Background Many methods of ocular hypertension modeling have been used before,but these models remain short-duration ocular hypertension only.A new method of elevating intraocular pressure in mice by anterior chamber injection of polystyrene microspheres was reported abroad.However,this model is rarely used in China.Objective This study was to evaluate the application value of anterior chamber injection of polystyrene microspheres to establish glaucoma model in mice.Methods Forty-two SPF adult female C57BL/6L mice were divided into three groups according to random number table.Polystyrene microspheres (2 μl) were injected into the anterior chamber monocularly in the microspheres group,and the equal amount of PBS was used in the same way in the PBS group.No intervene was performed in the normal control group.The eyes of mice were examined by slit lamp microscope,and the intraocular pressure (IOP) was measured with TonoLab rebound tomometer in a 3-day interval after injection.Ocular histological sections were prepared 2 and 4 weeks after injection,and the anterior chamber angle was examined under the optical microscope.Neurons retrograde labeling was performed by 4％ fluorogold to calculate the survival number of retinal ganglion cells (RGCs) and the nerve fiber density was detected to assess the degree of RGCs and axon damage in retinal flat mounts,and the β-Ⅲ-tubulin-positive cells in the RGCs layer were examined by immnofluorescence method.The use and care of the animals complied with the instruction of Association for Research in Vision Ophthalmology (ARVO).Results IOP was significantly higher in the mice of the microspheres group than that in the normal control group or PBS group 2 and 4 weeks (all at P＜0.05).In the microspheres group,IOP reached peak in 2 weeks after injection and was significantly higher than that of 4 weeks after injection ([29.67±2.34] mmHg versus[15.71±1.23] mmHg) (all at P＜0.05).In 2 and 4 weeks after the anterior chamber injection of polystyrene,corneal edema was found under the slit lamp microscope,and under the optical microscope,microspheres accumulated at the anterior chamber angle.Additionally,in 2 and 4 weeks after injection,the number of survival RGCs was (4 542.82 ± 653.72)/mm2 and (3 623.12 ± 628.79)/mm2,respectively in the microspheres group,which showed significantly decrease in comparison with (6 979.33 ± 678.49)/mm2 and (6963.91 ±497.29)/mm2 in the normal control group (t =17.729,28.569,both at P＜0.05) and (6 843.21 ±573.42)/mm2 and (6 937.53±465.24)/mm2in the PBS group (t =16.975,29.145,both at P＜0.05).The number of RGCs was significantly less in the fourth week compared with second week after injection (t =6.951,P＜0.05).The β-Ⅲ-tubulin positive RGCs were (4 576.36± 479.64)/mm2 and (3 712.90 ± 660.31)/mm2 in 2 and 4 weeks in the microspheres group,respectively,which were significantly decreased in comparison with (6 725.94 ± 619.42)/mm2 and (6 741.90±663.60)/mm2 of the normal control group (t =18.811,22.182,both at P ＜ 0.05) or (6 757.85 ±463.59)/mm2 and (6 773.17± 471.35)/mm2 in the PBS group (t =18.953,22.605,both at P＜0.05),and in the microspheres group,β-Ⅲ-tubulin positive cells in the fourth week were decreased than those in the second week after injection (t=7.253,P＜0.05).The neural fiber density in the microspheres group in 2 and 4 weeks after injection was (193.08 ±32.75)/mm2 and (139.O0 ±38.24)/mm2,respectively,with a significant decline in comparison with (305.57±81.21)/mm2 and (297.46±52.60)/mm2(t=8.900,16.883,both at P＜0.05) of the normal control group or (312.63±70.62)/mm2 and (269.37±61.63)/mm2 of the PBS group (t=7.731,15.959,both at P＜0.05),and the neural fiber density was significantly lower in the fourth week than that in the second week after injection (t =7.442,P＜0.05).Conclusions Single injection of polystyrene microspheres into the anterior chamber can induce chronic ocular hypertension in mouse,which leads to the progressive damage of RGCs and neural fibers.This animal model shows a similar chronic pathogenic process to human glaucomatous eye.%背景 以往国内关于青光眼研究的造模方法中大多存在高眼压维持时间较短的问题.目前前房内注射聚苯乙烯微球建立小鼠慢性高眼压模型的方法已在国外应用,但国内尚缺乏对该模型的评价.目的 评价前房内一次性注射聚苯乙烯微球建立小鼠慢性高眼压模型的方法学及应用价值.方法 将42只SPF级成年C57BL/6L雌性小鼠用随机数字表法随机分为3个组,正常对照组小鼠不进行任何处理;PBS组小鼠前房内一次性注射PBS溶液2μl;微球组小鼠左眼前房内一次性注射聚苯乙烯微球2μl.各组小鼠均于注射后2周和4周行裂隙灯显微镜检查,采用TonoLab回弹式眼压计测量眼压;并于相应时间点观察结束后处死动物,制备眼球的组织学切片和视网膜铺片,在光学显微镜下观察前房角情况;采用质量分数4％荧光金经上丘逆行标记视网膜神经节细胞(RGCs),在荧光显微镜下计数各组小鼠视网膜铺片中RGCs的存活密度和神经纤维的数量;采用免疫组织化学染色法检查视网膜铺片中β-Ⅲ-微管蛋白阳性细胞数. 结果 前房注射后2周和4周,微球组小鼠眼压均明显高于正常对照组和PBS组,差异均有统计学意义(P＜0.05).微球注射后2周小鼠平均眼压达高峰,为(29.67±2.34)mmHg(1 mmHg=0.133 kPa),之后眼压逐渐下降,4周时小鼠的平均眼压为(15.71±1.23)mmHg,但明显高于术前,差异均有统计学意义(P＜0.05).微球组小鼠前房注射后裂隙灯显微镜下可见角膜水肿,但前房内未见明显的炎症反应,眼球组织病理学检查可见微球聚集在前房角.前房注射后2周和4周,微球组RGCs存活密度分别为(4 542.82±653.72)个/mm2和(3 623.12±628.79)个/mm2,明显低于正常对照组的(6 979.33±678.49)个/mm2和(6 963.91±497.29)个/mm2,差异均有统计学意义(t=17.729、28.569,P＜0.05),亦明显低于PBS组的(6 843.21 ±573.42)个/mm2和(6 937.53±465.24)个/mm2,差异均有统计学意义(t=16.975、29.145,P＜0.05),且注射后4周RGCs的存活数量较2周时明显减少,差异有统计学意义(t=6.951,P＜0.05).前房注射后2周和4周,微球组小鼠视网膜β-Ⅲ-微管蛋白阳性细胞数为(4 576.36±479.64)个/mm2和(3 712.90±660.31)个/mm2,均少于正常对照组的(6 725.94±619.42)个/mm2和(6 741.90±663.60)个/mm2,差异均有统计学意义(t=18.811、22.182,P＜0.05),亦明显少于PBS组的(6 757.85±463.59)个/mm2和(6 773.17±471.35)个/mm2,差异均有统计学意义(t=18.953、22.605,P＜0.05),微球组注射后4周视网膜β-Ⅲ-微管蛋白阳性细胞数少于2周时,差异有统计学意义(t=7.253,P＜0.05).注射后2周微球组小鼠视网膜神经纤维密度为(193.08±32.75)个/mm2,4周时为(139.00±38.24)个/mm2,明显低于正常对照组的(305.57±81.21)个/mm2和(297.46±52.60)个/mm2,差异均有统计学意义(t=8.900、16.883,P＜0.05),亦明显少于PBS组的(312.63±70.62)个/mm2和(269.37±61.63)个/mm2,差异均有统计学意义(t=7.731、15.959,P＜0.05),微球组注射4周时视网膜神经纤维密度低于2周时,差异有统计学意义(t=7.442,P＜0.05).结论 前房一次性注射聚苯乙烯微球的方法可引起小鼠的持续性高眼压,并造成RGCs和神经纤维的进行性损害,与人类青光眼的病理变化过程类似.
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