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中华眼科医学杂志(电子版) ›› 2022, Vol. 12 ›› Issue (05) : 268 -274. doi: 10.3877/cma.j.issn.2095-2007.2022.05.003

论著

玻璃体腔注射低浓度碱溶液诱导小鼠视网膜内增殖模型的实验研究
赵一鸣1, 杨瑶2, 林晓峰2,†()   
  1. 1. 510060　广州，中山大学中山眼科中心2019级眼科学博士研究生，2022年8月至今首都医科大学附属北京朝阳医院眼科
    2. 510060　广州，中山大学中山眼科中心眼外伤科　眼科学国家重点实验室　广东省眼科视觉科学重点实验室
  • 收稿日期:2022-01-02 出版日期:2022-10-28
  • 通信作者: 林晓峰
  • 基金资助:
    国家自然科学基金项目(81974135)

A novel animal model of intraretinal proliferative vitreoretinopathy induced by intravitreally injected low-concentration alkali solution in mice

Yiming Zhao1, Yao Yang2, Xiaofeng Lin2,†()   

  1. 1. Doctoral degree 2019 major in Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China; Department of Ophthalmology, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100027, China (from August 2022)
    2. Ocular Trauma Department, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou 510060, China
  • Received:2022-01-02 Published:2022-10-28
  • Corresponding author: Xiaofeng Lin
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赵一鸣, 杨瑶, 林晓峰. 玻璃体腔注射低浓度碱溶液诱导小鼠视网膜内增殖模型的实验研究[J]. 中华眼科医学杂志(电子版), 2022, 12(05): 268-274.

Yiming Zhao, Yao Yang, Xiaofeng Lin. A novel animal model of intraretinal proliferative vitreoretinopathy induced by intravitreally injected low-concentration alkali solution in mice[J]. Chinese Journal of Ophthalmologic Medicine(Electronic Edition), 2022, 12(05): 268-274.

目的

探讨玻璃体腔注射低浓度碱溶液构建小鼠视网膜内增殖模型的方法。

方法

选用健康无特定病原体(SPF)级C57BL/6J小鼠共66只，6~8周龄，体重18~20 g，雌雄不限。以小鼠右眼为实验眼，左眼为空白对照眼。实验眼玻璃体腔注射0.3 M、0.5 M、0.75 M及1.0 M等4种浓度梯度的氢氧化钠溶液；分别于第7 d、14 d及28 d时摘取眼球；以苏木精－伊红染色后观察视网膜形态结构的变化，使用Image J软件测量全层视网膜、视网膜内核层及视网膜外核层的厚度；分别以胶质纤维酸性蛋白(GFAP)和波形蛋白(Vimentin)作为Müller细胞的标志物进行免疫荧光染色，观察视网膜内胶质反应和胶质瘢痕的情况，并使用Image J量化平均荧光强度。视网膜各层厚度和平均荧光强度以(±s)表示，采用配对t检验比较两组之间的差异。采用单因素方差分析比较多组之间的差异，并采用Tukey′s检验进行多重比较分析。

结果

造模后第28 d时，玻璃体腔注射0.5 M浓度氢氧化钠溶液小鼠实验眼后极部全层视网膜、视网膜内核层以及视网膜外核层的厚度均较对照眼降低，分别为(102.40±13.93)μm、(25.08±7.78)μm及(29.74±1.50)μm，实验眼全层视网膜及视网膜外核层厚度与对照眼的差异具有统计学意义(t＝8.56，2.09；P<0.05)；实验眼中周部全层视网膜、视网膜内核层及视网膜外核层的厚度均较对照眼降低，分别为(123.60±16.37)μm、(13.05±2.80)μm及(34.00±1.91)μm，其差异具有统计学意义(t＝5.01，11.49，7.923；P<0.05)；实验眼周边部全层视网膜、视网膜内核层以及视网膜外核层的厚度均较对照眼降低，分别为(108.50±24.45)μm、(12.80±4.66)μm及(34.45 ±2.90)μm，实验眼视网膜内核层厚度与对照眼的差异具有统计学意义(t＝4.76，P<0.05)。造模后7 d、14 d及28 d时，GFAP标记Müller细胞胶质反应的平均荧光强度均较对照眼增加，分别为(75.43±2.968)、(72.16±3.630)及(51.55±5.526)，其差异均具有统计学意义(q＝12.83，11.99，6.699；P<0.05)。造模后28 d时，GFAP的平均荧光强度比7 d及14 d时的强度降低，其差异具有统计学意义(q＝6.131，5.292；P<0.05)。造模后28 d时，Vimentin标记Müller细胞胶质反应和胶质瘢痕的平均荧光强度比GFAP更强，其差异均具有统计学意义(t＝4.563，15.72；P<0.05)。

结论

小鼠玻璃体腔注射0.5 M浓度氢氧化钠溶液可成功制作稳定的视网膜内胶质化模型。GFAP免疫荧光染色可作为早期反应性胶质增生的标志物，晚期的胶质反应及胶质瘢痕可通过Vimentin的免疫荧光染色进行评估。

关键词: 眼损伤, 视网膜内增殖, Müller细胞, 反应性胶质增生, 大规模胶质增生
Objective

To establish a mouse model of intraretinal proliferative vitreoretinopathy by intravitreal injection of low-concentration alkali solution.

Methods

66 healthy SPF C57BL/6J mice, aged 6~8 weeks, weighing 18~20 g, of both sexes were used. The right eye of mice was used as the experimental eyes, and the left eye was used as the blank control. 0.3 M, 0.5 M, 0.75 M and 1.0 M NaOH were injected into the vitreous cavity of the experimental eyes, respectively. After injection for 7, 14, and 28 days, eyeballs were enucleated and stained with hematoxylin and eosin staining to observe the changes of retinal morphology and structure. Image J software was used to measure the thickness of full-thickness retina, inner nuclear layer and outer nuclear layer. The glial fibrillary acidic protein (GFAP) and Vimentin were used as markers of Müller cells for immunofluorescence staining to observe the glial response and glial scar of the retina. Image J software was used to quantify the mean fluorescence intensity. The thickness of each retinal layer and the mean fluorescence intensity were described as (±s) and compared by paired t-test. One-way ANOVA was used to compare the differences between multiple groups, and Tukey′s test was used for multiple comparison analysis.

Results

After modeling for 28 days, the thickness of the full-thickness retina, inner nuclear layer and outer nuclear layer in the central retina of eyes injuried by 0.5 M NaOH were (102.40±13.93)μm, (25.08±7.78)μm, and (29.74±1.50)μm, respectively, which were lower than that of the control eyes. There were significantly differeces in the thickness of the full-thickness retina and outer nuclear layer between two eyes (t=8.56, 2.09; P<0.05). The thickness of full-thickness retina, inner nuclear layer and outer nuclear layer in the mid-peripheral retina of eyes injuried by 0.5 M NaOH were (123.60±16.37)μm, (13.05±2.80)μm, and (34.00±1.91)μm, respectively, which were significantly lower than those of eyes in the control group (t=5.01, 11.49, 7.923; P<0.05). The thickness of the full-thickness retina, inner nuclear layer and outer nuclear layer in the peripheral retina of eyes injuried by 0.5 M NaOH were (108.50±24.45)μm, (12.80±4.66)μm, and (34.45±2.90)μm, respectively. The difference of the inner retinal layer thickness between two eyes was statistically significant (t=4.76, P<0.05). After modeling for 7, 14, and 28 days, the average fluorescence intensities of GFAP were (75.43±2.968), (72.16±3.630), and (51.55±5.526), which were higher than those of the control eyes, and the differences were statistically significant (q=12.83, 11.99, 6.699; P<0.05). After modeling for 28 days, the average fluorescence intensity of GFAP was lower than those at 7 and 14 days, and the difference was statistically significant (q=6.131, 5.292; P<0.05). After modeling for 28 days, the average fluorescence intensity of Vimentin labeling Müller cells gliosis and glial scar were stronger than those of GFAP with the statistically significant differences (t=4.563, 15.72; P<0.05).

Conclusions

Intravitreal injection of 0.5 M NaOH in mice could successfully produce a stable model of intraretinal glioses. GFAP staining is a well-suited marker for the early reactive gliosis, while Vimentin staining is of great benifit to evaluate the gilal responses and glial scars in the later stage of injuries in the mice.

Key words: Eye Injuries, Intraretinal proliferative vitreoretinopathy, Müller cells, Reactive gliosis, Massive gliosis
图1 光学显微镜下玻璃体腔注射不同浓度氢氧化钠溶液后视网膜组织形态的显微结构及各时间点视网膜厚度对比图　图A示空白对照眼视网膜的形态结构；图B、图C、图D及图E分别表示玻璃体腔注射0.3 M、0.5 M、0.75 M及1.0 M浓度氢氧化钠溶液后7 d视网膜的显微结构；图F和图G示空白对照眼和玻璃体腔注射0.5 M浓度氢氧化钠溶液造模后低倍镜下14 d及28 d视网膜的显微结构图(H&E染色×4)；图H、图I及图J示玻璃体腔注射0.5 M浓度氢氧化钠溶液造模后28 d视网膜各层厚度比较的柱状图　 *与对照组相比，差异具有统计学意义
图2 激光扫描共焦显微镜下玻璃体腔注射0.5 M浓度氢氧化钠溶液后视网膜的免疫荧光染色图　图A~图D分别示对照眼与玻璃体腔注射0.5 M浓度氢氧化钠溶液后7 d、14 d及28 d后极部视网膜的兔抗鼠胶质纤维酸性蛋白和鼠抗鼠谷氨酰胺合成酶免疫荧光共染图(×40)；图E~图H分别示对照眼与玻璃体腔注射0.5 M浓度氢氧化钠溶液后7 d、14 d及28 d中周部视网膜的兔抗鼠胶质纤维酸性蛋白和鼠抗鼠谷氨酰胺合成酶免疫荧光共染图，图H中虚线框示视网膜内局部的胶质瘢痕(×40)；图I~图L分别示对照眼与玻璃体腔注射0.5 M浓度氢氧化钠溶液后7 d、14 d及28 d周边部视网膜的兔抗鼠胶质纤维酸性蛋白和鼠抗鼠谷氨酰胺合成酶免疫荧光共染图(×40)；图M和图N示玻璃体腔注射0.5 M浓度氢氧化钠溶液后28 d视网膜胶质瘢痕的兔抗鼠胶质纤维酸性蛋白和鼠抗鼠谷氨酰胺合成酶免疫荧光共染图(×40)；图O和图P示玻璃体腔注射0.5 M浓度氢氧化钠溶液后28 d视网膜胶质瘢痕的兔抗鼠波形蛋白和鼠抗鼠谷氨酰胺合成酶免疫荧光共染图，虚线示视网膜胶质瘢痕的范围，三角形示视网膜内胶质瘢痕，箭头示视网膜下胶质瘢痕(×10)
图3 激光扫描共焦显微镜下玻璃体腔注射0.5 M浓度氢氧化钠溶液后28 d后极部、中周部及周边部视网膜的兔抗鼠波形蛋白和鼠抗鼠谷氨酰胺合成酶免疫荧光染色图　图A~图C分别示对照眼后极部、中周部、周边部视网膜的免疫荧光染色图；图D~图F分别示玻璃体腔注射0.5 M浓度氢氧化钠溶液后28 d实验眼后极部、中周部及周边部视网膜的免疫荧光染色图(×40)
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