Cell Death – Apoptosis and Necrosis – Dissertation Sample

Necrosis vs. Apoptosis – An overview

Necrosis

Cell death takes place due to either of the two distinct phenomena – necrosis and apoptosis. These phenomena are collectively called cell death which may be accidental or programmed. Necrosis is death by accidental and unexpected cell damage and several physical damaging events can cause necrosis like exposure to toxins, excessive heat, trauma, radiation effects, lack of oxygen and a block in the normal flow of blood (Jacobson et al, 2002). Necrosis type of damage disrupts the cell organisation and contents and causes faster cell death than most other methods. As cell death proceeds, necrotic cells swell and holes and gaps begin to be seen on the plasma membrane. Intracellular materials begin spilling out of these holes when cell death occurs and there is a total disruption of the workings of the cell.

Normally the levels of Calcium and Ca2+ intracellular concentration are less than 10-7m whereas outside the cell it is generally ~ 1mM. High levels of calcium are found within the mitochondria and endoplasmic reticulum. When cell death or damage occurs, there is a rapid and sudden increase in the intracellular Ca2+ concentration. cell damage seems to draw out calcium to a great extent. Calcium is considered as an allosteric effector of proteins and changes protein organisation and activity of the cell. The release of this excess Calcium in the intracellular environment seems to generate many toxic chemicals within the cell environment and also induces harmful enzymes to activate the degradation and degeneration of cell structures and molecules. A release of excess calcium seems to be the initiating phenomenon leading to a host of changes within the cellular environment from activating enzymes and chemicals too degrading cellular structures.

With the initiation of cell breakdown, there is a subsequent breakdown of chemicals and new breakdown products are formed that are released into the cell environment (Potten and Wilson, 2004). The disassembling is accompanied by the break down of cellular substances to form certain new products such as phospholipids, such as arachidonic acid, free fatty acid or FFA. As son as these breakdown products are released into the intracellular environment, the neighbouring tissues and cells pick up signals of tissue damage and react to defend themselves from the process. The damaged and dead cells spew out calcium and in the process generate arachidonic acid and free fatty acids. arachidonic aicd acts as a substrate for several generating enzymes like the cyclo-oxygenases which form prostraglandins, and eicosanoids mediating inflammatory responses. This series of cell responses from the excess of calcium to generation of arachidonic acid and enzymes and eicosanoids leads to chronic inflammation in the body tissue.

Medical and mutation studies have pointed to the fact that the formation of eicosanoids is major reason for chronic inflammation so a way to stop the necrosis and chronic inflammation can be by stopping or inhibiting the production of enzymes generating these eicosanoids. With cell damage, vasoconstriction occurs which tends to stop the overall normal blood flow and the breakdown products that are released like the fatty acids also cause the capillaries to dilate. As this happens, the local blood flow within tissues seems to increase and this is accompanied by tissue redness and the release of histamines leading to stimulation of pain sensing neurons. As this happens, capillary permeability increases, there is an increase in the number of leukocytes and macrophages, white blood cells that come from the circulatory system to the damaged area and water from blood enters the tissues causing a swelling, inflammation or oedema (Gibson, 2004).

The capillaries within the tissues break and these are sealed from excess blood loss by forming a clot. These blood clots are formed by catalysed proteins that precipitates based on proteolysis. All bacteria, cellular remains, debris and other foreign media are engulfed and digested by the white blood cells. As a result of the presence of white blood cells in large amounts, the immune system gets activated and the area is properly sterilised. pus formation occurs when white blood cells fail to be adequately active and die leading to the damage of the tissues in the region. A drying up of pus and the tissue wound in general suggests that the area has begun to heal and there is general tissue re-growth and healing process under way.

Chronic inflammation has to be treated immediately otherwise there can be complete and irreparable damage of the cells in the inflamed area and tissues will fail to regenerate. One of the anti-inflammatory drugs given has cyclooxygenase inhibitors and these are found in the market popularly under the names of ibuprofen and aspirin.

Apoptosis

From a discussion of the general tissue damage as it happens in necrosis and the body defences that come into play, we turn to apoptosis another method of cell death and damage that is different from necrosis in certain fundamental features. Apoptosis has been originally described in plants and refers to the process of removing unwanted cells. The falling of leaves can be considered a sort of Apoptosis although it now means a wider range of cell deaths and include all non-traumatic and natural cellular death and damage (Wyllie, 1997). Thus whereas Necrosis refers to cell death and damage due to unnatural means, accidents and exposure to harmful substances of events, apoptosis is natural programmed cell death. Apoptosis is the method by which unwanted cells are removed from an organism. During this type of cell death, there is no release of calcium or such substances into the intracellular or extra-cellular environments, there is no rupture of cell membrane and no inflammation occurs.

The neighbouring cells engulf and destroy the apoptotic dead cells and they are removed without causing much pain or botheration to the body. Programmed cell death or apoptosis is the normal process of cell death. It is necessary in human body as dead cells are constantly replaced by new ones. Death and replacement of cells is a normal and necessary process in every organism and this process is programmed within the body and happens naturally at regular intervals (Gray, 2003). There is no eternal reason for this and cell death seems to be a natural body mechanism for regeneration of the body cells. 50% of neurons are regenerated and replaced during the development of the human vertebral system. During the formation of the foetus or metamorphosis is many insects and animals as in frogs or moth happen due to death of old cells and formation of new ones. This cell death and subsequent cell formation is caused by the natural process of cell death by apoptosis programmed within every living system.

Usually signals are transmitted for cell death and normal cells die after a period of time on receiving these signals. Entering a stage of apoptosis may be a very normal routine for most cells which die to be replaced by newer cells although cells can also enter apoptosis due to viral infection, DNA damage or cellular stress. The cell death is mediated by protein molecules that destroy all aberrant and deviant cells such as the cancer cells. The cell death of cancer cells are generally inactivated by mutations and by virtue of stopping cell death of aberrant cells by mutations, cancer cells can multiply fast with aberrant DNA (Lomo, 1998). This can be stopped with an apoptotic pathway and leads to cell death. The apoptotically dying cells can activate a group of potentially degradative enzymes also called the caspases and these enzymes mediate controlled disassembly and degeneration of the concerned cell.



Quite like the eukaryotic cells, the single celled bacteria are also capable of apoptotic cell death Apoptotic mechanisms seems to have some important functions for the bacterial cell. Bacterial cells generate an addiction module with a stable toxin and an unstable anti-toxin. When there is a block in the protein synthesis anti-toxin synthesis and toxin synthesis are both stopped. The unstable anti-toxins disappear slowly although the stable toxins are retained. With the gradual disappearance of the unstable anti-toxins along with the persistence of toxins, there is a complete shortage of anti-toxins. In this condition, the toxin takes control, becomes active and kills the cell. Bacterial programmed cell death thus seems to happen very much due to an imbalance in toxins and anti-toxins in the cell.

Differences between Apoptosis and Necrosis

Necrosis is uncontrolled unnatural cell death; it is not pre-programmed or programmed by the body in any way and has some distinguishing features:

1. It shows swelling, sometimes chronic inflammation,

2. there is considerable damage to the mitochondria and endoplasmic reticulum of the cells

3. there is a breakdown of homeostasis in the body

4. there is a rupture of the cell membranes, lyses leading to release of intercellular substances in the intracellular regions of the cell

5. formation of certain breakdown products and enzymes and subsequent formation of eicosanoids lead to oedema, and damage and death of cells.

6. In most cases, the formation of certain enzymes and release of free fatty acids send signals to surrounding cells and these are then able to defend themselves from damage

7. in case of neuronal necrosis sometimes, neurotransmitters are released and this can have excitatory properties that can cause excito-toxic injury to neighbouring cells.

Apoptosis on the other hand is controlled, programmed cell death and keeps all intracellular substances of the dying cell separate and sequestered so that one malfunction or cessation of function does not affect the other and unlike necrosis, in apoptosis one cell damage does not cause damage of surrounding tissues. The distinguishing features of apoptosis are:

1. There are many cellular changes that seem to happen in a internally regulated manner

2. the cell undergoing apoptosis shrinks in size, gets sequestered from surrounding cells and loses its connections with the surrounding intracellular matrix.

3. The cell displays intracellular proteins on its surface, the chromatin of the nucleus begins condensing and the DNA breaks into smaller fragments with several base pairs and lead to DNA laddering

4. the plasma membrane puffs up like bubbles and small bodies attached to the membrane break off carrying intracellular material like nuclear matter and cellular organelles. These organelles however remained unaffected by the process

5. the fragments of cellular material and cells which break off and disintegrate from the surrounding matrix are called apoptotic cells and these are quickly removed and destroyed by phagocytosis

6. If the removal of these apoptotic cells do not occur fast, there can be a process called secondary necrosis that results in the breakdown of intracellular organelles and plasma membrane causing lyses of the fragments.



Thus apoptosis and necrosis have different methods of cell death and completely different mechanisms, end results and physical outcomes. The differences are marked and we would take our discussion further on research studies emphasizing these two modes of cell death as well the distinct and different findings involved.

According to our preceding discussion, cellular mechanisms have two kinds of death responses

Necrosis – indicates an unnatural and pathological response of cell to external causes leading to cell damage and injury where the chromatin lumps up and rupture and lyses of the plasma membrane is followed by the swelling and rupture of mitochondria and cell contents spill out of the cell causing inflammation, oedema and enzyme reaction and a general overall tissue damage is triggered.

In Apoptosis however the chromatin condenses and migrates towards outer regions of the cell. The cytoplasm also sinks and the plasma membrane bubbles out and separate nodules are formed containing cellular organelles (Lavin and Watters, 1993). These separate nodules break off at a certain point and are engulfed by neighbouring phagocytic cells. There is no spillage, rupture of cell membrane or inflammation. Apoptosis represents the body's natural way of facing cell death so no negative signals or damage of whole tissue areas is seen. Here cell death occurs not due to toxins or unnatural means but simply due to a lack of receiving survival signals. Necrosis represents unnatural cell death with inflammation. Apoptosis represents natural cell death without any inflammatory response.

There are other processes by which cells are destroyed. One of them is autophagy. This occurs when there are inadequate nutrients within the cell and certain organelles within the cell body have to be used up for reuse of the components within the organelle. Double membranes form within the cell, the material marked is engulfed and an autophagosome is formed which fuses with the lysosome and the hydrolytic enzymes then degrade the materials.

Research Evidence on Cell Death

In a study on cell death, Majno and Joris (1995) reviewed the historical development of cell death and traced the origin of terms necrosis, coagulation necrosis, autolysis, physiological cell death and programmed cell death as also chromatolysis, karyorhexis, karyolysis and cell suicide. According to the authors there are three forms of cell death, by lysosomes, free radicals and genetic mechanism as in apoptosis. In contrast to blebbing and zeiosis typical features of apoptosis include budding, and the typical feature of necrosis, i.e. inflammation is also discussed. Cell death is categorised into two major divisions in this paper, either programmed cell death as in apoptosis or accidental cell death as in necrosis.

According to the authors, necrosis however is an incorrect term for cell death as it can indicate changes secondary to cell death which occurs only by apoptosis. So according to Majno and Joris, whereas apoptosis indicates primary cell death, necrosis refers to associated and secondary changes that follow apoptosis. Some suggest this as secondary necrosis which sees necrosis not as a different kind of cell death but a category only secondary to the main kind of cell death, namely apoptosis. One type of accidental cell death highlighted by Majno and Joris is Ischemic Cell death which is a category of its own caused mainly by a failure of ionic pumps of the plasma membrane. Ischemic cell death is accompanied by swelling and thus it is not called apoptosis but oncosis, derived from onkos, means ‘to swell’. Oncosis which is always accompanied by some form of swelling of tissue structure leads further to necrosis with karyolysis and this is in contrast to apoptosis leading to necrosis with karyohexis and cell shrinkage.

Afford and Randhawa (2000) discuss Apoptosis in greater detail. According to them apoptosis is a genetically related form of cell death and permits the safe disposal of cells at a point of time when their use to the body is over and the period for which they lived was long enough for them to complete their complete intended biological function. Apoptosis is seen in plant as well as animal tissues. It is considered as one of the vitally important processes and a normal process in development. Unlike necrosis which is pathological, apoptosis is an important part of normal development and adult life of many living organisms.

Within the human body any dysregulation or malfunction of the apoptotic mechanism can result in inflammatory, malignant, autoimmune and neurodegenerative diseases and can completely damage tissues and also the entire body. Along with these possible damaging effects, viruses and other infectious agents can exploit cellular apoptosis and host and invade the immune system. Afford and Randhawa's study give a brief description of some landmark discoveries in apoptosis research and covers the morphological and biochemical aspects of apoptosis and also discuss the implications of therapeutic intervention in treatments of diseases associated with apoptosis. The period of cell death or at the stage of apoptosis the tissues are vulnerable to certain infections and this can cause irreparable damage to the tissue structures of the system.

Franko et al (2000) have worked on the mechanism of apoptosis in some detail and noted future directions in apoptosis research. The rapidly developing research paradigms on cell death and the associated disorders has seen considerable progress in the last few decades and especially in the last five years this are of study has reached new horizons in medical research. According to studies by Franko and his colleagues, identification of different kinds of morphological and signalling aspects as well as the variances in requirement for energy aided them to construct a theory of three kinds of cell death mainly – apoptosis, necrosis and lysosomal cell death.

Oncoprotiens of the Bcl-2 family, mitochondria and certain catabolic enzymes that participate in the process of cell death serve as targets for pharmacological manipulation. In oncology, chronic inflammation and in ischemic, neurodegenerative and autoimmune disorders, the up regulation or down regulation of programmed cell death/ apoptosis is usually implicated. The study by Franko and his colleagues is an overview of genesis and development of theories on programmed cell death and apoptosis and follows the basic theoretical approach of three kinds of cell death as in apoptosis, necrosis and lysosomal cell death. Along with summarising the basic facts of apoptotic mechanisms, they also draw on the implications of their theoretical approach in medicine and surgery. Such categorisation as specified by them seems to be advantageous for medicinal, research and surgical purposes.

Laying emphasis on another aspect of apoptosis, Hashimoto (1997) mentioned that apoptosis is a form of cell death which is not only natural and necessary but also responsible for development and homeostasis of living bodies. Hashimoto relates apoptosis to induction factors and discusses the molecular mechanisms of induction and its relation to various diseases like cancer. The relation of apoptosis and necrosis to tumour growth and cancer is an important point which we will be discussing in later section in some detail.

Fadeel et al (1999) have identified the role of apoptosis in the genesis of diseases and emphasize that naturally occurring cell death or apoptosis is necessary for the maintenance of tissue homeostasis and helps in the removal of extraneous, unnecessary and dangerous cells in a very swift and unobtrusive manner. They pointed out that apoptosis has a significant role in a number of human diseases and pathological conditions. Dysregulation and a malfunction of apoptosis have been related to autoimmune diseases, acquired immune deficiency syndrome, and many viral and bacterial infections, as also neurodegenerative diseases and disorders like cancer.

When naturally damaged and unnecessary cells replicate with faulty DNA and do not get removed naturally, they can lead to cancerous cell growth implying defective apoptotic systems within the body and possibly a faulty immune system. Also a dysregulated apoptotic process can impinge on age related disorders like osteoporosis, atherosclerosis and the process of aging itself. Fadeel and his colleagues give an overview of human diseases associated with a defective or inadvertent apoptosis and in their analysis they give examples of pathological conditions in which putative apoptosis defects have been described successfully at the molecular level. As a recommendation they suggest some novel apoptosis modulating and regulating therapeutic strategies which according to them can promote an apoptotic process free from dysfunction or dysregulation.

Reiterating on the significant role of apoptosis in maintaining tissue homeostasis and proper working of an organism in general, Hetts (1998) indicate that the death of cells in tissues of humans and other multi-cellular organisms in neither always abnormal nor always detrimental ass in apoptosis cell death is completely normal, necessary, and regulated by an internal mechanism. Hetts describes this point further and writes that although necrosis ensues at sites of massive cellular injury, most body cells die through a subtle, non- inflammatory and energy-dependent form of cell death by the method of apoptosis. So the more common form of cell death is apoptosis as it is the most natural and painless and necessarily occurring phenomenon in the bodies of living organisms. Hetts claims that the number of cells in tissues of a living organism’s body is determined by the homeostatic balance between proliferation of new cells and death of old cells and the rates of proliferations and cell death by apoptosis vary widely form one tissue to another.

According to Hetts, recently conducted research delving into the molecular mechanisms of apoptosis has revealed that apoptosis is a genetically programmed cellular death and removal process that can become dysfunctional and deranged when components of the cellular apoptotic machinery are mutated or are present in inappropriate quantities leading to a lack or insufficiency in timely death signals for the cells. Although, cell death is in some cases harmful as seen in necrosis, it is a necessary process of cell growth and development of an organisms as seen in apoptosis and when there is a dysfunction or dysregulation of apoptosis, a pathogenesis occurs that may be associated with a wide variety of diseases such as cancer, neuro-degeneration, autoimmunity, heart diseases and other such disorders.

There are many genes and gene products which are involved in the regulation and execution of apoptosis and these genes are potentially excellent targets and elements for diagnosis and therapeutic intervention when diseases occur and identification of these genes are helpful as targets as they offer renewed hopes for cures and treatments of various types of diseases. Thus understanding the mechanism of certain neuro-degenerative and cancerous diseases can help identify the underlying gene processes involved and the treatments that could be developed as a result of such identification of the disease causing genes.

Giving an account of nuclear apoptotic death, Martelli et al (2001) suggests that apoptosis is a form of active cell death and is essential for morphogenesis, development and cell differentiation of all multi-cellular organisms. Taking a rather evolutionary approach in their explanation of their study, Martelli and his colleagues claim that the activation of genetically controlled pathways have been conserved in evolutionary history in the characteristic morphological features of all multicultural living organisms and these features are usually evident in the nucleus of a cell body. Within the cell, certain mechanisms are seen common in all living organisms associated with the process of apoptosis.

These include chromatin condensation, nuclear shrinkage and formation of nodules, budding of the membrane and formation of apoptotic bodies with their cell organelles. these morphological changes within the cell structure that are seen in apoptosis occur due to molecular alterations as in DNA and RNA cleavage, post transitional modifications of nuclear proteins and proteolysis of polypeptides which reside in the nucleus. Studies in the last five years has shed considerable new evidence on the workings of apoptosis and changed our understanding of this programmed nature of cell death considerably. Martelli et al claim although the mechanism of apoptosis itself has been made considerably clear in recent times due to increased research and evidence, the mechanism that lead to apoptotic changes within the nucleus is only particularly understood or clarified. Their study tries to address this issue and tries to delve deeper into why the nucleus shows the modifications that it dies during apoptosis. The authors also discuss those apoptotic events that act as a trigger for the generation of auto antibodies to the nuclear components.

Apoptosis can not only occur in isolation within a human body but can be associated with necrosis and there can be a cell death mode switch from the unnatural death of cells as in necrosis to the natural cellular deaths as in apoptosis. Ueda and Fujita (2004) report such a case in their study pointing out that in brain ischemia cell destructive necrosis occurs in the core of the brain tissue which in turn links to cell death that tends to expand to the vicinity.

Thus necrosis occurs at the centre, the main region of cell damage but cell damage expands and reaches the vicinity, the regions surrounding the core also known as the penumbra and thus apoptosis starts off several days after the initial necrosis. The authors further describe the process where cells showing apoptosis disappear by microglial phagocytosis in the brain, that is they are removed and digested by neighbouring cells and as this happens the cell death which has been induced by ischemia and brain ischemic stress is eventually terminated. This according to the authors is a self protective mechanism in the brain and their hypothesis is that a cell death mode switch in brain ischemia when necrosis at the core is slowly replaced by apoptosis at the periphery is an in vivo self protective mechanism.

The authors review the current understanding of the molecular mechanisms of both necrosis and apoptosis in relation to the ASTP hypothesis and introduce novel mechanisms by which they could explain the in vitro cell death mode switch. A cell death mode switch from necrosis or unnatural death of cells in the brain representing cell damage and inflammation to apoptosis or natural cell death due to a change in signals received by the brain represents a regulatory mechanism in the tissues that is capable of changing a potentially destructive process into a constructive and defensive body mechanism. This way the natural change from necrosis to apoptosis is represented as a self protective mechanism.

Stadelmann and Lassmann (2000) have studied extensively on the methods involved in the detection of apoptosis. During the last five years or so the detection of apoptosis has evolved from identifying the predominantly morphological basis to using the more specific techniques to understand its workings. The methods which are used widely to visualize DNA fragmentation in tissue sections are now supplemented by information on specific cell death pathways and the components involved. According to the authors the essential requirements for successful detection of apoptosis include detection techniques considering high sensitivity of apoptotic cells, the ability to differentiate between apoptotic and necrotic cell death and other different forms of DNA damage as also the detection of different stages of the cellular death process.

The recent technical advance in apoptosis detection covers improvement in DNA fragmentation techniques and also the new tools that are available for detection of apoptotic cells in the tissues. Sgonc and Wick (1994) detailed other methods of detection of apoptosis and suggested that apoptosis is central to many basic clinically oriented investigations and according to them the more frequently utilized methods for detection of apoptotic cells include the study of morphology, analysis of DNA degradation, DNA end labelling techniques, flow cytometric analysis and nuclease assays.

Apoptosis and necrosis have also been reported in fetus cell death due to intake of alcohol by mother during pregnancy. Maternal drinking leads to severe damages in the fetus and the potential mechanisms through which damage can occur are 'increased oxidative stress, damage to the mitochondria, interference with the activity of growth factors, effects on glial cells, impaired development and function of chemical messenger systems involved in neuronal communication, changes in the transport and uptake of the sugar glucose, effects on cell adhesion, and changes in the regulation of gene activity during development' (Goodlet and Horn, 2001).

As a result of the drinking, there are adverse effects in the offspring including cognitive impairment, growth deficiency, and CNS disorders. These associated changes happen because of inadequate development of the fetus due to cell death and damage within the fetal structures. This may be due to increase of toxins in the fetal blood.

Another factor that can be potentially dangerous to cells and cause cell death and damage is exposure to magnetic fields. A study by Lai and Singh (2004), found that acute magnetic field exposure increased apoptosis and necrosis of brain cells in the rat. The authors hypothesized that exposure to a 60-Hz magnetic field initiates an iron-mediated process or a reaction that increases free radical formation in brain cells, leading to DNA strand breaks and cell death. The authors suggest that their hypothesis could have important implications for health effects that are associated with exposure to extremely low-frequency magnetic fields in the public as well as occupational environments.

Exposure to metals, and toxins as well as magnetic fields lead to cell death and damage and we have cited several research studies to support this. Recent studies by Rahman et al (2002) provides further evidence on this as they found that inhalation of ultrafine titanium dioxide induces micronuclei and apoptosis in embryo fibroblasts as revealed by electron microscopy.

Although necrosis results from exposure to toxins and damaging external conditions, several recent studies have shown apoptosis is also induced by such mechanisms leaving the debate on the differences between the characteristics of necrosis and apoptosis open for further detailed research.

Yamashima (2000) discuss at length the implications of necrosis, apoptosis and cysteine proteases such as calpain, cathespin and caspase in ischemic neuronal death of primates. According to Yamashima, recent studies in ischemia and cell death have suggested that neuronal death after brief global ischemia occurs by apoptosis which according to him is an active and genetically controlled 'cell suicide' process. Apoptosis is thus often seen as cell suicide when a cell dies because of a genetically programmed and controlled process. Yamashima argues that studies on monkeys and humans support necrosis which is a calpain mediated release of lysosomal enzyme cathepsin.

This follows from the fact that ischemia contributes to cell degeneration of neurons. Yamashima presents an overview of neuronal cell death in his paper and presents the cascade of primate neuronal death keeping in mind the roles of cysteine proteases and indicates that selective cathepsin inhibitors is a novel neuro-protectant that works for this purpose. He also suggests the significance of a possible interaction among calpain, cathepsin and caspase within a cascade of ischemic neuronal cell death.

In another recent study on apoptosis and necrosis in the developing cerebellum and brainstem which is induced after focal-cerebral hypoxic ischemic injury, Peng et al (2005) discuss the associated cellular death and changes following focal cerebral hypoxia and ischemia. The authors highlight the fact that focal cerebral hypoxia-ischemia due to isolated vascular insufficiency is generally known to cause ipsilateral but not contra lateral cerebral apoptosis yet the hypoxic-ischemic damage to the cerebellum and brainstem has not been studied sufficiently.

Peng et al's experimental study on rodents demonstrates through DNA fragmentation and a labeling analysis (Peng et al, 2005) that neuronal cells in certain infratentorial regions also suffer from mild apoptosis and necrosis following a focal cerebral hypoxic ischemic injury in a newborn rat. This study is similar to a previous study where the damage of focal/ core brain cells due to necrosis is taken over by apoptotic damage of the peripheral brain cells. Peng et al's data definitely provide support to such studies and also provide additional insights into the mechanisms of neuronal injuries in the brain as a whole as also in specific areas as in brainstem and cerebellum areas resulting from a focal cerebral hypoxic-ischemic damage. Their study indicates and also experimentally demonstrates that all future therapeutic interventions for hypoxic-ischemic encephalopathic system must deal with the entire central system and consider cells at the peripheral regions of the CNS as also cells in the focus or core of the CNS that is the primary area subject to the injury.

The differences between Apoptosis and Necrosis at a more molecular level will be taken up in our discussions. Catelas et al (2005) suggest a qualitative analysis of macrophage apoptosis versus necrosis induced by cobalt and chromium ions and report some differences. Their study is based on the fact that the toxicity of metallic ions in tissues is a matter of concern for researchers and several studies are being conducted to know the exact effects of metal ions in cell mortality. Some previous studies demonstrated in laboratories suggested that Co2+ and Cr3+ ions induce TNF secretion in, macrophages as also in cell mortality. Catelas and his colleagues tried to quantify the rate of macrophages mortality either by apoptosis or by necrosis induced either by cobalt ions or by chromium ions. The researchers used electron microscopy, flow symmetry and ELISA cell detection methods to illustrate cell death differentiation between apoptotic and necrotic cells.

This study was performed experimentally on conventional cell culture conditions as J774 mouse macrophages were incubated in a growth medium for 24 to 48 hours. Cell culture indicated that cells which were exposed to low concentrations of Co2+ revealed a low degree of mortality whereas at highest concentrations of Co2+, late apoptosis occurred within 24 hours of placing the cell in the concentration. Although the initial 24 hours showed apoptotic cell death, after 48 hours there was clear evidence of an increased rate of necrosis and at this time apoptosis occurred at a much slower rate. In contrast macrophages which were exposed to Cr3+ demonstrated a predominance of apoptosis after the 24 hour period and very low concentrations of Cr3+ ions early and late apoptosis both occurred at the same rate. With higher concentrations the number of early apoptotic cells decreased and the number of late apoptotic cells increased considerably. After 48 hours the concentration of chromium when low induced a higher degree of early apoptosis and some necrosis also followed. At higher concentrations the percentages of early apoptotic cells decreased considerably and necrosis became the predominant process and replaced late apoptosis.

The study by Catelas and his colleagues demonstrates that macrophage mortality which is induced by metal ions depends on the types, concentrations of metal ions present and also the duration of the exposure of the cells to the concentration being studied. According to the authors, their study showed an overall predominance of apoptosis after the first 24 hours for both Co2+ and Cr3+, cobalt and chromium ions although higher concentration of these after 48 hours mainly induced the necrotic cell death process. This finding definitely suggests that when tissues have large concentrations of metal ions, after 48 hours there may a tendency to show tissue damage and inflammation as necrosis may start developing at this period.

This study has wider implications for research and understanding of tissue and cell damage and necrosis and apoptosis in general. There are two conclusions from this study. Apoptosis and necrosis are both possible with metallic ions and whereas apoptosis is seen in the first 24 hours of tissue exposure to metal ions, necrosis follows when high concentrations of metal ions are present near the tissue and the tissues are exposed for more than 48 hrs. The tissue culture results are generally important as it has implications for the role of metal ions in tissue death as well the differences of concentrations and exposure times of metal ions in necrosis and apoptosis. This is one further difference between the two types of cell death processes that we have identified and discussed.

The role of metal ions in inducing apoptosis and necrosis and the differential effects of Co2+ and Cr3+ ions have been studied further by Huk et al (2004). They justify the renewed interests in the use of metal-to metal (MOM) implants for total hip arthroplasty (THA) and reiterates that MOM articulation generates both metal particles and ions. However the exact physiological effects of these metallic ions are not completely known and their potential toxicity is according to many researchers a cause for concern as they are known to induce cell death.

In Huk et al's study, the researchers used murine J774 macrophages which were incubated with Co2+ and Cr3+ ions and the mode of the cell death whether apoptosis or necrosis after a period of time was evaluated in vitro by transmission fo electron microscopy and using cell death ELISA procedures. As according to the study by Catelas (2005), cell death was found to be dependent on ion concentration and the incubation time. According to these results, at short incubation times of 24 hours, the non inflammatory death and process of apoptosis was predominant. At longer incubation times of 48 hours and more, necrosis was found to be the predominant method of cell death when metal ion concentrations were high.

Vairetti et al (2004) discuss the role of apoptosis and necrosis in glutathione-mediated cell death. They suggest that hypothermia induces injury and lead to cell damage. Their study aimed to study the effects that glutathione (GSH) depletion induces on cell death in the isolated rat hepatocytes that was kept at 4°C for 20 hours. They modulated the intracellular GSH concentration using diethylmaleate and buthionine sulfoximine (DEM and BSO). The untreated hepatocytes showed Annexin V stained cells (AnxV+), scarce propidium, iodide stained cells (PI+) which were associated with LDH release within the incubation medium.

The addition of DEM and BSO during the re-warming phase caused a radical increase in cell death by apoptosis. Production of reactive oxygen species (ROS and the thiobarbituric species (TBARS) is associated with a decrease in GSH concentrations which was higher when DEM and BSO were added before cold storage. Cells which were treated with DEM and BSO before the cold storage showed much lower TP energy than the hepatocytes which were treated with DEM and BSO only during rewarming. Hepatocytes when pre-treated with deferoxamine were protected against apoptotic and necrotic morphological conditions and GSH depletion. According to Vairetti and her colleagues the results of their study suggests that pre-treatment of hepatocytes with DEM and BSO before cold storage can induce necrosis while the treatment of hepatocytes only during the re-warming period increases apoptosis. The findings suggest that in both the conditions during pre-treatment and during rewarming periods, iron represents the crucial mediator for cell death and the presence of iron is important in causing cell death either by apoptosis or by necrosis.

Borst and Rottenberg (2004) analyzed Zong et al's (2004) paper on the effects of alkylating DNA damage stimulating a regulated form of necrotic cell death. Zong and his colleges describe that alkylating agents kill cells by the process of something called 'programmed necrosis' which is induces by an excessive activation of PARP resulting in degradation of cystolic NAD+ and inhibition of glycolysis. According to Borst and Rottenberg, it cannot be sufficiently proved whether chemotherapy in patients can induce a sufficient NAD+ loss to affect glycolysis and it is also not evident in contrast to what argued by Zong et al, whether necrosis can be really programmed and whether there are mechanisms that make cancer cells hypersensitive to DNA damage other than the fact that they have a high rate of aerobic glycolysis.

Discussions and Conclusion

In this essay we saw various approaches to the study of cell death in accidental conditions as in necrosis or programmed cell death as in apoptosis. Although there are general and controversial discussions whether necrosis which is caused by toxins and other external agents can ever be programmed internally. Necrosis results in tissue damage irreparably at times whereas apoptosis is a natural and necessary way to rid the body of damaged and unwanted cells.

We discussed several issues here, how necrosis and apoptosis could be related and how they could be different going by the facts that apoptosis is considered genetically and internally timed , regulated and programmed and most cells die and are replaced by new ones. Apoptosis happens in a natural and painless way as there is much shrinking and budding off and finally breaking off of cell organelles which are later engulfed and removed by neighboring cells. In necrosis, accidental cell death leads to inflammation of cell tissues, painful conditions, swelling and enzymatic reactions in the body.

There have been several studies though that an initial necrosis can be later taken over by apoptosis leading to a natural removal of damaged cells and curing of the diseased condition. However the opposite is dangerous as natural cell damage when replaced by unnatural and repeated damage of surrounding tissues can cause severe harm to the body. Some researchers have claimed necrosis, apoptosis and lysosomal cell death related to autophagy are the three main types of programmed cell death. However in general apoptosis and necrosis remain largely the main types of cellular death and damage.

The differences of apoptosis and necrosis are not only seen in their structural and functional dissimilarities, they are also seen in the manifestations of these cell death modes in reactions to certain chemical concentrations of ions in the tissues. The presence of higher concentrations of metal ions like Cr3+ and Co2+ can cause apoptosis at lower incubation period of 24 hours and necrosis at higher exposure periods at 48 hours. This suggests that apoptosis begins sooner than necrosis in most cases of exposure to toxins or contaminants as well. The reason for this is not completely known, however it may be suggested that the metabolic and enzymatic reactions in apoptosis begins much faster than in necrosis.

Knowledge of the molecular mechanisms of apoptosis has led to better understanding of cell death processes in general and dysregulation of apoptosis has been found to lead to cancer and certain pathological conditions. A deeper understanding of apoptosis and necrosis will have to be attained to find out the exact mechanisms of cancerous cell growth and the means of counteracting such growth using further research in medicine and biochemical studies.