Cell

Injury & Adaptation, Part 3

Sept 2016

Morphology of Cell Injury &

Necrosis

Cell death
- reversible cell injury becomes irreversible
- in most cases, necrosis
- the point of no return is difficult to determine
2 important points that determine irreversible injury

i) inability to reverse mitochondrial dysfunction

ii) altered membrane function
Two types of cell death:
Necrosis
Apoptosis
Necrosis, is a spectrum of morphologic that follow cell
death in a living tissue
Apoptosis, is a self programmed cell death

Morphology of cell death

Necrosis:
- spectrum of morphologic changes that follow
cell death in a living tissue
- necrosis is the gross and microscopic features
of cell death

Necrosis
Morphology:
- necrotic cells are more eosinophilic, appear glassy, cytoplasmic
vacuolation
- finally calcification and formation of myelin figures remain
- nuclear changes; karyolysis, pyknosis, karyorrhexis
- always pathologic, associated with inflammation

Necrosis
Coagulation necrosis
- characterized by preservation
of cellular outlines
- increasing intracellular acidosis
leads to:
- *denaturation of enzymes and
proteins
- follows hypoxic death of most
cells except brain
- example: myocardial infarction
Remember: the earliest histologic
evidence of myocardial necrosis is
not noted until 4 to 12 hrs have
elapsed

Renal infarct: tombstone

appearance

coronary occlusion by thrombi - pale area of infarct

- coagulative necrosis
7

Necrosis
Coagulative: preservation of
the cell outlines
tombstone appearance
- increased eosinophilia
- partly due to loss of
cytoplasmic RNA (which
normally binds blue dye)
Examples: Myocardial infarction

Kidney: Gross and Microscopy associated with ischemia

A localized area of coagulative necrosis is called an infarct

Coagulative necrosis: Myocardial infarction

10

Osteonecrosis:
-is common and represents loss of blood supply to a region of bone
-common sites affected include the femoral head, scaphoid, lunate, and talus
-causes: most commonly related to trauma, corticosteroids, and idiopathic.
Femoral head: medial and lateral femoral circumflex vessels form a ring of vessels at the base
of the neck.
-Ascending cervical arteries give metaphyseal branches to the neck.
-A subsynovial ring formed at the junction of the neck and head gives epiphysial arteries,
which penetrate the head.
-Most of these arcades are superolateral with few branches anteriorly.
-Fractures of the femoral neck cause disruption of metaphyseal vessels and epiphysial
branches.

11

Bone necrosis.

12

Necrosis
Liquefactive:
1) focal bacterial or fungal
infection
2) hypoxic cell death in CNS
Pathogenesis:
- transformation of the tissue
into a liquid viscous mass
- lysosomal enzymes released
by necrotic cells,
neutrophils (infective)
Cut section lung showing abscess
(liquefactive necrosis)

13

Liquefactive necrosis: Cerebral abscess

14

Necrosis

Gangrenous: usually applied to a

limb that has lost its blood
supply (dry gangrene)
Bacterial infection when
superimposed: coagulative
necrosis is modified by
liquefactive action of bacteria
(wet gangrene)
Dry gangrene: toes in diabetics
Wet gangrene: small bowel
entrapment within inguinal
canal

Dry Gangrene: note line of demarcation

Bowel ischemia: note darkened segments of bowel

16

17

Necrosis
Caseous: distinctive form of
coagulative necrosis
- seen in Tuberculosis
- formation of caseating
granulomas
Gross:
- cheesy white
Micro:
- caseous granulomas
Fig: 1

Fig: 1

Pott disease, also known as tuberculous

spondylitis
-usually secondary to an extraspinal source of
infection
-anterior aspect of the vertebral body
adjacent to the subchondral plate is usually
affected
-Tuberculosis may spread from that area to
adjacent intervertebral disks
-progressive bone destruction leads to
vertebral collapse and kyphosis
-kyphotic deformity is caused by collapse in
the anterior spine
-lesions in the thoracic spine are more likely to
lead to kyphosis than those in the lumbar
spine
-cold abscess can occur if the infection
extends to adjacent ligaments and soft tissues
-abscesses in the lumbar region may descend
down the sheath of the psoas to the femoral
trigone region and eventually erode into the
skin
19

Fig. 2: Caseous Necrosis: TB lung

20

21

Tuberculosis
-Caused by Mycobacterium tuberculosis complex
-Rod shaped, gram positive, aerobic, non-spore forming & do not stain readily
-Usually affects the lung
-Lesion forming after infection is usually peripheral and accompanied by hilar & paratracheal
lymphadenopathy (GHON LESION)
-Transmission mostly by airborne spread of droplet nuclei produced by patients with
infectious pulmonary TB
-(there maybe 3000 infectious nuclei per cough)
-Clinical illness directly following infection produces Primary TB
-Dormant TB bacilli may persist for several years, when reactivated produces Secondary
(post primary) TB
-Most important risk factor for TB among infected individuals is HIV co-infection
-Extra-pulmonary TB: Lymph node (tuberculous lymphadenitis), pleural TB, Genito-urinary TB,
skeletal TB, TB meningitis & tuberculoma
22

Necrosis
Fat necrosis: areas of fat
destruction, following release of
activated pancreatic lipases into
substance of pancreas and
peritoneal cavity
activated lipases split triglyceride
esters
released fatty acids combine with
calcium to form chalky white
areas (fat saponification)

Red arrow: necrosis of fat cells. Black

arrow: inflammatory cells.

23

Pancreas: fat necrosis.

24

Pancreas: fat necrosis.

25

Acute Pancreatitis
-Most important cause- alcohol
-Others include- gall stones, post-operative states, blunt trauma, infections (mumps), drugs
-Pathogenesis- autodigestion following activation of pancreatic enzymes (trypsinogen,
chymotrypsinogen, proelastase, phospholipase A)
-These enzymes cause proteolysis, edema, interstitial hemorrhage, vascular damage, fat
necrosis
-Clinical features- abdominal pain located in epigastrium & periumblical region & radiating to
back, nausea & vomiting
-Lab investigation- increased serum amylase & lipase, leukocytosis
-X ray abdomen may show calcifications

26

Acute Pancreatitis: note calcification along the pancreas.

27

Necrosis
Fibrinoid Necrosis
1) immune reactions
- involves blood vessels
- Ag-Ab complex deposits on
blood vessel wall
- leads to inflammation, and
formation of fibrin
- vascular leakage permits cells
and fibrin to leak out
- imparts a bright pink color to the
involved areas
- important finding in vaculitis
syndromes
Immunofluoresence study on skin sections

28

29

Fibrinoid Necrosis: blood vessel damage (autoimmune)

30

Fibrinoid Necrosis: Autoimmune dis: RA

31

Necrosis
Fibrinoid Necrosis:
2)Hyperplastic Arteriolosclerosis:
-occurs in severe hypertension; vessels exhibit
concentric, laminated (onion-skin) thickening of the
walls with luminal narrowing
-laminations consist of smooth muscle cells with
thickened, reduplicated basement membrane; in
malignant hypertension, they are accompanied by
fibrinoid deposits and vessel wall necrosis: necrotizing
arteriolitis
-mostly affects kidney

32

Onion skin thickening

Fibrinoid necrosis: Hypertension

33

Necrosis
- ultimately, in the living patient most necrotic cells and
their contents disappear
- due to enzymatic digestion and phagocytosis of the
debris by leukocytes
- if necrotic cells and cellular debris are not promptly
destroyed and reabsorbed,
- they provide a nidus for the deposition of calcium
salts and other minerals and
- thus tend to become calcified
- this phenomenon, called dystrophic calcification
34

Cell injury
Ischemia-Reperfusion injury:
- if blood flow is restored to ischemic tissue, cell can
recover
- but sometimes cells are seen to die after blood flow
is restored
- new damage is seen during reoxygenation due to
production of reactive oxygen (injured mitochondria
causes incomplete reduction of O2) & nitrogen
species (endothelial cells)
- cell death in the form of necrosis and apoptosis
- this is referred to as reperfusion injury
35

Cell injury
How does reperfusion injury occur?
- new damage may occur during reoxygenation due to
generation of free radicals
1) free radicals promote mitochondrial permeability
transition
2) ischemic injury associated with inflammation:
cytokine mediated
3) activation of complement pathway
36

Cell death: apoptosis

Apoptosis
-programmed cell death, intracellular program
-cells destined to die activate enzymes that degrade the
cell
-apoptotic cells break up into fragments, called
apoptotic bodies, which contain portions of the
cytoplasm and nucleus
-cells plasma membrane is intact
-dead cell is rapidly cleared off
-not always pathological, not associated with
inflammation
37

Apoptosis
Causes of apoptosis
Physiologic situations:
- programmed cell destruction during embryogenesis
(loss of mllerian structures in male fetus)
- involution of thymus with aging
- hormone-dependent involution in adults: endometrial
cell breakdown in menstrual cycle
- cell deletion in proliferating cell population (immature
lymphocytes in bone marrow, intestinal crypt epithelia)
- removal of neutrophils in inflammation
- removal of harmful self-reactive lymphocytes

38

Apoptosis
Apoptosis in pathologic situations
- cell death following injury: radiation, cytotoxic
anticancer drugs
- viral induced cell injury: viral hepatitis
- accumulation of misfolded proteins: degenerative
diseases of brain
- pathologic atrophy in parenchymal organs following
duct obstruction: pancreas, parotid gland, kidney
39

Apoptosis
Apoptosis in pathologic situations (contd)
- cytotoxicT lymphocytes (CTLs): recognize foreign
antigens presented on the surface of infected host
cells
- on activation, CTLs secrete perforin,
- a transmembrane pore-forming molecule, which
promotes entry of
- the CTL granule serine proteases called granzymes
- granzymes cleave proteins at aspartate residues and
thus activate a variety of cellular caspases
40

Apoptosis
Morphology:
- cell shrinkage
- chromatin condensation
- cytoplasmic blebs
- phagocytosis of apoptotic cells or cell bodies

41

MORPHOLOGY OF APOPTOSIS

CELL INJURY

Chromatin condensation
Progressive cell shrinkage
Plasma membrane blebbing
Apoptotic bodies
Phagocytosis - no inflammation

42

Apoptosis
Biochemical features:
- protein cleavage: activation of caspases
- these are present in cells as inactive pro-enzyme
forms
- once activated, caspases cleave several vital cellular
proteins
- caspases activate DNAses, degrades DNA

43

Apoptosis
DNA breakdown:
- DNA is broken down to large pieces
- DNA is cleaved by calcium and magnesium dependent
endonucleases into oligonucleosomes
Phagocytic recognition:
- apoptotic cells express phosphatidylserine on the
surface of plasma membranes
- these are then recognized by macrophages
- no associated inflammation
44

Apoptosis
Mechanisms of Apoptosis
- two phases: i) initiation phase
ii) execution phase
Initiation phase:
- two pathways are involved

45

Mechanism of Apoptosis
Extrinsic (death-receptor) pathway
- death receptors are present on surface of many cells
- death receptors are members of Tumor Necrosis
Factor (TNF)
- death receptors: TNFR1 and Fas (CD95)
- Fas is expressed on many cell types
- ligand for Fas (FasL is expressed on T-cells that
recognize self-antigens)
- FasL then binds to Fas
46

Mechanism of Apoptosis
Extrinsic (death-receptor) pathway (contd)
- thus forming a binding site for another molecule
FADD (Fas associated death domain)
- when these death receptors are bound, leading to
activation of caspase 8, triggering a cascade
- this pathway can be blocked by FLIP, which binds to
pro-caspase 8
- (some viruses produce FLIP)

47

Mechanism of Apoptosis
The intrinsic (mitochondrial) pathway:
- it is a major mechanism of apoptosis
- results from increased mitochondrial permeability and
release of pro-apoptotic molecules
- growth factors stimulate production of anti-apoptotic
members of Bcl-2 family
- these reside within mitochondria membranes
- when cells lose stimuli or subjected to stress, Bcl-2 is
lost from mitochondrial membrane
- now, replaced by pro-apoptotic factors: Bak, Bax and
Bim
- leads to increased mitochondrial permeability
48

Mechanism of Apoptosis
- cytochrome c leaks out into the cytoplasm
- cytochrome c binds to Apaf-1(apoptotic protease
activating factor-1)
- activation of caspase 9, leading to apoptosis
The execution phase:
- final phase of apoptosis is mediated by a proteolytic
cascade
- caspases is divided into 2 groups:
- initiator caspases (caspase-8, caspase-9)
- executioners (caspase-3, caspase-6)
49

Mechanism of Apoptosis
Removal of dead
cells:
- dying cells secrete
soluble factors that
recruit phagocytes
- macrophages
engulf apoptotic
cells
Liver: apototic cells. Note, cell shrinkage, pyknotic
nuclei. These cells may appear larger, due to retraction
artifact (paleness around these cells). Retraction
artifact happens while tissue processing.
50

MECHANISMS OF APOPTOSIS

51

Liver biopsy in a patient with jaundice. Comment.

52

Differentiate these two liver biopsies

54

Enzyme markers for cell death

Aspartate aminotransferase diffuse liver cell necrosis,
(AST)
viral hepatitis
Alanine aminotransferase diffuse liver cell necrosis,
(ALT)
viral hepatitis
Creatinine kinase (CK-MB) acute myocardial infarction
or myocaditis
Amylase and lipase

acute pancreatitis

55

Troponins are regulatory proteins found in skeletal and cardiac muscle. Three subunits
have been identified: troponin I (TnI), troponin T (TnT), and troponin C (TnC)
They are very, very specific for myocardial muscle even more specific than CK-MB.
Troponins go up within 3-12 hours after the onset of MI (though the rise is more gradual
than the steep bump you see with CK-MB). They remain elevated for a long time (5-9 days
for troponin I and up to a couple weeks for troponin T), which means theyre great for
diagnosing MI in the recent past (even up to a couple weeks previous to the test) but not
so great for diagnosing re-infarction. Troponin I is more specific than troponin T (which can
be elevated rarely in skeletal muscle injury or renal failure).
Creatine kinase (MB fraction)
CK has three isoenzymes: MM, MB, and BB. CK-MM and CK-MB are both found in cardiac
and skeletal muscle, but CK-MB is much more specific for cardiac muscle. CK-BB is found in
brain, bowel, and bladder.
CK-MB is a very good test for acute myocardial injury. Its very specific (you dont see
elevations in CK-MB in other conditions very often), and it goes up very quickly and
dramatically after MI (within 2-8 hours). It returns to normal within 1-3 days, which makes
it a good test to use in diagnosing re-infarction.

56

Necroptosis:
this form of cell death is a hybrid that shares aspects of both necrosis and apoptosis. The
following features characterize necroptosis:
1) Morphologically, and to some extent biochemically, it resembles necrosis, both
characterized by loss of ATP, swelling of the cell and organelles, generation of ROS, release of
lysosomal enzymes and ultimately rupture of the plasma membrane
2) it is triggered by genetically programmed signal transduction events that culminate in cell
death. In this respect it resembles programmed cell death, which is considered the hallmark
of apoptosis. In sharp contrast to apoptosis, the genetic program that drives necroptosis does
not result in caspase activation and hence it is also sometimes referred to as caspaseindependent programmed cell death.

57

Intracellular accumulation

Lipids
Proteins
Glycogen
Pigments

Intracellular accumulations
- it can be: normal cellular constituent (water,
lipids, proteins and carbohydrates)
- abnormal substance (exogenous or
endogenous)
- pigment
59

Intracellular accumulations
Fatty change: abnormal
accumulation of triglycerides
within parenchymal cells
- liver, heart and muscle
- liver is enlarged, yellow and
greasy
- microvesicular,
macrovesicular fatty change
Clinical Features: mild elevation of LFT,
- Oil Red O stain

tenderness due to stretching of Glissons

capsule,
60

Intracellular accumulations
Alcoholic fatty change(Pathogenesis):
1) shunting of normal substrates away from catabolism
and toward lipid biosynthesis, as a result of increased
generation of NADH by the two major enzymes of
alcohol metabolism, alcohol dehydrogenase and
acetaldehyde dehydrogenase
2) impaired assembly and secretion of lipoproteins
3) increased peripheral catabolism of fat, thus releasing
free fatty acids into the circulation
61

Fatty Change
Causes for fatty change:
- Toxins (alcohol)
- Non-alcoholic fatty liver disease (NAFLD)
- protein malnutrition
- diabetes mellitus
- anoxia

62

Fatty Change
Non-alcoholic fatty liver disease (NAFLD)
-presence of hepatic steatosis (fatty liver) in individuals
who do not consume alcohol or do so in very small
quantities (less than 20g of ethanol/week)
-histologic hallmarks of NAFLD are most consistently
associated with the metabolic syndrome
Pathogenesis:
-insulin resistance gives rise to hepatic steatosis
-hepatocellular oxidative injury resulting in liver cell
necrosis and the inflammatory reactions to it

Fatty Change
Other organs showing fatty
change:
Heart
1) prolonged moderate
hypoxia: anemia
gross findings in heart:
tigered effect
2) myocarditis: Diphteria

Myocarditis

64

Bilirubin
Kernicterus: associated
with Rh incompatibility
in newborns
- fat soluble unconjugated
bilirubin deposits in
basal ganglia nuclei of
brain

degeneration of basal ganglia

- jaundice: scleral icterus

65

Kernicterus: neonate

66

Intracellular accumulations
Cholesterol and
cholesterol esters:
- atherosclerosis
- xanthomas: acquired
and hereditary
hyperlipidemias
- sites of inflammation
and necrosis

xanthoma: yellow to orange plaque-like

lesions. On biopsy show lipid-laden
macrophages. (eyelids, elbows, Achilles
tendon)

67

68

Note: cholesterol clefts (these are cholesterol laden macrophages).

69

70

Intracellular accumulations

Proteins:
appear as eosinophilic droplets,
vacuoles or aggregates in
cytoplasm
- reabsorption droplets in
proximal renal tubules: seen
in renal diseases
- immunoglobulin accumulation
in plasma cell: Russell bodies
- defects in protein folding:
amyloidosis

Fig: 1

Fig: 2

71

Protein resorption droplets within

tubular epithelial cells

Russell Bodies: Plasma cell

infiltrates

72

Intracellular accumulations
Hyaline change
- alteration within cells or in
extracellular spaces
- produced by a variety of
alterations and
- does not represent a specific
pattern of accumulation
Intracellular: includes protein
droplet, Russell body,
alcoholic hyaline (Mallory
body)
Extracellular: includes old scars, Hyaline change in blood vessels
long standing hypertension
73

74

Intracellular accumulations
Glycogen
- Diabetes mellitus: glycogen is
seen in epithelial cells of the
PCT (cells become insensitive
to insulin, leads to deposition
of glycogen)
- Glycogen storage
disease (Von Grierkes:
deficiency of glucose 6
phosphatase)
Special stain: Periodic Acid Schiff
(PAS)

Liver: normal architecture. PAS stain

highlights presence of glycogen.
75

Pigments
Exogenous pigments
- carbon: air pollution
- anthracosis: black
discoloration of
lungs
- coal workers
pneumoconiosis
Anthracosis of the lung

76

Raccoon eye

77

Pigments
Endogenous
Lipofuscin: wear and tear, aging
pigment
- composed of polymers of
lipids and phospholipids
complexed with protein
- importance lies in its being a
telltale sign of
- free radical injury and lipid
peroxidation
- no pathologic implications
- appears as a yellow-brown, Liver: hepatocytes show lipofuscin pigment
finely granular cytoplasmic,
often perinuclear, pigment
*must be differentiated from
hemosiderin

78

Pigments
Endogenous (contd)
Hemosiderin
hemoglobin derived, golden yellow
to brown
- when there is a local or systemic
excess of iron, ferritin forms
hemosiderin granules
local excess: hemorrhages in tissues
(hematoma)
systemic excess: iron overload
(hemosiderosis)
- hemosiderin pigment represents
aggregates of ferritin micelles
Special stain: Prussian blue stain

Hemosiderin: liver biopsy

79

Pigments
Endogenous (contd)
Melanin:
- endogenous, brown-black,
pigment formed
- when the enzyme tyrosinase
catalyzes the oxidation of
tyrosine to
dihydroxyphenylalanine in
melanocytes
Examples: Addisons disease,
melanotic skin lesions

Malignant melanoma

80

Pathologic Calcification

- is the abnormal tissue deposition of calcium salts,

together with smaller amounts of iron, magnesium,
and other mineral salts
Types:
Dystrophic calcification: occurs locally in dying tissues
- coagulative, caseous, or liquefactive type, and in foci
of enzymatic necrosis of fat
- present in the atheromas of advanced atherosclerosis
- commonly develops in aging or damaged heart valves
- normal serum levels of calcium
81

Pathologic Calcification
Types:
Metastatic calcification: deposition of calcium salts in otherwise
normal tissues
- always results from hypercalcemia secondary to some
disturbance in calcium metabolism
1. Increased secretion of parathyroid hormone (PTH) with
subsequent bone resorption, due to
- hyperparathyroidism: parathyroid tumors, and ectopic
secretion of PTH-related protein by malignant tumors
2. Resorption of bone tissue: secondary to primary tumors of
bone marrow (example: multiple myeloma)
3. Vitamin D related: vitamin D intoxication, sarcoidosis
4. Renal failure: which causes retention of phosphate, leading to
secondary hyperparathyroidism

82

Dystrophic calcification: demaged heart valve

in Rheumatic valvular disease

Dystrophic calcification: Psammoma

bodies: meningioma, papillary
carcinoma thyroid, papillary
carcinoma ovary
83

Hyperparathyroidism
-Characterized by excess production of PTH
-Common cause for hypercalcemia
-Mostly associated with autonomously functioning adenomas or hyperplasia
Primary hyperparathyroidism- disorder of calcium, phosphate, and bone metabolism
-Clinical presentation: recurrent nephrolithiasis, peptic ulcers, mental changes, less frequently
extensive bone resorption (osteitis fibrosa cystica), neuromuscular manifestation may include
proximal muscle weakness, easy fatigability, and atrophy of muscles
-Bone specific alkaline phosphatase, osteocalcin & type I procollagen peptides are abnormal in
these patients
Vitamin D related hypercalcemia
- Excessive ingestion or abnormal metabolism of the vitamin (granulomatous disorder such as
Sarcoidosis)

84

blaming others is nothing more than excusing yourself

Robin Sharma

85