Axonal Damage in Multiple Sclerosis - HISTOPATHOLOGICAL EVIDENCE OF AXONAL DAMAGE IN MULTIPLE SCLEROSIS
HISTOPATHOLOGICAL EVIDENCE OF AXONAL DAMAGE IN MULTIPLE SCLEROSIS
Morphological and Histopathological Characteristics of Axonal Damage
Axonal damage is a common feature of many neurodegenerative diseases. The morphological characteristics of early axonal damage include the presence of varicosities and spheroid structures,11 which are associated with impaired transport of proteins and organelles along the axon.12 Disrupted axonal transport can be detected by immunohistochemistry using antibodies specific for the amyloid precursor protein (APP) as a marker. In physiological conditions APP is rapidly transported along the axons and cannot be easily detected by immunoreactivity.13,14 In case of axonal dysfunction and altered transport, in contrast, APP accumulates in localized axonal enlargements that become immunoreactive and that can be detected in active, remyelinating, and inactive MS lesions.15,16 An additional marker of axonal damage is the nonphosphorylated form of neurofilament heavy chain (NFH). In healthy axons, NFH is phosphorylated and this correlates with fast axonal transport.17 In compromised axons, in contrast, the nonphosphorylated form of NFH, which can be identified by SMI-32 immunoreactivity, can be used as maker of neurodegeneration,18 ALS,19 and MS.20
Evidence for Axonal Damage as Consequence of Primary Demyelination
A correlation between axonal damage and demyelination has been determined from histopathological examination of MS postmortem tissue. Histopathological analysis of early MS lesions showed that most axonal transections occur during the process of active demyelination.21,22 Furthermore, regional axonal loss in the corpus callosum correlated with the cerebral white matter (WM) lesion volume distribution and was suggested to be a result of degenerated axons transected in demyelinated lesions.23 Axons in MS lesions also stained with antibodies specific for nonphosphorylated NFH and total axonal loss were correlated to the degree of inflammatory demyelination,21 suggesting that neurodegeneration occurs as the consequence of myelin loss.
Further evidence supporting this notion was the observation that the extent of remyelination extended lifespan of mice and provided a protective effect on axons.24 Perhaps the most striking evidence to substantiate axonal degeneration as a consequence of demyelination was obtained from animal models of MS, including experimental autoimmune encephalomyelitis (EAE) and cuprizone-induced demyelination. In EAE, animals are immunized with antigenic myelin extracts or peptides (eg, myelin-oligodendrocyte glycoprotein [MOG]), which elicit an immune T cell–mediated disease characterized by demyelination.25,26 After long-term demyelination in EAE, axonal loss is observed in rats immunized with MOG22 and in guinea pigs,27 substantiating the concept that axonal damage occurs following the loss of myelin support. Axonal loss can also be detected in the cuprizone-induced model of toxic demyelination in aged mice,28 which have less-efficient remyelination compared with young mice.29
Evidence in Favor of Hypothesis That Axonal Damage Can Occur Independently of Demyelination
The hypothesis that axonal damage in MS may occur independently of chronic demyelination has been suggested by several studies that will be reviewed in this manuscript. This section discusses recent neuropathological findings on gray matter (GM) lesions, meningeal infiltrates detected in human brain samples, and experimental evidence collected in genetically manipulated mouse mutants. The first evidence supporting the existence of alternative mechanisms of pathogenesis for axonal damage in MS were the description of GM lesions, characterized by neuronal loss and dendritic atrophy30 and the presence of axonal abnormalities in areas devoid of ongoing demyelination and thereby designated as normal appearing white matter (NAWM). Gray matter lesions differed from WM lesions31 – 33 and were characterized by a different composition of the inflammatory cell infiltrate34,35 and less-prominent antibody complement activation compared with WM tracts.36 Gray matter lesions have been correlated with cortical thinning and were considered a predictive index of disability in MS patients,37 – 39 including those with PPMS.40,41 In some studies, cortical demyelination in MS brain was detected during the late stages of disease pathology and interpreted as the consequence rather than the cause of neuronal loss.30 Neuronal cell death has also been detected in both cortical and thalamic lesion areas,34,42 and it could be mimicked by injection of neurofilament light chain (NFL) into mice, which produces a GM pathology with axonal loss and empty myelin sheaths, thereby suggesting an antibody-mediated primary axonal damage with a secondary involvement of myelin.43 This concept was supported by the detection of meningeal B-cell follicles in the brain of patients with a diagnosis of SPMS (but not PPMS) and by the correlation between the presence of these infiltrates and severe cortical pathology.44
The hypothesis that axonal damage in multiple sclerosis may occur independently of chronic demyelination has been suggested by several studies involving neuropathological findings on gray matter lesions and meningeal infiltrates in both human brain samples and genetically manipulated mouse mutants.
Very recent studies have further suggested a significant correlation between decreased axonal density in NAWM and diffuse parenchymal infiltration of major histocompatibility complex class II–positive and meningeal inflammatory infiltrates composed of CD3+ T cells in the cervical spinal cord of PPMS patients’ microglia.45 These intriguing data were consistent with other reports of inflammatory meningeal infiltrates in the brain of PPMS and SPMS patients with a high degree of axonal loss46 and provided a potential explanation for the axonal injury detected in the progressive course of the disease even in the absence of diffuse inflammatory infiltrates.47
A third line of evidence was provided by the description of axonal degeneration in mice with detectable myelin, even after genetic deletion of specific myelin proteins or oligodendrocyte components. Mice lacking the gene encoding for proteolipid protein (Plp), for instance, were characterized by progressive axonal degeneration.48 A similar phenotype was described for mice with genetic deletion of 2′, 3′-cyclic nucleotide phosphodiesterase,49 or of myelin-associated glycoprotein,50 which were characterized by relatively normal myelination and severe progressive axonal degeneration occurring with aging. Thus, even in the absence of morphological evidence of demyelination, microscopic imbalances of the oligodendrocyte-neuron unit may result in deregulation of energy metabolism, followed by destabilization and damage of the axon.51