Trans-Corneal Subretinal Injection in Mice and Its Effect on the Function and Morphology of the Retina.
Trans-Corneal Subretinal Injection in Mice and Its Effect on the Function and Morphology of the Retina.
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小鼠经角膜视网膜下注射及其对视网膜功能和形态的影响
DOI：
10.1371/journal.pone.0136523
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Pang J
中科院分区：
文献类型：
--
作者：
Qi Y;Dai X;Zhang H;He Y;Zhang Y;Han J;Zhu P;Zhang Y;Zheng Q;Li X;Zhao C;Pang J
目的介绍一种实用的小鼠视网膜下注射方法，并应用动态成像系统、电生理学和组织学评价注射诱导的视网膜脱离（RD）和损伤。方法将2月龄C57 BL/6 J小鼠瞳孔完全扩张后，用30 ½口径的一次性斜针头穿刺利姆布斯附近的角膜。将33 ½号钝针通过角膜穿孔插入前房，在更深地进入玻璃体腔之前避开透镜，并穿透内层视网膜以到达视网膜下腔。将小鼠分为四组：第1组中，约80-100%的视网膜充满视网膜下注射溶液;第2组中，约50-70%的视网膜充满注射溶液;第3组中，在溶液注射前停止程序;第4组中，未注射的眼睛用作阴性对照。光学相干断层扫描（OCT）成像系统用于监测注射后前三天的视网膜复位。光镜和视网膜电图（ERG）检查注射后5周的组织学和功能的变化。结果经过短期培训，成功率为70%，覆盖率为50%或以上（即，视网膜泡占据50%或更多的视网膜面积并充满注射的溶液），并且可以实现最小的注射相关损伤。滤过泡形成与神经视网膜和视网膜色素上皮（RPE）层之间的视网膜脱离（RD）相关。在注射后第1天可以观察到部分RD，并且到第2天，大部分视网膜已经复位。注射后5周，与未注射对照组4相比，第1组ERG的b波振幅降低22%，第2组降低16%，第3组降低7%;未注射组与50-70%或80-100%覆盖率组之间的b波振幅存在统计学差异。视网膜下注射诱导的RD在注射后5周重新附着并变得稳定，但在注射部位及其周围仍然可以观察到一些感光细胞损伤，特别是在80-100%覆盖组中。结论经角膜视网膜下注射是一种有效、实用的方法，但注射后视网膜下的损伤可能会造成一定的形态和功能损失。
Purpose To introduce a practical method of subretinal injection in mice and evaluate injection-induced retinal detachment (RD) and damage using a dynamic imaging system, electrophysiology, and histology. Methods After full dilation of a 2-month-old C57BL/6J mouse pupil, the cornea near the limbus was punctured with a 30 ½-gague disposable beveled needle. A 33 ½-gauge blunt needle was inserted through the corneal perforation into the anterior chamber, avoiding the lens before going deeper into the vitreous cavity, and penetrating the inner retina to reach the subretinal space. The mice were divided into four groups: in group 1, about 80–100% of the retina was filled with subretinally injected solution; in group 2, approximately 50–70% of the retina was filled with injected solution; in group 3, the procedures were stopped before solution injection; and non-injected eyes were used as the negative control in group 4. An optical coherence tomography (OCT) imaging system was used to monitor retinal reattachment during the first three days following the injections. Histological and functional changes were examined by light microscopy and electroretinography (ERG) at five weeks post-injection. Results After a short-term training, a 70% success rate with 50% or more coverage (i.e., retinal blebs occupied 50% or more retinal area and filled with the injected solution) with minimal injection-related damages can be achieved. Bleb formation was associated with retinal detachment (RD) between the neuroretina and the retinal pigment epithelium (RPE) layer. Partial RD could be observed at post-injection day 1, and by day 2 most of the retina had reattached. At 5 weeks post-injection, compared to uninjected control group 4, the b-wave amplitudes of ERG decreased 22% in group 1, 16% in group 2, and 7% in group 3; the b-wave amplitudes were statistically different between the uninjected group and the groups with either 50–70% or 80–100% coverage. The subretinal injection-induced RD reattached and became stable at five weeks post-injection, although some photoreceptor damage could still be observed in and around the injection sites, especially in 80–100% coverage group. Conclusions Trans-corneal subretinal injection is effective and practical, although subretinal injection-related damages can cause some morphological and functional loss.