Neural Injury at the Molecular Level

Summary of Key Points

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The molecular mechanisms of neural damage due to spinal cord injury and chronic spinal cord compression.
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Apoptosis is the primary force driving cellular loss following injury and an important determinant of morbidity.
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Oligodendrocytes are the primary cells affected by apoptosis in addition to smaller subpopulations of neurons and astrocytes.
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The cause of oligodendroglial apoptosis is multifactorial and includes oxidative damage, mechanical stress, and inflammatory signaling cascades.
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Following spinal cord injury, white matter loss continues to occur for several weeks in a more extensive axial section from the site of cord injury, a process known as secondary injury.
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TNF-alpha levels are known to increase following spinal cord injury, and interventions blocking TNF-alpha have reduced the extent of oligodendroglial apoptosis and improved functional outcomes in animal models.
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Further modulation of the inflammatory cascades following spinal cord injury can thus provide a potential therapeutic strategy to minimize the extent of secondary injury and preserve function.

The histopathologic appearance of acute traumatic spinal cord injury and chronic spinal cord injury has been well described. Chronic cervical spondylotic myelopathy includes the characteristic features of regional demyelination extending axially from the site of compression, preferential lateral column axonal loss, and anterior horn neuron dropout. Ongoing research projects are creating a better understanding of myelopathy on a molecular level, and studies indicate that a significant portion of cell loss appears to be caused by the process of programmed cell death, also known as apoptosis . Although the molecular pathways regulating apoptosis are extremely complex, programmed cell death affects restricted populations of spinal cord cells—including oligodendrocytes and some neuronal and astrocytic subpopulations—suggesting the possibility that targeted antiapoptotic therapy may be a reasonable goal for the treatment or prevention of myelopathy.

Microbiology of the Oligodendrocyte

The oligodendrocyte has been shown to play a pivotal role in several complex biologic processes, including development, injury repair, disease process modulation, and the formation and maintenance of myelin. During the early stages of human development, a large oligodendroglial population is generated, and an estimated 50% of these cells eventually disappear by the process of apoptosis. As the central nervous system matures, the oligodendroglia become responsible for the creation and maintenance of myelin sheaths. These sheaths, although formed directly from oligodendroglial cell membrane, demonstrate key biochemical differences from the parent cell membrane in terms of both chemical and protein composition. The biochemical and physiologic characteristics of the relatively small protein constituent are especially important, and absence or alteration of the major protein components (i.e., proteolipid protein or myelin basic protein) can lead to the advent of severe demyelinating disease. Another unique feature of the oligodendrocyte is the high concentration of microtubules, which contribute to formation of an elaborate cytoskeletal framework, allowing myelin sheath formation at remote distances from the cell karyon.

Considerable progress has been made in understanding the response of oligodendroglial cells to injury, and a more complete understanding of this complex process may lead to a greater appreciation of the mechanism of injury in processes such as cervical spondylotic myelopathy. Studies suggest that the oligodendrocyte is particularly sensitive to a wide range of oxidative, chemical, radiation-induced, and mechanical injuries. High iron content and relatively inefficient antioxidant defense mechanisms appear to render the oligodendrocyte vulnerable to oxidative stress. Injury-related release of intracellular iron may contribute to the generation of damaging hydroxyl radicals through the Fenton reaction. In addition, in vitro exposure of mature oligodendrocytes to hydrogen peroxide has been shown to induce apoptotic cell death, but preincubation of these cells with an iron chelator, such as deferoxamine, appears to confer some protection from oxidative cytotoxicity and apoptosis.

Toxins that impair mitochondrial respiration, such as cuprizone and ethidium bromide, have also been shown to trigger apoptosis in oligodendroglial cells. Subsequently, these chemicals have been used to develop experimental models of demyelinating disease and injury. It has been established that radiation exposure directly damages DNA and has been shown to lead to apoptotic cell death in many cell types. However, several studies of delayed neurologic injury after radiation therapy have revealed that oligodendrocytes are the most radiation-sensitive cell population in spinal cord tissue.

In addition to the previously mentioned causes of oligodendrocyte injury, mechanical stress has been repeatedly shown to trigger oligodendrocyte apoptosis. Mechanical injury appears capable of triggering a specific immune response with formation of antibodies and subsequent cytotoxicity directed against oligodendrocyte antigens. This immune-mediated injury may be caused by macrophage activity and appears to involve several different cytokines, such as tumor necrosis factor, lymphotoxin, and gamma-interferon. Immediately following injury, microglia are most active in clearing damaged tissue. Then activated macrophages become more prominent and also generate free radicals and nitric oxide, which have been shown to lead to apoptosis and secondary injury. Formation of the membrane attack complex through activation of the complement cascade is another consequence of macrophage activation and has been implicated in oligodendrocyte injury.

In addition to the macrophage, at least two specific subpopulations of T cells may also be involved in oligodendroglial apoptosis. CD4 ⁺T cells adhere to target cells through the Fas receptor identified on oligodendrocyte cells, thereby triggering apoptosis. Gamma-delta T cells have been found to co-localize with oligodendrocytes (expressing heat-shock protein 65) and may trigger cell death through production of gamma-interferon.

Apoptosis

Apoptosis, also known as “programmed cell death,” may be the primary cellular process underlying the disappearance of oligodendrocytes in the earliest histologic stages of traumatic spinal cord injury (SCI) and other processes such as cervical spondylotic myelopathy. The process of apoptosis is distinct from necrosis and involves a sequence of intracellular events that includes chromatin aggregation and internucleosomal DNA fragmentation, nuclear pyknosis, and subsequent cell shrinkage. Apoptosis ultimately results in phagocytic engulfment of cells without extracellular discharge of cytosolic contents and without generation of a local inflammatory response. A third form of cell death, autophagic cell death is less well studied but has been shown to play a role in SCI. Staining for light chain-3, a marker of autophagy, and electron microscopy to detect signs of autophagic cell death showed that these markers were increased in neurons, astrocytes, and oligodendrocytes following SCI.

In contrast to necrotic cell death, apoptosis is a much more abbreviated process, which has made its study relatively difficult. Apoptotic cells initially shrink and lose contact with adjacent cells, forming membrane blebs and expressing prophagocytic cell surface signals. The cell chromatin then condenses and fragments, and the process ends in compartmentalization of the entire cell into small, membrane-bound vesicles that are quickly phagocytized. By comparison, cell necrosis is a relatively prolonged affair that is characterized by cell membrane disruption, mitochondrial swelling, random DNA cleavage, and the generation of a local inflammatory reaction.

Several molecular biology assays have been developed for identification of apoptosis in various settings. A marker of DNA cleavage, such as the terminal deoxynucleotidyltransferase (TdT)-mediated nick-end labeling (TUNEL) technique, is a popular assay. Interpretation of studies relying solely on TUNEL staining has been criticized as possibly being limited by the observation that this method has been found to label cells undergoing necrosis as well and may not be as specific for apoptosis as once thought. Internucleosomal DNA cleavage, a hallmark of apoptosis, is demonstrated by a characteristic “laddering” pattern on gel electrophoresis, and this finding can reinforce the results of TUNEL staining. The most specific method for identifying apoptotic cells, however, remains direct histologic examination and the identification of chromatin condensation along the nuclear periphery, condensation of the cytoplasm with intact organelles, and membrane blebbing. A commercial assay is also available that uses monoclonal antibody to single-stranded DNA (Apostain; eBioscience, San Diego, CA). This method is purported to detect the earliest stages of apoptosis occurring before DNA fragmentation and supposedly has no cross-reactivity for necrotic cells.

Molecular Mechanisms of Apoptosis

The molecular pathways involved in apoptosis have been extensively examined but were initially studied in the roundworm, Caenorhabditis elegans . These studies led to the discovery of one of the first genes associated with apoptosis, which was appropriately named CED 3 in honor of this worm. Subsequently, a homologous family of apoptosis-related protein products has been identified in mammals and termed the CED 3/ICE (interleukin-1β-converting enzyme) family. These proteins, also known as caspases , serve as functional cysteine proteases. At least 10 distinct members of this gene family have been identified thus far, and at least 2 of these proteins, caspase-3 and caspase-9, have been strongly associated with apoptosis in human cells. The intracellular cascade involving caspase-3 ends in activation of specific endonucleases that cleave DNA strands into the characteristic internucleosomal fragments. Production of these 185 base-pair fragments results in the DNA laddering that is one of the histologic hallmarks of apoptosis. Activation of caspase-9 appears specifically to induce mitochondrial release of cytochrome c, which is one of the earliest intracellular events in apoptosis. Targeted inhibition of caspase-1 (ICE) and caspase-3 (CPP-32) in oligodendrocytes has been shown to prevent apoptotic death of these cells.

As previously described, numerous chemical and biologic triggers for apoptosis have been identified. Mature oligodendrocytes are particularly sensitive to oxidative stress. Experimental exposure of oligodendroglial cells to hydrogen peroxide leads to increased expression and nuclear translocation of transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), both implicated as critical elements in the apoptotic pathway.

One of the most important biologic triggers of oligodendrocyte apoptosis in SCI may be tumor necrosis factor-α (TNF-α). TNF-α has been shown to induce apoptosis in oligodendrocytes, both in vitro and in vivo. Designated death domains located on the intracellular side of the type I receptor for TNF-α (TNFR1) and related receptors have been associated with activation of caspase-3 and caspase-8, which subsequently leads to apoptosis. Gamma-interferon may further enhance susceptibility of oligodendrocytes to TNF-α-triggered apoptosis through up-regulation of the so-called death receptor, Fas. It has also been reported that the p38 and Jun N-terminal kinase (JNK) pathways play a role in the transmission of apoptosis signals following SCI. Further findings indicate that activation of JNK by TNF-α promotes expression of apoptosis signal-regulating kinase 1 (ASK1). The protein high-mobility group box-1 (HMGB-1), released from neurons as well as activated macrophages, has been identified as a potential upstream regulator of TNF-alpha, IL-1 beta, and IL-6. HMGB-1 expression occurs prior to TNF-alpha and does not peak until approximately 3 days after injury, suggesting a role of HMGB-1 in both the primary and secondary phases of spinal cord injury.

The oligodendrocyte apoptotic signal transduction pathway appears to begin with ligand binding to either Fas (CD95 or Apo1) or p75 (low-affinity neurotrophin receptors) cell surface receptors. These proteins are members of the TNFR family and have been shown to co-localize with cells undergoing apoptosis in a rat model of cervical SCI. Binding of Fas ligand (FasL) to the extracellular cysteine-rich domain of Fas results in formation of oligomers, which allows interaction of the intracellular death domain with Fas-associated death domain protein (FADD). Once the association is made, the death domain of FADD then interacts with procaspases 8 and 10 and triggers a caspase activation cascade that ultimately ends in activation of at least three different effector enzymes, caspases 3, 6, and 7. These effector molecules presumably interact with additional downstream targets, ultimately leading to cell apoptosis. FLICE (FADD-like interleukin-1β-converting enzyme) proteins are proteins demonstrating sequence homology with the caspases, but acting as inhibitors of the apoptosis-triggering pathway.

Another important apoptosis pathway involves the p53 tumor suppressor protein, as well as the proteins p21, Bcl-2, and Bax. In a rat model of SCI, p53 protein appeared within 30 minutes of injury, co-localizing with apoptotic glial cells and spreading in distribution over the course of 2 days. Cellular studies have further demonstrated that exposure of oligodendroglial cells to hydrogen peroxide leads to rapid translocation of p53 from the cytosol to the nucleus and cell death by apoptosis.

Apoptosis in Traumatic Spinal Cord Injury

It has been well established that cell loss in traumatic SCI occurs both at the time of injury and secondarily over a period of days to weeks after the event. At the epicenter of injury, the majority of cell death occurs through necrosis, with macrophages and microglia becoming actively engaged in phagocytosis of necrotic cell debris. However, cell loss in spinal cord white matter continues throughout a much more extensive axial section of the cord for up to several weeks in a process referred to as secondary injury . Although it has become apparent that this continued cell loss significantly worsens neurologic outcome in SCI, the underlying biologic mechanisms remain poorly understood. Several studies have suggested, however, that the primary process involves oligodendrocyte apoptosis.

Initial evidence that apoptosis contributes to ongoing cell death after acute SCI came from animal studies involving the rat. It was demonstrated that acute compressive cord injury leads to preferential apoptosis of oligodendrocytes along degenerating longitudinal white matter tracts. These initial findings were subsequently supported by similar results in other animal models, including primates. In most of these animal studies, visible signs of oligodendrocyte apoptosis appear within 24 hours and continue for at least 3 weeks after injury.

A histopathologic study of human SCI indicates that oligodendrocyte cell death by apoptosis can continue from 3 hours to at least 8 weeks after injury. In this study, oligodendrocyte apoptosis appeared to correlate with specific patterns of wallerian degeneration and was associated with intracellular activation of caspase-3. Apoptosis was more pronounced in ascending white matter tracts, and the authors speculated that this finding may reflect the histopathologic observation that wallerian degeneration affects ascending tracts before descending ones. The extent of oligodendrocyte apoptosis was shown to correlate with the severity of neurologic injury, being significantly less extensive in patients with incomplete neurologic deficits. This correlation of apoptosis and neurologic impairment is in agreement with previous findings from animal studies. Of note, neuronal apoptosis was not seen, suggesting that neuronal loss occurs through the process of necrosis.

The biochemical trigger for oligodendrocyte apoptosis related to traumatic SCI is currently unknown but is likely to be multifactorial. It has been observed that SCI is characterized by significant intracellular Ca ²⁺shifts, and several apoptotic processes are Ca ²⁺dependent, including DNA fragmentation and proteolysis. Similarly, acute SCI has been associated with hypoxia and free radical formation, which are also established triggers of apoptosis. Glutamate excitotoxicity has also been implicated in secondary SCI and appears to lead to apoptotic cell death.

Animal models have provided most of the information regarding biochemical responses to SCI. A rat model of SCI has demonstrated increased local TNF-α expression within 1 hour of injury, followed by increased nitric oxide levels at 4 hours. This model used a neutralizing antibody against TNF-α and significantly reduced nitric oxide levels as well as the extent of apoptosis. Similarly, addition of a nitric oxide synthase inhibitor, N-monomethyl- l -arginine acetate (L-NMMA) also reduced the number of apoptotic cells. These findings suggest that TNF-α signaling triggers apoptotic cell death after SCI, and that this effect is at least partly mediated by nitric oxide. Of note, the amount of decrease in apoptosis after administration of L-NMMA (42%) was less than half that observed after TNF-α antibody administration (89%), implying the existence of multiple parallel apoptotic pathways. A study by Genovese and colleagues demonstrated the neuroprotective effects of selective adenosine A _2Areceptor agonists, which act by decreasing the overall expression of myeloperoxidase, NF-κB, and inducible nitric oxide synthase (iNOS), and decreasing the activation of JNK mitogen-activated protein kinase (MAPK) in oligodendrocytes. Using a myeloperoxidase (MPO) mouse knockout model, MPO was shown to significantly increase the production of HOCl, a strong oxidant species, following SCI. In addition, another study found that mice with SCI, when treated with ethyl pyruvate, showed no increase of TNF-α expression and a decrease in oligodendrocyte apoptosis.

Several studies of development suggest that specific trophic factors are produced by axons and that absence of these factors results in oligodendrocyte apoptosis. Members of the neuregulin ligand family, in particular the glial growth factor (GGF), bind to the HER4 receptor on the surface of oligodendrocytes and appear to play an important role in cell differentiation and survival. Alternatively, the traumatic event may result in direct release of proapoptotic factors into spinal cord tissue. It is well established that activated microglia release several factors that may cause apoptosis, including TNF-α, reactive oxygen intermediates, and nitric oxide. Administration of exogenous thyroid hormone (triiodothyronine [T ₃]) during the early period after acute SCI has also been found to increase the population of apoptotic cells.

Apoptosis in Chronic Spinal Cord Compression

Several studies have suggested an important role for ischemic tissue injury in the pathogenesis of myelopathy in the setting of cervical spondylosis. On the cellular level, the sensitivity of oligodendrocytes to hypoxic injury is well established and appears to support the possibility of an ischemic cause. However, neurons are relatively more vulnerable to ischemic injury, and their sparing in early myelopathy makes a purely ischemic cause for cervical spondylotic myelopathy somewhat unlikely.

Although necrosis and apoptosis often occur simultaneously, distinguishing the two processes provides important information regarding the causes of specific disease processes. Although ischemia has been associated with apoptotic cell death, severe ischemia is characteristically thought to result in cell necrosis. Because oligodendrocyte disappearance in both trauma and chronic spondylotic myelopathy is apoptotic in nature, it is thought that mechanisms other than pure ischemia are involved.

Animal models strongly support a role for apoptotic cell death in the tissue degeneration seen in chronic, compression-related cervical myelopathy. The tiptoe-walking Yoshimura (twy) mouse is a specific strain of inbred mouse that has been useful as a model for chronic spinal cord compression. Twy mice become quadriparetic 4 to 8 months after birth because of the development of local hyperostosis along the dorsolateral margins of the C1 and C2 vertebrae, which results in severe cord compression at this level. Histologic examination of spinal cord tissue from these mice has revealed a characteristic pattern of descending degeneration affecting the anterior and lateral columns and ascending degeneration along the posterior columns. These findings are in addition to severe tissue damage at the level of compression. Cavity formation and myelin ovoids (myelin debris) were observed extending from the zone of compression into adjacent levels without gross deformation of the spinal cord. Detection of apoptotic cells using the TUNEL assay revealed a distribution of glial apoptosis that appeared to mirror the pattern of degeneration, whereas cell-specific staining confirmed that apoptotic cells were oligodendrocytes. The investigators included an autopsy study of a human patient dying with cervical myelopathy resulting from ossification of the posterior longitudinal ligament, in which a pattern of neuronal loss, demyelination, and apoptosis was observed that was similar to the findings in the twy mouse. Further studies of the twy mouse showed increased expression of TNFR1 and TNFR2 in chronically compressed spinal cord tissue, which further elucidates the effect of chronic compression on apoptosis and demyelination.

Oligodendrocyte survival depends on the presence of specific so-called survival factors produced by neighboring axons, leading to the possibility that oligodendroglial cell loss merely reflects prior neuronal injury. However, oligodendrocyte apoptosis likely precedes axonal degeneration in chronic myelopathy, as evidenced by both human and animal studies of spinal cord compression demonstrating apoptotic oligodendrocytes in the setting of intact demyelinated axons.

Prevention of Apoptosis

Oxidative stress has been shown to be a potent trigger for apoptotic death of oligodendrocytes. Antioxidant therapy with pyrrolidine dithiocarbamate (PDTC) and vitamin E appears to moderate this effect considerably. The asymmetric distribution of phospholipid polar-head groups across the plasma membrane bilayer may play a role in determining vulnerability to oxidative stress. Normally, there is an over-representation of choline phosphoglyceride and sphingomyelin in the outer leaflet, whereas the aminophospholipids, ethanolamine phosphoglyceride (EPG) and serine phosphoglyceride (SPG), are over-represented in the inner leaflet. Apoptosis has been associated with redistribution of SPG and EPG and loss of aminophospholipid asymmetry. The large, polyunsaturated, fatty acid content of both SPG and EPG makes them targets for propagating free radical reactions, leading to generation of lipid peroxides and apoptosis. It therefore makes sense that increasing polyunsaturated fatty acid content through addition of docosahexaenoic acid (DHA) enhances the sensitivity of oligodendrocytes to oxidative stress and thereby results in increased rates of apoptosis. Conversely, reducing EPG synthesis using N -monomethylethanolamine and N,N -dimethylethanolamine (DMEA) supplements appears to rescue cells from apoptotic death.

Methylprednisolone treatment has been shown to protect the spinal cord from injury. The protective effect of steroid therapy may be mediated in part by an inhibitory effect on oligodendrocyte apoptosis. Intraperitoneal injection of rats with dexamethasone after SCI significantly decreases the extent of apoptosis in both neurons and glial cells. At least part of this effect may be mediated through inhibition of TNF-α and NF-κB. A more recent study has also shown some additional benefit to the intraperitoneal administration of pregabalin in a post-SCI rat model. Treatment with pregabalin showed a significant decrease in expression of caspase-3, Bcl-2, and p38 MAPK compared with control and methylprednisolone treatment groups. All three of these factors have been shown to be key components of the inflammatory and apoptotic cascades.

The role of TNF-α in oligodendrocyte apoptosis appears complex and at times contradictory. Most studies have demonstrated primarily toxic effects, leading to apoptosis in several different models. This form of TNF-α-induced oligodendrocyte apoptosis can be inhibited in vitro by insulin-like growth factor 1 (IGF-1). However, a few studies have suggested that TNF-α may, in certain instances, protect oligodendrocytes from apoptosis.

The effects of TNF-α can be better understood through a description of its molecular mechanisms. TNF-α exerts its biologic effects through binding of two different cell surface receptors, the type 1 receptor (TNFR1) and the type 2 receptor (TNFR2). TNFR binding has been shown to prevent neuronal apoptosis in several studies. TNFR binding leads to increased NF-κB expression, and this TNFR-NF-κB signal transduction pathway has been identified as possibly a key endogenous, antiapoptotic cellular mechanism. NF-κB is a transcription factor that increases expression of several genes, resulting in increased production of cellular inhibitor of apoptosis protein 2 (c-IAP2). c-IAP2, in turn, inhibits apoptosis through binding TNFR-associated factor 2.

Protein inhibitors of apoptosis have been studied in baculovirus, and homologues to these proteins, referred to as inhibitors of apoptosis proteins (IAPs), have been identified in mammalian cells. IAPs appear to exert antiapoptotic effects through inhibition of the caspase cascade.

Evidence suggests that activation of the TNFR-NF-κB pathway is important in protecting spinal cord cells from apoptosis after SCI. In an animal model of SCI, rats lacking TNFR1 demonstrated decreased spinal cord tissue levels of NF-κB activity, lower levels of c-IAP2, and increased caspase-3 activity. Apoptosis was significantly increased, the overall lesion size was larger with more extensive demyelination and axonal disruption, and functional recovery was significantly worsened.

These studies suggested that pharmacologic modulation of TNF-α levels may yield benefits in patients with myelopathy or SCI. For example, interleukin-10 reduces TNF-α levels in the spinal cord and has been shown to improve functional recovery from SCI in rats. In addition to modulating TNF-alpha levels, IL-10 is known to act on a variety of other inflammatory signals to reduce inflammation, including IL-1beta, IL-6, interferon-gamma, iNOS, and MPO. Although untested in human SCI patients, IL-10 therapy has been used in the treatment of individuals with HIV, Crohn disease, rheumatoid arthritis, and several other conditions. Furthermore, the administration of etanercept, a TNF-alpha antagonist, following SCI resulted in a reduction of TNF-alpha, TNFR1, and TNFR2 as well as a decrease of oligodendrocyte and neuronal apoptosis.

Although inflammatory demyelinating disease represents a pathologic process distinct from traumatic injury, the generation of high levels of TNF-α leading to oligodendrocyte apoptosis in both demyelinating disease and SCI implies potentially useful biochemical similarities. Bcl-2 is a protein with antiapoptotic properties that is produced by certain types of cells, including oligodendrocytes. The activity of Bcl-2 has been studied in a rat model of human T-lymphocyte virus type I (HTLV-I)–associated myeloneuropathy. In this model, rats develop chronic progressive hind-limb weakness because of apoptotic oligodendrocyte death in the spinal cord. A study using this rat model has associated oligodendrocyte apoptosis with enhanced sensitivity to exogenous TNF-α and an associated down-regulation of Bcl-2 in affected cells. It is therefore conceivable that endogenous production of antiapoptotic proteins such as Bcl-2 can be up-regulated therapeutically as a treatment strategy for SCI and cervical myelopathy.

In the developing central nervous system, oligodendrocytes appear to be protected from apoptosis by molecular, and possibly electrical, signals provided through axonal contact. Several studies have demonstrated that exposure to specific cytokines protects oligodendrocytes from apoptosis. IGF-1 prevents TNF-α-triggered apoptosis in cell culture. A study of transgenic mice expressing high levels of IGF-1 demonstrated decreased oligodendrocyte death after exposure to the demyelinating toxin cuprizone. In addition, fibroblast growth factor triggers oligodendrocyte dedifferentiation and confers protection from apoptosis.

Serum growth factor deprivation has also been shown to lead to apoptosis in cultured oligodendrocytes. This model of apoptosis has been used to study the role of the complement system in apoptosis. Although assembly of the membrane attack complex, C5b-9, on cell membranes typically leads to formation of transmembrane channels and resultant cell death, sublytic levels of C5b-9 complement components activate the cell cycle and enhance cell survival by preventing apoptosis. This antiapoptotic effect appears to involve down-regulation of the proapoptotic cytosolic protein Bcl-2 antagonist of cell death (BAD). Studies suggest that a delicate balance exists between the protective antiapoptotic effects of the membrane-bound Bcl-2 and Bcl-X _Lproteins and the proapoptotic cytosolic proteins Bcl-2-associated X protein (BAX) and BAD. This balance appears to determine functional mitochondrial integrity and, consequently, whether a cell will undergo apoptosis.

Glutamate excitotoxicity represents yet another potential trigger for oligodendrocyte apoptosis. Oligodendrocytes express α-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid (AMPA)/kainite-type glutamate receptors and have been shown to be exquisitely sensitive to glutamate toxicity. The specific receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamyl-benzo(f)quinoxaline (NBQX) has been shown to protect oligodendrocytes from glutamate both in vitro and in vivo.

Finally, the process of apoptosis requires active protein synthesis. Inhibition of protein synthesis in animal models using the chemotherapeutic agent cycloheximide leads to a reduction in apoptotic cell death, less severe histopathologic changes, and improved clinical recovery.

Biomechanical Deformation as an Epigenetic Factor in Neuronal and Oligodendrocytic Apoptosis

Although the environmental sensitivity of oligodendrocytes has been stressed, it should be remembered that specific stresses will trigger apoptosis in neurons. In particular, biomechanical factors have been shown to be an important epigenetic factor in driving neuronal apoptosis. Stretch-related myelopathy and brainstem injury are substantiated in the literature. Neuronal strain (stretch) acts on the Na ⁺channel mechanoreceptors to increase Na ⁺influx, reverse cation exchange pumps, and depolarize voltage-gated Ca ²⁺channels, resulting in pathologic calcium influx. Sublethally damaged neurons also undergo up-regulation of N -methyl- d -aspartate receptors, resulting in heightened vulnerability to subsequent challenges of reactive oxygen species and peroxynitrites, concomitant mitochondrial dysfunction, and DNA fragmentation. Stretching neurons induces early calpain activation and contributes to progressive intra-axonal structural damage and apoptosis of neurons and oligodendrocytes. Stretch injury has been shown to induce phosphorylation of p38 MAPK and apoptosis in vascular, heart, and lung cells. The molecular events in neurons and oligodendrocytes should therefore be viewed within the matrix of environmental biomechanical stresses to which the organism is exposed.

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