General Pathology

9 reasons to perform a PM exam

1. Determine cause of death

2. Compare with clinical diagnosis

3. Increase diagnostic accuracy

4. Assess concurrent disease and management problems for loss of production

5. Efficacy of medical/surgical therapy

6. Efficacy and toxicity of therapeutic agents

7. Educate

8. Obtain forensic or legal information

9. ID emerging diseases, zoonoses and disease surveillance

When should PM be conducted? What should be done if delay is necessary? Why?

ASAP after death

• Chill the carcass to slow the rate of autolysis

• Freezing and thawing causes tissue damage leading to difficult histological interpretation

Steps of performing a PM examination (11)

1. External examination

2. Reflect front and back limb and abdominal skin

3. ID last rib and cut into abdominal cavity

4. Cut open thoracic cavity with bone cutters caudal to cranial

5. Remove pluck and examine: Trachea, oesophagus, lungs, thyroid glands, heart

6. Oral cavity

7. Abdominal cavity examination: Liver, gall bladder (squeeze to confirm patency), intestines, spleen, stomach

8. Sample intestine (tie off intestine with two pieces of string -bonbon)

9. Urinary system: Adrenal glands, kidneys (cut surface), open bladder

10. Joints: Palpate and incise to assess joint fluid

11. Remove head (for brain examination)

3 types of sampling (+ handling requirements)

1. Histology = Fix in formalin (10%) to prevent autolysis

   • Samples ≤1cm thick

   • 10 parts formalin : 1 part tissue

   • Whole brain → Large bucket of formalin (2 - 4d to fix)

     • Fix in-clinic, then transport in smaller container with just enough formalin to labeeeeeee

   • Use leak-proof screw top containers with wide necks

   • Can place multiple samples into SAME pottle

   • Formalin is hazardous (irritant gas affecting eyes and nose) → Make up in fume hood with mask

2. Microbiology, PCR, toxicology = Aseptic technique placed into sterile pottle/bag/swab

3. Parasitology

   • Whole parasites submitted in 70% alcohol

   • Faecal sample (≥5gm) for FEC

   • Whole abomasal contents + ≥10m small intestine for parasite counts → Mix with water to form aliquots for counting

7 details for sample submission form

1. Practice name, address and vet name

2. Owner's name

3. Animal ID and signalment

4. Date

5. Relevant clinical history

6. Nature and site of sample (diagram for histology and cytology)

7. Tests required

Describe 10 components of gross lesion description (+ examples)

1. Location = Tissue/organ, side of animal and which part of the organ

2. Arrangement = Grouping/distribution of lesion, scattered randomly? Symmetrical?

   • Multifocal vs. diffuse vs. coalesce

3. Number = Single vs. multiple

   • Miliary = Too many to count

4. Size

   • 3D estimate in mm

   • Compare with common reference item

   • % of tissue involved in lesion

5. Shape

   • Raised/bulging/nodular vs. depressed/flat

   • Well- vs. poorly-demarcated

6. Colour

7. Consistency = Compare to normal

   • Lesion = Rubbery, firm, spongy, semi-solid, pasty, fluid-like, watery etc.

   • Pus = Gritty, dry, cheesy

   • Fluid = Smooth, greasy

   • Organ surface = Wrinkled, smooth, moist and glistening, dry and leathery, pitted, dull, opaque

8. Cut surface

9. Content = Describe normal for pregnant female, GIT and urinary bladder

   • Amount, nature, appearance, parasites, foreign bodies

10. Odour

    • Black leg = Rancid butter smell

    • Ketosis = Acetone breath

Not ALL may be applicable

EXAMPLES: Provide a description and interpretation for the following images

Description (LUNGS): Within the lung parenchyma and affected all lung lobes, there are multifocal to coalescing, variably sized (8mm - 5cm), pale yellow nodules, that on cut surface exude and thick, viscous, yellow fluid

Interpretation: Embolic pneumonia

Description (KIDNEY): Within the renal parenchyma, there is a focal, triangular-shaped, well-demarcated, dark-red to brown area, that measures ~1 × 2cm and extends from the mid-medulla to the cortex

Interpretation: Renal infarct

Description (FEMUR): The marrow cavity is filled with a clear, pale yellow gelatinous material

Interpretation: Serous atrophy of fat

Description (LUNG): ~80% of the cranioventral portion of the left lung lobe and the ventral aspect of the caudal lung lobe are diffusely dark red to brown and firm. There is a single, focal 20 × 15mm dull grey, poorly-circumscribed area in the middle of the leading edge of the cranial lung lobe

Interpretation: Bronchopneumonia with focal abscessation

4 recommendations when photographing lesions

1. Good light to ensure image is clear and in focus

2. No glare over lesion in photo

3. Lesion in centre of photo

4. Include a scale (eg. ruler)

Death and Decay

• Define somatic death

• What is the order of cellular death (by organ)

Somatic Death: Disappearance of life from the entire individual (cardiac and respiratory function does NOT occur spontaneously)

• Not all cells in the body die at the same time (eventually due to anoxia)

Cellular Death Order: First → Last

1. Brain and heart cells

2. Kidneys, lungs and liver cells

3. Mesenchymal cells (bone, connective tissue, cartilage)

List and describe 4 PM changes (+ examples)

ONE: ALGOR MORTIS

• Definition: Cooling of body until it reaches ambient temperature

• Depends on initial body temperature (eg. fever), insulation (fat/wool) and ambient temperature

• Used to calculate range for time of death

TWO: RIGOR MORTIS

• Definition: Stiffening of muscle that occurs after death

• MoA: ATP is required to uncouple actin-myosin complexes. When ATP runs out after death, muscle fibres cannot relax and remain contracted

• Onset of rigor mortis depends on glycogen stores (higher BCS → delayed rigor mortis) and ambient temperature (higher temperature → faster rigor mortis)

  • Onset: 1 - 9hr

  • Disappearance: 12 - 30hr later (autolysis of muscle fibres)

• Order: Head → Heart → Diaphragm → Liver

  

THREE: BLOOD CHANGES

1. Hypostatic Congestion = Pooling of blood (dark) in dependent parts of body due to gravity

   • Blood clots and remains in place even when body is removed

   • Organs: Lungs and kidneys #1

   • DDx: Pneumonia

     

2. PM Clots

   • MoA: Heart stops beating → Blood stops flowing and clots → RBCs have time to sediment out to form pale clot of serum (“chicken fat clot”) over layer of red clot

   • Appearance:

     1. Conforms to vessel = smooth and glistening appearance

     2. Non-adherent (easy to remove from vessel)

     3. Chicken-fat clot OR dark red

     4. No lyaering

        

   • DDx: Thrombi = AM clot

     1. Rough and granular (conforms to damaged blood vessel)

     2. Does NOT separate into white and red layers

     3. Adheres to vessel wall at site of injury

     4. Colour depends on proportion of RBCs (usually dirty red-grey)

     5. Fibrin, cells and platelets laid down in layers

        

FOUR: PM DEGENERATION

1. Autolysis = Breakdown of cells/tissues by body's OWN cellular enzymes (decay of organelle membranes)

2. Putrefaction = Breakdown of cells/tissues by BACTERIA from environment or within body

   • Depends on:

     1. Temperature (warm = more active enzymes)

     2. Bacterial species present

        • Neonates are slower to putrefy as they have no intestinal flora

        • GIT decomposes rapidly

Pseudo-Lesions

• Definition

• Describe 9 examples

  • Cause

  • DDx

Definition; Lesion on PM caused by autolysis and putrefaction

Examples:

1. Paleness and friability

   • DDx: Fatty liver (floats in formalin)

2. Pseudonecrosis = Discolouration and softening within organs

   • Cause: Pressure from other organs OR bacterial proliferation

   • DDx: Necrosis (should have line of demarcation)

     

3. Pseudomelanosis = Black staining of tissues

   • Cause: Production of iron sulphide (reaction between hydrogen sulphide from anaerobic bacteria, and iron from RBCs)

     

4. Gas production = Foul-smelling gas accumulates in body cavities OR tissue (emphysema)

   • Cause: Anaerobic putrefactive bacteria

   • DDx: Bloat

     

5. Hb imbibition = Red staining with Hb pigment

   • Cause: Hb escapes dead RBCs

   • DDx: Bruising or haemorrhage

     

6. Bile imbibition = Yellow/green staining of tissues adjacent to gallbladder or bile ducts

   

7. Organ displacement

   • Cause: Diaphragmatic rupture

8. Mucosal sloughing in rumen

   • No sloughing = Metabolic issues eg. acidosis

9. Lens opacity

   • Cause: Low body temperature

   • DDx: Cataract (wait until fluid within eyes returns to ambient temperature → clear = PM change)

TWO Types of Cellular Damage

• Definition

• Cause

• MoA

• Morphological features on histology

ONE: CELLULAR DEGENERATION

Definition: Mild/brief cell damage which is REVERSIBLE once the stimulus has stopped

• Reversible if membrane is NOT ruptured

Cause: Damaged sodium-potassium pump OR loss of ATP (eg. hypoxia) necessary for the pump to function

• Required maintain cellular homeostasis by pumping out 3Na+ in exchange for 2K+ into the cell

MoA:

1. Damage to pump OR loss of ATP results in loss of function

2. Extracellular Na+ flows into the cell

3. Water is attracted to Na+ and follows it into the cell

4. Cell swells

5. Organelles swell = Ribosomes can detach from the ER

6. Impaired protein production and further compromised cell function results

Morphological Appearance: Hydropic degeneration

1. Cell swelling = Pale cells

2. Eosinophilia = Less contrast and pinker than normal

   • MoA: Ribosomes detach from ER → Loss of affinity of blue-staining from acidic RNA

3. Cells are pinker than normal as ribosomes detach from the ER resulting in lost affinity for blue staining from the acidic RNA Results in relative increase in pinkness ALSO fewer nuclei in swollen cells visible

TWO: NECROSIS

Definition: Irreversible cell injury resulting from severe or prolonged damage

Cause: Swelling becomes so severe → Points of no return

1. Mitochondrial damage preventing ATP function

2. Membrane rupture from lysed phospholipids (cellular AND organelle)

MoA: Membrane rupture is self-perpetuating through

1. Influx of intracellular Ca2+ activates endogenous phospholipidase which lyse the phospholipid membrane

2. Cytoskeletal damage (cell membrane detaches from cytoskeleton)

3. Free radical production

4. Lipid breakdown products have a detergent effect on the cell membrane

Morphological Appearance:

1. Eosinophilia (pinker than degeneration)

2. Shrunken cells

3. Loss of distinct cell membrane due to dissolution

4. Nuclear changes

   1. Pyknosis = Shrunken, dark stain nucleus

   2. Karyorrhexis = Fragmented nucleus

   3. Karyolysis = Faded nucleus from lost affinity for haematoxylin

Hypoxia OR ischaemia?

1. Cardiac arrest

2. Anaemia

3. Thrombus

4. Suffocation

5. High altitude

Hypoxia = Lack of O2 supply → Decreased ATP production

Ischaemia = Lack of blood supply

1. Ischaemia

2. Hypoxia

3. Ischaemia

4. Hypoxia

5. Hypoxia

What is haematoxylin and eosin?

Common routine stain for histology (NOT cytology)

• Haematoxylin is a basic dye which stains acidic structures blue (eg. nucleic acid)

• Eosin is an acidic dye that stains proteins pink

Necrosis

• Definition

• Local response

• Systemic response

Definition: Local death of tissue cells within a living individual caused by irreversible cell damage

Response: Mild local inflammatory reaction resulting from irritation (cellular debris within the ECF)

• Leads to an accumulation of WBCs and a line of demarcation

• Line of demarcation = Red rim hyperaemia (dilated blood vessels) that forms the border between living tissue and pale/friable necrotic tissue

  • Invaded by WBCs

Systemic Response: Release of inflammatory mediators (IL and PG) which cause fever, lethargy, depression and anorexia

FOUR types of necrosis

• Definition

• Causes

ONE: COAGULATIVE

Definition: Histological architecture is recognisable BUT cellular detail lost

• Beginning of every other type of necrosis

Causes:

1. Ischaemia

2. Burns

3. Caustic chemicals

TWO: CASEOUS NECROSIS

Definition: Affected areas form a pale, granular mass (cheesy)

• Loss of cellular AND architectural detail

Causes: Specific bacterial infection

• Mycobacterium spp. and Corynebacterium pseudotuberculosis

  

THREE: LIQUIFACTIVE

Definition: Disintegration of necrotic material into a liquid mass

• Loss of cellular AND architectural detail

Causes: CNS malacia

FOUR: FAT NECROSIS

Definition: Death of fat cells → Breakdown of fat into glycerol backbone and fatty acids

Cause: Fatty acids used in saponification = Combine with Ca2+/K+/Na+ salts to form soaps which precipitate

• Common in mesentery and pancreas

Appearance: Opaque, dull, firm, white which are slightly basophilic

• Some cases, peroxidation of lipids → deposition of ceroid (yellow pigment)

Sequelae of Necrosis

• Importance

• 4 (5) Examples

  • Indication

  • Outcome

Importance: Remove dead material to prevent infection and reduce inflammation

Examples:

ONE: LIQUEFACTION

Indication: Small areas of necrotic tissue which are rich in fluid and enzymes for neutrophil and macrophage infiltration

Outcome: Resulting products are absorbed into the blood/lymphatics thus creating a small hole

TWO: SEQUESTRATION

Indication: Dry necrosis (eg. coagulative or caseous)

• eg. cornea and bone

Outcome: Fibrous capsule surrounding a necrotic centre which can remain in the body (encapsulation without liquefaction)

THREE: ABCESSATION

Indication: Liquefaction of necrotic tissue → Suppuration and encapsulation

Outcomes:

1. Cold abscess (formation of thick, fibrous capsule around the pus)

2. Bursting to release necrotic, purulent material

FOUR: EROSION AND ULCERATION

Indication: Skin and luminal organs (bladder, ureter, bronchus, intestine)

Outcome: Sloughing (desquamate) of dead cells from underlying living tissue into surrounding lumen

• Erosion = Loss of epithelial cells ONLY

• Ulceration = Loss of epithelial cells AND basement membrane → Exposes underlying connective tissue (red due to vasculature)

  

FIVE: GANGRENE

Gangrene

• Definition/indication

• 3 Types

  • Definition

  • Example

• 6 Morphological features

• 3 Reasons why gangrene is dark/green in colour

• 2 Sequelae

• Treatment

Definition: Ischaemic necrosis of EXTREMITIES → Increased risk of bacterial infection due to lack of blood cells for normal defence

Types:

1. Dry Gangrene = Uncomplicated ischaemic necrosis eg. frostbite

   

2. Wet Gangrene = Ischaemic necrosis in areas of fluid/blood → Complicated by bacterial infection resulting in liquefaction of necrotic tissue (eg. mammary gland)

3. Gas Gangrene = Special type of moist gangrene in which ANAEROBIC, gas-producing, saprophytic bacteria (eg. clostridia) establish in necrotic tissue due to low O2. The bacteria also release exotoxins which also induce necrosis in nearby, healthy tissue (eg. black leg caused by Cl. chauvoei)

   

Morphological Features:

1. Cold to the touch

2. Unresponsive to stimuli

3. Does NOT bleed on incision

4. Dark/green colouration

5. Line of demarcation

6. Foul-smelling

Dark/Green Colour Due to:

1. Anoxic blood is dark red

2. Pigments (eg. haemosiderin) from RBC breakdown

3. Iron sulphide production (-SH containing AA breakdown and release H2S which reacts with iron from RBC breakdown to produce iron sulphide)

Sequelae:

1. Sloughing of dead tissue followed by scarring

2. Toxaemia = death

   • Bacterial toxins (septicaemia) and endogenous products from protein decomposition (endotoxaemia) are absorbed into the lymphatics and bloodstream of living areas to cause damage

Treatment: Debridement or amputation

Apoptosis

Programmed cell death that does NOT trigger an inflammatory process
Apoptotic bodies have markers to be targeted for phagocytosis

Physiological vs. pathological cellular adaptations

Physiological Cellular Adaptations = Changes to cells under NORMAL conditions in response to metabolic or physical demand

Pathological Cellular Adaptations = Changes to cells in response to sublethal, chronic cellular injury which enables the cell to survive

Types of Cellular Adaptation and Growth Changes

• Definition

• Examples

ONE: ATROPHY

Definition: Decreased cell SIZE (from normal size) which causes the whole organ/tissue to shrink

• Less cellular activity results in fewer cell organelles that produce less

Examples:

1. Disuse atrophy (eg. muscles)

2. Denervation atrophy (eg. sweeny)

3. Loss of nutrition/hormonal stimulation (eg. small thyroid)

TWO: HYPERTROPHY

Definition: Increase in cell SIZE which causes the organ/tissue to get bigger (can be specific cells)

• Cells are more active and require more organelles

• ONLY way for post-mitotic cells (eg. cardiac muscle) to increase in size as cells cannot divide

Examples:

1. Physiological

   1. Increased skeletal muscle bulk from prolonged exercise

   2. Enlarged smooth muscle cells in the uterus during pregnancy

2. Pathological = Increased ventricle wall thickness due to a stenotic valve

THREE: HYPOPLASIA

Definition: Incomplete growth of an organ/tissue that NEVER reached full size

• Fewer cells produced

• Caused by congenital conditions (NOT true cellular adaptations)

Examples:

1. Aplasia = Complete absence of tissue/organ (extreme hypoplasia) eg. aplasia of epididymis

2. Atresia = Absence of the lumen eg. atresia ani (no opening of anus)

FOUR: HYPERPLASIA

Definition: Increased NUMBER of cells which may increase gross size

• Stimulated in the same way as hypertrophy (can be both)

• Usually normal function

Examples:

1. Goitre (thyroid hyperplasia)

2. Nodular hyperplasia of the spleen

3. BPH

FIVE: DYSPLASIA

Definition: Lack of normal histological architecture in the tissue/organ

• Results from developmental/congenital condition OR acquired (scar tissue)

Examples:

1. Hip dysplasia

2. Kidney displasia

SIX: METAPLASIA

Definition: A fully differentiated cell type changes into another cell type from the same germline

• Usually changes into a more durable cell type in response to chronic irritation

Example: Squamous metaplasia

1. Change from secretory ciliated columnar epithelium in the bronchi (loss of mucociliary blanket)

2. Change from secretory columnar cells in the mammary glands

What is a hamartoma? Give an example

Congenital condition resulting in improper mix of tissues within an organ

• eg. Excess of vascular tissue in the skin causing strawberry birthmarks

• Resemble neoplasm grossly and microscopically

How to distinguish between atrophy and hypoplasia

Atrophy: Normal cells shrink resulting in disproportional size between organs (eg. testicle smaller, epididymis same size)

• Result in more irregular shape that is more dense due to more dense connective tissue Hypoplasia: Small version of the fully grown organ (normal structurally under microscope) PROPORTIONAL (eg. cryptorchidism

Overview of Haemolysis

• Normal function

• Normal MoA of extravascular haemolysis

• Structure of haemoglobin

• Outcomes of the components of RBC breakdown

Function: Membrane degradation of old and damaged RBCs that have reached the end of their lifespan → Allows their components to further broken down and recycled (spleen #1)

MoA:

1. Blood flows through the spleen

2. Damaged/old RBCs are targeted by splenic macrophages via receptors on their membrane surface

3. RBCs enter the macrophage's cytoplasm to undergo haemolysis by digestive enzymes

4. Haemoglobin (Hb) is released from lysed RBCs and broken down into heme, globin and iron

Haemoglobin Structure: 4 subunits of globin protein are each folded around a molecule of heme (each heme molecule contains a central atom of iron)

Components of RBC Breakdown:

1. Heme = Further broken down into bilirubin for excretion

   • Bilirubin = Yellow/orange

2. Globin = Broken down into AA for recycling

3. Iron = Incorporated into haemosiderin (or ferritin) for storage in macrophages

   • Haemosiderin = Yellow/brown

Extravascular vs. Intravascular Haemolysis

• Definition

• Outcomes

• 4 Causes

EXTRAVASCULAR HAEMOLYSIS

Definition: Haemolysis that occurs by splenic macrophages NOT within the blood vessels

• Physiological OR pathological

Outcome: Haemolytic anaemia and hyperbilirubinaemia

INTRAVASCULAR HAEMOLYSIS

Definition: Lysis of RBCs within the lumen of the blood vessels (fragile) resulting in Hb released directly into the plasma

Outcome:

1. Haemolytic anaemia

2. Hyperbilirubinaemia

3. Haemoglobinaemia = Free Hb in plasma (pink after centrifugation)

4. Haemoglobinuria = Free Hb in urine (pink after centrifugation)

Causes:

1. Complement-mediated immune reaction

2. Oxidative damage

3. RBC parasites

4. Congenital defects

Haemosiderin

• Definition

• Formation

• 4 Locations of accumulation

Definition: Yellow/brown insoluble pigment found in macrophages which can be broken down later to reuse iron

• Also, ferritin = Iron complexed with apoferritin protein (soluble storage pigment)

• Stains blue/black with Prussian blue-based stains (contains iron)

Formation: Made from iron (derived from Hb breakdown to heme) → Complexed loosely with protein

Locations:

1. Small amounts in splenic macrophages (NORMAL)

2. Local accumulation in old bruises and vascular congestion (eg. heart failure cells of lungs and liver)

3. Haemosiderosis = Systemic accumulation of excess haemosiderin in macrophages due to:

   1. Severe haemolytic disease

   2. Impaired iron utilisation

   3. After blood transfusions

4. Haemochromatosis = Rare iron storage disease due to defects in iron regulatory genes (i.e. excess iron in bird diets)

Bilirubin

• Formation

• Process of elimination

Formation: Yellow/orange pigment formed from the breakdown of heme AFTER iron is released

Bilirubin Excretion:

1. Free (unconjugated) bilirubin is released into the bloodstream following haemolysis and loosely coupled with albumin (still free)

2. It travels to the liver to be taken up by hepatocytes and converted into the more soluble: conjugated bilirubin (ester with 2 glucuronic acids)

3. Conjugated bilirubin is excreted into bile via the bile duct

4. It enters the small intestine to be converted to urobilinogens

5. Some urobilinogens are reabsorbed to oxidised to urobilin and excreted in the urine (yellow colour)

6. Most urobilinogens are oxidised to stercobilin in the intestine to be excreted in faeces (brown colour)

JAUNDICE

Definition: Abnormally yellow tissues and plasma resulting from high concentration of bilirubin in the blood and tissues Most evident in pale tissues such as the sclera and fat

Causes:

1. Haemolytic (pre-hepatic) jaundice = Excessive intra- or extravascular haemolysis which impairs liver uptake Results in more unconjugated bilirubin in the bloodstream Also, anaemia ± haemoglobinuria and haemoglobinaemia

   1. Examples:

      1. RBC parasites (eg. M. haemofelis)

      2. Incompatible blood transfusion

      3. Neonatal icterus (haemolytic disease of a newborn foal)

      
      

2. Toxic (hepatic) jaundice = Damaged hepatocytes are unable to conjugate and excrete bilirubin Results in build up of unconjugated bilirubin the bloodstream High liver enzymes in the blood would indicate liver damage

   1. Examples:

      1. L. copenhageni in dogs causing hepatocellular necrosis

      2. Hepatitis A and B in humans

      3. Copper poisoning the liver

      
      

3. Obstructive (post-hepatic) jaundice = Obstructed excretion of conjugated bile due to the blockage of the bile duct system Excess conjugated bilirubin enters the blood and faeces may be pale

   1. Complete obstruction = No bilirubin in the intestine resulting in pale faeces

   2. Cholestasis:

      1. Extra-hepatic: Obstruction in large bile ducts

      2. Intra-hepatic: Obstruction in bile canaliculi between hepatocytes (hepatocellular swelling)

   3. Examples:

      1. Gallstones

      2. Biliary tract tumours

      3. Parasites in the bile ducts

      
      

Overview of Melanin

• Definition

• Normal synthesis

• Cause of varied pigmentation

• Function

Definition: Yellow/brown granules secreted by melanocytes

• Responsible for the brown/black pigmentation of skin, hair, iris and choroid

Synthesis: Tyrosinase is used to oxidise and polymerise tyrosine which is complexed with sulphur-containing proteins to form melanin

• Tyrosinase = Copper-dependent enzyme necessary to produce melanin

Varied Pigmentation: Variation in EFFECTIVENESS of melanocytes to produce melanin (NOT the number of melanocytes)

Function: Transferred to epithelial cells of the skin and hair, or taken up by macrophages → Melanin surrounds the nucleus to protect the DNA from UV damage

TWO Aetiologies of Decreased Melanin Production (Pallor)

• Definition

• Examples

1. Albinism = Congenital mutation where no melanin is produced in the WHOLE body (usually a result in tyrosinase deficiency)

   • Determine as eyes are red

2. Acquired hypopigmentation

   1. Hormonal imbalance

   2. Local loss of melanocytes due to trauma or chemical injury (eg. scarring = increased fibrous connective tissue and freeze branding)

   3. Copper deficiency

Describe 4 causes of increased melanin production (darker)

1. UV light = UV light stimulates melanin production in exposed skin to protect nucleus from damage

2. Congenital = Uneven dispersion of melanocytes during embryonic development resulting in discrete focal areas of hyperpigmentation (eg. liver and lungs)

3. Moles = Dermal accumulations of melanoblasts which can transform into melanoma (neoplasm)

4. Plants = Accumulation of melanin precursors in various organs

Fat accumulation

• 3 Tissues fat normally accumulates in

• 4 Tissues fat should NOT accumulate in

Normal:

1. Adipose tissue

2. Adrenal glands

3. Sertoli cells

Abnormal:

1. Liver

2. Heart

3. Muscle

4. Kidney

Describe the process of fat metabolism

1. Lipids transported from the diet (as chylomicrons or FFAs) AND adipose tissue (as FFAs) to the liver

2. Most lipids converted to TAGs by hepatocytes (secreted from liver bound to apoproteins such as VLDLs)

3. Remainder is stored, or oxidised

Appearance of Fatty Liver

• Gross appearance

• Histological appearance

  • Acute

  • Chronic

Gross: Pale, greasy, friable and bulge out from the cut surface Liver may flat in water/formalin

Histology:

• Acute = Numerous small fat globules in the cytoplasm around the central nucleus

• Chronic = Globules coalesce which may displace the nucleus towards the edge of the cell

Terminal Vascular Bed

• Structure

• Function

• 2 Processes increasing blood to terminal vascular bed

• Main process decreasing blood to terminal vascular bed

Structure: All capillaries are patent, but some not completely filled with blood (functional reserve maintained)

Function: Exchange of nutrients and waste products between the arterioles and venules

Increasing Blood:

1. Hyperaemia

2. Congestion

Decreasing Blood: Ischaemia

Hyperaemia

• Definition

• Appearance

• 2 Aetiologies (+ examples)

Definition: More blood to tissues due to increased arterial blood flow and more reserve capillaries flowing with blood

• Usually an acute, active processes associated with inflammation

Appearance: Bright red tissues from accumulation of oxygenated Hb

Aetiologies:

1. Physiological hyperaemia

   1. Increased arterial blood flow in the stomach and intestine during digestion

   2. Exercise increases hyperaemia to muscles

2. Pathological hyperaemia = Hyperaemia as a result of inflammation

Congestion

• Definition

• Appearance

• 3 Types

  • Defintion

  • Examples

• 5 Effects of congestion

Definition: More blood to tissue caused by obstructed flow OUT of tissues at the venous side

• Passive process

Appearance: Dark red/blue tissues from accumulation of deoxygenated Hb

Types:

1. Localised = Local restriction of venous flow resulting in a BODY PART with bluish colour

   1. Internal occlusion (eg. thrombus)

   2. External compression (eg. tumour, tourniquet)

2. Systemic = Generalised congestion where the whole body is blue as a result of blood pooling in the great veins and affects a wide area of tissue eg. heart failure

3. Hypostatic = Blood pooling due to the force of gravity (PM examination)

Effects:

1. None (gradual onset of local congestion → time to develop adequate collateral circulation through other veins)

2. Oedema

3. Hypoxia and necrosis

4. Ischaemia and infarction

5. Diapedesis or rhexis

   • Diapedesis = Passage of blood cells through INTACT capillary walls

   • Rhexis = Haemorrhage caused by rupture of the vessel wall (usually due to trauma)

Describe the pathogenesis of left-sided heart failure (from congestion to heart failure cells)

LCHF:

1. Congestion of the left-side of heart leads to congestion of the pulmonary circulation and blood backs up in the lung

2. Increased pulmonary vascular pressure

3. Alveolar capillaries distend with blood resulting in increased permeability

4. Fluid and RBCs escape into alveolar spaces

5. Alveolar macrophages enter the alveolar space to engulf this material

6. Haemosiderin is produced and stored in alveolar macrophages as a result of RBC breakdown

Heart Failure Cells: Alveolar macrophages containing granules of haemosiderin formed from RBC breakdown and indicates haemorrhage into the alveolar space

RCHF:

1. Congestion of systemic circulation (esp. liver)

2. Dark red blood accumulates in the centrilobar veins of the liver which contrasts with the pale peripheral parts of the lobule (nutmeg liver)

3. As more blood accumulates, pressure in the central veins increases which causes adjacent hepatocytes to atrophy or die

4. Hepatocyte death results in cirrhosis (fibrosis of central vein)

5. Haemosiderin is also found within local macrophages as a result of RBC breakdown

Ischaemia

• 3 Consequences

• 6 Causes

• 4 Factors modifying the effects of ischaemia

Consequences:

1. Hypoxia → necrosis

2. Local nutrient deprivation (not limiting)

3. Failure to remove waste products (eg. muscle ischaemia and pain)

Causes:

1. Cardiac arrest

2. Arterial obstruction

   1. Thrombosis

   2. Embolism

   3. Arterial wall spasm (eg. ergot poisoning)

   4. Arteritis leading to thrombosis (eg. bovine MCF or Strongylus vulgaris)

   5. External occlusion (eg. bandage)

3. Venous obstruction

   • MoA:

     1. Occlusion of the venules prevents blood flow out of the terminal vascular bed

     2. Initially the arterial supply is not affected

     3. Later, occlusion causes increased pressure in the venous side of the capillaries

     4. Results in increased resistance to arterial blood flow and hence, ischaemia

4. Capillary damage/compression (eg. pressure sores)

5. Hypovolaemia

6. Severe vasodilation

Factors:

1. Specific tissue sensitivity to anoxia (brain vs. fibrous tissue)

2. Rate of development and duration (slow development = time to adapt by collateral circulation)

3. Well-oxygenated RBCs vs. anaemic (higher severity)

4. Temperature (low temp = decreased metabolic rate = reduced O2 demand)

Infarct

• Definition

• Most common type

• 2 Organs rarely affected (+ why)

• 7 Stages (artery occlusion → healing)

Definition: Localised are of coagulative necrotic tissue caused by ischaemia

Most Common: Arterial infarct (occlusion)

Organs Rarely Affected: Organs with dual blood supply

1. Lungs (pulmonary and bronchial arteries)

2. Liver (hepatic artery and portal vein)

Stages:

1. Artery occlusion causes decreased hydrostatic pressure within the capillary bed

2. Venous blood flows back into the tissue

3. Venous and capillary wall cells become hypoxic

4. Acute infarct becomes swollen and dark red due to haemorrhage and fluid loss

5. Subacute infarcts become paler due to necrosis caused by hypoxia

6. Neutrophils and macrophages invade the infarct to remove necrotic tissue (line of demarcation)

7. Healing occurs by a fibrotic scar which appears as a depressed scar in a chronic infarct

Provide definitions for the following terms

• Haematoma

• Petechial haemorrhage

• Ecchymotic haemorrhage

• Purpura

Haematoma = Local accumulation of blood (usually clotted) that causes swelling in tissues

Petechial Haemorrhage = Pin-point haemorrhagic spots usually caused by haemorrhage per diapedesis

Ecchymotic Haemorrhage = Slightly larger foci of haemorrhage usually caused by haemorrhage per diapedesis

Purpura = Skin, MM, viscera haemorrhage

What are 5 causes of haemorrhage?

1. Trauma (rhexis)

2. Vascular diseases (eg. vasculitis)

3. Abnormally weak blood vessels (eg. vitamin C deficiency = lack of collagen to strength blood vessels)

4. Local high BP

5. Abnormal clotting

Effects of Haemorrhage

• 2 Local effects

• 4 Systemic effects

Local Effects:

1. Small haemorrhage = little effect (can be absorbed) unless vial area

2. Large haemorrhage produce space occupying lesions which can have severe mechanical effects

   • eg. Cardiac tamponade (pericardium), asphyxiation (lungs), pain (joints)

Systemic Effects: Depends on total volume lost AND rate

1. Chronic, small amounts → Iron deficiency anaemia (iron required for oxygen transport)

   • Bleeding GIT ulcers

2. Acute, at least 1/3 of total → Hypovolaemic shock and death

3. At least 24 hr period, at least 1/3 of total → Compensatory mechanisms and no death

4. Acute, less than 20% lost → Small systemic effect (fast compensation)

Describe the 3 phases of compensatory mechanisms for haemorrhage

PHASE ONE: REDISTRIBUTION OF BLOOD TO VITAL CENTRES

Rapid response

1. Splenic contraction releases large stores of RBC into circulation to maintain

2. Selective arteriole constriction (adrenergic nerves stimulated) so blood is redistributed to the brain, heart and lungs

PHASE TWO: RESTORATION OF PLASMA VOLUME

• Arteriolar constriction causes intravascular pressure of capillaries to decrease which results in ECF moving into blood vessels

• Haemodilution results as the ECF does NOT contain blood cells

  • Complete 2d after original haemorrhage → PCV indicates amount of original blood loss

PHASE THREE: REPLACEMENT OF LOST RBCs

• Erythropoiesis is stimulated in bone marrow in response to RBC loss

• Maximum reticulocyte production 3 - 5 days post-haemorrhage and diminishes by 12 - 14 days

What is shock? How is it caused?

Failure of circulatory system to adequately perfuse organs

• Caused by disparity between the volume of circulating blood and the size of the vascular system

THREE Types of Shock

• Definition

• Examples

ONE: HYPOVOLAEMIA SHOCK

Definition: Dramatic decreased blood volume due to loss of fluid (dehydration) or whole blood (haemorrhage)

Examples:

1. Severe haemorrhage

2. Prolonged diuresis

3. Persistent emesis or diarrhoea

4. Burns

TWO: VASCULOGENIC SHOCK

Definition: Shock caused by widespread vasodilation or widespread endothelial damage

• aka. Distributive shock

Examples:

1. Septic shock (G- bacteria produce endotoxin causing vasodilation)

2. Hypoxia

3. Anaphylactic shock

4. Neurogenic shock (loss of sympathetic innervation)

THREE: CARDIOGENIC SHOCK

Defintion: Shock resulting from decreased cardiac output

Examples:

1. Acute myocardial dysfunction (eg. large infarct)

2. Diastolic dysfunction (decreased filling eg. cardiac tamponade)

3. Systolic dysfunction (decreased emptying eg. increased vascular resistance and ruptured chordae tendinae)

Irreversible Shock

• Definition

• 5 Effects on the body

Definition: When compensatory mechanisms are unable to sustain the patient leading to systemic hypoxia and death

Effects:

1. Widespread endothelial damage (leaking plasma from vessels causing sludging and stasis of blood flow)

2. Organs switch to anaerobic metabolism causing cellular and systemic acidosis

3. Hepatic and renal necrosis

4. Myocardial failure → Arrhythmia

5. Severe depression of the CNS → Loss of consciousness

Effect of Temperature on Shock

• 2 reasons why a shocked patient is cold

• Why is overwarming. shock patient bad?

Shock Patients Cold Because:

1. Selective arteriole constriction (warm blood away from peripheral structures)

2. Decreased heat output due to interference of -CHO metabolism

Overwarming Shock Patient is Bad: Causes dilation of skin vessels and further reduces blood volume to vital organs

Overview of Thrombosis

• Defintion

• 3 Causes (Virchow’s Triad)

Definition: Formation of a thrombus (blood clot) via coagulation

• Normal occurrences during life

Causes (Virchow’s Triad):

1. Endothelial Injury = Abnormal blood vessel wall #1

   • Causes: Trauma, inflammation, chemical irritants etc.)

   • MoA: Activation of tissue factor which triggers the clotting cascade

     • Causes a blood clot to attach to the vessel wall at the site of damage

2. Abnormal Blood Flow = Influences progression of thrombosis

   1. Blood stasis

      1. Varicose veins

      2. Aneurysm

      3. Poor peripheral circulation

   2. Turbulence = Disturbance in laminar flow (most commonly occurs at vessel branches or areas of irregularity in the vessel wall)

3. Hypercoagulability = Abnormal blood composition which influences progression of thrombosis

   1. Haemoconcentration (eg. dehydration)

   2. Higher conc. of procoagulants (genetic or contraceptive)

Occluding vs. Non-Occluding Thrombus

• Definition

• Sequelae

Occluding Thrombus

Definition: Blood clot fills the entire blood vessels which cannot lyse or recanalise

Sequelae: Leads to ischaemia and infarction

Non-Occluding Thrombus

Definition: Partially fills blood vessel

Sequelae:

1. Lysis = Lysed in SMALL blood vessels by plasminogen-plasmid system

   • Causes little damage to surrounding tissue

2. Retraction and recanalisation = Multiple canals form within the thrombus which is lined with endothelial cells (edge of the thrombus AND canals) for blood to flow to distal tissues

3. Organisation = Occluding thrombus in larger vessel shrinks over time and is lined with endothelial cells to allow passage of blood through

4. Thromboembolism = Blood flows over the thrombus which dislodges part of it to become an embolus

Embolism

• Define embolus vs. embolism

• 6 Types of emboli (+ examples)

• 3 Sequelae of emboli

Embolus: Abnormal mass circulating in the bloodstream

Embolism: Embolus arrested in a vessel which occludes it (vessel is same diameter as embolus)

Types:

1. Thromboembolic (bland or septic)

   • eg. IV catheter causing valvular endocarditis

   • eg. Saddle thrombus in cats

2. Gas ("The Bends")

3. Fat (common after orthopaedic surgery or trauma)

4. Neoplasm (can cause metastasis)

5. Foreign body (parasites eg. Dirofilaria spp., bacterial, pus)

6. Cartilage (intervertebral disc fragments lodge in spinal cord blood vessels)

Sequelae:

1. Septic emboli spreads infection (eg. valvular endocarditis caused by infected IV catheter)

2. Tissue infarction if embolus fully occludes vessel

3. Nothing

Provide definitions for the following terms

• Oedema

• Anasarca

Oedema: Excessive accumulation of ECF

Anasarca: Generalised oedema

Cat = Hydrothorax, dog = ascites, ruminant = SC

Describe the normal formation and drainage of tissue fluid within the terminal vascular beds (3 key steps)

1. HP (and osmotic pressure) in the arterial end of the capillary bed causes fluid and solutes (not protein) to flow OUT of the capillaries into the ECF (via gaps between the capillary endothelial cells) to supply tissues with nutrients, O2 and minerals

2. Some fluid reabsorbed at the venous end due to COP (and hydrostatic pressure)

3. Most fluid taken up by lymphatic system which is eventually returned to the bloodstream

FOUR Mechanisms of Oedema

• Definition/MoA

• Outcome (localised or systemic oedema)

• Examples

ONE: INCREASED HYDROSTATIC PRESSURE

Definition: Capillary pressure is determined by venous pressure, and not arterial pressure

1. Compression of venous end

2. Increased fluid in capillaries

3. Increase HP in capillaries

4. Overcomes COP of proteins

5. Net increased in fluid outflow in ECF

Outcome: Either localised OR systemic oedema

Examples:

• Localised Oedema

  1. Dystocia

  2. Tourniquet

  3. Tumour/abscess/thrombus occluding vein

  4. Torsion of luminal organ

• Generalised Oedema = Cardiac disease

  

TWO: DECREASED COLLOID OSMOTIC PRESSURE

Definition: COP = Osmotic pressure exerted by proteins (albumin) in circulation which draws fluid back into the capillaries

Outcome: Systemic oedema

Examples: Hypoalbuminaemia

1. Increased albumin loss

   1. Chronic haemorrhage (eg. Haemonchus contortus)

   2. PLE

   3. PLN (eg. nephrotic syndrome)

2. Decreased albumin production

   1. Protein malnutrition (insufficient AA to synthesise albumin)

   2. Chronic liver disease (hepatocytes cannot synthesise albumin)

THREE: LYMPHATIC BLOCKAGE

Definition: Decreased lymphatic drainage

Outcome: Localised oedema

Examples:

1. Extensive tumour invasion or regional lymph node

2. Removal of lymph nodes surgically

3. External mass compressing the lymphatic vessel

FOUR: INCREASED INTRAVASCULAR PERMEABILITY

Definition: Local inflammation → Widen gaps between endothelial cells to allow larger cells to enter tissues (eg. proteins and WBC)

Outcome: Localised oedema and exudate

Describe the pathogenesis of cardiac disease (from decreased CO to generalised oedema)

1. Decreased CO

2. Selective arteriole constriction shunts blood away from skin, GIT, kidneys to maintain blood supply to vital organs

3. Decreased renal blood flow causes the juxtaglomerular apparatus to secrete renin into circulation

4. Renin converts angiotensinogen to angiotensin I in the plasma

5. Angiotensin I to II in the lungs

6. Angiotensin II stimulates release of aldosterone from the adrenal cortex

7. Aldosterone acts on the kidney tubules to increase Na+ retention (and hence H2O retention)

8. Retained Na+ and H2O causes increased plasma volume and venous pressure

9. Increased movement of fluid into ECF

10. Oedema

Exudate vs. Transudate

1. Protein content

2. Fibrinogen present?

3. # of cells present

4. Specific gravity level

5. Colour

Exudate	Transudate
High total protein in plasma	Low protein content (<1g/100mL) most of which is albumin
Fibrinogen (exudate clots)	No fibrinogen (does not clot)
Many cells present	Few cells present
High specific gravity	Low specific gravity
May be turbid	Clear or straw-coloured

Describe the gross appearance of oedema in solid tissues vs. body cavities

Solid tissues = Wet, heavy and enlarged

• SC = Swellings that pit on pressure and gravity-dependent (bottle jaw and brisket oedema)

• Incised tissues may seep fluid

Body cavities = Free fluid

Overview of Inflammation

• Definition

• 4 Components required to induce an inflammatory response

• 5 Advantages

• 4 Disadvantages

• 5 Cardinal signs of acute inflammation

• 4 Outcomes

Definition: A complex cascade of LOCAL tissue reactions which consists of vascular and cellular responses

• Active response to inciting event which enables survival and tissue homeostasis

• Dynamic process with overlapping events

Components:

1. Inflammatory inducers (bacteria, activation of mast cells, trauma, necrosis)

2. Sensors to detect inflammatory inducers (dendritic cells, mast cells and macrophages eg. TLRs)

3. Inflammatory mediators induced by sensors (eg. TNF, ILs, histamine)

   • TLRs (Toll-like receptors) = MOST important family of pathogen recognition receptors (PRRs)

   • Specific TLRs interact with specific microbial products (PAMPs = pathogen associated molecular patterns) to activate a signalling pathway that stimulates production of inflammatory mediators

4. Target tissues that are affected by inflammatory mediators (cellular level OR tissue level)

Advantages:

1. Dilute and/or inactivate biologic and chemical toxins

2. Kill/sequestra microbes, foreign material, necrotic tissue, and neoplastic cells

3. Provide wound healing factors to damaged tissues

4. Restrict movement of appendages and joints to allow time for healing and repair

5. Increased temperature in body/locally to inhibit replication of some microbial agents

Disadvantages;

1. Tissue injury

2. Systemic effects of inflammation

3. Scar tissue formation

4. Loss of function (due to scar tissue formation)

Cardinal Signs of Inflammation:

1. Redness (rubor) via vasodilation

2. Swelling (tumor) via increased vascular permeability to allow larger healing proteins to enter site

3. Heat (calor) via vasodilation

4. Pain (dolor) via stimulation of neuronal pathways by inflammatory mediators

5. Loss of function (functio laesa)

Outcomes:

1. Return to normal

2. Abscessation

3. Fibrosis = Healing via connective tissue replacement

4. Progression to chronic inflammation

FOUR Classes of Inflammation (by Duration)

• Duration

• Cells present

• Vascular response

• Extracellular material

ONE: PERACUTE

Duration: Immediate

• Caused by potent stimulus → No time to respond morphologically

Cells: Few WBCs (neutrophils) but likely normal

Vascular Response: ± Hyperaemia

Extracellular Material:

1. Oedema

2. Haemorrhage

TWO: ACUTE

Duration: Minutes to 48hr

Cells: Variable WBC infiltration (neutrophils #1)

Vascular Response: Increased permeability, vasodilation and hyperaemia

Extracellular Material:

1. Oedema

2. Fibrin

THREE: SUBACUTE

Duration: Poorly defined between 3 - 4 days (transition between acute and chronic)

Cells: Proportion of WBC begins to change (neutrophils > macrophages > lymphocytes)

Vascular Response: Regressing hyperaemia and endothelial hypertrophy/hyperplasia

Extracellular Material:

1. Regressing oedema

2. Fibrin

3. No evidence of repair

FOUR: CHRONIC

Duration: 7d - years

• Caused by persistent inflammatory stimulus which the host failed to eliminate cause (eg. Mycobacterium hiding in macrophages)

Cells: Mononuclear #1 (macrophages, lymphocytes, plasma cells) and neutrophils

Vascular Response: Angiogenesis and neovascularisation

Extracellular Material: Collagen (fibrous granulation tissue)

Overview of Morphological Patterns of Inflammation

• Importance

• 2 Determinants of morphological classification

• Morphological classification based on (2)

Importance: Provides clues about aetiology, time course of disease, prognosis, treatment and DDx

Determinants:

1. Tissues involved

2. Type of aetiology inciting the inflammatory reaction

Morphological Classification:

1. Type of fluid leaking from vessels (exudate vs. transudate)

2. Cells and proteins migrating from vascular lumen to ECF

SEVEN Morphological Inflammatory Patterns

• Definition

• Location/Source

• Appearance

• Examples

ONE: SEROUS INFLAMMATION

Definition: Pattern of acute inflammation characterised by accumulation of fluid with low concentration of plasma proteins and rare WBC

Source: From plasma leaking from damaged blood vessels or secretions from mesothelial cells lining and serous glands in the peritoneal, pleural and pericardial cavities

Appearance: Clear, thin fluid which cannot be easily differentiated from non-inflammatory oedema (eg. heart failure, hypoalbuminaemia)

Examples:

1. Thermal injuries (burns)

2. Vesicles (skin blisters eg. FMD)

3. Acute allergic response

TWO: FIBRINOUS INFLAMMATION

Definition: Pattern of acute inflammation characterised by the accumulation of fluid with high concentration of plasma proteins and low to moderate numbers of WBCs

Source: Increased vascular permeability allows large proteins such as fibrinogen to travel between endothelial cells into the extravascular space

• Fibrinogen polymerises to form delicate strands of fibrin which promotes wound healing through formation of a meshwork

• Located around mucosa/serosa such as the mesenteric, pleural and pericardial surfaces

Appearance: Fluid is exudative and appears yellow due to the presence of fibrin

Outcomes:

1. Fibrous exudates removed by fibrinolysis and other debris by macrophages OR

2. Fibrin remains and stimulates proliferation and organisation of fibroblasts as well as neovascularisation and granulation tissue formation to form fibrous scar tissue

Examples:

1. Shipping fever

2. Peritonitis (eg. FIP)

THREE: DIPHTHERITIC INFLAMMATION

Definition: Fibrinous exudate (pseudomembrane) is so firmly attached to the underlying tissue in severe cases, that it cannot be removed unless torn off with superficial layer of bleeding tissue

• Pseudomembrane = False membrane formed from sheets of dense fibrin containing blood, bacteria and cellular debris (WBC and necrotic mucosal epithelium) Forms after severe cases of fibrinous inflammation

Appearance: Solid cores of fibrin in tubular organs

Examples: Infectious bovine rhinotracheitis (IBR) associated with herpesvirus

• Forms pseudomembranous plaques in the nasal cavity

FOUR: SUPPURATIVE/PURULE

Definition: Pattern of acute inflammation where tissues respond by accumulation of fluid with high concentration of plasma proteins AND neutrophils

Source: Pyogenic bacteria

Appearance: Milky white and foul-smelling fluid which may form and abscess

• Abscess

  1. Central necrotic region (necrotic leukocytes and tissue cells) = BLACK line in diagram

  2. Zone of preserved neutrophils (between yellow and black)

  3. Outer region of vascular dilation and parenchymal + fibroblastic proliferation (arrow)

Examples:

1. Cat bite abscess

2. Any condition with pyo- (eg. pyometra)

FIVE: CATARRHAL/MUCOID INFLAMMATION

Definition: Exudate contains mucus from cells which function to

1. Entrap bacteria

2. Stimulate ciliary movement to expel mucus and debris

3. May contain lysozyme and IgA

Source: MM (GIT and RT) with abundant goblet cells and mucus glands

Appearance: Thick and gelatinous fluid with abundant mucus and mucin

Examples:

1. Low-virulence bacterial/viral infection

2. Mildly irritating chemicals

3. Parasitic infections

SIX: GRANULOMATOUS INFLAMMATION

Definition: Distinctive morphological pattern of chronic inflammation in a several infectious and some non-infectious conditions defined by macrophage presence

Source: Cellular attempt to contain offending agent that is difficult to eradicate

• Strong activation of T lymphocytes leading to macrophage activation which may result in tissue injury

Appearance: Granuloma = Microscopic aggregation of macrophages which are transformed into epithelioid macrophages (epithelium-like cells)

• Surrounded by mononuclear WBCs (lymphocytes and plasma cells)

• The epithelioid macrophages may fuse to form giant cells present in the periphery or centre of the granuloma

Examples:

1. Fungal infection

2. Infection with Mycobacterium spp.

SEVEN: OTHER

1. Haemorrhagic

2. Erosive/ulcerative

3. Necrosis

Fibrinous vs. fibrosis

• Formed from what?

• Adherence to underlying tissue

• Acute or chronic process?

• Examples of each

Fibrinous	Fibrosis
From fibrinogen which leaks from blood vessels	From fibroblasts (tissue repair)
Separates and breaks off easily	Firmly adhered
Acute process	Chronic process
eg. acute peritonitis	eg. scar tissue

What is the morphological diagnosis?

A short phrase that summarises the most important aspects of the lesion (eg. fibrous synovitis)

Acute Inflammatory Response

• Function

• List 2 phases (+ definitions)

Function: To get the players (eg. leukocytes and proteins) from the vascular system to the site of injury

Phases:

1. Vascular Phase (histamine = #1 inflammatory mediator)

   1. Vasoconstriction (very short duration)

   2. Alterations in vascular caliber → Increased blood flow (vasodilation)

   3. Structural changes in microvascular which permit plasma proteins and leukocytes to leave the circulation

2. Cellular Phase = Emigration of leukocytes from microcirculation → Accumulate in area of injury → Activation of leukocytes to eliminate offending agent

   1. Marginalisation

   2. Rolling

   3. Adhesion to endothelium

   4. Emigration across endothelium

   5. Migration into tissues

THREE Processes of the Vascular Phase of Inflammation

1. Vasoconstriction = Very short duration to stop haemorrhage

2. Vasodilation = Arteriole dilation → Open new capillary beds in area → Hyperaemia

   • Induced by histamine (also nitric oxide, bradykinin, prostaglandins and leukotrienes)

3. Increased Vascular Permeability = Escape of protein-rich exudate into ECF → Oedema via

   1. Retraction of Endothelial Cells = Gaps between endothelial cells increase, allowing larger proteins and leukocytes to migrate into the tissue

      • Immediate, transient response

   2. Endothelial Injury = Direct damage to the endothelium (stimulated by severe injury, microbial toxins, endotheliotropic viruses etc.) which results in endothelial cell necrosis and detachment

      • Occurs rapidly after stimulus

      • Likely long-lived response (endothelium needs to regenerate and repair)

Describe how the vascular phase of inflammation causes blood stasis and the cellular phase

The vascular changes:

1. Increased blood vessel diameter

2. Loss of fluid

→ Slower blood flow, increased [RBC] in small vessels, increased blood viscosity (stasis) which

1. Causes leukocyte (neutrophil/macrophage) accumulation along vascular endothelium

2. Endothelial cells are activated by mediators produced at the site of inflammation → Increased adhesion molecules

3. Leukocytes adhere to endothelium and migrate through vascular wall into interstitial tissue

Describe the FIVE steps of leukocyte recruitment

ONE: MARGINALISATION

• Leukocytes take a peripheral position along the endothelial surface

• Occurs in venules

• Due to blood stasis caused by acute vascular changes

TWO: ROLLING

THREE: ADHESION TO ENDOTHELIUM

• Adhesion Molecules = Proteins present on the TWO cell types (leukocyte/platelet and endothelial cell) which mediate adhesion of the two cells

  • Selectins = Adhesion molecules which mediate the initial/weak interaction between the endothelium and leukocytes to allow rolling

    1. L-selectin (expressed on leukocytes)

    2. E-selectin (expressed on endothelial cells)

    3. P-selectin (expressed on platelets and endothelial cells)

  • Integrins = Proteins on leukocytes which mediate firm adhesion to the endothelial cells at the site of inflammation

    • Leukocytes normally express integrins in the low-affinity state

    • When the integrins are activated, they convert to the high affinity state to allow firm adhesion

• Adhesion molecule expression enhanced by cytokines (TNK, IL-1 and chemokines)

FOUR: EMIGRATION ACROSS ENDOTHELIUM

1. Chemokines stimulate cell migration through the inter-endothelial spaces (via adhesion molecules between the endothelial spaces) towards the site of injury (towards the chemical concentration gradient)

2. Leukocytes penetrate the basement membrane and enter the extravascular tissue

3. In connective tissue, leukocytes can adhere to the ECM via integrins to be retained at the site where needed

FIVE: MIGRATION INTO TISSUES

What is leukocyte adhesion deficiency (LAD)? What is the clinical consequence?

LAD is a defect in the sequence of steps leading to leukocyte migration into the inflammatory site (eg. lack of β2 integrin expression)
Results in NORMAL number of leukocytes in circulation but they cannot migrate out of the vasculature to the tissues where needed (results in sepsis and death)

Chemotaxis of WBC

• Definition

• 4 Chemoattractants

Definition: Mechanism responsible for the migration of leukocytes through tissue towards the inflammatory stimulus in the direct of the chemical gradient of locally produced chemoattractants

Chemoattractants:

1. Cytokines (esp. chemokine family = IL-8)

2. Components of complement system (eg. C5a)

3. Arachidonic acid metabolites (eg. leukotriene B4)

4. Exogenous = Bacterial products and some lipids

Describe the THREE steps of phagocytosis

ONE: RECOGNITION

Phagocytes have numerous receptors to recognise and bind specific microbes for phagocytosis

• Enhanced by opsonisation of foreign material (triggers phagocytosis)

• Opsonins = IgG, C3b, plasma lectins

TWO: ENGULFMENT

The phagocyte's plasma membrane forms a vesicle around the bound microbe to create a phagosome

• Phagosome fuses with a lysosome to form a phagolysosome which results in discharge of the lysosomal granules to breakdown and kill the microbe

THREE: KILLING AND DEGRADATION

1. Respiratory burst: More reactive oxygen and nitrogen species derived from nitric oxide produced upon phagocytosis

2. Degranulation of neutrophils which release lysozymes into the ECF to kill bacteria extracellularly

3. Neutrophil extracellular traps

3 instances when leukocytes may cause injury to NORMAL cells

1. Prolonged inflammation leading to collateral damage

2. Autoimmune disease (inflammatory response directed against host tissues)

3. Allergic reaction (excessive host reaction against harmless environmental substance)

What 2 factors result in termination of the acute inflammatory response?

1. Inflammatory mediators are produced in rapid bursts and have short half-lives

2. Development of inflammation also triggers signals to terminate the inflammatory reaction:

   1. Change of AA metabolites produced (from pro-inflammatory leukotrienes to anti-inflammatory lipoxins)

   2. Liberation of anti-inflammatory cytokines from macrophages

   3. Production of anti-inflammatory lipid mediators that inhibit production of TNF in macrophages

Overview of Mediators of Inflammation

• Definition

• 2 Types

  • Definition

  • Examples

Definition: Any messenger that acts on blood vessels, inflammatory cells or other types of cells to contribute to an inflammatory response

Types:

1. Cell-Derived = Mast cells, dendritic cells and platelets #1

   1. Pre-Formed = Present normally in intracellular granules and 1st secreted during inflammation

      1. Histamine

      2. Serotonin (PLT)

   2. Newly-Synthesised = Synthesised de novo (eg. arachidonic acid metabolites)

      1. Eicosanoids

         1. Cyclooxygenase (produce prostaglandins)

         2. Lipoxygenase (produced leukotrienes and lipoxins)

      2. Platelet-activating factor (PAF)

      3. Reactive O2 species

      4. Nitric oxide

      5. Cytokines

         1. Tumour necrosis factor (TNF0

         2. Interleukin 1 (IL-1)

2. Plasma-Derived = Inactive precursors synthesised by liver and may be activated

   1. Complement system

   2. Fibrinolytic system

   3. Coagulation system

   4. Kinin system

Histamine

• Sources

• Type of mediator

• Function

• Degranulation stimulated by

Source: Mast cell degranulation (also basophils and platelets)

Type: Cell-derived, pre-formed inflammatory mediator

Function: Immediate transient phase of vascular response

1. Vasodilation of arterioles

2. Increased vascular permeability

3. Endothelial activation

Stimulation:

1. Physical injury

2. Binding of antibodies and complement

3. Neuropeptides

4. Cytokines

Arachidonic Acid Metabolites (eg. Eicosanoids)

• Source

• Type of mediator

• 2 Enzymes responsible for production

• 5 Functions (+ example eicosanoids)

Source: Fatty acid stored in the phospholipid membrane and released by phospholipases

Type: Cell-derived, pre-formed inflammatory mediator

Enzymes: Synthesise eicosanoids from AA

1. Cyclooxygenase → Prostaglandins

2. Lipoxygenase → Leukotrienes and lipoxins

Functions:

Action	Eicosanoid
Vasodilation	PGI2 (prostacyclin), PGE1, PGE2, PGD2
Vasoconstriction	Thromboxane A2, leukotriene C4, D4, E4
Increased vascular permeability	Leukotriene C4, D4, E4
Chemotaxis, leukocyte adhesion	Leukotriene B4, HETE
Inhibit inflammation	Lipoxin

Platelet-Activating Factor (PAF)

• Sources

• Type of inflammatory mediator

• 3 Functions

Source: WBC and mast cells

Type: Cell-derived, pre-formed inflammatory mediator

Functions:

1. Platelet aggregation

2. Stimulates most vascular and cellular reactions of inflammation

3. Boosts synthesis of other mediators

Reactive O2 Species

• Source

• Type of inflammatory mediator

• Function

Source: WBC

Type: Cell-derived, pre-formed inflammatory mediator

Function: Amplify inflammatory response

Nitric Oxide (NO)

• Source

• Type of inflammatory mediator

• 5 Functions

Source: Endothelium and macrophages

Type: Cell-mediated, pre-formed inflammatory mediator

Functions:

1. Vasodilation

2. Inhibit cellular response to inflammation

3. Reduce platelet aggregation and adhesion

4. Inhibit WBC recruitment

5. Killing microbes

Cytokines

• Definition

• Type of inflammatory mediator

• 2 Examples

  • Source

  • Function

Definition: Proteins produced by many cell types that modulate functions of other cells

Type: Cell-derived, pre-formed inflammatory mediator

Examples:

1. Tumour Necrosis Factor (TNF)

   • Source: Macrophages, mast cells, T lymphocytes

   • Function: Stimulates expression of endothelial adhesion molecules and secretion of other cytokines (systemic effects)

2. Interleukin-1 (IL-1)

   • Source: Macrophages, endothelial cells, some epithelial cells

   • Function: As for TNF (greater role in fever)

Importance of factor XII

Triggers ALL four systems

1. Intrinsic pathway of coagulation

2. Kinin system

3. Fibrinolytic system

4. Complement system

Complement System

• Definition

• 4 Functions

Definition: Group of soluble proteins and membrane receptors which plays a key role in innate and adaptive immune defences

• Activated to become proteolytic enzymes that degrade other complement proteins to form an enzymatic cascade

Functions:

1. Inflammation (C3a, C4a and C5a = anaphylatoxin)

   • Anaphylatoxin = Stimulates histamine release from mast cells (increases vascular phase of inflammation) Similar to allergy antigens = anaphylaxis

2. Chemotaxis (C5a for neutrophils, monocytes, eosinophils and basophils)

3. Phagocytosis (C3b = opsonin)

4. Cell lysis (MAC: C5b - 9)

   • MAC = Membrane attack complex which makes bacteria permeable to water and ions = cell death

How does inflammation stimulate the coagulation system?

Inflammation increases production of several coagulation factors which makes the endothelial surface pro-thrombogenic and inhibits anticoagulation mechanisms = promotes clots

Describe THREE outcomes of acute inflammation (+ circumstances of each)

1. Complete Resolution = Tissue site restored to normal (removal of cellular debris and microbes by macrophages and resorption of oedema by lymphatics) which requires:

   1. Short-duration of stimulus

   2. Minimal tissue damage

   3. Damaged cells have high proliferative capacity and can regenerate

2. Healing by Fibrosis (Repair) = Tissue site repaired with fibrous connective tissue which requires:

   1. Substantial destruction (esp. to ECM or stromal framework or crypts or basal membrane)

   2. Destruction of cells incapable of regeneration (eg. heart and brain)

   3. Abundant fibrin in exudate that cannot be clear

3. Chronic Inflammation = Inflammation of prolonged duration in which tissue injury, inflammation and attempts at repair, coexist in varying combinations due to:

   1. Persistent infection of microorganisms that are difficult to eradicate (eg. Mycobacterium spp.)

      

   2. Immune-mediated inflammatory diseases (eg. type III hypersensitivity)

      

   3. Prolonged exposure to toxic agents

      

What are 3 morphological features of chronic inflammation?

1. Infiltration with mononuclear cells (macrophages, lymphocytes and plasma cells)

   

2. Healthy tissue destruction by inflammatory cells and persistent offending agent

   

3. Attempted healing by replacing damaged tissue with connective tissue (granulation tissue formation)

   • Proliferation of small blood vessels to vascularise new tissue formation

   
   

   

How does granulation tissue (immature) differ from fibrosis (mature granulation tissue) on histology?

Immature = Disorganised mesenchymal cells with tiny blood vessels and oedema in the tissue

Mature = Fibrosis with organised fibroblasts and blood vessels

Macrophage

• Importance

• 6 Functions

Importance: #1 Cell player in chronic inflammation as they have pro-inflammatory properties to stimulate leukocyte recruitment, phagocytosis and microbial killing AND anti-inflammatory properties to stimulate healing

Functions:

1. Highly phagocytic

2. Antimicrobial activity (NO)

3. Expresses tissue factor which may stimulate extrinsic coagulation pathway

4. Important Ag-presenting cell to drive the immune response

5. Stimulates chemotaxis and activation of inflammatory cells and fibroblasts

6. Secretes cytokines and growth factors to stimulate fibroblast infiltration and angiogenesis = REPAIR

What are 7 systemic effects of inflammation?

1. Fever

2. Leukocytosis = High WBC count

3. Acute-phase proteins

4. Increased pulse and BP

5. Shivering

6. Chills

7. Anorexia

3 Factors influencing balance of regeneration vs. repair

1. Proliferative capacity of the cells

   • Cells with high proliferative capacity continuously renew their cells and are able to regenerate after injury which means the organ is more likely to have restored normal function

2. Integrity of the ECM

3. Resolution OR chronicity of injury and inflammation

THREE classes of tissues based on proliferative capacity

• Definition

• Examples

ONE: LABILE TISSUES

Definition: Contain undifferentiated stem cells that are continuously diving and proliferate throughout their life, replacing those that are lost

Examples:

1. Mucosal epithelium

2. Bone marrow cells and haematopoietic tissues

TWO: STABLE TISSUES

Definition: Contain quiescent cells with low levels of replication

• The cells are usually in the rest phase of the cell cycle, but undergo rapid division in response to injury (stimulated by growth factors)

Examples:

1. Mesenchymal cells

2. Vascular endothelial cells

3. Parenchymal cells of the liver, kidneys and pancreas

THREE: PERMANENT TISSUES

Definition: Terminally differentiated non-proliferative cells

• Damage to these cells will result in loss of function as they repair by scar tissue formation

Examples:

1. Cardiac cells

2. Nervous tissue

Describe FIVE stages of healing by repair/scar tissue formation/fibrosis

1. Inflammation

2. Angiogenesis via vascular endothelial growth factor (VEGF)

   1. Branching and extension of adjacent PRE-EXISTING vessels

   2. Recruitment of endothelial progenitor cells from bone marrow

3. Migration and proliferation of fibroblasts

4. Scar formation = Deposition of connective tissue containing collagen and other ECM components

5. Connective tissue remodelling

What are the 3 phases of cutaneous wound healing?

1. Inflammation

2. Proliferation

3. Maturation

Granulation Tissue

• Appearance

• Structure

• Proud flesh

Appearance: Pink, soft, granular appearance due to the presence of new small vessels and proliferation of fibroblasts

• Oedema as new vessels are leaky to allow passage of plasma proteins into ECF

Structure:

1. Capillaries

2. Fibroblast

3. ECM (with collagen)

Proud Flesh: Exuberant granulation tissue formed on the distal limbs of horses which inhibits epithelialisation and potentially results in loss of function

Factors that Influence Wound Healing

• 4 Systemic factors

• 4 Local factors

Systemic Factors:

1. Poor nutrition (eg. protein or vitC deficiency)

2. Metabolic disorder (eg. diabetes mellitus = microangiopathy)

3. Poor tissue perfusion

4. Hormones (GC are anti-inflammatory and can inhibit collagen synthesis)

Local Factors:

1. Infection #1

2. Mechanical (compressing blood vessels and wound dehiscence)

3. Foreign bodies

4. Size, location and type of wound

What are five disadvantages of repair?

1. Deficient scar formation resulting in dehiscence

2. Ulceration due to inadequate vascularisation

3. Excessive formation of repair components (exuberant granulation or hypertrophic scars and keloids)

4. Deformed tissue due to excessive contracture

5. Fibrosis impairs function (depends on tissue type)

Overview of Plasma Proteins

• 2 Types

• 4 Functions

• Plasma vs. serum

• Age-related changes

Types:

1. Albumin

2. Globulin

Functions:

1. Exert COP

2. Participate in immune/inflammatory responses and clotting processes

3. Nutritive functions

4. Aid maintenance of acid-base balance

Plasma vs. Serum:

• Plasma = Liquid part of blood that has NOT clotted (contains fibrinogen) using an anticoagulant (eg. EDTA)

• Serum = Liquid part of blood that remains AFTER the blood has clotted (contains NO fibrinogen)

Age-Related Changes: Younger = Lower TP as colostrum is metabolised

• Consider use of adult RR

Albumin

• % of protein serum

• Source

• Half-life

• 6 Functions

Serum: 30 - 35% albumin (65 - 70% other protein)

Source: Synthesised in liver

Half-Life: Differs between species

• Dogs: ~8 days

• Horses: ~19 days

• Cattle: ~2 - 3 weeks

Functions:

1. Maintains COP (oncotic pressure)

2. Assists transport of sparingly soluble substances to prevent their loss through the kidney

3. Transports 40% total serum Ca

4. Source of amino acids in peripheral tissues

5. Antioxidant and buffer

6. Negative acute phase protein

THREE Groups of Globulins

• Source

• Function

1. Alpha-Globulins

   • Source: Liver

   • Function: Transport proteins and inactivate proteases (anti-inflammatory proteins)

2. Beta-Globulins

   • Source: Liver

   • Function: Transport, immunoglobulins (IgM and IgA) and complement (C3)

   • Examples: Fibrinogen, prothrombin and plasminogen (plasma NOT serum)

3. Gamma-Globulins

   • Source: B lymphocytes/plasma cells

   • Function: Increase IgG during immune response

Refractometry

• Function

• 4 Disadvantages

Function: Measures the total solid concentrations in different fluids (plasma, serum, urine, exudates)

• For plasma, it measures a refractive INDEX as an estimate for the total protein

-ve:

1. High concentration of glucose, urea, Na or Cl → falsely high TP

2. Lipaemia (milky serum) = turbidity alters light transmission → falsely high levels

3. Haemolysis (true or fractious animal or poor preservation of sample) → blurred line

4. Bilirubin (icteric) → altered colour NOT reading

What method of TP measurement is best for birds?

Biuret method

Serum Protein Electrophoresis (SPE)

• Function

• MoA

Function: Used to separate proteins into multiple bands (fractions) based on their size and charge → Determine cause of hyperglobulinaemia

1. Innate/acquired IMMUNE RESPONSE

2. Neoplastic lymphoid proliferation

MoA: When exposed to an electrical current across a gel, the proteins migrate across it at varying amounts depending on size and charge (eg. albumin is the smallest with the largest charge = further migration)

• Protein bands are then translated by a densitometer into tracing to produce an electrophoretogram