lymph node, transverse section, ox. A, The lymph has been replaced by a caseating granuloma. Note the caseous necrosis characterized by a pale yellow, crumbly exudate. B, Granulomatous inflammation in caseous necrosis. Cell walls are disrupted and tissue architecture is lost. Mineralization (not seen here) is common in this type of necrosis. H&E stain.

27 d) Gangrenous Necrosis definition = necrosis (usually ischemic) of extremities, eg digits, ear tips dry gangrene = coagulation necrosis of an extremity wet gangrene = when the coagulative necrosis of dry gangrene is modified by liquefactive action of saprophytic / putrefactive bacteria Frostbite of the hand in a person (above). Cat with sloughing of ear tips and paws following dry gangrene due to frostbite.

28 Calf, gangrenous necrosis of distal limbs due to ischemia; this ischemia could result from frostbite or sepsis or a mycotoxin such as ergot. Bovine, calf, gangrenous necrosis / dry gangrene with sloughing of distal limbs, ergot poisoning

29 Fig 1-21A (McGavin & Zachary), Moist gangrene, udder, sheep. The surrounding tissue is well vascularized, which contributes to the wet and bloody nature of the lesion. Often saprophytic bacteria and clostridia contaminate areas of necrosis. Bovine, mammary gland, wet gangrene; tissue starting to slough.

30 Bovine, lung, gangrenous pneumonia following aspiration of rumen content. [Although historically called gangrenous pneumonia, it is probably better called necrotizing pneumonia as a morphologic Dx or aspiration pneumonia as an etiologic Dx]

31 e) Fat Necrosis distinguished by its location within body fat stores etiology: inflammation (eg pancreatitis), Vit E deficiency, trauma, idiopathic

32 Canine, recurrent pancreatic necrosis ( pancreatitis ) with mesenteric fat necrosis

33 Canine, recurrent pancreatic necrosis ( pancreatitis ) with omental fat necrosis.

34 Canine, recurrent pancreatic necrosis ( pancreatitis ) with fat necrosis; note necrosis of fat (larger arrow) and pancreatic tissues (smaller arrow) some areas of coagulative type and other areas with significant enzyme degradation (liquefaction) of necrotic tissue.

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36 2) Apoptosis Greek = falling off death of single cells through activation of genetically programmed suicide pathways apoptosis indicates selective elimination of cells (either physiologic or pathologic), while necrosis points to widespread tissue injury (severe pathologic stimuli) apoptosis death of single cells / intact membranes / phagocytosed with no inflam. necrosis area of dead cells / damaged membranes / enzyme digest. & 2 o inflam. Figure (Molecular Biolology of the Cell, 4 th ed) Sculpting the digits in the developing mouse paw by apoptosis (A) The paw in this mouse embryo has been stained with a dye that specifically labels cells that have undergone apoptosis. The apoptotic cells appear as bright green dots between the developing digits (arrows). (B) This interdigital cell death eliminates the tissue between the developing digits, as seen one day later, when few, if any, apoptotic cells can be seen.

37 Incomplete tissue sculpting (ie incomplete apoptosis) of the digits results in syndactyly (from Greek syn = "together" + dactyly = digits ) It can be partial, as in the web toes (above left) or complete as seen in the childs hand (above right) and calves hooves (below left)

38 2) Apoptosis seen in many physiologic, adaptive and pathologic events: Physiologic causes: Cells undergoing programmed cell death during embryogenesis Cells undergoing normal turnover - hormone-dependent involution - cell deletion in proliferating population Immune System - deletion of autoreactive T cell in thymus - immune regulation

39 2) Apoptosis Pathologic causes: DNA damaged beyond repair eg radiation, toxins Misfolded proteins ER stress Specific infectious agents esp viruses Specific immune responses via cytotoxic T cells Pathologic atrophy of organs after duct obstruction

40 2) Apoptosis d) Morphology considerable apoptosis may occur in tissues before it is evident on histology Cell shrinkage - cytoplasm has packed organelles Chromatin condensation - dense aggregates of chromatin ± fragmentation Formation of cytoplasmic blebs / apoptotic bodies - with intact membranes (± nuclear fragments) Phagocytosis of apoptotic cells / bodies - usually by macrophages with no inflammation

41 Fig 1-8 (Robbins) Schematic illustration of morphologic changes in cell injury culminating in necrosis or apoptosis. Note: with necrosis cells swell, lose membrane integrity and incite an inflammatory response VS apoptosis where cells shrink (condense), membranes remain intact in forming apoptotic bodies and are removed with minimal inflammation.

42 SEM showing showing cytoplasmic blebbing / apoptotic body formation. Figure 3 23 (Junqueira s Basic Histology) Late apoptosis-formation of apoptotic bodies. TEM of a cell in late apoptosis shows that during this process the cell s shape changes radically and large cytoplasmic vesicles (blebs) are formed. These detach from the cell and often separate one from another, but remain contained within plasma membrane so that no cytoplasmic contents are released into the extracellular space.

43 Note, arrows indicate apoptotic epidermal keratinocytes ( sunburn cells ) due to UV-B radiation. These can be induced within 30 minutes of sun exposure.

44 Figure (McGavin & Zachary). Apoptosis, cytoarchitecture of cells, pancreas, rat. Individual acinar cells are shrunken and their chromatin condensed and fragmented (arrows). Cytoplasmic blebs are found in adjacent cells. Inflammation is absent. H&E. (note: blockage of glandular ducts typically results in apoptosis within the gland)

45 Biochemical Mechanisms Signaling pathways that initiate apoptosis ( Death Signals ) stimulate targets on cell surface or within the cell

46 Biochemical Mechanisms Control and integration balance of +ve / -ve regulatory molecules determines outcome of the affected cell

47 Biochemical Mechanisms Common execution phase actual death program accomplished by endonucleases & proteases (esp caspases)

48 Biochemical Mechanisms Removal of dead cells apoptotic bodies have ligands for phagocytic cells (efficient / no inflammation)

49 e) Consequences of too much or too little apoptosis Disorders associated with defective apoptosis (increased cell survival) increased survival of abnormal cells with neoplasia increased survival of autoreactive lymphocytes causing autoimmune disorder Disorders associated with increased apoptosis (excessive cell death) increased loss of cells in: neurodegenerative diseases ischemic injured cells viral infected cells

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