小鼠视神经损伤模型建立及重组人神经生长因子疗效评价

Establishment of an Optic Nerve Crush Injury Murine Model and Evaluation of Recombinant Human Nerve Growth Factor

目的:建立并评价小鼠视神经损伤(ONC)模型,在此基础上初步探讨重组人神经生长因子(rhNGF)的疗效。方法:采用夹伤法建立ONC模型,比较不同夹伤时间条件下建立小鼠ONC模型的差异。采用免疫荧光染色,观察ONC后不同时间点视网膜神经节细胞(RGCs)存活率,评价ONC模型建立。在此基础上,以rhNGF滴眼液为治疗组、生理盐水滴眼液为对照组,初步评价rhNGF治疗ONC模型的疗效。结果:采用夹伤5 s与夹伤10 s建立ONC模型,术后7 d视网膜免疫荧光染色RBPMS结果提示,夹伤5 s组RGCs存活率低于假手术组(P<0.01),夹伤10 s组RGCs存活率低于夹伤5 s组及假手术组(P<0.05),并且出现玻璃体浑浊。采用夹伤5 s为后续评价ONC模型,在ONC后第7、14、21、28 d,RGCs数量随时间延长而不断减少,且其密度均显著低于正常对照组(P<0.01)。在建立ONC模型基础上,治疗组和对照组分别连续13 d给予rhNGF滴眼液和生理盐水滴眼液,结果显示ONC后第14 d,治疗组RGCs存活率显著高于对照组(P<0.01)。结论:采用挤压法建立了一种稳定的小鼠ONC模型,并基于此初步评价了rhNGF治疗ONC的保护作用,为进一步探讨rhNGF治疗机制及药效评价奠定了基础。

Objective:The model of optic nerve crush(ONC)injury in mice was established.The therapeutic ef⁃fect for optic nerve crush by use of recombinant human nerve growth factor(rhNGF)was investigated.Methods:The ONC model was established by crushing the optical nerve in mice.ONC models with different injury time were first investigated.To evaluate the establishment of ONC model,the survival rate of retinal ganglion cells(RGCs)at different time points after ONC by immunofluorescence staining was used to evaluate the degree of in⁃jury on the ONC model.We further investigated the therapeutic effect of rhNGF by applying rhNGF eye drops in ONC mice.Results:We compared the ONC model with 5-second crushing and 10-second crushing.The retinal immunofluorescence staining of RNA binding protein with multiple splicing(RBPMS)7 days after operation was performed.The results showed that the survival rate of RGCs in 5-second group was lower than that in sham op⁃eration group(P<0.01).The survival rate of RGCs in 10-second group was lower than that in 5-second group and sham group(P<0.05).Vitreous opacity could be observed in 10-second group.The ONC model with 5-second crushing was used in the following work.Compared with normal control group,the number of RGCs decreased sig⁃nificantly on days 7,14,21 and 28 after ONC(P<0.01).Based on the ONC model,the ONC mice was treated with rhNGF eye drops and the control ONC mice were giventhe saline eye drops for 13 consecutive days,respec⁃tively.The survival rate of RGCs in the rhNGF-treated mice was significantly higher than that in the control mice 14 days after ONC(P<0.01).Conclusion:In this study,a stable murine crushing ONC model was estab⁃lished and the protective effect of rhNGF treatment on ONC was preliminarily evaluated,which lays a foundation for further study of the therapeutic mechanism of rhNGF.