Axonal Injury Heralds Virus-Induced Demyelination
2003; Elsevier BV; Volume: 162; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63922-3
ISSN1525-2191
AutoresIkuo Tsunoda, Li‐Qing Kuang, Jane E. Libbey, Robert S. Fujinami,
Tópico(s)Nerve injury and regeneration
ResumoAxonal 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. 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. Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Axonal damage in MS has recently attracted significant attention1Perry VH Anthony DC Axon damage and repair in multiple sclerosis.Philos Trans R Soc Lond B Biol Sci. 1999; 354: 1641-1647Crossref PubMed Scopus (57) Google Scholar even though the presence of axonal degeneration in MS has long been recognized.2Greenfield JG King LS Observations on the histopathology of the cerebral lesions in disseminated sclerosis.Brain. 1936; 59: 445-458Crossref Scopus (64) Google Scholar, 3Putman TJ Studies in multiple sclerosis. VII. Similarities between some forms of “encephalomyelitis” and multiple sclerosis.Arch Neurol Psychiatr. 1936; 35: 1289-1308Crossref Scopus (35) Google Scholar, 4Suzuki K Andrews JM Waltz JM Terry RD Ultrastructural studies of multiple sclerosis.Lab Invest. 1969; 20: 444-454PubMed Google Scholar, 5Ikuta F Zimmerman HM Distribution of plaques in seventy autopsy cases of multiple sclerosis in the United States.Neurology. 1976; 26: 26-28Crossref PubMed Google Scholar 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 protein6Ferguson B Matyszak MK Esiri MM Perry VH Axonal damage in acute multiple sclerosis lesions.Brain. 1997; 120: 393-399Crossref PubMed Scopus (1294) Google Scholar, 7Giometto B An SF Groves M Scaravilli T Geddes JF Miller R Tavolato B Beckett AA Scaravilli F Accumulation of β-amyloid precursor protein in HIV encephalitis: relationship with neuropsychological abnormalities.Ann Neurol. 1997; 42: 34-40Crossref PubMed Scopus (102) Google Scholar and neurofilament protein (NFP).8Trapp BD Peterson J Ransohoff RM Rudick R Mörk S Bö L Axonal transection in the lesions of multiple sclerosis.N Engl J Med. 1998; 338: 278-285Crossref PubMed Scopus (3422) Google Scholar These markers have made the identification of early and subtle changes possible in comparison to silver staining.7Giometto B An SF Groves M Scaravilli T Geddes JF Miller R Tavolato B Beckett AA Scaravilli F Accumulation of β-amyloid precursor protein in HIV encephalitis: relationship with neuropsychological abnormalities.Ann Neurol. 1997; 42: 34-40Crossref PubMed Scopus (102) Google Scholar Normally, NFP extends throughout the axon, dendrites, and soma of a nerve cell.9Vickers JC A cellular mechanism for the neuronal changes underlying Alzheimer's disease.Neuroscience. 1997; 78: 629-639Crossref PubMed Scopus (27) Google Scholar 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.10Meller 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: 162-166Crossref PubMed Scopus (27) Google Scholar, 11King 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: 548-552Crossref PubMed Scopus (15) Google Scholar, 12King 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: 31-40Google Scholar The presence of axonal degeneration has been shown in an experimental animal model of MS, experimental allergic encephalomyelitis.13Kornek 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: 267-276Abstract Full Text Full Text PDF PubMed Scopus (769) Google Scholar, 14Pender 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: 172-183Crossref PubMed Scopus (46) Google Scholar In MS and experimental allergic encephalomyelitis, damaged axons have been found in close proximity to perivascular inflammatory foci,15Bieger D White SR Anatomical evidence for bulbospinal monoamine axon damage in rats with experimental allergic encephalomyelitis.Neuroscience. 1981; 6: 1745-1752Crossref PubMed Scopus (21) Google Scholar 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)16Lovas G Szilagyi N Majtenyi K Palkovits M Komoly S Axonal changes in chronic demyelinated cervical spinal cord plaques.Brain. 2000; 123: 308-317Crossref PubMed Scopus (301) Google Scholar, 17Matthews 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: 327-336Crossref PubMed Scopus (166) Google Scholar and normal appearing gray matter in MS.18Sharma R Narayana PA Wolinsky JS Grey matter abnormalities in multiple sclerosis: proton magnetic resonance spectroscopic imaging.Mult Scler. 2001; 7: 221-226PubMed Google Scholar In addition, Dandekar and colleagues19Dandekar 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: 6115-6120Crossref PubMed Scopus (65) Google Scholar 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.20Tsunoda I Fujinami RS Two models for multiple sclerosis: experimental allergic encephalomyelitis and Theiler's murine encephalomyelitis virus.J Neuropathol Exp Neurol. 1996; 55: 673-686Crossref PubMed Scopus (161) Google Scholar, 21Tsunoda I Fujinami RS Theiler's murine encephalomyelitis virus.in: Ahmed R Chen I Persistent Viral Infections. John Wiley & Sons Ltd., Chichester1999: 517-536Google Scholar The latter are widely used as an animal model for MS.22Vanderlugt CL Miller SD Epitope spreading in immune-mediated diseases: implications for immunotherapy.Nat Rev Immunol. 2002; 2: 85-95Crossref PubMed Scopus (651) Google Scholar In TMEV infection, axonal degeneration23Dal Canto MC Lipton HL Primary demyelination in Theiler's virus infection. An ultrastructural study.Lab Invest. 1975; 33: 626-637PubMed Google Scholar and loss24Ure DR McGavern DB Sathornsumetee S Rodriguez M The contribution of axonal injury to neurologic dysfunction in Theiler's virus-induced inflammatory demyelinating disease.in: Hommes OR Clanet M Wekerle H Genes and Viruses in Multiple Sclerosis. Elsevier Science BV, Amsterdam2001: 79-88Google Scholar 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-Quiñones and colleagues25Rivera-Quiñones 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: 187-193Crossref PubMed Scopus (181) Google Scholar suggested a role for major histocompatibility complex class I-restricted CD8+ T cells in the development of axonal injury. 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. 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.26Kurtz CIB Sun XM Fujinami RS Protection of SJL/J mice from demyelinating disease mediated by Theiler's murine encephalomyelitis virus.Microb Pathog. 1995; 18: 11-27Crossref PubMed Scopus (21) Google Scholar Mice were inoculated under anesthesia intracerebrally with 20 μl 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. 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-μm-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 virus27Zurbriggen A Fujinami RS Theiler's virus infection in nude mice: viral RNA in vascular endothelial cells.J Virol. 1988; 62: 3589-3596PubMed Google Scholar and anti-CD3ε antibody (after trypsinization, 1: 30 dilution; DAKO Corporation, Carpinteria, CA).28Mason 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: 1194-1200Crossref PubMed Scopus (165) Google Scholar, 29Tsunoda 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: 402-418Crossref PubMed Scopus (78) Google Scholar 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.30Sternberger LA Sternberger NH Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.Proc Natl Acad Sci USA. 1983; 80: 6126-6130Crossref PubMed Scopus (1034) Google Scholar, 31Ulfig 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: 433-443Crossref PubMed Scopus (77) Google Scholar, 32Powell 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: 219-235Crossref PubMed Scopus (31) Google Scholar 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 communication with Dr. Sternberger) to nonphosphorylated NFP with autoclave pretreatment.33Shin 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: 693-702PubMed Google Scholar Microglia and macrophages were identified by biotinylated Ricinus Communis agglutinin (RCA) I (Vector Laboratories Inc., Burlingame, CA).34Suzuki 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: 221-227Crossref PubMed Scopus (88) Google Scholar, 35Tsunoda 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: 595-602Crossref PubMed Scopus (47) Google Scholar 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). 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. 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.20Tsunoda I Fujinami RS Two models for multiple sclerosis: experimental allergic encephalomyelitis and Theiler's murine encephalomyelitis virus.J Neuropathol Exp Neurol. 1996; 55: 673-686Crossref PubMed Scopus (161) Google Scholar, 35Tsunoda 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: 595-602Crossref PubMed Scopus (47) Google Scholar 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 neurons 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.36Strich 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: 443-448Abstract Scopus (345) Google Scholar, 37Oppenheimer DR Microscopic lesions in the brain following head injury.J Neurol Neurosurg Psychiatry. 1968; 31: 299-306Crossref PubMed Scopus (382) Google Scholar, 38Duchen LW General pathology of neurons and neuroglia.in: Adams JH Corsellis JAN Duchen LW Greenfield's Neuropathology. ed 4. Edward Arnold Publishers, Ltd., London1984: 1-52Google Scholar 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.25Rivera-Quiñones 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: 187-193Crossref PubMed Scopus (181) Google Scholar, 39Tsunoda 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: 481-493Crossref PubMed Scopus (93) Google Scholar 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.30Sternberger LA Sternberger NH Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.Proc Natl Acad Sci USA. 1983; 80: 6126-6130Crossref PubMed Scopus (1034) Google Scholar, 31Ulfig 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: 433-443Crossref PubMed Scopus (77) Google Scholar 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 balls2Greenfield JG King LS Observations on the histopathology of the cerebral lesions in disseminated sclerosis.Brain. 1936; 59: 445-458Crossref Scopus (64) Google Scholar 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 double-immunofluorescence 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.4Suzuki K Andrews JM Waltz JM Terry RD Ultrastructural studies of multiple sclerosis.Lab Invest. 1969; 20: 444-454PubMed Google Scholar 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.40Bjartmar 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: 1248-1252Crossref PubMed Scopus (240) Google Scholar 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.20Tsunoda I Fujinami RS Two models for multiple sclerosis: experimental allergic encephalomyelitis and Theiler's murine encephalomyelitis virus.J Neuropathol Exp Neurol. 1996; 55: 673-686Crossref PubMed Scopus (161) Google Scholar, 21Tsunoda I Fujinami RS Theiler's murine encephalomyelitis virus.in: Ahmed R Chen I Persistent Viral Infections. John Wiley & Sons Ltd., Chichester1999: 517-536Google Scholar 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). 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 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 Lipton41Dal Canto MC Lipton HL Ultrastructural immunohistochemical localization of virus in acute and chronic demyelinating Theiler's virus infection.Am J Pathol. 1982; 106: 20-29PubMed Google Scholar detected TMEV antigens in axons in the spinal cord gray matter 25 to 80 days after DA virus infection by immunoelectron microscopy. As seen in Figure 1, Figure 2, axonal injury was detectable in NAWM and no correlation was observed between axonal 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.34Suzuki 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: 221-227Crossref PubMed Scopus (88) Google Scholar, 35Tsunoda 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: 595-602Crossref PubMed Scopus (47) Google Scholar, 42Tsunoda I Kurtz CIB Fujinami RS Apoptosis in acute and chronic central nervous system disease induced by Theiler's murine encephalomyelitis virus.Virology. 1997; 228: 388-393Crossref PubMed Scopus (121) Google Scholar 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 r
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