Colivelin对视神经保护作用的初步研究

Preliminary study on protective effect of colivelin for optic nerve

目的初步探讨Colivelin对大鼠外伤性视神经损伤后的神经保护作用和机理,及其保护效果是否具有剂量依赖性。方法 40只健康2月龄Wistar大鼠,随机分为模型组、安慰剂组及Colivelin低剂量组(10-6 μmol·L-1 Colivelin)、Colivelin中剂量组(10-4μmol·L-1Colivelin)、Colivelin高剂量组(10-2 μmol·L-1 Colivelin),每组8只。40只大鼠均做单侧视神经损伤模型,模型组另一侧未做任何处理的8眼为空白对照组。应用无创血管夹建立大鼠视神经夹伤模型,造模后30 min Colivelin治疗组玻璃体内分别注射不同浓度Colivelin 5 μL,安慰剂组注射5μLPBS缓冲液,模型组和空白对照组不给予任何治疗。注药后7d,通过视网膜切片技术,进行HE染色观察视网膜神经节细胞(retinal ganglion cell,RGC)的形态及数目,TUNEL染色法检测RGC的凋亡以及免疫组织化学法检测视网膜中caspase-3的表达。结果 HE染色可见空白对照组视网膜各层细胞排列整齐密集,模型组、安慰剂组、Colivelin治疗组均可见部分RGC核固缩,但随着Colivelin注射浓度的增加,RGC发生核固缩的数量减少。RGC计数:空白对照组每400倍光镜视野下RGC数量为25.750±1.264,模型组为8.236±1.239,安慰剂组为8.514±1.222,Colivelin低剂量组为14.500±1.021,Colivelin中剂量组为16.250±1.319,Colivelin高剂量组为18.097±1.323,除模型组与安慰剂组之间差异无统计学意义(P>0.05)外,其他各组之间差异均有统计学意义(均为P<0.05)。TUNEL染色:模型组和安慰剂组注药后7 d TUNEL染色可见大量凋亡细胞,Colivelin治疗组凋亡细胞数量随Colivelin注射浓度的增加依次递减。RGC细胞凋亡率:模型组为(60.928±2.961)%,安慰剂组为(61.446±2.755)%,Colivelin低剂量组为(58.432±2.835)%,Colivelin中剂量组为(51.948±2.802)%,Colivelin高剂量组为(47.656±2.331)%。Caspase-3表达:模型组、安慰剂组及Colivelin低剂量组、中剂量组、高剂量组均可见caspase-3表达。平均光密度值显示,模型组和安慰剂组相比caspase-3表达无明显差异(分别为0.482±0.012和0.486±0.012),均高于Colivelin治疗组(均为P<0.05),并且Colivelin低剂量组、中剂量组、高剂量组组间差异亦均有统计学意义(分别为0.411±0.017、0.326±0.018、0.234±0.016;均为P<0.05)。结论 Colivelin能有效增加视神经损伤后RGC的数量,抑制其凋亡,减少caspase-3表达,且这一作用呈剂量依赖性。

Objective To investigate the protective effect and mechanism of colivelin for optic never after traumatic optic nerve injury in rat,and explore the relationship between the dosage and effect.Methods Forty healthy Wistar rats with 2 months old were randomly divided into model group,PBS control group,low-dose colivelin group（10-6 μmol·L-1）,medium-dose colivelin group（10-4 μmol·L-1）,high-dose colivelin group（10-2 μmol·L-1）,8 cases in each group.Forty rats were all unilateral optic never injury models,the opposite side of injury optic never in model group was treated nothing as normal control group.Animal models of optic never crush injury were created by microvascular clamp,5 μL colivelin and PBS were intravitreal injected in colivelin group and PBS control group after 30 minutes.The morphous and number of retinal ganglion cells（RGC）in all eyes were examined by HE staining at 7 days after operation through making retinal slices,RGC apoptosis and caspase-3 protein expression in retina were detected by TUNEL and immunohistochemistry.Results HE staining showed that every layer of retina was arranged orderly and densely in the normal control group,some RGC without nucleus could be seen in model group,PBS control group and colivelin group,but with the dose of colivelin increased,the RGC without nucleus were decreased gradually.Amount of RGC in normal control group,model group,PBS control group,low-dose colivelin group,medium-dose colivelin group and high-dose colivelin group were 25.750±1.264,8.236±1.239,8.514±1.222,14.500±1.021,16.250±1.319 and 18.097±1.323,respectively,except of model group,there were statistical differences compared with PBS control group（all P0.05）.TUNEL staining showed the apoptosis of RGC could be observed at 7 days after injury in the model group and PBS control group,the number of apoptosis of RGC was reduced at different degrees with the cure of colivelin and the effect was enforced with the increase of dosage.The apoptotic rate of RGC in model group,PBS control group,low-dose colivelin group,medium-dose colivelin group and high-dose colivelin group were（60.928±2.961）%,（61.446±2.755）%,（58.432±2.835）%,（51.948±2.802）% and （47.656±2.331）%,respectively.Expression of caspase-3 in model group,PBS control group,low-dose colivelin group,medium-dose colivelin group and high-dose colivelin group all could be detected,there was no statistical difference in the mean integrated optical density value between model group（0.482±0.012） and PBS control group（0.486±0.012,P0.05）,but they were all higher than colivelin group（both P0.05）,there were statistical differences among low-dose colivelin group（0.411±0.017）,medium-dose colivelin group（0.326±0.018） and high-dose colivelin group（0.234±0.016,all P0.05）.Conclusion Colivelin can increase the number of RGC after optic nerve injury,inhibit the RGC apoptosis and lessen the expression of caspase-3 with the dosage dependancy.