2009 Group 8 Project

From CellBiology

Cell Death: Necrosis

Introduction

Necrosis, derived from the Greek word neckros for ‘corpse’, is a type of irreversible cell death. It is mainly caused by early plasma membrane rupture , mitochondrial dysfunction, cell injury, infarction, inflammation and lysosomal rupture . Unlike apoptosis, it is considered as uncontrolled. Necrosis is irreversible and can be fatal. There are several types of necrosis classified based on morphological criteria, coagulative necrosis, liquefactive necrosis, caseous necrosis, fibrinoid necrosis and fat necrosis.


History


  • 1858 – cell death was first discussed in the lecture given by Rudolf Virchow, a German pathologist and biologist, he termed it “necrobiosis” which is described as “degeneration, softening,necrosis, and mortification”


  • 1877 – Carl Weigert and Julius Cohnheim identified coagulation necrosis, a white infract produced in cell death.


  • Up until 1971, the term “necrosis” was used for all types of cell death.


  • 1971 – the definition of cell death is modified to include a distinction between pathological and non-pathological forms.
    • In 1971 Kerr et al. first observed a form of non-pathologic cell death in certain tissues,terming it ‘shrinkage necrosis’. ‘Necrosis’ now refers to pathological forms of cell death.


  • 1972 - ‘Shrinkage necrosis’ is renamed ‘apoptosis’ as it became implicated in the control of organ homeostasis (Kerr et al. 1972). The two basic types of cell death were now termed apoptosis, defined as a non-pathological, ‘programmed’ form of cell death, and necrosis, defined as a pathological, non-programmed form of cell death.


  • Nomenclature Committee on Cell Death 2009: necrosis is defined as being ‘morphologically characterized by a gain in cell volume (oncosis), swelling of organelles, plasma membrane rupture and subsequent loss of intracellular contents’


  • Today there is increasing evidence to suggest that necrosis may not be just an accidental uncontrolled form of cell death. It is thought that necrosis may in fact controlled by various signal transduction pathways and catabolic mechanisms eg Toll-like receptors and death domain receptors



Apoptosis vs Necrosis

There are distinct morphological and biochemical differences between apoptosis and necrosis. Apoptosis is considered as a physiological event while necrosis is a pathological event.

Apoptosis is classified as programmed cell death and called “cell suicide”, the process is under genetic control and regulated by a variety of cellular signaling pathways. In Apoptosis, cell is shrinking and become denser. Condensation of chromatin to form sharply circumscribed, uniformly dense, cresentic masses that abut the nuclear envelope. Karyohexis takes place which means the breakdown of nucleus. DNA degeneration and cleavage of chromosomal DNA into internucleosomal fragments are the features of apoptosis. The cells are divided into many parts by "budding", forming apoptotic bodies. Apoptotic bodies contain cell organelles and nuclear materials, and are surrounded by an intact plasma membrane. These bodies are removed by neighbouring cells and macrophages. Caspase-1 and caspase-3 are the main proteases to mediate apoptosis. Cell death caused by apoptosis requires energy in the form of ATP.


Different from apoptosis, necrosis is not a programmed cell death and considered to be uncontrolled. It is initiated accidentally by ischemia, trauma or ATP depletion. Swelling cells can be found in the case of necrosis but no apoptotic bodies or DNA fragment is present. The plasma membrane integrity is not maintained in necrosis, necrotic cell released cellular contents including lysosomal enzymes due to the breakdown of the plasma membrane into the extracellular fluid, and this cellular leakage promote inflammation.Unlike apoptosis, there is no energy is required for the process of necrosis. Necrosis is also known as caspase-independent cell death that caspase is not involved in this type of cell death.

The differences in apoptotic and necrotic death illustrated in the table below can be viewed in this animated video

Apoptosis vs Necrosis
Apoptosis Necrosis
Regulation genetic programmed ischemia,trauma or ATP depletion
Control controlled uncontrolled
Cell sharp skrinkage, condensed swelling
Plasma membrane integrity maintained collasped
Cellular process budding blebbing
Cellular content packaged in apoptoic bodies leakage to extracellular fluid
DNA fragmentation, chromatin condensation no fragmentation
Energy ATP required not required
Inflammatory response absent present
Mediator caspase caspase-independent

Diagram shows difference between apoptosis and necrosis


Cellular Processes

The term ‘necrosis’ is used for the presence of dead tissues or cells and is the total of the transformations which have occurred in cells after they have died. It is currently thought to be a type of cell death that is accidental as opposed to programmed. Necrosis this therefore thought of as a passive form of cell death because it does not require the complex regulatory mechanisms such as protein synthesis that are characteristic of programmed cell death. In addition to this, the process of necrosis has minimal energy requirements, whereas cell death by apoptosis requires energy in the form of ATP.

Figure 1. Condensed chromatin and dilated organelles of necrotic cells

The full process of necrosis is complete only after 12-24 hours. Therefore, cells are dead long before any necrotic changes can be seen through a light microscope. Necrosis, therefore, refers to morphological features noticed after a cell has already died and attained equilibrium with its surroundings. In general, the death of a cell is accompanied by severe swelling, distension of organelles, condensed nuclear chromatin, “plasma membrane endocytosis and autophagy” [1]. However, the morphology of cells that are dying is fairly diverse. Necrosis is typified by rapid cell swelling and eventually full lysis of the cell. The cell membrane becomes permeable fairly early in the course of necrosis with loss of cell membrane integrity and the subsequent leakage of cytoplasmic contents and generation of an inflammatory response. This specific form of cell death is also characterised morphologically by intracellular swelling with the dilated organelles (Figure 1.) and the ribosomes dissociating from the endoplasmic reticulum (Ziegler & Groscurth, 2004). Clarke (1990) defined two different types of necrosis based on morphology and involvement of lysosomes, one being autophagic and the other being nonlysosomal disintegration [2] . Autophagic cell death is characterised by “numerous vacuoles in the cytoplasm filled with cellular remnants. The nonlysosomal cell death displays characteristic dilation of organelles and formation of unfilled spaces with degeneration proceeding without any measureable participation of lysosomes.

Morphology of necrosis

Detection of necrotic cells employs the distinguishing characteristic of a loss of cell membrane integrity. This subsequently allows the passage of small charged molecules that would not normally traverse the membrane and enter the cell.

Indicators of Necrosis

Light microscopy level

  • Early mitochondrial swelling
  • Loss of plasma membrane
  • Nuclear membrane preserved


Ultrastructural level

  • Proliferation of the endoplasmic reticulum
  • Disaggregation of polyribosomes
  • Dilation of organelles
  • Intranuclear vacuoles
  • Breakdown of cell membrane (plasmalemma)


Changes in the nucleus

  • Clumping and swelling of chromatin
  • Pyknosis
  • Kayorrhexis
  • Karyolysis

The nucleus is one major cell feature that disintegrates late in the process of necrosis. The morphological changes that occur within the nucleus however, can be observed over time. The nuclear changes appear in three patterns (as indicated above) and all due to nonspecific breakdown of DNA.

The first pattern is identified as karyolysis. This is essentially the fading of basophilia of the chromatin which results in the dissolution of the nucleus by action of deoxyribonucleases. The second pattern is pyknosis, characterised by nuclear shrinkage and condensation of the nucleus which is mainly due to increased basophilia. In the third pattern, karyorrhexis, the nucleus which has already undergone pyknosis then undergoes fragmentation. Over time, the nucleus eventually disappears. However, pyknotic and fragemented nuclei although observable, are not a common feature in necrotic cell death (REFERENCE- Ziegler).



Causes

(IN PROGRESS) Necrosis is best understood when compared to apoptosis. Necrotic cells have distinct morphological features.

  • Cells first swell (oncosis)
  • Plasma membrane collapses
  • Cells are rapidly lysed and cytotoxic components are released from the plasma membrane causing inflammation

These morphological features differ from apoptotic cells. Apoptotic cells undergo regulated catabolism where enzymes digest the cytosolic components and nuclear DNA. The cells shrink into fragmented apoptotic bodies which are phagocytized by neighboring cells. Apoptosis has a regulated mechanism leading to death of the cell and necrosis has been long accepted to be an unregulated series of events leading to the death of the cell. However, recent research shows that the cell does undergo a regulated series of events leading to necrosis. Cells can die through unregulated processes of membrane and cytosolic destruction under extreme conditions. These extreme conditions tend to me physico-chemical stresses and thus necrosis is said to be accidental and uncontrolled. These stresses arise from various bacterial toxins and viruses, inadequate secretions of cytokines, nitric oxide and reactive oxygen species and calcium. A cell may undergo two different processes leading to necrosis. The first, primary necrosis, is characterized by external chemical and physical damage to the cell. The second, secondary necrosis occurs in late apoptotic cells which fail to be engulfed by macrophages. Cells undergoing this process are not phagocytosed and thus lose membrane integrity, cease to be metabolically active and release their cytoplasmic content out into the extracellular matrix, or if in vitro, into the culture medium. Double staining procedures are used to stain necrotic cells to identify if they are undergoing primary or secondary necrosis.

Cytokines

Cytokines are secreted by affected tissues during infection and inflammation and are capable of initiating necrosis. This is demonstrated in the pancreatic cells of diabetic patients where exposure to cytokines such as TNF-α caused both apoptotic and necrotic cell death.

Mitochondria and ROS

Mitochondrial Dysfunction: It is one of the major causes of necrosis due to ATP depletion. The mitochondria is site of cellular oxygen consumption and responsible for generating ATP by oxidative phosphorylation. When there are insufficient oxygen supply to the cell, it causes the uncoupling of oxidative phosphorylation that makes the mitochondria cannot generate ATP and this may lead to cell death. At the early stage of oxygen deprivation, there is a formation of protrusion of the plasma membrane called blebs, it casuse the mitochrondria cell start to swell. If this condition persists, the growth of blebs accelerated and the mitochrodrial membrane continues to swell. Once the plasma membrane of blebs disrupted, this cell injury is irreversible and lead to necrosis.[3]

Oxidative Stress: In the case of ischemia, there is lack of oxygen and a raise of Ca2+. In prolonged ischemia, the Ca2+ overloaded mitochondria produce oxygen free radicals (OFR) such as superoxide, singlet oxygen and hydroxyl radical. These OFR reacts with the protein and lipid inside the cell and form oxidized protein and peroxidized lipid which then disrupt the membrane structure. However, recovery from ischemia by reintroduction of oxygen will not prevent the formation of OFR but causing a more serious condition since excess production of OFR is developed when oxygen is reintroduced.[http://www.ncbi.nlm.nih.gov/pubmed/18974775

It is clear that oxygen depletion is a major factor in mitochondrial dysfunction leading to necrosis. Oxygen depletion also leads to energy depletion and as a consequence the maintenance of the cell membrane potential is neglected. Early depolarization leads to excitotoxicity through excessive upregulation of glutamate and an increase in intracellular calcium concentration. Oxygen depletion can also lead to acidification. Acidosis activates calcium permeability and can result in glutamate receptor-independent neuronal injury due to calcium toxicity. This increase in calcium concentration results in apoptotic and necrotic cell death.

Elevated intracellular Calcium is a constant feature of both apoptotic and necrotic death. From the information stated above, we note that elevated calcium levels leads to downstream irreversible reactions such as oxidative stress and mitochondrial dysfunction. It can also lead to calcium dependent protease activation.

Proteases and necrosis

Proteases play an important role in the death of cells. They are considered the “executioners” of cell death because once a proteolytic cascade is set in motion, death is the only outcome. There are a variety of proteases that are involved in cell death processes.

  • Cytosolic cysteine proteases (Caspases and Calpains)
  • Lysosomal proteases (Cathepsins)

Cytosolic cysteine Proteases

  • Caspases

Caspases are cysteine proteases that cleave aspartic residues in target proteins. The are produced as inactive proteases calles "zymogens" and are activated both during necrotic and apoptotic cell death. Apoptotic caspases are classified as initiator caspases or effector caspases. Their activation triggers apoptotic caspase cascades that are tightly regulated. Once activated, they proteolytically cleave a large range of celular targets leading to cell death. Caspase mediated proteolysis is also present in necrotic cell death.

Lysosomal proteases

  • Cathepsins

--Gurkiran Flora 20:48, 17 May 2009 (EST)




Patterns of Necrosis

As a result of necrotic cell death, the affected tissues or organs display morphological changes. Based on this morphology, necrosis can be categorized into several distinct types or patterns. Such types, when observed at both the gross and microscopic level, can provide pathologists and clinicians with clues about its underlying cause.


Coagulative necrosis

Figure 1: Coagulative necrosis in the liver

Coagulative necrosis is the most common pattern of necrosis occurring in tissues or organs. Its morphological pattern is primarily a consequence of protein degradation frequently caused by ischemia where lack of oxygen causes cell death in a localized area. Coagulative necrosis can occur in all tissues, except the brain (liquefactive necrosis is the pattern observed here) and is characteristic of infarcts in solid organs, for example a myocardial infarction in the heart or a renal infarct in the kidney.

The gross morphology of an area of coagulative necrosis is observed as being a pale yellow-whitish colour with diminished transparency compared to the surrounding non-affected areas which have a good vascular supply. Initially foci may be swollen due to an inflammatory response elicited by the necrotic cells which release inflammatory factors such as cytokines and interleukin-2. Later the tissue becomes very firm.

When observed under a light microscope, the microscopic morphology of coagulative necrosis shows that despite the cells are dead and anucleate their basic cell shape, or ‘ghost’ outline (a term commonly used to describe their appearance), remains preserved for several days. An acute inflammatory response develops and this too is observable under the microscope with an influx of phagocytic leukocytes such as macrophages which remove the necrotic cells.

Provided there is an adequate amount of labile cells around the affected tissue, regeneration of this tissue can occur. Labile cells which are constantly dividing can replicate and replace the cells that were killed to restore the tissue back to normal. This is in contrast to stable or permanent cells (eg cardiac myocytes) which are not constantly replicating and so will not replace the affected tissue. Healing by fibrosis therefore follows, first with granulation tissue which is eventually replaced by fibrosis after a few months. During this time, fibroblasts can be microscopically observed in the affected tissue and the fibrosis is attributable to the firm gross appearance observed on the organ.

Thus the end result of this necrosis is a whitish-yellow area of firm tissue and a histological structure barely recognizable to the original.



Liquefactive necrosis

Figure 2: Liquefactive necrosis in the brain

Liquefactive necrosis is a pattern of necrosis which occurs as a consequence of enzymatic degradation. This is in contrast to coagulative necrosis which occurs as a result of protein degradation.

Liquefactive necrosis typically occurs in tissues which have a high lipid content, particularly the brain. Mentioned previously, whilst hypoxic cell death in most tissues normally results in coagulative necrosis, this pattern of necrosis is not usually typical to tissue in the central nervous system. Because these tissues have little structural framework, hypoxic cell death as in a cerebral infarct for example, results in disintegration of the necrotic cells and ultimately, transformation of the affected tissue into a liquid viscous mass. By taking a section through the brain, the macroscopic appearance of the liquefactive necrosis typically reveals the affected tissue to be gelatinous-looking. Microscopically, the tissue is edematous, with the neurons undergoing chromatolysis, with loss of cytological detail. An infiltrate of neutrophils, shortly replaced by macrophages, can also be observed, as well as microglia, phagocytes of the central nervous system. Removal of the necrotic cells is usually seen after two weeks concurrent with the repair process which is termed gliosis. A gross examination of the brain will now show destruction where the necrosis was and surrounding gliosis.

Liquefactive necrosis is also observed within abscesses caused by focal bacterial, and sometimes fungal, infections. Such bacterial or fungal infections result in the migration of polymorphonuclear leukocytes, specifically neutrophils and macrophages, to the site of insult. The leukocytes release enzymes to eliminate the offending microbes; however this comes to the detriment of the surrounding tissue which is also enzymatically digested, and the tissue is transformed into a viscous liquid material. Whenever liquefactive necrosis is caused by acute inflammation, the ‘liquefied’ tissue characteristically has a creamy-yellow colour and is called pus.

Caseous necrosis

Figure 3: Caseous necrosis in the kidney

Caseous necrosis is a form of necrosis characteristic of mycobaterial infections and is most commonly observed in tuberculosis lesions. The term ‘caseous’ (meaning cheese-like) arises from the gross morphology of the necrotic tissue, described to resemble crumbly yellow-white cottage cheese.

A microscopic examination of the necrotic area shows the architecture of the affected tissue to be completely destroyed: the cells are fragmented or lysed and have an amorphous and granular appearance. This is in contrast to the microscopic morphology of coagulative necrosis, where despite the cells being dead, the tissue architecture is preserved. Often the lesion is observed microscopically as a granuloma whereby there a focal area of necrosis is surrounded by a distinct inflammatory border. The granuloma comprises four recognisable layers; it has a centre of caseous necrosis which is surrounded by a layer of giant and epithelioid cells (activated macrophages). The third layer consists of leucocytes and the outer layer is a layer of fibrosis.



Fibrinoid necrosis

Figure 4: Fibrinoid necrosis of polyarteritis nodosa in arteries of the kidney

Fibrinoid necrosis is a type of necrosis that occurs specifically in blood vessels. It is associated with injury often caused by immunologically mediated reactions to blood vessels by the formation of immune complexes in the circulation. Immune complexes, which are essentially complexes of an antigen and an antibody, are deposited on the walls of blood vessels, along with fibrin, which leaks out from the blood vessels. These depositions result in its characteristic fibrinoid (or ‘fibrin-like’) appearance when viewed under the light microscope. Fibrin is easily identified by the fact it stains brightly with eosin. Following deposition of the immune complexes, an inflammatory response ensues with attempted phagocytosis of the immune complexes. The immune complexes can in addition cause platelet aggregation which enhances the inflammatory response and also initiates the formation of microthrombi contributing to local ischemia. This process can consequently culminate in the characteristic fibrinoid necrosis.

Polyarteritis nodosa, manifested by systemic vasculitis, is an example of a disease in which fibrinoid necrosis is observed.



Fat necrosis

Figure 5: Fat necrosis of the mesentery

Fat necrosis is another type of necrosis, which like fibrinoid necrosis, occurs in specific tissues. As the name suggests, fat necrosis occurs in tissues with a high lipid content and is primarily characterised by focal areas of fat destruction. It is usually caused by acute pancreatitis (inflammation of the pancreas) or by direct physical trauma to fat (for example as a result of surgery or by a physical blow).

The necrosis is ultimately due to the release of pancreatic lipases (enzymes that digest fat) out of the acinar cells and ducts of the pancreas and into the peritoneum. Here the lipases act on adipose tissue, particularly fat cell membranes, splitting triglyceride esters and causing free fatty acids to be released. The fatty acids may then combine with calcium to produce white-chalky deposits. This is called fat saponification and is visible at a gross level. A microscopic view of fat necrosis shows the shadowy outline of the adipocytes (fat cells) with a surrounding inflammatory reaction that is similar to coagulative necrosis but has the extra addition of calcium deposits.

Glossary

Abscess - a localised collection of pus

Eosinophilia - increased numbers of eosinophils circulating in the blood

Fibrosis - the extensive deposition of connective tissue, particularly collagen, in an organ or tissue, that occurs as a part of the reparative process after substantial tissue destruction

Gliosis - the process of repair and scar formation in the brain

Granuloma - a focal collection of chronic inflammatory immune cells which forms when the immune system tries to wall of foriegn material but cannot eliminate. Necrosis is often found within the centre of a granuloma

Hypoxic - an oxygen deficiency to the tissues or organs of the body

Infarct - a circumscribed area of necrosis in a tissue or organ resulting from obstruction to blood flow

Inflammation - a local response to cellular injury that serves as a mechanism to eliminate noxious agents and of damaged tissue and protect from further injury; may be acute or chronic - however when unqualified the term usually tends to refer to acute inflammation which is characterised by vascular dilatation, leukocyte infiltration, redness, heat, pain, swelling, and often loss of function

Ischaemia - a state occurring when there is an insufficient supply of blood to a tissue or organ

Karyolysis – dissolution of the nucleus by action of deoxyribonecleases

Kayorrhexis – fragmentation of the nucleus

Leukocyte - a white blood cell

Macrophages - phagocytic white blood cells that function in the destruction of foreign antigens, including microbes, as well as the removal of necrotic debris. They also function as antigen-presenting cells

Microthrombi - small thrombi. A thrombus is a solid or semi-solid mass derived from the constituents of blood within a blood vessel and remains attached to the place in which it was formed

Morphology - the structure of a tissue or organ; can be described at macroscopic(gross) and microscopic levels

Necrosis - a type of cell death 'morphologically characterized by a gain in cell volume (oncosis), swelling of organelles, plasma membrane rupture and subsequent loss of intracellular contents'

Neutrophils - are described as polymorphonuclear granulocytic white blood cells, in that they have a multi-lobed nucleus and a granulocytic cytoplasm. These cells function as phagocytes, capable of ingesting microbes and particulate matter. They are also one of the first cell types to be seen at a site of acute inflammation

Phagocytosis - the engulfment, ingestion and usually destruction of particulate or foreign matter by phagocytic cells

Proteases - Enzymes that hydrolyse peptide bonds and are highly involved in the degradation of the cell

Pus - a thick yellowish white semi-fluid matter consisting of both necrotic and living neutrophils, exudate, microbes and tissue debris

Pyknosis – condensation and skrinkage of the nucleus

Zymogens - Inactive enzyme precursor that requires a biochemical change to be activated. They are synthesised to protect the cell from unwanted enzymatic activity. They are activated only when required


References

  1. Syntichaki P, Tavernarakis N. (2002) Death by necrosis. Uncontrollable catastrophe, or is there order behind the chaos? EMBO Rep. Jul;3(7):604-9. [1] PMID: 12101090]
  2. Clarke PG. (1990) Developmental cell death: morphological diversity and multiple mechanisms. Anat Embryol (Berl).;181(3):195-213. Review. [2] PMID: 2186664


Celsi F, Pizzo P, Brini M, Leo S, Fotino C, Pinton P, et. al(2009), Mitochondria, calcium and cell death: A deadly triad in neurodegeneration, Biochim Biophys Acta, 1787(5):335-344 PMID 19268425


http://www.copewithcytokines.de/cope.cgi?key=necrosis

Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, Blagosklonny MV, El-Deiry WS, Golstein P, Green DR, Hengartner M, Knight RA, Kumar S, Lipton SA, Malorni W, Nuñez G, Peter ME, Tschopp J, Yuan J, Piacentini M, Zhivotovsky B, Melino G; Nomenclature Committee on Cell Death 2009. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ. 2009 Jan;16(1):3-11. Epub 2008 Oct 10. PMID: 18846107

Kumar, V., Abbas, A., Fausto, N. & Mitchell, R., ‘Robbins Basic Pathology’, 8th Edition, Saunders Elsevier, China, 2007.


Majno G. and Joris I. (1995), Apoptosis, Oncosis, and Necrosis, an Overview of Cell Death, American Journal of Pathology, 146(1):3-15 PMID: 7856735

Nagańska E, Matyja E. Folia Neuropathol, Ultrastructural characteristics of necrotic and apoptotic mode of neuronal cell death ina model of anoxia in vitro, 2001;39(3):129-39. PMID: 11770123


Syntichaki P, Tavernarakis N. (2002), Death by necrosis. Uncontrollable catastrophe, or is there order behind the chaos? EMBO Rep. 3(7):604-9. PMID: 12101090

Power. C., Fanning Noel and Redmond. P. (2002), CELLULAR APOPTOSIS AND ORGAN INJURY IN SEPSIS: A REVIEW, Shock, 18(3),197-211


Proskuryakov SY, Konoplyannikov AG, Gabai VL. (2003), Necrosis: a specific form of programmed cell death? Exp Cell Res, 283(1):1-16. Review. PMID: 12565815


Van Cruchten S, Van Den Broeck W. (2002), Morphological and biochemical aspects of apoptosis, oncosis and necrosis. Anat Histol Embryol. 31(4):214-23, PMID: 12196263



2009 Group Projects

--Mark Hill 14:02, 19 March 2009 (EST) Please leave these links to all group projects at the bottom of your project page.

Group 1 Meiosis | Group 2 Cell Death - Apoptosis | Group 3 Cell Division | Group 4 Trk Receptors | Group 5 The Cell Cycle | Group 6 Golgi Apparatus | Group 7 Mitochondria | Group 8 Cell Death - Necrosis | Group 9 Nucleus | Group 10 Cell Shape