Recent pathological studies have re-emphasized that axonal injury is present in patients with multiple sclerosis, the most common demyelinating disease of the CNS in humans. However, the temporal profile of demyelination and axonal loss in multiple sclerosis patients and their independent contributions to clinical and electrophysiological abnormalities are not completely understood. In this study, we used the Theiler's murine encephalomyelitis virus model of progressive CNS inflammatory demyelination to demonstrate that demyelination in the spinal cord is followed by a loss of medium to large myelinated fibres. By measuring spinal cord areas, motor-evoked potentials, and motor coordination and balance, we determined that axonal loss following demyelination was associated with electrophysiological abnormalities and correlated strongly with reduced motor coordination and spinal cord atrophy. These findings demonstrate that axonal loss can follow primary, immune-mediated demyelination in the CNS and that the severity of axonal loss correlates almost perfectly with the degree of spinal cord atrophy and neurological deficits.
In this study we demonstrate perforin-mediated cytotoxic effector function is necessary for viral clearance and may directly contribute to the development of neurologic deficits after demyelination in the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. We previously demonstrated major histocompatability complex (MHC) class I-deficient (2m-deficient) mice with an otherwise resistant genotype develop severe demyelination with minimal neurologic disease when chronically infected with TMEV. These studies implicate CD8ϩ T cells as the pathogenic cell in the induction of neurologic disease after demyelination. To determine which effector mechanisms of CD8 ϩ T cells, granule exocytosis or Fas ligand expression, play a role in the development of demyelination and clinical disease, we infected perforin-deficient, lpr (Fas mutation), and gld (Fas ligand mutation) mice with TMEV. Perforin-deficient mice showed viral persistence in the CNS, chronic brain pathology, and demyelination in the spinal cord white matter. Perforin-deficient mice demonstrated severely impaired MHC class I-restricted cytotoxicity against viral epitopes, but normal MHC class II-restricted delayed-type hypersensitivity responses to virus antigen. Despite demyelination, virus-infected perforin-deficient mice showed only minimal neurologic deficits as indicated by clinical disease score, activity monitoring, and footprint analysis. Perforin-and MHC class II-deficient mice (with functional CD8 ϩ T cells and perforin molecules and an H-2 b haplotype) had comparable demyelination and genotype, however, only the latter showed severe clinical disease. Gld and lpr mice demonstrated normal TMEVspecific cytotoxicity and maintained resistance to TMEVinduced demyelinating disease. These studies implicate perforin release by CD8ϩ T cells as a potential mechanism by which neurologic deficits are induced after demyelination.
Following intracerebral infection with Theiler’s murine encephalomyelitis virus (TMEV), susceptible strains of mice (SJL and PLJ) develop virus persistence and demyelination similar to that found in human multiple sclerosis. Resistant strains of mice (C57BL/6) clear virus and do not develop demyelination. To resolve the controversy about the role of CD4+ and CD8+ T cells in the development of demyelination and neurologic deficits in diseases of the central nervous system, we analyzed TMEV infection in CD4- and CD8-deficient B6, PLJ, and SJL mice. Genetic deletion of either CD4 or CD8 from resistant B6 mice resulted in viral persistence and demyelination during the chronic stage of disease. Viral persistence and demyelination were detected in all strains of susceptible background. Although genetic deletion of CD8 had no effect on the extent of demyelination in susceptible strains, deletion of CD4 dramatically increased the degree of demyelination observed. Whereas strains with deletions of CD4 showed severe neurologic deficits, mice with deletions of CD8 showed minimal or no deficits despite demyelination. In all strains, deletion of CD4 but not CD8 resulted in a decreased delayed-type hypersensitivity response to viral antigen. We conclude that each T-cell subset makes a discrete and nonredundant contribution to protection from viral persistence and demyelination in resistant strains. In contrast, in susceptible strains, CD8+ T cells do not provide protection against chronic demyelinating disease. Furthermore, in persistent TMEV infection of the central nervous system, neurologic deficits appear to result either from the absence of a protective class II-restricted immune response or from the presence of a pathogenic class I-restricted response.
Spinal cord pathology, such as demyelination and axonal loss, is a common feature in multiple models of central nervous system (CNS) injury and disease. Development of methods to quantify spinal cord pathology objectively would aid studies designed to establish mechanisms of damage, correlate pathology with neurologic function, and assess therapeutic interventions. In this study, we describe sensitive methods to objectively quantify spinal cord demyelination, remyelination, atrophy, and axonal loss following the initiation of a progressive inflammatory demyelinating disease with Theiler's murine encephalomyelitis virus (TMEV). Spinal cord demyelination, remyelination, and atrophy were quantified from representative 1-microm-thick cross sections embedded in Araldite plastic using interactive image analysis. In addition, this study demonstrates novel, automated methodology to quantify axonal loss from areas of normal-appearing white matter, as a measure of secondary axonal injury following demyelination. These morphologic methods, which are applicable to various models of CNS injury, provide an innovative way to assess the benefits of therapeutic agents, to determine mechanisms of spinal cord damage, or to establish a correlation with sensitive measures of neurologic function. J. Neurosci Res 58:492-504.
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