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A. M. Fontainhas, R. J. Clarke, E. Townes-Anderson; Axon Retraction in Pig Retina After Detachment Depends on RhoA Activation. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1537.
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Retinal detachment leads to the disruption of synaptic connections in the outer plexiform layer (OPL) due to retraction of rod axon terminals into the outer nuclear layer (ONL). This remodeling occurs over a 24hr period in human, cat and pig retina. We hypothesize that terminal retraction is due, in part, to activation of a small GTPase, RhoA.
To examine for changes in RhoA activation, RhoA activation assays were performed on 8mm buttons of trephined pig retina (n=3 animals) that was either undetached, or detached for 30 seconds, 2 and 24hrs. Resulting bands on Western blots were analyzed with NIH image for intensity of immunostaining for RhoA. Next, morphological analysis was performed to determine if active RhoA affects axon terminal retraction. Retinas (n=3 animals) were treated with a ROCK inhibitor, Y27632. ROCK is a downstream effecter of active RhoA. Y27632 (100µM) was applied in MEM fortified medium to detached pig retinas incubated at 37°C for 24hrs. Untreated, detached retina was used to measure retraction at 24hrs. At the end of the incubation, retinas were fixed with 4% paraformaldehyde, embedded in gelatin, sectioned and stained with antibody for synaptic vesicle protein 2 (SV2) and propidium iodide for nuclei. One µm retinal sections were obtained with confocal microscopy. SV2 staining was measured in the ONL with MetaMorph software.
The total amount of retinal RhoA, determined by Western blot analysis, remained constant before and after detachment. However, the proportion of active RhoA increased immediately after detachment to almost double the level of active RhoA in undetached retina. Active RhoA dropped by 2 and 24hrs in comparison to immediately detached and undetached retina. Thus, retinal detachment leads to a quick, transient rise in active RhoA. In retina, SV2 staining is sequestered in the OPL. After detachment, in contrast, SV2 staining is found in the ONL, an indication of axon terminal retraction. In detached retinas, SV2 staining was present in 13% of the ONL area after 24hrs. Y27632 significantly reduced the amount of SV2 staining found in ONL after detachment to only 3.3%. Thus, blocking the RhoA pathway decreased the amount of morphological retraction.
Based on these findings, we conclude that retinal detachment leads to RhoA activation and that active RhoA contributes to retraction of axon terminals into the ONL. Blocking RhoA activity may preserve normal structure at the photoreceptor synapse after injury and help preserve vision after detachment.
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