Bis(7)-tacrine protects retinal ganglion cells against excitotoxicity via NMDA receptor inhibition

Abstract

Aim: To investigate whether bis(7)-tacrine, a multifunctional drug, inhibits N-methyl-D-aspartate (NMDA) -activated current in retinal ganglion cells(RGC) and provides neuroprotection against retinal cell damage.

Methods: Purified RGC 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. In animal experiments, RGCs were damaged after intravitreal injection of NMDA (5µL, 40nmol) in adult rats. Bis(7)-tacrine(0.05, 0.1, 0.2mg/kg) or memantine(20mg/kg) was intraperitoneal administered to the rats fifteen minutes before intravitreally injection of NMDA. RGC damage was analyzed by histologic techniques, TUNEL and retrograde labeling techniques.

Results: Whole-cell patch-clamp recordings demonstrated that NMDA (30µmol/L) resulted in approximately -50 pA inward currents that were blocked by bis(7)-tacrine(1µmol/L). 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 hours after injection.

Conclusion: Bis(7)-tacrine possesses remarkable neuroprotective activities against retinal excitotoxicity through inhibition of NMDA receptors.

Keywords: N-methyl-D-aspartate receptors; bis(7)-tacrine; excitotoxicity; neuroprotection.

Figure 1. Inhibition of NMDA-activated current by bis(7)-tacrine in RGC in vitro

Bis(7)-tacrine: 30µmol/L; NMDA: 1µmol/L

Figure 2. Protective effects of bis(7)-tacrine on retinal damage induced by intravitreal injection of NMDA(40nmol)

^aP<0.05, ^bP<0.01 vs NMDA; Scale bar, 100µm

Figure 3. Bis(7)-tacrine reduces NMDA-induced apoptosis of ganglion cell layer(GCL) cells in vivo

A: Representative photographs of retina in the vehicle group, the NMDA group and the NMDA group with peritoneal injection of bis(7)-tacrine(0.2mg/kg) or memantine(20mg/kg); B: The number of TUNEL-positive cells in GCL was counted. NMDA treatment plus NMDA receptor blocker was tested vs NMDA treatment alone ^bP<0.01 vs NMDA; Scale bar, 100µm

Figure 4. Protective effects of bis(7)-tacrine on retinal ganglion cells(RGC) following intravitreal injection of NMDA (40nmol)

^aP<0.05, ^bP<0.01 vs NMDA; Scale bar, 100µm