改良视网膜墨汁灌注法观察先天性静止性夜盲对视网膜新生血管的影响

Effect of congenital stationary night blindness on oxygen induced retinal neovascularization by improved Chinese ink perfusion method

背景先天性静止性夜盲（CSNB）是一种常见的眼科遗传性疾病。CSNB较少见到视网膜新生血管，其如何影响视网膜新生血管尚需深入探讨。目的改进视网膜墨汁灌注方法，并用于观察CSNB对视网膜新生血管生成的影响。方法选取清洁级7日龄SD大鼠和CSNB大鼠各18只，任意选取其中12只制作氧诱导视网膜病变（OIR）模型作为OIR模型组，其余6只作为正常对照组。OIR模型组SD大鼠和CSNB大鼠各9只按照随机数字表法随机分为体积比1∶1墨汁灌注组、体积比2∶1墨汁灌注组和单纯墨汁灌注组，分别灌注体积比1∶1墨汁灌注液、体积比2∶1墨汁灌注液和单纯墨汁灌注液各10 ml。取单侧眼球制作视网膜铺片，取另一侧眼球制作石蜡切片。观察并比较不同体积比墨汁灌注组视网膜血管成像质量。另取SD大鼠和CSNB大鼠OIR组各3只幼鼠与正常对照组幼鼠均按照此法灌注体积比2∶1墨汁灌注液。取各组石蜡切片行组织病理学观察并计数每张切片中突破内界膜细胞核的数量。免疫组织化学法检测视网膜中血管性血友病因子（v-WF）的表达。结果体积比2∶1墨汁灌注组视网膜铺片中血管网成像质量较体积比1∶1墨汁灌注组和单纯墨汁灌注组高。组织病理学检查显示，正常对照组大鼠视网膜各层组织结构未见明显异常，SD大鼠和CSNB大鼠均未见突破内界膜的细胞核；OIR模型组可见大量内皮细胞核突破内界膜，OIR模型组中SD大鼠和CSNB大鼠突破内界膜的细胞核数量分别为（23.08±2.99）个/切片和（41.12±9.36）个/切片。CSNB大鼠突破内界膜的细胞核数量略高于SD大鼠，但差异无统计学意义（q＝1.70，P＝0.50）。免疫组织化学检查结果显示，SD大鼠和CSNB大鼠突破内界膜的细胞v-WF均表达阳性。结论体积比2∶1墨汁明胶灌注液对大鼠视网膜血管成像质量优于单纯墨汁灌注液，是一种重复性好、操作简单的视网膜铺片方法。高氧可诱导CSNB大鼠视网膜新生血管生成。

BackgroundCongenital stationary night blindness （CSNB） is a common genetic eye disease.Retinal angiogenesis is rarely obtained in retinal degeneration animal.The effects of CSNB on retinal angiogenesis require further study.ObjectiveThis study was to improve Chinese ink perfusion technology and to explore the effect of CSNB on oxygen induced neovascularization.MethodsEighteen clean 7-day old SD rats and 18 clean 7-day old CSNB rats were included, twelve SD rats and twelve CSNB rats were chosen randomly for oxygen induced retinopathy （OIR） modeling, and served as OIR group, six SD rats and six CSNB rats were chosen as normal control.Nine rats were chosen randomly from both SD rats and CSNB rats in OIR group, respectively.The rats were separated into 1∶1 ratio ink group, 2∶1 ratio ink group and conventional ink group, which were perfused with 1∶1 ratio ink perfusate, 2∶1 ratio ink perfusate and conventional ink, respectively.The unilateral eyes of the rats were prepared for whole-mount retina, the other eyes were performed for paraffin imbedding.The quality of retinal vascular imaging were compared among different ink perfusate groups.The normal control rats, three SD rats in OIR group and three CSNB rats in OIR group were perfused with 2∶1 ratio ink perfusate.Histopathology examination was performed on the paraffin section, and the number of nuclei breakthrough the inner limiting membrane were counted.Immunocytochemistry were performed on the paraffin section for detecting the expression of von Wilebrand factor （v-WF）.ResultsCompared with 1∶1 ratio ink perfusion and conventional ink perfusion, 2∶1 ratio ink perfusion showed the full vascular net clearly.Histopathology showed that the structure of retina was normal in the normal control group, and there were no endothelial nuclei breakthrough the inner limiting membrane.A large number of endothelial nuclei breakthrough the inner limiting membrane in the OIR group, the number of endothelial nuclei breaking through the inner limiting membrane were （23.08±2.99）/slide and （41.12±9.36）/slide for SD rats and CSNB rats, respectively.The number of endothelial nuclei breakthrough inner limiting membrane was higher in the CSNB rats than that in the SD rats, with no difference between the two group（q=1.70, P=0.50）. Immunocytochemistry results showed that v-WF was positive expressed in the cells breakthrough inner limiting membrane.ConclusionsImproved ink perfusion method was an easy-to-use whole-mount retina method with good repeatability.Hyperoxia can induce retinal neovascularization of CSNB rats.