SF090

Types of stress that can lead to cell injury (6)

Time course (3)

1. Physical factors (e.g. heat, cold, mechanical)
2. Environmental factors (radiation, infectious, toxins,
   deficient nutrition)
3. Reduced aerobic respiration or energy supply (e.g.
   ischemia or anoxia)
4. Oxygen free radical reactions (e.g. secondary to
   inflammation, ischemia, radiation)
5. Genetic factors (e.g. enzyme deficiencies,
   inappropriate storage of substances)
6. Aging (reduced ability to regenerate tissues)

Rapid (seconds to minutes) – ischemia, physical trauma

Intermediate (days to weeks) – inflammatory / infection

Slow (months to decades) – nutritional deficit, age-related degenerative disease

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How do we recognize cell injury by microscopy?

By Electron Microscopy?

1. Stain and look for...

Cytoplasmic protein degradation

Nuclear degradation

Storage material

2. Immunohistochemistry (add Antibodies) and look for..

Protein Aggregates or Loss

3. In situ hybridization to detect DNA or RNA
from a virus

4. Enzyme histochemistry

Look at mitochondria and look for inclusions (eg Viruses in nucleus) in the mitochondria

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Cellular response to stress (3)

Disuse -> Atrophy
Overuse/Abuse -> Swelling, Hypertropy, Hyperplasia, Metaplasia, Neoplasia, Storage, Death

Some changes are reversible if the stressor is removed

eg. cell swelling or atrophy

Some changes are compensatory – allow improved function of
an individual cell

e.g. altered cytoskeleton for strength or increased enzymes for toxin processing

or organ

e.g. more and larger muscle fibers to increase strength of muscle

Some changes are irreversible but not fatal to the cell

e.g. storage material related to aging

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Types of cell death (3)

Necrosis – sudden failure of cell homeostasis with loss of energy supply, swelling, membrane rupture, and uncontrolled release of cellular contents. Necrosis is “messy” and can damage adjacent cells

“clean” allowing controlled removal of cells without damage to surroundings
Apoptosis – extracellular signals (pathological or genetic in development) initiate a variety (± mitochondrial involvement) of enzyme activation cascades (caspases) that eventually lead to controlled cell fragmentation and nuclear condensation (endonucleases).

Apoptosis can be activated through multiple pathways.

• Intrinsic pathway (e.g. energy withdrawal) operates through mitochondria. Increased permeability to cytochrome c can activate caspases in the cytoplasm.
• Extrinsic pathways (e.g. related to inflammation or cytokine signaling) may bypass the mitochondria. The caspase activation cascade in turn activates endonucleases that digest DNA.

Autophagy – regulated degradation of cell components through its own the lysosomal machinery; organelles are sequestered in membranes which then fuse with lysosomes (starving cells degrade materials to provide nutrients for more essential processes -> ie passed onto surrounding cells)

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Ischemia mechanism

SF090

Reversible changes associated
with energy supply reduction (4)

• Cell and organelle swelling due to changes in ion gradients
• Separation of ribosomes from ER
• Calcium release from ER causes...
• Calcium accumulation in mitochondria
• Cytoskeletal protein aggregation due to activation of calpains

SF090

Mechanisms & manifestations of acute (sudden) cellular injury (3)

Physical Injury:

Cells and tissues that are directly smashed, cut, crushed,
frozen, or cooked die rapidly

Radiation:
Ionizing: alpha, beta, X rays, gamma rays (DNA, Pr. and cell memb damage)
Non-ionizing electromagnetic: ultraviolet (DNA, collagen), microwaves(thermal FX, possible DNA damage)

-Potential for acute and cumulative

Anoxia/Ischemia:

• Deprivation of oxygen or blood flow deprives tissues of main energy substrates (oxygen and glucose), which may lead to cell death.
• Rapid restoration of blood flow or oxygen may avert the damage; late restoration of blood flow can increase the amount of damage (greater inflammation and oxygen free radical production)
• Cooler temperature reduces metabolic demand leading to increased tolerance of ischemia (hence hypothermic surgery)

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2 Types of tissue necrosis (explanation and diagram of both)

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Oxygen free radicals

Cellular manifestations of chronic stress or injury, and aging leading to Cell Storage, 3 FX

• produced by radiation, inflammation, and chemical toxins
• O₂^- (superoxide), ·OH (Hydroxyl Radical)
• Lead to Membrane damage, Pr. damage and DNA damage

1. Altered Metabolism (Dietary/toxic) = Potentially reversible
2. Enzyme Deficieny (Inherited) = Potentially Toxic
3. Indigestible material Phagocytosed (eg Hemosiderin) or accumulated (eg lipofuscin) = Indicator of aging or stress but no adverse FX

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Aging of cells and tissues:

1. Genetic Factors

2. Env. Factors

3. Outcomes

1. Genetic Factors

• DNA repair
• Telomere status
• Antioxidant defense
• Stress response

2. Env. Factors

• Caloric intake
• Nutrition
• Diseases
• Exogenous stresses (radiation, toxins, etc.)

3. Outcomes

• Metabolic activity
• Oxidative stress
• DNA damage
• Protein damage
• Lipid peroxidation
• Failure to repair or replace
• Aging

SF076

How do changes in body building occur?

What happens to bone marrow after it is radiated?

Muscle is stressed and hypertrophies, Satellite (stem) cells proliferate, merge with existing myofibers, and individual muscle fibers enlarge

It is less cellular with loss of nuclei (fewer blue spots) and cell debris (pink smears). This happens because Bone marrow contains germinal blood cells that proliferate continuously. Dividing cells are susceptible to radiation.

SF076

Look at the rest of the Cases

SF052

1. In a thermal injury to the skin results in the “exudation” of fluid onto the surface. How does the formation of this fluid differ from that of normal extracellular fluid?

2. What is hypovolemic shock and how would it occur in a patient with a severe burn?

1. Normal ECF has no protein, but blister fluid is more sticky than normal fluid because of xs protein. Normally there are tight junctions that prevent protein going into ECF usually, but in this case the protein can via 1 of 2 mechanisms:

A) Contraction of endothelial cells causing formation of intercellular gaps
B) Direct injury to endothelium

This changes the oncotic pressure so that water is pulled out.

Note: Arterial Hydrostatic P isn’t affected by high BP, arterioles act as sphincters to ensure that AHP doesn’t go too high. Not the same for V(HP) because the venules don’t have the same controls.

2. Losing blood volume because of proteins outside of plasma and pulling out fluid leading to low volume of blood. It is a chronic wasting away of blood volume.

SF115

Events in the Resolution of Inflammation

Definition of Chronic Inflammation

& causes

1) Return to normal vascular permeability
2) Drainage of edema fluid and proteins into lymphatics
3) Pinocytosis of fluid and proteins by macrophages
4) Phagocytosis of apoptotic (dead) neutrophils
5) Phagocytosis of debris by macrophages
6) Disposal of macrophages

Note: Macrophages are key in this process

Chronic Inflammation:
Inflammation of prolonged duration (weeks-months) in which active inflammation, tissue destruction and attempts at repair are proceeding simultaneously

Causes:

1. Persistant infections (e.g. TB, Treponema Pallidum (syphilis), fungi)
2. Persistance of a foreign body (e.g. suture)
3. Prolonged exposure to potentially toxic agents, either endogenous or exogenous (e.g. silica-silicosis, plasma lipid components-atherosclerosis)
4. Autoimmunity (e.g. rheumatoid arthritis, SLE)

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Histological Features of Chronic Inflammation

Maturation steps for Macrophages

• Infiltration by mononuclear cells (macrophages, lymphocytes and plasma cells) -> Would be all neutrophils if damage was acute
• Tissue destruction (by inflammatory cells)
• Attempts at healing by connective tissue replacement of damaged tissue (angiogenesis and fibrosis i.e. granulation tissue)

Stem Cell to Monoblast (in bone marrow)

Monoblast becomes Monocyte (now in blood)

Monocytes becomes Macrophage (now in tissue)

Macrophage can go through:

1. Activation into Activated Macrophages, Epitheliod Cells, Giant Cells

or

2. Differentiation into Microglia (CNS), Kupffer Cells (Liver), alveolar macrophages (lung), Osteoclasts (bone)

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When and Why do Macrophages appear?

Activated Macrophage responds to...

Mechanisms of Macrophage Accumulation

Macrophages appear shortly after the beginning of an acute inflammatory response and predominate after 48 hours.

Immune Activation: T cell secrete factors (IFN-y classically) and activates macrophage

Non-immune activation: endotoxin (bacterial cell walls), fibronectin...

Activated Macrophage responds to:

1. Tissue Damage

2. Fibrosis

Mechanism occurs in 3 ways:

1. Unlike neutrophils, they can divide and proliferate (mitosis)

2. Recruitment from elsewhere in body
3. Immobilization (via cytokines) to keep the macropages in the tissue

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Macrophage-Lymphocyte interactions in chronic inflammation (cycle diagram)

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Non Macrophage cells present in Chronic Inflammation (4)

Granulomatous Inflammation

Types of Granulomas (2)

1. Lymphocytes
2. Mast Cells
3. Eosinophils
4. Neutrophils (persistent bacterial infections) -> not always

Granulomatous Inflammation:

• Distinctive pattern of chronic inflammation in which the predominant cell type is an activated macrophage with a modified epithelial-like (epithelioid) morphology
• Genesis is linked to cell mediated immune reactions
• Always present in fungal infections, also Tb, Leprosy, Syphilis, Cat scratch disease

Types:

1. Foreign Body (e.g. talc, sutures, fibers large enough to preclude phagocytosis but immunologically inert)

2. Immune (insoluble particles that induce a cell-mediated immune response) -> Ex Tb

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Components of a Granuloma (4)

1. Epitheloid cell (mod macrophage)
2. Lymphocyte
3. Multinucleated giant cells
4. Fibroblasts and connective tissue

[image]

They cause tissue necrosis around where they form

SF115

Tissue Repair with chronic inflammation: Two Distinct Processes

Imptnt Growth Factors

1. Regeneration - replacement of injured cells by cells of the same type leaving no trace of previous injury (only possible when stem cells available)
   
   
2. Replacement by connective tissue (fibroplasia/fibrosis) Replacement of injured cells with scar tissue (CT) -> especially in Hair, Sweat glands

Often both processes are involved

GFs: Note these can be Cytokines, but not always

Fibroblast growth factor (FGF) - new blood vessel formation, recruitment of macrophages fibroblasts and endothelial cells, embryogenesis, hematopoiesis. Granulation Tissue formation to get blood supply to area.

FGF1 and FGF2 stimulate angiogenesis and the proliferation of fibroblasts

FGF7 and FGF10 (Keratinocyte Growth Factors) stimulate repair of skin epithelium and
mucosal tissues

Vascular endothelial growth factor (VEGF) - promote blood vessel formation/angiogenesis

TGF-Beta - can both stimulate or inhibit growth of fibroblasts and smooth muscle cells depending on concentration and stimulates fibroblast secretion of collagen

SF115

Repair by Connective Tissue Steps (4)

Fibrosis Steps involved (3)

Wound Healing Steps (5)
(skipped in lec, may not be imptnt)

CT repair:

1. Formation of new blood vessels (granulation tissue)
2. Migration and proliferation of fibroblasts
3. Deposition of extracellular matrix (collagen -> scar tissue)
4. Maturation and organization of fibrous tissue also called tissue remodeling

Fibrosis:

1. Emigration, proliferation and maturation of fibroblasts
2. Deposition of extracellular matrix (collagen etc) by fibroblasts
3. Net collagen accumulation depends on both synthesis and degradation

Wound Healing Steps:

1. Induction of acute inflammatory response by initial injury
2. Regeneration of parenchymal cells
3. Migration and proliferation of parenchymal and connective tissue cells
4. Synthesis of extracellular matrix proteins
5. Remodelling of connective tissue and parenchymal components
6. Collagenization and acquisition of wound strength

SF061

General principals of tissue healing and repair

Creation of new cells (3 methods)

Some heal well (gut, skin) others don't (heart, brain). Healing depends on:

1. Ability of cells to respond to trophic factors (=Growth Factors)
2. Presence of progenitor (stem) cells
3. Complexity of the organ

Creation of new cells (methods)

1. Continuously cycling (dividing) cells can regenerate well -> e.g. skin, intestine
2. Quiescent cells can re-enter cell cycle when needed; e.g. hepatocytes, fibroblasts, endothelium, astrocytes in brain (GFs cause cells to move from G0
   (resting) phase of cell cycle to G1 phase)
3. Stem cells may be activated to produce new cell populations -> e.g. skeletal muscle
   
   Note: Terminally differentiated non-dividing cells are not replaced -> e.g. cardiac myocytes, neurons (rare exceptions)

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Major Collagen Types (5)

Synthesis of Collagen Steps

Five most common types: 28 in total though
Collagen I: skin, tendon, vascular ligature, organs, bone;
90% of collagen in body
Collagen II: cartilage
Collagen III: reticulate, with type I
Collagen IV: basement membrane
Collagen V: hair and placenta

Synthesis of Collagen Steps (unlikely to be tested):

1. Pre-pro-peptide of collagen is synthesized and inserted into endoplasmic reticulum
2. Cleaved to pro-peptide via:
   - hydroxylation of proline and lysine (Vit C dep.)
   - glycosylation and helix formation (procollagen)
   - transported to Golgi apparatus
3. Procollagen is modified by addition of oligosaccharides then excreted
4. Collagen peptidase removes ends of procollagen, forming tropocollagen
5. Lysyl oxidase produces OH-lysine residues which then undergo covalent bonding to form cross-linked collagen fibril

SF061

Wound healing sequence

Re-epithelialization

Wound healing sequence

Coagulation (blood clotting) - (Hours)

Inflammation (Hours - Days)

Fibroblast migration/proliferation & angiogenesis (Days)
Epithelialization/ECM production/contraction (Days-Wks)
Remodeling/scar (Weeks-months)

Re-epithelialization

• Epidermal covering (keratinocytes) from wound margin and hair follicle remnants (progenitor/stem cell population)
• Migration across wound
• Gradual differentiation
• Skin healing aided by moist environment

Angiogenesis:

• Formation of new blood vessels
• Angiogenic stimuli from macrophages et al.
• Endothelial cell buds progress toward wound following oxygen deficit gradient
• Differentiate into arterioles, capillaries, venules

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Fibroplasia

Tissue Remodeling

Multiple functions of fibroblasts:

• Migration and proliferation influenced by growth factors (PDGF, FGF et al.)
• Extracellular matrix protein production and subsequent remodeling
• Produce growth factors (EGF et al.)
• Support angiogenesis

Tissue Remodeling

• Replacement of granulation tissue with scar tissue
• Involves metalloproteinases, enzymes that degrade extracellular matrix components:
• interstitial collagenases (degrade Types I, II and III)
• gelatinases (degrade Type IV and fibronectin)
• stromelysins (degrade laminin, fibronectin, etc.)
• membrane bound matrix metalloproteinases

SF061

Healing is impaired by... (7)

Pathologic Aspects of Wound Repair

1. Ischemia
2. Dry environment
3. Infection
4. Foreign material
5. Anti-inflammatory therapy (e.g. steroids)
6. Nutritional deficiency (esp. vitamin C)
7. Genetic factors

Chronic wounds occur with impairment of any cellular component, repeated injury, excess inflammation, systemic disease (e.g. diabetes)

Pathologic Aspects of Wound Repair:

• Deficient scar formation (e.g. wound dehiscence, ulceration of wounds in an area of poor blood flow)
• Keloid (hypertrophic scar)
• Contractures (deformities of wound and surrounding tissues, esp. burns)

SF114

Following the phagocytosis of Tb bacilli?

Macrophages  activate T helper cells. This involves the following:

1. presenting processed peptide antigen in class II MHC molecules
2. co-­stimulatory signals to T helper (Th) cells
3. secreting IL-­12, which promotes the secretion of interferon gamma (IFN-­γ).
   
   This leads to the development of a Th1-­mediated immune response, which contributes to tissue injury (hence “hypersensitivity”).

Macrophages can produce bactericidal molecules that may kill the intracellular bacilli.

Examples include tumor necrosis factor alpha (TNFα), nitric oxide and active oxygen intermediates. These factors also contribute to tissue injury in the lung.

IFN-­γ secreted by Th1 cells activates macrophages to do this. Activated macrophages secrete chemokines, which recruit other monocytes to the area. When the antigen persists, macrophages differentiate to form epithelioid cells and multinucleated giant cells, which are present within granulomas. Note that Granulomas also contain sensiitzed T cells.

SF114

What is the Mantoux test?

Tuberculin test (also known as a Mantoux test):
A positive reaction is the result of a recall immune response to a soluble antigen. The antigen is tuberculin, a mixture of antigens from a culture of tubercle bacilli.
This bump develops as a result of the response of Th cells and macrophages to the soluble antigen.

It is an example of a delayed type hypersensitivity (DTH) reaction. It does not include granuloma formation because that would require persistent antigen (as in the case of an actual tuberculosis infection).

SF141

Barotrauma (2 main types)

Neurogenic shock vs Hemorrhagic shock

1. Local high pressure trauma (blast injury)
2. Altitude-related illness

Neocurogenic shock results in hypotension, this is attributed to the disruption of the autonomic pathways within the spinal cord.

In more simple terms: the trauma causes a sudden loss of background sympathetic stimulation to the blood vessels. This causes them to relax (vasodilation) resulting in a sudden decrease in blood pressure.

Hemorrhagic shock is a condition of reduced tissue perfusion, resulting in the inadequate delivery of oxygen and nutrients that are necessary for cellular function.

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Types of Injuries

1. Abrasion = Graze/Scratch = superficial
   
   
2. Contusion = Bruise
   = Collection of blood in tissues due to rupture of vein, venule, and small arteries
   
   
3. Laceration = Cut/Tear
   Full thickness tear of skin where basal tissue strands/bridges, nerves, tendons affected. Hair is intact.
   
   
4. Incised Wound = Cut
   Clean margin with sharp edges
   Could cut nerves, tendons and tissues
   
   
5. Stab Wound
   
   
6. Fracture - compound is through skin

[image]