IRREVERSIBLE CELL INJURY

Irreversible Cell Injury (cell death) comes about by one of two mechanisms.
1. Apoptosis - in which the cell actively participates in its own death (referred to as programmed
cell death).
2. Necrosis - in which the damage to cell membranes and organelles becomes lethal (cell death).
In other words sublethal injury may progress (rapidly or otherwise) to lethal injury and cell
death. Necrosis refers to the morphological changes that occur to the cell after cell death.

APOPTOSIS
 Apoptosis is a form of cell death designed to eliminate unwanted host cells. It occurs in
the following general settings:- during development; as a homeostatic mechanism to
maintain cell populations in tissues; as a defense mechanism such as in immune
reactions; when cells are damaged by disease; in ageing.
Morphological Features
 Apoptosis usually involves single cells or small clusters of cells that appear in H & E
stained sections as round or oval masses with intensely eosinophilic cytoplasm. The
following morphological features characterize cells undergoing apoptosis
(1) Cell shrinkage: The cell is smaller in size. The cytoplasm is dense, and the organelles are
more tightly packed.
(2) Chromatin condensation: The nuclear chromatin is condensed, and it aggregates
peripherally, under the nuclear membrane, into well defined dense masses of various shapes
and sizes. The nucleus itself may break up, producing two or more fragments.
(3) Formation of cytoplasmic blebs (buds) and apoptotic bodies: The apoptotic cells
rapidly shrink, then show extensive surface blebbing (bud formation), and finally undergo
fragmentation into a number of membrane-bound apoptotic bodies (vesicles) composed of
cytosol (fluid portion of the cytoplasm) and tightly packed organelles, with or without
nuclear fragments The plasma membranes remain intact during apoptosis.
(4) Phagocytosis of apoptotic cells or bodies by nearby healthy cells, either parenchymal
cells or macrophages. Because apoptotic bodies are quickly extruded (thrown out) and
phagocytosed and degraded within lysosomes, even significant apoptosis may be
microscopically not visible. Also, the nearby cells migrate or proliferate to replace the space
earlier occupied by the apoptotic cell.
 Moreover, apoptosis, in contrast to necrosis, does not produce inflammation. This makes
it more difficult to detect apoptosis microscopically.
 Apoptosis can be induced by both physiological and pathological processes. Mammalian
cells have a complex system of positive and negative controls regulating apoptosis.

Apoptosis is important in the following circumstances:

1. Programmed destruction of cells in embryogenesis, including implantation,
organogenesis, developmental involution and metamorphosis
2. Hormone-dependent physiological involution, such as involution of the
endometrium during menstrual cycle in humans, or the lactating breast (mammary
gland in animals) after weaning, or pathological atrophy, as in the prostate after
castration in humans.
3. Cell deletion in proliferating cell populations, such as in intestinal crypt
epithelium
 4. Cell death in tumours
5. Death of neutrophils during an acute inflammatory response
6. Death of immune cells - both B and T cells. More than 95% of B-cells die in the
bursa of Fabricius in the chicken (bone marrow in mammals) and thymocytes in
the thymus during maturation
7. Cell death induced by cytotoxic T cells
8. Pathologic atrophy in parenchymal organs after duct ligation
9. Certain viral diseases e.g. viral hepatitis
10. Low level injurious stimuli that normally cause necrosis at higher levels. For
example, heat, radiation, cytotoxic anti-cancer drugs, and hypoxia can induce
apoptosis if the irritant is mild, but large doses of the same stimuli result in
necrosis. The mild injurious stimuli cause irreparable DNA damage. This, in turn,
triggers cell suicide pathway.
.
NECROSIS
Necrosis is the local death of tissue within the living individual. It refers to a sequence of
morphological changes that follow cell death in living tissue or organ, resulting from the
progressively degradative action of enzymes on the irreversibly injured cell. It is a long
established term used to describe the overall process that occurs when cells die. The
morphological changes that we can observe microscopically, only occur after cell death. Initial
degradative changes are autolysis.
The morphological appearance of necrosis is the result of two concurrent processes:
(1) Enzymatic digestion of the cell, and
(2) Denaturation of proteins
The digestive enzymes are derived either from the lysosomes of the dead cells themselves, in
which case the digestion is referred to as autolysis, or from the lysosomes of infiltrating
leukocytes, termed heterolysis.
Aetiology
(1) Poisons:
(a) Chemical poisons: These may be strong acids and alkalis, drugs, insecticides, fungicides,
and other toxic chemicals like lead arsenate (an insecticide), phenol, mercuric chloride, uranium
nitrate, and others. They cause cell death either by coagulation of proteins or by poisoning the
enzymatic systems.
(b) Toxins of pathogenic organisms: The toxins (harmful products) produced by bacteria,
viruses, fungi, rickettsiae, protozoa, and metazoan parasites may cause necrosis. For example,
bacteria of various types (Salmonella, Fusobacterium and Staphylococcus) commonly cause
necrosis of tissues.
(c) Plant poisons: Alkaloids produce necrosis. For example, plants of Senecio species in large
amounts are hepatotoxic and produce necrosis of hepatic cells in cattle. Mushrooms contain a
toxic glycoside, phallin, which causes renal tubular necrosis. Croton oil causes necrosis of
epithelium in the skin and mucous membrane.
(d) Animal poisons: Cantharidin from beetles, and bee stings, cause focal necrosis of tissue.
Parasites of many types (e.g., Histomonas meleagridis in chickens) also cause necrosis.
(2) Disturbances in circulation:
(a) Ischaemia or loss of blood supply results in necrosis and infarction. It is produced by
thrombus and embolism; compression of the artery by tumour, abscess, cyst, ligature, tourniquet;
ergot poisoning which causes contraction of the smooth muscles of the arteries.
(b) Passive hyperaemia causes necrosis when it persists. It is commonly observed in torsion,
volvulus, and strangulation of the intestine.
(c) General anaemia may result in necrosis (brain and liver) if the amount of oxygen and
nutrient is not sufficient to maintain cellular metabolism.
(3) Mechanical injuries cause necrosis when tissues are crushed or when blood supply is injured
or destroyed.
(4) Thermal changes: Both heat and cold can coagulate protein and cause necrosis. The
coagulation of tissues with a branding iron and freezing of extremities are common causes of
necrosis. Frostbite causes thrombosis of blood vessels resulting in necrosis of the affected parts.
(5) Electric currents produced by lightning or generators may coagulate or char tissues. High
voltage electric currents cause coagulation of protoplasm.
(6) X-ray and nuclear fission substances cause protoplasmic alterations that result in necrosis.
Morphological Changes
Macroscopic changes
 Lighter colour than surrounding tissues attributable to coagulation of cytoplasmic proteins
and to reduced blood flow to the necrotic area. Haemorrhage may also be possible.
 Swollen tissue because of cellular swelling
 Reduced volume because of degradation
 Malacia - softer to the touch
 Surrounding zone of inflammation.. The ability to provoke an inflammatory reaction
differentiates necrosis from apoptosis
Microscopic changes
Cytoplasm
 Cytoplasmic swelling can involve locally extensive areas of tissue (as opposed to
apoptosis)
 Increased eosinophilia of the cytoplasm due to altered proteins and loss of ribosomes
 Cytoplasm may become hyalinised (homogenous glassy appearance)
 Cytoplasmic vacuolation
 Swollen mitochondria may be evident as eosinophilic cytoplasmic granules
 Vacuolation and 'moth-eaten' appearance as cytoplasmic contents undergo autolysis
 Calcification is possible under the right conditions

Nucleus patterns during necrosis

Nuclear changes appear in the form of various patterns, all due to non-specific breakdown of
DNA:
 Clumping of chromatin early in reversible and irreversible cell injury
 Pyknosis - nuclear shrinkage and intense basophilia. The DNA condenses into a solid
shrunken mass.
 Karyolysis - a sure sign of necrosis and prior cell death. Basophilia of the chromatin may
fade (karyolysis), presumably due to the action of DNases of lysosomal origin.
 Karyorrhexis, the pyknotic nucleus undergoes fragmentation. In a day or two, nucleus in
the necrotic cell completely disappears.

CLASSIFICATION OF NECROTIC LESIONS

The gross and histological appearance may vary somewhat depending on local tissue conditions
especially how much fluid or blood is present.

COAGULATION NECROSIS:
This is the most common type of necrosis. It is characterized by preservation of the basic
structural outline of the coagulated cells or tissue, for at least some days. The nucleus, however,
is usually lost, but the basic cellular shape is preserved permitting recognition of the cell outlines
and tissue architecture.
Coagulative necrosis results most commonly from sudden severe ischaemia, that is, hypoxic
death of cells in such organs as the heart, kidney, and adrenal gland. Thus, infarcts are the best
example. Fusobacterium necrophorum is a common cause of coagulative necrosis in the livers
of cattle. Other examples include muscular dystrophy in cattle and sheep associated with vitamin
E deficiency; renal tubular epithelium in mercury, thalium, or uranium poisoning.
Macroscopically, the necrotic area is firm and rather dry in consistency. It has a homogeneous,
opaque, cooked appearance, and is grey, white, or tan. Dead cells take on the appearance of an
eosinophilic shadow of the original cells, cellular detail is lost but cell shape and tissue
organisation remain. There is eventual phagocytosis of these dead cells. Microscopically, the
architectural outline of the tissue or organ is maintained but the cellular details are lost.

LIQUEFACTION NECROSIS:
This type of necrosis is characterized by transformation of the tissue into a liquid mass in which
cellular and architectural detail is lost. Whatever the pathogenesis, liquefaction completely
digests the dead cells. It results from the action of powerful lysosomal enzymes. Liquefactive
necrosis is characteristic of focal bacterial infections, because these agents constitute powerful
stimuli to the accumulation of neutrophils. Enzymes of bacterial and leukocytic origin contribute
to the digestion of dead cells.
Hypoxic death of cells in brain it produces liquefactive necrosis. Brain tissue undergoing
liquefactive necrosis is ultimately converted into a cystic structure filled with debris and fluid. In
the central nervous system, liquefactive necrosis occurs in areas of infarction and traumatic
injury. It is also associated with hypoxia; carbon monoxide and cyanide poisoning; thiamine
deficiency in cat; vitamin E deficiency in chickens.
Macroscopically, the tissue in the area of necrosis is liquefied; and may be watery, tenacious or
semisolid in consistency. The softening is called malacia. The colour is usually white, yellow, or
red.
Microscopically, no architectural or cellular detail is visible in the area of necrosis. The dead
tissue is homogeneous and stains pink with eosin. If bacteria are present, neutrophils in varying
degrees of disintegration are found. The entire necrotic mass is surrounded by a zone of acute or
chronic inflammation depending on the length of time necrosis has been present.

Caseous Necrosis:
Caseous necrosis is characterized by the conversion of dead tissue into a homogeneous granular
mass resembling cheese, and by the absence of both architectural and cellular detail. The term
'caseous' means cheesy and is derived from the white and cheesy appearance of the area of
necrosis. Caseous necrosis is encountered mainly with tuberculosis in animals, and caseous
lymphadenitis in sheep. The cheesy appearance in tuberculosis has been attributed to the capsule
of the tuberculous bacillus Mycobacterium tuberculosis, which contains lipopolysaccharides.
Oesophagostomiasis in sheep produces multiple focal areas of caseous necrosis.
Macroscopically, the area of necrosis is a granular amorphous material resembling cheese. The
mass is dry but creamy in consistency. It is soft, friable, and white-grey in colour. Calcium salts
are frequently deposited in the dead tissue. The caseous mass is enclosed within a connective
tissue capsule. Microscopically, the necrotic focus is composed of structureless, amorphous
granular debris enclosed within a distinctive ring of granulomatous inflammation. Neither
architectural nor cellular detail is present. Calcification commonly occurs in the necrotic areas,
especially in sheep and cattle.
TISSUE RESPONSE FROM NECROSIS
 The surrounding tissue response to necrosis is an inflammatory response, including
vascular engorgement and congestion +/- haemorrhage.
 The vascular reaction gives rise to the gross appearance of a red zone surrounding a
necrotic lesion such as an infarct. There is also infiltration of the tissue by neutrophils and
macrophages. These provide hydrolytic enzymes which help breakdown dead tissue and
act to phagocytose necrotic material.
 Subsequently, there is a healing process. This can take the form primarily of fibrosis with
scar formation or primarily of parenchymal replacement.

The diagrammatic representation illustrates the morphological differences between cell death by
apoptosis and that by oncosis. Also emphasised is the end result that the parts of cells that die by
apoptosis are phagocytosed by surrounding cells and tissue macrophages, whereas cells that die
by oncosis release cellular constituents into the surrounding tissue and induce a localised
inflammatory reaction (as described above).

Consequences of Cell Injury

The effect of cell death are many and varied as disease itself. Examples:
 Relatively small numbers of dysfunctional cells within the myocardium can substantially
affect the way the whole tissue is able to function and therefore has a major impact on the
health of the whole animal.
  Large numbers of cells need to be affected in skeletal muscle or the liver for the function
of the whole tissue to be significantly affected.
 The effect of cell injury depends upon the degree of damage (i.e. sublethal or lethal cell
injury), the extent of damage (i.e. the number of cells injured) and the particular
tissue affected.
 A diagnostically useful feature of specialised cellular function is the predominance of
different enzymatic pathways in different tissues.
 When cells are injured, the cell membrane becomes leaky and these enzymes leak out
into the surrounding tissue and disperse through the extracellular fluid and eventually
enter the circulation. The clinician can measure the levels of some of these different
enzymes (clinical pathology).
 Normal cell turnover causes normal circulating levels of enzymes, therefore any
measurement must be compared to the expected level in a normal healthy animal (i.e.
normal range / controls). Creatine kinase (CK) and alanine aminotransferase (ALT) are
two such enzymes that can be diagnostically useful.

The consequences of necrosis

 Necrosis will cause more than just loss of function in the affected area of tissue. For a
start, the ensuing inflammatory response around the necrotic area is likely to interfere
with surrounding viable tissue.
 Impairment of organ function is likely to be much greater than just the grossly visible
necrosis would suggest.
 Very few tissues are able to replace necrotic tissue with new parenchyma, however this
does not necessarily mean impaired function results because most organs have a
considerable amount of functional reserve. The clinical consequences of necrosis will
also depend on which organ is involved.

GANGRENE
What is Gangrene?
Gangrene is simply an old term for a specific case of necrosis - the term refers to necrosis on a
grand scale such as a limb or an entire lung lobe. Gangrene is the invasion and putrefaction of
necrotic tissue by saprophytic bacteria. It most commonly results from vascular obstruction, with
a variable role played by bacteria.
Aetiology: Gangrene is seen most often in the lungs, intestine, mammary gland, heavy muscles
of the thigh and shoulder, and the extremities.
(1) Lungs: Gangrene occurs most commonly from faulty drenching of medicines. The medicine
is often quite irritating and injures the lung. In addition, it carries bacteria with it into the lungs.
As a result of the lung injury and invasion of the necrotic tissue by saprophytic organisms,
gangrene occurs. In paralysis and infectious diseases of the throat, food may pass into the trachea
and produce gangrene.
(2) Intestine: In the horse infarction results from a verminous thrombus in the anterior
mesenteric artery, or the acute local passive hyperaemia associated with a malposition of the
viscera (torsion, volvulus, or intussusception) may cause gangrene of the intestinal tract. The
vascular disturbance causes necrosis of the intestinal wall, and the necrotic tissue is then invaded
with saprophytic bacteria found in the intestinal contents.
(3) Extremities: Gangrene of the leg, ear, tail, wattle, or comb is associated with freezing.
Freezing temperatures cause coagulative necrosis of tissue that is later invaded with saprophytic
bacteria. Certain drugs or plants (eg., ergot and mould) contain active principles which cause
arterial spasms and restrict the blood flow. Because of the ischaemia, necrosis of the extremities
occurs, and later the tissues are invaded with bacteria.
Diabetic gangrene also occurs from narrowing of arteries. Bacteria grow well in the tissue due to
their high sugar content.

CLASSIFICATION GANGRENE
Gangrene may be broadly divided into three types related largely to the role played by bacteria:
(i) Dry gangrene: seen typically with vascular occlusion to an extremity such as foot or ear tip;
bacteria play no significant role - the necrotic tissue dries and sloughs off. Essentially, this is an
example of coagulative necrosis.
Since bacteria require moisture for growth, invasion and spread of bacteria in the area are slow.
Macroscopically, the area is dry, shrivelled and appears mummified as a result of dehydration. It
is reddish brown, green, grey or black in colour. The colour depends on the amount of iron
sulphide formed. The disintegrating erythrocytes liberate haemoglobin. Bacterial putrefaction of
the dead tissue produces hydrogen sulphide. When hydrogen sulphide comes in contact with iron
of haemoglobin, iron sulphide (a black pigment) is produced. The area also has a very putrid
odour due to hydrogen sulphide. Microscopically, a structureless necrotic area, stained pink, is
seen with numerous bacteria. A few gas bubbles are evident by clear spaces. An acute
inflammatory reaction is present at the junction of the living and dead tissue.

(ii) Wet gangrene [Moist gangrene]: necrosis (usually of vascular origin) is followed by
massive proliferation of bacteria normally resident at the site e.g. twisted loop of gut, skin
bacteria on necrotic leg. Bacterial toxin production may result in shock and death of the animal.
Essentially, this is an example of liquefactive necrosis.
Moist gangrene occurs in the internal organs eg., intestinal tract where there is an abundance of
moisture and the temperature is higher. With optimum conditions of moisture and warmth, the
growth and spread of saprophytic bacteria are very rapid. When the intestinal wall gets necrotic,
rupture occurs and the faecal contents are discharged into the peritoneal cavity, spreading
microorganisms throughout the viscera. The course of events is extremely rapid, and death
occurs from septicaemia, toxaemia, and shock.
Macroscopically, the gangrenous tissue is moist; and red, green, grey or black in colour as a
result of iron sulphide formation. The odour is extremely offensive because of the abundance of
hydrogen sulphide and other decomposition products associated with putrefaction. The
putrefaction process produces gas, and numerous gas bubbles are present within the tissues. The
intestine is distended with large quantities of gas. There is no sharp line of demarcation between
the living and dead tissue.

(iii) Gas gangrene [Malignant Oedema and Blackquarter - Blackleg):

These are specific types of moist gangrene that are due to invasion of the tissue by various
clostridial organisms (Clostridium perfringens, C. chauvoei, C. septicum, and C. novyi). These
organisms are inhabitants of the soil and the digestive tract. They enter into the wounds of
various types, e.g., shearing, castration, docking, and ear notching. The injury to tissue results in
necrosis, and in this necrotic tissue these organisms grow and multiply. They produce toxins that
kill the surrounding tissue. They then invade the necrotic tissue, spread throughout the body and
bring about the death. The same series of events occurs in blackquarter and malignant oedema in
cattle and sheep. Cattle and sheep are injured by other animals by kicks, horn thrusts, or bunts
(head thrusts).

In contrast to wet gangrene, here the bacteria are directly responsible for the necrosis. Gas
gangrene is rapidly fatal. Essentially, this is an example of liquefactive necrosis.

DEATH
 Death is traditionally taken to be the moment when the heart is irreversibly stopped, but
of course tissues and even whole organs may remain viable for several hours beyond this
point.
 Life as we know it, however, is dependent on brain activity and so attention may be more
appropriately directed toward assessment of this rather than merely noting presence or
absence of a heart beat.
 Some parts of the brain are capable of maintaining activity longer than others and this
ability is roughly related to the importance of the function they control.

BEYOND (post mortem changes)

Several processes contribute to the decomposition of a carcass after death. These processes (or
post mortem changes) are of considerable practical significance because they may:
(i) obscure or obliterate gross and microscopic lesions
(ii) mimic true lesions
(iii) adversely affect the quality of laboratory specimens, especially samples for bacteriology
and histopathology.

Factors influencing the rapidity of the onset of postmortem changes:

(1) Environmental temperature: Since increased summer temperatures increase the rate of
enzymatic and bacterial activity, animals decompose very rapidly in the summer. Animals can be
preserved for long periods with modem refrigeration.
(2) Size of the animal: The larger the animal, the more rapidly will postmortem changes occur.
This is because the large animals require a longer period of time before the body heat is
dissipated.
(3) External insulation: Thick cutaneous coverings of fur, feathers, hair, or wool prevent heat
dissipation. As such, sheep decompose very rapidly because of insulating wool.
(4) Nutritional state of the animal: Fat is an insulating substance. The fatter the animal the
slower will be the loss of heat and the more rapid will be the rate uf decomposition. Rigor mortis
occurs more rapidly in fat animals than in emaciated animals.
(5) Species of animal: Apart from the size and insulation, the species of animal also determines
the character of the flesh. Pig flesh is soft, moist, and contains fat. Therefore, the rate of
decomposition is rapid.

 Common post mortem changes in approximate order of appearance include:

Rigor mortis:
Rigor mortis is the shortening and contraction of muscles that occur after death, and result in
stiffness and immobilization of the body. It is due to oxygen and energy depletion in muscle
fibres results in a contraction-like state. It begins in the anterior portion and progresses in a
posterior direction (head, neck, trunk, and limbs). It usually appears 1 to 8 hours after death. The
muscular immobility usually disappears 20 to 30 hours after death.
Congestion:
Severe congestion in many organs is very common following barbiturate euthanasia. Affected
organs appear dark red/purple and the spleen is enlarged; the lungs are often severely affected by
this process, and care must be taken not to confuse this change with reddening due to pneumonia.
The effect of gravity will also cause congestion after death: blood is drawn to the lower
(dependent) regions of the carcass; this is called hypostatic congestion and is most obvious in the
lower lung lobes and kidney.

Bile staining: diffusion of bile after death will cause yellow discolouration of tissues adjacent to
the gall bladder and also in loops of bowel which contained bile before death.
Haem staining:
This is usually the most obvious visual change affecting the organs after death. A pink to red
discolouration occurs due to diffusion of haem-containing compounds through tissues following
lysis of red cells or breakdown of myoglobin. Haem staining starts to become pronounced about
24 hrs after death. Imbibition with haemoglobin is the staining of tissue with haemoglobin. After
death, the erythrocytes are haemolysed by cellular and bacterial enzymes, and haemoglobin in
liberated. Being soluble in body fluid, it diffuses into the surrounding tissues and stains them red.

Autolysis:
Liberation of lysosomal enzymes leads to breakdown of cells and softening of tissues after death.
The absence of a red border around areas of advanced autolysis should help distinguish this
change from liquefactive necrosis. When tissue is fixed or embalmed cellular enzymes are
inactivated by formalin or alcohol and autolysis is prevented.
Bacterial invasion:
Autolysis causes destruction of physical barriers fairly quickly, allowing normal bacterial flora
(mostly from the gut) to invade distant tissues. The four major effects of this all begin with S:
softening of tissue (by bacterial enzymes), smell production, staining of tissue green or black
due to bacterial hydrogen sulphide reacting with haemoglobin, and sponginess of tissue and
distension of hollow organs due to gas production. The overall process is known as
putrefaction.
Postmortem clotting of blood: It is the coagulation of blood in vessels after death. In dead
animals the endothelial cells begin to degenerate and liberate thromboplastin which then clots the
blood within the heart, arteries, and veins. In anthrax, however, no clotting occurs because
fibrinolysin produced by the bacteria liquefies fibrin. In sweet clover poisoning also clotting does
not occur since prothrombin activity is inhibited.

Postmortem emphysema: is the accumulation of gas in tissues as the result of bacterial

fermentation.
PIGMENTATIONS AND TISSUE DEPOSITS
Tissue deposits are divided into: those seen without staining (i.e. have an intrinsic colour –
Pigments) and those that have no intrinsic colour and need to be demonstrated by use of special
stains (tissue deposits).

PIGMENTS
 Pigments are coloured substances. They are either endogenous, synthesized within the
body, or exogenous, coming from outside.
 Pigments are divided into exogenous and endogenous pigments, dependent on their
origin. Exogenous pigments include carbon (anthracosis, pneumoconiosis), tattoo ink,
plant pigments (carotenids) and others. These are often contaminants from the
environment.

PNEUMOCONIOSIS
 is the term used to refer to the group of diseases caused by the entrapment of inhaled
particles in the lung. The amount of dust retained.in the lungs is determined by its
concentration in the air, the duration of the exposure, and the effectiveness of
clearance mechanisms
ANTHRACOSIS:
o Is the deposition of carbon particles and is more a problem for humans or animals
that live in the city. These inhaled particles are often found in macrophages in the
lungs and draining lymph nodes. In the case of anthracosis, the pigment gives rise
to a black discolouration of the tissues. Most are relatively harmless unless
present in large amounts in the lung when they may result in pulmonary fibrosis.
o Tattooing is a form of localized pigmentation of the skin. Carbon may be
introduced through cutaneous abrasions. Various pigments in the form of
tattooing inks are used to identify animals. The tattooing is usually done in the
ear. The pigment is located in the macrophages in the dermis and subcutaneous
tissue. Some of the pigment is carried to the regional lymph nodes.
o Macroscopically, the carbon appears as black pigment in the tissues. Carbon in
the lungs appears as focal accumulations or as a diffuse infiltration. Lungs are
black in colour. The regional lymph nodes are also involved, pigment being
present in the medulla. The carbon is only mildly irritating
o Microscopically, particles of carbon are contained within macrophages. The
particles are just black in colour, extremely small and clustered together into
globular masses. Since carbon is insoluble in tissue fluids, the pigmentation
persists for the life. Extensive deposits in the lung may cause slight respiratory
distress, but if silicon is also present, then a serious pulmonary disease may result.
SIDEROSIS
 red pigmentation, which is focal in the beginning due to the local accumulation of
macrophages containing iron dust. The pigmentation becomes diffuse as more and more
dust enters the lungs. Iron is mildly irritating causing only slight fibrosis.
 Microscopically, the iron occurs as brown or black irregularly shaped granules (usually
spherical masses) within macrophages.
  Siderosis is usually of no clinical significance. When iron is present in large amounts or if
silicon is also present, extensive chronic inflammation of the lungs results which may
lead to chronic general passive hyperaemia.

ENDOGENOUS PIGMENTS
Are predominantly divided into three groups:
1. Those pigments derived from haemoglobin
2. Phenolic compounds derived from tyrosine metabolism (melanin)
3. Lipid breakdown pigments (lipofuscin, ceroid)

PIGMENTS DERIVED FROM HAEMOGLOBIN

 Haemoglobin is the oxygen-carrying pigment of erythrocytes. It is a combination of
globin and haem. Haem is the iron-containing porphyrin molecule responsible for the red
colour of haemoglobin and myoglobin.
 When red blood cells are lysed, the haem molecule is broken down in macrophages and
the iron portion is bound to the protein apoferritin and sequestered as particles of ferritin.
Ferritin is accumulated in the cytoplasm until it moves to the lysosome to be converted to
haemosiderin. The porphyrin component of the haem molecule is broken down mainly to
bilirubin.
 Bruises are focal areas of haemorrhage in a tissue often due to trauma. As the red blood
cells free in the extravascular tissues, and those phagocytosed by macrophages,
breakdown the bruise displays various colours. These represent various intermediate
porphyrins and haemosiderin from the breakdown of haem.

Haemosiderin
 Haemosiderin is commonly seen in areas of congestion or haemorrhage or where there is
excessive breakdown of red blood cells. This is a yellow-brown, granular, intracellular
pigment frequently observed histologically in tissue sections.
 The best example of localized haemosiderosis is the common bruise [See above] .
 If present in sufficient amounts it may also be visible grossly (e.g. as brown speckles in
lymph nodes draining a site of haemorrhage). It is typically observed in macrophages in
the normal spleen (the spleen is the graveyard for old red cells). It may be observed in
any tissue in which there has been haemorrhage since macrophages enter to clean up the
debris (macrophages are the garbage collectors and recyclers).
 In animals in which haemolysis (breakdown of red blood cells) is occurring, damaged
circulating red blood cells are taken up by macrophages in organs such as liver and
spleen as they ‘filter’ the blood. In the systemic derangement, haemosiderinis deposited
in many organs and tissues, a condition called haemosiderosis.

Jaundice
Jaundice is the most clinically important pigmentation. Accumulation of bilirubin in the
circulation = hyperbilirubinaemia = jaundice

Bilirubin
  Bilirubin is an endogenous yellow-green pigment. It is the normal breakdown product of
the porphyrin component of haem. Since erythrocytes are continually being destroyed
and replaced in health, there is continual production of bilirubin.
 It is produced in macrophages principally within the spleen (but can occur elsewhere). It
is then transported bound to albumin in the blood until it reaches the liver.
 The albumin-bilirubin complex dissociates at the hepatocyte plasma membrane and
bilirubin then enters the hepatocyte where it is conjugated to glucuronide. It is now
water-soluble and is secreted in biliary canaliculi and ultimately excreted as bile in the
duodenum.
 Within the duodenum, bile is converted by bacteria into urobilinogen. In the small
intestine urobilinogen is either reabsorbed back into the blood or excreted in the faeces.
 Bilirubin may accumulate in the blood (hyperbilirubinaemia) when there is increased
breakdown of erythrocytes (as in haemolytic anaemia), or when there is hepatic or biliary
disease.
 Accumulation of bilirubin in the blood ultimately results in an accumulation of bilirubin
in the tissues giving them a yellow discolouration known clinically as ‘jaundice’ or
‘icterus’. Jaundice is an important alarm bell warning the clinician that there is a disease
of the red cells, liver or biliary tract. Measuring the relative concentrations of conjugated
or unconjugated bilirubin in the serum of jaundiced animals may help to determine the
cause of the disease.

Haemoglobin and Myoglobin in Tissues

 If there is widespread haemolysis or muscle damage, haemoglobin and myoglobin will be
released into the serum giving it a red/brown discoloration observed when the red cells
are spun down.
 Haemoglobin and myoglobin can then also enter the urine resulting in renal damage as
they pass through the kidney. Haemoglobin can be observed in renal tubules after
intravascular haemolysis of erythrocytes tissue as reddish brown casts.

ACID HAEMATIN
 This is a derivative of haemoglobin produced in tissues fixed improperly in buffered
formalin and is therefore an artefact and of no pathological significance. Superficially it
resembles haemosiderin but usually overlies cells instead of being within them. You may
see this sometimes in practical class sections.
MELANIN
 Melanin is a brown-black pigment formed by melanocytes when the enzyme tyrosinase
catalyzes oxidation of amino acid tyrosine to dihydroxyphenylalanine. It is found in
melanocytes, in epidermal cells, and in the pigmented epithelial cells of the eye
 In the skin, melanin is produced in melanocytes and transferred from these cells to basal
cells of the epidermis where is provides some protection from UV rays.
 Microscopically, melanin appears as very minute, uniformly regular dirty-brown,
spherical granules.
 In chronic dermatitis, the skin may become thickened (hyperplastic). This may be
associated with increased deposition of pigmentation (hyperpigmentation) and a failure of
transfer of melanin to the epidermal cells.
  Hyperpigmentation may also be seen in those endocrine disorders with manifestations in
the skin (e.g. hyperadrenocorticism and hypothyroidism). Grossly, the skin becomes
darker than the surrounding normal skin. Histologically, there are increased numbers of
melanin granules in melanocytes, epidermal cells and dermal macrophages (called
melanophages).
 Hypopigmentation is usually genetic and is mainly observed in the hair coat or the eye.
 Melanosis is the aberrant accumulation of melanin and may occur in as a congenital
condition in tissues such as the lung or liver. It is of no pathological significance.
Acquired melanosis the liver of sheep may occur due to the ingestion of certain plants.

LIPOFUSCIN
 In tissue, lipofuscin appears as a golden brown, finely granular, intracellular pigment that
consists of a complex of lipids, phospholipids, and some protein.
 It is derived chiefly from the non-degradable materials that result from the breakdown of
lipids, usually those derived from organelle membranes. It is therefore found most
commonly in aged stable cells (such as neurons or myocardial fibres) or in chronically
injured cells and is often referred to as ‘wear and tear’ pigment.
 It may also be increased in cells in some species as a consequence of vitamin E or
selenium deficiency.
 Lipofuscin can also be increased in animals fed on a diet rich in unsaturated fatty acids. It
is usually of no pathological significance.
COPPER
 Copper may be stored in tissues, particularly hepatocytes in animals with chronic copper
poisoning (seen most commonly in sheep) or as a consequence of chronic liver disease.
 Hepatic copper accumulation may also occur in some dog breeds (e.g. Bedlington
terriers)
 Storage of large amounts of copper is potentially toxic to the hepatocyte and may result
in acute onset hepatocellular necrosis.
CALCIFICATION
 Calcification is a common manifestation of lethal cell injury, but may also occur in
normal tissues. When calcification occurs in injured tissues it is said to be dystrophic, and
when it occurs in apparently normal tissues it is said to be metastatic.
 Dystrophic calcification occurs in necrotic cells injured by a variety of mechanisms
including vascular, toxic, metabolic, or inflammatory causes. Other sites include old
abscesses, degenerative and necrotic portions of tumours, dead parasites, and parasitic
lesions (Trichinella, Oesophagostoma, Sarcosporidia), necrotic renal tubules, areas of
infarction, foot pads of old dogs, and thrombi.
 Metastatic calcification is commonly associated with elevated levels of calcium in the
blood (called hypercalcaemia).
 Abnormalities of calcium homeostasis are often associated with increases in parathyroid
hormone secretion (hyperparathyroidism) resulting in the reabsorption of calcium from
bones.
 Hyperparathyroidism may occur due to a dietary calcium deficiency or Ca:P imbalance in
the diet (nutritional secondary hyperparathyroidism); abnormal phosphorous/calcium
regulation by the kidney in renal failure (renal secondary hyperparathyroidism).
  : Macroscopically (for both dystrophic and metastatic calcification), the calcium salts
appear as fine, white granules, or as yellowish-white or grey chalky masses within the
tissue, often felt as gritty 'deposits. Calcified tissue has a firm consistency, and when cut
with a knife, a definite grit can be detected against the knife blade. Calcification is
especially common in cattle, sheep, and rabbit.
 Microscopically, with the usual H & E stain, calcium salts appear as deep blue
(basophilic) granules or masses. They can be intracellular, extracellular, or in both
locations

AMYLOID
 Amyloid is a protein that can be deposited in tissues, and a disease process resulting from
deposition of this material is known as amyloidosis. It is deposition of amyloid in the
interstitial tissues in a wide variety of clinical disorders. Most common sites are spleen,
liver, kidneys, lymph nodes and adrenals.
 On clinical grounds, the systemic pattern is further classified, both in humans and
animals, into:
(1) Primary amyloidosis when associated with some immunological disorder
(immunocyte dyscrasia), and
(2) secondary amyloidosis when it occurs as a complication of an underlying chronic
inflammatory or tissue destructive process.
 Histologically, amyloid is lightly eosinophilic, amorphous, hyaline material that is
deposited extracellularly. A special stain called congo red stains amyloid orange red.

URATES
 Deposits of uric acid crystals and urates in the tissues due to excessive production or
insufficient excretion of uric acid are observed in the disease termed gout. Deposition is
due to an increase in the concentration of uric acid in blood (hyperuricaemia) and body
fluids. Gout is only seen in humans, birds and reptiles. There are two forms of gout:
1. Visceral gout is the more common form in birds. It is a common sequela to dehydration and
results in urates being deposited in kidney, liver, joints and pericardium.
2. Articular gout refers to deposition of urate crystals in joints and tendon sheaths resulting in a
painful inflammatory response. A similar disease is observed in humans where the urate is
derived from purine metabolism not amino acids. Precipitation of the crystals in joints triggers a
chain of events that end in joint injury.