Session 1 - Cell injury and death

Question 1

What is disease?

Answer 1

Disease: Failed homeostasis. Pathological condition of a body part, an organ, or a system characteristed by an identifiable group of signs or symptoms.

Question 2

Histology vesus cytology - give examples and pros and cons of each

Answer 2

Question 3

How does a histopathologist arrive at a diagnosis?

Answer 3

• Normal or not?
• Inflammatory or neoplastic?
• Benign or malignant?
• Primary or secondary site?

Question 4

Fixation process

• Why is it necessary?

Frozen sections

Answer 4

OFESS

Obtain
Fixation: use formalin (formaldegyde in water), usually fixes in 24-48 hrs
Then cut out the tissue, place in casettes
Embed: first, need to remove the water from the tissue, dehydrate using alcohol, then replace alcohol with xylene which can mix with wax, then add paraffin wax (hardening agent)
Section: (microtomy) cut into very thin slices using microtome
Stain: usually H+E stain (haematoxylin stain nuclei purple and eosin stain cytoplasm and connective tissue pink)
Mounting: Mounting medium applied, coverslip added on top, mouting medium dries and hardens, preserving the tissue and attaching the coverslip

Necessary:
Autolysis - the cell and tissue architecture will be destroyed. Role of fixatives is: inactivate tissue enzymes and denature proteins, prevent bacterial growth, harden tissue

Frozen sections:
Urgent, method of hardening tissue quickly, intra-operative, 10-15 mins, accuracy is 96%
Rapidly frozen -> sliced -> stained

Question 5

Immunohistochemistry

Answer 5

Any substance that is antigenic can be demonstrated by labelling them with specific antibodies. Antibody is joined to an enzyme that catalyses a colour-producing reaction.

• actin
• cadherins
• hormone receptors
• microorganisms e.g. HPV
• her2 receptor (growth factor receptors)
• cytoketatin e.g. can give info about primary site of a carcinoma… CK7+/CK20- lung, breast, endometrium…

Question 6

Molecular pathology

Answer 6

Studies how diseases are caused by alterations in normal cellular molecular biology, e.g. can be seen by FISH. Sequencing of DNA purified from tumour tissue can show if a mutation is present in a particular gene

Question 7

Cell injury

1. Degree of injury depends on…
2. Spectrum of change…
3. Example of cell/tissue respose to injury…

Answer 7

Degree of injury depends on:
Type of injury, severity of injury, duration of injury, type of tissue

Spectrum of change:
homeostasis, cellular adaptation, cell injury, cell death
reversible irreversible

Example of cell/tissue respose to injury:
cardiac myocytes and their response to hypertension.. increased workload on the heart, hypertrophy, increased weight and size of heart, if workload is not reduced and additional stress, leads to cell injury and death (myocardial infarction)

Question 8

Causes of cell injury
1. Brief list

2. Hypoxia - all the different types
3. List some toxins
4. How does the immune system damage body’s cells?

Answer 8

Brief list: Hypoxia, toxins, physical agents (e.g. trauma, temperature extremes), radiation, micro-organisms, nutitional deficiencies)

Hypoxia:
- Hypoxaemic - arterial content of oxygen is low…
reduced inspired p0₂ at altitude, reduced absorption secondary to lung
disease, pneumonia etc
- Anaemic - decreased ability of hb to carry oxygen… anaemia, CO poisoning
- Histiotoxic - inability to utilise oxygen in cells due to disabled oxidative phosphorylation enzymes… cyanide poisoning
- Ischaemic - interruption to blood supply… blockage of a vessel, heart failure (global low blood pressure)

Toxins: poisons, pollutants, herbicides, alcohol, medicines…

Immune system: Hypersensitivity reactions (host tissue is injured secondary to an overly vigorous immune reaction e.g. hives), autoimmune reactions (immune system fails to distinguish self from non-self)

Question 9

How does an injured cell deal with cell injury?
1. Most susceptible components

2. Consequences of reduced ATP during cell injury (..hypoxia)
3. Consequences of influx on Ca2+

Answer 9

1. Cell membrane. nucleus, protein, mitochondria
2. See attached STILL REVERSIBLE AT THIS STAGE
3. See attached IRREVERSIBLE

Question 10

Free radicals
What are they? Examples? Formation?

What can free radicals damage?

What is oxidative stress?

Body control of free radicals?

Answer 10

Free radicals - Reactive oxygen species, single unpaired electron in very outer orbit, unstable configuration, very reactive
Examples: OH (hydroxyl), O2- (superoxide), H2O2 (hydrogen peroxide)
Formation: Normal metabolic reactions e.g. oxidative phosphorylation, radiation, transistion metals, drugs and chemicals, normal part of inflammatory response (oxidative burst of neutrophils)

Damage: Lipids, proteins, nucleic acid

Oxidative stress: Imbalance between free radical generation and free radical scavenging systems

Control systems include:
- Decay spontaneously, anti-oxidants (donate electrons to the free radicals, vitamins A, C, E), enzymes that neutralise free radicals (glutathione peroxidase, catalase, superoxide dismutase), heat shock proteins (these mend mis-folded proteins and maintain cell viability)

Question 11

Definitions of cellular dying and cell death

Answer 11

Oncosis: cell death with swelling, the spectrum of changes that occur in injured cells prior to death

Apoptosis: Programmed cell death

Necrosis: In a living organism the morphological changes that occur after a cell has been dead for some time (seen after 12-24 hours)

• Coagulative
• Liquefactive
• Caseous
• Fat necrosis
• Fibrinoid necrosis

Question 12

Light microscope - changes that can be seen

Electron microscope - Reversible injury, irresersible injury

Answer 12

Light:
Pyknosis - shrinking of nucleus and chromatin
Karyorrhexis - fragmentation
Karyologysis - complete disolution of the nucleus

Electron:
Reversible -
Blebbing; ribosome detach; swelling: generalised, mitochondria, endoplasmic reticulum; clumping of chromatin in nucleus
Irreversible -
Nuclear changes: pyknosis, karyorrhexis, karyolysis; rupture of lysosomes; lysis of ER, defects of cell membrane

Question 13

Types of necrosis

• Coagulative necrosis
• Liquefactive necrosis

Answer 13

Necrosis is the change in appearance seen when there has been cell death. It takes several hours for microscopic changes are visible. Necrotic tissues are enzymatically broken down and then phagocytosed by white blood cells.

There are two main types of necrosis:

• Coagulative necrosis: Occurs in solid organs such as the liver. The appearance in coagulative necrosis is due to denatured proteins coagulating. Under a light microscope you can see the ‘ghost outline’ of the cells in the tissue
• Liquefactive necrosis: Happens in loose and soft tissues such as the lungs and the brain. The appearance is due to autolysed proteins. No evidence of cells is visible under the microscope, only debris.

Question 14

Types of necrosis cont.

• Caseous necrosis
• Fat necrosis

Answer 14

- Caseous necrosis

• Caseous Necrosis (COVERED WHEN TALKING ABOUT CASEOUS NECROSIS GRANALOMATA’S)
  
  • A form of cell death in which the tissue has a cheese-like appearance, seen under the microscope as a mix of coagulative and liquefactive states. This is seen in certain diseases such as tuberculosis.
• Fat necrosis
• Fat necrosis is characterised by the action of digestive enzymes on fat released from adipocytes. In fat necrosis the enzyme lipase releases fatty acids from triglycerides. The fatty acids then complex with calcium to form soaps. These soaps appear as white chalky deposits.

Question 15

Summary of different necrosis

Answer 15

• when/where does it take place?
• microscopically what do you see?

Question 16

Apoptosis

1.

• What is this?
• Describe it’s overarching characteristics

2.
- Two types of apoptosis and when these occur

Answer 16

1.

• Apoptosis is programmed cell death with shrinkage (cell suicide)
• Non-random (whereas oncosis and necrosis - chopped up into random pieces)
• Regulated intracellular program where a cell activates enzymes that degrades it’s own nuclear DNA and proteins

2. Physiological apoptosis - to maintain a steady state (e.g. bone marrow, removed when no longer needed), embryologenesis (e.g. synovial joints, developing digits on the hand)
   Pathological apoptosis - Occurs when a cell is damaged, particularly DNA. Cytotoxic T cell killing of virus-infected or neoplastic cells, graft versus host diseases, cells are damaged

Question 17

Stages of Apoptosis (list)

Describe the two pathways for initiation

Answer 17

1. Initiation and execution (triggered by intrinsic or extrinsic pathway) - results in the activation of capases, when these enzymes then act they are called ‘executioners’. Capases enzymes control and mediate apoptosis -> cause cleavage of DNA and cleavage of protein cytoskeleton into membrane bound apoptotic bodies
2. Degradation: Condensation and fragmentation
3. Apoptotic body (the apoptotic bodies express proteins on their surface, they can now be recognised by phagocytes or neighbouring cells)
4. Phagocytosis

• *Intrinsic pathway** - intitating signals comes from within the cell, e.g. due to irreparable DNA damage, withdrawal of growth factors or hormones
• *Extrinsic pathway** - initiated by extracellular signals, e.g. due to tumour cells, virus-infected cells
• • Both intrinsic and extrinsic pathways cause the cells to shrink and break up into apoptotic bodies

Question 18

Comparisons of Apoptosis versus Oncosis

Answer 18

Question 19

Define:

• Gangrene
• Infarction
• Infarct

Answer 19

Gangrene - necrosis VISIBLE to the naked eye (an appearance of necrosis)

Infarction - necrosis caused by reduction in arterial blood flow (a cause of necrosis, can result in gangrene)

Infarct - An area of necrotic tissue which is the result of loss of arterial blood supply (an area ischaemic necrosis)

Question 20

Gangrene
- types

Infarction
- what is it?

Answer 20

Types of gangrene:

• Dry gangrene (necrosis, exposure to the air, coagulative necrosis)
• Wet gangrene (necrosis infection, lots of bacteria, liquefactive necrosis)

Infarction:
Reduction in arterial blood flow e.g. artherosclerosis, occlusion by thrombus, twisting e.g. testes, compression
…heart, brain, lungs, kidneys, limbs, testicles, gastrointestinal system

Question 21

Infarction
- types of infarct

• complications of infarction

Answer 21

White infarct/anaemic infarct:
• ‘Solid organs’ e.g. spleen / kidney / heart • Occlusion of an end artery • Often wedge-shaped • Microscopically you see coagulative necrosis

Red infarct/haemrrhagic infarct:
• = haemorrhagic infarct • Loose tissue e.g. lung / bowel • Dual blood supply (the secondary blood supply is insufficient to rescue the tissue, but will allow some blood to enter this space - creating red area of infarction) • Numerous anastomoses • Prior congestion • Raised venous pressure • Re-perfusion

Complications of infarction:
Ranges from none to death
Depends on: Alternative blood supply, speed of ischaemia, tissue involved, oxygen content of the blood

Ischaemic-reperfusion injury

Question 22

Complications of infarction

• Ranges from
• Depends on

What is ischaemia-reperfusion injury?

Answer 22

Range: None to death

Depends on:

• Alternate blood supply e.g. collateral blood supply
• Speed of ischaemia
• Tissue involved (heart/brain i.e. vital organs)
• Oxygen content of the blood

Ischaemia-reperfusion injury:

• Increased production of oxygen free radicals with reoxygenation.
• Increased number of neutrophils resulting in more inflammation and increased tissue injury.
• Delivery of complement proteins and activation of the complement pathway

Question 23

1. Cell injury can lead to -> _____ cellular accumulations
   - Occurs when…
   - Accumulations can be…
2. Mechanisms of intracellular accumulations

Answer 23

1.
ABNORMAL

Occurs when: there is deranged metabolism, e.g. if a cell can’t metabolise something, it will remain WITHIN the cell

Accumulations can be:

• Normal cell components - build up to excessive levels e.g. water, lipids, proteins, carbohydrates
• Abnormal components - endogenous e.g. product produced by dysfunctional synthesis or metabolism, exogenous e.g. minerals, infections
• Pigment

2. • Abnormal metabolism
     - Alterations in protein folding and transport
     - Deficiency of critical enzymes
     - Inability to degrade phagocytosed particles

Question 24

Types of cellular accumulations:
- Fluid/water
causes, name when entire cell is swollen

• Lipids - Triglycerides
  name of a fatty change, caused by…, complications
• Cholesterol
  quick explanation, disease processes
• Proteins
  microscopically what does this look like, diseases associated
• Accumulation of pigment
  coloured substance, exogenous list, how got into lungs

Answer 24

Fluid/water:
Severe cellular distress e.g. hypoxia
Osmotic disturbance
HYDROPHIC SWELLINGS (entire cell swollen)

Lipids/Triglycerides:
Steatosis (fatty change) = accumulation of triglycerides
Commonly seen in liver
Caused by: Chronic alcohol misuse, obsesity, diabetes mellitus
Complications: Liver failure, Liver cirrhosis, Liver and metabolic dysfunction

Cholesterol:
• Insoluble and can’t be broken down in the body • Eliminated through the liver • If cant, its stored in intracellular vesicles. • Caused by: (see semester 1 modules) Mutations in LDL- R, abnormalities in apoproteins and lipoproteins.
• Disease processes: Atherosclerosis – cholesterol builds up in smooth muscle cells and macrophages / histiocytes (foam cells) = atherosclerotic plaque (see session 6). Xanthoma – cholesterol in macrophages within skin/ tendons, associated with Hereditary hyperlipidaemias. Inflammation and necrosis – site of cell injury due to phagocytosis of cells Cholesterolosis – accumulation of cholesterol laden macrophages in the gallbladder Niemann Pick disease type C (lysosomal storage disease)

• *Proteins:
• ** Intracellular accumulation leads to eosinophillic (pink) droplets
• Diseases: Alcoholic liver disease (Mallory’s hyaline), alpha-1-antitrypsin deficiency (liver produces incorrectly folded alpha-1 antitrypsin proteins (a protease inhibitor), cannot be packaged by ER, accumulate within ER and is not secreted, systemic deficiency - proteases in lung act unchecked resulting in emphysema
• *Accumulation of pigment:**
• Coloured substance = pigment
• Exogenous - carbon/soot, dust, coal
• Carbon/soot inhaled into lungs, phagocytosed by alveolar macrophages, see black pigment on the surface and within the lungs and draining lymph nodes

Question 25

Types of cellular accumulations:
- Tattooing

• Haemosiderin
• Bilirubin

Answer 25

Tattooing
Tattooing - pigments picked into skin
Phagocytosed by macrophages in the dermis and remain there.
Some pigment will reach the draining lymph nodes

Haemosiderin
Iron storage molecule
Derived from haemoglobin
Yellow/brown
Local excess of iron e.g. bruise
Systemic excess/overload of iron = haemosiderosis

Bilirubin
RBC/proteins contain Haem (cytochromes), broken down to:
Biliverdin -> Bilirubin (yellow)
Binds to albumin and transported to the liver.
Conjugated in liver
Excreted from liver in bile:
(1) Gut - bacteria - stercobilinogen - faeces its colour
(2) Blood stream - urobilinogen - urine
If bile flow obstructed / overwhelmed: Jaundice Bilirubin in blood rises Deposited in tissues extracellularly or in macrophages
TOXIC

Question 26

Cell injury can lead to permeable (leaky) membranes

• Local and systemic effects
• Three important molecules that often accumulate

Answer 26

Local and systemic effects:

• Can cause local inflammation
• General toxic effects on body
• May appear in high concentrations in blood and can aid in diagnosis

Three important molecules:
Potassium, Enzymes, Myoglobin

Question 27

Molecules that accumulate:
- Potassium

• Enzymes
• Myoglobin

Answer 27

• Potassium: look at diagram to see how it accumulates
• Enzymes: Can indicate the organ involved i.e. cellular damaged from that organ. Clinically: Cardiac enzymes and Liver enzymes (e.g. transaminases, ALK) Indicator of cardiac or liver damage
• Myoglobin: • Released from dead myocardium and striated muscle. • If excessive amounts (rhabdomyolysis)– damages the kidneys by plugging up the renal tubules. • Results in acute renal failure/injury

Question 28

Calcium accumulation

Answer 28

• Calcium:
• *Localised - Localised in dying tissue (dystrophic)** Most common e.g. atheromatous plaques, aged/damaged heart valve, tuberculous lymph node, malignancy etc. Nothing to do with calcium metabolism

Generalised (metastatic) - Deposition in otherwise normal tissue Due to hypercalcaemia (elevated calcium) due to dysfunction in calcium disturbance.
•Hypercalcaemia secondary to disturbances in calcium metabolism •Hydroxyapatite crystals are deposited in normal tissues throughout the body •Usually asymptomatic but it can be lethal •Can regress if the cause of hypercalcaemia is corrected • Causes: Increased secretion of parathyroid hormone (PTH) resulting in bone resorption: • Primary hyperparathyroidism- due to parathyroid hyperplasia or tumour (adenoma) • Secondary hyperparathyroidism– due to renal failure and the retention of phosphate (low calcium therefore induce PTH) • Tertiary hyperparathyroidism i.r. ectopic production - secretion of PTHrelated protein by malignant tumours (e.g., carcinoma of the lung) Destruction of bone tissue: • Primary tumours of bone marrow, e.g., leukaemia, multiple myeloma • Diffuse skeletal metastases • Paget’s disease of bone – when accelerated bone turnover occurs • Immobilisation