视网膜缺血-再灌注损伤中小胶质细胞对微循环的破坏作用

Retinal microglia activation and its effects on microcirculation in experimental retinal ischemia reperfusion injury

目的探讨视网膜缺血-再灌注损伤(retinal ischemia-reperfusion injury,RIRI)中小胶质细胞活化与视网膜微循环损伤的关系及作用机制。方法 160只雄性C57BL/6小鼠右眼均采用前房灌注建立RIRI模型为RIRI组,左眼不作处理为正常对照组。在损伤后24 h、48 h、72 h分别进行视网膜冰冻切片、视网膜铺片免疫荧光染色检测小胶质细胞的活化情况,检测相关缺氧因子及炎症因子的表达,与正常对照组比较,研究小胶质细胞的激活状态与微循环损伤的关系,并初步分析其作用机制。结果视网膜微循环结构损伤观察结果显示,与正常对照组相比,RIRI后24 h组大部分血管仍呈正常形态;RIRI后48 h组,闭塞的血管数量增多;RIRI后72 h组,血管损伤明显加重。浅层毛细血管密度正常对照组,RIRI后24 h、48 h、72 h组间两两比较差异均无统计学意义(均为P>0.05)。而深层血管网毛细血管密度RIRI后72 h组与其余3组相比明显减少,差异均有统计学意义(均为P<0.05),其余3组间差异均无统计学意义(均为P>0.05)。视网膜冰冻切片检测显示,RIRI后24 h组与正常对照组各层视网膜中抗离子钙接头蛋白(ionized calcium bindingadaptor molecule-1,Iba-1)阳性细胞数差异无统计学意义(P>0.05)。RIRI后48 h组各层中Iba-1阳性细胞数与正常对照组及24 h组差异均有统计学意义(均为P<0.05)。RIRI后72 h组各层中Iba-1阳性细胞数与正常对照组、24 h组、48 h组差异均有统计学意义(均为P<0.05)。视网膜铺片结果显示,RIRI后48 h组和72 h组活化的小胶质细胞数明显增加,与正常对照组和RIRI后24 h组在两个层次毛细血管中差异均有统计学意义(均为P<0.05),而72 h组小胶质细胞的活化达到高峰值,与其余组差异均有统计学意义(均为P<0.05)。RT-PCR检测结果显示:血管内皮生长因子和缺氧诱导因子-1α的缺血缺氧因子,肿瘤坏死因子-α和白细胞介素-1β的炎症因子在正常对照组与RIRI后24 h组、48 h组、72 h组,组间比较差异均有统计学意义(均为P<0.05)。结论激活的小胶质细胞在RIRI中发挥了对微循环的破坏作用,损伤早期抑制小胶质细胞的活化可能成为此类疾病治疗的新思路。

Objective To investigate retinal microglia activation and its effect on microcirculation in experimental retinal ischemia reperfusion injury(RIRI).Methods The animal model of IRI was established by anterior chamber perfusion.The retinal frozen sections and retinal flat mount immunofluorescence staining were used to detect the activation of microglia in the 24 h,48 h and 72 h groups.The expression of hypoxia factor and inflammatory factor was compared with the normal control group to study the relationship between the activation state of microglia and microcirculation injury,and to analyze its mechanism.Results The results of retinal microcirculatory structure damage showed that most of the blood vessels in the 24 h after RIRI were normal in the normal control group(normal mouse);the number of occluded vessels increased in the 48 h after RIRI;the vascular injury was obvious in the 72 h after RIRI.The results of retinal superficial capillary density showed that there was no significant difference between the two groups in the normal control group and the 24 h,48 h and 72 h after injury(all P>0.05).The capillary density of the deep vascular network was significantly reduced compared with the other three groups at 72 h after injury,and the difference was statistically significant(all P<0.05).There was no significant difference between the other three groups(all P>0.05).The number of activated microglia in the frozen section of the retina showed that there was no significant difference in the number of Ionized calcium binding adaptor molecule-1(Iba-1) positive cells in the groups between 24 h group and the normal control(P>0.05).The number of Iba-1 postive cells in each retinal layer of 48 h group was significantly different between the normal control and 24 h group(all P<0.05).The number of Iba-1 positive cells in each retinal layer of 72 h after injury was significantly different between the normal control group,24 h group and 48 h group(all P<0.05).The results of retinal flat mount staining showed that the number of activated microglia in the 48 h and 72 h groups increased significantly,and there was significant difference between the normal control group and the 24 h group in the superficial and deep capillary(all P<0.05).The activation of microglia in the 72 h group reached peak,and the difference between other groups was statistically significant(all P<0.05).RT-PCR detected the ischemia-induced hypoxia factor of vascular endothelial growth factor(VEGF) and hypoxia inducible factor-1α(HIF-1α) and the inflammatory factors of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in normal control group,24 h,48 h and 72 h groups.There were significant differences between each group(all P<0.05).Conclusion Activated microglial cells play a damaging role in microcirculation during retinal ischemia-reperfusion injury.Inhibition of microglial activation at early stage of injury may be a new idea for the treatment of such diseases.