Optic neuritis is a major cause of untreatable and recurrent visual loss. It is often the first clinical sign of multiple sclerosis (MS). During the past grant period, our magnetic resonance imaging (MRI) studies showed blood-brain (BBB) disruption and demyelination of the optic nerve in patients with optic neuritis or MS and in the animal model of experimental allergic encephalomyelitis (EAE). The PI showed that detoxification of reactive oxygen species (ROS) has a protective effect in EAE; it suppresses disruption of the BBB and demyelination of the optic nerve. These results support the hypothese that ROS play a major role in the vascular alterations of immune-mediated demyelination, and that poor cellular defenses against ROS are a likely component to the vulnerability of the optic nerve in both EAE and MS. He now proposes to determine whether the optic nerve can be protected in EAE from free radical mediators of the inflammatory response by genetic amplification of specific cellular defenses against ROS.
The specific aims are to test the following hypotheses: Hypothesis 1: Protective genes can be transferred to axons, glia, and endothelia of the optic nerve via viral mediators. The ability of promoters to drive cellular expression of anti-ROS genes will be determined. The cellular localization and duration of expression of transfected proteins and mRNAs will be analyzed using immunohistochemistry and in situ RT PCR. The safety of viral mediated transfer of genes in ocular tissues will be assessed by MRI, fundus photography and histopathology. Hypothesis 2. BBB disruption and demyelination of the optic nerve can be suppressed in EAE animals by amplification of cellular defenses against ROS through viral mediated transfer of protective genes. Transfected guinea pigs will be sensitized for EAE, then evaluated by serial MRI and histopathologic analysis of the optic nerve. The mechanisms of modulation of free radical mediators of the the inflammatory response by the gene transfer will be assessed by measurements of in vivo superoxide, nitric oxide, peroxynitrite, and hydrogen peroxide. Hypothesis 3. BBB disruption and demyelination can be reduced in EAE by transgenic modulation of specific defenses against ROS. Mice transgenic for SOD and mice with nitric oxide synthase knockout will be sensitized for EAE. BBB disruption and demyelination will be assessed by serial MRI and histopathologic analysis.
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