Defibrinogenation Ameliorates Retinal Microgliosis and Inflammation in A CX3CR1-Independent Manner

Abstract

Microglia-mediated inflammation plays a significant role in neuronal and vascular damage in diabetic retinopathy (DR), but the mechanism linking inflammation, neurodegeneration, and impaired vascular integrity is still unclear. Previous studies from diabetic mouse models showed accumulation of fibrinogen at vessel lesions surrounded by perivascular microglial clusters. The purpose of this study was to evaluate whether the pathological hallmarks of gliosis and vascular aberrations characterized in diabetic animal models are consistent with those in diabetic human retinas, and to assess the effects of the defibrinogenating agent ancrod in retinal pathology and visual acuity in a two-hit inflammatory diabetic mouse model. Post-mortem human eyes were assessed for retinal and inflammatory gene expression by quantitative PCR. Immunohistochemical analyses in human and murine retinas were performed using markers of gliosis, vascular integrity, and fibrinogen deposition. An inflammatory microenvironment, with microgliosis and microaneurysms, was found in the diabetic human eye. Microglial activation, fibrinogen deposition, and axonal loss were also observed in the diabetic murine retina. Ancrod treatment correlated with reduced microgliosis, less fibrinogen deposition, and reduced pro-inflammatory cytokine levels in diseased retinal tissues. Together, these data suggest that fibrinogen contributes to microglia-mediated inflammation in the diabetic retina. Since retinal microgliosis, vascular pathology, and vision deficits manifest in diabetic mice irrespective of CX3CR1 genotype, our results indicate that defibrinogenation can dampen systemic neuroinflammation and vascular insults, thereby improving vision at early stages of diabetes. Summary Statement Diabetic human and murine retinas revealed pronounced microglial morphological activation and vascular abnormalities associated with inflammation. Pharmacological fibrinogen depletion using ancrod dampened microglial morphology alterations, resolved fibrinogen accumulation, rescued axonal integrity, and reduced inflammation in the diabetic murine retina.

Models of microglia depletion and replenishment elicit protective effects to alleviate vascular and neuronal damage in the diabetic murine retina

“…6 F, I) revealed a reduction in microglial activation associated gene clusters ( Ly86 , Cx3cr1 , Siglech , Clec7a , Trem2 , Fcgr3 , Csf1r , Fcrg1 ) indicative that these changes in microglia morphology correlate with transcriptional reprograming. Additionally, it was shown that fibrinogen depletion using the defibrinogenating agent Ancrod, ameliorated reactive microgliosis and release of proinflammatory cytokines in the diabetic retina [ 45 ]. Complementary to these findings, depleting microglia reduced fibrinogen deposition in the diabetic retina (Figs.…”

Pharmacological depletion of microglia alleviates neuronal and vascular damage in the diabetic CX3CR1-WT retina but not in CX3CR1-KO or hCX3CR1I249/M280-expressing retina

“…Four months diabetic mice ( Figure 2 ) did not receive LPS treatment, due to their increased sensitivity to cachexia at this more chronic stage of diabetes. Acute LPS treatment can cause hypoglycemia and induce large variation in circulating glucose levels in diabetic mice ( 37 , 38 ). Therefore, to avoid large fluctuations in glucose levels and to prevent masking hyperglycemia, 10 weeks diabetic mice, ( Figures 3 – 5 and Supplementary Figures 2 – 6 ) received one daily injection of 0.08mg/kg LPS for 2 consecutive days prior to euthanasia as previously characterized ( 26 ).…”

Fractalkine isoforms differentially regulate microglia-mediated inflammation and enhance visual function in the diabetic retina

“…A visual acuity assessment, featuring a two-choice discrimination task (Fig. 5 A, B) was utilized to determine if mice could distinguish between two distinct visual cues [ 5 , 29 ]. ND WT and FKN KO PBS-treated animals were tested under visible and infrared light to establish a baseline of visual acuity readouts.…”

“…Individually housed mice were food deprived to 85% of their ad libitum body weight to increase motivation for the task. During the food deprivation period, mice were shaped to search for food reward and a cocoa puff (Cocoa Krispies, Kellogg’s) buried in a 30 mL cup, which limited their food intake prior to the commencement of the experiment [ 5 ]. Both the food pellet and the cocoa cereal crumb were hidden under cumin-scented, fine-grain woodchip bedding (Sani-Chip 146 IRR, LabSupply), to prevent the use of olfactory/reward associations.…”

“…Diabetic retinopathy (DR), a common and severe complication of diabetes, leads to vision impairment and ocular morbidity in roughly 200 million patients globally [ 1 ]. Our group and others have shown that metabolic disease, partly due to the release of lipopolysaccharide (LPS), which induces low-grade systemic inflammation, influenced the severity of diabetes, [ 2 – 5 ] and exacerbated retinal damage in experimental DR [ 4 ]. Other studies suggest that high serum endotoxemia precedes the development of type 2 diabetes [ 6 , 7 ].…”