两种常用大鼠视神经钳夹伤模型造模效果的比较

背景 视神经钳夹伤(ONC)动物模型是外伤性视神经损伤致病机制及治疗方法相关基础研究的主要工具,目前常用的造模方法有经眼眶上缘开神经鞘膜视神经钳夹法和经球结膜外眦部夹伤视神经法,但关于2种模型优劣评价的研究很少. 目的 比较2种常用大鼠ONC模型的造模效果,为相关的实验研究中造模方法的选择提供依据.方法 采用随机分配方法将8～10周龄健康成年雄性SD大鼠24只分为经眶上缘开神经鞘膜ONC组和经眶上缘开神经鞘膜ONC 20 s、40 s、60 s组,分别采用经眶上缘开神经鞘膜视神经夹伤法(20 s)及经球结膜外眦部夹伤视神经法在大鼠的一侧眼建立ONC动物模型,各组大鼠的正常对侧眼作为对照.于造模后14 d记录各组大鼠闪光视觉诱发电位(F-VEP)P1波;制备大鼠视神经组织切片,采用苏木精-伊红染色法观察大鼠视神经的组织病理学改变;采用免疫荧光法观察并计数大鼠视网膜组织中Brn-3α阳性视网膜神经节细胞(RGCs).对2种造模方法的造模过程和检测结果进行比较.结果 造模后14d,经眶上缘开神经鞘ONC组和经球结膜ONC 20 s组、40 s组、60 s组大鼠的F-VEP P1波潜伏期较各自的正常对侧眼均明显延长,差异均有统计学意义(t=-11.64、-8.04、-6.50、-10.84,均P＜0.01);经眶上缘开神经鞘ONC组大鼠P1波潜伏期与经球结膜ONC 20 s、40 s、60 s组大鼠比较均明显延长,差异均有统计学意义(P=0.01、0.02、0.05);各组大鼠术眼P1波振幅与其正常对侧眼比较差异均无统计学意义(均P＞0.05).造模后14 d,免疫荧光检测显示各组大鼠模型眼视网膜上Brn-3α表达阳性RGCs数均较正常对侧眼明显减少,经眶上缘开神经鞘ONC组大鼠视网膜上Brn-3α表达阳性RGCs数为(13.60±2.14)个/视野,为其正常对侧眼的47.49％,经球结膜ONC 20 s、40 s和60 s组中Brn-3α阳性RGCs数分别为(18.74±3.61)、(15.84±2.31)和(14.58±3.23)个/视野,分别为其正常对侧眼的67.70％、56.69％和50.17％,经眶上缘开神经鞘ONC组大鼠视网膜中Brn-3α阳性RGCs数与经球结膜ONC 40 s和60 s组比较,差异均无统计学意义(均P＞0.05).组织病理学检查显示,各组大鼠造模眼神经胶质细胞核排列紊乱,细胞基质空泡化,可见大量炎性细胞浸润,以眶上缘开神经鞘ONC组更为严重.结论 与经球结膜ONC模型鼠比较,经眶上缘开视神经鞘ONC模型大鼠视神经形态结构损害更为严重,视神经的传导功能更为迟缓,RGCs的存活率更低.

A comparative study of two common optic nerve crush models in rats

Background Optic nerve crush (ONC) model is an available tool in the basic research on the mechanism and treatment of optic nerve injury.The opening optic nerves sheath crushing and via bulbar conjunctiva lateral canthus optic nerves crushing are frequently used ONC modeling methods.However,the comparison between these models is not elaborated.Objective This study was to compare the outcomes between opening sheath ONC model and via bulbar conjunctiva lateral canthus ONC model.Methods Twenty-four male SD rats were randomized into four groups.ONC models were established via superorbital rim opening sheath to crushing optic nerve for 20 seconds in the opening sheath ONC group,or via bulbar conjunctiva lateral canthus cutting to crush optic nerve for different time in the via bulbar conjunctiva ONC 20-second,40-second and 60-second groups,respectively.All models were monocular created in the rats,and the fellow eyes served as controls.In 14 days after modeling,flash visual evoked potential (F-VEP) were recorded,optic nerve and retinal sections were prepared in the rats.The histopathology of the samples was examined by hematoxylin and eosin staining.The expression of Brn-3α in the retinal ganglion cells (RGCs) was detected by immunofluorescence technique and the number of Brn-3α+ RGCs was counted.The modeling procedure and outcomes were compared between the two approaches.Results The latencies of P1 waves were significantly extended in the opening sheath ONC group,via bulbar conjunctiva ONC 20-second,40-second and 60-second groups in comparison with the corresponding control eyes (t =-11.64,-8.04,-6.50,-10.84,all at P＜0.01).The P1 latencies were longer in the opening sheath ONC group than those in the via bulbar conjunctiva ONC 20-second,40-second and 60-second groups (P =0.01,0.02,0.05),but no significant differences were found in the amplitudes of P1 waves between model eyes and corresponding control eyes (all at P＞0.05).The Brn-3α+ RGCs numbers were evidently decreased in the model eyes in comparison with the fellow control eyes.The Brn-3 α + RGCs numbers were (13.60 ± 2.14),(18.74 ± 3.61),(15.84 ± 2.31) and (14.58 ± 3.23)/field in the opening sheath ONC group,via bulbar conjunctiva ONC 20-second,40-second and 60-second groups,which reduced to 47.49％,67.70％,56.69％ and 50.17％ of the fellow eyes,respectively.No significant differences were seen in the Brn-3α+ RGC numbers between the opening sheath ONC group and via bulbar conjunctiva ONC 40-second or 60-second groups (both at P＞0.05).The disorder of glial cell arrangement,vacuolization of the cell matrix and infiltration of inflammatory cells were displayed in various model groups,with the prominent findings in the opening sheath ONC group.Conclusions Compared with the via bulbar conjunctiva ONC models,the morphological and functional damage of optic nerve is more obvious,and the survival rate of RGCs is lower in the opening sheath ONC models.