12/7/2023

LECTURE
CELLULAR NECROSIS AND APOPTOSIS

Masum Shahriar
Professor, Department of Pharmacy

Reading Assignment
Robbins: Pathologic Basis of Disease, Saunders, 9th edition,
Chapter 1 (ISBN 0-7216-0187-1)

Learning Objectives:
At the end of this lecture, candidates should be able to:
* Define Necrosis and list the gross histological, electron
microscopic and nuclear changes that commonly occur with this
form of cell death.
* Define irreversible Cell injury and know the mechanisms
* Define and describe Coagulate Necrosis, Liquefactive Necrosis,
Caseous Necrosis, and Fat Necrosis.
* Define and describe apoptosis and the gross histological, electron
microscopic and nuclear changes that commonly occur with this
form of cell death.
* Describe the basic biochemical mechanisms involved in
programmed cell death.

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Cell death

With continuing

damage, the injury

becomes irreversible,

at which time the cell

cannot recover and it

dies.

Irreversible cell injury

Associated with:
*Severe swelling of the mitochondria
* Large amorphous deposits appear in the mitochondrial matrix.
*Extensive damage to cell membranes
* Massive influx of calcium occurs into the cytoplasm
* Causes leakage of cell components into the extracellular space
*Swelling of lysosomes and damage to lysosomal membranes
* Causes leakage of lysosomal enzymes (proteases, lipases, DNases and
RNases, etc...) into the cytoplasm

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Irreversible cell injury (the point of no return)

Stages of cell injury and death

There are two types of cell death—

Injurious Stimulus

Reversible Reversible
Cell Injury Stage ?

Necrosis Apoptosis

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Necrosis

Necrosis refers to a spectrum of morphologic changes that

follow cell death, largely resulting from the progressive
degradative actions of enzymes on the injured cell.

When damage to membranes is severe, enzymes leak out of

lysosomes, enter the cytoplasm, and digest the cell, resulting
in necrosis. Necrosis is the major pathway of cell death in
many commonly encountered injuries, such as those
resulting from ischemia, exposure to toxins, various
infections, and trauma.

Gross cellular histological changes common to early

necrosis
Necrosis is characterized by changes in the cytoplasm and nuclei of
the injured cells
Cytoplasmic changes.
• Increased "eosin" (acid) staining due to loss of basophilic mRNA
and increased eosin binding to denatured proteins (eosin is a red
dye used in histology)
• Glassy homogeneous appearance of cytoplasm due to loss of
glycogen particles
• Vacuolated or "Moth Eaten" cytoplasm from enzymes that have
digested intracellular organelles
• Dead cells may be replaced by large, whorled phospholipid
masses, called myelin figures, that are derived from damaged
cellular membranes.
• Calcification due to accumulation of calcium products

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Necrotic cells under electron microscopy

Discontinuous membranes

* For both organelles and cell plasma membrane

* Dilation of mitochondria
* appearance of intra-mitochondrial amorphous
accumulations

* Intracytoplasmic "myelin" bodies

* Aggregates of "fluffy material in cytoplasm
* Most likely denatured protein

Nuclear changes due to breakdown of DNA

Nuclear changes assume one of three patterns, all due to breakdown

of DNA and chromatin.
Karyolysis
Basophilia of nuclei fade due to chromatin changes and DNase
activity.
Pyknosis
Nuclear shrinkage and increased basophilia
DNA condenses into solid shrunken basophilic mass
Karyorrhexis
Includes fragmentation of the pyknotic or partially pyknotic
nucleus

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Fates of necrotic cells.

Necrotic cells may persist for some time or may be

digested by enzymes and disappear. Dead cells may

be replaced by myelin figures, which are either

phagocytosed by other cells or further degraded into

fatty acids. These fatty acids bind calcium salts,

which may result in the dead cells ultimately

becoming calcified.

Morpho1ogical Changes in Necrosis

* Swelling of the cell

* Swelling and disruption of
lysosomes
* Large amorphous densities
in swollen mitochondria
* Disruption of cellular
membranes
* Nuclear condensation
(PYKNOSIS)
* Nuclear fragmentation
(KARYORRHEXIS)
* Dissolution of the nucleus
(KARYOLYSIS).

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Patterns of Tissue Necrosis

There are several morphologically distinct patterns of tissue

necrosis, which may provide clues about the underlying
cause. Different cells shows different morphologic changes
after they undergo necrosis. Base on that necrosis can be
classified into:
1. Coagulative necrosis
2. Liquefactive necrosis
3. Caseous / granulamatous necrosis
4. Fat necrosis
5. Gangrenous Necrosis

Coagulative necrosis
Typically occur in solid organ; heart, kidney, adrenal glands except the brain.
Coagulative necrosis is a form of tissue necrosis in which the component cells
are dead but the basic tissue architectureis preserved for at least several days.
Results from injury and following acidosis that denatures proteins and enzymes
(blocks proteolysis).

Wedge-shaped kidney infarct (yellow) with Microscopic view of the edge of the infarct, with
preservation of the outlines normal kidney (N) and necrotic cells in the infarct (I).

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Liquefactive necrosis
Liquefactive necrosis is seen in focal bacterial or, occasionally, fungal
infections, because microbes stimulate the accumulation of inflammatory cells
and the enzymes of leukocytes digest (―liquefy‖) the tissue. Hypoxic death of
cells within the central nervous system often evokes liquefactive necrosis

liquefactive necrosis in the Brain liquefactive necrosis in the kidney

If the process was initiated by acute inflammation, the material is frequently

creamy yellow and is called pus

Caseous necrosis
The term ―caseous‖ (cheeselike) is derived from the friable yellow-white
appearance of the area of necrosis. Caseous necrosis is encountered most
often in foci of tuberculous infection.

Unlike coagulative necrosis, the tissue Pulmonary tuberculosis

architecture is completely obliterated

and cellular outlines cannot be
discerned. Caseous necrosis is often
enclosed within a distinctive
inflammatory border; this appearance
is characteristic of a focus of
inflammation known as a granuloma

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Fat necrosis
Fat necrosis, refers to focal areas of fat destruction, typically
resulting from release of activated pancreatic lipases into the
substance of the pancreas and the peritoneal cavity.
pancreatitis
In this disorder, pancreatic enzymes
that have leaked out of acinar cells and
ducts liquefy the membranes of fat
cells in the peritoneum, and lipases
split the triglyceride esters contained
within fat cells. The released fatty
acids combine with calcium to produce
grossly visible chalky white areas (fat
saponification).

Gangrenous Necrosis

* Extensive tissue necrosis

* Divide into two; dry and wet
* Dry – occurs in extremities as a
results of ischemic coagulative
necrosis due to arterial obstruction
* Wet – occurs in extremities and
internal organ as a results of
liquefactive necrosis due to bacterial
infection.
* Gas – necrotic tissue infected by
clostridium perfringes

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Fibrinoid necrosis
Fibrinoid necrosis is a special
form of necrosis usually seen in
immune reactions involving
blood vessels. This pattern of
necrosis is prominent when
complexes of antigens and
antibodies are deposited in the
walls of arteries. Deposits of
these ―immune complexes,‖
together with fibrin that has
leaked out of vessels, result in a
bright pink and amorphous
appearance in H&E stains, called Polyarteritis nodosa
―fibrinoid‖

Apoptosis
Apoptosis is a pathway of cell death in
which cells activate enzymes that
degrade the cells’ own nuclear DNA and
nuclear and cytoplasmic proteins.
Fragments of the apoptotic cells then
break off, giving the appearance that is
responsible for the name (apoptosis,
“falling off”).

Apoptosis is the end-point of an energy-

dependent cascade of molecular events
initiated by certain stimuli.

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Causes of Apoptosis

Apoptosis occurs in many normal situations and

serves to eliminate potentially harmful cells and
cells that have outlived their usefulness. It also
occurs as a pathologic event when cells are
damaged beyond repair, especially when the
damage affects the cell’s DNA or proteins; in these
situations, the irreparably damaged cell is
eliminated.

Apoptosis in Physiologic Situations

Death by apoptosis is a normal phenomenon that serves to
eliminate cells that are no longer needed and to maintain a constant
number of cells of various types in tissues. It is important in the
following physiologic situations:

• Development - During embryogenesis and organogenesis

• Hormone dependent - As occurs to endometrium in the
menstrual cycle
• Cell loss in proliferating cell populations- such as intestinal
crypt epithelia, in order to maintain a constant number
• Elimination of cells that have served their useful purpose -
neutrophils in an acute inflammatory response
• Elimination of potentially harmful self-reactive lymphocytes-in
order to prevent reactions against the body’s own tissues

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Apoptosis in Pathologic Conditions

Apoptosis eliminates cells that are genetically altered or injured beyond
repair and does so without eliciting a severe host reaction, thereby
keeping the extent of tissue damage to a minimum. Death by apoptosis
is responsible for loss of cells in a variety of pathologic states:

• DNA damage -If repair mechanisms cannot cope with the injury, he cell
triggers intrinsic mechanisms that induce apoptosis.

• Accumulation of misfolded proteins- Excessive accumulation of these proteins

in the ER leads to a condition called ER stress, which culminates in apoptotic
death of cells.

• Cell injury in certain infections- Viral hepatitis

• Pathologic atrophy in parenchymal organs - after duct obstruction, such as

occurs in the pancreas, parotid gland, and kidney

* Apoptotic pathways

– The decision Phase: Cells receive a stimulus and depending

on both its internal and external environments may or may not
trigger to die

– The commitment Phase: Cells are committed to death and

cannot recover

– The execution Phase: Cells activate apoptotic machinery

leads to morphological changes that define apoptotic cells

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* Biochemical Features and Mechanisms

• Protein Cleavage - Involves activation of

cysteine proteases called caspases, which
hydrolyze proteins especially the nuclear scaffold DNA Fragmentation

and cytoskeletal proteins

• Protein Cross Linking - Extensive crosslinking
converts cytoplasmic proteins into covalently
shrunken cells
• DNA Breakdown - Characteristic breakdown of
DNA into large, 50-300 kilobase pieces followed
eventually by DNA lysis into oligonucleosomes in
multiples of 180-200 base pairs

Morphologic Changes in Apoptosis

• Cell shrinkage - Organelles are normal, but
more tightly packed.
• Chromatin condensation - Chromatin
aggregates peripherally under nuclear
membrane
• Formation of cytoplasmic blebs and apoptotic
bodies - Blebbing of cell membrane which
eventually break off as apoptotic bodies.
• Phagocytosis of apoptotic cells or bodies - By
surrounding healthy cells, either parenchymal
or macrophages.
• Absence of inflammation - Considerable
apoptosis can occur in tissues before it
becomes apparent in histological sections.
• Cell membranes - Generally remain intact for
Morphologic appearance of apoptotic cells.
a long time

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Mechanisms of apoptosis
Apoptosis results from the activation of enzymes called caspases.
The caspase family can be divided on the order in which they are activated
during apoptosis into two groups— initiator caspases include caspase-8
and caspase-9 and — executioner caspases includes caspase-3 and
caspase-6, serve as executioners. Like many proteases, caspases exist as
inactive pro-enzymes, and must undergo an enzymatic cleavage to become
active.

The activation of caspases depends on a finely tuned balance between

production of pro- and anti-apoptotic proteins. Two distinct pathways
converge on caspase activation: the mitochondrial (intrinsic) pathway
and the death receptor (extrinsic) pathway.

Mechanisms of apoptosis

(1) Major inducers of

apoptosis.
(2) Control and regulation
are influenced by
members of the Bcl-2
family of proteins
(3) Executioner caspases
activate cytoplasmic
endonucleases and
proteases
(4) Formation of
apoptotic bodies and
phagocytosis

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The intrinsic pathway of apoptosis

Mitochondria contain several proteins that are capable of
inducing apoptosis; these proteins include cytochrome c and
other proteins that neutralize endogenous inhibitors of
apoptosis. The choice between cell survival and death is
determined by the permeability of mitochondria, which is
controlled by a family of more than 20 proteins, the prototype of
which is Bcl-2 . When cells are deprived of growth factors and
other survival signals, or are exposed to agents that damage
DNA, or accumulate unacceptable amounts of misfolded
proteins, a number of sensors are activated.

The intrinsic pathway of apoptosis

Bind to and Neutralize

Mitochondria
Other Pro-apoptotic Inhibitors of
Proteins (AIF) apoptosis
(IAPs)

Inner membrane Pro Caspase-9

Bax,
MPT Bak Apaf-1

MITOCHONDRIAL
MATRIX Cyto C Caspase-9
Bcl-2
Bcl-xL
Outer Membrane Executioner Caspases
(PARP-1)

APOPTOSIS

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The extrinsic pathway of apoptosis

* This pathway is initiated by engagement of plasma membrane
death receptors on a variety of cells.

* Death receptors are members of the TNF receptor family that

contain a cytoplasmic domain involved in protein-protein
interactions that is called the death domain because it is
essential for delivering apoptotic signals.

* The best-known death receptors are the type 1 TNF receptor

(TNFR1) and a related protein called Fas (CD95).

* Fas ligand (FasL) is a membrane protein expressed mainly on

activated T lymphocytes.

The extrinsic (death receptor) pathway of apoptosis

When FasL binds to Fas, three or more
molecules of Fas are brought together,
and their cytoplasmic death domains FasL Fas
form a binding site for an adapter
protein that also contains a death
domain and is called FADD (Fas-
associated death domain). Death Domain FADD
FADD that is attached to the death
receptors in turn binds an inactive form
of caspase-8. Multiple pro-caspase-8 Pro Caspase-8
molecules are thus brought into
proximity, and they cleave one another Autocatalytic
to generate active caspase-8. Caspase
Activation
Executioner
The enzyme then triggers a cascade of Caspases Caspase-8
caspase activation by cleaving and
thereby activating other pro-caspases,
and the active enzymes mediate the APOPTOSIS
execution phase of apoptosis

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The Execution Phase of Apoptosis

* The two initiating pathways converge to a cascade of caspase activation,
which mediates the final phase of apoptosis.
* As we have seen, the mitochondrial pathway leads to activation of the
initiator caspase-9, and the death receptor pathway to the initiators
caspase-8 and -10.
* After an initiator caspase is cleaved to generate its active form, the
enzymatic death program is set in motion by rapid and sequential
activation of the executioner caspases. Executioner caspases, such as
caspase-3 and -6, act on many cellular components.
* For instance, these caspases, once activated, cleave an inhibitor of a
cytoplasmic DNase and thus make the DNase enzymatically active; this
enzyme induces the characteristic cleavage of DNA into nucleosome-sized
pieces.
* Caspasesalso degrade structural components of the nuclear matrix, and
thus promote fragmentation of nuclei.

Clearance of Apoptotic Cells.

Apoptotic cells undergo several changes in their membranes that

promote their phagocytosis. In normal cells phosphatidylserine is
present on the inner leaflet of the plasma membrane, but in
apoptotic cells this phospholipid ―flips‖ out and is expressed on the
outer layer of the membrane, where it is recognized by
macrophages. Cells that are dying by apoptosis also secrete soluble
factors that recruit phagocytes. This facilitates prompt clearance of
the dead cells before they undergo secondary membrane damage
and release their cellular contents

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Features of Necrosis and Apoptosis

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