CELLULAR INJURY

NECROSIS &APOPTOSIS
DR OMONIYI-ESAN G.O

INTRODUCTION
The cell is the basic living unit of an
organism.
A living cell must maintain an organization
capable of producing energy.
Thus the most pressing need for a living cell
is to establish a structural and functional
barrier between its internal milieu and an
hostile environment. i.e. it must maintain a
normal homeostasis

Cell injury
However, in the presence of a severe
adverse condition, cellular function
becomes abnormal.
Subsequently, the structure of the cell may
also become abnormal, and cell injury is
said to have occurred.

Response of cell to injury.

This depends on the following
Type, duration and severity of injury
Type , state and adaptability of the injured cell

The response varies from

Cellular adaptation
Intracellular accumulations
Sub-lethal alterations
Reversible injury (Cell swelling and fatty change)
to
Irreversible injury (or cell death)

Causes of cell injury

Hypoxia: Loss of blood supply, anaemia
,air starvation, metabolic poison
Physical agents: heat cold, trauma,
radiation
Chemical agents: poisons,hyperbaric
O2,excess glucose
Infectious agents: microbial agents and
the effects of toxins e.g viruses,
bacteria,parasites,fungi etc

Causes of cell injury

Immunologic reactions: Ag-Ab rxn
hypersensitivity
Genetic derangements: enzyme defects
e.g Down syndrome,
Nutritional imbalances : starvation, obesity

Cellular adaptation.

Hypertrophy
Atrophy
Metaplasia
Hyperplasia
Dysplasia

Biochemical mechanism of cell

injury
Whatever the cause of injury, there are five
main foci of biochemical mechanisms
affected in the cell.
Decreased generation of ATP.
Increased production of free radicals
Intracellular accumulation of calcium and
loss of calcium homoeostasis
Defects in membrane permeability
Mitochondrial dysfunction and damage

Loss of ATP generation

ATP source of energy for many synthetic and
degradative processes within the cell such as
membrane transport, protein synthesis, lipogenesis
, and deacylation-reacylation reactions for phospholipid
turnover

ATP is generated in two ways

Oxidative phosphorylation of ADP in mitochondria
Gylcolytic pathway in the absence of oxygen

ATP DEPLETION
Tissues that have a greater gycolytic pathway
such as liver are at an advantage when level of
ATP is failing due to an inhibition of the oxidative
metabolism by injury
ATP depletion is a common consequence of
ischemic and toxic injury such as cyanide
poisoning which inactivates cytochrome oxidase,
fluoroacetate which interferes with citric acid
cycle.

Generation of reactive oxygen

species.
Some amount of partially reduced reactive oxygen
species are produced during the process of
generating energy in the cell by reduction of
molecular oxygen to water.
Under normal conditions, only small amounts are
produced, and the body has the ability to prevent
injury.
An imbalance in the amount results in oxidative
stress, a condition associated with cell injury

 Reactive oxygen species plays a critical

role in
ischemia,
radiation,
inflammation,
oxygen toxicity,
chemicals,
reperfusion injury

Intracellular accumulation of
calcium and loss of calcium
homeostasis
Levels of cytosolic calcium are usually maintained at low
levels < 0.1mol and sequestered in the mitochondrion
and ER. (extra cellular calcium level is 1.3mmol ).
Plasma level 2.25-2.75mmol/L.
These gradients are maintained by the energy
dependent Ca+,Mg2+- ATPases.
Increase in cytosolic calcium result when there is
decreased ATP production such as occurs in ischemia
and with some toxins.

 Increased calcium activates the following

enzymes
phoshpholipases,
proteases,
ATPases,
endonucleases.

Defects in cell membrane

permeability.
Early loss of cell membrane permeability is a
consistent feature in cell injury
The defects may arise from a series of events
such as
ATP depletion and
calcium modulated activity of enzymes

This type of damage affects all membrane

systems in the cell

CELL MEMBRANE DAMAGE

Plasma membrane damage can also
occur directly by
action of bacteria toxins,
viral proteins,
lytic complement components,
products of cytolytic lymphocytes
(perforins) and
other physical and chemical agents

Mitochondrial dysfunction and

irreversible mitochondrial damage.
Directly or indirectly the mitochondria is an important
target in all forms of injury.
They can be damaged by increase in cytosolic calcium,
oxidative stress, breakdown of phospholipids through
action of phospholipase A2 and sphingomyelin pathway,
with production of toxic fatty acids and ceremides.
This results in the formation of high conductance
channel the mitochondrial permeability transition (MPT)
in the inner mitochondrial membrane, a nonselective
high conductance channel.

MITOCHONDRIA DYSFUNCTION
Irreversible mitochondrial permeability is
death to the tissues.
MPT is associated with the leakage of
cytochrome c, an integral component of
the electron transport chain that can
trigger the apoptotic pathway.

Ischemic /hypoxic injury

Ischemic and hypoxic injury are the commonest causes
of cell injury in clinical medicine.
Ischemia is a decrease in the blood flow to a tissue or
organ while hypoxia is a decrease in oxygenation of the
tissues.
Ischemia results in injury faster than hypoxia because
anaerobic energy generation will stop after gylcoytic
substrates are exhausted or it is inhibited by
accumulation of metabolites that would have been
carried away in the blood stream.

Types of hypoxia
Histiotoxic
Stagnant
Hypoxic
Anemic

Types of ischemic injury

Reversible
Irreversible
Reperfusion injury

Hypoxic tissue injury/reversible

Hypoxia is a common pathway for most
causes of injury.
Results in reduction of ATP
Na+/ATPase pump is affected, increase
influx of ca2+ &water, exflux of k+
Cellular swelling, loss of microvilli, ER
swelling, myellin figures.

Decrease ATP ,
increase glycolysis,
lactic acidosis,
decrease PH &glycogen, this leads to clumping
of nuclear chromatin
ER swelling, detachment of ribosome, decrease
protein synthesis and lipid deposition.
All these are reversible if the insult is removed.

Morphology of reversible injury

Cellular swelling hydropic change or vacuolar
degeneration
Macroscopy
Pallor, turgid tissue
Increased weight
Bulging cut surface.

Microscopy
Clear vacuoles in cytoplasm
Takes up less stains

Ultra structural changes

Plasma membrane alterations

Mitochondrial changes
Dilatation of ER
Nuclear alterations

Irreversible injury.
Occurs when hypoxia/ischemia persist.
It is associated with extensive damage to cell
membrane
Large amorphous densities in mitochondria
membrane indicative of irreversible injury in
myocardium and occurs as early as 30mins after
ischemia
Massive influx of calcium into the cell

 Loss of proteins, enzymes, ribonucleic

acid and other metabolites
Breakdown of lysosomal membranes and
leakage of enzymes
*Acidosis protects against lethal injury due
to the inhibitory effects of low pH on
enzymatic reactions and MPT.

Mechanism of irreversible injury

The two suspect biochemical events that are
thought to be responsible for the cell going to
the point of no return are
1. Irreversible mitochondrial dysfunction resulting
in loss of ATP production
2. Development of profound disturbances in
membrane function.
Evidences clearly indicate that membrane
damage is a central factor in the pathogenesis
of irreversible injury and it is contributed to by

Mechanism of irreversible injury

Mitochondrial dysfunction
Loss of membrane phospholipids from activation
of endogenous phospholipases by cytosolic
calcium
Cytoskeletal abnormalities-activation of
proteases
Reactive oxygen species-peroxidation of lipids

 Lipid breakdown products such as acyl

carnitine, FFA, lysophospholipids-lipid
degradation from detergent effect &
electrophysiologic alterations by insertion
into the lipid bilayer.
Loss of amino acids e.g. glycine helps
ATP depleted cells to withstand the lethal
effects of high calcium

Ischemia/ re-perfusion injury

This occurs after restoration of blood flow to an
ischemic tissue.
It occurs days after and the affected tissues
show neutrophilic infiltrate .
It occurs in cells that are structurally intact but
biochemically compromised that lose their
integrity after reperfusion.
New damaging processes are set in motion
during reperfusion causing death of cells that
may have recovered.

Necrosis

1.
2.

This refers to the spectrum of morphologic changes

that follow cell death in living tissues.
Its most common manifestation is coagulative
necrosis.
The morphologic appearance of necrosis is the result
of two essentially concurrent processes
Enzymatic digestion of the cell which can be autolysis
(self digestion) or heterolysis (enzymes from
lysosomes of leucocytes) and
Denaturation of proteins
Features of necrosis are not immediately apparent
after cell death.

Types of Necrosis

Coagulative necrosis
Liquefactive necrosis
Caseous necrosis
Gangrenous necrosis
Fat necrosis
Others
Fibrinoid necrosis.

Necrosis-cytoplasmic changes.
LM

Increased eosinophilia
Glassy appearance
Vacoulation of cytoplasm
Calcification

EM
Discontinuity of plasma and organelle membranes
Dilatation of mitochondria with large amorphous
densities, intra-cytoplasmic myelin figures
Amorphous osmiophilic debris

Necrosis nuclear changes.

There are three main patterns
Karyolysis- dissolution of the nucleus by
the action of DNAses
Pyknosis- nuclear shrinkage and increased
basophilia
Karyorrhexis- the pkynotic or partially
pkynotic nuclear undergoes fragmentation.
Within a day or two, the nucleus of the
necrotic cell disappears.

Coagulative necrosis
the basic outline of the coagulated cell is
maintained for a span of some days.
It results when the structural and enzymatic
proteins are denatured by acidosis that ensues
from the injury thus preventing enzymatic
digestion of the cells.
The necrotic cells are removed by scavenger
white blood cells
This form of necrosis occurs in all tissues
following hypoxic injury except in the brain. It is
exemplified in infarcts of solid organs such as the
heart, the spleen and the kidney.

Coagulative necrosis contd

This form of necrosis occurs in all tissues
following hypoxic injury except in the
brain.
It is exemplified in infarcts of solid organs
such as the heart,

the spleen and

the kidney.

Coagulative necrosis-3

Macroscopy.
Following infarcts of solid organs,
it appears as a firm raised pale area,
wedged shape with apex at the centre and base
towards the surface of the tissue.
The tissue shows loss of normal markings.

 Microscopy. The cells retain their normal

outlines.
The nucleus shows one of two changes
karyolysis and karyorrhexis
The cytoplasm becomes opaque and strongly
eosinophilic

EM shows disorganization and

disintegration of the cytoplasmic organelles
and severe damage to plasma membrane

Colliquative(liquefactive) necrosis.
This occurs when the rate of dissolution of the
necrotic cells is faster than the rate of repair.
In the brain following an infarct, there is rapid
dissolution of dead tissue due to the presence of
abundant lysosomal enzymes or different
hydrolases in the brain tissue.
If extensive cyst formation results that may
persist for life

Colliquative necrosis
Colliquative necrosis will occur in area of
coagulative necrosis as a result of
secondary change seen in
Suppuration e.g. septic infarct
Liquefaction of caseous material

Caseous necrosis
This is characteristic of Tuberculosis
In caseous necrosis, the cells fail to maintain their outline
like in coagulative necrosis and they do not disappear by
lysis like in colliquactive necrosis.
Rather the cells persist indefinitely as amorphous
coarsely granular eosinophilc material.
Also unlike coagulative necrosis, the tissue architecture
is obliterated

caseous necrosis
Grossly the debris appears as greyish
while friable material resembling clumpy
cheese hence the name caseous necrosis
Microscopically, it appears as amorphous
esinophilic material bordered by epitheliod
cells, Laghans giant cells and fibrocytes

Apoptosis-1.
Definition.Literally means dropping off
This can be defined as programmed cell
death or individual cell necrosis,
designed to eliminate unwanted host cells
through activation of a coordinated internally
programmed series of events effected by
dedicated set of gene products.
It is an important process in both health and
disease.

Apoptosis in Health and normal

development.
Health
Loss of auto reactive response of T cells in thymus
preventing auto immune attack
In atrophy and involution e.g. menstruation, ovarian
follicular atresia in menopause, regression of the
lactating breast after weaning, prostatic atrophy
after castration
Normal cell turnover .e.g. Intestinal crypt epithelium
During embyogenesis e.g separation of digits
during limb development.

Apoptosis in disease

Irradiation
Cytotoxic anti-cancer drugs
Heat
Hypoxia
Virus infections e.g. councilman bodies in viral
hepatitis
Action of cytotoxic T cells. e.g. rejection of
transplanted organ
In tumors
Pathological atrophy in parenchyma cells after
duct obstruction

Stages of Apoptosis

During apoptosis, the cells go through

the following morphologic stages which
can be categorized into
A. The drying process
B. The elimination process.

The drying process

Cell shrinkage. The cell is smaller in size,
cytoplasm dense, organelles relatively normal
though more tightly packed
Chromatin condensation. Most characteristic
feature of apoptosis. Chromatin aggregates
under nuclear membrane. Nucleus make break
up into fragments.

 Formation of cytoplasmic blebs and

apoptotic bodies.
The apoptotic cell shows extensive surface
blebbing then undergoes
fragmentation into a number of membrane
bound apoptotic bodies composed of
cytoplasm and tightly packed organelles
with or without nuclear fragments

The elimination process.

The apoptotic bodies are phagocytosed by
either healthy cells in the parenchymal or
macrophages where they are readily
degraded in the lysosome.
The adjacent cells migrate or proliferate to
replace the apoptotic cell.

Morphology of Apoptosis
The apoptotic cell appears round or oval with
eosinophilic cytoplasm and condensed nuclear
chromatin
Because of the rapid rate of elimination,
considerable apoptosis would have occurred in
the cell before it becomes apparent on sections
Apoptosis does not elicit an inflammatory
reaction, making it more difficult to appreciate
histologically

Morphology of Apoptosis

Biochemical features of
apoptosis
Protein cleavage
Involves action of the caspases, a member of the
cysteine proteases which cleaves the nuclear
scaffolding and cytoskeletal proteins
The caspases also triggers the endonucleases

Protein cross linking

This is carried out by transglutaminase activity
and converts cytoplasmic proteins into covalently
linked shrunken shells that break into apoptotic
bodies

Biochemical features of apoptosis-2

DNA breakdown
DNA broken down into 50-300kilobase pieces.
This is followed by internucleosomal cleavage of DNA
into oligonucleomes by Ca 2+ and Mg 2+ dependent
endonucleases

Phagocytic recognition
Express phosphatidylserine on outer layers of plasma
membrane.
Occasionally thrombospondin is expressed
This permits early recognition of dead cells by
macrophages and adjacent cells without the release
of pro inflammatory substances

Mechanism of apoptosis
The mechanism of apoptosis has several keys stages .
This include
Signalling pathways,
Transmembrane signals that can be positive or negative. E.g. TNFR
Intracellular signaling e.g. binding of glucocorticoids to nuclear
receptors, heat , radiation hypoxia and viral infections

The control and integration stage

Adapter proteins with death domain e.g Fas-Fas ligand
Bcl-2 family of proteins---,regulates mitochondrial function
while p-53, c-myc gene promotes

Execution stage - activation of caspases that perform terminal

proteolysis.
Caspases are zymogens and need to be cleaved before apoptosis
occurs. Cleavage is autocatalytic
Phagocytosis Apoptotic bodies are finally removed by.