For studies using differentiated RGC cells, cells were incubated in media containing nM staurosporine for hours to allow cells to differentiate before further treatments.Microscope fields were randomly sampled and all photos were taken at the same exposure settings.RGC cells were seeded onto the glass coverslips in well tissue culture plates. After differentiation, cells were treated with various agents for hours.Cells were then stained with calceinAM in PBS for minutes at C in humidified CO.Coverslips were rinsed in PBS and fixed in paraformaldehyde for minutes before rinsing times with PBS.Four random microscope fields were captured for each coverslip for analysis.An observer masked to treatment groups manually counted the number of live cells in each microphotograph.Briefly, anesthetized rats were placed in a stereotactic apparatus and the cortex surface was exposed by drilling the parietal bone to facilitate dye injection.Rats with abnormal retinal vessels or retinal edema were not used further.Figures A and D show the <a href="https://www.ncbi.nlm.nih.gov/pubmed/9681662"></a>
different injection time points relative to transection and imaging for different experiments.Briefly, rats were anesthetized and their pupils dilated with phenylephrine and atropine.To ensure the plane of focus was in the ganglion cell layer, the CSLO polarization filter was used to focus onto the nerve fiber layer and then adjusted posteriorly.Retinal images were captured using a field of view lens with realtime averaging of at least images.The same four retinal quadrants immediately adjacent to the optic nerve were imaged over time in each rat.The reaction approaches maximal fluorescence at minutes or bovine erythrocyte SOD, we calculated the slope from the first minutes of each reaction and compared the rate of superoxide production in the presence or absence of scavenger.The slope with the positive control SOD was significantly less than the noscavenger control, and indicated scavenging of of available superoxide.Quantification of the nuclear fluorescence therefore is an indirect measure of superoxide levels. Clinically, patients with genetic vitamin B metabolism disorders have significantly greater levels of markers for oxidative stress in their urine, possibly due to the lack of superoxide scavenging by vitamin B.Together with the results from our study demonstrating that vitamin B scavenges superoxide and is neuroprotective in neuronal cells, these findings suggest that vitamin B contributes to the maintenance of cellular redox status, in addition to its several known functions as a cofactor in metabolic processes.Such findings provide a pathophysiological explanation for an uncommon but potentially devastating disease, the optic neuropathy caused by vitamin B deficiency.This deficiency has wellknown neurological manifestations, with peripheral neuropathy, subacute combined degeneration of the spinal cord, cognitive changes, and optic neuropathy being the most prominent.Therefore, a unifying explanation for the pathophysiology of vitamin B deficiency in the eye necessarily has to include why it is restricted to the RGC.Drugs that scavenge superoxide decrease RGC death, while induction of elevated superoxide by knockdown of superoxide dismutase isoforms induces neuronal or RGC death. These endogenous superoxide scavengers would have to be overwhelmed in vitamin B deficiency, which would occur in situations where there was high levels of mitochondrial oxygen consumption and consequent superoxide leak from the mitochondrial electron transport chain.