小鼠视神经损伤后的视功能评估

Evaluation of visual function after optic nerve injury in mice

目的 通过闪光-视网膜电图(f-ERG)和视动反应(OMR)评估小鼠视神经钳夹(ONC)损伤后视功能的变化。方法 30只6~8周龄健康雄性C57BL/6J小鼠,按照随机数字表法分为正常对照(NC)组、ONC 3 d组、ONC 5 d组、ONC 7 d组以及ONC 14 d组,每组小鼠各6只。NC组不做特殊处理,ONC组小鼠在同一天进行双眼ONC 10 s造模。在相应时间点进行f-ERG和OMR检查,然后处死小鼠取视网膜进行视网膜免疫荧光染色铺片实验,观察各组小鼠视网膜神经节细胞(RGC)存活率的变化。比较NC组与ONC 3 d组、ONC 5 d组、ONC 7 d组以及ONC 14 d组小鼠在不同光刺激强度下f-ERG的a波与b波波幅变化,以及随着视神经损伤时间延长,OMR测得的小鼠视力变化,评估小鼠视神经损伤后的视功能。结果 与NC组相比,ONC 3 d组、ONC 5 d组、ONC 7 d组以及ONC 14 d组小鼠RGC存活率均下降,差异均有统计学意义(均为P<0.05)。NC组、ONC 3 d组、ONC 5 d组、ONC 7 d组以及ONC 14 d组小鼠f-ERG的a波与b波波幅均随光刺激强度增大而升高。在光刺激强度为0.1 cds·m^(-2)、0.3 cds·m^(-2)、1.0 cds·m^(-2)、3.0 cds·m^(-2)时,ONC各组小鼠a波与b波波幅均呈波动性下降;在光刺激强度为10.0 cds·m^(-2)时,ONC各组小鼠a波与b波波幅随ONC时间延长而持续下降。与NC组相比,在0.3 cds·m^(-2)、1.0 cds·m^(-2)、3.0 cds·m^(-2)、10.0 cds·m^(-2)光刺激强度下,ONC 3 d组、ONC 5 d组、ONC 7 d组以及ONC 14 d组小鼠a波与b波波幅均显著下降(均为P<0.05);在光刺激强度为0.1 cds·m^(-2)时,ONC 5 d组小鼠a波与b波、ONC 7 d组和ONC 14 d小鼠b波波幅也显著下降(均为P<0.05)。OMR记录的视锐度,随着ONC时间延长而逐渐下降,并在短期内(14 d)丧失。在ONC 14 d组,小鼠RGC存活率降低至不足10%,各光刺激强度下小鼠a波与b波波幅降低,小鼠OMR也完全丧失,小鼠RGC存活率、波幅、视锐度具有较好的一致性。结论 小鼠视神经损伤后,RGC存活率与视功能迅速下降。OMR和f-ERG的联合运用,可作为评估小鼠视神经损伤后视功能变化的有效手段。

Objective To evaluate the change in visual function of mice after optic nerve crush(ONC) through the flash electroretinogram(f-ERG) and optomotor response(OMR). Methods Totally 30 healthy male C57BL/6J mice aged 6 to 8 weeks were divided into the normal control(NC) group, ONC 3 d group, ONC 5 d group, ONC 7 d group, and ONC 14 d group according to the random number table method, with 6 mice in each group. Mice in the NC group were not given special treatment, and the model was established by performing binocular ONC on mice in the ONC group for 10 s on the same day. F-ERG and OMR were performed at the corresponding time points. Then the mice were sacrificed, and the retinas were taken out for immunofluorescence experiments on the retinal flat-mount preparation to observe the change in the survival rate of retinal ganglion cells(RGC). The change in a-wave and b-wave amplitude of f-ERG were compared among the NC group and ONC 3 d group, ONC 5 d group, ONC 7 d group, and ONC 14 d group under different light stimulation intensities, and the change in visual acuity was measured by the OMR with the extension of optic nerve injury time to evaluate the visual function of mice after optic nerve injury. Results The survival of RGC in the ONC 3 d group, ONC 5 d group, ONC 7 d group and ONC 14 d group decreased compared with the NC group, and the differences were statistically significant(all P<0.05). The amplitudes of a-wave and b-wave of f-ERG in the NC group, ONC 3 d group, ONC 5 d group, ONC 7 d group and ONC 14 d group increased with the increase of light stimulation intensity. When the intensity of light stimulation was 0.1 cds·m^(-2), 0.3 cds·m^(-2), 1.0 cds·m^(-2)and 3.0 cds·m^(-2), the amplitude fluctuation of a-wave and b-wave in each group decreased. When the intensity of light stimulation was 10.0 cds·m^(-2), the amplitude of a-wave and b-wave continued to decrease with the extension of the ONC time. When the intensity of light stimulation was 0.3 cds·m^(-2), 1.0 cds·m^(-2), 3.0 cds·m^(-2)and 10.0 cds·m^(-2), the amplitude of a-wave and b-wave in the ONC 3 d group, ONC 5 d group, ONC 7 d group and ONC 14 d group decreased significantly compared with the NC group(all P<0.05). When the intensity of light stimulation was 0.1 cds·m^(-2), the amplitude of a-wave and b-wave in the ONC 5 d group and b-wave in the ONC 7 d group and ONC 14 d group also decreased significantly(all P<0.05). The visual acuity recorded by the OMR decreased gradually with the extension of the ONC time and was lost within a short time(14 d). In the ONC 14 d group, the survival rate of RGC reduced to less than 10%, the amplitude of a-wave and b-wave in mice decreased under different light stimulation intensities and the OMR of the mice was completely lost. The RGC survival rate, amplitude and visual acuity showed good consistency. Conclusion After optic nerve injury, the RGC survival rate and visual function of mice decrease rapidly. The combined application of OMR and f-ERG can be used as an effective method to evaluate the changes in visual function after optic nerve injury in mice.