Cellular Pathologypeople.upei.ca/hanna/CELL PATH 4/CellPath-L4WEB-18.pdfRaw egg white (albumin protein) is a transparent / clear viscous liquid Adding heat, acid or alkali will denature

Cellular Pathology

(VPM 152)

Lecture 4 (Web)

Paul Hanna Jan 2018

• 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 2o or 3o structure)

IRREVERSIBLE CELL INJURY

1) Necrosis

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

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

• 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

1) Necrosis

• most common manifestation of cell death

• characteristic of hypoxic / ischemic death of cells in all tissues (except brain)

• necrotic cells eventually removed by leukocyte proteolysis & phagocytosis

• on LM, basic outline of the coagulated cell persists at least a few days

(protein denaturation predominates over enzymatic digestion)

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

a) Coagulation Necrosis

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.

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.

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).

Bovine, heart, myocardial necrosis (coagulative);

note irregular area of pallor within myocardium.

Microscopic Appearance

• original cell shape & tissue architecture is preserved (ie eosinophilic "shadow“)

• cytoplasm:

increased eosinophilia (H&E stain)

usually hyalinized (homogeneous / glassy)

may be mineralized

a) Coagulation Necrosis

• nucleus:

karyolysis

pyknosis

karyorrhexis

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)

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.

White muscle disease, skeletal muscle, calf. Note coagulation necrosis of myofibers characterized

by fragmentation and hyalinization; also note extensive mineralization (blue-purple staining)

Path Basis of Vet Disease, 8th ed

Ultrastructural Appearance of Irreversible Injury

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.

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. http://www.nal.usda.gov/awic/newsletters/v8n3/graphics/8n3afg1c.gif

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 Ca2+) and macromolecules from the ECF

- cells debris removed by phagocytosis or broken into fatty acid residues

Irreversible Injury Irreversible Injury

• 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

b) Liquefactive Necrosis

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.

Porcine, kidney, suppurative pyelonephritis, note

purulent exudate (again an example of liquefactive

necrosis).

Cytology of an aspirate from abscess / suppurative

exudate. The predominate cell type is neutrophils

and there are fewer scattered macrophages.

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.

• 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)

c) Caseous Necrosis

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)

Equine, lung tissue, pyogranulomatous

pneumonia due to Rhodococcus equi;

note caseous exudate (arrows).

Figure 01-19. 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.

• 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

d) Gangrenous Necrosis

Frostbite of the hand in a person (above). Cat with sloughing

of ear tips and paws following dry gangrene due to frostbite.

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

Bovine, mammary gland, wet gangrene;

tissue starting to slough.

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, 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]

• distinguished by its location within body fat stores

• etiology: inflammation (eg pancreatitis), Vit E deficiency, trauma, idiopathic

e) Fat Necrosis

Canine, recurrent pancreatic necrosis (“pancreatitis”) with mesenteric fat necrosis

Canine, recurrent pancreatic necrosis

(“pancreatitis”) with omental fat necrosis.

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.

• 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. & 2o inflam.

2) Apoptosis

Figure 17-35 (Molecular Biolology of the Cell, 4th 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.

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)

Physiologic causes:

• seen in many physiologic, adaptive and pathologic events:

Cells undergoing normal turnover

- hormone-dependent involution

- cell deletion in proliferating population

2) Apoptosis

Cells undergoing programmed cell death during embryogenesis

Immune System - deletion of autoreactive T cell in thymus

- immune regulation

Specific immune responses – via cytotoxic T cells

Pathologic atrophy of organs after duct obstruction

2) Apoptosis

Pathologic causes:

Misfolded proteins – ER stress

DNA damaged beyond repair – eg radiation, toxins

Specific infectious agents – esp viruses

d) Morphology

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

2) Apoptosis

• considerable apoptosis may occur in tissues before it is evident on histology

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.

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.

www.skin-science.com

Note, arrows indicate apoptotic epidermal keratinocytes (“sunburn cells”) due to UV-B radiation.

These can be induced within 30 minutes of sun exposure.

Figure 01-33 (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)

Signaling pathways that initiate apoptosis (“Death Signals”)

• stimulate targets on cell surface or within the cell

Biochemical Mechanisms

Control and integration

• balance of +ve / -ve regulatory molecules determines outcome of the affected cell

Biochemical Mechanisms

Common execution phase

• actual death program accomplished by endonucleases & proteases (esp caspases)

Biochemical Mechanisms

Removal of dead cells

• apoptotic bodies have ligands for phagocytic cells (efficient / no inflammation)

Biochemical Mechanisms

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