The Roles Of Reactive Macroglial Cells In Retinal Synaptic Plasticity Following Acute High Intraocular Pressure

Objective To detect the effects of acute high intraocular pressure (HlOP)-induced injury on retinal synapses and investigate the possible roles and the potential molecular mechanisms of glial cells in synaptic changes after HIOP injury.Methods The acute HIOP animal model was established by injecting normal saline into the anterior chamber of the left eyes.The intraocular pressure was gradually elevated to110mmHg and maintained for60minutes. All animals survived for2hours,6hours,12hours,1day,3days,7days and14days, respectively. The unoperated right eyes served as normal controls. The expression of presynaptic marker synaptophysin (SYN) and postsynaptic marker synaptic associated protein102(SAP102) was performed by immunohistochemistry. SYN protein expression was also determined by western blot assay. Retinal sections were labeled with antibodies to PKCa, rhodopsin, paravalbumin, calbindin which served as the specific markers for bipolar cells, rod photoreceptor cells, amacrines cells and horizontal cells, respectively. The double-labeling of SYN with these neuronal markers was detected by immunofluorescent histochemistry. As the marker of macroglial activation, GFAP expression was determined by immunohistochemistry and western blot and real-time PCR assay. The distribution and specific cellular localization of pro-synaptogenetic molecules thrombospondins (TSPs) and their receptor α2δ1was also determined in healthy adult retina and HIOP retinas by immunofluorescent histochemistry. After the HIOP animal model was established successfully, fluorocitrate was injected intravitreally to inhibit the metabolism of activated macroglial cells in the retina, while gabapentin was administrated by intraperitoneal injection to block the interaction of TSPs and their receptor α2δ1. The expression of SYN, GFAP and TSP1/2in the retina was detected after being treated with fluorocitrate or gabapentin. Results1. Western blot and immunofluorescence assay showed SYN expression had a distinct spatiotemporal tendency in the rat retina after acute HIOP. SYN expression was increased in the inner plexiform layer (IPL) within1day and its distribution was widened in the outer plexiform layer (OPL) after3days with a peak on the seventh day extending into the outer nuclear layer (ONL). The distribution pattern and fluorescent intensity of SAP102had no apparent change at various time points after acute HIOP injury and had no direct correlation with the increased SYN expression in the IPL and OPL2. Rod bipolar cells processes labeled with PKCa was initially increased after acute HIOP injury and colocalized with SYN in the IPL, while PKCa immunoreactivity did not change in the OPL when SYN expression was widened in the OPL and ONL on day3and day7of HIOP induction. Rod photoreceptor cells labeled with rhodopsin colocalized perfectly with the increased immunoreactivity of SYN in the OPL and ONL after3days. Paravalbumin-positive amacrine cells somata and processes were initially slightly increased and partially colocalized with SYN in the IPL, while both the somata and processes were greatly decreased after3days of HIOP induction. Calbindin-positive horizontal cells did not change at various time points after injury and did not correlate directly with the increased expression of SYN in the OPL3. GFAP expression was greatly increased after3days in HIOP retinas with a peak on the seventh day when GFAP-positive glial processes reached the ONL and outer limited membrane and colocalized with GS, the marker of muller cells. The enhanced GFAP expression in the retina was apparently inhibited after being treated with fluorocitrate. Even if on the seventh day after injury, increased glial processes were not detectable. With the inhibition of glial activation, SYN expression in the rat retina was obviously decreased in the IPL within1day and its widened distribution disappeared in the OPL after3days.4. TSP1and TSP2had a distinct differential distribution in normal adult rat retina. TSP1was expressed by retinal neurons while TSP2was expressed by retinal glial cells. Retinal ganglion cells, bipolar cells and horizontal cells expressed a high level of α2δ1. After acute HIOP injury, the distribution of TSP1in the retina did not change only TSP1-positive cells in the GCL was decreased with the death of ganglion cells after3days but the ratio of TSP1-positive cells to neurons in the GCL had no statistical difference compared to normal control. After being treated with fluorocitrate, TSP1expression was similar to that of HIOP retinas. However, TSP2was greatly increased after3days of HIOP induction and perfectly colocalized with glial cell markers GFAP and GS. After being treated with fluorocitrate, TSP2expression was apparently decreased with the inhibition of glial activation. Following acute HIOP injury, a281immunoreactive somata in the GCL were decreased but α2δ1-positive processes and the fluorescent intensity were increased in the outer retina.5. After being treated with gabapentin, SYN expression was decreased in the IPL within1day and its widened distribution also disappeared in the OPL after3days. After being treated with gabapentin, the expression of TSP1, TSP2and GFAP at the various time points was similar to that of acute HIOP retinas.Conclusion1. Synapses in the retina might undergo plastic changes internally to externally after acute HIOP injury that presynaptic marker SYN had a distinct spatiotemporal tendency, while postsynaptic elements might not company in the process. Bipolar cells and rod photoreceptor cells in the direct pathway and partial amacrine cells in the lateral pathway might participate in the presynaptic spatiotemporal alterations.2. Macroglial cells were greatly activated in the rat retina following acute HIOP and reactive macroglial cells in the retina might be responsible for the synaptic plasticity induced by acute HIOP injury by secreting TSP2binding to its neuronal receptor α2δ1.