Neuroprotective Effects Of Bis(7)-tacrine Against Excitoxins-induced Retinal Ganglion Cells Damage In Rats And Its Mechanism

Purpose:Glutamate-mediated excitotoxicity, primarily through N-methyl-D-aspartate (NMDA) receptors, may be an important cause of retinal ganglion cells (RGCs) death in glaucoma and several other retinal diseases. Bis(7)-tacrine is a noncompetitive NMDA receptors antagonist that can prevent glutamate-induced hippocampal neurons damage. In this study, we investigate whether bis(7)-tacrine inhibits NMDA-activated current in retinal ganglion cells(RGCs) and test the effects of bis(7)-tacrine against excitoxins-induced rat RGCs damage in vitro and in vivo.Methods:Purified RGCs cultures were obtained from retinas of 1-3 days old Sprague-Dawley(SD) rats, following a two-step immunopanning procedure. After 7 days of cultivation, the inhibition of NMDA-activated current by bis(7)-tacrine was measured by using patch-clamp recording techniques. Cultured RGCs were exposed to glutamate for 6-24 h in the presence or absence of bis(7)-tacrine(0.01-1μM) or memantine (1-10μM). Treated cells were assayed for cell viability using the 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, for apoptosis by Annexin V-FITC/PI assays. In animal experiments, RGCs were damaged after intravitreal injection of glutamate (5μl,20nmol) or NMDA (5μl,40nmol) in adult rats. Bis(7)-tacrine(0.05,0.1,0.2mg/kg) or memantine(20mg/kg) were intraperitoneal administered to the rats fifteen minutes before intravitreally injection of glutamate or NMD A. RGCs damage was analyzed by histologic techniques, TUNEL and retrograde labeling techniques.Results:Whole-cell patch-clamp recordings demonstrated that NMDA (30μM) resulted in approximately-50 pA inward currents that were blocked by bis(7)-tacrine(1μM). The MTT assay showed that glutamate induced a concentration-and time-dependent toxicity. Bis(7)-tacrine and memantine prevented glutamate-induced cell death in a concentration-dependent manner with IC50 values of 0.028μM and 0.834μM, respectively. The anti-apoptosis effects of bis(7)-tacrine were confirmed by annexin V-FITC/PI staining. In vivo, TUNEL analysis and retrograde labeling analysis found that pretreatment with bis(7)-tacrine(0.2mg/kg) induced a significant neuroprotective effect against glutamate-induced RGCs damage. Histological examination and retrograde labeling analysis revealed that bis(7)-tacrine induced a significant neuroprotective effect against NMDA-induced cell damage 7 days after NMDA injection. TUNEL staining showed that pretreatment with bis(7)-tacrine was effective in ameliorating NMDA-induced apoptotic cell loss in the retinal ganglion cell layer 18 h after injection.Conclusions:Our results showed that bis(7)-tacrine had neuroprotective effects against excitoxins-induced RGCs damage in vitro and in vivo through inhibition of NMDA receptors. These findings make bis(7)-tacrine potentially useful for treating a variety of ischemic or traumatic retinopathies inclusive of glaucoma.