Introduction To Pathology

Dr. Wubshet Assefa (MD,

Pathologist)
 Pathology
• Greek (pathos - suffering,
logos -study).
• A scientific study of
disease
• Bridging discipline which
encompasses both basic
science and clinical
practice.
• Disease: dis-ease
Never think of leaves
without the the stem.
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• Rudolf virchow – father
of modern pathology
• ‘’cell injury a basis for
diseases’’
• Other discoveries
– Virchow cell (lepra cell)
– Virchow node
– Virchow traid etc …
• From 1821-1902
german physcician

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It is divided into
General pathology - cellular and tissue responses to pathologic
stimuli
Systemic pathology - responses of specialized organs pathologic
stimuli

• Pathology gives explanations of a disease by

studying the following four aspects of the
disease.
• 1. Etiology,
• 2. Pathogenesis,
• 3. Morphologic changes and
• 4. Functional derangements and clinical
significance.
1. Etiology - means the cause of the disease.
• Can be known (1ry) or unknown (idiopathic)
• primary cause is a back bone for the diagnosis and
treatment development
• Etiologic factors
– genetic/intrinsic
– Acquired
– multifactorial (Env’t & genetics)
Knowledge or discovery of the primary cause remains the
backbone on which a diagnosis can be made, a disease
understood & a treatment developed.
The pathogenic mechanisms could take place in the latent or
incubation period.
• Environmental agents:
– Physical
– Chemical
– Nutritional
– Infections
– Immunological
– Psychological
– … Multifctorial
• Genetic Factors:
– Age
– Genes
– Immunological
– Psychological
– ……

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 2. Pathogenesis – means mechanism for
development of the disease
• Sequence of events that leads to morphologic
changes.
3. Morphologic changes –
refer to the structural alterations in cells or tissues that
occur following the pathogenetic mechanisms
Can be gross or microscopic change
The changes may be specific to a disease, that help
pathologist diagnose the disease
 Cirrhosis/ኮምትርa of the liver ,as is well known ,
consists of hepatic nodules and vascular septa , but
in post-hepatic or alcoholic cirrhosis the shape of
the liver is mantained ,while it is distorted in
autoimmune rare syphilitic (hepar lobatum
cirrhosis.
A , thick bands of collagen separate rounded
cirrhotic nodeles. (Masson trichrome stain)
4. Functional derangements and clinical
features
• are consequences of morphologic changes

Out come and prognosis

– Cure/resolution
– Disability/permanent damage
– Death
• In summary, pathology studies:-
• Etiology → Pathogenesis → Morphologic
changes → Clinical features → outcome &
Prognosis of all disease
• Understanding of the above core aspects of
disease (i.e. understanding pathology) will
help one to understand how the clinical
features of different
• diseases occur & how their treatments work

i.e =that is
Course of diseases
• exposure→ biologic onset →clinical onset→
permanent damage →death
• natural course of disease-course without
intervention
• The out comes can occur at any course of
diseases

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 Diagnostic techniques used in pathology
• The pathologist uses the following techniques to the
diagnose diseases:
– Histopathology
– Cytopathology
– Hematopathology
– Immunohistochemistry
– Microbiological examination
– Biochemical examination
– Cytogenetics
– Molecular techniques
– Autopsy
For most diseases ,diagnosis is based on a combination of
pathological investigations.
A.Histopathological techniques
• studies tissues abnormalities under
microscope.
• The gold standard for pathologic diagnosis.
• Tissues obtained by biopsy.
• Biopsy
– a tissue sample from a living person to identify
the disease.
– can be either incisional or excisional.
Tissue removed from the patient immediately
fixed (putting it into adequate amount of 10%
formaldehyde (10% formalin) before sending it to
the pathologist. The purpose of fixation.
• Steps
– tissue removed from the patient (biopsy)→
fixation (eg. 10% formalin)→macroscopic
examination→ tissue processing →
embedding in to paraffin→ sectioned (cut)
into thin slices→ staining → microscopic
examination→ look for abnormal structures
in the tissue.
• Staining
– Routine stains :
Hematoxylin/Eosin(H&E)
stain
• It gives nucleus a blue
& cytoplasm & ECM
pinkish color.
– Special stains : such as
PAS/p-aminosalicylic acid,
Immunohistochemistry,
etc.
Normal breast histology
• B. Cytopathologic methods
• study of cells from various body sites to
determine the cause or nature of disease.
• Advantages :
– it is cheap, takes less time and needs no anesthesia to
take specimens.
– Therefore, it is appropriate for developing countries
– it is complementary to histopathological examination.
• Cytopathologic methods includes:
• 1. Fine-needle aspiration cytology (FNAC)
– cells are obtained by aspirating the diseased
organ uses a very thin needle
– the aspirated cells are then stained and studied
under the microscope.
– Superficial organs (e.g. thyroid,breast, LNs/lymph
nodes/, skin and soft tissues)
• can be easily aspirated.
– Deep organs; such as the lung, liver, pancreas,
kidney
• aspirated with guidance by fluoroscopy, ultrasound or
CT scan.
• 2. Exfoliative cytology
– examination of cells that are shed
spontaneously into body fluids or secretions.
– Includes sputum/ አክታ, CSF, effusions/እዥ in
body cavities (pleura, pericardium,
peritoneum)

3. Abrasive cytology
Refers to methods by which cells are dislodged by various
tools from body surfaces (skin, mucous membranes, and
serous membranes).
E.g. Pap smears: preparation of cervical smears with a spatula
or a small brush to detect cancer of the uterine cervix at early
stages.
• Applications of cytopathology:
1.Screening or early detection of asymptomatic cancer.
2. Diagnosis of symptomatic cancer
3. To diagnose cysts, inflammatory conditions and
infections of organs
4. Surveillance of patients treated for cancer
• periodic urine cytology to monitor the recurrence of
cancer of the UT
• C. Hematological examination
– abnormalities of the cells of the blood and their
precursors in BM are investigated
– Used to diagnose different kinds of anemias & leukemias.
• D. Immunohistochemistry
– used to detect a specific antigen in the tissue in order to
identify the type of disease.
• E. Microbiological examination
– Identifying micro-organisms from body fluids,cells and
excised tissues
– Uses microscopy,cultural and serological techniques

F. Biochemical examination
Assessment of metabolic disturbances of disease
Using assay of various normal and abnormal compounds in the
blood, urine, etc.
G. Molecular techniques
– used to detect genetic diseases.
– The techniques such as fluorescent in situ
hybridization, Southern blot, PCR etc...
H. Cytogenetics,
– Asses chromosomal abnormalities in the cells using of
molecular techniques
A method in which inherited chromosomal abnormalities
in the germ cells or aquired chromosomal abnormalities
in somatic cells

I. Autopsy
▪ Examination of the dead body to identify the
cause of death.
▪ can be done for forensic or clinical purposes.
 Cellular Responses to Stress and Noxious Stimuli

• Homeostasis
– it is the steady state that cells exist in normally
– an equilibrium of cells with their environment
for adequate function
–disturbance of it leads to disease onset
Increased workload = Adaptation

Stress == reversible injury

Increased/persistent stress = Irreversible injury

• Example: heart muscle
• Increased hemodynamic loads→ the heart
muscle becomes enlarged (adaptation)→ the
blood supply to the myocardium is inadequate→
reversible injury → Eventually irreversible injury
and die
• Increased hemodynamic loads → the heart
muscle becomes enlarged ( adaptation) → if the
blood supply to the myocardium is inadequate→
the muscle first suffers reversible injury
,manifested by certain cytoplasmic changes
(described later. Eventually ,the suffer
irreversible injury and die.
• Figure 2-2 The relationship between normal, adapted, reversibly injured, and
dead myocardial cells.
• All three transverse sections of the heart have been stained with
triphenyltetrazolium chloride, an enzyme substrate that colors viable
myocardium magenta.
• The cellular adaptation shown here is myocardial hypertrophy(lower left),
caused by increased blood pressure requiring greater mechanical effort by
myocardial cells.
• This adaptation leads to thickening of the left ventricular wall (compare with
the normal heart).
• In reversibly injured myocardium (illustrated schematically, right), there are
functional alterations, usually without any gross or microscopic changes but
sometimes with cytoplasmic changes such as cellular swelling and fat
accumulation.
• In the specimen showing necrosis, a form of cell death (lower right), the light
area in the posterolateral left ventricle represents an acute myocardial infarction
caused by reduced blood flow (ischemia)
• Stresses may also induce the
following changes in cells and
tissues
–intracellular accumulations,
–pathologic calcification, and
–Cell aging
 CELLULAR ADAPTATIONS TO STRESS
• Adaptations
– reversible functional and structural responses
– a new but altered steady states is achieved
– allow the cell to survive and continue to function during changes in
physiologic states (e.g., pregnancy) and some pathologic stimuli
– It is a response by cells for physiologic stresses or pathologic stimuli

KEY POINTS: ADAPTATIONS

1. Adaptations are usually reversible changes that result in increased,
decreased, or altered functions of cells, tissues, and organs.
2. Adaptations can be physiologic or pathologic.
3. Adaptations may have many causes.
• Physiologic adaptations
– responses of cells to normal stimulation by
hormones or endogenous chemical mediators
e.g., the hormone-induced enlargement of the
breast and uterus during pregnancy
• Pathologic adaptations
– responses to stress that allow cells to modulate
/modify/ their structure and function and thus
escape injury
• Adaptation can be by:-
–Hypertrophy
–Atrophy
–Hyperplasia
–Metaplasia
 Hypertrophy
• it is increase in cell size resulting in increase in
the size of the organ
• Cellular enlargment is due to increased
synthesis of cell structural components and
organelles leads to an increase in organ size and
function.
• Causes
a. increased functional demand
• b. specific hormonal stimulation
Physiologic hypertrophy
• In the nonpregnant woman, the uterus is an almost-
solid structure weighing about 70 g and with a cavity of
10 mL or less.
• During pregnancy, the uterus is transformed into a
relatively thin-walled muscular organ of sufficient
capacity to accommodate the fetus, placenta, and
amnionic fluid.
• The total volume of the contents at term averages
about 5 L but may be 20 L or more.
• By the end of pregnancy, the uterus has achieved a
capacity that is 500 to 1000 times greater than in the
nonpregnant state.
• The corresponding increase in uterine weight is such
that, by term, the organ weighs approximately 1100 g.
 Pathologic hypertrophy
Pathological Hypertrophy

mechanical triggers, such as stretch, and trophic triggers, such as

activation of α-adrenergic receptors → induction of a number of
genes→stimulate synthesis of numerous cellular proteins, including
GF and structural proteins→synthesis of more proteins and
myofilaments per cell,➔ which achieves improved performance and
thus a balance between the demand and the cell's functional capacity
▪ Pure hypertrophy without any hyperplasia occurs only in the heart
and the skeletal muscle.
▪ Because the heart muscle is composed of terminally differentiated
myocytes that cannot divide, an increased demand for action can be
met only by enlarging the size of the muscle cells.
▪ Hyperplasia cannot occur in the heart.
▪ Skeletal muscles contain reserve cells that could theoretically divide
and contribute to the muscle mass, but this never occurs in normal
circumstances.
▪ Thus, a demand for additional work is met by muscle cell hypertrophy
 Hyperplasia
• it is an increase in the number of normal cells
that leads to an increase in the size of the organ
• Causes
a. Hypersecretion of trophic hormone
(e.g., GH →acromegaly)
b. Chronic irritation
(e.g.smoking→bronchial mucous gland hyperplasia)
c. Chemical imbalance
(e.g., hypocalcemia →parathyroid gland hyperplasia)
physiological - eg. enlargement of the breast during pregnancy
pathological - eg. endometrial hyperplasia.
• Hyperplasia can be physiologic or pathologic.
physiologic hyperplasia includes
• (1) hormonal hyperplasia
– eg. Enlargment of female breast at puberty and during
pregnancy
• (2) compensatory hyperplasia
- a type of hyperplasia that occurs when a
portion of the tissue is removed or diseased
eg. Hepatocyte hyperplasia when a liver is resected
pathologic hyperplasia are caused by excessive
hormonal or GF stimulation,
• Examples Growth factor
1. in balance between estrogen and progesterone
→ endometrial hyperplasia →abnormal
menstrual bleeding
2.the growth factors may be produced by papilloma
viruses or by infected cell→ hyperplastic
epithelium → skin warts and mucosal lesions
• Pathologic hyperplasia , if untreated may
developed to cancer
e \g. Endometrial hyperplasia to endometrial
cancer.
reaching 90% by the
eighth decade of life
excessive androgen-
dependent growth of
stromal and glandular
elements has a central
role. BPH does not
occur in males
castrated before the
onset of puberty or in
men with genetic
diseases that block
androgen activity.

BPH:-benign prostatic hyperplasia

 Atrophy
• Shrinkage in the size of the cell by the loss of cell substance
is known as atrophy.
• When a sufficient number of cells involved, the entire
tissue or organ diminishes in size, becoming atrophic
• atrophic cells may have diminished function, they are not
dead. Causes :
• 1. disuse
• 2. denervation
• 3. diminished blood supply /undernutrition
• 4. loss of endocrine stimulation
– .g., hypopituitarism causing atrophy of target
organs such as the thyroid
• 5. aging (senile atrophy).
 Cellular mechanisms of Atrophy
• 1. decreased protein synthesis because of
reduced metabolic activity
• 2. increased protein degradation due to
ubiquitin-proteasome pathway
• Nutrient deficiency and disuse → activate
ubiquitin ligases, → attach ubiquitin peptide
to cellular proteins→ proteasomes target
these proteins and degrade them
 Pupil intake factor

Necrotizing facility Tumor necrosis factor

 Metaplasia
• replacement of one fully differentiated cell type by another
• cells sensitive to a particular stress are replaced by other cell
types better able to withstand the adverse environment
• Metaplasia is thought to arise by genetic "reprogramming" of
stem cells rather than transdifferentiation of already
differentiated cells.
Is metaplasia reversible?
In most cases, metaplasia is reversible and the tissue reverts to its
normal state after the stimulus or irritant has been removed. If the
adverse circumstances persist, metaplasia may progress to dysplasia
and then to frank neoplasia, which is irreversible
• Types of metaplasia
• a. Squamous: replacement of columnar
epithelium by squamous epithelium e.g.,
squamous metaplasia of mainstem bronchus
• b. Glandular: replacement of squamous
epithelium with intestinal cells (goblet cells,
mucus secreting cells)
Eg . Barrett's esophagus
Barrett's esophagus:-squamous to columnar epithelium
due to chronic inflammation
It is replacement of squamous metaplasia of the
esophagus with intestinal cells (goblet cells, mucus
secreting cells)
• Advantage:
–Protective against inciting/inflaming
stimuli
• Disadvantage:
–Loss of functional capability of
original cell type
–Risk of cancerous transformation
Atrophy Hypertrophy Hyperplasia
• Increase in number of cells in tissues/
• Reduced size of an
• Increase in size and organ.
organ or tissue
function of cells • Results due to increase in growth
resulting from a
• Results due to factors, increased expression of growth
decrease in cell size
increase in growth promoting genes and increased DNA
and number.
factors or trophic synthesis.
• Caused by ischemia,
stimuli. • It persists so long as the stimulus is
ageing, malnutrition etc.
• Includes puberty, present.
• May result due to
lactating breasts and • e.g. breast development at puberty,
chronic absence of
skeletal muscle fibres endometrial hyperplasia, benign
stimulus (disuse
(in body builders). hyperplasia of prostate, hyperplasia of
atrophy).
liver cells after partial hepatectomy.

Metaplasia Dysplasia

• Reversible change in which one

differentiated cell type (epithelial or
• Abnormal multiplication of cells
mesenchymal) is replaced by another cell
characterized by change in size, shape
type.
and loss of cellular organization
• Results from “reprogramming” of stem
• The basement membrane is intact
cells that are known to exist in normal
• Can progress to cancer
tissues, or of undifferentiated
mesenchymal cells in connective tissue.
Features of Reversible cell
Features of irreversible cell injury
injury

• Large flocculent, amorphous densities in swollen

• Cellular swelling
mitochondria due to increased
• Loss of microvilli
calcium influx.
• Formation of cytoplasmic
• Swelling and disruption of lysosomes and leakage
blebs
of lysosomal enzymes in cytoplasm
• Endoplasmic reticulum
• Decreased basophilia
swelling
• Severe damage to plasma membranes
• Detachment of ribosomes
• Nuclear changes include
• Myelin figures
*Pyknosis (Nuclear condensation OR shiriking)
• Clumping of nuclear
*Karyorrhexis (Nuclear fragmentation)
chromatin
*Karyolysis (Nuclear dissolution)
 Cell injury
• Results when cells failed or unable to adapt to
stresses, injurious agents or intrinsic
abnormalities
• Injury may progress through a reversible stage
→ become irreversible → culminate /terminate/ in
cell death
 CAUSES OF CELL INJURY
A. Hypoxia: is decreased oxygen supply to tissues
• It is the most cause of cell injury.
❑It can be caused by:
• 1. Ischemia (decreased blood flow)
– most common cause to hypoxia
• 2. Anemia: reduction in O2 carrying RBCs
• 3. CO poisoning : decreases the oxygen-capacity
of RBCs by chemical alteration of hemoglobin
• 4. pulmonary disease: Poor oxygenation of blood
The causes of cell injury range from the gross physical trauma of a
motor vehicle accident to the single gene defect that results in a
defective enzyme underlying a specific metabolic disease.
B. Chemical Agents
includes: - Excess of innocuous substances
- poisons
- potentially toxic agents
- therapeutic drugs
• Mechanisms -- by altering
-membrane permeability
-osmotic homeostasis, or
-integrity of an enzyme or cofactor
C. Infectious Agents
– Range from submicroscopic viruses to meter-
long tapeworms
D.Immunologic Reactions
– Examples include autoimmune reactions and
allergic reactions against environmental
substances in genetically susceptible
individuals
E. Genetic Defects
• leads to cell injury by resulting deficiency of
functional protein
• It can result in gross pathologic changes
(eg.Down syndrome ) or microscopic (eg.sickle
cell anemia) alteration

Down syndrome(trisomy 21):- caused by the presence of all or

part of a third copy of chromosome 21.
 • F. Nutritional Imbalances
– deficiencies or excesses
• G . Physical Agents
– Trauma, extremes of temperatures, radiation,
electric shock …
• H. Aging
-Cellular senescence →decreased replicative and repair abilities of
individual cells and tissues→ diminished ability to respond to damage
→the death of cells and of the organism
▪ obesity markedly increases the risk for type 2 diabetes mellitus.
▪ Nutritional deficiencies remain a major cause of cell injury
▪ Excesses of nutrition can are also causes of morbidity and mortality
example : diets rich in animal fat →atherosclerosis
 MECHANISMS OF CELL INJURY
• GENERAL PRINCIPLES
1. The cellular response to injurious stimuli
depends on:
-type of injury,
-its duration, and
-severity
– low doses of toxins or a brief duration of
ischemia -> reversible cell injury.
– larger toxin doses or longer ischemic intervals
-> irreversible injury and cell death.
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 2 .The consequences of an injurious stimulus depend on
characterstics of cell
– A.Cell type : Sk.muscle Vs cardiac muscle
– B. Nutritional (or hormonal) status of cell:
• glycogen-replete hepatocyte tolerate ischemia
– C. Genetically makeup of cell
• individuals with polymorphisms in enzyme
genes → catabolize toxins with different
efficacies → different outcomes
▪ skeletal muscle show not irreversible injury after
complete ischemia for 2 to 3 hrs.
▪ But cardiac muscle dies after only 20 to 30 minutes.

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 3. Cellular function is lost far before cell death
occurs, and the morphologic changes of cell
injury (or death) lag far behind both.
• Onset of Injury→ viable but nonfunctional
cells → persistent injury →irreversible
biochemical alterations leading to cell death→
ultrastructural→ light microscopic→ grossly
visible morphologic changes.
4. The structural and biochemical components of a cell are so
integrally connected that regardless of the initial locus of injury,
multiple secondary effects rapidly occur.
poisoning of aerobic respiration with cyanide -> diminished activity of
the sodium-potassium ATPase -> cell swelling and rupture.
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Sequential development of biochemical and morphologic changes
• Figure 2-7 Sequential development of biochemical
and morphologic changes
in cell injury. Cells may become rapidly
nonfunctional after the onset of injury,
although they may still be viable, with potentially
reversible damage; a longer
duration of injury may lead to irreversible injury and
cell death. Note that
irreversible biochemical level alterations may cause
cell death→ ultrastructural changes→ light
microscopic changes→ grossly visible morphologic
changes.
• Example
• myocardial cells –
– non contractile after 1 to 2 minutes of
ischemia = function loss
– die after 30 minutes of ischemia= chemical level
– do not appear dead by ultrastructural
evaluation (electron microscopy) for 2 to 3
hours
– by light microscopy for 6 to 12 hours.

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4. Five intracellular systems are particularly vulnerable:
1. cell membrane integrity,
• critical to cellular ionic and osmotic homeostasis;
2. mitochondria or ATP generation,
• largely via mitochondrial aerobic respiration;
3. protein synthesis;
4. the integrity of the genetic apparatus.
5. the cytoskeleton

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The principal biochemical
mechanisms and sites of
damage in cell injury
Biochemical mechanisms of cell injury
• Cell injury results from different biochemical
mechanisms
• The biochemical alterations resulted
– Following damages of essential cellular
components; or
– simultaneously triggered by injurious agent that
causes the cellular damage
 Biochemical basis of cell injury…
1. Depletion of ATP
• Depletion of ATP to < 5% to 10% of normal levels has
widespread effects on many critical cellular systems.
• major causes :
– reduced supply of oxygen and nutrients
– mitochondrial damage, and
– the actions of some toxins (e.g., cyanide/CN-).
• Effect depends on glycolytic capacity of the tissue
(e.g. liver better survive than the brain)
Effects of ATP depletion
A. Anaerobic glycolysis is used for ATP synthesis and is
accompanied by:
i. Activation of phosphofructokinase caused by low
citrate levels and increased AMP
ii. Excess of lactate →decrease in intracellular pH
→decreased activity of many cellular enzymes
B. Impaired Na, K+ -ATPase pump, resulting in diffusion of
Na+ and H20 into cells and causing cellular swelling
 C. Impaired calcium (Ca 2+)-ATPase pump,
resulting in increased cytosolic Ca2+
D. Decreased protein synthesis, resulting in the
detachment of ribosomes from the rough EPR

Prolonged or worsening depletion of ATP causes structural

disruption of the protein synthetic apparatus, manifested as
detachment of ribosomes from the rough endoplastic
reticulum (RER) and dissociation of polysomes into
monosomes, with a consequent reduction in protein
synthesis. Ultimately, there is irreversible damage to
mitochondrial and lysosomal membranes, and the cell
undergoes necrosis.
2.Influx of Calcium
• Increased cytosolic Ca2+ leads to:
• Enzyme activation
– Phospholipase: increases membrane permeability
– Proteases: damages membrane & structural proteins
– Endonucleases: damages nuclear chromatin
– ATPases : hastens/accelerate/ ATP depletion
• Reentry of Ca2+ into mitochondria:
– increases mitochondrial membrane permeability, with
release of cytochrome C (activates apoptosis)
The importance of Ca2+ in cell injury was established by the finding that depleting
extracellular Ca2+ delays cell death after hypoxia and exposure to some toxins. Cytosolic
free calcium is normally maintained by ATP-dependent calcium transporters at
concentrations that are as much as 10,000 times lower than the concentration of
extracellular calcium or of sequestered intracellular mitochondrial and ER calcium
 Ionized calcium amplifies the adverse effects of hypoxia
by activating several enzymes:
Lytic ATPase: Degrades ATP & further reduces the energy store
Phospholipases: These enzymes remove phospholipids
from the plasma or mitochondrial membranes, further
impairing their functions
Proteases: These enzymes degrade cell membrane
or cytoskeletal proteins.
Endonucleases: These enzymes act on the RNA and DNA.
All of these changes are initially reversible, but if prolonged
or intensified they may lead to irreversible cell injury
 Figure 1-2. Sources and consequences of increased cytosolic calcium in cell injury. ATP,
adenosine triphosphate. (From Kumar V, Cotran RS, Robbins SL: Robbins and Cotran
Pathologic Basis of
Disease, 7th ed. Philadelphia, Saunders, 2005, p. 8.)

Where come from these enzymes?

3. Damage to Mitochondria
• There are two major consequences of
mitochondrial damage :
A. Formation of mitochondrial permeability
transition pore
– which leads to the loss of mitochondrial membrane
potential and pH changes → failure of oxidative
phosphorylation→ progressive depletion of ATP, →
culminating/ending in necrosis of the cell
B. leakage of cytochrome C into the cytosol →death
by apoptosis
4. Free radicals accumulation
• Free radicals are chemical species with a single
unpaired electron in an outer orbital
• Such chemical states are extremely unstable and
readily react with inorganic and organic chemicals
mainly
• When generated in cells they avidly attack nucleic
acids as well as a variety of cellular proteins and
lipids
➢ The role of reactive oxygen species (ROS) in cell injury
O2 is converted to superoxide by oxidative enzymes in the
endoplasmic reticulum, mitochondria, plasma membrane,
peroxisomes, and cytosol ,is converted to H2O2 by
dismutation and thence to OH• by the Cu2+/Fe2+-catalyzed
Fenton reaction.
➢ H2O2 is also derived directly from oxidases in
peroxisomes.
➢ The major antioxidant enzymes are superoxide
dismutase (SOD), catalase, and glutathione peroxidase.
➢ Cell injury in many circumstances involves damage by free
radicals; these situations include ischemia-reperfusion ,
chemical and radiation injury, toxicity from oxygen and
other gases, cellular aging, microbial killing by phagocytic
cells, and tissue injury caused by inflammatory cells.
• Reactive oxygen species (ROS)
– oxygen-derived free radical
– has a well established role in cell injury
– produced normally in cells during mitochondrial
respiration and energy generation, but they are
degraded and removed by cellular defense systems.
• When the production of ROS increases or the
scavenging systems are ineffective, the result is
an excess of these free radicals, leading to a
condition called oxidative stress
Reactions responsible for generation of free radicals includes:
A. The reduction-oxidation (redox) reactions that occur during
normal mitochondrial metabolism
– eg partial reduction of oxygen; these include superoxide
radicals , H2O2 and OH•.
B.Transition metals such as Cu and iron accept or donate free
electrons during certain intracellular reactions and thereby
catalyze free-radical formation,
C. The absorption of Ionizing radiation (e.g., UV light, x-rays) can
hydrolyze water into hydroxyl (OH•) and hydrogen (H•) free radicals
H2O UV, X-rays OH• +O•
D. The enzymatic metabolism of exogenous chemicals e.g., carbon
tetrachloride(CCl4;
E. Inflammation: free radicals are produced by leukocytes that enter
tissues
 Mechanisms to remove free radicals
1.Superoxide dismutases neutralize superoxides
– o2.- → H2O2 + O2
2. Catalase
– directs the degradation of hydrogen peroxide 2H2O2 → O2 + 2H2O
3.Glutathione peroxidase
– catalyzing free-radical breakdown:
+ 2GSH → 2H2O + GSSG (glutathione homodimer)
4. Endogenous or exogenous antioxidants (e.g., vitamins E, A, and C, and β-
carotene)
– may either block formation of free radicals or scavenge them once they
have formed.
5. Storage and transport proteins (e.g., transferrin, ferritin, lactoferrin, and
ceruloplasmin)
– bind, iron and copper ions thereby decreasing formation of ROS.
 Consequences of free radical injury
• Three reactions are relevant to cell injury mediated by free
radicals:
1. Lipid peroxidation of membranes.
• Double bonds in membrane polyunsaturated lipids.
• The lipid-radical interactions -> peroxides.
2. DNA fragmentation.
– reactions with thymine -> single-strand breaks.
– cell death, aging, and malignant transformation of cells.
3. Cross-linking of proteins.
– sulfhydryl-mediated protein cross-linking,
• degradation or loss of enzymatic activity.
– directly cause polypeptide fragmentation.

90
Mechanisms of reperfusion injuries
1. increased generation of ROS :
reoxygenation → incomplete reduction of oxygen by
the damaged mitochondria of parenchyma cells and
endothelial cells and from infiltrating leukocytes.
2.Ischemic injury is associated with inflammation, which
may increase with reperfusion because of increased
influx of leukocytes and plasma proteins
– The products of activated leukocytes may cause
additional tissue injury
• Free radical damage also underlies
– Chemical and Radiation injury,
– Toxicity from oxygen and other gases,
– Cellular aging,
– Microbial killing by phagocytic cells,
– Inflammatory cell damage,
– Tumor destruction by macrophages,etc
➢Ischemia-Reperfusion Injury
• If cells are reversibly injured by ischemia, the
restoration of blood flow has two possible effects
• 1. cell recovery-- in most cases
• 2. a paradoxical exacerbated /worsened/ injury-
occasionally
– This is so-called ischemia-reperfusion injury
– It contribute significantly to tissue damage in
myocardial and cerebral infarctions.
THE MORPHOLOGY OF CELL AND TISSUE INJURY

• Noxious stimuli → molecular or biochemical

alterations → cellular function lost →
morphologic changes of cell injury or death
• For example, :
• ischemic myocardial cells →become non
contractile → then die → appear dead by EM
then LM
➢ All stresses and noxious influences exert their effects first at the
molecular or biochemical level Cellular function may be lost long
before cell death occurs, and the morphologic changes of cell injury
(or death) lag far behind both
For example,
▪ myocardial cells become noncontractile after 1 to 2 minutes of
ischemia,
▪ Although they do not die until 20 to 30 minutes of ischemia have
elapsed.
▪ These myocytes do not appear dead by electron microscopy for 2
to 3 hours, and
▪ By light microscopy for 6 to 12 hours.
NB:- cells may rapidly become nonfunctional after the onset of injury,
although they are still viable, with potentially reversible damage; a
longer duration of injury may eventually lead to irreversible injury and
cell death.
NB:- cell death typically precedes/come before
Morphologic correlates of reversible cell injury
A. Cellular swelling
• Also called hydropic change or vacuolar degeneration
• The 1st manifestation of almost all forms of injury to cells
• it is the result of failure of energy-dependent ion pumps in
plasma membrane, leading to an inability to maintain ionic and
fluid homeostasis
• difficult to appreciate it with the light microscope.

• Grossly
– pallor, increased turgor, and increase in weight of the organ.
• Microscopic examination may reveal
– small, clear vacuoles within the cytoplasm;
• Which are distended and pinched-off segments of ER
Injured cells may also show increased eosinophilic staining, which
becomes much more pronounced with progression to necrosis
B. Fatty change
• manifested by the appearance of lipid
vacuoles in the cytoplasm.
• It occurs mainly in cells involved in
and dependent on fat metabolism,
such as hepatocytes and myocardial
cells.
• Ultrastructural changes of reversible cell injury
– (1) Plasma membrane alterations
• such as blebbing, loosening of
intercellular attachments
– (2) Mitochondrial changes such as swelling
– (3) Dilation of the ER with detachment of
ribosomes and dissociation of polysomes;
– (4) Nuclear alterations, with chromatin
clumping
 Sub cellular Alterations in Cell Injury
• It is distinctive alterations involving only sub cellular
organelles
• Some forms of cell injury affect particular organelles
and have unique manifestations
• The causes includes:
– acute lethal injury,
– chronic forms of cell injury, and
– adaptive responses

Subcellular Alterations in Cell Injury: Effects of Injurious Agents on Organelles and

Cellular Components.
Thus far we have mainly focused on the whole tissue or the cell as a unit
Sub cellular Alterations in Cell Injury
1. Lysosomal aaccumulations
2. Induction of Smooth ER
3. Mitochondrial Alterations
4.Cytoskeletal Abnormalities
1. Lysosomal aaccumulations
• 2ry lysosomes, or phagolysosomes with
– Lipofuscin pigment granules (during
autophagy)
– indigestible pigments,
– Hereditary lysosomal storage disorders

104
Autophagy
• It is lysosomal digestion of the cell's own component
• Intracellular organelles and portions of cytosol are first
sequestered from the cytoplasm in an autophagic vacuole formed
from ribosome-free regions of the RER
• The vacuole fuses with lysosomes to form an autophagolysosome,
and the cellular components are digested by lysosomal enzymes.
• it is initiated by several proteins that sense nutrient deprivation
• Lysosomes with undigested debris may persist within cells as
residual bodies or may be extruded
Examples
-- Lipofuscin pigment : indigestible material resulting from free
radical-mediated lipid peroxidation
2. Induction of Smooth ER
• SER is involved in chemicals metabolism thus cells exposed to
chemicals may respond with an adaptive hypertrophy of the ER
• Example:
– barbiturates metabolized in liver by the cytochrome P-450 in the
SER
– Prolonged use of barbiturates leads to a state of tolerance due to
increased volume (hypertrophy) of the SER of hepatocytes and
increased P-450 enzymatic activity

Abundant SER can be seen in the light

microscope as :
a ground-glass appearance on
cytoplasm of affected hepatocytes.
3. Mitochondrial Alterations
• Nonlethal pathologic conditions may result alterations in the
- number,
- size,
- shape, of mitochondria.
- function
➢ For Example,
– Cellular hypertrophy-increase no. of mitochondria
– cellular atrophy - decrease number of mitochondria
– nutritional deficiencies and alcoholic liver disease –
megamitochondria of hepatocytes
4.Cytoskeletal Abnormalities
– Nonpolymerized and nonfilamentous contractile proteins -> cellular
scaffolding.
• Actin and myosin filaments, Microtubules, and Intermediate filaments;
– Functions
• Maintenance of basic cell architecture
• Conveying cell-cell and cell-extracellular matrix signals to the nucleus
• Cell mobility ,Phagocytosis,..
• increase or decrease in cytoskeletal elements.
– Hypertrophy and atrophy
• functional organization abnormalities
– hypertrophic cardiomyopathy,
– aberrant movements of intracellular organelles,
– defective cell locomotion,etc

110
 Morphologic correlates of irreversibility
• Two phenomena consistently characterize irreversibility:
1 .The inability to reverse mitochondrial dysfunction (lack of oxidative
phosphorylation and ATP generation) even after resolution of the original
injury, and
2. profound disturbances in membrane function
Cell death
 Necrosis
• Necrosis is death of groups of cells, often
accompanied by an inflammatory infiltrate

• largely resulting from the degradative action of

enzymes on lethally injured cells derived from
– the lysosomes of the dying cells themselves or
– from the lysosomes of leukocytes that are recruited as
part of the inflammatory reaction to the dead cells

• Necrotic cells are unable to maintain membrane

integrity, and their contents often leak out
Morphology of necrotic cells
• They show increased eosinophilia
• have a more glassy homogeneous appearance
• Cytoplasm becomes vacuolated
• myelin figures accumulated, that are derived
from damaged cellular membranes replace the
dead cells.
• Calcifications result from membrane
phospholipid precipitates and degraded into
fatty acids results in the generation of calcium
soaps calcification of dead cells
• Ultrastructure morphologic changes of necrotic cells
– Discontinuities in plasma and organelle membranes
– Marked dilation of mitochondria with the appearance of large
amorphous densities
– Disruption of lysosomes
– Intracytoplasmic myelin figures, and
– Profound nuclear changes includes karyolysis, pyknosis, karyorrhexis

Nuclear changes: - due to nonspecific DNA

breakdown
• Pyknosis = nuclear
condensation(shrinkage) and ↑
basophilia
• Karyorrhexis [rhexis, rupture]= nuclear
fragmentation
• Karyolysis= loss of DNA → ↓ basophilia
 Patterns of Tissue Necrosis
1. Coagulative necrosis
• when component of cells are dead but the basic
tissue architecture is preserved
• Injury→ denatures enzymes (in addition to
structural proteins)→ so blocks the proteolysis of
the dead cells→ anucleated cells may persist for
days or weeks→ architecture preserved.
• Coagulative necrosis is characteristic of infarcts
(areas of ischemic necrosis) in all solid organs
except the brain
Figure 1-10 Coagulative necrosis A, A wedge-shaped kidney
infarct (yellow) with preservation of the outlines. B,
Microscopic view of the edge of the infarct, with normal
kidney (N) and necrotic cells in the infarct (I). The necrotic
cells show preserved outlines with loss of nuclei, and an
inflammatory infiltrate is present (difficult to discern at this
magnification). i. most often results from sudden interruption
of blood supply to an organ, especially to the heart.
2. Gangrenous necrosis
• not a distinctive type of necrosis but commonly
used in clinical practice to a limb that has lost its
blood supply and undergone coagulative necrosis
• when bacterial superinfection is superimposed
coagulative necrosis is modified by the liquefactive
action of the bacteria and the attracted leukocytes
(so-called wet gangrene).
➢Gangrenous necrosis can be either
- dry
- wet

118
 1. Dry Gangrene
• Is associated with obstruction of the arterial blood supply with out
interference of the venous return
• Is confined to the extremities
• The involved part become dry, skin wrinkle
• The color change to dark brown or black
• The spread is slow
• There is a line of demarcation
• Can be changed to wet gangrene, if bacterial infection

2. Wet Gangrene
• Is primarily due to interference of the venous return
• There is bacterial invasion
• Can affect the extremities or even the internal organs
• The involved part is cold, swollen & pulseless
• The skin is moist, foul smelling & black
• No line of demarcation
• Spread of tissue is rapid
• Can be fatal
 Diabetic
gangrene

Bowel-gangrenous

120
3. Liquefactive necrosis
• It Is necrotic degradation of tissue that softens and becomes liquified
• Mechanism
• microbes → stimulate the accumulation of inflammatory cells → enzymes of
leukocytes digest the tissue→ formation of liquid viscous mass
Examples
• 1. CNS infarction: autocatalytic effect of hydrolytic enzymes generated by
neuroglial cells produces a cystic space
2. Abscess in a bacterial infection: hydrolytic enzymes generated by neutrophils
liquefy dead tissue
4. Caseous necrosis
• The necrotic area appears friable yellow-white or cheese-like thus
called "caseous"
– formed by the release of lipid from the cell walls of Mycobacterium
tuberculosis and systemic fungi (e.g., Histoplasma) after destruction
by macrophages.
• tissue architecture is completely obliterated /eliminate and cellular
outlines cannot be discerned/separated
• Microscopic features:
– The acellular caseous material in the center usually surrounded by
distinctive inflammatory border known as a granuloma
5. 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
• It is associated with acute pancreatitis
• Mechanisms
(1) Activation of pancreatic lipase (e.g., alcohol excess): hydrolysis of triacylglycerol in fat
cells
(2) Conversion of fatty acids into soap (saponification): combination of fatty acids and
calcium
• Gross appearance:
– chalky yellow-white deposits
(fat saponification)
– primarily located in
- peripancreatic
-omental adipose tissue.
• Microscopic appearance: pale outlines
of fat cells filled with basophilic-
staining calcified areas
• Surrounded by an inflammatory
reaction Shadowy outlines of necrotic fat cells,
with basophilic calcium deposits,
surrounded by an inflammatory reaction
• Non enzymatic fat necrosis
– results from hypoxic necrosis or mechanical injury to fat cells.
– The fat liquefies at body temperature and is released as an oily
mass, resulting in formation of oil cysts.
Eg. -In the breast following trauma
-In subcutaneous tissue

6. 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 "fibrinoid" (fibrin-like) by pathologists
• The immunologically mediated diseases (e.g., polyarteritis nodosa).

124
 Types of necrosis
Coagulative Liquefactive Caseous Fibrinoid Gangrenous
Fat necrosis
necrosis necrosis necrosis necrosis necrosis
(Surgically used
term; necrosis of
tissue with
superadded
– Most common – Complexes of putrefaction)
type of antigens and –Dry gangrene is
necrosis antibodies are similar to
Enzymatic Combination of
– Loss of deposited in coagulative
destruction of coagulative – Action of
nucleus with vessel wall necrosis
cells and liquefactive lipases on – Wet gangrene
the cellular – Abscess necrosis fatty tissue
with leakage
outline being of fibrinogen is similar to
formation –Characteristic – Seen in liquefactive
preserved – Pancreatitis of TB breast,
out of vessels
–Associated – Seen in PAN necrosis and is
– Seen in brain – Cheese like omentum
with ischemia Aschoff due to secondary
– Seen in - Wet gangrene appearance and
bodies (in infection
of extremities of the necrotic pancreatitis – Noma is
organs (heart, rheumatic
material. gangrenous
liver, kidney heart disease)
etc.) except and malignant lesion of vulva or
BRAIN. hypertension. mouth(cancrumor
is)
– Fournier’s
gangrene is seen
in scrotum
Figure 2-15 Fibrinoid necrosis in an artery. The wall of the artery
shows a circumferential bright pink area of necrosis with
inflammation (neutrophils with dark nuclei).
 Enzyme markers of cell death
• Leakage of intracellular proteins through the damaged cell membrane and ultimately into
the circulation provides a means of detecting tissue-specific necrosis using blood or
serum samples
– Cardiac muscle contains a unique isoform of the enzyme creatine kinase and troponin(contractile
protein),
– hepatic bile duct epithelium contains alkaline phosphatase
– hepatocytes contain transaminases
• Tissues release certain enzymes that indicate the type tissue involved and
extent of injury.
• Irreversible injury and cell death in these tissues are reflected in increased
serum levels of such proteins, and measurement of serum levels is used
clinically to assess damage to these tissues
• Aspartate aminotransferase(AST)
-- Marker of diffuse liver cell necrosis (e.g., viral hepatitis)
• Alanine aminotransferase (ALT)
-- Marker of diffuse liver cell necrosis (e.g., viral hepatitis)
-- More specific for liver cell necrosis than AST
• Creatine kinase MB (CK-MB)
-- Isoenzyme elevated in acute MI or myocarditis
• Amylase and lipase
-- Marker enzymes for acute pancreatitis
-- Lipase more specific than amylase for pancreatitis
 What is the outcome of necrosis?

• Complete restitution: This process is called regeneration, and the

dead cells are replaced by almost parenchymal cell. Regeneration
occurs in organs composed of facultative mitotic cells, such as the
kidneys or liver.3
• Repair: The dead cells are replaced by fibrous tissue forming
microscopic or macroscopic scars. For example, in the heart dead
myocardial cells are removed by phagocytes and replaced by a
fibrous scar.
• Calcification: In some instances, the necrotic tissue is impregnated
with calcium salts (dystrophic calcification).
• Resorption of necrotic tissue: In the brain, the necrotic tissue is
removed by macrophages, and the infarct is transformed into a
fluid-filled pseudocyst
 APOPTOSIS
• a pathway of cell death that is induced by a tightly
regulated suicide program
• cells destined to die activate enzymes capable of
degrading the cells' own nuclear DNA and cellular
proteins.
• (apoptosis, "falling off").
– fragments of the apoptotic cells then break off,
Apoptosis may be initiated through several pathways. The two most important
pathways are:
Extrinsic pathway: This pathway is activated by the activation of the so-called
death receptors on the surface of the cell membrane.
• Ligands for these receptors are proteins such as tumor necrosis factor or Fas
ligand.
Intrinsic mitochondrial pathway: This pathway is initiated by an increased
permeability of mitochondria, which release pro-apoptotic molecules, such as
cytochrome, that act on the initiator caspases, such as the extrinsic pathway.
• Many other mechanisms can initiate apoptosis, such as radiation, drugs,
hormones, immune mechanisms (e.g., cytotoxic T lymphocytes). Withdrawal of
hormones and growth factors essential for the survival of the cell can also
cause apoptosis.
• For example, castration is accompanied by a decrease of testosterone
concentration in the blood, which leads to apoptosis of prostatic cells.
• The plasma membrane of the apoptotic cell
– remains intact but altered in such a way that the cell
and its fragments become avid targets for phagocytes.
Therefore
• dead cell is rapidly cleared before its contents have leaked out
• cell death by appoptosis does not elicit an inflammatory
reaction in the host.
Apoptosis differs from necrosis
• Necrosis; which is characterized by loss of membrane
integrity, enzymatic digestion of cells, leakage of cellular
contents, and frequently a host reaction
• However, apoptosis and necrosis sometimes coexist, and
apoptosis induced by some pathologic stimuli may
progress to necrosis.
 Irreversible cell injury may be necrosis or apoptosis (Programmed cell death)

Necrosis Apoptosis

• May be physiological or
• Always pathological
pathological
• Important for development,
• Associated with disruption of cellular
homeostasis
homeostasis
& elimination of pathogens & tumor
(e.g. ischemia, hypoxia & poisoning)
cells

• Affects contiguous (adjacent) group of cells • Affect single cells

• Cell size is increased • Cell size is shrunken

• Passive • Active

• Causes inflammatory reaction • No inflammatory reaction

• Plasma membrane is disrupted • Plasma membrane is intact

• ’Smear pattern’ on electrophoresis • Step ladder pattern is seen

Differences between apoptosis and necrosis

Feature Necrosis Apoptosis

Cell size Enlarged (swollen) Reduced (shrink)

Fragmentation into nucleosome-

Nucleus Pyknosis/Karyorrhexis/Karyolysis
sized fragments

Intact; altered structure,

Plasma membrane Disrupted
especially orientation of lipids

Enzymatic digestion; may leak Intact; may be released in

Cellular contents
out of cell apoptotic bodies

Adjacent inflammation Frequent No

Often physiologic. May be

Invariably pathologic (culmination
pathologic after cell injury as
Role in body of
DNA
irreversible cell injury)
damage.

Bak; Bax; Bim; P53 gene;

Caspases TNFRI; Fas [CD95];
Proapoptotic
FADD
(Fas associated death domain)

Bcl-2/bcl-X; FLIP; Apaf-1

Anti-apoptotic (Apoptosis activating factor-1)
Cytochrome C
 Physiologic role of apoptosis:
1. The programmed destruction of cells during embryogenesis, .
2. Involution of hormone-dependent tissues upon hormone deprivation
3. Cell loss in proliferating cell population to maintain a constant number,
4.Death of cells that have served their useful purpose,
such as neutrophils and lymphocytes at the end of an immune response. In these
situations, cells undergo apoptosis because they are deprived of necessary
survival signals, such as growth factors
5. Elimination of potentially harmful self-reaction lymphocytes.
6. Cell death induced by cytotoxic T lymphocytes,
– to kill and eliminate virus-infected and neoplastic cells.
➢Apoptosis in Pathologic Conditions :
1. DNA damage
– Radiation, cytotoxic anticancer drugs and hypoxia can damage DNA,
– either directly or via production of free radicals
– If repair mechanisms cannot cope with the injury, the cell triggers intrinsic
mechanisms that induce apoptosis
NB :-larger doses of the same stimuli result in necrosis.
2. Accumulation of misfolded proteins ,resulted due to:
-mutations in the genes encoding proteins or free radicals damage
-Excessive accumulation of these proteins in the ER → ER stress→ apoptotic
death of cells
3. Cell injury in certain infections,particularly viral infections,
– Apoptosis induced by
• the virus (as in adenHIV infections) or
• the host immune response (as in viral hepatitis)
4. Pathologic atrophy in parenchymal organs after duct obstruction,
– such as occurs in the pancreas, parotid gland, and kidney
 EXAMPLES Of APOPTOSIS

Physiological conditions Pathological conditions

1. Endometrial cells 1. Councilman bodies:

(Menstruation) Viral hepatitis
2. Cell removal during 2. Gland atrophy following
embryogenesis duct obliteration as in
3. Virus infected cells and cystic fibrosis
Neoplastic cells by 3. Graft versus host
cytotoxic T cells disease (GVHD
 Morphology of Apoptosis
• In H&E-stained tissue sections, apoptotic cells
appear as :
– round or oval masses with intensely eosinophilic
cytoplasm
– The cells rapidly shrink, form cytoplasmic buds,
and fragment into apoptotic bodies composed of
membrane-bound vesicles of cytosol and
organelles
– Nuclei show chromatin condensation and
aggregation and, ultimately, karyorrhexis;
Apoptosis of an
epidermal cell in an
immune reaction.
The cell is reduced
in size and
contains brightly
eosinophilic
cytoplasm and a
condensed nucleus
 Mechanism of Apoptosis
• The fundamental event in apoptosis is the activation of caspase
enzymes
• Activated caspases cleave numerous targets,
culminating/ending/ in activation of nucleases that degrade
DNA and other enzymes that presumably destroy
nucleoproteins and cytoskeletal proteins
• the activation of caspases depends on a finely tuned balance
between pro- and anti-apoptotic molecular pathways.
• Two distinct pathways for caspase activation
– the mitochondrial pathway and
– the death receptor pathway
• Both differ in their
- induction and
- regulation,
But both culminate/end/ in the activation of "executioner“/killer/ caspases
 Mitochondrial (Intrinsic) Pathway of Apoptosis
MECHNISMS or steps
→ Injurious agent damage DNA or accumulation of misfolded ptn
→ activation of pro apoptotic members of BCL2 family (Bax,Bak)
→ inserted into mitochondrial membrane to form channel
→ cytochrom C and other ptns escape to cytosol
→ activate caspases
→ nuclear fragmentation.
Death Receptor (Extrinsic) Pathway of Apoptosis
• Many cells express surface molecules,called death receptors, that trigger apoptosis.
• Most of these are members of the tumor necrosis factor (TNF) receptor family
• Fas-ligand (FasL) is a membrane protein expressed mainly on activated T
lymphocytes.
• When these T cells recognize Fas-expressing targets, Fas molecules are cross-linked
by the FasL and they bind adapter proteins, which in turn bind caspase-8.
• Fas ligand expressed on activated T cells --> bind to death receptor (fas and
TNF)expressing cells --> bind caspase 8--> clustering of many caspases-->initiate
caspase cascade
TRAIL:-TNF-related apoptosis
inducing ligand
 Clearance of Apoptotic Cells
• The mechanisms facilitates clearance of
apoptotic calls by phagocytes
– expression of phosphatidylserine PL on the outer
layer of cell membrane →recognized by
macrophages.
– apoptotic cells secrete soluble factors that recruit
phagocytes
– macrophages may produce proteins that bind to
apoptotic cells (but not to live cells) and target the
dead cells for engulfment.
 Reading assignment
• Necroptosis and Pyroptosis

• Intracellular Accumulations

• aging
 Intracellular Accumulations
• Occur either in the cytoplasm (typically lysosomes),
or in the nucleus.
• harmless or may cause varied degrees of injury.
• The substance may be synthesized by the affected
cells or may be produced elsewhere.
• Three general pathways for abnormal intracellular
accumulations :
1. Abnormal or inadequate metabolism. ex. Fatty liver
2. genetic or acquired defects in packaging, transport, or
secretion of the proteins. Ex, protein accumulation
3. Ingestion of abnormal indigestible exogenous substance.
–leads to accumulation of exogenous substance 147
Substances accumulating inside the cells can be classified as exogenous or
endogenous.
Exogenous substances include, for example, carbon particles inhaled from polluted
air accumulating in macrophages in anthracosis or pigment used for tattooing of
skin.

Endogenous substances accumulating in cells are as follows:

Lipids: Accumulation of triglycerides in hepatocytes leads to
formation of fatty liver (hepatic steatosis).
Proteins: Accumulation of a1-antitrypsinin hepatocytes results in the
formation of cytoplasmic globules.
Glycogen: Accumulates in the liver and many other organs in
congenital glycogenoses.
Lipofuscin: This lipid-rich brown pigment of aging accumulates in
many organs during aging and in chronic diseases.
Hemosiderin: This iron-rich brown pigment accumulates in the liver
and many other organs in congenital hemochromatosis or in patients
who have received multiple transfusions.
 Fatty Change (Steatosis)
• abnormal accumulation of TG within
parenchymal cells.
• seen in liver, heart, sk.muscle,kidney
• may be caused by
– toxins, protein malnutrition, DM, obesity, and
anoxia.
• alcohol abuse is most common cause of
fatty change in liver (fatty liver) in west

151
Free fatty acids from adipose tissue or
ingested food

152
• May result from defects at any step from
fatty acid entry to lipoprotein exit.
• Decreasing synthesis of apoprotein: eg. CCl4 and protein
malnutrition
• Inhibition of fatty acid oxidation: eg. Anoxia
• Increased fatty acid mobilization from peripheral stores:
Starvation, DM /diabetes mellitus
• Alcohol lead to
- increased synthesis
-reduced breakdown of lipids

153
• The significance of fatty change depends on the
cause and the severity of accumulation.
• mild - no effect on cellular function.
• severe fatty - transiently impair cellular function.
• fatty change is reversible.
NB:- In a severe form, fatty change may precede cell
death
MORPHOLOGY
• clear vacuoles within parenchymal cells.
• Sudan IV or oil red O - stain fat orange-red.
• PAS - stains glycogen red-violet.
• If vacuoles don't stain for either fat or glycogen -> water.

154
 Morphology….
• Fatty accumulations identified by staining with Sudan IV or oil red
O (these stain fat orange-red)
• Glycogen may be identified by staining for polysaccharides using
the periodic acid-Schiff stain (which stains glycogen red-violet)
• If vacuoles do not stain for either fat or glycogen, they are
presumed to be composed mostly of water
• Early fatty change is seen by light microscopy as small fat vacuoles
in the cytoplasm around the nucleus.
• In later stages, the vacuoles coalesce to create cleared spaces that
displace the nucleus to the cell periphery
• Occasionally contiguous cells rupture, and the enclosed fat
globules unite to produce so-called fatty cysts
• With increasing accumulation, the organ enlarges and appear
bright yellow, soft, and greasy.

155
 FATTY CHANGE OF THE LIVER
• Gross - weigh 3-6 kg
and appear bright
yellow, soft, and
greasy.
• Light microscopy –
microvesicles or
macrovesicles.
the THREE common causes of fatty liver, also called fatty
metamorphosis, also called fatty change, also called steatosis:
1)Obesity
2)Alcoholism
3)Diabetes
156
157
 Cholesterol and Cholesteryl Esters.
• Scavenger macrophages phagocytozed lipid debris
of necrotic cells or abnormal (e.g., oxidized) forms
of plasma lipid.
• Atherosclerosis
• Xanthoma:-deposition yellowish cholesterol rich material appearing any w/r in the
body in various diseased state

• Gross – the lesion is yellow in

color in both atherosclerosis
and xanthoma.
• Microscopy – macrophages
filled with minute, membrane-
bound vacuoles of lipid(foam
cells).
158
 Proteins accumulation
• Less common than lipid accumulations
• occur because:
-excesses are presented to the cells
- the cells synthesize excessive amounts.
– Nephrotic syndrome
– Plasma cells
Histology
• Nephrotic syndrome
– pink, hyaline cytoplasmic droplets
• Plasma cells,
– rounded, eosinophilic Russell bodies.
• Alcoholic liver disease
– the Mallory body, or "alcoholic hyalin“
• Alzheimer disease
– neurofibrillary tangle

➢ In some disorders, such as certain forms of amyloidosis,

abnormal proteins deposit primarily in extracellular spaces 159
 Protein reabsorption droplets in the renal
tubular epithelium. (Courtesy of Dr. Helmut Rennke, Department of Pathology,
Brigham and Women's Hospital, Boston, Massachusetts.)
 Glycogen accumulation
• abnormalities in metabolism of either glucose or
glycogen.
• In poorly controlled diabetes mellitus,
– aberrant glucose metabolism,
– renal tubular epithelium, cardiac myocytes,
and beta cells of the islets of Langerhans.
• Glycogen storage diseases, or glycogenoses
– enzymatic defects in the synthesis or
breakdown of glycogen
– secondary injury and cell death.

161
 Pigments accumulation
• Pigments are colored substances
• exogenous,
–Carbon
–tattoo
• endogenous.
–Lipofuscin
–melanin
–hemosidern
162
 A. Carbon ( eg. coal dust)
• The most common exogenous pigment from polluted air.
• When inhaled, it is phagocytosed by alveolar macrophages and
transported through lymphatic channels to the regional
tracheobronchial lymph nodes.
• Aggregates of the pigment blacken the draining lymph nodes and
pulmonary parenchyma (anthracosis)
• Heavy accumulations may induce emphysema or a fibroblastic
reaction that can result in a serious lung disease called
coal workers' pneumoconiosis
Anthracosis
• Inhaled carbon -> phagocytosed by alveolar macrophages
and transported through lymphatic channels to the regional
tracheobronchial lymph nodes -> aggregates of the pigment -
> blackenening the draining lymph nodes and pulmonary
parenchyma.
• Heavy accumulations may induce coal workers‘
pneumoconiosis. 163
 ANTHRACOSIS

Why does anthracosis look worse along the pleural surface than on
cut sections?
Why are MANY extrathoracic lymph nodes also anthracotic?
What in the body is black and NOT due to EXOGENOUS pigments?
164
 ንቅሳት

TATTOO, MICROSCOPIC

• This tiny amount of microscopic tattoo pigment can make white skin
look quite black!
➢ Is this exogenous or endogenous?
▪ The pigments inoculated are phagocytosed by dermal macrophages,
in which they reside for the remainder of the life of the embellished.
• sometimes with embarrassing consequences for the bearer of the
tattoo when proposing to Mary while the tattoo says Valerie!
165
 B. Melanin
• an endogenous, brown-black pigment
• synthesized exclusively by melanocytes by oxidation of
tyrosine to dihydroxyphenylalanine(DOPA).
Freckles- ↑ melanocytes
Nevus neoplasias & hyperplasia of melanocytes
melanoma – malignant growth of melanocytes

Vitiligo – ↓melanocytes

Albinism defected melanocytes

• Acts as a screen against harmful ultraviolet radiation.

• Although melanocytes are the only source of melanin,
adjacent basal keratinocytes in the skin can accumulate
the pigment (e.g., in freckles), as can dermal
macrophages. 166
 Slide -- Melanoma

The number of melanocytes in normal skin is constant in all races,

the ratio being one melanocyte for every 4 to 10 basal keratinocytes.
Thus, the color of the skin is determined by the number and size of melanosomes
present both in keratinocytes and melanocytes”and not by the No’ of melanocytes.
➢ The number of melanocytes decreases with age.
Skin becomes lighter in color and the incidence of skin cancer increases.
Melanocytic disorders
A decrease or absencent number of melanocytes == vitiligo.
An increase in pigment donation to adjacent keratinocytes == freckle
A defect in the synthesis of melanin (normal number) == albinism
Melanocytic hyperplasia seen In lentigo, benign and malignant melanocytic neoplasm,

168
169
 Lipofuscin
• pigmented material formed of an oxidized
membrane lipid
• periodic acid-Schiff (PAS) and fat positive
• accumulates with age thus known as "wear and
tear" pigment.
• Its Presence implies that the cell is
– slow growing and benign
 Hemosiderin
• a hemoglobin-derived granular pigment that is
golden-yellow to brown.
• represents large aggregates of ferritin micelles.
• accumulates in tissues when there is a local or
systemic excess of iron.
• Local excesses of iron - hemorrhage.
• systemic overload of iron (hemosiderosis)
– (1) increased absorption of dietary iron,
– (2) impaired utilization of iron,
– (3) hemolytic anemias, and
– (4) transfusions 171
Figure 14-22 Regulation of iron absorption
Hemosiderin -- micro

173
 Hemosiderin/Melanin/etc.

Why would somebody order a prussian blue

stain, or a S-100 immunoperoxidase stain or a
HMB-45 stain? 174
 Hemosiderosis
• first - the mononuclear phagocytes of the
liver, bone marrow, spleen, and lymph
nodes and macrophages in other organs.
• Later - parenchymal cells ( the liver,
pancreas, heart, and endocrine organs)
• No organ dysfunction.
• Extensive accumulations of iron + tissue
injury (liver fibrosis, heart failure, and
diabetes mellitus) -> hemochromatosis.
175
 endogenous Pigments:

Lipofuscin (wear and tear

Melanin Hemosiderin
pigment)

- Perinuclear, brown coloured

pigment
- Only naturally - Golden yellow
- Responsible for brown atrophy
occurring pigment
of liver and heart
endogenous black - Seen at sites of
- It is derived through lipid
pigment derived from hemorrhage or
peroxidation of polyunsaturated
tyrosine bruise
lipids of subcellular membranes
- Responsible for - Also seen in
and is indicative of free radical
pigmentation of skin and hemochromatosis
injury to the cell
hair (Ironoverload)
- Seen in aging, protein energy
malnutrition and cancer cachexia.
 PATHOLOGIC CALCIFICATION
• An abnormal deposition of calcium salts, together
with smaller amounts of iron, magnesium, and
other minerals
• Of two type
1. Dystrophic calcification
• in the absence of calcium metabolic derangements
2. metastatic calcification
• almost always reflects hypercalcemia
➢ NB. hypercalcemia is not a prerequisite for
dystrophic calcification but it can exacerbate it.

Worsening/↑symptomes
 Dystrophic Calcification
• deposition of calcium at sites of cell injury and necrosis
• It is virtually inevitable/አቀሪ/ in atheromas of advanced atherosclerosis,
• It may be an incidental finding indicating insignificant past cell injury
• it may also be a cause of organ dysfunction.
- For example, calcification can develop in aging or damaged heart valves

• Morphology
• Grossly calcium salts seen as
– fine white granules or clumps,
– often felt as gritty deposits.
– In time, heterotopic bone may be
formed
• Histologically,
– appears as intracellular and/or
extracellular basophilic deposits.
 Metastatic Calcification
• Can occur in normal tissues whenever there is hypercalcemia.
• The four major causes of hypercalcemia are :
1. Increased secretion of PTH , Parathyroid hormone
– due to either primary parathyroid tumors OR
– production of parathyroid hormone-related protein by other malignant tumors;
2. destruction of bone
Due to the effects of accelerated turnover (e.g., Paget disease), immobilization, or
tumors (increased bone catabolism associated with multiple myeloma)
3. vitamin D-related disorders
including vitamin D intoxication and sarcoidosis (in which macrophages
activate a vitamin D precursor)
4). renal failure
In which phosphate retention leads to secondary hyperparathyroidism.
Morphology
• The calcium deposits morphologically resemble those described in dystrophic calcification.
• extensive calcifications may lead to
– remarkable radiographs and respiratory deficits in the lungs
– Nephrocalcinosis leading to renal damage.
Dystrophic Metastatic

- Seen in living tissues also

- Seen in dead tissues
- Association with elevated serum
- Serum calcium is normal
Ca2+
- Seen at sites of necrosis
- Does not cause clinical dysfunction
- Often causes organ dysfunction
- Seen in
- Examples include:
• Hyperparathyroid
R – Rheumatic heart disease (in cardiac
• Renal failure
valves)
• Vitamin D intoxication
A – Atheromatous plaque
• Sarcoidosis
T – Tubercular lymph node Tumors (MOST
• Milk alkali syndrome
for PG)
• Multiple myeloma
• M – Meningioma, Mesothelioma
• Metastatic tumors to bone
• O – Papillary carcinoma of Ovary (serous
- Found in organs which loose acid
ovarian cystadenoma
and have alkaline environment inside
• S – Papillary carcinoma of Salivary gland
them [like lungs (most commonly),
• T – Papillary carcinoma of Thyroid
kidneys,
• Prolactinoma
stomach, systemic artery, pulmonary
• Glucagonoma
veins etc
 Cellular aging
• represents a progressive accumulation of sublethal injury.
• Lead to
– compromised cellular function
– cell death, or
– diminished capacity to respond to injury.
• Cellular functions decline:
– Mitochondrial oxidative phosphorylation
– synthesis of structural,
– enzymatic, and
– receptor proteins.
– diminished capacity for nutrient uptake
– repair of chromosomal damage.
 በጣም ማርጀት
• The morphologic alterations in senescent cells:
– irregular nuclei,
– pleomorphic vacuolated mitochondria,
– diminished endoplasmic reticulum, and
– distorted Golgi apparatuses.
– a steady accumulation of lipofuscin pigment
– abnormally folded proteins, and
– advanced glycosylation end products (AGEs)
 Mechanisms cellular aging
Aging process

▪ Cellular senescence is multifactorial.

➢ Intrinsic molecular clock of cellular
aging
▪ Cells with dividing capacity
▪ Doesn’t work in post mitotic cells
➢ Extrinsic stressors of the cellular
environment (wear and tear).
▪ Free radical damage
▪ Advanced glycosylation end-products
The intrinsic cellular aging theories
• cell senescence occurs because of predetermined genetic
programming.
– adult human fibroblasts in culture have a finite life span;
– stop dividing and become senescent after about 50
doublings.
– Fibroblasts from neonates go through about 65
doublings before they cease dividing,
– fibroblasts from patients with progeria, exhibit only
35 doublings.

how cells "know" the number of divisions they've undergone?

 Mechanisms intrinsic aging
1. Incomplete replication of chromosome ends (telomere shortening).
– every normal cell division results in a slightly truncated copy of each
chromosome.
• somatic cells replication
– a small section of each telomere array is not duplicated -> telomeres
progressively shortened
-> After multiple cell division -> severely truncated telomeres -> signal the
process of cellular senescence.
Telomeres: short, multiply repeated sequences of nontranscribed DNA (TTAGGG)
ends of chromosomes. a buffer of nontranscribed DNA can be repeatedly shortened
without affecting the replication of functional genes, protect chromosome termini
from fusion and degradation.
• Germ cells and stem cells
– indefinite rounds of replication are required,
– telomere length is restored after each cell division by telomerase.
– telomerase is also activated in immortal cancer cells.
2. Clock genes.
– genetic timers control the tempo of aging is supported by the identification
of clock genes, in lower life forms.
Extrinsic (wear-and-tear) theories
• despite robust cellular repair mechanisms long-term adverse
exogenous influences eventually prevail and cells senesce.
• Preservation of the cellular genetic apparatus is particularly
crucial in maintaining cell longevity,
• cells recognition and repair of damaged DNA is extremely efficient,
• occasional errors persist and accumulate as cells age.
• the rate at which such errors accumulate strongly correlates with
cellular senescence.

Werner syndrome - show premature aging;

• a defective helicase, a DNA-unwinding protein involved in DNA
replication and repair.
Cockayne syndrome &ataxia telangiectasia.
• accelerated aging.
• Defects in DNA repair.
causes of cellular wear and tear involves :
• free radical damage,
– exposure to influences as ionizing radiation,
– a progressive reduction of antioxidant defense
mechanisms (e.g., vitamin E, glutathione
peroxidase), or
– both.
Consistent with this theory of aging are the following observations:
(1) longevity among different species is inversely/in reverse correlated
with the rates of mitochondrial generation of superoxide radicals;
(2) overexpression of the antioxidative enzymes superoxide dismutase
and catalase extends life span in experimental models of aging; and
(3) restriction of caloric intake lowers steady-state levels of oxidative
damage, slows age-related changes, and extends the maximal life span in
mammals.
A second wear-and-tear mechanism involves
• post-translational modifications of
intracellular and extracellular proteins.
• free radical oxidation or nonenzymatic
glycosylation, leading to the formation of
AGEs capable of cross-linking adjacent
proteins.
• Age-related glycosylation of lens proteins
underlies senile /aging/ cataracts.
 Reading assignments !!
• Necroptosis and pyroptosis
• Roles of caloric restriction in
increasing longevity (delaying aging
process)
 Necroptosis
• Necroptosis is a programmed form of necrosis, or inflammatory cell death.
• Conventionally, necrosis is associated with unprogrammed cell death
resulting from cellular damage or infiltration by pathogens, in contrast to
orderly, programmed cell death via apoptosis.
• The discovery of necroptosis showed that cells can execute necrosis in a
programmed fashion and that apoptosis is not always the preferred form of
cell death.
• Furthermore, the immunogenic nature of necroptosis favors its participation
in certain circumstances, such as aiding in defence against pathogens by the
immune system.
• Necroptosis is well defined as a viral defense mechanism, allowing the cell
to undergo "cellular suicide" in a caspase-independent fashion in the
presence of viral caspase inhibitors to restrict virus replication.
• In addition to being a response to disease, necroptosis has also been
characterized as a component of inflammatory diseases such as Crohn's
disease, pancreatitis, and myocardial infarction.
▪ The signaling pathway responsible for carrying out necroptosis is generally understood.
▪ TNFα leads to stimulation of its receptor TNFR1.
▪ TNFR1 binding protein TNFR-associated death protein TRADD and TNF receptor-
associated factor 2 TRAF2 signals to RIPK1 which recruits RIPK3 forming the necrosome
also named ripoptosome.
▪ Phosphorylation of MLKL by the ripoptosome drives oligomerization of MLKL, allowing
MLKL to insert into and permeabilize plasma membranes and organelles.
▪ Integration of MLKL leads to the inflammatory phenotype and release of damage-
associated molecular patterns (DAMPs), which elicit immune responses.
Functions
• Necroptosis is specific to vertebrates and may have originated as an additional defense to
pathogens.
• Necroptosis also acts as an alternative "fail-safe" cell death pathway in cases where cells
are unable to undergo apoptosis, such as during viral infection in which apoptosis
Pyroptosis
 pyroptosis
• Pyroptosis is a highly inflammatory form of lytic programmed cell death that occurs most
frequently upon infection with intracellular pathogens and is likely to form part of the
antimicrobial response.
• This process promotes the rapid clearance of various bacterial, viral, fungal and protozoan
infections by removing intracellular replication niches and enhancing the host's defensive
responses.
• Pyroptosis can take place in immune cells and is also reported to occur in keratinocytes
and some epithelial cells.
• The process is initiated by formation of a large supramolecular complex termed the
inflammasome (also known as a pyroptosome) upon intracellular danger signals.
• The inflammasome activates a different set of caspases as compared to apoptosis, for
example, caspase-1/4/5 in humans and caspase-11 in mice.
• These caspases contribute to the maturation and activation of several proinflammatory
cytokines and pore-forming protein gasdermins.
 • Formation of pores causes cell membrane rupture and release of cytokines, as well as
various damage-associated molecular pattern (DAMP) molecules such as HMGB-1, ATP
and DNA, out of the cell. These molecules recruit more immune cells and further
perpetuate the inflammatory cascade in the tissue.
• However, in pathogenic chronic diseases, the inflammatory response does not eradicate the
primary stimulus.
• A chronic form of inflammation ensues that ultimately contributes to tissue damage.
Pyroptosis is associated with diseases including cancer, neurodegeneration and those of the
cardiovascular system.
• Some examples of pyroptosis include Salmonella-infected macrophages and abortively
HIV-infected T helper cells.signaling proteins are blocked by the virus.
Mechanism
The innate immune system, by using germ-line encoded pattern recognition receptors (PRRs),
can recognize a wide range of pathogen-associated molecular patterns (PAMPs) and damage-
associated molecular patterns (DAMPs) upon microbe infection. Classic examples of PRRs
include toll-like receptors (TLRs) and NOD-like receptors (NLRs). Recognition of PAMPs and
DAMPs triggers the formation of multi-protein complex inflammasomes, which then activates
caspases to initiate pyroptosis. The inflammasome pathway may be canonical or noncanonical,
with the former using caspase-1-activating inflammasomes and the latter using other
caspases.
 caloric restriction
Calorie restriction (caloric restriction or energy restriction) is a dietary regimen that reduces
food intake without incurring malnutrition.
• "Reduce" can be defined relative to the subject's previous intake before intentionally
restricting food or beverage consumption, or relative to an average person of similar
body type.
• Calorie restriction is typically adopted intentionally to reduce body weight. It is
recommended as a possible regimen by US dietary guidelines and scientific societies for
body weight control.
Life extension
According to scientific reviews, accumulating data suggests dietary restriction (DR) – mainly
intermittent fasting and caloric restriction – results in many of the same beneficial changes
in adult humans as in studied organisms, potentially increasing health- and lifespan beyond
the benefits of healthy body weight.
Which protocols of and combinations (e.g. see caloric restriction mimetic and AMPK) with
DR are effective or most effective in humans is largely unknown and is being actively
researched.
A geroscience field of "precision nutrigeroscience" is proposed that also considers the
potential need for adjustments of nutritional interventions per individual (e.g. due to
differences in genetics and age).
The mechanisms of these effects include autophagy and a decline in inflammaging.
Intermittent fasting refers to periods with intervals during which no food but only e.g.
water and tea/coffee are ingested – such as a period of daily time-restricted eating with a
window of 8 to 12 hours for any caloric intake – and could be combined with:
overall caloric restriction and variants of the Mediterranean diet which usually has
benefits of long-term cardiovascular health and longevity.
Side effects
People losing weight during calorie restriction risk developing side effects, such as
- cold sensitivity -menstrual irregularities
-infertility - hormonal changes.