小鼠视神经再生研究动物模型的建立

Techniques and Experience in Developing Animal Model for Study on Mice Optic Nerve Regeneration

目的 总结制作小鼠视神经完全截断性动物模型作为视神经再生研究的经验和体会。方法 将雄性Bcl-2高表达转基因小鼠（Bcl-2 transgenic mice）和受GFAP启动子控制表达疱疹病毒-胸苷激酶转基因雌性小鼠（GFAP-TK）交配产生的4只8～12周成年小鼠（20～30g），Bcl-2／GFAP-TK双转基因小鼠作为实验组，同周龄4只Bcl-2转基因小鼠作为对照组。其中Bcl-2／GFAP-TK双转基因小鼠皮下植入缓释泵，连续7d释放更昔洛韦（GCV）100mg·kg^-1·d“^-1选择性地去除视神经损伤后激活的星形胶质细胞。更昔洛韦缓释泵植入术后2d在两组动物中制作右侧单眼标准完全性视神经钳夹损伤模型，视神经钳夹10d后获取组织标本。采用免疫荧光染色特异性检测再生轴突纤维并进行定量分析；结合罗丹明的霍乱毒索B亚单位（CTB-R）或增强表达绿色荧光蛋白的复制缺陷型腺相关病毒（AAV-EGFP）用作顺行性标记物以显示再生轴突是否到达大脑靶器官。结果 在Bcl-2／GFAP-TK双转基因小鼠中存在免疫荧光阳性的再生视神经轴突，再生轴突计数为71．99±24．04，并可见生长锥（growth cone）样结构，但是再生轴突纤维未能延伸达到大脑靶器官。在对照组Bcl-2转基因小鼠中未见明显再生迹象。结论 小鼠视神经完全截断性动物模型可用于视神经病变的再生研究。

Objective To summarize techniques and experience in developing mice optic nerve crushing model for studying optic nerve regeneration. Methods 4 Bcl-2/GFAP-Tk double transgenic mice were obtained by mating male Bcl-2 transgenic mice with female mice expressing herpes simplex virus-thymidine kinase transgenic mice controlled under GFAP promoter （GFAP-Tk） and 4 Bcl-2 transgenic mice were used as controls, and ganciclovir was administered through slow-releasing osmotic pump to selectively eliminate activated astrocytes in Bcl-2/GFAP-Tk transgenic mice. The right optic nerve crush was carded out according to the standard procedure in these mice aged 8～ 12 weeks （20 ～ 30g） on the second day after implantation of the osmotic pump. The tissue samples were taken on the tenth day after the optic nerve crush, and the axon regeneration was analyzed qualitatively and quantitatively by GAP43 immunofluorescence staining. Rodamine conjugate CTB or replication defective adeno associate virus carrying enhanced green fluorescence protein （AAV-EGFP） was injected in vitreous to label axon anterogradely and to determine whether the regenerated axons reach the brain targets. Results The remarkable optic nerve regeneration and the growth cone-like structure were found and the regenerated axon count was 71.99 ± 24.04 in Bcl-2/GFAP-Tk double transgenic mice after GCV administration and optic nerve crushing, but the axon reached into the brain was found. No axonal regeneration was identified in Bcl-2 transgenic mice. Conclusion Complete optic nerve crushing in the mice is an ideal mode for studying neural regeneration, but stable techniques of developing the mode and specific labeling methods for axonal regeneration are imperative.