腺相关病毒介导的脑源性神经营养因子对DBA／2J小鼠视网膜神经节细胞的保护作用

背景神经营养因子的缺乏与青光眼的视神经损害密切相关。外源性神经营养因子的补充具有短暂的保护作用。腺相关病毒（AAV）介导的神经营养因子可在眼内长期表达，但是否对青光眼动物的视神经具有持久的保护作用有待研究。目的评估AAV介导的脑源性神经营养因子（BDNF）基因在DBA／2J小鼠眼内的表达及其对视网膜神经节细胞（RGCs）的保护作用。方法健康清洁级DBA／2J小鼠10只从4月龄起，每月使用Tonolab眼压计测量眼压。6月龄时左眼玻璃体腔内注射AAV介导的BDNF和绿色荧光蛋白（GFP）基因（AAV—BDNF—GFP）1μ1，右眼注射等量的生理盐水作为对照。注射后3个月心脏灌流后取出视网膜，荧光显微镜下观察GFP在视网膜中的表达，免疫组织化学法计算存活的RGCs数目并进行比较。结果DBA／2J小鼠4月龄时AAV—BDNF—GFP眼眼压平均为11．90mmHg，对照眼眼压为11．40mmHg。实验眼与对照眼眼压5月龄时均开始升高，8月龄时达到高峰。从4月龄到9月龄，实验组和对照组眼压比较差异无统计学意义（t=-1．78～0．61，P=0．11—0．90）。玻璃体腔注射AAV—BDNF—GFP3月龄后视网膜可以观察到GFP阳性细胞，转染率为46．33％±8．08％。AAV—BDNF—GFP组实验眼的平均RGCs的密度为（3168．13±1319．33）mm^2，对照眼为（2024．81±796．38）mm^2，差异有统计学意义（t=2．75，P=0．02）。结论AAV介导的BDNF对DBA／2J小鼠RGCs有保护作用。

Neuroprotection of adenoassociated vira-mediated brain-derived neurotrophic factor on retinal ganglion cells in DBA/2J mice with glaucoma

Background Deficiency of neurotrophic factor is associated with the damage of optic nerve in glaucoma.Reaserches showed that ectopically applied neurotrophic factor has a transient neuroprotective effect in glaucoma model,and the viral expression of adeno-associated neurotrophie factor may provide long-term supplementation of neurotrophic factor and neuroprotection in tissues.Objective The present study Was to investigate the neuroproteetive effect of adeno-associated viral(AAV)-mediated brain-derived neurotrophic factor (BDNF)on retinal ganglion cells(RGCs)in DBA/2J mice with experimental glaucoma. Methods 10 clean DBA/2J mice were administered intravitreal injection with 1 microliter of AAV-BDNF-GFP in the left eyes at the age of 6 months,and the right eyes were injected with the same volume of saline solution as control.Intraocular pressure (IOP)was measured with Tonolab in the mice every month.Retinas were obtained after 3 months for the investigation of GFP expression in RGCs using fluorescence microscopy.Immunohistochemistry Was performed by applying TUJ1 and Cy3 antibodies to identify surviving RGCs. Results The IOP of DBA/2J mice were 11.90 mmHg and 11.40 mmHg in the right eyes and left eyes,respectively,at 4 months.The IOP of DBA/2J mice began to rise at 5 months and reached its peak in 8 month-old mice.There was no statistically significant difference in IOPs between the right eyes and the left eyes from 4 month-through 9 month-old mice(t=-1.78-0.61,P=0.11-0.90).Three months after intrlavitreal injection of AAV-BDNF-GFP,GFP was positively expressed in RGCs of retinas with the expression rate of 46.33%±8.08%.The over-expression of BDNF led to more RGCs survival than the control eyes (3168.13±1319.33/mm2 vs 2024.81±796.38/mm2,t=2.75,P=0.02). Conclusion These data suggest that BDNF can exert a protective effect in DBA/2J glaucoma mice.