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

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 2 μl of sterile water.In the cx3cr1 injection group, the RIR model was established at 4 hours after the intravitreal injection (2 μl) of 0.2 μg/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.

Retina; Ischemia-reperfusion injury; Microcirculation; Microglia; Cytokines; Inbred C57BL mice