改良的氧诱导小鼠视网膜病变模型及其评价

Modification and evaluation of ameliorative oxygen-induced retinopathy mouse model

背景视网膜新生血管是多种视网膜血管病变的共同病理改变，探讨其发病机制对临床治疗有重要意义。氧诱导视网膜病变（OIR）模型是研究视网膜新生血管性疾病常用的动物模型，但在实际造模过程中存在动物死亡率高、成模率低且不稳定等缺点。目的对传统的OIR造模方法进行改良，建立方法简单、模型稳定、成模率高的小鼠OIR模型。方法将80只SPF级1周龄C57BL／6J小鼠按随机数字表法随机分为正常对照组和OIR组，每组40只。正常对照组新生鼠与哺乳母鼠共同在正常空气环境下饲养，OIR组小鼠于出生后2天（P2）每2窝合笼饲养至鼠龄P7时。将2窝P7小鼠与1只母鼠放人含体积分数80％氧的氧氮混合气体的氧箱中饲养5d，然后放回至正常空气环境中饲养，另1母鼠在正常室内空气中饲养，每日更换放人氧箱的母鼠，评价指标包括成模率、母鼠死亡率和幼鼠成活率。OIR组和正常对照组分别取P12、P14、P17、P21小鼠各10只。5只小鼠行心脏异硫氰酸葡聚糖（FITC）荧光素与质量分数4％多聚甲醛的PBS混合液灌注，制备视网膜铺片，评估各组小鼠视网膜血管分布。5只小鼠双眼行组织病理学检查，计算视网膜中突破内界膜的血管内皮细胞核数目。结果正常对照组母鼠和幼鼠成活率为100％。OIR组2只母鼠出氧箱后死亡，幼鼠生长情况良好，母鼠成活率为85．7％，幼鼠成活率为100％；所有OIR组小鼠均出现视网膜新生血管，造模成功率为100％。视网膜铺片显示，正常对照组P14小鼠视网膜血管由视盘向四周均匀分布，视网膜血管发育基本成熟。OIR组P12小鼠视网膜后极部出现无灌注区；P14小鼠视网膜后极部可见大片无灌注区，周边部出现新生血管；P17小鼠视网膜血管区与无血管区交界处可见大量新生血管形成和荧光素渗漏；P21小鼠视网膜周边部可见少量无灌注区，新生血管明显减少。正常对照组各鼠龄小鼠视网膜内界膜连续、平整，仅少数切片中出现突破内界膜的血管内皮细胞核；OIR组P12小鼠少量血管内皮细胞突破内界膜，P17小鼠血管内皮细胞核数达到高峰，P21小鼠仅见少数突破内界膜的血管内皮细胞核。OIR组P14、P17、P21小鼠视网膜突破内界膜的内皮细胞核数分别为（11．44±2．01）、（31．24±1．50）和（9．23±1．12）／切片，明显高于正常对照组相应鼠龄小鼠的（0．27±0．14）、（0．30±0．11）和（0．32±0．16）／切片，差异均有统计学意义（t=47．90、61．30、40．70，均P〈0．05）。结论采用母鼠交换、80％的氧氮混合气体环境喂养及幼鼠合笼喂养建立OIR模型的方法操作简单，造模期间母鼠死亡率低，能构建出稳定、典型的小鼠视网膜新生血管模型。

Background Retinal neovascularization is associated with various disorders. Studying the pathogenesis of retinal neovascularization is of important significance. Oxygen-induced retinopathy（OIR） mouse model is a common animal model for the study of retinal neovascular diseases. However, conventional modeling methods usually cause high animal mortality and low rate of success. Objective This study aimed to establish a modified method of mouse OIR model. Methods Eighty 1-week-old SPF C57BL/6J mice were randomly divided into normal control group and OIR group with 40 mice for each. The newborn mice of the normal control group were kept in a normal air environment with their breast-feeding mothers, but the mice of postnatal 2 days （P2） in the OIR group were raised with two litters per cage until PT. The P7 mice exposed to oxygen tank containing 80% oxygen together with one or another mother mouse alternately daily for 5 days and then returned to the normal air environment. The success rate of modeling,mortality rate of maternal mice and survival rate of immature mice were evaluated. The mixed solution of fluorescein isothiocyanate （FITC） and PBS with 4% paraformaldehyde was infused into the hearts of P12,PI4,PI7 and P21 mice and the eyeballs were obtained after the mice were sacrificed fur histopathological examination of retinas and preparation of retinal flatmounts. The number of vascular endothelial ceils extending inner limiting membrane was counted and the distribution of retinal vessels was evaluated. The use and care of the animals complied with the Statement of ARVO. Results The survival rate of the neonatal mice was 100% both in the normal control group and the OIR group,and the survival rate of maternal mice was 85.7% in the OIR group. Retinal new vessels were found in the mice of the OIR group,with the success rate of modeling 100%. The retinal vessels distrihuted from optical disc toward periphery in PI4 mice in the normal group. However,in the OIR group,non-perfusion area at the posterior pole was seen in P12 mice,new blood vessels at the periphery were found in P14 mice, neovaseularization at the junction area between vascular area amt non-perfusion area as well as leakages were exhibited in PI7 mice,and less non-perfusion area and new vessels were seen in P21 mice. Retinal inner limiting membrane was smooth in the mice of the normal group, and the vascular endothelial cell nucleus extending inner limiting membrane were seen in P12 mice and peaked in PI7 mice. The vascular endothelial cell nucleus were （ 11.44±2. 01 ） , （31.24±1.50） and （9.23±1.12）/slide in P14, PI7 and P21 mice in the OIR group,which were significantly more than （0.27±0. 14）,（0.30±0. 11） and （0.32±0. 16）/slide in P14, PI7 and P21 mice in the normal group （t=47.90,61.30,40.70,all at P〈0.05）. Conclusions The method of OIR modeling is modified by ahernating maternal mice,exposing to 80% oxygen-nitrogen mixture gas and eohabitating immature mice. Modified modeling method is simple with the low death rate of maternal mice and stable OIR phenotype.