Fractalkine signaling in microglia is neuroprotective in the diabetic retina following lipopolysaccharide-induced activation

Abstract

Purpose: Microglia are known to mediate inflammation and neuronal damage during CNS inflammation, however their role in diabetic retinopathy remains unclear. Previous studies from our laboratory showed that CX3CR1 plays neuroprotective roles in models of acute innate immunity and neurodegeneration via inhibition of microglia activation. We hypothesize that in the diabetic retina and brain, absence of CX3CR1 induces rapid activation of microglia leading to pathogenic inflammation via release of inflammatory cytokines and reactive oxygen species potentiating neuronal cell death.

Methods: To understand the function of CX3CR1 in diabetes, we crossed the insulin2^akita strain (type 1 diabetic mouse model) with our Cx3cr1^gfp reporter mice to generate diabetic Cx3cr1^+/+ (Akita-WT) and diabetic Cx3cr1^-/- (Akita-KO) mice. To extend these studies, we challenged 8-10 week old non-diabetic WT or Cx3cr1^+/- (HET), Cx3cr1^-/- (KO) and Akita-WT and Akita-KO mice with a low dose of lipopolysaccharide (LPS; 20 µg/day) for four consecutive days to establish a low level endotoxemia.

Results: Confocal analysis of retinal whole mounts revealed that LPS induced a global hyper-activation and pro-inflammatory phenotype in KO-retinal microglia represented by amoeboid-cellular morphology, round-phagocytic-like cell clusters around blood vessels, and increased iNOS expression when compared to HET-retinas. This phenotype correlated with a significant increase in TUNEL+ neurons and microglia in the retina of KO mice (P<0.05). The degree of microglial activation and inflammation was exacerbated in Akita-KO mice following LPS challenge. Conversely, microglia in Akita-WT retinas resemble naive microglial morphology represented by a branched cellular appearance, few to no round-phagocytic-like cells and no cell clustering. Interestingly, this global activation was also observed in the brain of KO mice, which correlated with a decrease in NeuN+ cells in the visual and lateral cortices, superior colliculus and hippocampus (N=12 mice per genotype) .

Conclusions: These data support our hypothesis that fractalkine signaling is neuroprotective in the diabetic retina and brain, whereas microglia unable to control their effector function following an acute infection, can promote neuronal pathology which can lead to visual impairments.