Axonal Injury Heralds Virus-Induced Demyelination

American Journal of Pathology, Vol. 162, No. 4, April 2003 Copyright © American Society for Investigative Pathology

Axonal Injury Heralds Virus-Induced Demyelination

Ikuo Tsunoda, Li-Qing Kuang, Jane E. Libbey, and Robert S. Fujinami From the Department of Neurology, University of Utah School of Medicine, Salt Lake City, Utah

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 Iⴙ 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. (Am J Pathol 2003, 162:1259 –1269)

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Axonal damage in MS has recently attracted significant attention1 even though the presence of axonal degeneration in MS has long been recognized.2–5 Axonal damage is a major problem in MS, because axonal loss can significantly contribute to permanent functional deficit. Innovations in pathological and neuroimaging techniques have contributed to the detection of mild axonal pathology, which has been overlooked by classical methods. Axonal injury has been detected immunohistologically using two markers: amyloid precursor protein6,7 and neurofilament protein (NFP).8 These markers have made the identification of early and subtle changes possible in

comparison to silver staining.7 Normally, NFP extends throughout the axon, dendrites, and soma of a nerve cell.9 The NFP subunits, which localized to the soma and dendrites, are nonphosphorylated on their carboxy-terminal domains, whereas NFP within the axon are predominantly phosphorylated. After a traumatic episode, reactive axonal swelling or a bulb can be labeled with antibodies to nonphosphorylated NFP epitopes, which normally are found exclusively within the cell bodies and dendrites.10 –12 The presence of axonal degeneration has been shown in an experimental animal model of MS, experimental allergic encephalomyelitis.13,14 In MS and experimental allergic encephalomyelitis, damaged axons have been found in close proximity to perivascular inflammatory foci,15 and the extent and severity of the inflammation is to some degree related to the amount of axonal damage. Axonal damage has been believed to occur secondarily only after the severe destruction of the myelin sheath by vigorous inflammatory responses. In contrast, axonal injury has been detected not only in inflammatory demyelinating lesions, but also in normal appearing white matter (NAWM)16,17 and normal appearing gray matter in MS.18 In addition, Dandekar and colleagues19 demonstrated that axonal injury occurred not only in areas of demyelination but also in adjacent areas devoid of myelin damage in a viral model for MS, murine hepatitis virus infection. Therefore, this raises the possibility that axonal injury might be induced by an independent mechanism and, at least in a subtype of MS, demyelination is secondary. Theiler’s murine encephalomyelitis virus (TMEV) belongs to the genus Cardiovirus, family Picornaviridae. TMEV is divided into two subgroups: high-neurovirulent strains, including GDVII and FA, which cause fatal encephalitis, and low-neurovirulent strains, such as DA and BeAn, which cause persistent infection and demyelination in mice.20,21 The latter are widely used as an animal model for MS.22 In TMEV infection, axonal degeneration23 and loss24 have been reported during the late chronic phase of DA virus infection. The onset and development of axonal injury during the early stage of infection, as well as the precise mechanisms of axonal injury in TMEV, are unknown. Rivera-Quin˜ones and colleagues25 suggested a role for major histocompatibility complex class I-restricted CD8⫹ T cells in the development of axonal injury. Supported by the National Institutes of Health (grant 2 R01 NS34497). Accepted for publication January 6, 2003. Address reprint requests to Robert S. Fujinami, Ph.D., Department of Neurology, University of Utah, 30 North 1900 East, 3R330 SOM, Salt Lake City, UT 84132-2305. E-mail: [email protected].

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Using antibody against nonphosphorylated NFP, we performed time-course studies measuring the extent and location of axonal injury in both DA and GDVII virus infections. In DA virus infection, axonal injury was detected as early as 1 week after infection. The number of damaged axons increased throughout time. During the subclinical phase, 2 and 3 weeks after infection, axonal injury was often associated with parenchymal infiltration of microglia and T cells, and viral antigen. Using confocal microscopy, we found damaged axons present within intact myelin sheaths. However, vigorous inflammatory demyelinating lesions were not seen until the chronic phase (4 weeks after infection). In GDVII virus infection, extensive axonal injury was noted 1 week after infection without association with inflammation, virus, or demyelination. It appeared that axonal injury was induced without overt perivascular cuffing or demyelination in TMEV infection. Interestingly, the distribution of injured or damaged axons in both GDVII virus infection and the early phase of DA virus infection corresponded to regions where subsequent demyelination occurred during the chronic phase of DA virus infection. Our results suggest that axonal injury heralds or triggers the demyelinating disease.

communication with Dr. Sternberger) to nonphosphorylated NFP with autoclave pretreatment.33 Microglia and macrophages were identified by biotinylated Ricinus Communis agglutinin (RCA) I (Vector Laboratories Inc., Burlingame, CA).34,35

Materials and Methods

Image Processing

Animal Experiment

To compare the distribution of axonal injury among mice infected with GDVII and DA viruses, we used thoracic segments of the spinal cord sections that were immunostained with SMI 311. Using Image-Pro Plus (Media Cybernetics, Silver Springs, MD) and Adobe Photoshop (Adobe Systems Inc., San Jose, CA), we superimposed thoracic images of five mice from each group at 1 week after GDVII virus infection and 2 and 3 weeks after DA virus infection.

Four-week-old female SJL/J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Working stocks of DA and GDVII strains of TMEV were prepared and titrated in baby hamster kidney (BHK)-21 cells and used for all experiments.26 Mice were inoculated under anesthesia intracerebrally with 20 ␮l of either 2 ⫻ 105 plaque-forming units (PFU) of DA virus or 1 ⫻ 103 PFU of GDVII virus. Mice were killed 1 week after GDVII virus infection, and 1, 2, 3, 4, 8, and 12 weeks after DA virus infection. Each group included 8 to 13 mice.

Immunohistochemistry and Lectin Histochemistry Mice were euthanized with halothane and perfused with phosphate-buffered saline, followed by a 4% paraformaldehyde phosphate-buffered solution. Spinal cords were cut either longitudinally or transversely and embedded in paraffin by standard methods. Four-␮m-thick tissue sections were stained with Luxol fast blue for myelin visualization. TMEV antigen and T cells were visualized by the avidin-biotin peroxidase complex technique, using hyperimmune rabbit serum to DA virus27 and anti-CD3⑀ antibody (after trypsinization, 1: 30 dilution; DAKO Corporation, Carpinteria, CA).28,29 Normal axons were visualized with SMI 312, a cocktail of monoclonal antibodies (SMI 31, 34, 35, 36, and 310; personal communication with Dr. Ludwig Sternberger, Sternberger Monoclonal, Inc., Baltimore, MD) to phosphorylated NFP.30 –32 Normal neurons and dendrites and damaged axons were visualized with SMI 311 (Sternberger Monoclonal, Inc.) a cocktail of antibodies (SMI 32, 33, 37, 38, and 39; personal

Immunofluorescent Microscopy Myelin was visualized by polyclonal rabbit anti-myelin basic protein (MBP) antibody (1:100 dilution, DAKO). We dual labeled axons for myelin, using anti-MBP antibody, and TMEV, using anti-DA serum, for myelin and phosphorylated NFP, using SMI 312, for myelin and nonphosphorylated NFP, using SMI 311, for TMEV and phosphorylated NFP, and for TMEV and nonphosphorylated NFP. Fluorescein isothiocyanate-conjugated anti-mouse IgG (1:128 dilution; Sigma Immunochemicals, St. Louis, MO) and tetramethyl-rhodamine isothiocyanate-conjugated anti-rabbit IgG (1:100 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were used as secondary antibodies. Fluorescent images were collected and analyzed by laser- scanning confocal microscopy (Cell Imaging Facility, University of Utah, Salt Lake City, UT).

Results Spinal Cord White Matter Appeared Normal during the Subclinical Stage with Significant Inflammatory Demyelination Detected 1 Month after DA Virus Infection Clinically, DA virus is known to cause a biphasic disease. Although DA virus-infected mice develop acute polioencephalomyelitis 1 week after infection (acute phase), all mice recovered without sequelae and showed no obvious clinical signs (subclinical phase, 2 to 3 weeks after infection). One month after infection, however, the mice developed spastic paralysis (chronic phase). Using Luxol fast blue stain, studies were initiated to determine the time course of general pathology in the spinal cord during DA virus infection. During the acute phase, 1 week after infection, DA virus is known to induce significant inflammatory lesions without demyelination mainly in the gray matter of the brain.20,35 At this time, lesions in the spinal cord were inconspicuous; however, mild meningitis and neuronophagia were detected in a few segments (Figure 1a). Neuronophagia, dying neu-

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Figure 1. a: One week after DA virus infection, the white matter of the spinal cord appeared normal. In the gray matter, an anterior horn neuron was surrounded by microglia, the process of neuronophagia (arrow; inset, normal neuron and neuronophagia). b: Two weeks after infection the spinal cord appeared primarily normal with a possible increase of cellularity in the NAWM (double arrows; inset, glial star). c: Three weeks after infection meningitis and mild cell infiltration in the parenchyma were evident (arrowhead). Mild vacuolar degeneration of the white matter, possible onset of demyelination, was seen in the VREZ of the white matter (arrowhead). d: One month after infection, significant perivascular infiltration of mononuclear cells and demyelination were detected in the anterior and lateral funiculi of the white matter (large arrow). Luxol fast blue stain. Original magnifications: ⫻40 (a– d); ⫻180 (inset in a); ⫻90 (inset in b).

rons invaded by microglia and macrophages, was seen in the anterior horn of the gray matter (Figure 1a, inset). The white matter of the spinal cord appeared normal. Two weeks after infection, the acute polioencephalomyelitis primarily subsided and no lesions were detected in the white matter except an increase of cellularity was occasionally suspected in the ventral root exit zone (VREZ) and the lateral funiculus (Figure 1b). Morphologically, this appeared similar to glial stars, clumps of microglia, which have been described as the only evidence of damage or cell loss either after axonal injury or after viral infection.36 –38 Three weeks after infection, significant meningitis and perivascular cuffing were detected, but cellular infiltration in the white matter parenchyma was mild. Mild vacuolar changes, which can be the onset of demyelination, were detected in the VREZ and the lateral and anterior funiculi in the white matter (Figure 1c). Significant perivascular cuffing with mononuclear cells and demyelination was not evident until 1 month after infection (Figure 1d). The anterior and lateral funiculi were involved, and the posterior funiculus was relatively preserved.25,39

Axonal Injury in the Absence of Vigorous Inflammation during the Subclinical Phase of DA Virus Infection SMI 311, a cocktail of antibodies against nonphosphorylated NFP, detects axonal injury. Normal neurons and dendrites and injured axons bind SMI 311, whereas normal axons do not react.30,31 In the spinal cord of control mice, neurons and dendrites in the gray matter were positive for SMI 311 staining, whereas no positive binding was detectable in the white matter (Figure 2a). In DA virus

infection, SMI 311⫹ axons were detectable as early as 1 week after infection in NAWM of the anterior and lateral funiculi (Figure 2b). Although the total number of SMI 311⫹ axons was small, axonal swelling was obvious, and retraction balls2 were seen easily in longitudinal sections. The number and extent of swelling of SMI 311⫹ axons increased throughout time (Figure 2; c, d, and e). They were detected in the anterior and lateral funiculi and the VREZ, but not in the posterior funiculus. Although SMI 311⫹ axonal swelling was seen around the glial stars, it was also detectable in the white matter regions where increases in cellularity were not noted. Three weeks after infection, beaded, fragmented, and degenerating axons were detected reactive for SMI 311. During the chronic phase, more than 1 month after infection, axonal loss was clearly evident by a lack of immunostaining with SMI 312 (Figure 2f), a cocktail of antibodies against phosphorylated NFP, specific for normal axons (Figure 2g). These results contradict the conventional belief that axonal injury is caused secondarily to severe inflammatory demyelinating lesions. However, this does not discount the possibility that axonal injury might be caused secondarily to minimal local myelin injury. Using doubleimmunofluorescence confocal microscopy, we tested whether injured axons co-localized with demyelination at a single fiber level. We found that SMI 311⫹ distended axons were wrapped with MBP⫹ myelin sheaths in the white matter of the spinal cord during the subclinical phase of DA virus infection (Figure 3; a to f). Distended axons with intact myelin have also been observed in MS.4 In addition, as early as 1 week after infection, MBP⫹ myelin sheaths lacking SMI 312⫹ axons were found suggesting that the axons were lost or injured and thus became negative for SMI 312 (Figure 3; g to j). This empty myelin, axonal degeneration in the absence of myelin loss, was similar to empty myelin profiles in NAWM in MS described by Bjartmar and colleagues.40

Axonal Injury Did Not Correlate with Virus Persistence Because induction of axonal injury in DA virus infection did not accompany obvious demyelinating lesions, possible effector mechanisms were investigated that could initiate axonal injury during the acute and subclinical phases. Because direct virus infection can cause tissue damage in the brain during the TMEV infection, we tested whether virus persistence correlated with axonal injury in DA virus infection. It is known that 1 week after infection, DA virus antigens can be detected mainly in the gray matter of the brain and spinal cord involvement is mild.20,21 As reported previously, viral antigens were detected in only a few neurons in the gray matter and no viral antigen-positive cells were found in the white matter in the spinal cord (Figure 4a). At 2 weeks after infection, a small number of viral antigen-positive cells were present in the white matter (Figure 4b). Although a slight increase in viral antigen-positive cells was seen 3 weeks after infection and thereafter, their number remained at low levels during the chronic phase (Figure 4, c and d).

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Figure 2. a– e: Nonphosphorylated NFP immunostain with antibody SMI 311. a: In control mice, nonphosphorylated NFP was detected in neurons and dendrites in the gray matter (GM), but not in the white matter (WM) of the thoracic segment of the transverse section. b: One week after DA virus infection, SMI 311⫹ axonal swelling (arrows) was detected in the NAWM and SMI 311⫹ axonal retraction balls were visible in the longitudinal section (inset). c: Two weeks after infection, the number of SMI 311⫹ axons increased. d: Three weeks after infection, the extent of swelling was severe and coarse varicosities on axons (inset) were seen in the longitudinal section. e: One month after infection, significant axonal swelling occurred in the anterior and lateral funiculi. SMI 311⫹ fragmented and degenerating axons were shown in the longitudinal section (inset). f and g: Immunostain against phosphorylated NFP showed axonal loss in the white matter (arrowhead) during the chronic phase of DA virus infection (f), but not in the control mice (g). Original magnifications: ⫻110 (a– e); ⫻45 (f); ⫻30 (g); ⫻300 (insets in b, d, e).

Using dual labeling for viral antigen with NFP, investigation as to whether viral antigens were detectable either in normal or in damaged axons was done. During the subclinical and chronic phases of DA virus infection, in most cases viral antigens co-localized with neither SMI 311- nor SMI 312-positive axons (Figure 4e). Usually, viral antigens in oligodendrocytes and their myelin processes were detected. Occasionally, TMEV⫹ myelin sheaths

wrapped multiple axons. Some axons appeared normal and were positive for SMI 312, and others were negative for SMI 312, resulting in the empty myelin profile (Figure 4f). However, in rare instances, a few viral antigen-positive axons were detected in the white matter of the spinal cord during the subclinical and chronic phases (Figure 4; g, h, and i). In summary, the kinetics of the increase in viral antigen- positive cells did not correlate with axonal

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Figure 3. Double labeling of axons in the white matter of the spinal cord 2 or 3 weeks after DA virus infection with one label detecting MBP⫹ myelin (rhodamine, red). a–f: Injured axons were detected with antibody against nonphosphorylated NFP (fluorescein isothiocyanate, green). Axonal injury (a) (green, arrowhead) was detected in MBP⫹ NAWM (b) (red). c–f: Merged images demonstrated injured axons wrapped with MBP⫹ myelin sheath. g: Phosphorylated NFP labeling (green) visualized not only normal axons but also distended axons (double arrows) that were wrapped with MBP⫹ myelin sheath (red) (h). i and j: Merged images demonstrated that some myelin sheaths lacked axonal staining (empty myelin, arrow) among intact myelinated axons. Original magnifications: ⫻230 (a– c); ⫻100 (d); ⫻380 (e); ⫻120 (f); ⫻230 (g–i); ⫻710 (j).

injury and viral antigen was rarely found in axons. Therefore, direct attack by TMEV on axons seemed unlikely as the cause of axonal injury. In addition, axons appear not to be the favorite site for virus persistence, whereas axonal transfer of virus is likely during the subclinical phase of DA virus infection. Dal Canto and Lipton41 detected TMEV antigens in axons in the spinal cord gray matter 25 to 80 days after DA virus infection by immunoelectron microscopy.

Axonal Injury Coincided with Microglia/Macrophage Infiltration, but Not with Perivascular Cuffing during the Subclinical Phase of DA Virus Infection As seen in Figures 1 and 2, axonal injury was detectable in NAWM and no correlation was observed between ax-

onal injury and perivascular cuffing. However, this does not discount the possibility of insidious activation or infiltration of resident microglia and/or macrophages that were not detected by conventional staining. Therefore, RCA I lectin histochemistry was used to detect monocyte/ macrophage lineage cells.34,35,42 One week after DA virus infection, small round RCA I⫹ cells were seen predominantly in the gray matter of the spinal cord. In the anterior horn, RCA I⫹ microglia congregated around dying neurons (neuronophagia; Figure 5a, inset). Two weeks after infection, the poliomyelitis subsided and RCA I⫹ cells disappeared from the gray matter. However, in the white matter, small clusters of RCA I⫹ cells appeared (Figure 5b). Although some RCA I⫹ cells were round, other RCA I⫹ cells had dendritic processes, thus morphologically appearing to be microglia. Consecutive tissue sections showed RCA I⫹ microglia proliferation within glial stars by Luxol fast blue staining and around SMI 311⫹ axonal swelling (data not shown). Three weeks after infection, RCA I⫹ cells grew plumper and seemed to develop into more rounded phagocytes (Figure 5c). At this stage, some RCA I⫹ cells appeared to be bloodderived macrophages that infiltrated into the parenchyma from either the meninges or blood vessels. During the chronic phase, RCA I⫹ cells localized with the demyelinating lesions of the white matter (Figure 5d). We also detected T-cell infiltration using an anti-CD3 antibody. One week after infection, T cells were detected predominantly in the gray matter of the spinal cord. A small number of T cells were also seen sporadically in the white matter, although they could not be detected without using CD3 immunohistochemistry. Two weeks after infection, T cells were detected in glial stars and the VREZ, where they were scattered in the parenchyma or presented as small cell clusters in the white matter (Figure 5e). Perivascular inflammation was not seen at this time. Perivascular cuffs with T cells were not detected in the white matter until the chronic phase (Figure 5f). Thus, axonal injury was occasionally accompanied by insidious microglia and T-cell infiltration but not by perivascular cuffing during the subclinical phase.

In GDVII Virus Infection, Axonal Injury Was Not Associated with Virus-Infected Cells, T-Cell Infiltration, or Microglia Activation GDVII virus, from the highly neurovirulent subgroup of TMEV, is known to infect neurons in the gray matter of the CNS and induces an acute fatal polioencephalomyelitis. However, the white matter appears normal by conventional staining (Figure 6, a and c). Because we found axonal injury in NAWM in DA virus infection, we investigated whether there is axonal damage in NAWM of the spinal cord, 1 week after GDVII virus infection. Using antibody against nonphosphorylated NFP, we found severe axonal swelling and degeneration in NAWM in GDVII virus-infected mice to a similar extent as the axonal injury seen during the chronic phase of DA virus infection (Figure 6, b and d).

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Figure 4. a– d: Time-course study of DA virus persistence. e and f: Double labeling of axons with one label detecting DA virus antigen. a: One week after infection virus antigen was not detected in the spinal cord. b: A small number of virus antigen-positive cells (arrow) were detected in the VREZ, 2 weeks after infection. c: A slight increase of virus antigen-positive cells was noted at 3 weeks after infection. d: DA virus persistence remained at low levels during the chronic phase. e: Usually viral antigen (rhodamine, red, large white arrow) did not co-localize with SMI 311⫹-damaged axons (fluorescein isothiocyanate, small arrow). f: Viral antigen⫹ oligodendrocyte (red, rhodamine, center) had processes that wrapped SMI 312⫹ axons (arrowhead). An empty myelin profile (double arrowhead) was seen in the top center, where viral antigen-positive myelin sheath contained no axon or an SMI 312-negative axon. g–i: In rare instances, viral antigen was seen in axons. In the white matter of the spinal cord, we detected SMI 311 single-positive axons (fluorescein isothiocyanate, green, white arrow), viral antigen single-positive cells (rhodamine, red, large white arrow) and SMI 311/TMEV double-positive axons (yellow, double arrows). Original magnifications: ⫻110 (a– d); ⫻230 (e); ⫻620 (f); ⫻140 (g–i).

We next tested whether viral antigen-positive cells, T cells, and macrophages around axonal injury were evident. Viral antigen-containing cells were detected in the gray matter, but not in the white matter (Figure 6e). Using consecutive sections, we found that axonal injury was seen not only in spinal cord segments that contained viral

antigen, but also in those without viral antigen (Figure 6d). Also, the distribution of T cells did not correlate with axonal damage. Usually, a small number of T cells was found in the meninges and the gray matter (Figure 6f).43 Although fewer T cells were also scattered in the white matter in only a few segments of the spinal cord, consec-

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Figure 5. Kinetics of microglia/macrophage (a– d) and T-cell (e, f) infiltration in the spinal cord in DA virus infection. a: During the acute phase, 1 week after infection, RCA I⫹ microglia were detected in the gray matter (GM) (arrow), but not in the white matter (WM). In the anterior horn, RCA I⫹ cells marked the positions of dead neurons (neuronophagia, large arrow, inset). b: Two weeks after infection a cluster of RCA I⫹ cells (glial star, arrow), which had dendritic processes (inset), was detected in the white matter. c: Three weeks after infection RCA I⫹ cells developed into rounded phagocytes (inset) and some RCA I⫹ cells appeared to infiltrate into the parenchyma from the perivascular space and meninges. d: During the chronic phase, a large number of RCA I⫹ cells were seen in demyelinating lesion of the white matter. e: A consecutive section of (b) showed that CD3⫹ T cells (arrow) also infiltrated sporadically into the white matter. No perivascular cuffing was detected at this stage. f: Typical perivascular cuffings (arrowhead) and meningitis with CD3⫹ T cells were seen during the chronic phase, 1 month after infection. a– d: RCA I lectin histochemistry; e and f: anti-CD3 immunohistochemistry. Original magnifications: ⫻60 (a– d); ⫻120 (e, f); ⫻300 (insets in a– c).

utive sections showed that axonal injury was not accompanied by T-cell infiltration. Rather T cells were detected in areas where no axonal swelling was detected (data not shown). RCA I⫹ microglia/macrophages were found predominantly in the gray matter (Figure 6g). Therefore, neither viral antigen nor T cell and macrophage infiltration was associated with axonal injury in GDVII virus infection. To compare the distribution of axonal damage between GDVII (1 week after infection) and DA (2 and 3 weeks after infection) virus infections, sections immunostained with SMI 311 were superimposed using an image processing software. We found similar lesion distribution in all groups. Injured axons were seen in the anterior and the lateral funiculi and the VREZ, but not in the posterior funiculus (Figure 7; b, c, and d). Interestingly, the lesion

distribution corresponded to regions where subsequent demyelination occurs during the chronic phase of DA virus infection (Figure 7e).25,39

Discussion In MS, the mechanism(s) of axonal injury is primarily unknown, although inflammatory demyelinating events are believed to cause axonal damage secondarily.1 Our results indicate that axonal injury in TMEV infection was associated neither with a vigorous perivascular inflammatory response nor with demyelination as seen in GDVII virus infection and during the early stage of DA virus infection. Similar to our results, Bitsch and colleagues44

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Figure 6. Spinal cord pathology, 1 week after GDVII virus infection (a and b, longitudinal section; c– g, transverse section). a and c: By Luxol fast blue stain, mild meningitis and a few pyknotic neurons in the gray matter (GM) were the only pathological findings; the white matter (WM) appeared normal. b and d: The consecutive sections demonstrated numerous axonal swellings and degeneration positive for nonphosphorylated NFP (arrowhead). Distribution of axonal injury did not correlate with the distribution of viral antigen (e), T cells (f), or microglia/macrophages (g). e: Viral antigen was not detectable by immunohistochemistry in this segment. f: T cells were detected only in the meninges by immunohistochemistry against CD3. g: RCA I⫹ microglia/macrophages (arrow) were detected in the gray matter. Original magnifications: ⫻80 (a, b); ⫻40 (c– g).

found that axonal injury in MS did not correlate with the number of CD3⫹ T cells. Injured axons were identified not only in active demyelinating lesions, but also in remyelinating lesions and in NAWM, suggesting axonal injury was independent of demyelinating activity. Lovas and colleagues16 also demonstrated that acute inflammatory demyelination did not correlate with axonal injury in secondary progressive MS. Injured axons in NAWM in MS were also demonstrated by magnetic resonance spectroscopy.45,46 In addition, although it was mild and rare, Mancardi and colleagues47 have reported axonal damage in NAWM in experimental allergic encephalomyelitis. The precise mechanism of axonal injury in the early stage of TMEV infection is not clear. The relative preservation of the posterior funiculus suggests that axonal injury in the white matter of the spinal cord might result from wallerian degeneration induced by neuronal death in the gray matter. The posterior funiculus is mainly comprised of ascending tracts, whereas the anterior and lateral funiculi contain both ascending and descending tracts. During the acute phase, TMEV infects neurons in the gray matter in the brain and anterior horn cells in the spinal cord, leading to apoptosis of neurons.42 Thus, neuronal loss in the brain could lead to wallerian degeneration of the descending tracts in the anterior and lateral funiculi, and neuronal death in the anterior horn could

lead to wallerian degeneration in the VREZ. This cannot explain, however, the preservation of the corticospinal tract that occupies a position in the ventral portion of the posterior funiculus in rodents (Figure 7a).48 This is in contrast to higher mammals whose corticospinal fibers are in the lateral funiculus. McGavern and colleagues49 showed no decrease in small axon fibers and a loss of medium to large axon fibers 195 to 220 days after infection in mice infected with DA virus. Because the fibers in the corticospinal tract are smaller in diameter,48 the fiber size of axons might be important in susceptibility and/or resistance to damage. Although the distribution of injured axons is similar between GDVII and DA virus infections, axonal injury developed much more slowly in DA virus-infected mice versus GDVII virus-infected mice (mean number of SMI 311⫹ axons with swelling in the lateral funiculi of the spinal cord ⫾ SEM/mm2: GDVII virus 1 week after infection, 62.4 ⫾ 15.9; DA virus 2 weeks after infection, 11.5 ⫾ 4.3; DA virus 3 week after infection, 36 ⫾ 4.3). This observation could be partly because of the fact that neuronal death during the acute phase is more severe in GDVII virus infection than in DA virus infection,42 resulting in different degrees in or extent of wallerian degeneration. Alternatively, we found microglia and T-cell parenchymal infiltration around damaged axons at 2 and 3 weeks after DA virus infection. Therefore, microglia and/or T cells could be required for full-blown axonal damage in DA virus infection. Distribution of injured axons during the early stage of DA virus infection corresponded to regions where subsequent demyelination occurred during the chronic phase. This suggests that axonal injury might play an important role in recruiting inflammatory cells and inducing demyelination. Indeed, axonal injury and subsequent microglia activation have been shown to drive encephalitogenic cells to home to sites of wallerian degeneration in the CNS.50 –52 These investigators demonstrated activation of microglia at the site of wallerian degeneration that was induced by axonal transection in rats. Ten days after transection, Konno and colleagues51 adoptively transferred encephalitogenic cells into the rats and found the recipient rats developed inflammatory lesions at the sites of wallerian degeneration. Spinal cord injury induces oligodendrocyte apoptosis, but there is a marked delay in oligodendrocyte death until after the acute phase, ⬃2 weeks after injury.53–55 Although the pathomechanism is unclear, Shuman and colleagues56 have proposed two hypotheses. First, axonal breakdown within regions of wallerian degeneration promotes microglial cell activation. The activated microglia triggers apoptosis of oligodendrocytes through the secretion of cytotoxic factors such as tumor necrosis factor-␣.57 This is further supported by accumulation and activation of microglia during wallerian degeneration.58 Second, the disruption of the intimate relationship between oligodendrocytes and intact axons leads to the loss of axon-derived trophic signals that could provide a trigger for inducing oligodendrocyte apoptosis.59 – 61 Because oligodendrocyte apoptosis is also seen in TMEV infection,42 the above two hypotheses provide potential

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Figure 7. a: Section through a thoracic segment of the mouse spinal cord to demonstrate the subdivisions of the white matter. In transverse section, the spinal cord consists of a butterfly-shaped region of central gray matter and a surrounding mantle of white matter. The white matter of the spinal cord is divided into three funiculi, which are anterior, lateral, and posterior. The corticospinal tract (**) is located in the ventral most portion of the posterior funiculus in rodents. b– d: The distribution of axonal injury was similar for GDVII virus infection at 1 week after infection and DA virus infection at 2 and 3 weeks after infection. Axonal injury in the spinal cord was detected by immunohistochemistry against nonphosphorylated NFP. Thoracic segment images from five mice in each group were superimposed using Image-Pro plus and Adobe Photoshop. e: Luxol fast blue staining showed that demyelination was seen in the anterior and lateral funiculi and the VREZ (arrowhead), but not in the posterior funiculus during the chronic phase of DA virus infection. The distribution of demyelination correlated with regions of axonal injury during the subclinical phase, 2 and 3 weeks after infection. Original magnifications: ⫻30 (b– d); ⫻10 (e).

mechanisms for the initiation of demyelination in TMEV infection. A possible relationship between axonal injury and demyelination is also suggested by our results showing that the distribution of axonal injury during the subclinical phase of DA virus infection was similar to regions where subsequent demyelination occurred during the chronic phase (Figure 7). Conversely, apoptosis of oligodendrocytes could contribute to the exacerbation of axonal injury because myelinated axons have been reported to require local oligodendrocyte support.62 We are currently investigating the association of axonal injury and oligodendrocyte apoptosis during TMEV infection. Our working hypothesis of lesion development in TMEV infection is as follows. TMEV first infects neurons in the gray matter and spreads axonally.63,64 Infected neurons die by apoptosis, and axons are damaged by wallerian degeneration and/or by a local self-destruct program,65 which activates resident microglia and recruits macrophages and T cells that can produce molecules cytotoxic

to axons and/or myelin. Virus being transported within axons would also contribute to axonal degeneration and infect the myelin sheaths that wrap infected axons leading to infection of the oligodendrocytes. Inflammatory responses, infection, and a lack of axonal support1 would contribute to oligodendrocyte death, leading to demyelination. Myelin debris and virus antigens are phagocytosed and presented by microglia/macrophages to T cells, generating myelin and virus-specific immunity. This will lead to demyelination by anti-myelin immune responses or damage because of bystander effects.22 This secondary inflammatory response exacerbates axonal injury and leads to additional cycles of autosensitization and disease progression.40 An alternative interpretation is that axonal injury can be beneficial for the host in certain instances. Recently, Raff and colleagues65 suggested that neurons have two selfdestruct programs, which are apoptosis and axonal degeneration. Here, axonal injury may occur through a local self-destruct program designed to disconnect the neuron

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from its target cell, thereby conserving energy. Because TMEV has been shown to be transported along axons,63,64 axonal injury might be induced by the neuron to limit the spread of virus. Transection of infected axons would efficiently limit transport and spread of viruses from the gray matter to the white matter. This might help explain why GDVII virus does not persistently infect cells in the white matter of the spinal cord in rare survivor mice.66 In GDVII virus infection, we find a more severe and rapid degeneration of axons than during DA virus infection. In other words, slower axonal degeneration would favor virus spread to the white matter of the spinal cord in DA virus infection. In addition, during the subclinical phase, microglia appear to differentiate into activated phagocytes that are equipped with a battery of tools for destroying invading microorganisms.58 This might also reflect an innate immune response to eradicate viruses from the CNS. As we have discussed, axonal injury can induce activation of microglia/macrophage and apoptosis of oligodendrocyte, leading to demyelination. After this type of CNS destruction, it is possible that myelin antigens can be released and presented by antigen-presenting cells to T cells, further exacerbating demyelination by autoimmunity. Clinically, however, the role of brain and spinal cord trauma in causing or aggravating MS has been controversial. Most, but not all,67 case-control studies have failed to show a significant relationship between trauma and MS onset and none of the studies have shown a 100% trauma history in MS patients.68,69 Therefore, CNS injury alone cannot explain the pathogenesis of all MS patients. However, considering that MS likely has a multifactorial etiology, this would not preclude the possibility that CNS injury may contribute to the onset or exacerbations of MS in a small percentage of cases.70 Indeed, anecdotal case histories of head and spinal cord trauma have been reported to precede MS exacerbations.71 In this article we showed axonal injury during the early stage of TMEV infection. We suggested that axonal injury might herald and trigger demyelination, and hypothesized that axonal injury and demyelination propagate a vicious cycle or initiate a cascade of destructive events. Therapeutic strategies that interfere with the sequence of cellular responses to axonal injury and demyelination may prove clinically beneficial.

Acknowledgments We thank Dr. Ludwig A. Sternberger and Dr. Mauro C. Dal Canto for their helpful discussions; Dr. Warren Davis, Duane S. Doyle, Isaac Z. M. Igenge, and Kenneth B. Scott for their technical assistance; and Ms. Kathleen Borick for preparation of the manuscript.

4. 5.

6. 7.

8.

9. 10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23. 24.

References 1. Perry VH, Anthony DC: Axon damage and repair in multiple sclerosis. Philos Trans R Soc Lond B Biol Sci 1999, 354:1641–1647 2. Greenfield JG, King LS: Observations on the histopathology of the cerebral lesions in disseminated sclerosis. Brain 1936, 59:445– 458 3. Putman TJ: Studies in multiple sclerosis. VII. Similarities between

25.

26.

some forms of “encephalomyelitis” and multiple sclerosis. Arch Neurol Psychiatr 1936, 35:1289 –1308 Suzuki K, Andrews JM, Waltz JM, Terry RD: Ultrastructural studies of multiple sclerosis. Lab Invest 1969, 20:444 – 454 Ikuta F, Zimmerman HM: Distribution of plaques in seventy autopsy cases of multiple sclerosis in the United States. Neurology 1976, 26:26 –28 Ferguson B, Matyszak MK, Esiri MM, Perry VH: Axonal damage in acute multiple sclerosis lesions. Brain 1997, 120:393–399 Giometto B, An SF, Groves M, Scaravilli T, Geddes JF, Miller R, Tavolato B, Beckett AA, Scaravilli F: Accumulation of ␤-amyloid precursor protein in HIV encephalitis: relationship with neuropsychological abnormalities. Ann Neurol 1997, 42:34 – 40 Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mo¨rk S, Bo¨ L: Axonal transection in the lesions of multiple sclerosis. N Engl J Med 1998, 338:278 –285 Vickers JC: A cellular mechanism for the neuronal changes underlying Alzheimer’s disease. Neuroscience 1997, 78:629 – 639 Meller D, Eysel UT, Schmidt-Kastner R: Transient immunohistochemical labelling of rat retinal axons during Wallerian degeneration by a monoclonal antibody to neurofilaments. Brain Res 1994, 648:162–166 King CE, Adlard PA, Dickson TC, Vickers JC: Neuronal response to physical injury and its relationship to the pathology of Alzheimer’s disease. Clin Exp Pharmacol Physiol 2000, 27:548 –552 King CE, Dickson TC, Jacobs I, McCormack GH, Riederer BM, Vickers JC: Acute CNS axonal injury models a subtype of dystrophic neurite in Alzheimer’s disease. Alzheimer’s Rep 2000, 3:31– 40 Kornek B, Storch MK, Weissert R, Wallstroem E, Stefferl A, Olsson T, Linington C, Schmidbauer M, Lassmann H: Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions. Am J Pathol 2000, 157:267–276 Pender MP, Stanley GP, Yoong G, Nguyen KB: The neuropathology of chronic relapsing experimental allergic encephalomyelitis induced in the Lewis rat by inoculation with whole spinal cord and treatment with cyclosporin A. Acta Neuropathol (Berl) 1990, 80:172–183 Bieger D, White SR: Anatomical evidence for bulbospinal monoamine axon damage in rats with experimental allergic encephalomyelitis. Neuroscience 1981, 6:1745–1752 Lovas G, Szilagyi N, Majtenyi K, Palkovits M, Komoly S: Axonal changes in chronic demyelinated cervical spinal cord plaques. Brain 2000, 123:308 –317 Matthews PM, De Stefano N, Narayanan S, Francis GS, Wolinsky JS, Antel JP, Arnold DL: Putting magnetic resonance spectroscopy studies in context: axonal damage and disability in multiple sclerosis. Semin Neurol 1998, 18:327–336 Sharma R, Narayana PA, Wolinsky JS: Grey matter abnormalities in multiple sclerosis: proton magnetic resonance spectroscopic imaging. Mult Scler 2001, 7:221–226 Dandekar AA, Wu GF, Pewe L, Perlman S: Axonal damage is T cell mediated and occurs concomitantly with demyelination in mice infected with a neurotropic coronavirus. J Virol 2001, 75:6115– 6120 Tsunoda I, Fujinami RS: Two models for multiple sclerosis: experimental allergic encephalomyelitis and Theiler’s murine encephalomyelitis virus. J Neuropathol Exp Neurol 1996, 55:673– 686 Tsunoda I, Fujinami RS: Theiler’s murine encephalomyelitis virus. Persistent Viral Infections. Edited by R Ahmed, I Chen. Chichester, John Wiley & Sons Ltd., 1999, pp 517–536 Vanderlugt CL, Miller SD: Epitope spreading in immune-mediated diseases: implications for immunotherapy. Nat Rev Immunol 2002, 2:85–95 Dal Canto MC, Lipton HL: Primary demyelination in Theiler’s virus infection. An ultrastructural study. Lab Invest 1975, 33:626 – 637 Ure DR, McGavern DB, Sathornsumetee S, Rodriguez M: The contribution of axonal injury to neurologic dysfunction in Theiler’s virusinduced inflammatory demyelinating disease. Genes and Viruses in Multiple Sclerosis. Edited by OR Hommes, M Clanet, H Wekerle. Amsterdam, Elsevier Science BV, 2001, pp 79 – 88 Rivera-Quin˜ones C, McGavern D, Schmelzer JD, Hunter SF, Low PA, Rodriguez M: Absence of neurological deficits following extensive demyelination in a class I-deficient murine model of multiple sclerosis. Nat Med 1998, 4:187–193 Kurtz CIB, Sun XM, Fujinami RS: Protection of SJL/J mice from de-

Axonal Injury in TMEV Infection 1269 AJP April 2003, Vol. 162, No. 4

27. 28.

29.

30.

31.

32.

33.

34.

35.

36.

37. 38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

myelinating disease mediated by Theiler’s murine encephalomyelitis virus. Microb Pathog 1995, 18:11–27 Zurbriggen A, Fujinami RS: Theiler’s virus infection in nude mice: viral RNA in vascular endothelial cells. J Virol 1988, 62:3589 –3596 Mason DY, Cordell J, Brown M, Pallesen G, Ralfkiaer E, Rothbard J, Crumpton M, Gatter KC: Detection of T cells in paraffin wax embedded tissue using antibodies against a peptide sequence from the CD3 antigen. J Clin Pathol 1989, 42:1194 –1200 Tsunoda I, Kuang L-Q, Theil DJ, Fujinami RS: Antibody association with a novel model for primary progressive multiple sclerosis: Induction of relapsing-remitting and progressive forms of EAE in H2S mouse strains. Brain Pathol 2000, 10:402– 418 Sternberger LA, Sternberger NH: Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ. Proc Natl Acad Sci USA 1983, 80:6126 – 6130 Ulfig N, Nickel J, Bohl J: Monoclonal antibodies SMI 311 and SMI 312 as tools to investigate the maturation of nerve cells and axonal patterns in human fetal brain. Cell Tissue Res 1998, 291:433– 443 Powell HC, Garrett RS, Brett FM, Chiang C-S, Chen E, Masliah E, Campbell IL: Response of glia, mast cells and the blood brain barrier, in transgenic mice expressing interleukin-3 in astrocytes, an experimental model for CNS demyelination. Brain Pathol 1999, 9:219 –235 Shin R-W, Iwaki T, Kitamoto T, Tateishi J: Hydrated autoclave pretreatment enhances tau immunoreactivity in formalin-fixed normal and Alzheimer’s disease brain tissues. Lab Invest 1991, 64:693–702 Suzuki H, Franz H, Yamamoto T, Iwasaki Y, Konno H: Identification of the normal microglial population in human and rodent nervous tissue using lectin-histochemistry. Neuropathol Appl Neurobiol 1988, 14: 221–227 Tsunoda I, Iwasaki Y, Terunuma H, Sako K, Ohara Y: A comparative study of acute and chronic diseases induced by two subgroups of Theiler’s murine encephalomyelitis virus. Acta Neuropathol (Berl) 1996, 91:595– 602 Strich SJ, Oxon DM: Shearing of nerve fibres as a cause of brain damage due to head injury. A pathological study of twenty cases. Lancet 1961, 2:443– 448 Oppenheimer DR: Microscopic lesions in the brain following head injury. J Neurol Neurosurg Psychiatry 1968, 31:299 –306 Duchen LW: General pathology of neurons and neuroglia. Greenfield’s Neuropathology, ed 4. Edited by JH Adams, JAN Corsellis, LW Duchen. London, Edward Arnold Publishers, Ltd., 1984, pp 1–52 Tsunoda I, Tolley ND, Theil DJ, Whitton JL, Kobayashi H, Fujinami RS: Exacerbation of viral and autoimmune animal models for multiple sclerosis by bacterial DNA. Brain Pathol 1999, 9:481– 493 Bjartmar C, Kinkel RP, Kidd G, Rudick RA, Trapp BD: Axonal loss in normal-appearing white matter in a patient with acute MS. Neurology 2001, 57:1248 –1252 Dal Canto MC, Lipton HL: Ultrastructural immunohistochemical localization of virus in acute and chronic demyelinating Theiler’s virus infection. Am J Pathol 1982, 106:20 –29 Tsunoda I, Kurtz CIB, Fujinami RS: Apoptosis in acute and chronic central nervous system disease induced by Theiler’s murine encephalomyelitis virus. Virology 1997, 228:388 –393 Theil DJ, Tsunoda I, Libbey JE, Derfuss TJ, Fujinami RS: Alterations in cytokine but not chemokine mRNA expression during three distinct Theiler’s virus infections. J Neuroimmunol 2000, 104:22–30 Bitsch A, Schuchardt J, Bunkowski S, Kuhlmann T, Bru¨ck W: Acute axonal injury in multiple sclerosis. Correlation with demyelination and inflammation. Brain 2000, 123:1174 –1183 Husted CA, Goodin DS, Hugg JW, Maudsley AA, Tsuruda JS, de Bie SH, Fein G, Matson GB, Weiner MW: Biochemical alterations in multiple sclerosis lesions and normal-appearing white matter detected by in vivo 31P and 1H spectroscopic imaging. Ann Neurol 1994, 36:157–165 Davie CA, Barker GJ, Thompson AJ, Tofts PS, McDonald WI, Miller DH: 1H magnetic resonance spectroscopy of chronic cerebral white matter lesions and normal appearing white matter in multiple sclerosis. J Neurol Neurosurg Psychiatry 1997, 63:736 –742 Mancardi G, Hart B, Roccatagliata L, Brok H, Giunti D, Bontrop R, Massacesi L, Capello E, Uccelli A: Demyelination and axonal damage in a non-human primate model of multiple sclerosis. J Neurol Sci 2001, 184:41– 49 Brown Jr LT: Projections and termination of the corticospinal tract in rodents. Exp Brain Res 1971, 13:432– 450

49. McGavern DB, Murray PD, Rivera-Quin˜ones C, Schmelzer JD, Low PA, Rodriguez M: Axonal loss results in spinal cord atrophy, electrophysiological abnormalities and neurological deficits following demyelination in a chronic inflammatory model of multiple sclerosis. Brain 2000, 123:519 –531 50. Konno H, Yamamoto T, Iwasaki Y, Suzuki H, Saito T, Terunuma H: wallerian degeneration induces Ia-antigen expression in the rat brain. J Neuroimmunol 1989, 25:151–159 51. Konno H, Yamamoto T, Suzuki H, Yamamoto H, Iwasaki Y, Ohara Y, Terunuma H, Harata N: Targeting of adoptively transferred experimental allergic encephalitis lesion at the sites of wallerian degeneration. Acta Neuropathol (Berl) 1990, 80:521–526 52. Molleston MC, Thomas ML, Hickey WF: Novel major histocompatibility complex expression by microglia and site-specific experimental allergic encephalomyelitis lesions in the rat central nervous system after optic nerve transection. Adv Neurol 1993, 59:337–348 53. Abe Y, Yamamoto T, Sugiyama Y, Watanabe T, Saito N, Kayama H, Kumagai T: Apoptotic cells associated with wallerian degeneration after experimental spinal cord injury: a possible mechanism of oligodendroglial death. J Neurotrauma 1999, 16:945–952 54. Crowe MJ, Bresnahan JC, Shuman SL, Masters JN, Beattie MS: Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nat Med 1997, 3:73–76 55. Warden P, Bamber NI, Li H, Esposito A, Ahmad KA, Hsu CY, Xu XM: Delayed glial cell death following wallerian degeneration in white matter tracts after spinal cord dorsal column cordotomy in adult rats. Exp Neurol 2001, 168:213–224 56. Shuman SL, Bresnahan JC, Beattie MS: Apoptosis of microglia and oligodendrocytes after spinal cord contusion in rats. J Neurosci Res 1997, 50:798 – 808 57. Gehrmann J, Matsumoto Y, Kreutzberg GW: Microglia: intrinsic immuneffector cell of the brain. Brain Res Brain Res Rev 1995, 20:269 –287 58. Aldskogius H, Kozlova EN: Central neuron-glial and glial-glial interactions following axon injury. Prog Neurobiol 1998, 55:1–26 59. Barres BA, Hart IK, Coles HSR, Burne JF, Voyvodic JT, Richardson WD, Raff MC: Cell death and control of cell survival in the oligodendrocyte lineage. Cell 1992, 70:31– 46 60. Frost EE, Buttery PC, Milner R, ffrench-Constant C: Integrins mediate a neuronal survival signal for oligodendrocytes. Curr Biol 1999, 9:1251–1254 61. Raff MC, Barres BA, Burne JF, Coles HSR, Ishizaki Y, Jacobson MD: Programmed cell death and the control of cell survival: lessons from the nervous system. Science 1993, 262:695–700 62. Griffiths I, Klugmann M, Anderson T, Yool D, Thomson C, Schwab MH, Schneider A, Zimmermann F, McCulloch M, Nadon N, Nave KA: Axonal swellings and degeneration in mice lacking the major proteolipid of myelin. Science 1998, 280:1610 –1613 63. Rustigian R, Pappenheimer AM: Myositis in mice following intramuscular injection of viruses of the mouse encephalomyelitis group and of certain other neurotropic viruses. J Exp Med 1949, 89:69 –92 64. Martinat C, Jarousse N, Prevost MC, Brahic M: The GDVII strain of Theiler’s virus spreads via axonal transport. J Virol 1999, 73:6093– 6098 65. Raff MC, Whitmore AV, Finn JT: Axonal self-destruction and neurodegeneration. Science 2002, 296:868 – 871 66. Lipton HL: Persistent Theiler’s murine encephalomyelitis virus infection in mice depends on plaque size. J Gen Virol 1980, 46:169 –177 67. McAlpine D, Compton SR: Some aspects of the natural history of disseminated sclerosis. Part I. The incidence, course and prognosis. Part II. Factors affecting the onset and course Q J Med 1951, 21: 135–167 68. Compston A: Distribution of multiple sclerosis. McAlpine’s Multiple Sclerosis. Edited by DA Compston, WB Matthews, A Compston, G Ebers, H Lassmann. London, Churchill Livingston, 1998, pp 63–100 69. Cook SD: Trauma does not precipitate multiple sclerosis. Arch Neurol 2000, 57:1077–1078 70. Poser CM: Trauma to the central nervous system may result in formation or enlargement of multiple sclerosis plaques. Arch Neurol 2000, 57:1074 –1077 71. Goodin DS, Ebers GC, Johnson KP, Rodriguez M, Sibley WA, Wolinsky JS: The relationship of MS to physical trauma and psychological stress: report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology. Neurology 1999, 52:1737–1745