Cell injury and introduction to autopsy Flashcards
Give another term for autopsy.
Post-mortem
What is a Coroner?
Most autopsies performed in the UK are undertaken on behalf of HM Coroner. The Coroner is a judicial officer employed by a local authority and is responsible for
investigating certain categories of death.
What categories of death would be investigated by the coroner?
- Deceased unknown
- Deceased not seen by a doctor within 14 days of death
- Attending doctor not able to give cause of death
- Obviously unnatural death (murder, accident, suicide)
- Death related to occupational disease or accident
- Death related to medical treatment or procedure
Why do autopsies on behalf of HM coroner not require the consent of family members?
Autopsies order by HM Coroner are a legal requirement.
What are consent or hospital autopsies?
Consent or hospital autopsies are conducted with consent from the next of kin or other ‘qualifying persons’ as determined by the Human Tissue Act. These examinations may be undertaken to determine the cause of death but are also performed in order to determine extent of disease or effects of treatment. These autopsies may be limited to part of the body or an organ system by the family. On some occasions needle core samples may be all that is permitted.
What do most autopsies include?
Most autopsies will include a full external and internal examination with dissection of the major organ systems. The naked eye appearances may be sufficient to provide a cause of death. However, in some cases additional investigations are required in order to determine the cause of death, e.g. histology, toxicology, biochemistry or microbiology.
What may post-mortem radiology be used for?
There is increasing use of imaging as an adjunct to the autopsy and some Coroners will now accept a cause of death based only on imaging. The techniques are not,
however, fully validated and research is continuing.
Which pathologists perform which type of autopsy?
Most routine Coroner’s autopsies are performed by histopathologists working in the NHS but they are currently not part of routine NHS work. Some categories of death, particularly suspicious deaths, are dealt with by forensic pathologists.
Perinatal and paediatric autopsies are performed by paediatric pathologists.
How are autopsies regulated?
All autopsies must be conducted in premises licensed by the Human Tissue Authority, which also regulates the retention and disposal of samples taken at autopsy.
What is diagnostic pathology?
Diagnostic pathology involves studying the structural and functional alterations in cells and tissues e.g., by microscopy, in order to arrive at a diagnosis.
Why is diagnostic pathology used clinically?
The morphological changes in cells and tissues and their
distribution within an organ results in the symptoms and signs of disease.
All disease starts with molecular or structural alterations in cells and this is why it is important to understand what injures cells and the processes that occur in cell injury.
Why might cells undergo morphological and physiological adaptations?
To overcome more severe environmental changes in order to remain viable.
Cells may respond with an increase or decrease in activity (hyperplasia or atrophy).
What happens when cells reach the limits of their adaptive response?
When cells reach the limits of their adaptive response they may show evidence of reversible injury or become irreversibly injured and die. The degree of cell damage depends on the type, duration and severity of an injury and the type of tissue that is involved.
In disease, where does the abnormality ultimately lie?
In the cell.
What are the causes of cell injury?
Hypoxia Physical agents Chemical agents/ drugs Microorganisms Immune mechanisms Dietary insufficiency/ deficiencies/ dietary excess Genetic abnormalities
What can the causes of hypoxia be classified as?
-Hypoxaemic: arterial content of oxygen is low, e.g., reduced inspired pO2 at altitude
-Anaemic: decreased ability of haemoglobin to carry oxygen, e.g., anaemia, carbon monoxide poisoning
-Ischaemic: interruption to blood supply, e.g., blockage of a
vessel, heart failure
-Histiocytic: inability to utilise oxygen in cells due to disabled oxidative phosphorylation enzymes, e.g., cyanide poisoning.
What is ischaemia?
Loss of blood supply due to reduced arterial supply (e.g., obstruction of an artery, hypotension) or reduced venous drainage. This causes a reduced supply of oxygen and metabolic substrates, e.g., glucose for glycolysis, and the resultant injury therefore occurs more rapidly and is more severe than that seen with hypoxia.
How do cells react to hypoxia?
Hypoxia results in decreased aerobic oxidative respiration (although glycolytic energy production can continue) which if persistent will cause cell adaptation (e.g., atrophy), cell injury or cell death. It is a very common and important
cause of cell injury and cell death. The length of time that a cell can tolerate hypoxia varies; some neurones can only tolerate a few minutes while dermal fibroblasts can tolerate a number of hours.
Give examples of physical agents that can cause cell injury.
Direct trauma, extremes of temperature (burns and severe cold), sudden changes in atmospheric pressure, electric
currents, radiation.
Give examples of chemical agents that can cause cell injury.
Glucose or salt in hypertonic solutions, oxygen in high concentrations, poisons, insecticides, herbicides, asbestos, alcohol, illicit drugs, therapeutic drugs.
Which microorganisms can cause cell injury?
Viruses, bacteria, fungi, other parasites.
How do immune mechanisms cause cell injury?
These cause injury principally by two mechanisms; hypersensitivity reactions where the host tissue is injured secondary to an overly vigorous immune reaction, e.g.,
urticaria (= hives) and autoimmune reactions where the immune system fails to distinguish self from non-self, e.g., Grave’s disease of the thyroid.
Give an example of genetic abnormality that can result in cell injury.
Inborn errors of metabolism
How many cell components are the principal targets of cell injury and what are they?
There are four essential cell components that are the principal targets of cell injury:
- Cell membranes – the plasma membrane, effectively the skin of the cell, which plays an essential role in homeostasis and the organellar membranes which compartmentalise organelles such as lysosomes (particularly important as they contain potent enzymes that can themselves cause cell damage).
- Nucleus which contains the genetic material of the cell.
- Proteins – the structural proteins forming the cytoskeleton and enzymes involved in the metabolic processes of the cell.
- Mitochondria where oxidative phosphorylation and production of ATP occurs.
What are the most common causes of cell injury? Briefly, what do they do to the cell?
- Hypoxia and ischaemia are the most common cause of cell injury and as it is relatively straightforward to induce hypoxia experimentally, cell injury during hypoxia has been studied in detail. Many of the changes seen in hypoxia also occur with other causes of cell injury although the sequence of events might be different.
- As the cell becomes deprived of oxygen there is decreased production of ATP by oxidative phosphorylation in mitochondria. When the levels of ATP in the cell drop to less than 5-10% of normal concentrations vital cellular functions become compromised.
How do cellular functions become compromised during reversible hypoxia?
- There is loss of activity of the Na+/K+ plasma membrane pump (which is energy dependent). As the intracellular concentration of Na+ rises water enters the cell and the cell and its organelles swell up (oncosis). Ca++ also enters the cell and this results in damage to cell components.
- With the lack of oxygen the cell switches to the glycolytic pathway of ATP production. This results in the accumulation of lactic acid which reduces the pH within the cell. The low pH affects the activity of many enzymes within the cell. Chromatin clumping is seen.
- Ribosomes detach from the endoplasmic reticulum (as energy is required to keep them attached) and protein synthesis is disrupted. This can result in intracellular accumulations of substances such as fat and denatured proteins.
When does hypoxia become irreversible?
At some point, which is not well understood, the injury becomes irreversible and the cell will eventually die. Most cells in hypoxia die by oncosis and eventually the tissue will appear necrotic. It is not clear what actually kills the cell but a key event is the development of profound disturbances in membrane integrity and therefore an increase in membrane permeability followed by a massive influx of Ca++ into the cytoplasm.
What is the role of calcium in irreversible hypoxia?
Cytosolic free Ca++ is usually at very low levels as it is kept within mitochondria and the endoplasmic reticulum. When cells are severely damaged, Ca++ enters the cell from outside across the damaged plasma membrane and is released from stores in the endoplasmic reticulum and
mitochondria. This is important because calcium ions are biologically very active and high concentrations within the cytoplasm results in the activation of an array of potent enzymes such as ATPases (decrease the concentrations
of ATP further), phospholipases (cause further membrane damage), proteases (breakdown membranes and cytoskeletal proteins) and endonucleases (damage DNA). When lysosomal membranes are damaged their enzymes leak into the cytoplasm further damaging the cell.
What happens to cellular contents during irreversible damage?
Whilst Ca++ enters cells whose membranes are irreversibly damaged, intracellular substances leak out into the circulation. These can be detected in blood samples and the type of substances detected indicates where the
cellular damage is occurring, e.g., if liver cells are injured transaminases will be detected in the blood.
What are the 14 steps of hypoxic injury?
- Cell is deprived of oxygen.
- Mitochondrial ATP production stops.
- The ATP-driven membrane ionic pumps run down.
- Sodium and water seep into the cell.
- The cell swells, and the plasma membrane is stretched.
- Glycolysis enables the cell to limp on for a while.
- The cell initiates a heat-shock (stress) response (see below), which will probably not be able to cope if the hypoxia persists.
- The pH drops as cells produce energy by glycolysis and lactic acid accumulates.
- Calcium enters the cell.
- Calcium activates:
• phospholipases, causing cell membranes to lose phospholipid,
• proteases, damaging cytoskeletal structures and attacking
membrane proteins,
• ATPase, causing more loss of ATP,
• endonucleases, causing the nuclear chromatin to clump. - The ER and other organelles swell.
- Enzymes leak out of lysosomes and these enzymes attack cytoplasmic components.
- All cell membranes are damaged and start to show blebbing.
- At some point the cell dies, possibly killed by the burst of a bleb.
What is ischaemia-reperfusion injury?
If blood flow is returned to a tissue which has been subject to ischaemia but isn’t yet necrotic, sometimes the tissue injury that is then sustained is worse than if blood flow was not restored.
What can cause iscaemia-reperfusion injury?
- Increased production of oxygen free radicals (see below) with reoxygenation.
- Increased number of neutrophils following reinstatement of blood supply resulting in more inflammation and increased tissue injury.
- Delivery of complement proteins and activation of the complement pathway.
How do chemicals cause cell injury?
Some chemicals act by combining with a cellular component, e.g., cyanide binds to mitochondrial cytochrome oxidase and blocks oxidative phosphorylation
What are free radicals?
Free radicals are reactive oxygen species. They have a single unpaired electron in an outer orbit. This is an unstable configuration and because of this free radicals react with other molecules, often producing further free
radicals.
How are free radicals produced?
Free radicals are particularly produced in chemical and radiation injury, ischaemia-reperfusion injury, cellular aging, and at high oxygen concentrations.
Some are produced by leucocytes in the body, which are used physiologically.
What do free radicals do?
They attack lipids in cell membranes and cause lipid
peroxidation. They can also damage proteins, carbohydrates and nucleic acids. These molecules become bent out of shape, broken or cross-linked.
Free radicals are also known to be mutagenic.
Free radicals are also involved in many pathological events but they are also involved in many physiologic events – we could not live without free radicals. They are produced by leucocytes and used for killing bacteria and are also used in cell signalling.
Which free radicals are of biological significance in cells?
Three free radicals are of particular biological significance in cells: OH• (hydroxyl- the most dangerous), 02- (superoxide) and H202 (hydrogen peroxide).
How can hydroxyl free radicals be formed?
OH• can be formed in a number of ways:
• Radiation can directly lyse water → OH•
• The Fenton and Haber-Weiss reactions produce OH•.
The Fenton reaction is important in injury where bleeding occurs as when blood is around iron is available for the production of free radicals.
Why is it important to rapidly remove superoxide and hydrogen peroxide free radicals?
H202 and 02- are substrates for the Fenton and Haber-Weiss reactions. This is one reason why it is important to rapidly remove 02- and H202 so that the more dangerous OH• cannot be generated.
What is the Fenton reaction?
Fe2+ + H2O2 → Fe3+ + OH- + OH•
What is the Haber-Weiss reaction?
O2- + H+ + H2O2 → O2 + H2O + OH•
How does the body defend itself against free radicals?
The body has defence systems to prevent injury caused by free radicals.
These are known as the anti-oxidant system.
If there is an imbalance between free radical production and free radical scavenging, free radicals build up and the cell or tissue is said to be in oxidative stress. This causes
cell injury.
What does the antioxidant system consist of?
Enzymes, free radical scavengers and storage proteins.
What role do enzymes play in the antioxidant system?
o Superoxide dismutase (SOD) catalyses the reaction
O2- →H202. H2O2 is significantly less toxic to cells.
o Catalases and peroxidases complete the process of free
radical removal: H202 → 02 + H20.
What role do free radical scavengers play in the antioxidant system?
Free radical scavengers neutralise free radicals. Vitamins A, C and E and glutathione are free radical scavengers.
What role do storage proteins play in the antioxidant system?
Storage proteins sequester transition metals in the extracellular matrix. Transferrin and ceruloplasmin sequester iron and copper, which catalyse the formation of free radicals.
What are heat shock proteins? Give an example.
Heat shock proteins are stress proteins, unfoldases and chaperonins.
They are present in lower concentrations in unstressed cells.
In stressed cells, heat shock proteins are a mechanism used by the cell to protect itself against the effects of injury.
An example of a heat shock protein is ubiquitin.
How is heat shock triggered?
- Heat shock or the cellular stress response is triggered by any form of injury, not just heat.
- All cells from any organism when submitted to stress turn down their usual protein synthesis and turn up synthesis of HSPs.
What do heat shock proteins do?
- As all organisms utilise HSPs they must play a key role in survival.
- HSPs aren’t secreted, they remain within the cell where they are concerned with protein repair (analogous to DNA repair).
- They are important when the folding step in protein synthesis goes astray or when proteins become denatured during cell injury.
- They recognise proteins that are incorrectly folded and repair them by ensuring they are refolded correctly. If this isn’t possible then the mis-folded protein is destroyed.
- HSPs are important in cell injury, as the heat shock response plays a key role in maintaining protein viability and thus maximising cell survival.
Which morphological changes to cells from oncosis can be seen under a light microscope?
- Cytoplasmic changes – there is reduced pink staining of the cytoplasm due to accumulation of water (reversible change). This may be followed by increased pink staining due to detachment and loss of ribosomes from the endoplasmic reticulum and accumulation of denatured proteins (irreversible change).
- Nuclear changes - chromatin is subtly clumped (reversible change). This may be followed by various combinations of pyknosis, karryohexis and karryolysis of the nucleus (irreversible change).
- Abnormal intracellular accumulations
What is pyknosis?
Shrinkage of the nucleus
What is karryohexis?
Fragmentation of the nucleus
What is karryolysis?
Dissolution of the nucleus
Can histological sections give time of death of cells?
No. It is hard to identify cells that died minutes to hours ago and hard to distinguish reversible injury from recent death. There is also little to be seen by the naked eye around the time of cell death.
How is time of cell death determined?
The diagnosis of cell death is probably best made on functional rather than morphologic criteria, e.g., increased
permeability of the cell membrane. This can be assessed by the dye exclusion technique where dye is put into the cells’ medium. If it doesn’t enter the cell, the cell is alive. If they soak it up they are dead.
What is oncosis?
Cell death with swelling, the spectrum of changes that occur prior to death in cells injured by hypoxia and some other agents.
What is the main cause of oncosis?
Hypoxia/ ischaemia
Summarise cell swelling.
In summary, cell swelling is due to the failure of ionic pumps in the cell membrane through lack of an energy supply. Therefore as well as occurring with hypoxia oncosis would also be expected to occur with poisons that interfere with a cell’s energy metabolism or the integrity of the cell membrane. As cells undergo oncosis they (and the tissue as a whole) increases in weight.
