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J.A. Kammer, L. Pan, B. Carlson, D.J. Calkins; Localization of CD95 and FasL in the Rodent Retina . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4203.
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Purpose: There is strong evidence implicating Fas System involvement in neuronal apoptotic death, in the pathogenesis of multiple neurodegenerative conditions and in ocular pathology like macular degeration. Before setting out on our goal of determining Fas involvement in glaucomatous retinal ganglion cell death, we sought to establish the basal state of Fas elements in the retina. The goal of this set of assays is to determine whether CD95 and FasLigand are present in normal rat and mouse retina. Methods: The retinal samples were obtained by dissecting the retina from the ret of the eye under a dissecting microscope. Primers for CD95 was then used in conjunction with the rat retina as RT–PCR was performed using standard reagents. The PCR products were then loaded onto the gel for electrophoresis. For Western Blot analysis, the rat retinal tissue was lysed and then subjected to electrophoresis. The proteins were then incubated with primary antibodies to FasL and CD95 to identify the proteins in question. For immunocytochemistry (ICC), adult normal mice were enucleated and the eyes were immersion fixed in 4% paraformaldehyde, cryoprotected, embedded and sectioned on a cryostat. Immunohistochemistry reactions were then performed using standard protocols with antibodies to CD95 Results: The PCR results clearly demonstrate a band that represents CD95, based on comparison to the Jurkat cell control. The Western Blot assay produced bands for both CD95 and FasL that were present, albeit with a lighter density than their corresponding controls. Finally, ICC cryostat sections of murine retina revelaed CD95 within the choroid, in the basal portion of the RPE cells and in the intravascular system within the inner plexiform complex, the outer plexiform complex and the retinal ganglion layer. Conclusions: Our results demonstrate that (1) CD95 mRNA is being actively expressed in the normal rat retina and (2) CD95 and FasL proteins are present within the rat retina. Immunocytochemistry localizes the CD95 protein within the retina and demonstrates that while the preferential location of the Fas Receptor is the RPE, there is basal expression within the vasculature of three retinal layers, including the retinal ganglion cell layer. These results establish a baseline against which we can compare future results as we work to determine the role that the Fas System plays in glaucomatous retinal ganglion cell death.
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