Cx3cr1抗体玻璃体腔注射对视网膜缺血-再灌注损伤模型小鼠视网膜微循环的保护作用及其机制

Protective effect and mechanism of cx3cr1 antibody intravitreal injection in microcirculation of retinal ischemia-reperfusion injury in mice

目的探讨小胶质细胞活化抑制剂C-X3-C基序趋化因子受体1(cx3cr1)抗体玻璃体腔注射对视网膜缺血—再灌注损伤(RIR)过程中视网膜微循环的保护作用及其可能机制。方法采用随机数字表法将150只成年健康C57BL/6小鼠随机分为空白对照组、模型组和cx3cr1抗体注射组,每组各50只。空白对照组小鼠仅玻璃体腔注射无菌注射用水2μl,模型组小鼠采用前房灌注升高眼压法建立RIR模型,cx3cr1抗体注射组小鼠玻璃体腔注射0.2μg/μl cx3cr1抗体2μl,注射后4 h建立RIR模型。于造模后3 d采用免疫荧光染色法检查各组小鼠实验眼冰冻切片中各层视网膜结构Iba-1阳性表达以评估小胶质细胞活化情况;采用视网膜铺片血管染色法观察视网膜深层和浅层血管密度变化及活化小胶质细胞数以评估视网膜微循环改变;采用FITC-dextran造影法测定视网膜血管渗漏面积;采用实时荧光定量PCR法测定视网膜中缺氧相关因子及炎性因子mRNA表达变化。结果眼球冰冻切片免疫荧光染色结果显示,空白对照组中Iba-1阳性小胶质细胞稀疏分布于视网膜神经节细胞层和内丛状层,呈分支形态;模型组Iba-1阳性细胞数量明显增多,呈阿米巴样或球形,并明显向外丛状层、外核层等视网膜外层移动;cx3cr1抗体注射组球形或阿米巴样的Iba-1阳性细胞数较模型组明显减少;模型组小鼠视网膜各层活化小胶质细胞数明显多于空白对照组和cx3cr1抗体注射组,差异均有统计学意义(均P<0.05)。与模型组比较,cx3cr1抗体注射组小鼠视网膜血管周围活化小胶质细胞数量明显减少。视网膜铺片血管与活化小胶质细胞共染色结果显示,空白对照组和cx3cr1抗体注射组小鼠视网膜深层血管密度均明显高于模型组,cx3cr1抗体注射组浅层及深层视网膜血管周围小胶质细胞数量较模型组明显减少,差异均有统计学意义(均P<0.05)。空白对照组、模型组和cx3cr1抗体注射组血管相对渗漏率分别为(100.0±4.7)%、(162.1±10.6)%和(130.5±9.5)%,总体比较差异有统计学意义(F=128.66,P<0.01),cx3cr1抗体注射组小鼠视网膜血管相对渗漏率明显低于模型组,差异有统计学意义(P<0.05)。实时荧光定量PCR结果显示,与模型组比较,cx3cr1抗体注射组和空白对照组小鼠视网膜中血管内皮生长因子A(VEGF-A)、缺氧诱导因子-1α(HIF-1α)、肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)mRNA相对表达量均明显降低,差异均有统计学意义(均P<0.05)。结论Cx3cr1抗体玻璃体腔注射可对RIR模型小鼠的视网膜微循环系统血管完整性发挥保护作用。

Objective To investigate the protective effect of C-X3-C motif chemokine receptor 1(cx3cr1)antibody,a microglia activation inhibitor,on microcirculation during retinal ischemia reperfusion(RIR)and its possible mechanism.Methods One hundred and fifty healthy adult C57BL/6 mice were randomized into blank control group,model group and cx3cr1 injection group by random number table method,with 50 mice in each group.The RIR model was established by anterior chamber infusion to elevate intraocular pressure in this study.Mice in the blank control group were intravitreally injected with 2μl of sterile water.In the cx3cr1 injection group,the RIR model was established at 4 hours after the intravitreal injection(2μl)of 0.2μg/μl cx3cr1 antibody.Immunofluorescence staining of frozen eyeball sections was performed to assess the microglia activation by observing the Iba-1 positive expression in different retinal layers three days following the model establishment.Retinal preparation vascular staining was carried out to observe the changes in the density of deep and shallow retinal blood vessels and the number of activated microglia to evaluate the changes in retinal microcirculation.FITC-dextran contrast method was used to determine the retinal vascular leakage area.Real-time fluorescent quantitative polymerase chain reaction(qPCR)method was employed to detect the mRNA expression changes of hypoxia-related factors and inflammatory factors in the mice retina.The study protocol was approved by an Ethics Committee of Kunming Medical University(No.20180106).The use and care of the animals complied with the Regulations of the Administration of Affair Concerning Experimental Animals.Results The immunofluorescence staining result of eyeball frozen section showed that in the blank control group,Iba-1 positive microglial cells were sparsely distributed in the retinal ganglion cell layer and inner plexiform layer,presenting branched state.In the model group,Iba-1 positive microglial cells were increased and moved outward to the outer retinal plexiform layer and outer nuclear layer obviously,showing globular or amoeba-like.The number of globular or amoeba-like Iba-1 positive cells was significantly reduced in the cx3cr1 injection group in comparison with the model group(P<0.05).The number of activated microglial cells in different retinal layers of the model group was significantly larger than that of the cx3cr1 injection group and the blank control group(both at P<0.05).Compared with the model group,the number of activated microglial cells around the retinal blood vessels was reduced significantly in the cx3cr1 injection group.The double fluorescence result of retinal vascular staining and activated microglial cells showed that the density of deep blood vessels in the blank control group and cx3cr1 injection group was significantly higher than that of the model group,and the number of microglial cells around superficial and deep retinal vessels was significantly larger in the model group than that of the cx3cr1 injection group(all at P<0.05).The relative vascular leakage rate of the blank control group,model group and cx3cr1 injection group were(100.0±4.7)%,(162.1±10.6)%and(130.5±9.5)%,respectively,and the overall difference was statistically significant(F=128.66,P<0.01).The relative vascular leakage rate in the cx3cr1 injection group was significantly lower than that in the model group(P<0.05).The qPCR result showed that the relative expression levels of vascular endothelial growth factor-A(VEGF-A),hypoxia inducible factor-1a(HIF-1α),tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)mRNA were significantly reduced in the retina of the cx3cr1 injection group in comparison with the model group(all at P<0.05).Conclusions Intravitreal injection of cx3cr1 can protect the vascular integrity of the retinal microcirculation system in RIR mice.