The Preliminary Study Of Receptor Interacting Protein3Expression On Retinal Ganglion Cells Following Acute High Intraocular Pressure

OBJECTIVE:To study the distribution of Receptor interacting protein3(RIP3) in adult rat retina, investigate its temporal and spatial variation following acute high intraocular pressure (HIOP). Preliminarily, we intend to explore the possible injury mechanism of retinal ganglion cells.METHOD:(1) Six adult Sprague-Dawley rats were selected randomly to study the distribution of RIP3in retina, three rats’retinas were homogenized for western blot to exam the antibody specificity of RIP3which we have already used in this study. Three rats’retinas were fixed and sliced for antibody absorption test. Immunofluorescence double labeling of RIP3and several retina neuronal markers had been investigated including Neuron-specific nuclear antigen (NeuN, retinal ganglion cell marker), Calbindin (CB, horizontal cell marker), Protein kinase C alpha (PKC-a, biopolar cell marker), Parvalbumin (PV, amacrine cell marker), Rhodopsin (Rho, phtoreceptor marker), Synaptophysin (Syn, pre-synapse marker). The glial markers (Glutamine synthetase; Glial fibrillary acidic protein; Integrin alpha M) had also been detected with RIP3double labeling.(2) Eighteen Sprague-Dawley rats were divided randomly into6hr groups,12hr groups,24hr groups, six rats each. Those rats were subjected to HIOP in one side of the eyes and the other side was set to normal control. The eyes with high intraocular pressure were punctured a needle into anterior chamber to elevate pressure gradually and maintained110mm Hg for60minutes. After survival for6hrs,12hrs or24hrs, three rats’retina tissues of each group were homogenized for western blot to evaluate RIP3protein level, the remaining were fixed in paraformaldehyde for RIP3immunohistochemistry.RESULTS:(1) The immunohistochemistry shows RIP3express in nerve fiber layer (NFL), ganglion cell layer (GCL) and inner nuclear layer (INL) in normal rat retina;(2) The double labeling of RIP3and NeuN, Parvalbumin, Calbindin suggests a preferential localization of RIP3in ganglion cells, partial amacrine cells and horizontal cells; The double labeling of RIP3and GS, GFAP, CD11b indicates a localization of RIP3in Muller cells, glial cells in the NFL, GCL and INL; There is no distinctly double labeling between RIP3and PKC-α Rhodopsin, which suggests RIP3does not express in bipolar cells and photoreceptor cells.(3) There are no changes of RIP3distribution between normal and HIOP retinas by immunofluorescence. RIP3positive products are increased in GCL at6hr,12hr, but decrease in24hr. Western blot densitometric analysis of RIP3bands demonstrate that RIP3protein levels are increased in6hr,12hr, but decrease in24hr. And there is a statistically significant increase in the HIOP-treated retinas in12hrs.CONCLUSION:RIP3is expressed in NFL, GCL and INL, furthermore, it is distributed in ganglion cells, partial horizontal cells, amacrine cells, Muller cells, glial cells. RIP3expression is increased following acute HIOP in early stage.