超声微泡造影剂联合美金胺增强视神经损伤大鼠视网膜神经节细胞保护作用

目的 探讨超声微泡造影剂联合美金胺对视神经损伤大鼠视网膜神经节细胞( RGC)的保护作用.方法 将Sprague-Dawley(SD)雄性成年大鼠40只随机分为正常对照组(A组),假手术组(B组),空白对照组(C组),玻璃体腔单独注射美金胺组(D组),玻璃体腔注射美金胺加超声微泡组(E组)5个组,每组8只大鼠,再将各组随机分为视神经损伤后1、2周2个亚组,各亚组4只大鼠.A组不做任何处理；B组只暴露视神经,不进行钳夹,玻璃体腔注射生理盐水,立即用超声波辐照大鼠眼球；C～E组建立视神经钳夹伤模型后,处理方式分别为C组玻璃体腔注射生理盐水,D组玻璃体腔注射美金胺,E组玻璃体腔注射超声微泡造影剂及美金胺,立即用超声波辐照大鼠眼球.视神经损伤1、2周时,各组行逆行荧光金标记RGC并计数；闪光视觉诱发电位(F-VEP)检测,记录P100波潜伏期及振幅；荧光电子显微镜下观察视网膜细胞形态学改变.结果 逆行荧光金标记RGC结果显示,各处理组视网膜定向铺片上均可见金黄色着染的RGC.A、B组RGC数间差异无统计学意义(q=0.018,0.011；P=0.986,0.873)；C～E组RGC数均较A组减少,差异具有统计学意义(F=85.944,P=0.012)；D组RGC数多于C组,差异具有统计学意义(q=1.721,1.924；P=0.043,0.037)；E组RGC数明显高于C、D组,差异具有统计学意义(q=1.128,1.482,P=0.027,0.008;q=1.453,1.855,P=0.031,0.010).F-VEP检测发现,A、B组P100波潜伏期及振幅间差异无统计学意义(q=0.008,0.019,P=0.981,0.946；q=0.072,0.052,P=0.737,0.851) ;C～E组P100波潜伏期较A组延长,振幅较A组降低,差异具有统计学意义(F=134.312,106.312；P=0.017,0.009).荧光电子显微镜下观察发现,A、B组大鼠视网膜各层结构完整,排列整齐,RGC排列紧密整齐,细胞核均匀深染,胞核大小一致.C～E组大鼠的视网膜不同程度水肿变厚,RGC有不同程度的排列紊乱,空泡化及细胞数目减少.结论 超声微泡造影剂联合美金胺能抑制视神经损伤后大鼠RGC的丢失,促进其视功能的恢复,对视神经损伤大鼠的RGC具有保护作用.

Ultrasonic microbubbles and memantine can protect rat retinal ganglion cells after optic nerve injury

Objective To observe the protection effects of ultrasonic microbubbles combined with memantine on rat retinal ganglion cells (RGCs) after optic nerve injury.Methods Forty Sprague-Dawley adult male rats were randomly divided into normal control group (group A),sham operation group (group B),blank control group (group C),memantine group (group D) and memantine and ultrasonic microbubbles group (group E),8 rats in each group.Then A - E groups were randomly divided into 1 week subgroup and 2 weeks subgroup after the optic nerve injury,4 rats in each subgroup.Group A had no interference treatment.The optic nerves in group B eyes were exposed but not clamped.Normal saline was injected into the vitreous,and those eyes were immediately radiated with ultrasound.The optic nerves in Group C- E were exposed and clamped to establish the optic nerve clamped models.Normal saline was injected into the vitreous of group C eyes; memantine was injected into the vitreous of group D eyes;ultrasonic microbubble and memantine was orderly injected into the vitreous of group E eyes and those eyes were immediately radiated with ultrasound.One week and 2 weeks after the optic nerve injury,RGC was labeled by retrograde fluorogold to count the RGC number; flash visual evoked potential (F-VEP) was used to record the incubation period and amplitude of P100 wave; fluorescence microscopy was used to observe the pathological morphology change of retinal cell.Results There were gold-labeled RGCs on the retina of group A - E.The difference of RGC count was not statistically significant between group A and B (q=0.018,0.011; P=0.986,0.873).Compared to group A,the RGC count in group C - E were decreased significantly (F=85.944,P=0.012).The RGC count in group D was significantly higher than that in group C (q=1.721,1.924; P=0.043,0.037).The RGC count in group E was significantly higher than that ingroup C and D (q=1.128,1.482,P=0.027,0.008; q=1.453,1.855,P=0.031,0.010).F-VEP showed that there was no statistically significant difference of incubation period and amplitude of P100 wave between group A and B (q=0.008,0.019,P=0.981,0.946; q=0.072,0.052,P=0.737,0.851).Compared to group A,the incubation period were lengthened and the amplitude were decreased in group C - E with statistically significant (F=134.312,106.312; P=0.017,0.009).Observed under the electron microscope,the retinal structure of group A,B eyes was normal,but there were varying degrees of edema and thickening,RGC loss in group C - E eyes.Conclusions Memantine and ultrasonic microbubble can inhibit the rat RGC loss after optic nerve injury,and improve the visual function.