Disruption of Fractalkine Signaling Leads to Microglial Activation and Neuronal Damage in the Diabetic Retina.
Cardona Sandra M,Mendiola Andrew S,Yang Ya-Chin,Adkins Sarina L,Torres Vanessa,Cardona Astrid E
Fractalkine (CX3CL1 or FKN) is a membrane-bound chemokine expressed on neuronal membranes and is proteolytically cleaved to shed a soluble chemoattractant domain. FKN signals via its unique receptor CX3CR1 expressed on microglia and other peripheral leukocytes. The aim of this study is to determine the role of CX3CR1 in inflammatory-mediated damage to retinal neurons using a model of diabetic retinopathy. For this, we compared neuronal, microglial, and astroglial densities and inflammatory response in nondiabetic and diabetic (Ins2(Akita)) CX3CR1-wild-type and CX3CR1-deficient mice at 10 and 20 weeks of age. Our results show that Ins2(Akita) CX3CR1-knockout mice exhibited (a) decreased neuronal cell counts in the retinal ganglion cell layer, (b) increased microglial cell numbers, and (c) decreased astrocyte responses comparable with Ins2(Akita) CX3CR1-Wild-type mice at 20 weeks of age. Analyses of the inflammatory response using PCR arrays showed several inflammatory genes differentially regulated in diabetic tissues. From those, the response in Ins2(Akita) CX3CR1-deficient mice at 10 weeks of age revealed a significant upregulation of IL-1β at the transcript level that was confirmed by enzyme-linked immunosorbent assay in soluble retinal extracts. Overall, IL-1β, VEGF, and nitrite levels as a read out of nitric oxide production were abundant in Ins2(Akita) CX3CR1-deficient retina. Notably, double immunofluorescence staining shows that astrocytes act as a source of IL-1β in the Ins2(Akita) retina, and CX3CR1-deficient microglia potentiate the inflammatory response via IL-1β release. Collectively, these data demonstrate that dysregulated microglial responses in absence of CX3CR1 contribute to inflammatory-mediated damage of neurons in the diabetic retina.
The effects of age and Cx3cr1 deficiency on retinal microglia in the Ins2(Akita) diabetic mouse.
Kezic Jelena Marie,Chen Xiangting,Rakoczy Elizabeth P,McMenamin Paul G
Investigative ophthalmology & visual science
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 Ins2(Akita) 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(+) microglia was examined ex vivo using whole mount immunofluorescence staining and confocal microscopy. RESULTS:clinical examination of the fundus, vasculature, or GFP(+) 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 Ins2(Akita) 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(+) 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 fractalkine- Cx(3)cr1 interactions may have a role in early neuronal changes in preproliferative DR.