低浓度氯喹保护成年小鼠视网膜神经节细胞抵抗N-甲基-D-天冬氨酸的兴奋性毒性作用

目的:观察不同浓度氯喹对N-甲基-D-天冬氨酸(NMDA)损伤模型小鼠RGC的保护作用及可能调控机制。方法:健康雄性C57/BL6小鼠54只,随机分为氯喹低浓度组、氯喹高浓度组、PBS对照组,每组均为18只。氯喹低浓度组、氯喹高浓度组小鼠分别按体重10、100 mg/kg剂量腹腔注射氯喹;PBS组小鼠腹腔注射等体积PBS。腹腔注射1次/d,连续2d后,氯喹低浓度组、氯喹高浓度组小鼠左眼玻璃体腔注射5 nmol/L NMDA,对侧眼注射等体积PBS。建模后7d,视网膜铺片RGC染色,计数RGC存活数目;建模后9、10d,分别行视动反应和ERG检查;建模后11 d,行视网膜免疫荧光染色检测各组小鼠视网膜胶质纤维酸性蛋白(GFAP)荧光表达和实时荧光定量PCR(RT-PCR)检测各组小鼠视网膜GFAP、TNF-α、IL-6 mRNA表达。各组间RGC密度、视敏度、光负性反应(PhNR)波振幅比较采用单因素方差分析。结果:建模后7d,与PBS对照组小鼠RGC密度比较,氯喹低浓度组显著升高,差异有统计学意义(F=54.41,P<0.01);氯喹高浓度组降低,但差异无统计学意义(F=1.18,P>0.05)。氯喹低浓度组小鼠视敏度、PhNR波振幅均高于PBS对照组,差异有统计学意义(F=9.10、17.60,P<0.05、<0.01)。氯喹低浓度组小鼠视网膜GFAP绿色荧光表达明显少于PBS对照组、氯喹高浓度组,差异有统计学意义(F=23.66,P<0.05)。低浓度氯喹组小鼠视网膜GFAP(F=110.20)、IL-6(F=167.60)、TNF-α(F=17.78)mRNA表达较高浓度氯喹组、PBS对照组显著下降,差异有统计学意义(P<0.010、<0.001、<0.010)。结论:低浓度氯喹对NMDA损伤所致RGC丢失有保护作用,其作用机制可能与抑制胶质细胞活化与炎症反应有关;高浓度氯喹会加重RGC凋亡。

Low-dose chloroquine mediated neuroprotection against n-methyl-d-aspartate induced excitotoxicity in adult mice

Objective To investigate the protective effects of different concentrations of chloroquine on RGC in n-methyl-d-aspartate(NMDA)injured mice and its possible mechanisms.Methods Fifty-four healthy male C57/BL6 mice were randomly divided into three groups,18 in each group.The mice in low-dose chloroquine group were intraperitoneally injected with chloroquine solution at a dose of 10 mg/kg daily.Mice in high-dose chloroquine group were intraperitoneally injected with chloroquine solution at a dose of 100 mg/kg,and the mice in control group were intraperitoneally injected with the same volume of PBS.NMDA intravitreal injection was performed 2 days after intraperitoneal injection,5 nmoles NMDA was injected into the left eye,and the same volume of PBS was injected into the right eye as a control.The RGC staining of retinal plaques were performed 7 days after NMDA injection,and the number of alive RGC was calculated.The visual acuity and electroretinogram were used to evaluate the electrophysiological functions of RGC at 9 and 10 days after modeling.Real-time quantitative PCR and retinal frozen sections and glial fibrillary acidic protein(GFAP)immunofluorescence staining were performed 11 days after NMDA injection to evaluate the glial activation of the retina.The density,visual acuity,and the amplitude of PhNR-wave of RGC between groups were compared by one-way analysis of variance.Results At 7 days after NMDA injection,the density of RGC in retinal patch of low-dose chloroquine group was significantly higher than that of intraperitoneal injection of PBS control group(F=54.41,P<0.01).The density of RGC in retinal patch of high-dose chloroquine group was lower than that of control group(F=1.18,P>0.05).The visual acuity was higher than control group,and the difference was statistically significant(F=9.10,P<0.05).The amplitude of PhNR-wave was significantly higher in low-dose chloroquine group than that of the control group(F=17.60,P<0.01).The mRNA level of inflammatory factor and GFAP positive signal was also significantly lower than that of the control group(F=23.66,P<0.05).The amplitude of PhNR-wave,the expression of GFAP(F=110.20,P<0.01)and the mRNA level of inflammatory factors(F=167.60,17.78;P<0.01)in the high-dose chloroquine group were higher than the other two groups,and the differences were statistically significant.Conclusions In NMDA injury retinal model,low-dose chloroquine significantly increased the survival and physiological function of RGC,and the mechanism may be related to the inhibition of glial activation and inflammatory response.High-dose of chloroquine would aggravate the apoptosis of RGC.