The Effects of Age and Cx₃cr1 Deficiency on Retinal Microglia in theIns2^AkitaDiabetic Mouse

Abstract

PURPOSE. Diabetic retinopathy (DR) is a major cause of visual impairment in developed countries. While DR has been described classically as a microvascular disease, recent evidence suggests that changes to retinal microglia are an early feature of retinopathy. In our study, we assessed changes in microglial distribution and morphology in vivo and ex vivo in a mouse model of non-proliferative DR, and further examined effects of age and the absence of the functional chemokine receptor Cx 3 cr1 on the progression of these changes. METHODS.To isolate the effects of the three variables: diabetic status, age, and role of Cx 3 cr1, the Ins2Akita mouse was crossed with Cx 3 cr1-eGFP reporter mice. Eyes were assessed clinically in vivo at 10, 20, 30, and 46 weeks of age, and the retinal structure and arrangement of GFP þ microglia was examined ex vivo using whole mount immunofluorescence staining and confocal microscopy.RESULTS. Clinical examination of the fundus, vasculature, or GFP þ microglial distribution did not reveal any macroscopic changes related to diabetic status: however, ex vivo microscopic analysis revealed alterations in microglial network organization, and evidence of cell shape changes regarded classically as signs of activation, in Ins2Akita mice from 10 weeks of age. These changes were exacerbated in older diabetic mice whose microglia lacked Cx 3 cr1 (Ins2 Akita Cx 3 cr1 gfp/gfp mice). Diabetic status and Cx 3 cr1 deficiency led to accumulations of Iba-1 þ hyalocytes (vitreal macrophages) and subretinal macrophages.CONCLUSIONS. These data showed that changes to murine retinal microglia occur in response to systemic diabetic status in the absence of overt retinopathy and inflammation. These changes are exaggerated in mice lacking Cx 3 cr1, suggesting fractalkineCx 3 cr1 interactions may have a role in early neuronal changes in preproliferative DR. (Invest Ophthalmol Vis Sci.

Bilateral retinal microglial response to unilateral optic nerve transection in rats

“…Though diverse descriptions of layer separation have been shown by other groups with different investigative methods (Santos et al, 2008, Bosco et al, 2011, Kezic et al, 2013, Choe et al, 2014, microglia were found, in the present study, to be mainly located in three separate layers by epifluorescence microscopy on retinal flat mounts: the RGC layer (GCL, including the nerve fiber layer), inner plexiform layer (IPL), and outer plexiform layer (OPL). Studies have shown that the most obvious microglial proliferation is detected in the GCL after ON transection, whereas microglia in the OPL remain undisturbed (Garcia-Valenzuela et al, 2005).…”

Glia–neuron interactions in the mammalian retina

“…In the optic nerve, microglia ramifications appear to be orientated along the fibres. This orientation is disorganized in an experimental model of diabetes , and the distribution and organization of microglia within the retinal layers is altered in a variety of pathological states, when changes in cell shape can be observed Kezic et al, 2013). Moreover, when RGCs are labelled with a retrograde vital fluorescent dye in an experimental axotomy, microglia containing RGC debris can be detected months after the injection of marker, indicating both the phagocytotic capacity of microglia and their relative longevity (Thanos et al, 1994).…”

Comprehensive analysis of mouse retinal mononuclear phagocytes

“…In fact, most recent in situ analyses of ramified and reactive retinal microglia rely on Iba1 staining [26][27][28][29][30] as the marker allows consistent and reliable staining of microglia somata and their processes in a broad spectrum of different activation phenotypes [31][32][33] . In general, a combined in situ analysis of retinal microglia using sections and flat mounts as presented in our protocol is recommended.…”