小鼠视神经损伤对视网膜神经节细胞存活率的影响

Survival rate change of retinal ganglion cells following optic nerve injuries in mice

目的探讨小鼠视神经钳夹损伤(ONC)后视网膜神经节细胞(RGCs)存活率变化。方法选取97只6~8周龄雄性C57BL/6J小鼠,按照随机数字表法分为正常组(5只)、假手术组(5只)、ONC组(87只)。正常组双眼不进行任何操作;假手术组左眼不进行任何操作,右眼行假手术操作;ONC组左眼行ONC,右眼行假手术对照。正常组分别计算左右眼RGCs密度,比较其差异;假手术组计算假手术眼RGCs密度,比较其与正常组平均RGCs密度的差异;ONC组中,以左眼(ONC眼)与右眼(假手术眼)RGCs密度的比值计算RGCs存活率,然后比较不同钳夹持续时间(5,10,20,30 s)的RGCs存活率差异(取材时间统一为钳夹后7 d),以及不同取材时间(3,4,5,7,14,30,60,90,180 d)的RGCs存活率差异(钳夹持续时间统一为20 s)。结果正常组左右眼RGCs密度分别为(5167.3±55.6)个/mm^(2)和(5199.6±44.8)个/mm^(2),差异无统计学意义(P>0.05)。正常组与假手术组RGCs密度分别为(5183.5±33.4)个/mm^(2)和(5151.5±87.6)个/mm^(2),差异无统计学意义(P>0.05)。ONC组不同钳夹时间(5,10,20,30 s)的RGCs存活率分别为(37.6±1.1)%、(34.0±0.9)%、(33.6±1.6)%、(30.3±0.6)%(P<0.01)。钳夹5 s与30 s相比,ONC组小鼠RGCs存活率差异有统计学意义(P<0.01);其余不同钳夹时间相比,ONC组小鼠RGCs存活率差异均无统计学意义(P>0.05)。ONC组不同取材时间(3,4,5,7,14,30,60,90,180 d)的RGCs存活率分别为(85.4±2.0)%、(67.6±3.1)%、(43.0±1.0)%、(33.6±1.6)%、(22.7±2.0)%、(12.8±0.6)%、(10.4±0.8)%、(8.6±0.5)%、(6.7±0.2)%(P<0.01),尤其3~5 d,RGCs存活率大幅下降,30 d后RGCs存活率呈平稳下降趋势。结论在ONC小鼠模型中,不同钳夹持续时间对RGCs造成的损伤程度不同,且ONC后RGCs死亡呈进行性发展,提示在原发性损伤(钳夹)后,小鼠的RGCs经历了继发性损伤。因此,有效控制RGCs的继发性损伤,可能成为治疗视神经损伤性疾病的关键。

Objective To investigate the survival rate change of retinal ganglion cells(RGCs)in a mouse of optic nerve crush(ONC).Methods Ninety-seven male C57BL/6J mice(6 to 8 weeks)were selected and divided into normal group(n=5),sham-operation group(n=5)and ONC group(n=5)according to the random number table.In normal group,both eyes of the mice did not receive any intervention.In sham-operation group,the right eye of the mice received sham operation,while the left eye reveived no intervention.In ONC group,the left eye received ONC,and the right eye received sham operation.In normal group,the density of RGCs in both eyes was quantified and compared.In sham-operatioin group,the density of RGCs in the sham operation eye was calculated and then compared to the average density of RGCs in normal group.In ONC group,the survival rate of RGCs was set as the ratio between the left eye(ONC eye)and the right eye(sham-operation eye).The survival rate of RGCs in ONC group was compared after crush injury for (5,10,20,30 seconds)(the sacrifice time was set at day 7),and was compared after sampling on days3,4,5,7,14,30,60,90,180(the duration of crush injury was set as 20 seconds).Results In normal group,the density of RGCs in the right eye was(5,167.3±55.6)cell/mm^(2),with no statistical difference from that in the left eye[(5,199.6±44.8)cell/mm^(2)](P>0.05).The density of RGCs in normal group and sham-operation group was(5,183.5±33.4)cell/mm^(2) and(5,151.5±87.6)cell/mm^(2),showing no statistical difference(P>0.05).The survival rate of RGCs in ONC group after crush injury for 5,10,20,30 seconds was(37.6±1.1)%,(34.0±0.9)%,(33.6±1.6)%and(30.3±0.6)%(P<0.01).In comparison,there was statistical difference in the survival rate of RGCs between crush injury for 5 seconds and for 30 seconds(P<0.01),but not among other duration of crush injury(P>0.05).The survival rate of RGCs in ONC group after sampling on days 3,4,5,7,14,30,60,90,180 was(85.4±2.0)%,(67.6±3.1)%,(43.0±1.0)%,(33.6±1.6)%,(22.7±2.0)%,(12.8±0.6)%,(10.4±0.8)%,(8.6±0.5)%and(6.7±0.2)%(P<0.01),showing the most obvious drop from day 3 to day 5.Additionally,the curve became flattened after 30 days.Conclusions In a mouse model of ONC,varying durations of crushing will lead to different damage to RGCs in a progressive mode,indicating that following the primary injury(ONC),the RGCs suffer secondary injury as well.Therefore,effectively controlling the secondary injury may be the key point of treating optic nerve injuries.