April 10, 2003
Current Neurology and Neuroscience Reports
Volume 3, Number 3: May 2003 • Demyelinating Disorders • pp. 231-237
by Kottil W. Rammohan, MD
The pivotal role of axons in the pathophysiology and pathogenesis of multiple sclerosis (MS) is increasingly becoming the focus of our attention. Axonal injury, considered at one time to be a late phenomenon, is now recognized as an early occurrence in the inflammatory lesions of MS. There is converging evidence from histopathologic, as well as magnetic resonance imaging and magnetic resonance spectroscopy studies, that axons play a crucial and dynamic role during the evolution of MS pathology and the development of clinical disability. It has been repeatedly demonstrated that neurologic functional impairment correlates best with axonal, rather than myelin, injury. The pathophysiology of axonal injury remains speculative. Although generally considered to be sequelae of demyelination, it is possible that axonal injury in MS is indeed a primary event. The discovery that axonal injury can be reversible has provided an impetus to institute early therapy. The finding that irreversible axonal transection occurs in early lesions has underscored now, more than ever before, the need to curtail inflammation and the need to institute early treatment with disease-modifying agents. The axon will undoubtedly remain the focus of our attention regarding research on MS now and in the future.
Demyelination with relative preservation of axons has been considered to be the pathologic hallmark of multiple sclerosis (MS) since the description of this disorder by Charcot in 1868 . Although axonal injury was recognized to occur in MS even in the descriptions that antedate Charcot, the problem has always been considered to be a late event or a consequence of astrogliosis. Recent studies have brought to our attention the presence of axonal injury in the early acute lesions of multiple sclerosis [2, 3]. Recognition of such injury has reiterated the need for aggressive early treatment so that irreversible injury and its adverse functional consequences can be avoided.
It is now becoming evident that a critical understanding of the pathophysiology of MS is only possible when not only the impact of demyelination, but also of axonal injury, is taken into consideration. Paradoxes that eluded explanations previously now become self evident when the impact of axonal injury and demyelination are considered concomitantly. In this review, current information regarding axonal injury in MS is examined, and the impact of this information on our understanding of all aspects of MS is critically evaluated. This includes an examination of current concepts regarding pathology, immunopathogenesis, clinicopathologic correlations, and magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS).
Normal Myelinated Axons
Myelination is a feature of large axons that conduct rapidly using saltatory conduction. Myelination influences axonal structure and function. An understanding of axon-myelin interactions can elucidate why demyelination can be associated with subsequent axonal dystrophy. Actin, tubulin, and neurofilaments form the major structural proteins of all axons. From a functional standpoint, the major proteins of the axon are tubulin and neurofilaments. Tubulin, which comprises 20% of the proteins of the brain , creates the microtubule channels that mediate fast axoplasmic transport. Phosphorylation of microtubule-associated proteins, necessary for the integrity of the microtubule cytoskeleton, is influenced by myelination [5, 6].
Neurofilaments (NFs) provide the framework of the axonal structure and are made up of phosphorylated subunits of the heavy (NF-H, approximately 200 kD), medium (NF-M, approximately 160 kD) or light (NF-L, approximately 68 kD) subunits. Phosphorylation of the NF subunits leads to negative charges that repel to expand the axonal diameter. Myelination leads to greater numbers of NFs and more phosphorylated subunits, and demyelination adversely effects phosphorylation . Phosphorylation is impaired in the diseased axons, a phenomenon that leads to collapse of the axon and disruption of the axonal architecture. NF-L forms the core of the axons whereas NF-M and NF-H form the axonal periphery. Following axonal injury, release into the cerebrospinal fluid of NF-L occurs in MS as well as a number of other degenerative disorders of the brain, a finding that may serve as a marker of axonal injury in MS as well as other neuronal or axonal degenerative disorders . Conformational determinants unique for nonphosphorylated NFs have been identified using monoclonal antibodies. These antibodies have made it possible to identify diseased axons in immunohistochemistry of MS plaques .
Excitation of the axolemma leads to the nerve action potential. In the unmyelinated nerve and in the fetal myelinated nerves prior to myelination, the axonal ion channels are randomly distributed along the axolemma. Following myelination and the development of the nodes of Ranvier, distinct clustering of ion channels occurs, so that the voltage gated sodium channels are localized for the most part to the nodes of Ranvier and the non-voltage gated fast potassium channels to the internodal axolemma underlying the myelin [9, 10]. Following demyelination, these potassium channels are bared or unmasked, resulting in a loss of intracellular potassium, which renders the demyelinated segments of axolemma less excitable. During functional recovery, which is seldom due to remyelination, there occurs a reorganization of the ion channels such that the voltage gated sodium channels rearrange from the nodes to the internodal axolemma, and the loss of intracellular potassium through the non-voltage gated potassium channels is curbed by mechanisms yet to be defined. This endogenous blockade or masking of potassium channels is necessary before function can be restored. There is little information regarding whether additional functional sodium channels are produced to populate the demyelinated segments of axolemma, but most studies would suggest that fresh channels are recruited to these sites. This would imply that a demyelinated but conducting axon will function, but at a price, namely sodium loading with resultant osmotic considerations that may result in long-term injury and axonal degeneration [11, 12, 13, 14].
Axonal Injury: Pathologic Studies
Is the resurgence of interest in axonal injury a "rediscovery" of the observations by Charcot and others before him? Although at first glance it may appear to be a rediscovery, there are important differences. The earliest descriptions of the pathology of MS from the times of Charcot reported selective loss of myelin with relative preservation of axons. This suggested a selective injury to myelin in the absence of injury to the axon. Accordingly, Charcot and his contemporaries and investigators for the next century concluded that axonal injury, when present, is a late phenomenon. In recent studies, Ferguson et al.  identified the presence of axonal injury in early lesions of MS by using amyloid precursor protein (APP) as a marker for dystrophic axons. Shortly thereafter, Trapp et al.  identified a marker for injured axons by use of confocal microscopy evidence of axonal injury by staining acute plaques for nonphosphorylated NFs. Their elegant studies identified in early and acute lesions the presence of transected axons characterized by the presence of "terminal axon ovoids." It should be noted that similar if not identical lesions were described by Doinikow, a contemporary of Charcot, who coined the term "end-bulbs" . However, his observations were eclipsed by the more influential Charcot, whose dogma regarding the lack of involvement of the axon until late has prevailed. The fact that axonotomy occurred early was unknown until the studies of Ferguson et al.  and Trapp et al. .
Axonal injury has been demonstrated in acute and chronic lesions, and resultant loss of axons has been demonstrated in normal-appearing white matter of the corpus callosum, internal capsule, and cervical spinal cord [16*, 17, 18]. Of interest is the observation that ongoing axonal injury was identified in normal-appearing white matter remote from lesion site, as well as chronic demyelinated cervical spinal cord plaques . Similarly, loss of as much as 50% of axons was demonstrated in the corpus callosum remote from any areas of acute or chronic plaques [16*]. Although there is generally a tight coupling of demyelination and axonal injury , there is no consensus that demyelination is a prerequisite for axonal injury. In a recent study that examined axonal injury in early biopsied lesions of MS, it was concluded that damage to axons as evidenced by the expression of APP could be demonstrated independent of the stage of demyelination [21**]. Thus, APP was present in axons in areas remote from demyelination, in areas of demyelination, and also in areas of remyelination. Further, axonal injury correlated with the presence of macrophages and CD8 T cells, but not T cells of the CD4 phenotype [21**]. These observations have particular relevance to mechanisms of axonal injury because a direct injury to axons by CD8 T cells can be speculated, as class 1 antigens are expressed by diseased axons but not by myelin. Therefore, it would appear that axonal injury might well be an early event, independent of demyelination in the pathogenesis of injury in multiple sclerosis.
Demyelination and Clinical Impairment
It is well recognized that irrespective of their clinical presentations, the MRI abnormalities of MS patients are strikingly similar between patients. In most patients, lesions predominate in the periventricular white matter, corpus callosum, juxtacortical white matter and the brain stem, and infratentorial white matter. Unlike in stroke, a one to one correlation of lesions to clinical symptoms is almost never present in MS. It has long been recognized that the extent of demyelination seldom correlated with the extent of disability in MS. The paradox of the lack of relationship of demyelination to function was recently elegantly demonstrated in the Theiler's encephalomyocarditis virus murine model of demyelination . Knockout mice deficient in b2 microglobulin (major histocompatibility complex [MHC] class 1), when infected with Theiler's virus, showed severe demyelination but little clinical disease. In contrast, mice that were deficient in MHC class 2 molecules had demyelination as well as clinical disease, including paralysis and death. Demyelination alone was clearly inadequate to explain the presence of clinical impairment. The basis of difference between these models remains a mystery.
Quantitation of demyelination, as evidenced by the total T2 disease burden by MRI, correlated poorly with clinical disability [23, 24]. Much of the T2 disease burden is demyelination, and what has become apparent from these observations is that loss of myelin is not always equal to loss of function. This is particularly evident in the optic nerves, where demyelinated plaques from remote optic neuritis can be identified in one or both optic nerves at autopsy in most MS patients. Yet, normal vision was present during life in most of these individuals, some of who experienced a clinical bout of optic neuritis decades earlier. It has been shown that although conduction block occurs during the acute event, recovery of conduction in the absence of myelin routinely occurs in these individuals with restoration of normal or near-normal vision .
After the advent of MRI, it became apparent through imaging of patients with MS that silent "attacks" of demyelination occurred routinely [25, 26, 27]. Although the explanation for a lack of concomitant clinical findings usually invoked the involvement of noneloquent brain, it has become evident that such silent lesions can also occur in critical eloquent areas, including the spinal cord. It would appear that the lack of symptoms is once again best explained by the fact that the axonal functions were spared in these silent plaques. A positive correlation of clinical disability to T1 hypointensities and brain atrophy (both measures of axonal loss) has been reported [28*, 29]. There is good evidence from pathology that the lesions underlying black holes involve severe tissue injury and disruption, including severe axonal loss . It has become clear in recent years that conventional MR imaging is useful for detecting demyelination, but is woefully inadequate for detection of axonal dysfunction or axonal loss.
Axonal Injury and Clinical Disability
N-acetyl aspartate (NAA) is a well-established marker for neurons, dendrites, and axons. This substance is identifiable as a specific peak by MRS and has been used as a marker for axons in the white matter because axons are the only source of NAA in normal and abnormal white matter. When spectroscopic studies were performed in the plaques as well as in seemingly unaffected white matter in MS, the following findings became apparent [31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42]: 1) NAA levels are decreased in an acute lesion, from 30% to sometimes as much as 70%; 2) NAA levels can return to normal or near-normal values during recovery (ie, a decrease in NAA can be a result of impaired reversible neuronal function or reversible impairment of axonal function); 3) NAA levels are normal when known benign lesions are examined by MRS (recent lesions on MRI with manifest clinical symptoms and normal NAA recover without sequelae); 4) NAA levels can be decreased in the normal-appearing white matter in MS patients, indicating that the normal-appearing white matter is not normal; and 5) decreased whole brain NAA levels correlate well with clinical disability.
Axonal dysfunction (reversible) or loss (irreversible) seems to correlate better with presence of clinical symptoms (reversible or irreversible) or clinical disability (irreversible) than loss of myelin.
Is Multiple Sclerosis a Primary Disorder of the Axon with Secondary Demyelination?
Although early injury to the axons has been identified in lesions of MS, such injury is generally considered to be a consequence of demyelination. The concept of primary axonal injury with secondary demyelination as the basis of MS is contrary to a century of thinking regarding the pathogenesis of this disorder. However, in reality, everything that we currently know about MS, from clinical studies, MRI, or observations from pathology, cross-validates best when this disorder is viewed as a primary disorder of the axon with secondary demyelination, as opposed to the prevailing view of primary demyelination with secondary axonal injury. Pathologically, demyelination is the most striking abnormality, and, therefore, demyelination has occupied our attention during the past century. The more subtle axonal injury is only becoming evident in recent years. However, the concept that at least in some cases this disorder may well be a primary axonal disorder has not gained much support.
Figure 1: Postulated events in the development of reversible and irreversible axonal injury. The cartoon depiction of the axonal "blebs" is based on a single axon with multiple swellings seen. A, Normal myelinated axon. B, Injury to nodes of Ranvier with development of axon ovoids. Clinical impairment is evident without evidence of demyelination. Magnetic resonance imaging (MRI) reflects no abnormalities. C, Complete recovery without sequelae. Ovoids resolve without demyelination. MRI remains normal. D, Paranodal demyelination with potential for remyelination. Function can return and MRI is normal. E, Axonotomy with irreversible injury and no potential for remyelination. N-acetyl aspartate levels decreased on magnetic resonance spectroscopy, and MRI remains abnormal. (Adapted from Trapp et al. .)
I would like to propose the following hypothesis. If MS was a disorder of the axon, where would the disease begin? MS is a disorder of nerve conduction and the most logical location of involvement would be the node of Ranvier, the location that generates the nerve action potential. The initial event in the pathogenesis of MS may be an immune-mediated attack at the node, and this event may be antibody- and complement-mediated, or alternately CD8 T cell-mediated. The CD8 and not the CD4 T cell may be the mediator of axonal injury because the axolemma at the node of Ranvier has the potential to express MHC class 1 molecules. Because CD8 T lymphocytes can recognize axonal antigens presented on class 1 human leukocyte antigen molecules, CD8 T-cell-mediated cytotoxicity can occur with resultant axonal injury. Any disruption of the node of Ranvier will be associated with neurologic dysfunction, with or without demyelination. This initial event could be transparent to MRI because MRI may not detect nodal injury unless paranodal demyelination has occurred. This functional disruption of the node may not be detectable even by NAA spectroscopy because changes in NAA may not be evident at this early stage of the lesion evolution. The injury at the node may be reversible. Recovery of nodal function can lead to resolution of the attack, with or without paranodal demyelination. When demyelination occurs, it may be a secondary event of minimal functional consequence. Of course, if the magnitude of injury at the node is severe, axonotomy can occur with irreversible consequences. In some patients, the initial event may be a primary demyelinating event. In these patients, functional disruption may occur only when axonal function is impaired as a consequence of demyelination. In these patients, the MRI is always abnormal because MRI is sensitive to myelin loss.
In their seminal work on axonal transection in lesions of MS, Trapp et al.  reported the occurrence of bulbous ends of transected axons that they termed "terminal axonal ovoids." Are terminal axonal ovoids always a terminal event, namely a consequence of axonal transection, or do axonal ovoids represent reversible changes in the axon? A critical examination of their work would point out that although most of the bulbous ends were from transected axons, multiple ovoids were sometimes seen on a single axon. The ovoids could, therefore, represent areas of injury on the axon rather than areas of transection. The demonstration of a single axon with three bulbous lesions (Fig. 1) could suggest that these bulbous lesions may be enlarged nodes of Ranvier. Why then were so many axons with transected bulbous ends evident in their study? It is possible that the axons became severed during fixation and snapped at the point of greatest fragility, namely at the enlarged bulbous nodes of Ranvier. This would imply that the presence of the ovoids is not sine qua non with axonal transection, and indeed may represent sometimes even reversible injury to the node of Ranvier. In support of this observation, it should be noted that Wallerian degeneration, although present in MS, can not explain the magnitude of early loss of axons in this disorder. This would also suggest that the number of axons actually transected in vivo may be less than what the Trapp et al.  study would suggest. Unfortunately, there are no ways to control for this situation because any attempt to fix the tissue and improve permeation of the reagents will require expansion and contraction of the tissues, which can cause these axons with swollen nodes of Ranvier to become transected. Figure 1 shows the postulated different possibilities if the bulbous lesions are indeed swollen nodes of Ranvier.
Mechanisms of Axonal Injury
Specific mechanisms of axonal injury are unknown. Injury to the axon may occur by several mechanisms. Exposure of axons following demyelination can make the axon vulnerable to injury by immune cells such as T cells and microglia. The close association of microglia to the axon ovoids in histopathologic studies would suggest that such mechanisms might well be operational. There is good evidence from animal studies that the presence of nonspecific inflammation in the brain can lead to axonal injury. For example, mice sensitized with bacille Calmette-Guérin (BCG) immunization can develop axonal injury when intracerebral inoculation of BCG is carried out to induce non-brain antigen-related inflammation .
Although there is no direct evidence that inflammation can lead to axonal injury in vivo, there is evidence that many of the same mediators that can cause demyelination may also cause axonal injury, agents like tumor necrosis factor a, interferon g, proteases, nitric oxide (NO), and reactive free radicals. In this regard, NO and peroxynitrites may be particularly important because these molecules not only cause tissue injury, but also induce reversible conduction block of otherwise conductive but demyelinated axons [44, 45, 46, 47]. This is of particular interest because normal myelinated axons are impervious to the conduction-blocking properties of NO, unlike demyelinated axons, which are vulnerable
Demyelination can contribute to axonal injury and retardation of axonal growth in several ways. First, in the absence of myelin, phosphorylation of NF proteins becomes impaired and axonal cytoarchitecture tends to collapse. The collapse of the axons impairs axoplasmic transport and organelles, and mitochondria accumulate. Demyelination promotes glial proliferation of not only oligodendrocytes, but also astrocytes. The resultant gliosis is detrimental to the growth of neuritis, and any attempt at axonal regeneration thereby fails.
Demyelinated axons rarely remyelinate and the cause of this is unknown. In demyelinated plaques from MS patients, re-expression of a polysialylated form of neural cell adhesion molecule was demonstrated . This molecule is seldom seen in the adult brain because its expression is limited to the brain during development. This molecule, which is expressed on the axonal membranes, must be removed before myelination can be initiated during development. The re-expression of this molecule during adult life during active demyelination may serve as a retardant to remyelination of lesions of MS. Strategies aimed at removal of these molecules may be a method to facilitate remyelination in MS.
Because myelin does not express MHC class 1 or 2 molecules, it has been postulated that the damage to myelin mediated by T lymphocytes is indirect, namely through secretion of noxious cytokines by activated microglia and T cells, which then indirectly mediate demyelination. Can T lymphocytes cause direct axonal injury, or do they mediate injury indirectly through secretion of noxious cytokines? There is recent evidence that although MS has been considered to be mostly a CD4 T cell-mediated injury, there are many CD8 T cells in the inflammatory lesions as well [21**]. Because axons, especially during injury, express b2 microglobulin (MHC class 2 molecule), it is possible that the CD8 T cells may mediate direct injury to axons through interactions of the CD8 T-cell receptors with MHC class 1 molecules on the axons that are expressing an unidentified axonal antigen. The presence of APP expression in damaged axons in areas of macrophages and CD8 T cells, and not CD4 cells, would suggest that the previously postulated mechanisms are indeed plausible [21**].
Can axonal ion channels be the targets of axonal autoimmunity? In other words, is MS a disorder of autoimmunity to the voltage gated sodium channel similar to myasthenia gravis, which is a disorder of autoimmunity to a ligand gated ion channel, namely the acetylcholine receptor? In preliminary experiments, we immunized mice to a fusion protein generated from the extracellular toxin-binding region of the rat type-2 voltage gated sodium channel. The mice developed autoimmune encephalitis characterized predominantly by ataxia, irritability, lethargy, and seizures [49, 50]. Large perivascular and meningeal infiltrates were seen in the cerebellum with routine histology, but there was no evidence of demyelination when brains were examined by luxol-fast-blue staining. The spinal cord and the cauda equina (peripheral nervous system) were uninvolved. By immunohistochemistry, the majority of the inflammatory cells were identified to be macrophages, with some CD4 and CD8 T-cell clusters. Large titers of antibody to the voltage gated sodium channel and the fusion protein could be demonstrated. Further studies are needed to determine if this model of axonal injury will help to define the type of axonal abnormalities and injury seen in multiple sclerosis.
Sodium Loading of Axons
It has been known for a long time that demyelinated axons can regain the ability to conduct without remyelination. The mechanism of such repair appears to be a consequence of generation of new voltage gated sodium channels as well as redistribution of the sodium channels from the nodes of Ranvier to the internodal axolemma. Additionally, the non-voltage gated potassium channels previously covered by myelin, but open after demyelination, are capped by as yet unknown mechanisms, resulting in restoration of the resting membrane potential. The demyelinated axolemma once again becomes excitable but at a cost, namely sodium loading of the axon because considerably larger numbers of sodium channels must be depolarized to conduct across such segments. Although this may not be a problem under most circumstances, repeated conduction through such a region can be associated with considerable sodium loading and resultant edema and degeneration of the axon. As a result, when large areas of demyelinated axons are subjected to repeated conduction, edema of the axons can occur that can contribute to further axonal injury. Further, demyelinated but electrically conducting axons are particularly vulnerable to irreversible injury from concentrations of NO generally seen at sites of inflammation [51**]. This should be kept in mind when strategies to restore function through demyelinated segments are developed, namely when using potassium channel blockers.
Recent histopathologic studies have identified the presence of transection of axons in early acute lesions of MS. Subsequent studies using MRI and MRS have extended the findings on histopathology further, by the identification of not only the irreversible, but also reversible, axonal injury in this disorder. The observation that low NAA levels can normalize during recovery showed that axonal dysfunction is not always irreversible. Mechanisms of axonal dysfunction and injury are unknown. The possibility that such injury may be primary rather than secondary to demyelination is a novel concept that needs additional investigation. Such evaluation is certain to open up new antigens on the axon that serve as targets of immune-mediated injury. New therapeutic options may become available from such studies and allow exploration of strategies aimed at preserving the axons.
References and Recommended Reading
Recently published papers of particular interest have been highlighted as:
* Of importance
** Of major importance
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51. ** Smith KJ, Kapoor R, Hall SM, Davies M: Electrically active
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49: 470-476 (An important study that demonstrates injury to conducting
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