Axon Reactive B Cells Clonally Expanded in the Cerebrospinal Fluid of Patients with Multiple Sclerosis

Demyelination and axonal loss have been described as the histological hallmarks of inflammatory lesions of multiple sclerosis (MS) and are the pathological correlates of persistent disability. However, the immune mechanisms underlying axonal damage in MS remain unknown. Here, we report the use of single chain-variable domain fragments (scFv) from clonally expanded cerebrospinal fluid (CSF) B cells to show the role of an anti-axon immune response in the central nervous system (CNS) in MS. The cellular and subcellular distribution of the antigen(s) recognized by these CSF-derived clonal scFv antibodies (CSFC-scFv Abs) was studied by immunochemical staining of brain tissues obtained at autopsy from patients with MS. Immunochemistry showed specific binding of CSFC-scFv Abs to axons in acute MS lesions. The stained axons showed three major types of axonal pathological changes: 1) linear axons, axonal ovoid formation, and axonal transection were seen in the myelinated white matter adjacent to the lesion; 2) accumulation of axonal ovoid formations and Wallerian degeneration were seen at the border between demyelinated lesions and the adjacent white matter; and 3) Wallerian degeneration occurred at the center and edge of acute demyelinated lesions. These findings suggest a B cell axonal specific immune response in the CNS in MS.     

Axonal Pathology and Loss Precede Demyelination and Accompany Chronic Lesions in a Spontaneously Occurring Animal Model of Multiple Sclerosis

Axonal damage has been highlighted recently as a cause of neurological disability in various demyelinating diseases, including multiple sclerosis, either as a primary pathological change or secondary due to myelin loss. To characterize and quantify axonal damage and loss in canine distemper demyelinating leukoencephalomyelitis (DL), formalin‐fixed paraffin‐embedded cerebella were investigated histochemically and immunohistochemically using the modified Bielschowsky''s silver stain as well as antibodies against nonphosphorylated (n‐NF), phosphorylated neurofilament (p‐NF) and β‐amyloid precursor protein (β‐APP). Injured axons characterized by immunoreactivity against n‐NF and β‐APP were detected in early distemper lesions without demyelination. In subacute and chronic demyelinating lesions the number of injured axons increased. Moreover, a significant decrease in axonal density was observed within lesions and in the normal appearing white matter in DL as determined by morphometric analyses using Bielschowsky''s silver stain and p‐NF immunohistochemistry. Summarized, the observed findings indicate that axonal damage (i) occurs early in DL; (ii) can be detected before myelin loss; and (iii) represents a pivotal feature in advanced lesions. It must be postulated that axonal damage plays an important role in the initial phase as a primary event and during progression of nervous distemper as a result of demyelination.     

Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions

Recent magnetic resonance (MR) studies of multiple sclerosis lesions indicate that axonal injury is a major correlate of permanent clinical deficit. In the present study we systematically quantified acute axonal injury, defined by immunoreactivity for beta-amyloid-precursor-protein in dystrophic neurites, in the central nervous system of 22 multiple sclerosis patients and 18 rats with myelin-oligodendrocyte glycoprotein (MOG)-induced chronic autoimmune encephalomyelitis (EAE). The highest incidence of acute axonal injury was found during active demyelination, which was associated with axonal damage in periplaque and in the normal appearing white matter of actively demyelinating cases. In addition, low but significant axonal injury was also observed in inactive demyelinated plaques. In contrast, no significant axonal damage was found in remyelinated shadow plaques. The patterns of axonal pathology in chronic active EAE were qualitatively and quantitatively similar to those found in multiple sclerosis. Our studies confirm previous observations of axonal destruction in multiple sclerosis lesions during active demyelination, but also indicate that ongoing axonal damage in inactive lesions may significantly contribute to the clinical progression of the disease. The results further emphasize that MOG-induced EAE may serve as a suitable model for testing axon-protective therapies in inflammatory demyelinating conditions.     

Axonal injury heralds virus-induced demyelination

Axonal pathology has been highlighted as a cause of neurological disability in multiple sclerosis. The Daniels (DA) strain of Theiler's murine encephalomyelitis virus infects the gray matter of the central nervous system of mice during the acute phase and persistently infects the white matter of the spinal cord during the chronic phase, leading to demyelination. This experimental infection has been used as an animal model for multiple sclerosis. The GDVII strain causes an acute fatal polioencephalomyelitis without demyelination. Injured axons were detected in normal appearing white matter at 1 week after infection with DA virus by immunohistochemistry using antibodies specific for neurofilament protein. The number of damaged axons increased throughout time. By 2 and 3 weeks after infection, injured axons were accompanied by parenchymal infiltration of Ricinus communis agglutinin I(+) microglia/macrophages, but never associated with perivascular T-cell infiltration or obvious demyelination until the chronic phase. GDVII virus infection resulted in severe axonal injury in normal appearing white matter at 1 week after infection, without the presence of macrophages, T cells, or viral antigen-positive cells. The distribution of axonal injury observed during the early phase corresponded to regions where subsequent demyelination occurs during the chronic phase. The results suggest that axonal injury might herald or trigger demyelination.     

Alleviation of extensive visual pathway dysfunction by a remyelinating drug in a chronic mouse model of multiple sclerosis

Visual deficits are among the most prevalent symptoms in patients with multiple sclerosis (MS). To understand deficits in the visual pathway during MS and potential treatment effects, we used experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS. The afferent visual pathway was assessed in vivo using optical coherence tomography (OCT), electroretinography (ERG), and visually evoked cortical potentials (VEPs). Inflammation, demyelination, and neurodegeneration were examined by immunohistochemistry ex vivo. In addition, an immunomodulatory, remyelinating agent, the estrogen receptor β ligand chloroindazole (IndCl), was tested for its therapeutic potential in the visual pathway. EAE produced functional deficits in visual system electrophysiology, including suppression of ERG and VEP waveform amplitudes and increased signal latencies. Therapeutic IndCl rescued overall visual system latency by VEP but had little impact on amplitude or ERG findings relative to vehicle. Faster VEP conduction in IndCl‐treated mice was associated with enhanced myelin basic protein signal in all visual system structures examined. IndCl preserved retinal ganglion cells (RGCs) and oligodendrocyte density in the prechiasmatic white matter, but similar retinal nerve fiber layer thinning by OCT was noted in vehicle and IndCl‐treated mice. Although IndCl differentially attenuated leukocyte and astrocyte staining signal throughout the structures analyzed, axolemmal varicosities were observed in all visual fiber tracts of mice with EAE irrespective of treatment, suggesting impaired axonal energy homeostasis. These data support incomplete functional recovery of VEP amplitude with IndCl, as fiber tracts displayed persistent axon pathology despite remyelination‐induced decreases in latencies, evidenced by reduced optic nerve g‐ratio in IndCl‐treated mice. Although additional studies are required, these findings demonstrate the dynamics of visual pathway dysfunction and disability during EAE, along with the importance of early treatment to mitigate EAE‐induced axon damage.     

Axonal loss and neurofilament phosphorylation changes accompany lesion development and clinical progression in multiple sclerosis

Neuroaxonal damage and loss are increasingly recognized as disability determining features in multiple sclerosis (MS) pathology. However, little is known about the long-term sequelae of inflammatory demyelination on neurons and axons. Spinal cord tissue of 31 MS patients was compared to three amyotrophic lateral sclerosis (ALS) and 10 control subjects. MS lesions were staged according to the density of KiM-1P positive macrophages and microglia and the presence of myelin basic protein (MBP) positive phagocytes. T cells were quantified in the parenchyma and meninges. Neuroaxonal changes were studied by immunoreactivity (IR) for amyloid precursor protein (APP) and variably phosphorylated neurofilaments (SMI312, SMI31, SMI32). Little T cell infiltration was still evident in chronic inactive lesions. The loss of SMI32 IR in ventral horn neurons correlated with MS lesion development and disease progression. Similarly, axonal loss in white matter (WM) lesions correlated with disease duration. A selective reduction of axonal phosphorylated neurofilaments (SMI31) was observed in WM lesions. In ALS, the loss of neuronal SMI32 IR was even more pronounced, whereas the relative axonal reduction resembled that found in MS. Progressive neuroaxonal neurofilament alterations in the context of chronic inflammatory demyelination may reflect changes in neuroaxonal metabolism and result in chronic neuroaxonal dysfunction as a putative substrate of clinical progression.     

Axonal loss in the multiple sclerosis spinal cord revisited

Preventing chronic disease deterioration is an unmet need in people with multiple sclerosis, where axonal loss is considered a key substrate of disability. Clinically, chronic multiple sclerosis often presents as progressive myelopathy. Spinal cord cross‐sectional area (CSA) assessed using MRI predicts increasing disability and has, by inference, been proposed as an indirect index of axonal degeneration. However, the association between CSA and axonal loss, and their correlation with demyelination, have never been systematically investigated using human post mortem tissue. We extensively sampled spinal cords of seven women and six men with multiple sclerosis (mean disease duration= 29 years) and five healthy controls to quantify axonal density and its association with demyelination and CSA. 396 tissue blocks were embedded in paraffin and immuno‐stained for myelin basic protein and phosphorylated neurofilaments. Measurements included total CSA, areas of (i) lateral cortico‐spinal tracts, (ii) gray matter, (iii) white matter, (iv) demyelination, and the number of axons within the lateral cortico‐spinal tracts. Linear mixed models were used to analyze relationships. In multiple sclerosis CSA reduction at cervical, thoracic and lumbar levels ranged between 19 and 24% with white (19–24%) and gray (17–21%) matter atrophy contributing equally across levels. Axonal density in multiple sclerosis was lower by 57–62% across all levels and affected all fibers regardless of diameter. Demyelination affected 24–48% of the gray matter, most extensively at the thoracic level, and 11–13% of the white matter, with no significant differences across levels. Disease duration was associated with reduced axonal density, however not with any area index. Significant association was detected between focal demyelination and decreased axonal density. In conclusion, over nearly 30 years multiple sclerosis reduces axonal density by 60% throughout the spinal cord. Spinal cord cross sectional area, reduced by about 20%, appears to be a poor predictor of axonal density.     

An animal model of cortical and callosal pathology in multiple sclerosis

The pathological and radiological hallmarks of multiple sclerosis (MS) include multiple demyelinated lesions disseminated throughout the white matter of the central nervous system (CNS). More recently, the cerebral cortex has been shown to be affected in MS, but the elucidation of events causing cortical demyelination has been hampered by the lack of animal models reflecting such human cortical pathology. In this report, we have described the presence of cortical gray matter and callosal white matter demyelinating lesions in the chronic experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Similar to the pathological lesions of MS patients, EAE lesions have been classified as type I-leukocortical, type II-intracortical and type III-subpial. All of these lesions had varying degrees of demyelination, inflammatory cells and reactive astrocytes. Similar to MS, cortical layers during EAE showed demyelination, microglia activation, synaptic protein alterations and apoptotic cells. In addition, the callosal white matter during EAE had many inflammatory demyelinating lesions and axon degeneration. Functional electrophysiological conduction analysis showed deficits in both myelinated and unmyelinated callosal axons during early and late EAE. The chronic EAE mouse model has features that mimic cortical and callosal pathology of MS, and can be potentially used to screen agents to prevent these features of disease.     

Elevated myeloperoxidase activity in white matter in multiple sclerosis

Recent studies have revealed extensive axonal damage in patients with progressive multiple sclerosis (MS). Axonal damage can be caused by a plethora of factors including the release of proteolytic enzymes and cytotoxic oxidants by activated immune cells and glia within the lesion. Macrophages and microglia are known to express myeloperoxidase (MPO) and generate reactive oxygen species during myelin phagocytosis in the white matter. In the present study we have measured MPO levels in post-mortem homogenates of demyelinated and non-demyelinated regions of white matter from nine patients with MS and seven controls, and assessed MPO immunoreactivity within MS brain. In homogenates of MS white matter, demyelination was associated with significantly elevated MPO activity when compared to controls. Immunohistochemistry showed MPO to be expressed mainly by macrophages within and adjacent to plaques. Demyelination in MS is associated with increased activity of MPO, suggesting that this production of reactive oxygen species may contribute to axonal injury within plaques.     

Microglia‐Derived Macrophage Colony Stimulating Factor Promotes Generation of Proinflammatory Cytokines by Astrocytes in the Periventricular White Matter in the Hypoxic Neonatal Brain

Inflammation in the periventricular white matter (PWM) of hypoxic neonatal brain causes myelination disturbances. In this connection, macrophage colony‐stimulating factor (M‐CSF) has been reported to regulate release of proinflammatory cytokines that may be linked to PWM damage. We sought to determine if M‐CSF derived from amoeboid microglial cells (AMC) would promote proinflammatory cytokine production by astrocytes in the PWM following hypoxic exposure, and, if so, whether it is associated with axon degeneration and myelination disturbances. In 1‐day hypoxic rats, expression of M‐CSF was upregulated in AMC. This was coupled with increased expression of CSF‐1 receptor, tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) in astrocytes, and TNF‐receptor 1 and IL‐receptor 1 on the axons. Neurofilament‐200 immunopositive axons and myelin basic protein immunopositive processes appeared to undergo disruption in 14‐days hypoxic rats. By electron microscopy, some axons showed degenerative changes affecting the microtubules and myelin sheath. Primary cultured microglial cells subjected to hypoxia showed enhanced release of M‐CSF. Remarkably, primary cultured astrocytes treated with conditioned‐medium derived from hypoxic microglia or M‐CSF exhibited increased production of TNF‐α and IL‐1β. Our results suggest that AMC‐derived M‐CSF promotes astrocytes to generate proinflammatory cytokines, which may be involved in axonal damage following a hypoxic insult.     

Autoreactive T Cells in Multiple Sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by infiltration of T lymphocytes and macrophages into white matter leading to demyelination [1-2]. This pathology is frequently associated with disability of neurological function, in particular sensory deficits, visual problems and paralysis. The acute MS plaques are markered by the presence of activated T cells expressing the IL-2 receptor as well as activated, class II MHC positive macrophages [3-4]. In addition, cytokines such as TNF and oligoclonal immunoglobulin have been found in the brain and cerebrospinal fluid (CSF) of patients with MS [5-7]. This active inflammatory process is confined to the CNS, not affecting either the peripheral nervous system or other organs. Although it is generally accepted that this CNS inflammatory process causes demyelination and the resulting neurologic disability in MS, the mechanism(s) by which the inflammation is initiated and maintained is unknown.     

Fluoxetine increases cerebral white matter NAA/Cr ratio in patients with multiple sclerosis

Axonal degeneration in multiple sclerosis (MS) may be caused by mitochondrial dysfunction and is associated with decreased levels of N-acetylaspartate (NAA) as measured with 1H-magnetic resonance spectroscopy (MRS). Fluoxetine stimulates astrocytic glycogenolysis, which serves as an energy source for axons. Eleven patients with MS received fluoxetine orally 20 mg a day during the first week, and 40 mg a day during the second week. The mean NAA/Creatine ratio in cerebral white matter of the MS patients increased from 1.77 at baseline to 1.84 at the end of the second week (p=0.007). These findings show evidence for a reversible axonal dysfunction in patients with MS and provide a rationale for investigating whether fluoxetine has neuroprotective effects in MS.     

Retrograde and Wallerian axonal degeneration occur synchronously after retinal ganglion cell axotomy

Axonal injury and degeneration are pivotal pathological events in diseases of the nervous system. In the past decade, it has been recognized that the process of axonal degeneration is distinct from somal degeneration and that axoprotective strategies may be distinct from those that protect the soma. Preserving the cell body via neuroprotection cannot improve function if the axon is damaged, because the soma is still disconnected from its target. Therefore, understanding the mechanisms of axonal degeneration is critical for developing new therapeutic interventions for axonal disease treatment. We combined in vivo imaging with a multilaser confocal scanning laser ophthalmoscope and in vivo axotomy with a diode-pumped solid-state laser to assess the time course of Wallerian and retrograde degeneration of unmyelinated retinal ganglion cell axons in living rats for 4 weeks after intraretinal axotomy. Laser injury resulted in reproducible axon loss both distal and proximal to the site of injury. Longitudinal polarization-sensitive imaging of axons demonstrated that Wallerian and retrograde degeneration occurred synchronously. Neurofilament immunostaining of retinal whole-mounts confirmed axonal loss and demonstrated sparing of adjacent axons to the axotomy site. In vivo fluorescent imaging of axonal transport and photobleaching of labeled axons demonstrated that the laser axotomy model did not affect adjacent axon function. These results are consistent with a shared mechanism for Wallerian and retrograde degeneration.     

Diverse changes in microglia morphology and axonal pathology during the course of 1 year after mild traumatic brain injury in pigs

Over 2.8 million people experience mild traumatic brain injury (TBI) in the United States each year, which may lead to long‐term neurological dysfunction. The mechanical forces that are caused by TBI propagate through the brain to produce diffuse axonal injury (DAI) and trigger secondary neuroinflammatory cascades. The cascades may persist from acute to chronic time points after injury, altering the homeostasis of the brain. However, the relationship between the hallmark axonal pathology of diffuse TBI and potential changes in glial cell activation or morphology have not been established in a clinically relevant large animal model at chronic time points. In this study, we assessed the tissue from pigs subjected to rapid head rotation in the coronal plane to generate mild TBI. Neuropathological assessments for axonal pathology, microglial morphological changes, and astrocyte reactivity were conducted in specimens out to 1‐year post‐injury. We detected an increase in overall amyloid precursor protein pathology, as well as periventricular white matter and fimbria/fornix pathology after a single mild TBI. We did not detect the changes in corpus callosum integrity or astrocyte reactivity. However, detailed microglial skeletal analysis revealed changes in morphology, most notably increases in the number of microglial branches, junctions, and endpoints. These subtle changes were most evident in periventricular white matter and certain hippocampal subfields, and were observed out to 1‐year post‐injury in some cases. These ongoing morphological alterations suggest persistent change in neuroimmune homeostasis. Additional studies are needed to characterize the underlying molecular and neurophysiological alterations, as well as potential contributions to neurological deficits.     

Axonal pathology in myelin disorders

Myelination provides extrinsic trophic signals that influence normal maturation and long-term survival of axons. The extent of axonal involvement in diseases affecting myelin or myelin forming cells has traditionally been underestimated. There are, however, many examples of axon damage as a consequence of dysmyelinating or demyelinating disorders. More than a century ago, Charcot described the pathology of multiple sclerosis (MS) in terms of demyelination and relative sparing of axons. Recent reports demonstrate a strong correlation between inflammatory demyelination in MS lesions and axonal transection, indicating axonal loss at disease onset. Disruption of axons is also observed in experimental allergic encephalomyelitis and in Theiler's murine encephalomyelitis virus disease, two animal models of inflammatory demyelinating CNS disease. A number of dysmyelinating mouse mutants with axonal pathology have provided insights regarding cellular and molecular mechanisms of axon degeneration. For example, the myelin-associated glycoprotein and proteolipid protein have been shown to be essential for mediating myelin-derived trophic signals to axons. Patients with the inherited peripheral neuropathy Charcot-Marie Tooth disease type 1 develop symptomatic progressive axonal loss due to abnormal Schwann cell expression of peripheral myelin protein 22. The data summarized in this review indicate that axonal damage is an integral part of myelin disease, and that loss of axons contributes to the irreversible functional impairment observed in affected individuals. Early neuroprotection should be considered as an additional therapeutic option for these patients.     

Inside-Out versus Outside-In models for virus induced demyelination: axonal damage triggering demyelination

The primary target in multiple sclerosis (MS) is believed to be either myelin itself (myelinopathy) or the myelin-forming cell, the oligodendrocyte (oligodendrogliopathy). Although axonal injury occurs in MS, it is regarded as a secondary event to the myelin damage. Here, the lesion develops from myelin (outside) to the axon (inside) (Outside-In model). Recently, gray matter lesions and axonal injury in normal-appearing white matter have also been reported in MS. This raises two questions. 1) Is axonal injury exclusively secondary to myelin damage or from a direct insult to the axon or neurons (axonopathy)? (2) Is the injured axon regarded as only an end result of pathology or disease, or can axonal injury contribute to the spread of secondary damage, including demyelination? The former is raised from the fact that axonal damage has been reported in several virus infections, including human immunodeficiency virus, human T-lymphotropic virus 1, herpes simplex virus and coronavirus, which also cause demyelination. The latter possibility where axonal injury leads to other changes is raised from the rather unexpected similarity between spinal cord injury (SCI) and MS where axonal injury, oligodendrocyte apoptosis and demyelination are all present. In SCI, transection of axons leads to delayed oligodendrocyte apoptosis with secondary demyelination. Neurofilament immunostaining of spinal cord sections demonstrates that axonal injury with oligodendrocyte apoptosis also precedes demyelination in an animal model for MS, Theiler's murine encephalomyelitis virus infection. This implies that axonal injury could trigger demyelination. In this instance, lesions develop from the axon (inside) to the myelin (outside) (Inside-Out model).     

Early and extensive spinal white matter involvement in neuromyelitis optica

ObjectivesStudies of longitudinally extensive spinal cord lesions (LESCLs) in neuromyelitis optica (NMO) have focused on gray matter, where the relevant antigen, aquaporin‐4 (AQP4), is abundant. Because spinal white matter pathology in NMO is not well characterized, we aimed to clarify spinal white matter pathology of LESCLs in NMO.MethodsWe analyzed 50 spinal cord lesions from eleven autopsied NMO/NMO spectrum disorder (NMOSD) cases. We also evaluated LESCLs with three or fewer spinal cord attacks by 3‐tesla MRI in 15 AQP4 antibody‐positive NMO/NMOSD patients and in 15 AQP4 antibody‐negative multiple sclerosis (MS) patients.ResultsPathological analysis revealed seven cases of AQP4 loss and four predominantly demyelinating cases. Forty‐four lesions from AQP4 loss cases involved significantly more frequently posterior columns (PC) and lateral columns (LC) than anterior columns (AC) (59.1%, 63.6%, and 34.1%, respectively). The posterior horn (PH), central portion (CP), and anterior horn (AH) were similarly affected (38.6%, 36.4% and 31.8%, respectively). Isolated perivascular inflammatory lesions with selective loss of astrocyte endfoot proteins, AQP4 and connexin 43, were present only in white matter and were more frequent in PC and LC than in AC (22.7%, 29.5% and 2.3%, P ^corr = 0.020, and P ^corr = 0.004, respectively). MRI indicated LESCLs more frequently affected PC and LC than AC in anti‐AQP4 antibody‐seropositive NMO/NMOSD (86.7%, 60.0% and 20.0%, P ^corr = 0.005, and P ^corr = 0.043, respectively) and AQP4 antibody‐seronegative MS patients (86.7%, 73.3% and 33.3%, P ^corr = 0.063, and P ^corr = 0.043, respectively). PH, CP and AH were involved in 93.3%, 86.7% and 73.3% of seropositive patients, respectively, and in 53.3%, 60.0% and 40.0% of seronegative patients, respectively.ConclusionsNMO frequently and extensively affects spinal white matter in addition to central gray matter, especially in PC and LC, where isolated perivascular lesions with astrocyte endfoot protein loss may emerge. Spinal white matter involvement may also appear in early NMO, similar to cerebral white matter lesions.     

Magnetic resonance imaging correlates of neuro‐axonal pathology in the MS spinal cord

In people with multiple sclerosis (MS), the spinal cord is the structure most commonly affected by clinically detectable pathology at presentation, and a key part of the central nervous system involved in chronic disease deterioration. Indices, such as the spinal cord cross‐sectional area at the level C₂ have been developed as tools to predict future disability, and—by inference—axonal loss. However, this and other histo‐pathological correlates of spinal cord magnetic resonance imaging (MRI) changes in MS remain incompletely understood. In recent years, there has been a surge of interest in developing quantitative MRI tools to measure specific tissue features, including axonal density, myelin content, neurite density, and orientation, among others, with an emphasis on the spinal cord. Quantitative MRI techniques including T₁ and T₂, magnetization transfer and a number of diffusion‐derived indices have all been applied to MS spinal cord. Particularly diffusion‐based MRI techniques combined with microscopic resolution achievable using high magnetic field scanners enable a new level of anatomical detail and quantification of indices that are clinically meaningful.     

Noninvasive detection of brainstem and spinal cord axonal degeneration in an amyotrophic lateral sclerosis mouse model

Degeneration of motor neurons and their associated axons is a hallmark of amyotrophic lateral sclerosis, but reliable noninvasive lesion detection is lacking. In vivo diffusion tensor imaging was performed to evaluate neurodegeneration in the brainstem and cervical spinal cord of wild-type and G93A-SOD1 transgenic mice, an animal model of amyotrophic lateral sclerosis. A statistically significant reduction in the apparent diffusion coefficient was observed in the motor nuclei VII and XII of G93A-SOD1 transgenic mice relative to wild-type mice. No significant difference in diffusion anisotropy was observed in dorsal white or gray matter in cervical and lumbar segments of the spinal cord. In contrast, statistically significant decreases in axial diffusivity (diffusivity parallel to the axis of the spinal cord) and apparent diffusion coefficient were found in the ventrolateral white matter of G93A-SOD1 mice in both the cervical and lumbar spinal cord. The reduction in axial diffusivity, suggestive of axonal injury, in the white matter of the spinal cord of G93A-SOD1 mice was verified by immunostaining with nonphosphorylated neurofilament. The present study demonstrates that in vivo diffusion tensor imaging-derived axial diffusivity may be used to accurately evaluate axonal degeneration in an animal model of amyotrophic lateral sclerosis.