1. Cell Death
Dr (Prof) Vishal Saxena
MBBS, MD(Path)

2. Cell Death Some important terms !
Autolysis: degradation of cell and its constituents caused by its own enzymes.
Heterolysis: degradation of cell and its constituents by enzymes derived from sources extrinsic to the cell (e.g. neutrophils, macrophages).
Putrefaction: lysis of dead tissue by bacterial enzymes.
Irreversible injury inevitably leads to cell death.
Types of cell death:
Necrosis
Apoptosis

3. Necrosis (Greek - Nekrosis = deadness)
is defined as the spectrum of gross and light-microscopic appearances that indicate cell death in living tissue,
Caused by action of hydrolytic enzymes on the injured cells.
Is accompanied by inflammation.
Cell expands and nucleus becomes pyknotic ( or is lost)
Always pathological.

4. How do you tell a cell is dead??
Necrotic cells show certain changes:
Cytoplasm becomes hypereosinophilic due to:.
Loss of RNA
Binding of eosin to coagulated proteins
Loss of glycogen: Cell smaller, more pink.
Nuclear changes:
Karyorrhexis, Karyolysis and pyknosis.
After these early changes, mass of necrotic cells may have different appearances.
Appearance depends upon two processes:
Denaturation of proteins
Digestion by enzyme (Enzymatic digestion)
If Denaturation is predominant  coagulation necrosis develops
If enzymatic digestion is predominant  liquefactive necrosis develops.

5. Morphologic types of necrosis
Coagulation necrosis
Liquefactive necrosis
Caseous necrosis
Fat necrosis
Traumatic
Enzymatic
Gangrenous necrosis
Gummatous necrosis

6. Coagulative necrosis Is secondary to
denaturation and coagulation of structural and enzymatic proteins due to intracellular acidosis.
Denaturation of lysosomal enzymes by intracellular acidosis prevents autolysis.
Preserving cell outlines and tissue architecture.
Most common cause: ischemia (secondary to atherosclerosis thrombus formation).
Also produced by denaturation of proteins in
Heavy metal poisoning (lead) and
Irradiation.
Commonly seen in solid organs like:
Heart, kidney, spleen

7. Infarction:
Refers to a localized area of tissue necrosis resulting from loss or reduction in blood supply (= ischemic necrosis).
The dead tissue is called an infarct.
Coagulation necrosis is the type of necrosis associated with infarction except in infarction of brain.
In Brain:
Lack of good structural support and
cells rich in lysosomal enzymes and lipid result in liquefactive necrosis.

8. Coagulation necrosis GROSS APPEARANCE:
Dead tissue: appears dense and dry
Cut surface: white-grey
MICROSCOPY:
Loss of the nucleus but preservation of cellular shape.
Increased cytoplasmic eosinophilia.

9. Preservation of cellular shape.
Area of necrosis
Area of
Necrosis
Preservation of cellular shape.
In this gross photograph of the heart , note the area of pale gray myocardial infarction (arrows) in the anterior portion of the left ventricle and extending into the anterior portion of the interventricular septum.
This high-power photomicrograph contains normal myocytes (1), vacuolated myocytes (2), and infarcted myocytes (3).
Increased
cytoplasmic
eosinophilia

10. Liquefaction necrosis
Definition: necrosis with complete loss of cell and tissue structure.
Primarily results due to
Destruction of dead cells by hydrolytic enzymes.
Enzymes derived from either :
Cell’s own lysosomes (autolysis) or from
neutrophils and macrophages (heterolysis).

11. Liquefaction necrosis
Seen in
Suppurative bacterial infections: characterized by formation of pus (liquefied necrotic debris and neutrophils).
In ischemic injury to brain tissue and abscess of brain.
Enzymatic destruction of brain tissue leaves behind a cystic cavity.
Gross appearance
Necrotic area: soft and filled with fluid.
Microscopy:
Structure-less tissue debris (no cellular outlines)
Neutrophils
NOTE:
Liquefaction by leukocyte enzyme is called Suppuration and the resultant fluid is called pus

12. Lung Abscesses Necrotic material with loss of cellular architecture
The two lung abscesses seen here are examples of liquefactive necrosis in which there is a liquid center in an area of tissue injury. One abscess appears in the upper lobe and one in the lower lobe.
Liquefactive necrosis is typical of organs in which the tissues have a lot of lipid (such as brain) or when there is an abscess with lots of acute inflammatory cells whose release of proteolytic enzymes destroys the surrounding tissues.
Necrotic material
with loss of cellular
architecture
Lung Abscesses

13. Neutrophils
Necrotic tissue

14. liquefactive necrosis
Cerebral infarction:
liquefactive necrosis
Grossly, the cerebral infarction at the upper left here demonstrates liquefactive necrosis. Eventually, the removal of the dead tissue leaves behind a cavity.
At high magnification, liquefactive necrosis of the brain demonstrates many macrophages at the right which are cleaning up the necrotic cellular debris. The job description of a macrophage includes janitorial services such as this, particularly when there is lipid.

15. Coagulative Vs liquefactive necrosis
Coagulative and liquefactive necrosis.
A, Kidney infarct exhibiting coagulative necrosis, with loss of nuclei and clumping of cytoplasm but with preservation of basic outlines of glomerular and tubular architecture.
B, A focus of liquefactive necrosis in the kidney caused by fungal infection. The focus is filled with white cells and cellular debris, creating a renal abscess that obliterates the normal architecture.
Coagulative necrosis
Focus of liquefactive necrosis

16. Caseous necrosis
Definition: a distinct form of necrosis without preservation of cellular outlines and tissue architecture, but firm in consistency.
A combination of Coagulative and liquefactive necrosis.
Most commonly associated with
Formation of granulomas in tuberculosis and
Systemic fungal infection like histoplasmosis.

17. Yellowish white, “cheese like” material. Cheese like appearance
GROSS:
Yellowish white, “cheese like” material.
Cheese like appearance
due to release of lipid from the cell walls of M.tuberculosis and systemic fungi (histoplasma).
(Latin “caseous” – cheese)
MICRO:
Eosinophilic material surrounded by
inflammatory cells, activated macrophages, multinucleated giant cells and helper T cells (= a granuloma).
Granuloma: a lesion seen in conditions referred to as granulomatous disease.
Examples of granulomatous diseases:
tuberculosis
systemic fungal infections like Histoplasmosis
leprosy
sarcoidosis
berylliosis
The main cell which forms the granuloma is modified macrophage (also known as epithelioid cell).
In other words granuloma is a localized collection of modified macrophages or epithelioid cells.
Granulomas may be associated with caseous necrosis (caseating granulomas) : e.g. tuberculosis, histoplasmosis or
May no be associated with caseous necrosis (non caseating granulomas) e.g. sarcoidosis.

18. Caseous necrosis Lymphnode: histoplasmosis Lung: tuberculosis B
B: Lymph node, mediastinal, histoplasmosis - Gross, cut surface  The caseous necrosis in this case of fungal infection has a white, cheese-like appearance. Caseous necrosis has a similar appearance, irrespective of the tissue involved. B
Lymphnode: histoplasmosis
Lung: tuberculosis

19. Caseous necrosis Langhan’s giant cells
Lung, pulmonary tuberculosis - Low power :
The area of caseous necrosis appears as amorphous, granular debris without preservation of the tissue architecture. At its edge, granulomatous inflammation with multinucleated giant cells is apparent.
Lung, pulmonary tuberculosis - High power  Caseous nodules are pink and structureless, and unlike coagulation necrosis, architecture and cell outlines have been largely destroyed. Once again, Langhans giant cells are prominent
Langhan’s giant cells

20. Granuloma
Epithelioid cell
Langhan’s
giant cell

21. Enzymatic fat necrosis
Definition: a focal areas of fat destruction.
Occurs due to action of pancreatic enzyme on fatty tissue located around pancreas.
Occurs as a complication of Acute pancreatitis.
Acute pancreatitis:
Release of lipases and amylase from pancreas.
Lipases break triglycerides into fatty acids (FA).
FA combine with Ca++ via the process of saponification
to form chalky white calcified deposits
in the pancreas as well as in the omental fat.
Precipitating factors for Ac. Pancreatitis:
Alcoholism.

22. Enzymatic fat necrosis
Clinical significance:
Deposits visible on abdominal X ray
Marker of pancreatitis.
Consequence of extensive enzymatic fat necrosis
Tetany (due to lowering of the Ca levels)
Gross appearance
Chalky white calcium deposits (calcium soaps)
Pancreas and adjacent tissues
Microscopy:
Necrotic fat cells – pale outlines.
Cytoplasm filled with basophilic staining calcified material.
Hematoxylin stains calcium blue.

23. Area of necrosis with blue discoloration
White nodules on the
surface of pancreas
Foci of fat necrosis with saponification. The areas of white chalky deposits represent calcium soap formation at sites of lipid breakdown.
Area of necrosis with
blue discoloration

24. Normal Fat tissue Vs. Fat necrosis
Left: control; right: fat necrosis: the cellular outlines vaguely remain, the fat cells have lost their peripheral nuclei and their cytoplasm has converted to a mass of basophilic amorphous necrotic material.

25. Traumatic fat necrosis
Differs from enzymatic fat necrosis by being
Secondary to trauma.
Trauma to fatty tissue  acute inflammatory reaction (neutrophils)  healing by fibrous tissue and dystrophic calcification.
Commonly seen in women with pendulous breasts.
Clinical significance:
Scar tissue feels firm, retracts the overlying skin and shows calcifications on mammography.
“these findings also seen in breast cancer”.
Traumatic fat necrosis of breast: differential diagnosis breast cancer.

26. A famous surgeon went on a safari in Africa
A famous surgeon went on a safari in Africa. When he came back, his colleagues asked him how it had been. 'Oh, it was very disappointing,' he said. 'I didn't kill a thing. I’d have been better off staying here in the hospital.'

27. Gangrenous necrosis
Most often occurs in lower limbs and bowel secondary to loss of blood supply.
Two types:
Dry gangrene:
Is a form of infarction that results from ischemia.
Characterized primarily by coagulative necrosis without liquefaction.
Dead tissue has mummified appearance.
Characteristic finding in diabetic foot.
Dry gangrene
If the blood flow is interrupted for a reason other than severe bacterial infection, the result is a dry gangrene. Persons with impaired peripheral blood flow, such as diabetics, are at greater risk for dry gangrene.
The early signs of dry gangrene are a dull ache and sensation of coldness in the area, along with pallor of the flesh. If caught early, the process can sometimes be reversed by vascular surgery. However, if necrosis sets in, the affected tissue must be removed just as with wet gangrene.

28. Dry Gangrene A
A: This is gangrene, or necrosis of foot. In this case, the toes were involved in a frostbite injury. This is an example of "dry" gangrene in which there is mainly coagulative necrosis from the anoxic injury.
B: Foot, dry gangrene - Clinical presentation  Gangrenous necrosis usually implies coagulative necrosis and is commonly seen in a limb that has lost its blood supply. Wet gangrene is necrosis combined with bacterial infection.   B

29. Gangrenous necrosis Wet gangrene:
Refers to necrosis with superimposed bacterial infection.
Liquefactive necrosis is the primary type of necrosis in wet gangrene.
Associated Acute inflammatory reaction leads to
Ulceration
Gas production and
Oozing of purulent material.

30. This is gangrene of the lower extremity
This is gangrene of the lower extremity. In this case the term "wet" gangrene is more applicable because of the liquefactive component from superimposed infection in addition to the coagulative necrosis from loss of blood supply. This patient had diabetes mellitus.
Wet Gangrene

31. Gummatous necrosis
Refers to a type of coagulative necrosis seen in tertiary syphilis.
It is associated with formation of Gummas (soft rubbery masses composed of plasma cells, macrophages, lymphocytes etc.)
Gummas are very destructive lesions and can be found in any organ, esp. in skin and bones.
Necrosis in syphilis is due to endarteritis obliterans (the arterial lumen is obliterated leading to ischemic damage to tissue and necrosis).

32. Apoptosis From Gk. Meaning falling off
Is genetically controlled programmed single cell death by activation of particular enzyme pathways.
Helps eliminate unwanted cells.
Plays role in
embryogenesis, normal physiologic and pathologic cell death.
Different from necrosis!!
Absence of inflammation
Cell shrinks and nucleus becomes pyknotic
An active process and involves RNA and protein synthesis.
Apoptosis: refers to individual cell death without an inflammatory response.
Main function is to remove unwanted cell selectively with minimal disturbance to surrounding cells.

33. Remember ! IN APOPTOSIS: Cells actually expend energy in order to die.
The cell membrane does not rupture,
The cell contents are not released into the extracellular space, and
Inflammation does not occur.

34. Physiologic examples of apoptosis
Embryogenesis
Disappearance of Mullerian and Wolffian duct structures.
Development of lumen within hollow organs (e.g bowel and heart)
Hormone-dependent involution in adults
Endometrial breakdown in menstruation
Post-lactational atrophy of breast
Prostate atrophy following castration.
Involution of Thymus in the adult.
Cells that are programmed to die; for example,
the cells of the outer layers of epidermis,
cells in the gut epithelium.
Destruction of inflammatory cells

35. Pathologic examples of apoptosis
Toxin induced injury
Diphtheria toxin  myocarditis
Councilman bodies= dead hepatocytes in viral hepatitis.
Psammoma bodies: apoptosis of neoplastic cell with subsequent calcification.
Tumor cell death by cytotoxic T cells
Neurons that are lost in Alzheimer's disease
HIV-positive T-lymphocytes die by apoptosis.
Psamomma bodies: seen in
Papillary carcinoma of thyroid
papillary cystadenocarcinoma of the ovary
meningioma

36. Mechanism Divided into 3 phases
Initiation phase:
Execution phase:
Removal of dead cell:

37. Initiation phase Characterized by activation of caspases.
Activation of caspases occurs by a death signal from:
Outside the cell (Extrinsic or receptor initiated pathway)
Inside the cell (Intrinsic or mitochondrial pathway)
Regulated by genes
Death signal

38. Initiation phase A: Extrinsic pathway: Receptor-ligand interactions:
Binding of Tumor necrosis factor (TNF) to its receptor(TNFR1)
Fas binding to the Fas ligand.
Result: activation of caspases
B: Intrinsic pathway:
Withdrawal of hormones or growth factors
Damage to DNA:
viruses, radiation, free radicals.
Result: increased permeability of mitochondrial membrane  release of cytochrome c  activate caspases

39. Initiation phase
Whatever the signal for apoptosis ; the ultimate result is activation of enzymes k/a caspases.
Caspases
Death Signal

40. Execution of apoptosis
Mediated by caspases (group of proteases).
Caspases:
Activate enzymes endonuclease and protease
Activation of proteases:
leads to breakdown of the cytoskleton.
Activation of endonucleases:
DNA degradation (size base pairs)  laddering appearance of DNA on electrophoresis.

41. Execution phase
Endonuclease
Caspases
Protease
Death Signal

42. Execution phase Result is: DNA breakdown
Cytoplasmic buds forming apoptotic bodies.
Endonuclease
DNA breakdown,
Cytoplasm budding
= apoptotic bodies
Caspases
Protease
Death Signal

43. Lane A: Control Lane B: Apoptosis Lane C: Necrosis Ladder pattern
of DNA
DNA electrophoresis

44. Removal of dead cells
Formation of cytoplasmic buds on the cell membrane containing
Nuclear fragments, mitochondria and protein fragments.
Breaking off of cytoplasmic buds  apoptotic bodies.
Phagocytosis of apoptotic bodies by neighboring cells or macrophages.

45. Proapoptotic molecules Formation of apoptotic bodies
Receptor ligand
Interaction
FAS
TNF receptor
Withdrawl of
GF and Hormones
Proapoptotic molecules
Cytochrome c
Adaptor proteins
Initiator caspases
Injury
Rad,FR,Toxin
(DNA damage)
Execution caspases
Activation of
Endonuclease
& proteases
Cytoskeletal
breakdown
DNA
fragmentation
Formation of apoptotic bodies
Phagocytosis

46. Apoptosis is regulated by genes
Bcl-2 gene (inhibits apoptosis)
Manufactures gene products that inhibit apoptosis by
Preventing release of cytochrome c from mitochondria
p53 : Tumor suppressor gene (Stimulates apoptosis).
Elevated by DNA injury and arrests the cell cycle to repair DNA damage.
If DNA repair is impossible p53 stimulates apoptosis by activating BAX apoptosis gene.
Bax: stimulates apoptosis

47. Morphologic appearance
Apoptotic cells:
Have deeply pink staining cytoplasm.
Have pyknotic nucleus which fragment.
Are smaller in size
Breakdown into fragments (apoptotic bodies)

48. Apoptosis of epidermal Apoptotic cell in liver
A, Apoptosis of epidermal cells in an immune-mediated reaction. The apoptotic cells are visible in the epidermis with intensely eosinophilic cytoplasm and small, dense nuclei. H&E stain.
B, High power of apoptotic cell in liver in immune-mediated hepatic cell injury.
Apoptosis of epidermal
cells
Apoptotic cell in liver

49. Apoptotic cell with eosinophilic Cytoplasm

50. Councilman bodies
Apoptosis is a more orderly process of cell death in which there is individual cell necrosis, not necrosis of large numbers of cells. In this example, liver cells are dying individually (arrows) from injury by viral hepatitis. The cells are pink and without nuclei.

51. What if apoptosis is too little or too much!! = dysregulated apoptosis
Two groups of disorders:
Decreased apoptosis with increased cell survival can give rise to:
Cancers
Autoimmune disorders.
Increased apoptosis with decreased cell survival:
Neurodegenerative diseases
Death of virus infected cells
Lymphocyte depletion as in AIDS

52. A pipe burst in a doctor's house. He called a plumber
A pipe burst in a doctor's house. He called a plumber. The plumber arrived, unpacked his tools, did mysterious plumber-type things for a while, and handed the doctor a bill for $ The doctor exclaimed, "This is ridiculous! I don't even make that much as a doctor!." The plumber waited for him to finish and quietly said, "Neither did I when I was a doctor."

53. Apoptosis Vs. Necrosis

54. The sequential ultrastructural changes seen in necrosis (left) and apoptosis (right).
In apoptosis, the initial changes consist of
nuclear chromatin condensation and fragmentation, followed by
cytoplasmic budding and
phagocytosis of the extruded apoptotic bodies.

55. Apoptosis Vs. Necrosis Feature Necrosis Apoptosis Cell size
Enlarged (swelling)
Reduced (shrinkage)
Nucleus
Pyknosis  karyorrhexiskaryolysis
Fragmentation
Cellular contents
Enzymatic digestion ; may leak out of cell
Intact, may be released in apoptotic bodies.

56. Feature
Necrosis
Apoptosis
Adjacent inflammation
Frequent No
Physiologic or pathologic role
Always pathologic
Often physiologic, may be pathologic

57. Enzyme markers of cell death

58. Enzyme markers of cell death
Diagnostic use
Aspartate aminotransferase (AST)
Marker of diffuse liver cell necrosis (e.g. viral hepatitis)
Also increased in alcohol induced liver disease
Alanine aminotransferase (ALT)
More specific for liver cell necrosis than AST

59. Enzyme markers of cell death
Diagnostic use
Creatine kinase MB (CK-MB)
Elevated in AMI or myocarditis
Marker enzyme for acute pancreatitis.
Lipase more specific than amylase for pancreatitis.
Amylase also increased in salivary gland inflammation (e.g. mumps)
Amylase and lipase

60. Self study : extra images

61. Gross appearance: Caseous necrosis
Necrotic area

62. Microscopic appearance of caseous necrosis

63. Caseous Necrosis

64. Granulomas

65. Granuloma 3 1 2 Granuloma: 1: modified macrophage = epithelioid cell
2: Langhans giant cell
3: rim of lymphocytes

66. Kidney infarct showing coagulation necrosis
Preservation of
tissue architecture.
Kidney infarct showing coagulation necrosis
Kidney infarction showing coagulation necrosis with preservation of tissue architecture.

67. Splenic infarction: coagulation necrosis
Splenic infarction showing coagulation necrosis

68. General Practitioners know nothing and do little
General Practitioners know nothing and do little. Surgeons know little and do everything. Internists knows everything and do nothing. Pathologists know everything and can do everything, but it's usually too late.

69. She told me that she loved me
She said her love was true
But what is the use in loving
When “Path” has got you blue?

70. Thank You !