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Table of Contents
(The Yellow Pages)

1. Cellular injury: an overview (no web lesson).....2

2. Mechanisms of Cell Injury..............7
3. Morphology of Cell Death....13
4. Injury to Cell Membranes..25
5. Cell Injury from DNA Damage.... 29
6. Consequences of Cell Injury......34
7. Intracellular Accumulations, Fatty Change, Pigments and Tissue Deposits .....37
8. Extracellular Accumulations .....48
9. Postmortem Change ...54
10. Microscopic Images ...59

An Overview
Before we start our discussion of cell injury, please read this review. There is no
corresponding web lesson

Learning Objectives for this lesson

1) Know that there are more than one type of cell death
2) Understand how the types of cell death occur
3) Understand that apoptosis is a type of cell death
4) Understand the difference between apoptosis and necrosis
5) Understand the difference between necrosis and autolysis


1. If injury to cells is severe enough they die. Remember thrombosis?

2. Cell death can occur in a living animal in one of two ways:

Necrosis cell dies due to lack of energy
o Coagulative necrosis the default- cell dies, proteins coagulate
o Caseous necrosis when giant cells and multinucleated giant cells get
o Liquefactive necrosis when enzymes from neutrophils break down
coagulated protein OR in tissue with relatively little protein such as the brain
o Enyzmatic fat necrosis when lipases break down fat
o Non-enyzymatic fat necrosis loss of blood supply to fatty tissue
o Gangrenous necrosis when saprophytic bacteria get involved

Apoptosis- bunch of special genes and enzymes within the cell get activated and kill
the cell
o Also called programmed cell death or cell suicide because the dying cells
themselves activate the special genes and enzymes that end up causing the cell

3. Autolysis when the whole body dies and the cells die with it

Cell Injury
The cell is under constant bombardment by injurious agents. These injurious agents include
anything from trauma, chemicals, bacteria and viruses, to antibodies
produced by our own bodies (immune-mediated diseases).

This bombardment is the cause of disease and is broadly referred to

as Cell Injury. Cell injury is defined as an alteration in cell
structure or biochemical functioning resulting from some stress that
exceeds the ability of the cell to compensate through normal
physiologic adaptive mechanisms.

When cells are exposed to a stress that has the potential for producing injury, they typically
respond in one of two ways:
1. They can adapt by altering their structure
and/or biochemical processes in an attempt to
achieve a new "steady state" and maintain
near-normal physiologic functions
(homeostasis). For example, chronic exposure
to sunlight causes melanocytes in the skin to
synthesize more melanin to protect cells from
potentially injurious UV radiation tanning
(epidermal cell adaptation).
2. If the limits of adaptation are exceeded so
that critical systems within the cell cannot
maintain normal function, the cell is said to be
injured. For example, acute prolonged
exposure to the sun may lead to "sunburn"
(epidermal cell injury).

If the limits of adaptive capability are exceeded, or when the protective systems are
overwhelmed, the cell is "injured". The injury of the cell may range from mild and fully
reversible to severe and lethal. These changes are referred to as irreversible. And what do
cells with irreversible cell injury do??? Yup - they die.

Adaptation, reversible injury and cell death can therefore be considered as a continuum of
increasing and progressive "challenge" of the cell's structure and function.

Cell death
Simply stated, cell death is that point at which then injured cell cannot return to viable state (the
point of no return). Depending on the nature of the injury and the type of cell injured we may or
may not see morphologic evidence of cell death.

Types of cell death

For every cell, there is a time to live and a time to die. In general, there are three ways in which
cells die:
A. They are killed by injurious agents (necrosis)
B. They are induced to commit suicide (apoptosis)
C. They die when the animal dies (autolysis)

Necrosis Apoptosis Autolysis

A) Death by Injurious agents - Necrosis
Following necrosis, the sequence of events varies according to the environment, the inflammatory
response, the type of cell/tissue, the presence of a biological agent.

Types of necrosis:
Coagulative necrosis is most common. When the cell dies, the cytoplasmic proteins become
denatured and the cell is literally "coagulated or clotted". In coagulative necrosis, the outline of
the cells remains, as does the overall structure of the tissue. Grossly, the tissue will usually
appear pale compared to the normal tissue, and microscopically, the cells demonstrate loss of
nuclei, increased cytoplasmic eosinophilia, and loss of internal detail without losing the overall
cell shape or tissue organization.

Gangrenous necrosis is usually more of a clinical term rather than a pathological one. It refers to
necrosis affecting a limb or extremity, usually due to ischemia and associated with superimposed
infection. Gangrenous necrosis is coagulative necrosis with secondary invasion with saprophytic
(dead tissue loving) bacteria.

Liquefactive necrosis happens when necrotic tissue liquefies. This can happen for two different
1) Powerful hydrolytic enzymes from neutrophils and macrophages overpower the protein
denaturation. This occurs classically in many bacterial infections (abscesses).
2) Brain tissue that has very little protein to coagulation (your brain is mostly lipid) falls apart
and looks soft and gooey.

Enzymatic fat necrosis usually occurs in adipose tissue and is due to the release of enzymes
(lipases) from dead cells. The best example is when pancreatic cells die (acute pancreatitis).
Lipases act on triglycerides to generate free fatty acids, which react with intracellular ions like
Na+, K+, and Ca++ and produce soaps (saponification). Macroscopically these look like white
chalky nodules in the fat surrounding the pancreas or in the abdominal cavity. Microscopically
they consist of dead fat cells, inflammatory cells and phagocytes filled with fat.

Non-enymatic fat necrosis usually occurs in lipid-filled tissue (omentum) that lose their blood
supply. Like brain tissue, there is little protein to coagulate. The appearance is just like enzymatic
fat necrosis!

Caseous necrosis: Characteristic of tuberculosis, this is actually a combination of coagulative and

liquefactive necrosis. Grossly, the necrotic areas have a cheesy appearance (caseous means
cheese), and microscopically they necrotic areas are surrounded by macrophages and
multinucleated giant cells.

B) Death by suicide - Apoptosis


Cells can also die by being induced to commit suicide. The pattern of events in death by suicide
is so orderly that the process is often called programmed cell death or PCD. Programmed cell
death is also called apoptosis. (There is no consensus yet on how to pronounce it; some say A

POP toe sis; some say ah puh TOE sis.) This is often seen in some viral diseases, neoplastic
cells, and cells that the body has no need for anymore (like certain cells in the brain as an animal
undergoes normal development).

C) Death when the animal dies - Autolysis

When an animal dies, there is no longer the ability for cellular respiration to take place. When
cellular respiration ceases, the pH in the cell is altered. The destruction of a cell after its death is

usually caused by the action of its own (endogenous) enzymes, which are triggered by this

alteration in intra-cellular pH. This process is called autolysis (lysis of self).

2. Mechanisms of cell injury
(corresponds to the web lesson of the same name)

Learning objectives:
1. Understand the mechanisms leading to the four main causes of cell injury
2. Understand the differences in the response of different types of cells to injury
3. Relate the ultrastructural changes in reversible and irreversible cell injury


There are four reasons that cells get injured:

Hypoxia not enough oxygen - usually due to ischemia, but other causes like severe
anemia, carboxyhemoglobinemia, methemoglobinemia and cyanide poisoning
Injury by free radicals (including activated oxygen) these do most of their damage at
the cell membrane, disrupting homeostasis
Chemical/Traumatic injury Chemicals include a lot of toxins that act like free radicals,
but in addition, there are specific chemicals that get in and mess up the organelles.
Injury by infectious agents This might be the most common form of cell injury and it
happens in all kinds of ways. Viruses get in and mess up the cells normal function;
bacterial toxins can also damage cells. Some of the bacterial toxins act like free radicals.

Injury by hypoxia refers to loss of oxygen in a cell or tissue. Loss of oxygen can occur
either through ischemic injury or reduced oxygen carrying capacity of blood.

Ischemia = interruption of blood flow

Remember from the previous unit (Disturbances of Circulation) that thrombosis of an artery can
result in ischemia of the tissue supplied by that artery. If the tissue is totally dependent on the
thrombosed artery for nutrients and oxygen (single vessel supply), the tissue supplied by that
artery will become hypoxic, ischemic, and eventually dies. The coronary arteries, renal arcuate
arteries, and cerebral arteries are all examples of vessels that are the sole blood supply to a region
of tissue.

The brain, heart, and kidney are all organs that are severely damaged following vascular
thrombosis. But they are not all affected equally. What are some factors that influence the
response of a cell to injury?

If the tissue has good collateral circulation, (parallel supply), as in skeletal muscle and the
digestive tract, the tissue is less likely to experience the degree of ischemic damage.
The cell type can influence the degree of damage. Neurons in the brain can be deprived
of oxygen and nutrients for only 2 or 3 minutes before irreversible damage occurs.
Myocardial fibers can survive longer, perhaps 30-60 minutes.
The ability of cells to switch to alternate energy pathways, such as anaerobic glycolysis,
is also a primary factor in determining the ability of the cells to withstand a brief episode
of ischemia.

Eventually, regardless of the cells ability to respond to injury, cell damage can occur. Many
cells can withstand deprivation of oxygen and nutrient for brief periods; however, if the ischemia
persists, even cells that can meet energy needs by anaerobic glycolysis of stored glycogen will
eventually become depleted of their reserves.

Remember, also, an organ is made of several types of tissues, and some are less susceptible to
injury than others. For example, mesenchymal cells such as fibroblasts, which make up the
stromal framework of many parenchymal organs, are less susceptible to ischemic injury than the
epithelial cells of the organ. This is fortunate, because if the stromal framework survives, the
epithelial cells can proliferate to regenerate organs such as liver following episodes of ischemia.

Ischemic injury is probably the best-studied form of hypoxia. In ischemic damage, the changes
are divided into reversible and irreversible.

Reversible changes are known to occur when the duration of the ischemia is short and in the
following sequence:

Because ATP is needed to keep cell membrane integrity and for active sodium transport
(Na+/K+ pump), in hypoxia, the cell is unable to keep Na+ out of the cell and cell swelling
(reversible) occurs.

If cell swelling persists, there is accumulation of lactate and inorganic phosphate. This is associated
with dilation of the endoplasmic reticulum. The next effect is detachment of ribosomes from RER.
Without ribosomes on the RER, there is no protein made. Cells cant live if they cant make
protein (Irreversible).

Impaired aerobic respiration (mitochondria) decreased ATP (energy) anaerobic glycolysis

glycogen depletion accumulation of lactic acid DEATH.

Irreversible changes occur if hypoxia persists.

The intracellular pH changes produces injury to
lysosomal cell membranes, leakage of their
powerful enzymes into cytoplasm and cell
digestion including nuclear changes and cell
death. It is believed that the critical changes that
make injury irreversible and lethal in hypoxia
have to do with the inability of the
mitochondria to recover from injury (after re-
oxygenation) producing permanent damage on
cell membrane function and integrity. The
biochemical pathways involved in such cell
membrane dysfunction include:

Loss of surface phospholipids

Cytoskeletal alterations
Free radicals (more on this later)
Toxic effect of lipid breakdown products
(excessive release of free fatty acids have a
detergent effect on cell membranes).

If blood supply is restored suddenly and re-

oxygenation takes place in a cell so
structurally damaged, this in itself can cause
cell injury. Denaturation of membrane
proteins, release of inflammatory mediators
and production of large amounts of oxygen-
derived free radicals occurs. These all further
damage the cells and this additional injury is
known as re-perfusion injury.


If the blood is not getting oxygenated properly, as occurs in cardiorespiratory failure, or if the
oxygen carrying capacity of blood is diminished, as in carbon monoxide poisoning, hypoxia will
occur. The mechanisms of cell injury in this form of hypoxia are very similar to that of ischemia

with one major difference

Q. What is the difference between hypoxia and ischemia?

A. The damage tends to be more generalized and not localized as in ischemia.

Severe anemia would reduce the oxygen carrying capacity of the blood since the level of
hemoglobin would be drastically reduced. This will have a generalized effect on the animal.

Cyanide poisoning
Even if oxygen is adequately delivered to cells, the mitochondria must be able to use it.
Interruption of the electron transport chain for any reason would prevent the continued reduction
of oxygen to form water. Cyanide is an inhibitor of the electron transport chain, causes
intracellular hypoxia, even though the blood appears bright red due to the super-saturation of
hemoglobin with unusable oxygen.

Cyanide interferes with oxidative phosphorylation by blocking the terminal step in the electron
transport chain. It does this by forming a stable, irreversible complex with the cytochrome a3
subunit of cytochrome C oxidase, making it unable to transport elections.

M Carboxyhemoglobinemia/ Methemoglobinemia
Even if adequate hemoglobin is
present in the blood, it must be
able to carry the oxygen, and
formation of compounds such
as carboxyhemoglobin (as in
carbon monoxide poisoning), or
as methemoglobin (in nitrate
poisoning for example), would
interfere with the ability to
transport oxygen throughout the
entire body.

Injury by Free Radicals

Free radicals are substances produced by cells during normal daily activities. There are many
systems in place that dispose of (scavenge) these free radicals before they can damage the cell
membrane. There are also many agents that can cause cell damage by messing with the ability
of the body to maintain the integrity of cell membranes and triggering a lethal sequence of events
(lipid peroxidation). Activated oxygen radicals are now known to be the common mechanism to
cell in injury in many conditions, i.e. aging, chemical and radiation injury, bacterial infections,
inflammation, tumor necrosis, etc. These free radicals combine with inorganic or organic
substances in cell membranes (lipid peroxidation) and nucleic acids (mutations).

Remembered, however, that the body has in place a series of protective mechanisms to protect the
cells from these free radicals (antioxidants, enzymes such as catalase, glutathione peroxidase and
superoxide dismutase).

One of the most common causes of free radical damage occurs when the protective mechanisms
fail due to nutritional imbalances. One of the most common syndrome seen in association with
nutritional imbalances is selenium or vitamin E deficiency. Grossly, white muscle disease
produces pale patches or streaks in the muscle, and can involve skeletal muscles such as those of
the thigh, shoulder (even the tongue) as well as the heart giving the disease the name White Muscle

Selenium and vitamin E deficiency cause accumulation of free radicals that damage vital cell
components. Vitamin E and selenium-containing enzymes are required in order to get rid of free
radicals the body produces. Free radicals may initiate cellular injury by causing peroxidation of
membrane lipids and by causing physical and chemical damage to protein molecules.

Infectious agents are the most common cause of cell injury and death. They can have a
direct cytopathic (cell killing) effect or cause injury indirectly through one of the other three
above mentioned causes. Infectious agents include viruses, bacteria, fungi, yeasts and

Other forms of cell injury which are less common include immune-mediated cell
reactions and genetic derangements.
Immune-mediated reactions

Immunological reactions can be a cause of

cell death. As you will learn later, there are
four types of immune mediated causes of cell
death. One of these types (Type II) can cause
autoimmune hemolytic anemia.

Individuals with autoimmune hemolytic

anemia produce antibodies to their own red
blood cells which are then destroyed by
phagocytosis due to opsonization (coating
with antibody). This occurs primarily in the
spleen, where the red blood cells are
recognized as foreign due to surface

Genetic derangements
Genetic derangements can also be responsible for the death of cells. An example of a genetic
disorder would be the lysosomal storage diseases, such as globoid cell leukodystrophy (Krabbes
disease), which affects people as well as Irish Setters, Cairn Terriers, and West Highland White
terriers and several other breeds of dog (more on this disease later). With lysosomal storage
diseases, the cell lacks an enzyme necessary to break down used cellular molecules. The
deficient enzyme determines the lysosomal storage disease. (e.g. in globoid cell leukodystrophy,
the enzyme lacking is -galactocerebrosidase aka galactosylceramidase). In globoid cell
leukodystrophy, lysosomes become full of non-degraded cellular molecules, and get very large,
giving the affected cell a foamy globoid appearance.

3. Morphology Of Cell Death
(corresponds to the web lesson of the same name)

Learning objectives:

1. Recognize the different types of cell death

2. Distinguish apoptosis from necrosis and autolysis


In general, three different forms of cell death are recognized

Necrosis - Necrotic cells are usually found in contiguous sheets and often associated with
a striking acute inflammatory reaction.
o The types of necrosis
Coagulative usually involves ischemia
Liquefactive involves hydrolytic enzymes (like bacteria and neutrophils
Enzymatic fat necrosis happens only in fat, usually due to anti-fat
enzymes (like lipases) from the pancreas leaking out
Caseous type that happens with specific bacteria that hydrolytic enzymes
cant touch
o Nuclear changes characteristic of necrosis, usually in this order
Pyknosis chromatin is clumped, dense, nucleus is small
Karyorrhexis chromatin is breaking apart and scattered
Karyolysis all the nuclear material is gone, cant find it!
o Cytoplasmic changes characteristic of necrosis
Often depends on the insult
The cytoplasm gets MORE eosinophilic, and uniform, because proteins
are denaturing
Apoptosis Cell DECIDES to die. The apoptotic cells are usually individualized and are
phagocytized by macrophages or adjacent cells; no significant inflammatory reaction
appears to occur.
Autolysis death of cells after death of animal; because there is no more energy to maintain
membranes, enzymes leak out and start to degrade everything. Also, bacteria move into
the tissues, usually from the GUT, and have a fun time, and utilize stuff from the cells,
making them look awful. Plus, everything smells really bad

Necrosis results from severely disturbed extracellular environmental conditions, and it is
associated with the characteristic morphologic changes of cell injury, including
increased cytoplasmic eosinophilia
pyknosis (shrinkage of the nucleus to a black blob)
karyorrhexis (breaking up of the nucleus into fragments)
karyolysis (lysis of nucleus and/or loss of nuclear staining).

Types of necrosis:
Coagulative necrosis is most common. This is when the
cell is literally "coagulated or clotted". The proteins of the
cell are denatured and the cell becomes an opaque,
eosinophilic mass and often the nucleus disappears. This
usually occurs in conditions of ischemia. Not only the
structural proteins are denatured but also the enzymes
necessary for their degradation. Ultimately such areas of
coagulative necrosis are removed by phagocytosis and
enzymatic destruction by inflammatory cells. Gangrenous
necrosis refers to necrosis affecting a limb or extremity,
usually due to ischemia and associated with superimposed
saprophytic bacterial invasion. So there is coagulative
necrosis plus yucky bacteria that like to eat rotting flesh!

Liquefactive necrosis: Powerful hydrolytic enzymes

play a predominant role in protein denaturation. This
occurs classically in bacterial infections (like those
that make pus!). It also occurs readily in brain,
because the brain has relatively little protein to

Enzymatic fat necrosis usually occurs in adipose
tissue and is due to the release of enzymes (lipases)
from dead cells. The best example is when
pancreatic cells die (acute pancreatitis). Lipases act
on triglycerides to generate free fatty acids, which
react with K+. Na+, and Ca++ and produce soaps
(saponification). Macroscopically these look like
white chalky nodules in the fat. Microscopically
they consist of dead fat cells, inflammatory cells and
phagocytes filled with fat. (Non-enzymatic fat
necrosis looks the same but is due to ischemia)

Caseous necrosis: Characteristic of diseases

such as caseous lymphadenitis, this is actually a
combination of coagulative and liquefactive
necrosis. The necrotic areas macroscopically
have a cheesy appearance. Caseous = cheese.
Microscopically, they are infiltrated with
macrophages and multinucleated giant cells

Necrotic cells are characterized microscopically by both cytoplasmic and nuclear changes.

Nuclear changes include

o pyknosis
o karyorrhexis
o karyolysis

In pyknosis, the cell nucleus becomes

small, dark and rounded.

In karyorrhexis, the nucleus begins to

disintegrate and appears as multiple
little pieces. (Rhexis = BREAK)

Finally karyolysis occurs. In this

process, the nucleus is difficult to
discern. (Lysis = DISSOLVE)

Cytoplasmic changes are also evident.

In coagulative necrosis, the outline of the cells remains, as does the overall structure of the
tissue. Grossly, the tissue will usually appear pale compared to the normal tissue, and
microscopically, the cells demonstrate loss of nuclei, increased cytoplasmic eosinophilia, and
loss of internal detail without losing the overall cell shape or tissue organization.


Apoptosis is often also called
programmed cell death. This form
of cell death is heavily involved in
cellular development, aging, and
normal cell turnover in the adult

Apoptosis is a highly regulated

process involving veritable maze of
interactive enzymes and pathways.

Apoptotic cells die by design.

Necrotic cells die by accident.

Abnormal regulation of apoptosis

contributes to well-known
disorders, such as autoimmune
diseases, neoplasia, and viral

Mechanisms of Apoptosis
Apoptosis means the "falling off" (like leaves from a
tree) or a selective removal of individual cells without
complete disruption of the rest of the tissue or organ
from which they came. There is usually no overt
inflammatory response and dead cells are phagocytized by
macrophages or adjacent cells

Apoptosis can be divided into three phases:

an induction phase
an effector phase
a degradation phase

Note that in real life these are non-distinct overlapping phases.

Apoptosis is characterized morphologically by cell shrinkage and other characteristic changes

such as:
nuclear chromatin condensation,
fragmentation of nucleus,
fragmentation of cell into membrane bound bodies (apoptotic bodies).

In the nucleus, the chromatin condenses, the nuclear envelope breaks down,
and the DNA is fragmented by enzymes called nucleases into 180-200 base
pair fragments that can be detected on an agarose gel. The fragments form a
distinct ladder appearance (lane B), while necrotic cells from a smear (lane

Necrosis vs. Apoptosis
Necrosis can be distinguished from apoptosis. In
necrosis, which is cell death due to some kind of severe cell
injury, and the normal tissue is quite disrupted. In
apoptosis, which is genetically programmed cell death, there
is an orderly disassembly of cellular proteins and DNA with
minimal disruption to normal tissue.
Apoptosis can be a normal physiologic process
designed to eliminate unwanted, functionally abnormal, or
senescent (old and worn out) cells. It plays an important role
in the developing embryo, certain hormone-dependent
tissues, and in aging. However, in some instances, apoptosis
may be a pathologic process induced by cell injury (e.g.,
viral infection, radiation injury, etc.).


Autolysis is auto + lysis = self digestion. After death, organs decompose due to enzymes
released by tissues and by bacterial contaminants. So, the short answer is, yes, this is cell death.
BUT does it really matter, because the cell is only dying because the whole animal died. However,
there is information that can be gleaned from autolysis.

The rate of autolysis is variable - it depends upon temperature and other environmental factors.
Since autolysis is variable, forensic pathologists have difficulty determining the exact time of

Here is an overview of necrosis?
Coagulative grossly it is pale; histologically architecture is preserved but ghost-like
Liquefactive grossly it is gooey; histologically there is no detail, everything is
destroyed. One type = abscess; other type = CNS
Caseous grossly it is cheesy or crumbly; histologically, it is messy, might have a lot of
Gangrenous grossly it is usually dry and dark; histologically it is ghost-like, but often
with a lot of big saprophytic bacteria
Fibrinoid this is only a histologic term, and refers to changes in blood vessels when
they are so damaged that proteins leak into the walls making them homogeneous and
Enzymatic fat necrosis grossly it is chalky; histologically it is amorphous pink and blue
with lots of dead and dying fat cells
Non-enzymatic fat necrosis grossly it is chalky; histologically it is amorphous pink and
blue with lots of dead and dying fat cells

Here is an in-depth view of necrosis
Coagulative necrosis
Coagulative necrosis is the most common and is when proteins of the cell are denatured and the
cell becomes histologically an opaque, acidophilic (reddish) mass. Usually the nucleus
disappears. This usually occurs in conditions of ischemia in high protein tissues. Not only the
structural proteins are denatured but also the enzymes necessary for degradation of such.
Ultimately such areas of coagulative necrosis are removed by phagocytosis and enzymatic
destruction by inflammatory cells. Necrotic foci are pale compared to adjoining parenchyma.
Microscopically, tissue architecture is preserved and while the identity of the necrotic cells is
still recognizable, the cells are ghost-like. The nuclei of necrotic cells have often disappeared.
Their cytoplasm is usually homogeneous and eosinophilic, due to the coagulation of cytoplasmic
proteins. As the cell dies, pH drops preventing autolytic enzymes from working. It also causes
the proteins in the cytoplasm to coagulate. It is the coagulation of these proteins that prevents
the rapid destruction of the cell, and preserves the outline of the cell.

Liquefactive necrosis
Liquefactive necrosis occurs when powerful hydrolytic enzymes play a predominant role over
protein denaturation. This occurs classically in brain tissue and in some bacterial infections.

Abscesses are classical examples of liquefactive necrosis. An abscess contains lakes of degenerate
neutrophils with intracellular bacteria (pus involved). The original tissue has been digested away
by proteases. These proteases are primarily from neutrophils; hence liquefactive necrosis is often
seen associated with purulent (pus containing) inflammation. Except for some phagocytized
debris, none of the original tissue would remain.

Another type of liquefactive necrosis occurs in the central nervous system. This type of
liquefactive necrosis typically develops following anoxic cell injury and death (no pus involved).
The CNS is high in lipid and low in protein therefore there is not a lot of protein to coagulate. This
necrosis can be produced by a variety of mechanisms and agents including infarction, toxins,
nutritional deficiencies, and trauma. Malacia is the term used for the gross appearance of

liquefactive necrosis in the CNS, but not the microscopic appearance.

Liquefactive necrosis - malacia

necrosis - pus

Nigropalladial encephalomalacia in horses

is caused by ingestion of yellow star thistle, a
common weed in the Western states. The
typical histological lesion is a large cavitated
defect in the brain surrounded
by and containing gitter cells,

which are histiocytes

(macrophages) of the CNS.
Above is a great example of
liquefactive necrosis in the

Caseous necrosis
Caseous necrosis occurs when the bacteria causing the necrosis are covered with a thick capsule
and cannot be easily broken down by neutrophils. The necrotic areas macroscopically have a
cheesy-crumbly appearance.

Caseous necrosis is usually associated with granulomatous inflammation (inflammation with


macrophages and giant cells). The typical lesion is seen in caseous lymphadenitis, a disease of
sheep caused by Corynebacterium pseudotuberculosis. Large portions of the lymph nodes are
occupied by thick, pasty white material. It may be difficult to tell this material from abscesses,
but remember; pus is liquid (usually unless it gets dried up).
The material in caseous lymphadenitis is like dried
toothpaste, and crumbly and is the hallmark of caseous

Histologically, tissue cells are no longer recognizable in

caseous necrosis and tissue architecture is lost. In many
cases, the only recognizable cells are inflammatory cells,
and the cellular debris is surrounded by a zone of
lymphocytes, epitheliod (epithelial-looking) macrophages
and multinucleated giant cells. Occasionally, in cases of
caseous lymphadenitis, the causative bacteria, Corynebacterium pseudotuberculosis, can be
found at the center of the areas of necrosis, but this is the exception rather than the rule. These
bacteria have a thick waxy coat and are not easily broken down by neutrophils, so the next line
of defense, the macrophages and the multinucleated giant cells rush in to the rescue.

Gangrenous necrosis
Gangrenous necrosis is usually a clinical term rather than a pathological one. It refers to
coagulative necrosis affecting a limb, usually due to ischemia and associated with superimposed
infection. The term gangrene generally refers to ischemic necrosis of extremities, with invasion
of saprophytic bacteria. In fact, it is not a specific type of necrosis, but coagulative necrosis with

saprophytic bacterial invasion superimposed. Gangrenous tissues have an odor of rotting flesh.
Tissues which are particularly susceptible to developing gangrene include limbs, mammary glands,
and male genitalia. The term is also applied to aspiration pneumonia and occasionally to
saprophytic invasion of preexisting upper GI lesions.

Fibrinoid necrosis
Fibrinoid necrosis is most often seen in association with blood vessels.
It the result of coagulative necrosis of the blood vessel wall
superimposed with leaky fibrinogen from the damaged blood vessels and
immune complex deposition. The affected blood vessels appear very
eosinophilic and the deposition can obscure the coagulative necrosis of
the vessel wall. Sometimes, there are accumulations of inflammatory
cells, usually neutrophils, in association with the fibrinoid necrosis.

Enzymatic Fat Necrosis

Enzymatic fat necrosis usually occurs in adipose tissue and is due to the release of enzymes such
as lipases from dead cells. The best example is when pancreatic cells die (acute pancreatitis).
Injured pancreatic tissue releases amylases, and proteases, which further attack the pancreatic cell
membranes. Damage to the cell membrane activates lipases, which catalyze the production of
free-fatty acids from triglycerides. The free fatty acids react with calcium, sodium and potassium
ions to produce soaps. The process is referred to as saponification. Grossly, these look like
"chalky" nodules in the fat surrounding the pancreas or in the abdominal cavity. Microscopically
they consist of dead fat cells, inflammatory cells and phagocytes filled with fat.

In most cases in small animals, enzymatic fat necrosis is secondary to pancreatitis or pancreatic
overstimulation (obesity).

Non-enzymatic fat necrosis
In cattle, another kind of fat necrosis can be often seen in the abdominal cavity. The mesenteric
fat around the small intestine can undergo necrosis and it becomes dominated by chalky white to
yellow deposits. This is not enzymatic fat necrosis but rather fat necrosis secondary to ischemia
due to an endophyte fungus (Acremomium coenophialum) infecting pasture (predominantly tall
fescue pasture).

The pathogenesis of non-enzymatic fat necrosis in cattle is as follows; cattle feed on the pasture
and ingest the fungus. A toxin in the fungus called ergotamine causes vasoconstriction and reduced
blood flow to the intra-abdominal fat. This causes loss of blood supply to the fat which leads to
non-enzymatic fat necrosis. This condition can be subclinical or can be fatal if large amounts of
ergotamine are ingested. In most cases, it does cause decreased rate of growth.
Okay, now cover the bottom half of the page and uncover it after you answer the question below.

Question. The fungus can also cause decreased blood supply to the feet, teats and tips of the ears.
What kind of necrosis could occur then?

Answer? Gangrenous necrosis

Did you get it right?

(corresponds to the web lesson of the same name)

Learning objectives:
1. Understand the function of the Na/K pump
2. Understand how free radicals cause damage to cell membranes
3. Know the pathogenesis of lipid peroxidation


Maintenance of the cell membrane is essential for maintaining homeostasis.

How does the cell membrane get damaged?
By free radicals these molecules are really good at damaging the lipids in cell
By disrupted cytoskeleton not so many things do this, but if the cytoskeleton is
damaged, so are the cell membranes because they are all connected
Consequences of cell membrane damage
- Because the sodium pump is in the membrane, when it is damaged, water rushes into the
cell, causing the cell to swell.
- Calcium ions come in also, which cause serious damage to the mitochondria, so there is
no energy to run the cell.
- Ribosomes fall off the endoplasmic reticulum, which means the cell cant make any
protein, and that is most definitely a bad thing.

First, a review of normal cell membrane structure and function

The cell membrane serves as a semipermeable barrier. Injury to the cell membrane has
immediate and severe consequences. Even in anoxic injury, the common pathway to irreversible
cell injury and necrosis involves damage to the cell membrane. The cell membrane is the site
of the sodium ion pump which maintains cell volume by pumping sodium out of the cell.

Consequences of membrane damage. When cell membranes are exposed to noxious agents
both outer plasma membrane and the membranes of organelles are at risk. The consequences
Loss of the sodium/potassium ion pump and cell swelling
Abnormal calcium fluxes occur, leading to mitochondrial damage and activation of
Injury to the rough endoplasmic reticulum results in disassociation of ribosomes and protein
synthesis ceases.
Injury to mitochondrial membranes and sequestration of calcium inhibits oxidative
phosphorylation, starving the cell of energy in the form of ATP.

HOW does the cell membrane get injured? Really, there are only two ways -

Free radical injury

One of the most important mechanisms of membrane damage results from the action of free
radicals on the unsaturated lipids in membranes. This generates an autocatalytic chain reaction
called lipid peroxidation which causes widespread damage.

Free radicals are highly unstable chemical species that have a single unpaired electron in an outer
orbit. This unpaired electron makes them extremely reactive with proteins, lipids, and
carbohydrates. Where do free radicals come from? Some are from inside normal cells. Lots come

from the white blood cells, especially neutrophils and macrophages. This is part of the bodys
defense against invaders. Free radicals can also come in from outside especially from ionizing
radiation, or the reaction of the bodys enzymes on ingested chemicals or drugs.

Free radicals produced by endogenous metabolic reactions include

superoxide anion (O2-),
hydrogen peroxide (H2O2)
hydroxyl radical (-OH).

Lipid Peroxidation
Once oxidation of fatty acids begins, further reactions form lipid radicals that combine with oxygen
to form hydroperoxyl radicals that in turn form lipid peroxides. This reaction becomes self-
propagating resulting in widespread membrane damage due to lipid peroxidation of any cell
membrane that is high in unsaturated fatty acids.

As would be expected, the cell has a bunch of defensive mechanisms against this kind of damage.
Endogenous antioxidants such as vitamin E, ascorbate, ceruloplasmin, transferrin, and cysteine,
help protect membranes from oxidative injury. Enzymes such as superoxide dismutase,
glutathione peroxidase and catalase neutralize free radicals. Any interference with these
protective mechanisms can result in membrane damage by oxidants.

Cytoskeletal abnormalities lead to membrane damage.
The cytoskeleton composed of microfilaments,
intermediate filaments and microtubules serves as a
structural support system and transport system for the
cell. Detachment of the cytoskeleton from the plasma
membrane caused by cell swelling or certain toxins
(e.g. microcystin found in blue-green algae) results in
membrane blebs and renders the cell membrane
much more susceptible to lysis.

5. Cell Injury from DNA Damage
(corresponds to the web lesson of the same name)

Learning objectives:
1. Understand the various ways that DNA can be damaged and the pathogenesis of each.
2. Know the mechanisms of DNA repair
3. Know the consequences of failure of accurate DNA repair


DNA can be damaged by many causes.

There is an inherent risk of DNA damage all the time, from simple errors in replication
that occur spontaneously, and also from exogenous attacks. Sometimes these get
Repair mechanisms are important.
cell cycle checkpoints are activated to arrest cell cycle progression to allow time for
repair before the damage is passed onto daughter cells
induction of transcriptional programs
enhancement of DNA repair pathways, and when the level of damage is severe,
initiation of apoptosis

DNA is a double-stranded molecule twisted into a

helix (think of a spiral staircase). Each spiraling
strand, comprised of a sugar-phosphate backbone
and attached bases, is connected to a
complementary strand by non-covalent hydrogen
bonding between paired bases. The bases are
adenine (A), thymine (T), cytosine (C) and guanine
(G). A and T are connected by two hydrogen bonds.
G and C are connected by three hydrogen bonds.

The maintenance of genome of the cell is essential for the proper function and survival of all
organisms. This task is housed in the molecule DNA (deoxyribonucleic acid) the so-called
building block of the cell. This task is particularly daunting due to
constant assault on the DNA by genotoxic agents (both endogenous and exogenous)
nucleotide mis-incorporation during DNA replication, and the fact that DNA is weirdly
biochemically unstable and has to be constantly repaired!!!

Cell damage can be the direct result of DNA damage. DNA like all other biomolecules, can be
damaged in numerous ways. Both intrinsic and extrinsic damage can occur.

Intrinsic damage occurs due to internal replication errors like deamination, depurination,
or oxidation.

Extrinsic damage occurs due to external causes like X-radiation, u-v light, and a bunch of
bad chemicals.

Either way, when the DNA is damaged, it has to get repaired or there might be consequences.

Damaged DNA can be either repaired or replicated and the resultant damage incorporated into
the genome as a mutation. The resultant mutation can increase the viability of the cell. Most
often however, the mutation is either a null or neutral mutation that results in a normal variation
(blue eyes vs. brown eyes) or a mutation that results in cell damage or even cell death. DNA
damage has been shown to be involved in a variety of genetically inherited disorders, in aging,
and, as you will see later on in this course, in carcinogenesis (cancer causing).

Many types of DNA damage are generated spontaneously, in some cases at very high frequency.
There are enzyme systems in place to repair this DNA damage. However, damage to these
enzyme systems can also occur. These defects in DNA repair systems are likely to occur even in
the absence of additional damage caused by environmental factors.

Types of DNA damage:

Base loss
Loss of one of the bases in DNA can occur. This
loss is usually repaired by enzymes called
endonucleases that cut the molecule on either
side of the area of base loss and sew it back
together again. Damage to these enzymes results
in lack of repair hence damaged DNA.

Base modification/deamination
Cytosine can react with water and undergo domination to uracil. Uracil is
not normally found in DNA so its presence is abnormal and results in DNA
damage. This is the most common pro-mutagenic change seen in DNA.

Chemical modification
The nucleic acid bases are susceptible to numerous modifications by a wide variety of
chemical agents. Free radicals (generated during normal metabolism or by ionizing
radiation can damage DNA. Environmental chemicals, including "natural" ones
(frequently in the food we eat) can modify DNA bases, frequently by addition of a
methyl or other alkyl group (alkylation).

The Target theory and the Indirect hit theory

You dont have to live near Chernobyl to experience effects of radiation. The most common source of
radiant energy-induced damage to animals and humans is the sun.

There are two theories as to the mechanism of

radiation induced cell injury. The target theory
proposes that radiant energy can make either a
direct or an indirect effect on DNA.

A direct hit directly damages bonds within DNA

of a cell. This attack on DNA may lead to
mutation having genetic or cancerous potentials
(watch that suntan) or to inhibition of cell division
and cell death.

The indirect hit theory proposes that radiant

energy exerts its affect by acting on water and
producing free radicals which interfere with
critical cellular components, such as membranes,
nucleic acids, and enzymes, leading to cell death.

Replication errors
Mismatches or small insertions or deletions during DNA replication cause replication errors.
DNA polymerases are enzymes that fix these errors. Although DNA polymerases are
moderately accurate, and most of their mistakes are immediately corrected by polymerase-
associated proofreading enzymes called exonucleases, this fix is not always perfect.

Inter-strand cross-links
Alkylating agents such as psoralen can cross-link
DNA strands. Psoralen does most of its action by
getting between the two strands of the DNA
double-helix, specifically between adenine and
thymine bases. Without activation, this is not a
problem as proteins can push the unbound psoralen
aside and get to the DNA. When exposed to UV
light like that found in sunlight, or UV lamps, a
chemical reaction takes place that binds the
psoralen molecule on both sides, effectively tying
the DNA together. Any process that would need
the DNA, like cell reaction or division, can't
happen since the DNA is no longer usable. This
cross-linking can lead to cancer!!
DNA-protein cross-links
Enzymes known as DNA topoisomerases generate cross-links between themselves and their
DNA substrates during the course of their enzymatic action. Usually these cross-links are
transient and are reversed as the topoisomerase action is completed. Occasionally something
interferes with reversal, and a stable topoisomerase-DNA bond is established. Alkylating
agents and radiation can also create crosslinks between DNA and protein molecules.

Strand breaks
X-rays can cause DNA strand breaks that lead to mutations that can also lead to cancer.

DNA repair mechanisms

There are many ways that DNA molecules can be damaged. Since repair systems must be
capable of recognizing and dealing with each type of damage, it is not surprising that there are a
large number of different types of repair systems.

All cells have evolved a multifaceted

response to counteract the bad effects of
DNA damage. Upon sensing DNA damage
or stalls in DNA replication, several
mechanisms go into action -
1. cell cycle checkpoints are activated to
arrest cell cycle progression to allow time
for repair before the damage is passed onto
daughter cells
2. induction of transcriptional programs
3. enhancement of DNA repair pathways,
and when the level of damage is severe,
4. initiation of apoptosis.

(corresponds to the web lesson of the same name)

Learning objectives:
1. Understand the difference between regeneration and scarring (fibroplasia)
2. Understand the difference between an ulcer and an erosion


When cells die, and the animal continues to live, there are only two outcomes:
Cells regenerate and fill in the gap left by the dead cells
o This can only happen in organs that can regenerate.
o What does it look like histologically?
New cells are usually bigger because they are busy making new materials
There may be obvious mitosis evident in cells that can regenerate
Cells dont regenerate and instead the gap is filled by scarring
o This is the only option in some tissues, like heart and brain
o It also happens in tissues that ARE capable of regenerating but when the gap is so
big, that there is no framework left on which regenerating cells can work.
o What does this look like? Starts out as granulation tissue (fibroblasts actively
working and capillaries growing; progresses to fibrous tissue which is the scar)

Eventually all cells die. It has been said that life is a fatal disease - eventually we all die. Thus
in cell injury, we are really concerned with premature dysfunction and cell death.

There are two outcomes of cell injury - recovery or death. Purists might add another - incomplete
recovery (the cell does not return to complete normality).

Outcomes of Necrosis

In cases of necrosis, the damaged tissue can recover from the necrotic process if:
the cause of the necrosis is neutralized or removed AND
the animal remains alive during the recovery period AND
new cells are produced to replace the necrotic cells.

If the agent is not controlled, the animal often dies. The cause of the cell injury may be difficult
or impossible to remove. If so, recovery cannot occur and the organ may be severely damaged
and the animal destroyed! It is analogous to the situation where For lack of a nail, a shoe was
lost, for lack of a shoe a horse was lost, and for lack of a horse the battle was lost.

There are two possible outcomes for tissue replacement in the case of focal areas of necrosis

Dead cells must be replaced. In some tissues, necrotic cells are replaced by regeneration. Certain
organs are very capable of regeneration. The liver is an example. A big section of liver can be
destroyed, but if the framework is still there, it can regenerate. Some other tissues can never
regenerate. For instance, heart and brain once those cells have been lost they are GONE.
One proof of cell replacement is mitosis. Another is increased cytoplasmic basophilia (cells
appear bluer on hematoxylin and eosin stains). This is because there are more protein-producing
ribosomes in the regenerating cell. When cells are replaced by new cells, the replacement cells
(new cells) are larger and more basophilic because the ribosomes are geared up to produce
protein and other needed products.

Fibroplasia (Fibrosis, scarring)

It is desirable to replace the cells with the same type that was destroyed. Some tissues have no
capacity to regenerate. You have all the cells you are ever going to have in that organ on the day
you are born. Examples here include heart and brain. Any brain cell you lose is gone forever.

In some tissues that are quite capable of regeneration, if the damaged area is very large, there may
be no template or framework for the regenerating cells to build on. Examples would be intestine
and skin. Both of these organs have tremendous capabilities of regeneration. But they need a
basement membrane to work on. If the necrotic gap is too big, those basal cells just cant move
across it to do their thing. This is why with serious skin injuries, such as burns, it requires a skin
graft. The remaining skin cells just cant stretch across that far, without a nice framework to move
on. In these cases, the connective tissue (collagen, fibroblasts) proliferate to replace the dead cells
in the tissue.

Ulcers and erosions

ULCERS occur ehen the epithelium is lost and the basement membrane
or underlying tissue is exposed. Ulcers are often the result of focal
necrosis of epithelium through the basement membrane. In ulcers, the

epithelial cells are irreversibly injured and cell death ensues. The
necrotic cells are sloughed and an ulcer results. One concern with ulcers
is hemorrhage. Another is that the necrosis may not stop with the
epithelium, but continue and destroy the entire wall of the organ or tissue and result in perforation.
Incomplete loss of the epithelium in which only the surface epithelium is lost is different. These

are called EROSIONS. They do not extend through the basement membrane. Erosions are easier
to repair as there is no damage to the basement membrane! Erosions usually do not bleed because
the blood vessels below the basement membrane are not damaged.

The stomach is very susceptible to ulceration due to the gastric acids that are secreted. If the
normal protection provided by mucus and buffering bicarbonate secretions is lost, the acids attack
the epithelium and cause cell death. Thus erosions that would heal quickly on the skin may become
an ulcer in the stomach.

(corresponds to the web lesson of the same name)

Learning objectives:
1. Understand the causes and results of fatty change
2. Differentiate a pigment from a deposit
3. Identify the pathogenesis of pigments and other deposits in tissues


Things that accumulate within cells

Lipids - this is called fatty change
Lipofuscin brown, granular, these are breakdown products of lipids and indicate that
a cell is old or has been chronically injured.
Melanin normal in dark tissues
Bilirubin liver cells that cant work so well will have too much of this because
export is impaired.
Iron from breakdown of red blood cells; can accumulate in macrophage around
areas of hemorrhage. When it occurs in the lung due to severe congestion (heart
failure) and leakage of red cells into alveoli, it is called hemosiderosis.
Lysosomes can fill with things that cant be degraded, usually because of specific
enzyme deficiencies. NOT NORMAL. VERY BAD. These constipated lysosomes
accumulate and cause damage to the cell, inhibiting the function. There are a number
of lyososomal storage diseases and they affect primarily muscle and brain.
Viral inclusion bodies
Things that accumulate within tissues
o Dystrophic mineralization there is damage to the tissue and the circulating
mineral is deposited in damaged areas
o Metastatic mineralization there is excess circulating mineral and it deposits
in lots of places
Porphyria an error of metabolism where porphyrin gets deposited in teeth and bone,
giving it a pink color. The metabolic error is involved with hemoglobin.

Postmortem changes that are colored:
Hemoglobin imbibition hemoglobin leaks out of autolyzed
blood vessels staining the tissue around it red.
Pseudomelanosis - bacteria replicating postmortem can
produce hydrogen sulfide which combines with the iron in
hemoglobin to form iron sulfide which is black
Bile imbibition Bile leaks out of the gall bladder, staining
tissues around it green.
Whose flag is this?

Intracellular Accumulations

A cell may accumulate:

normal cellular components in excess (i.e. lipid, glycogen, some proteins), because either
the metabolism is inadequate (lack of enzyme) or the cell is overwhelmed by the stuff
coming in.
an abnormal substance (because the cell lacks the enzyme required or is unable to transport
a pigment (unable to digest or transport).

These accumulations can occur in both the cytoplasm or the nucleus, although cytoplasmic

accumulation is more common. Are the accumulations significant? Well, it depends. Read on.

Lipids (Fatty change or hepatic lipidosis)

Fat vacuoles within cells other than adipocytes (fat cells) indicate lipid accumulation. The best
example is seen in the liver and heart but practically any tissue can be affected. This can be the
result of several types of cell injury.

The MAJOR causes of fatty change:

Overload of the liver with free fatty acids (starvation, diabetes, diet)
Inability of the liver to conjugate with protein (starvation, toxins)
Decreased utilization of FFA along pathways other than TG synthesis - examples:
anemia (hypoxia), choline or methionine deficiency.
Consequences of Fatty Change:
If it is severe, function is impaired

Fatty change is a reversible change seen primarily in injured hepatocytes and renal tubular

What is the basic mechanism? In order to understand that, we need to review how fats are
metabolized in the liver:

Remember the circulation in the liver. There are two blood supplies. MOST of the blood comes
in through the portal vein, which carries nutrient-rich, but not well-oxygenated blood from the
intestine. The portal vein comes in at the portal triad and the blood percolates through the
sinuses, working its way toward the central vein. As it moves through the sinusoids, hepatocytes
take the good stuff out and process it for the body. The hepatic artery also comes in at the portal
triad but this blood (which is only about 30% of liver blood flow) brings oxygen and other things
the hepatocytes need. Hepatic arterial blood also flows from portal triad to central vein. In
contrast, the bile flow goes the OTHER way. Hepatocytes process the bilirubin and send it into
canaliculi which flow CENTRIFUGALLY, from the central vein to the portal triad, to be carried
out and taken to the gall bladder for safe removal from the liver and use in fat digestion.

Okay, so what do the hepatocytes do with the free fatty acids that are coming in from the intestine
and the portal vein? They turn them into triglycerides (TG) that can then be conjugated with an
apoprotein (protein that binds fat) and sent out to the body for safe keeping in the fat, or utilized
for energy.

Here are the important points in

the chain:
1 free fatty acids come in to the
liver from the circulation
2 Most are esterified in the liver
into triglycerides
3 protein gets conjugated to
4 these lipoproteins are

Cells (and consequently organs) get fatty because of an imbalance at one of these critical points.

Lets look at some specific examples of fatty change in the liver and explore the pathogenesis of
the lesion in each case. Make sure you understand the pathogenesis of all the causes of fatty


During starvation, there is widespread mobilization of body fat, resulting increased delivery of free

fatty acids (FFA) (1) into hepatocytes Most of these FFA are used in the synthesis of triglycerides.
In addition, protein synthesis, including the synthesis of apoprotein (3), is decreased as the supply
of amino acids normally derived from the diet is lacking. So, along with increased synthesis of
triglycerides there is also decreased fat export, resulting in fatty change.

Starvation causes increased mobilization of FFA from fat stores > increased synthesis of TG plus
decreased export of TG > fatty liver.
Inhibition of protein synthesis-> decreased apoprotein production. Apoprotein is needed for movement of
TG out of hepatocytes so -> fatty liver.

Diabetes mellitus

Another disease in which fatty liver is often seen is uncontrolled diabetes mellitus. In this disease,
glucose cannot be used for energy, so cells send signals that they are starving and adipose tissue
is mobilized, leading to an increase in FFA (1) presented to the liver.

In pigs fed moldy feed containing aflatoxin, the toxic metabolites of aflatoxin bind to nucleic acids
and nucleoproteins, effectively inhibiting protein synthesis (3). Other toxins, such as carbon
tetrachloride, phosphorus, and methionine, also inhibit protein synthesis, leading to fatty liver
(hepatic lipidosis) by a similar mechanism.

Anemia leads to hepatocyte hypoxia, another cause of triglyceride accumulation. Lack of oxygen
prevents FFA oxidation, which prevents degradation of fatty acids. Histologically fatty change is
present in hepatocytes around the central vein because these are the cells furthest from the
oxygenated blood.

The centrilobular zone is furthest away from the arterial (oxygenated) blood supply, and so those
hepatocytes closer to the central vein are exposed to progressively less well-oxygenated blood.
When arterial oxygen levels drop in anemia, the centrilobular hepatocytes are not adequately
oxygenated and fatty acids are diverted to triglycerides. Fatty acids are also liberated from cellular
membranes due to hypoxic injury.

Hypoxia inhibits use of FFA (2) and export of lipid from cell (4). This leads to an increase in the
amount of FFA available for TG synthesis (2). In addition to increasing the amount of TG
produced, hypoxia also inhibits the export of lipid from the cell by inhibiting lipoprotein synthesis
and transport (3 and 4).

Cats normally store large quantities of lipid in their renal tubular epithelium, and the kidneys are

normally yellow-tan in color. For other species, if the renal tubular epithelium gets fatty, it is a
real lesion. Normal cat kidney yellow; normal dog, goat, horse, cow liver brown!

Significance of fatty change WHAT DOES IT MEAN FOR THE ANIMALS HEALTH?

When mild, fatty change may have no effect on cell function. It is simply a reversible indication
that the cell has been damaged. More severe fatty change may impair cellular function. In the
liver, the enlargement of hepatocytes due to fatty change may compress adjacent bile canaliculi,
leading to cholestasis. In addition, if lipid continues to accumulate, the cell may pass the point
of no return and venture into the territory of irreversible cell injury or necrosis.

Lipofuscin (a lipid rich pigment derived from degraded cell membranes) is commonly
found in aging or chronically injured tissues. This substance gives these tissues a
characteristic yellow-brown color.

Melanin granules may be found in melanocytes, macrophages and other cells. Melanin is
responsible for the pigment characteristics of tissues and cells. Melanin can serve a protectant
against free radicals in the skin.

Occasionally melanin is deposited in
abnormal locations (such as the liver and
lung). This is called melanosis - it is not

neoplastic and does not interfere with

function of the involved tissues or organs.
However affected tissues are condemned as
food for aesthetic reasons (who wants to eat
spotted liver? Heckwho wants to eat

Cattle and sheep that are black (Angus,

Hampshire, Suffolk) often have melanin
deposits in the meninges of the brain too.
These deposits should not be considered
abnormal in these animals. The Chow-Chow
normally has a black tongue. Pigment may
also be found normally in other parts of the
oral cavity in this breed and their crosses.

Depigmentation, a different kind of color change:

As stated, melanin is responsible for the pigmentation and color seen in
tissues or cells. Sometimes pathologic processes (e.g. immune-mediated
disease) remove the melanin and depigmentation is then seen.

The mucous membranes can be
discolored due to icterus (which
signifies problems with hemolysis or
liver function). The yellow color is
due to bilirubin, a normal pigment that
may be present in excessive amounts.
Bilirubin pigment is a breakdown
product of hemoglobin. Anything that
causes excessive erythrocyte
destruction can cause excessive
formation of bilirubin. This kind of
bilirubin is unconjugated bilirubin.
Conjugated bilirubin is another bile
pigment that accumulates in tissues
when there is blockage of the outflow
of bile from the liver. The bilirubin
backs up into the blood and the bile pigments are deposited in tissues.

Hemosiderosis (iron)

In cases of chronic congestive heart failure, blood backs up into the lungs and is phagocytized by
macrophages. When enough iron collects in the macrophages, the lungs can appear brown!!! The
lungs appear tan to brown due to the pigment in the hemosiderin. When hemosiderin is present in
large amounts, the condition is called hemosiderosis.

Many deposits cause no harm, but in larger quantities they may induce fibrosis. Fibrosis may or
may not be harmful - depending on the organ involved. Apparently in the spleen, hemorrhage
occurs over a chronic period of time resulting in deposits of blood pigments (hemosiderin,
hematoidin and iron). Fibrosis is a consequence. These nodules are harmless on the spleen and
are called siderofibrotic plaques or Gamna-Gandy bodies. Note that fibrosis would interfere with
function in other organs especially the lung. For instance silicosis or asbestos may cause
pulmonary fibrosis and marked alteration in pulmonary function.


A deposit is a substance (usually abnormal) which

accumulates inside cells or in the interstitium.
If these deposits are colored then they are also pigments.

A tattoo is BOTH a pigment and a deposit. The basis of a tattoo is

that the ink (deposit) will remain in a certain body location and is
colored (pigmented) so it is visible. Tattoos are used in
thoroughbred horses to ascertain identity at horse races.

After you get a tattoo, some of the ink will be deposited in the
regional lymph nodes in lymphatic spaces or within the cytoplasm
of macrophages. One of the functions of macrophages is to be the
scavenger of deposits and pigments.

Accumulation of abnormal substances within cells

Lysosomal Storage Diseases
These are a group of genetic diseases caused by an inherited lack or
deficiency of an enzyme necessary to break down normal
intracytoplasmic substances. The result is an accumulation of these non-
degraded substances in lysosomes. Lysosomal accumulations are seen in
the neurons of dogs with Krabbes disease also known as globoid cell
leukodystrophy. These dogs lack the enzyme -galactocerebrosidase
which results in an accumulation of galactocerebroside, a component of
myelin within lysosomes. This disrupts the cells that normally produce
myelin, a fatty substance that coats nerve cells, serves as an electrical
insulator and is crucial to the normal conduction of nerve impulses. The

progressive loss of myelin in the white matter tracts of the nervous system (brain, spinal cord
and/or peripheral nerves) causes a variety of clinical signs such as lack of coordination,
tremors, and weakness. This condition is inherited in West Highland White and Cairn Terriers
as an autosomal recessive condition. It is also inherited in Irish setters, Miniature Poodles,
Beagles, Blue Tick and Basset Hounds, and Pomeranians.

Viral inclusion bodies

When viruses replicate in cells, sometimes they produce so much nucleic acid or protein, that they
form a rounded mass that can be seen histologically. These can be eosinophilic (mostly viral and
or host protein) or basophilic (mostly viral or host nucleic acid). They can be intra-nuclear or
intra-cytoplasmic. What color they are and where they are depends on the causative virus.

At the left is a hepatocyte infected with infectious

canine hepatitis virus. This is an adenovirus, it
replicates in the nucleus, so you see the big old
basophilic nuclear inclusion body filling most of
the nucleus (arrow). Viral inclusion bodies in the
nucleus typically have a halo of chromatin
clearing surrounding them. This helps to
differentiate them from just plain nucleoli. The
basophilia suggests that this inclusion body is
made up primarily of viral or host nucleic acid.

Neurons contain intracytoplasmic inclusion bodies

(DEPOSITS) caused by RABIES virus infection.
Host and viral protein comprise this eosinophilic
inclusion body. These are used to diagnose the
disease. This picture shows a neuron with an
inclusion body (arrow). First described by Dr.
Negri, hence the name, Negri body.

Canine distemper virus infection also has
resultant inclusion body deposits in some
cells. Here is the stomach lining of a
skunk that died of canine distemper.
This virus likes a whole lot of cells
stomach lining, lung, bladder, and brain.
The cytoplasmic eosinophilic inclusions
are abundant in the cytoplasmic of the
epithelium. A couple of inclusion bodies
are highlighted with arrows.

Abnormal accumulations within TISSUES:


Injury and the death of cells may result in the release of intracellular phosphate ions and fatty
acids into the extracellular environment. These compounds react with calcium ions forming
insoluble calcium salts which are precipitated in tissues. This type of calcification is particularly
common in atherosclerosis in people and diseases associated with chronic inflammation such as
tuberculosis. Abnormal calcifications can also occur in conditions associated with hypercalcemia
excess levels of calcium in the blood (e.g., hyperparathyroidism).

Mineralized deposits are often composed of combination of calcium and phosphate. Deposition
of minerals is a last ditch body defense to wall off or isolate an injury or agent. Calcium
deposits (calcification) often appear white in tissues and are granular when palpated. Calcification
occurs by two mechanisms:
Dystrophic calcification results when cells or tissue are injured
Metastatic calcification results when calcium or phosphate blood levels are excessive
However many experts believe all calcification is dystrophic since high levels of calcium are
injurious to cells.

There are certain types of rodenticides that cause excessive calcification in tissues.
Cholecalciferol-based rodenticides induce hypervitaminosis D. This results in hypercalcemia due
to increased absorption from the gastrointestinal tract and can cause renal failure due to renal


Porphyria is caused by deposition of an endogenous pigment when certain enzymes responsible

for breaking down hemoglobin are deficient. This disease has been reported in cattle and cats, and
the enzyme deficiency may be inherited in these animals. Porphyrins are then deposited in bones
and teeth resulting in discoloration that fluoresce under UV light. The lay term is pink tooth

Postmortem changes that could be confused with accumulations of abnormal
Hemoglobin Imbibition results when blood vessels become leaky after death. The blood
vessels leak blood and hemoglobin from the erythrocytes leaks out and stains surrounding
tissues. If the post mortem interval is short, the hemoglobin imbibition appears as pink to
red feathering around blood vessels. Animals that have been dead for longer have more
diffuse staining of tissues with hemoglobin making them appear uniformly pink or red.

Pseudomelanosis or false melanosis is a common postmortem finding. Sections of

intestine in contact with tissue can produce the black color.

Bacteria in the intestine form hydrogen sulfide. This gas diffuses across the intestinal wall and
comes into contact with the iron in hemoglobin in tissues. The iron in the hemoglobin reacts with
the sulfide and forms a black precipitate, iron sulfide, which is the basis of pseudomelanosis. Note
this is a POSTMORTEM event - it does not occur in the live animal! Remember too that the wall
of the intestine may be black because it too contains hemoglobin and iron.

Bile Imbibition

The bile in the gall bladder will stain adjacent tissues after death and result in green to black
discoloration of the tissues. This postmortem change is called bile imbibition. Bile imbibition is
a post mortem change. A similar discoloration of tissues can be seen if there is rupture of the gall
bladder with release of bile into the peritoneum. This results in a chemical peritonitis and the
peritoneum is often stained green.

(corresponds to the web lesson of the same name)

Learning objectives:
1. Understand the reasons amyloid is deposited in tissue
2. Understand the meaning of the word hyaline
3. Differentiate amyloid from other hyalin extracellular deposits
4. Understand the causes of amyloid formation and deposition
5. Understand how amyloid causes azotemia and proteinuria


Amyloid was a normal protein, got misfolded, now it is a problem

Gets deposited in tissues and doesnt go away; causes dysfunction by replacing normal
tissue or just clogging it.
Appears microscopically as homogeneous, pink
o From SAA this is the most common form of amyloid in animals
Liver produces an acute phase protein called Serum Amyloid A (SAA). Acute
phase proteins are important in the early inflammatory response. If there is
inflammation that occurs for a long time, there are too many SAAs circulating and
some will misfold and deposit.
o From AL immunoglobulin light chains, when produced in overabundance, can
misfold to create amyloid
o From IAPP protein found in islet cells in the pancreas that can, if processed
incorrectly, lead to diabetes
Favorite spots for amyloid to deposit are renal glomeruli, pancreatic islet cells and the
Other extracellular deposits
Tamm-Horsfall proteins in the kidney
Sclerosis this is excessive collagen
Fibrin especially in vessels (fibrinoid necrosis - remember)

32BAmyloidosis is a disease of abnormal protein folding. During their life cycle, proteins undergo
many types of conformational changes. The first type of conformational change is the process of
protein folding. The protein must fold into its active three-dimensional structure before it is
degraded by cellular enzymes. Most amyloid is caused by a normal protein that, for one of several
reasons, is abnormally folded into structures known as beta-pleated sheets. These sheets cannot
be degraded and aggregate to form fibrils which are then deposited in extracellular tissues. The
extracellular amyloid fibrils often interfere with function if they are deposited in a critical tissue.

Amyloidosis may result from several mechanisms. It can be induced in certain avian species
(ducks, geese) simply by crowding them in pens. Certain breeds have a greater incidence of
amyloidosis. This includes Sharpei dogs and Abyssinian cats. Thus genetic influences may be


Pathogenesis of Amyloidosis
Amyloid fibrils are formed by macrophage cleavage of certain precursor proteins. There are at
least 8 precursor proteins but immunoglobulin light chains (AL) and serum Amyloid A protein
(SAA) are two of the many precursor proteins which may result in systemic amyloidosis.

SAA Amyloidosis
SAA is the most common cause of amyloidosis in animals! Horses were once injected with specific
antigens so that antisera (serum high in specific antibodies) could be harvested. These horses often
developed amyloidosis of the spleen and liver. These organs enlarged and frequently ruptured
with resultant exsanguination and death of the horse. It is now known that these antigens caused
certain proteins to be formed by the liver. These proteins (called acute phase reactants) are part of
the normal response to antigens and inflammation. One acute phase reactant produced by the liver
is SAA (Serum Amyloid A protein)

SAA is an acute phase reactant produced by the liver to modulate inflammatory reactions. The
stimulus (inflammation, antigen, etc) causes macrophages to secrete Interleukin 1(IL-1) which
interacts with hepatocytes. The hepatocytes produce SAA which is secreted into the blood. There
are at least 4 forms of SAA and some may be amyloidogenic - these would be cleaved by
proteolytic factors from macrophages and subsequentially become AA fibrils. These AA (amyloid
A) fibrils would be deposited with Protein P (another acute phase reactant) in various locations in
the body to form amyloid deposits. Renal amyloidosis in domestic animals is usually the result of
deposition of these AA fibrils in the sub-endothelial area of glomerular capillaries.

AL Amyloidosis
A second kind of amyloidosis can occur from overproduction of immunoglobulin light chains.
These get misfolded and deposited as amyloid. This form of amyloidosis is less common in
animals than the SAA form.

IAPP Amyloidosis (yet another kind of amyloidosis)

Islet amyloid polypeptide (IAPP) is a substance
deposited in the islets of Langerhans in the pancreas.
Normal IAPP is a 37 amino acid peptide which is
co-stored and co-secreted with insulin from the beta-
cell secretory granules of the pancreatic islets. IAPP
is derived from a larger molecule called proIAPP
and processed by enzymes which are also
responsible for the conversion of proinsulin to
insulin. Defects in this processing in the islet beta
cells results in diabetes in cats, and type 2 diabetes
in people This impairment leads to the reduced
conversion of proinsulin to insulin and
accumulation of an incompletely cut IAPP molecule within the beta-cells. IAPP aggregates within
the beta-cells, ultimately contributing to cell death.

Diagnosis of Amyloidosis
Amyloid means starch-like. The name comes from the early mistaken identification of the
substance as starch (amylum in Latin), based on the fact that grossly, iodine would stain amyloid
deposits in tissues. In some organs, such as kidneys, amyloid deposits stained with iodine can be
seen as red to black dots in the cortex corresponding to glomerular deposits of amyloid. There is
a glycoprotein associated with the amyloid deposits which stains with iodine. However, iodine
staining is a crude test and it is not accurate (there are many false negatives).

A more accurate method of diagnosis is by histopathology. Histologically amyloid is said to be


a hyaline substance. Hyaline means homogeneous and eosinophilic (pink with H&E stain).

Amyloid tends to be deposited in aggregates. Eventually the entire tissue may be filled with the
deposits. However other substances may have a similar appearance - thus special stains are

Amyloid in the liver. Arrows labeled (1) point to the amyloid. Arrows Congo red stained liver. Arrows point to orange staining which is
labeled (2) point to hepatocytes positive for amyloid

Congo Red is a dye used to stain fabric. It has been found to be a good stain for amyloid too. The
amyloid deposits stain red to orange. Congo Red will also stain connective tissue. To be certain
the red material is amyloid, you can place the stained slide under polarized light. With polarized
light, the Congo Red stained fibrils will be birefringent. This birefringent color has been described
as apple green birefringence.

The absolute definitive diagnosis is made using electron

microscopy, but most pathologists rarely go to this level.
Ultrastructurally, amyloid looks like a bunch of pick-up
sticks (amyloid fibrils).

Electron micrograph of amyloid fibrils

There are several diseases that are the result of amyloid deposition.
Diabetes mellitus
Cats often develop diabetes mellitus secondary to amyloid
deposition in the pancreas. This kind of amyloid is due to
abnormal accumulation of an abnormal protein called islet
amyloid polypeptide. The amyloid fibrils are deposited in the
Islets of Langerhans and compress the normal cells in the
Islets. If beta cells are destroyed and insulin is not produced,
diabetes mellitus results.

Pancreas with an Islet of Langerhans obliterated by eosinophilic amyloid (arrow)

Renal amyloidosis
Renal glomeruli are a common site for deposition of amyloid
fibrils. Renal function may be severely affected if amyloid is
deposited in the glomeruli. A favorite spot for amyloid to
deposit in the kidney glomerulus is in the subendothelial space
the space between the endothelium and the basement
membrane of the renal epithelium (podocyte). There they
interfere with filtration and the glomerulus becomes leaky, with
resultant leakage of plasma proteins into the urine. Proteinuria

Amyloid deposits in the results in thickening capillary wall. This will alter blood flow through
glomerular capillaries and cause decreased glomerular filtration rate (GFR). This results in
azotemia. The foot processes of the podocyte are usually fused or effaced due to alteration/loss
of the sialic acid covering the foot processes. If the sialic acid is lost, the negative charge on the
surface of the foot processes and basement membrane is also lost and albumin and other proteins
are more readily able to diffuse through the capillary wall resulting in proteinuria.

Alzheimers Disease
A protein known as amyloid beta (or beta amyloid)
is consistently seen in patients with Alzheimers
disease. This amyloid beta is found in the brain in
abnormal tangles called plaques. The "amyloid
hypothesis", that the plaques are responsible for the
pathology of Alzheimer's disease, is accepted by the
majority of researchers but is by no means
conclusively established.

Amyloid deposits in the interstitial tissues can cause dysfunction in other organs such as the thyroid
gland. Interstitial amyloid deposits may compress thyroid follicles. However the fact that the
deposits are in the interstitium and not inside follicles is indicative of less interference with
function. Dysfunction would occur only if all follicles were compressed.

Summary of Amyloidosis

Amyloidosis results from the cleavage of certain proteins, many of which are associated with
immune function such as serum amyloid A, and formation of inert amyloid fibrils which are
deposited extracelluarly. These fibrils stain with Congo red. They also polarize light and have a
distinctive shape on electron microscopy. They produce disease by crowding tissue or cells.
Common sequelae are edema, proteinuria and death from organ rupture or organ failure. The
disease is not reversible.


Other extracellular deposits

We need to discuss some other deposits that may be confused with amyloid. These deposits are
all are termed hyaline materials since they appear smooth and shiny.

Tamm-Horsfall Protein
Tamm-Horsfall protein is a protein normally produced in the
kidney to aid the counter current mechanism. The ascending limb
must be impermeable to water if the counter current mechanism
is to function properly. Tamm-Horsfall protein lines this
segment of the tubules and prevents water loss into the
interstitium from the ascending portion of the tubules. When
renal injury occurs, Tamm-Horsfall protein aggregates
Tamm- (precipitates) and thus becomes a cast.

Casts are tubular lumen precipitates - they are a cast of the

tubular lumen. Hyaline casts are composed of Tamm-Horsfall protein and are one component of
the sediment in urine. They are a protein precipitate and may be seen as a sequela to dehydration,
tubular injury, proteinuria, electrolyte imbalance and other conditions. Tamm-Horsfall protein is
the component of the brightly eosinophilic casts seen in amyloidosis.

Sclerosis (Collagen)
Collagen is a protein product of fibroblasts. It is deposited as part of the extracellular matrix for
support and healing. In tissue sections, it stains pink and is shiny.


Hyaline material can be found extracellularly in diseased vascular walls. This is similar to
fibrinoid necrosis in vascular walls. In fact it is fibrin and it is due to leakage of fibrinogen from
the lumen into the wall of the vessel. The fibrinogen polymerizes to fibrin - and appears hyaline.

This hyaline material or fibrinoid change indicates the wall has been severely injured. This may
occur as a result of viruses, fungi, toxins (uremia) or other insults. In addition to fibrinogen leaking
into the wall and polymerizing to fibrin, immune complexes can also be deposited.

Hyaline deposits within vessel lumens are thrombi and are also composed of fibrin.

(corresponds to the web lesson of the same name)

1. Know the 3 types of mortis
2. Understand the pathogenesis of rigor mortis
3. Distinguish between autolysis and necrosis


Three gross changes occurring after death:

Algor mortis cooling of the body; rate depends on the ambient temperature, the size of
the body, amount of subcutaneous fat, and the temperature of the animal at the time of
death ,depends on the ambient temperature
Livor mortis Blood moves with gravity after death
Rigor mortis Energy is required to unlock muscles, so body with normal energy stores
will be stiff at about 6-8 hours postmortem. Then autolysis breaks down proteins, and
this happens at 1-2 days, resulting in rigor relaxing
How to determine antemortem vs. postmortem changes:
Autolysis is uniform and not associated with inflammation
Hemorrhage and bruising cannot occur postmortem
Specific postmortem changes:
Hemoglobin imbibition Red cells lyse postmortem and hemoglobin leaks into
surrounding tissues, giving them a pink appearance
Bile imbibition Bile leaks out of the gall bladder and into the surrounding tissues,
making them green
Bloat Bacteria in the intestinal tract replicate very well after death, thank you, releasing
lots of gas that can distend the gastrointestinal tract with gas.
Agonal froth The last few gasps as an animal is dying can make froth that can extend up
the trachea
Pseudo-prolapse Postmortem bloat leaves little room for the viscera in the abdominal
cavity, with the result that rectum or vulva can get pushed to the outside.

The Family Mortis (see website for pictures of the brothers)

Once upon a time there were three brothers. They were all members of the
MORTIS family
Algor was the coolest
Livor was the lazy son - he gravitated to the lowest spot
Rigor was oldest and best known - he was rapid and inflexible

Algor Mortis is cooling of the body. After death, the body temperature declines to the

environmental temperature. The rate of cooling depends upon the ambient temperature, body fat,
and amount of hair or wool covering the animal.

*The white lenses seen in the eyes of some young animals are an artifact of cold, but not algor mortis. When an
animal is placed in a cold room or cooler, the lenses become opaque - thus these are not cataracts.

Livor Mortis is settling of blood in the most dependent parts of the body after death. Gravity
38B U U

pulls blood to dependent portions of the vascular bed resulting in pooling of blood in the most
ventral areas. Note this would not occur if clotting was present or the animal was exsanguinated
(bled out).

Hypostatic Congestion is also pooling of the blood in dependent body parts. Hypostatic congestion
can occur antemortem or postmortem. This is the reason you turn animals over periodically when
they are recovering from anesthesia. Hypostatic congested lungs are more prone to develop

Rigor Mortis is the stiffening of a dead body, accompanying the depletion of ATP in the muscle
fibers. ATP is necessary to unlock the contraction units in
myofibers. If ATP is depleted, the contraction units remain
locked and the body becomes stiff or in rigor. Rigor is
usually complete by 6 to 8 hours after death and may last 1 to
2 days. The times are variable depending on environment
conditions and carcass conditions.

An animal in good body condition would have abundant stores

of glycogen & ATP. Rigor mortis would be delayed unless it
ran (or exercised strenuously) just before death. Such exercise
would deplete ATP stores and induce rapid rigor mortis.

An animal with less body stores of glycogen would go in to rigor rapidly. Emaciated animals may
be in rigor mortis within an hour after death due to lack of glycogen and inability to form ATP.

Extensor rigidity is seen in rigor mortis and in live animals with tetanus. This occurs because
the extensor muscles are more powerful than the flexure muscles. In the heart, there is a difference
in contraction during rigor mortis between the left and right ventricles. The left ventricle contacts
more vigorously than the right. Rigor mortis begins in the heart before the skeletal muscle too.
Because contraction during rigor mortis is stronger in the left heart, the blood is expressed from
the chamber (squeezed out). However, since the contraction is weaker in the right ventricle, blood

remains and a clot is normally found there. A clot in the right ventricle of a dead animal is normal

A clot in the left ventricle, however, is not normal. This usually indicates a weakened ventricular
muscle (and thus an indication of a diseased heart). Blood can be found in the left ventricle after

the animal has gone out of rigor. This blood is usually NOT clotted and is from blood falling back
into the left ventricle out of the relaxing aorta.

Muscle contraction in general, is the result of release of calcium ions from the sarcoplasmic
reticulum, binding of calcium ions to troponin,
formation of cross-linkages between actin and
myosin with resultant sliding of filaments and
myofiber shortening (contraction).

When ATP is generated during the relaxation cycle,

calcium is pumped back into the sarcoplasmic
reticulum and is released from troponin. Relaxation
occurs and actin and myosin slide apart.

In rigor mortis, ATP production continues for a little

while after death, but eventually the glycogen stores
are used up and no more ATP can be produced. The
calcium pump fails and contraction remains until autolytic changes cause the actin-myosin cross-
bridges to breakdown. At that time rigor is over. Similarly animals that die following strenuous
exercise have rapid onset of rigor mortis because there is depletion of muscle glycogen and ATP
cannot be generated. Since ATP is absent, calcium remains bound to troponin and relaxation
cannot occur.

Rigor mortis ends as autolysis advances.

Antemortem vs. Postmortem

A major factor in differentiation between postmortem and antemortem events is whether we can
determine a response by the host. Antemortem events usually have evidence of a host response -
e.g. redness in a pig with a septicemic disease. Active terms such as hemorrhage and bruising
are all antemortem events, not postmortem. Reddening of tissues after death are likely due to
postmortem leakage of hemoglobin due to processes such as hemoglobin inhibition (see below
for description). As clinicians, you will also rely on the cardinal signs of inflammation - this
response may not always be evident on gross postmortem examination - thus microscopic
examination is needed too.

Two criteria used to differentiate autolysis (postmortem) from necrosis (antemortem) are
lack of an inflammatory response
bland uniformity

Remember that there is always a certain amount of autolysis since autolysis begins when the
animal dies! Necrosis and autolysis are often present in the same microscopic section. Those
tissues like the gastrointestinal tract, pancreas, liver, and kidney autolyse fastest as certain bacteria
proliferate after death and speed up the autolytic processes. Since these bacteria are present the
cadaver (carcass), they are also known as cadaver bacilli.

Some postmortem changes
1) Hemoglobin imbibition occurs when the red blood cells have lysed and released their pigment
- hemoglobin. Hemoglobin imbibition begins adjacent to vessels which contain the red blood
cells. This initial appearance is feathering - the red pigment leaches into surrounding tissue and
can adopt a feather-like appearance around autolytic blood vessels.
Eventually, the hemoglobin diffuses into all the surrounding tissue (the tissue has imbibed the
hemoglobin!) and changes its color to a pinkish red. This change may mask other color changes
in these tissues, and as the postmortem interval increases, the hemoglobin imbibition intensifies.

2) Bile imbibition occurs in tissues such as the liver (where it was in contact with the gall bladder).
Bile pigment leaks out of the gall bladder after death and stains adjacent tissues.

The black discoloration seen in some tissues after death is called pseudomelanosis (false melanin)
and it may be seen during postmortem examination of animals. Often it is quite extensive. A
common factor is that a section of intestine was in contact with the black portion of the organs.

Hydrogen sulfide is formed by bacteria in the intestine. This gas diffuses across the intestinal wall
and comes into cataract with the kidney and with hemoglobin in the kidney. The iron in the
hemoglobin reacts with the sulfide and forms the black precipitate, iron sulfide, which is the basis
of pseudomelanosis. Note this is a postmortem event - it does not occur in the live animal!
Remember too that other organs in contact with the intestines may be black due to
pseudomelanosis (in fact the wall of the intestine may be black because it too contains hemoglobin
and iron).

3) Postmortem Bloat
Bloat occurs in animals after death due to gas formation in the gut. The microorganisms present
in the gut during the animals life, persist after death and continue to ferment food. However the
gas produced cannot be expelled and thus accumulates. The gut then becomes distended with the
trapped gas. Live animals can bloat due to inability to expel fermented gasses too.

Bacteria can begin a putrefaction processes in tissues other than the gut with resultant gas
formation. Since the cutaneous tissue is intact, the gas accumulates and causes gross swelling of
the carcass. This is called postmortem emphysema.

4) Agonal Froth
Foam or froth extending into the upper trachea is an indication of true pulmonary edema. This
must be distinguished from froth seen in the lower third of the trachea in agonal animals. This is
the result of labored breathing coupled with the impaired venous return of blood in a terminally
hypoxic animal. The result is fluid mixing with air in the lower trachea. The result is agonal

5) Pseudo-prolapse or rupture
In ruminants, the pressure from the gas formation in the expanding rumen compresses all the
abdominal viscera. The abdomen runs out of space to contain them so the rectum and or vagina

may pseudo prolapse. This is not a real antemortem prolapse but due to postmortem bloat. In
horses, the post mortem accumulation of gas in the stomach can cause the stomach to rupture
postmortem. In an ante-mortem rupture, hemorrhage and signs of inflammation are often seen
around the ruptured edges, while in a postmortem rupture, these changes are not present.

Because of the forensic impact of the interpretation of these lesions, it is important that due
diligence is paid to the assessment and interpretation of these postmortem findings.

Veterinary Forensic Pathology (a brief introduction)

Veterinarians are being asked more and more frequently to testify in court on medicolegal cases.
When handling a forensic case, your observations are a key component. You must carefully
document all observations, provide an objective scientific pathological evaluation, and able to
defend your observations and conclusions in a court of law. In these cases, the veterinarian is
often asked their expert opinion about the following;

1. The cause of death - the disease/s, injury/ies or abnormality/ies that alone or in combination
is responsible for initiating the sequence of functional disturbances that ended in death.

2. The mechanism of death -the structural or functional change that makes independent life no
longer possible.

3. The manner of death - the way in which the cause of death came about.

4. The time of death - the length of time between the discovery of the carcass and the death of the
animal. (Body temperature may have some use in determining time of death in recently dead
animals and birds, however, many factors must be considered in estimating time of death,
including nutritional state, stage of decomposition, insect infestation, scavenger impact, and

It is very important to be able to distinguish post mortem from antemortem changes. Remember,
changes such as hemorrhage ONLY occur antemortem and should be distinguished from changes
such as post mortem excoriation (due to dragging, for example) and hemoglobin imbibition.

Finally, you must retain objectivity. Remember, the veterinarian is not an advocate for either
the prosecution or the defense, but a fact finder and interpreter. In a forensic necropsy
examination, it is not enough to place a label or diagnosis on a case. It is your responsibility to
reconstruct the event as best you can within the constraints of the available evidence without
assumption or speculation or prejudice.


1. Cat (12 years old)

This is a section of liver. At lowest magnification, the lobules are "mottled" and some have dense
red material in the central vein area.
At higher magnification, this red
material is seen to be erythrocytes.
Whether this is congestion
(centrilobular) or hemorrhage is a
difficult decision because blood is
normally seen in sinusoids around
hepatocytes. However these areas have
lost some hepatocytes and thus this is
most likely hemorrhage due to necrosis.
Did you spot the golden to brown
pigmented deposits in the Kupffer cells?
This is probably hemosiderin due to
breakdown of erythrocytes in these
A major change is seen in the
hepatocytes. How would you describe them and what are your rule-outs?

Many hepatocytes have variably sized, clear vacuoles in their cytoplasm. In some
hepatocytes, the nuclei are displaced from their
central location due to these vacuoles. The
diagnosis is hepatic vacuolar change and the rule-
outs would be glycogen deposition or fatty change.
Special stains would be needed to confirm the
vacuole content. Because the vacuoles are variably
sized and occasional displace the cell nucleus, this
is most likely fatty change.

It was confirmed clinically that this cat had diabetes

mellitus. A probable mechanism for fatty change
would include increased demand for body fat stores
increased mobilization of body fat stores
presentation of excess free fatty acids to the liver
inability to export all fatty acids fatty liver.

This change is reversible. If the cause is corrected, the hepatocytes will return to normal.
2. Aborted calf

This is a section of liver that stains "pink and poorly". The hepatocytes are often present as single
cells rather than cords of cells. These changes (hepatocyte "individualization", uniform
eosinophilic staining, and lack
of basophilic staining) are
compatible with autolysis.
In addition to these changes,
there are multiple, round foci
which stain more intensely pink.
These foci are randomly
scattered throughout the liver
and at higher magnification are
seen to be NECROTIC.

Lesion diagnosis: multifocal

hepatic necrosis

The disease that caused the

necrosis caused the animal to
die, so we have autolysis AND
necrosis in the same tissue.

These necrotic foci have cells

with solid to granular pink
cytoplasm and nuclei that are
pyknotic, karyorrhectic or
karyolytic. Some of these cells
are inflammatory cells in
addition to hepatocytes

Do you see a cause? With

diligent searching of the more
normal hepatocytes at the margin
of the necrotic foci, you can find
small pink bodies in the nuclei of
some hepatocytes. These are the
intranuclear inclusion bodies of infectious bovine rhinotracheitis (IBR) virus. This virus causes an
upper respiratory tract disease in adult cattle and may cause abortion in pregnant animals.

Why is autolysis so prevalent in this liver? The
calf died while still in the uterus and remained
in the uterus for several days. The cow's body
temperature promoted autolytic processes and
thus postmortem change was advanced by the
time the calf was expelled (aborted).

3. Puppy (5 months old)

Look at this section of liver. With low power

microscopic examination, there appears to be
very little normal appearing hepatic
parenchyma left. Portal areas can be detected
based on the bile ducts found there. What else
can be found in the portal areas?
The rest of the liver contains hepatocytes that
stain intensely eosinophilic (pink). The nuclei
of these hepatocytes are shrunken (pyknotic),
fragmented (karyorrhectic) or missing

Why do these hepatocytes stain more

eosinophilic? The eosin (acidic dye) has
increased affinity for all the (basic) proteolytic
enzymes that are breaking the cell down.

Morphologic diagnosis: Centrilobular hepatic necrosis

What type of necrosis is this? This is coagulative necrosis - note you can still recognize the dead
cells as having been hepatocytes.

What caused this necrosis? This puppy ate

mushrooms probably Amanita sp. These
mushrooms found all over the southern USA
and can pop up suddenly especially after a
rainstorm. They contain potent hepatotoxins
(aminatoxins) that inhibit RNA polymerase B
and hence protein synthesis. Usually within
24-48 hours after then the mushroom is
ingested, sever liver damage and death results.

4. Sheep (aged ewe)

This is a section of lymph node, but it is difficult to say for sure that it is lymph node because the
normal architecture has been all but obliterated by a central core of eosinophilic amorphous (no
shape) material, which is surrounded by a margin of fibrous connective tissue.

Can you recognize any cells in the debris? No this is caseous necrosis. Caseous is Greek for
cheesy and it implies that the material is a homogeneous necrotic mass whose origin cannot be
determined morphologically.

There are numerous macrophages at

the interface of the fibrous
connective tissue and necrotic
debris. There are also occasional
neutrophils. The outermost
concentric ring is composed of
fibroblasts and connective tissue.
This process was due to infection
with Corynebacterium
pseudotuberculosis, the cause of
caseous lymphadenitis.

5. Dog (Afghan, 6 months old)

This is a section of spinal cord. If you hold this section against a white background, you will
notice there are areas of pallor (poor staining) in the ventral white matter and in the dorsal central
segment of the cord. Look at the dorsal central region under the microscope. There is absence of
neural tissue in this area. In fact this is a cystic area (microcavitation). Note there are macrophages
(gitter cells) with foamy
cytoplasm in this lesion. Gitter
cells are actively phagocytizing
neural material and thus have
cytoplasm distended with
foamy stuff.

Morphologic diagnosis:
Myelomalacia (liquefactive

Malacia is the gross

(macroscopic) term for necrosis
of nervous tissue. It is
characterized as a liquefactive
type of necrosis. The lipid
material in the myelin is released and degraded. The gitter cells remove debris. A hole
(microcavity) is left in the neural tissue.

This disease is hereditary

myelopathy of the Afghan
hound. It is inherited as an
autosomal recessive disorder.
Signs are first seen between 3
and 12 months, beginning with
posterior ataxia and progressing
to paraplegia and eventually
phrenic paralysis and death. The
exact mechanisms of disease are

(H&E and special stain) Dog (adult)

This is a section of kidney. Most glomeruli contain eosinophilic hyaline material. Some tubules
are dilated and contain eosinophilic hyaline material too.

The glomerular lesions are really spectacular. Many of the glomerular tufts are just chock full of a
homogeneous eosinophilic hyaline material that is, in all probability, amyloid, making this a case
of glomerular amyloidosis. This glomerular lesion has then led to the tubular lesion.

The tubular epithelium is "flattened" in some tubules. This is a response to injury, in this case,
hypoxia. Some cells have been lost and the remaining cells "spread out" to cover the tubular
lumen. The cells are lost due to ischemia and necrosis. Also, many of the tubules are filled with
eosinophilic material this is protein leaking through the damaged glomerular tufts.

Why would ischemia occur in the

tubular epithelium? Remember the
blood to the tubules must flow through
the glomerular capillaries first. If the
amyloid deposits hinder this flow and
results in poor perfusion, tubular
epithelial cells become ischemic.

What causes amyloid? Amyloidosis

can be a primary disorder or it can be
secondary due to chronic inflammation.

7. Cat (adult)

Find the pancreas on this slide. Within the pancreas there are focal areas of necrosis
characterized by pyknotic nuclei and loss of acinar cells. Neutrophils are numerous in the
pancreatic tissue. There are lots of neutrophils in the fat.

Most of these neutrophils have pyknotic nuclei. Intermixed with the fat cells and neutrophils is
"foamy" or vacuolated debris that has areas of both pink and blue staining.

The pink areas represent release of plasma proteins and perhaps some fibrin. The blue areas
represent a mineralized focus fatty acids released from the fat deposits combine with calcium
and other salts to form a soap which stains blue. This is called saponification.

Morphologic diagnosis: acute necrotizing pancreatitis with enzymatic fat necrosis

The enzymatic fat necrosis is secondary to the pancreatitis. Enzymes released from the necrotic
pancreatic cells cause lysis of the fat cells and fat deposits. The fatty acids are then able to
combine with calcium and other salts => fat necrosis and saponification.

There is another lesion in this slide. Note

the large blood vessel near the area of fat
necrosis? The vessel wall is thickened
with eosinophilic fibrillar material. This
is fibrin and the term is fibrinoid

Fibrinoid necrosis occurs when

coagulative necrosis occurs in a blood
vessel and then fibrin and immune
complexes from the leaky blood vessels
are superimposed

8. 5-year-old Irish Setter

The tissue on the slide is skeletal

muscle. There are many normal
appearing skeletal muscle fibers.
However, some appear narrower
and more lightly staining than

Morphologic diagnosis: skeletal

muscle atrophy

What happens to the cell in

muscle atrophy? Remember, the
cellular organelles become
diminished in number due to a
decreased workload.

This was a case of neurogenic atrophy. This dog had loss of innervation to his hind limbs and the
muscle fibers atrophied. Note that there are also a few swollen, hypereosinophilic fibers in this
slide too. Why do you think they are there?

Remember, the muscle fibers can only compensate for a certain time by atrophying. After a while,
if the loss of innervation continues, the fibers will die and undergo coagulative necrosis.