Irreversible cell injury:

Dr. Huda Elsheikhei (2019-2020)

*Necrosis (Pathologic death):

- Necrosis is defined as a localised area of death of tissue that associated
with loss of cell membrane integrity and leakage of cellular contents
followed by degradation of tissue by hydrolytic enzymes liberated from
dead cells; it`s accompanied by inflammatory reaction.

- It occurs either directly or following reversible injury.

- Remember that the Necrosis is always pathologic .

*Causes:
- It is caused by various agents such as hypoxia, chemical and physical
agents, microbial agents, immunological injury, etc.

- Two essential changes characterise irreversible cell injury in necrosis of

all types:

i) Cell digestion by lytic enzymes. Digestion of cells by lysosomal

enzymes of dying cells ( autolysis) and leukocytes (heterolysis).

ii) Denaturation of proteins. a change in protein structure caused by

changes in pH, temperature. It can be reversible at the beginning but
then it will not be reversible if denaturation is severe.

- Usually denaturation and enzymatic digestion occur together.

*Morphology of necrosis:
- There are cytoplasmic and nuclear changes. -

*Cytoplasmic changes: increased eosinophilia (pink staining from the

eosin dye). Increased eosinophilia is due to :

1- Increased binding of eosin to denatured proteins and

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2- The loss of basophilia (decreases hematoxylin binding to RNA),
because RNA in the cytoplasm decreases.

-Due to these changes cells appear homogenous and glassy.

*Nuclear changes in necrosis: One of three patterns:

1) karyolysis: decreased chromatin basophilia (decreased hematoxylin

binding to DNA) secondary to deoxy ribonuclease (DNase) activity.

2) pyknosis: nuclear shrinkage and increased basophilia (DNA condenses

into a solid shrunken mass).

3) karyorrhexis: fragmentation then disappearance of nucleus.

- One of the three patterns could happpen, but usually nuclear necrosis
must end by karyorrhexis.

* EM changes:

1)Discontinuities in plasma and organelle membranes.

2) Marked dilation of mitochondria and large amorphous densities

(small densities is seen in reversible injury).

3)Disruption of lysosomes.

4) Intracytoplasmic myelin figures.

*Myelin figures:

- It`s an aggregates of damaged cell membranes (phospholipids), it

attracts calcium and can be a nidus for calcium deposition and
calcifications might occur..

- it is seen as a morphologic change in reversible and irreversible cell

injury.

- Fate of Myelin figures: phagocytosed by other cells ,or further -

degraded into fatty acids and calcify.

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*Types of necrosis:
- Morphologically, there are five types of necrosis: coagulative,
liquefaction, caseous, fat, and fibrinoid necrosis.

- Denaturation of protein predominates…. Coagulative necrosis.

- Enzymatic digestion predominates… liquefactive necrosis.

- Special circumstances: caseous necrosis, fat necrosis and fibrinoid

necrosis.

1- Coagulative necrosis:
- This is the most common type of necrosis caused by irreversible focal
injury, mostly from sudden cessation of blood flow (ischaemia), and less
often from bacterial and chemical agents.

- The organs commonly affected are the heart, kidney, and spleen.

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* Morphology of coagulative necrosis:

* Grossly, foci of coagulative necrosis in the early stage are pale, firm,
and slightly swollen. With progression, they become more yellowish,
softer, and shrunken.

* Microscopically, the hallmark of coagulative necrosis is the conversion

of normal cells into their ‘tombstones’ i.e. outlines of the cells are
retained so that the cell type can still be recognised but their
cytoplasmic and nuclear details are lost.

- The necrosed cells are swollen and appear more eosinophilic than the
normal, along with nuclear changes described before. But cell digestion
and liquefaction fail to occur ( liquefaction necrosis).

-Eventually, the necrosed focus is infiltrated by inflammatory cells and

the dead cells are phagocytosed leaving granular debris and fragments
of cells.

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 -

2- Liquifactive necrosis:
- Occurs commonly due to Ischaemic injury and bacterial or fungal -
infections. It occurs due to degradation of tissue by the action
hydrolytic enzymes resulting into a liquid jelly-like mass .

- The common examples are hypoxic death in central nervous -

system (infarct brain) and abscess.

* Morphology of liquifactive necrosis: -

*Grossly, the affected area is soft with liquefied centre containing *

necrotic debris. Later, a cyst wall is formed.

*Microscopically, the cystic space contains necrotic cell debris and

macrophages filled with phagocytosed material. The cyst wall is formed
by proliferating capillaries, inflammatory cells, and gliosis (proliferating
glial cells) in the case of brain and proliferating fibroblasts in the case of
abscess cavity.

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3- Caseous necrosis:
White cheese like friable necrosis found in the centre of foci of
tuberculous infections. It combines features of both coagulative and
liquefactive necrosis.

* Morphology of caseous necrosis:

* Grossly, foci of caseous necrosis, as the name implies, resemble dry

cheese and are soft, granular and yellowish.

This appearance is partly attributed to the histotoxic effects of

lipopolysaccharides present in the capsule of the tubercle bacilli,
Mycobacterium tuberculosis.

*Microscopically, the necrosed foci are structureless, eosinophilic, and

contain granular debris.The surrounding tissue shows characteristic
granulomatous inflammatory reaction consisting of epithelioid cells with

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interspersed giant cells of Langhans’ or foreign body type and peripheral
mantle of lymphocytes.

4- Fat necrosis:
- Used as a clinical terms and not a specific type (necrosis of fat). -

- Typical example: acute pancreatic necrosis, trumatic fat necrosis -

commnly in breasts.

- In case of pancrease, there is liberation of pancreatic lipases from

injured or inflamed tissue that results in necrosis of the pancreas as well
as of the fat deposts throughout the peritoneal cavity, and sometimes,
eve affecting the extraabdominal adipose tissue.

- Fat necrosis hydrolyses neutral fat present in adipose cells into glycerol
and free fatty acids. The damaged adipose cells assume cloudy
appearance. The leaked out free fatty acids complex with calcium to
form calcium soaps (saponification).

- The normal fat appears in yellow, necrotic fat cells appear in white.

* Morphology of fat necrosis:

*Grossly, fat necrosis appears as yellowish-white and firm deposits.

Formation of calcium soaps imparts the necrosed foci firmer and chalky
white appearance.

*Microscopically, the necrosed fat cells have cloudy appearance and are
surrounded by an inflammatory reaction. Formation of calcium soaps is
identified in thetissue sections as amorphous, granular and basophilic
material.

5- Fibrinoid necrosis:
- Characterised by deposition of fibrin-like material which has the
staining properties of fibrin.

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- It is encountered in various examples of immunologic tissue injury (e.g.
in immune complex vasculitis, autoimmune diseases, arterioles in
hypertension, peptic ulcer etc.

*Microscopically, fibrinoid necrosis is identified by brightly eosinophilic,

hyaline-like deposition in the vessel wall.

- Necrotic focus is surrounded by nuclear debris of neutrophils

(leucocytoclasis).

- Local haemorrhage may occur due to rupture of the blood vessel.

* Fate of necrosis:
• Phagocytosis. • Replacement by scar.

• Regeneration. • Calcification.

*Apoptosis (programmed cell death):

- Also named:, cell suicide, individual cell death.

- Apoptosis is a Greek word which means “falling off”, the name is given
due to the appearance of fragments of the apoptotic cells break off .

- Apoptosis induced by a tightly regulated suicide program in which cells

activate enzymes capable of degrading the cells' own nuclear DNA and

cytoplasmic proteins.

- Can be pathologic or physiologic (the majority is physiologic).

*Physiologic apoptosis: -

- The causes of Physiologic apoptosis are to eliminate cells that are -

no longer needed OR to maintain a steady number of various cell
populations in tissues.

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-Physiologic situations: -

•Embryogenesis, the embryo starts as one block, some cells need to die
in order to shape the body.

•evolution of hormone-dependent tissues upon hormone withdrawal.

(endometrium and breast after pregnancy)

• Cell loss in proliferating cell populations. (gastrointestinal tract, skin…)

• Death of host cells after serving their useful function. (neutrophils and
lymphocytes in inflammation)

• Elimination of potentially harmful self-reactive lymphocytes.

•Cell death induced by cytotoxic T lymphocytes (tumor cells and virally

infected cells).

* Pathologic apoptosis:

- Pathologic situations:

•DNA damaged cells; if DNA damage is severe and cannot be repaired

the cell dies by Apoptosis.

•Cells with accumulation of misfolded proteins,

•Certain infections (viral ones): may be induced by the virus (as in

human immunodeficiency virus infections) or by the host immune
response (as in viral hepatitis).

•Pathologic atrophy in parenchymal organs after duct obstruction

(pancreas, parotid and kidney).

*Mechanismes of Apoptosis:

-Two main pathways:

1- Mitochondrial pathway (intrinsic): everything happens inside the cell.

2- Death receptor pathway (extrinsic): first message is from outside the

cell, then all the process happens inside the cell.

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1. Intrinsic pathway (mitochondrial pathway):

- Mitochondria contains several proteins that can induce apoptosis, the

most important of these is cytochrome C .

-Intrinsic pathway starts when cytochrome C exits the mitochondria,

how does it cross the mitochondrial membrane?

Mitochondrial permeability is controlled by a family of more than 20

proteins (Bcl2 family).

-A Sensor called BH3 protein (part of the Bcl2 family) senses any -
stress inside the cell ( e.g: When cells are deprived of growth
signals, or exposed to severe DNA damage or have misfolded
proteins) and become active.

- BH3 now activates members of the family : Bax and Bak ,these proteins
increase the permeability of the membrane and called (pro-apoptotic
members).

-BH3 also inhibit anti inhibitory members of the family: BCL-2 and BCL-xl
, these proteins decrease the permeability of the membrane and called
(anti-apoptotic members).

- When bax and bak are stimulated they dimerize and insert into the
mitochondrial membrane

They form channels through which cytochrome c escapes into cytosol.

-So BH3 stimulates proapptotic and inhibits antiapoptotic signals.. Net

result is the leakage of cytochrome c from the mitochondria to cytosol .

- Once cytochrome c is in the cytosol it stimulates caspase 9 which

stimulates other caspases called (executioner caspases), these caspases
lead to nuclear fragmentation.

*Summary of intrinsic pathway:

•BH3 stimulates pro-apoptotic (bax, bak), and inhibits anti apoptotic

proteins (bcl-2, bcl-xl) .

•Cytochrome c leaks out.

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•Stimulates caspase 9.

•Stimulates executioner caspases that degrade cell components.

2. Extrinsic pathway (death receptor pathway):

- Cells have many receptors, called death receptors, these receptors are
involved in the extrinsic pathway, and the most important types of death
receptors are: TNF type 1 receptor and Fas receptor (CD95)

- Fas receptor has a ligand called (Fasl ).

- FasL = fas ligand is a membrane protein expressed mainly on T

lymphocytes.

- When T cells recognize fas expressing target, fas molecules are cross
linked by FasL to activate caspase 8.

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- Caspase 8 activates executioner caspases that degrade cell
components.

* Morphology of apoptosis:

- The characteristic morphologic changes in apoptosis seen in histologic

and electron microscopic examination are:

1. Involvement of single cells or small clusters of cells in the background

of viable cells.

2. The apoptotic cells are round to oval shrunken masses of intensely

eosinophilic cytoplasm (mummified cell) containing shrunken or almost-
normal organelles.

3. The nuclear chromatin is condensed or fragmented (pyknosis or

karyorrehexis).

4. The cell membrane may show convolutions or projections

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on the surface. ›

5. There may be formation of membrane-bound nearspherical bodies on

or around the cell called apoptotic bodies containing compacted
organelles.

6. Characteristically, unlike necrosis, there is no acute inflammatory

reaction around apoptosis

7. Phagocytosis of apoptotic bodies by macrophages

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