The Effects Of Age, Cx3cr1 Deficiency And Hyperglycaemia On Retinal Macrophages In Ins2Akita Mice

Abstract

Purpose: : Diabetic retinopathy (DR) is a major cause of visual impairment, being the third commonest cause of blindness in developed countries. Whilst DR has classically been described as a microvascular disease, recent evidence suggests that changes to resident retinal macrophages are an early feature of retinopathy. Here, we sought to characterize the retinal microglial changes associated with hyperglycaemia across various ages in a mouse model of non-proliferative or background DR. We further sought to investigate whether these changes are influenced by the absence of the monocytic chemokine receptor Cx3cr1.

Methods: : Ins2^Akita mice bear a point mutation in the insulin2 gene, resulting in spontaneous development of hyperglycaemia by 4 weeks of age. For this study, Ins2^Akita mice were crossed with C57BL/6 Cx3cr1^+/gfp and Cx3cr1^gfp/gfp mice, allowing for the clinical (Micron III Fundus Camera) and microscopic visualization of Cx3cr1⁺ microglia. Eyes were clinically examined at 10, 20 and 30 weeks of age and processed for whole mount immunoflurescence staining and confocal microscopy. Analysis of changes in microglia, Iba-1⁺ hyalocytes and subretinal macrophages were performed across genotypes and age groups.

Results: : Whilst there were no overt changes to retinal microglia observed by clinical assessment, analysis of retinal whole mounts by confocal microscopy revealed a marked disruption of the regular microglial network in the inner and outer plexiform layers of the retina in Ins2^Akita mice from 20 weeks of age. These microglial changes were similar in both Cx3cr1^+/gfp and Cx3cr1^gfp/gfp Ins2^Akita mice, however, the absence of Cx3cr1 accelerated the age-related accumulation of Iba-1⁺ subretinal macrophages. Both Cx3cr1-deficiency and hyperglycaemia significantly increased the cell density of Iba-1⁺ hyalocytes.

Conclusions: : Prolonged hyperglycaemia leads to disruption of retinal microglial networks which is independent of Cx3cr1 signaling. However, Cx3cr1 deficiency influences the density of hyalocytes and subretinal macrophages prior to the detection of retinopathy in the Ins2^Akita mouse. Thus, changes in these macrophages populations at the retinal interface may be predictive of functional activation of retinal parenchymal microglia in a hyperglycaemic retinal microenvironment.

Keywords: microglia • diabetic retinopathy • imaging/image analysis: clinical