Dietary Regulation of Microglia Recruitment in Retinas From Omega-3 or Omega-6 Lcpufa Fed Mice

Abstract

Purpose: : Ocular neovascularization is the most common cause of blindness in all age groups and evidence suggests that this pathology is mediated in part through inflammation. Mammals must rely on a dietary supply of ω-3 long-chain polyunsaturated fatty acids (LCPUFAs), which are essential nutrients known to have anti-inflammatory effects. We have previously reported that ω-3 LCPUFAs (docosahexaenoic acid and eicosapentaenoic acid) have a significant protective effect in retinopathy and that retinal microglia play a key role in this process. Mice given a ω-3 LCPUFA diet exhibited enhanced vessel regrowth and reduced neovascularization following oxygen exposure. To further investigate the dietary regulation of microglia recruitment, we performed Illumina microarray analysis on retinas from mice on diets enriched in either ω-3 or ω-6 LCPUFAs.

Methods: : Beginning at postnatal day 0 (P0), mothers were fed diets enriched with either ω-3 or ω-6 LCPUFAs. To induce vessel loss, and subsequent pathological neovascularization, nursing mothers and pups were exposed to 75% oxygen from P7 to P12 and returned to room air. Retinas from each group were isolated and flash frozen using RNase-free techniques. Total RNA was extracted and prepared for Illumina microarray analysis using the Mouse-ref 6 chip. Data were acquired using the Illumina BeadStudio software and further analysis was performed using Significance Analysis of Microarray (SAM), and J-Express Pro 2.7 software. Differentially expressed genes were validated using real-time PCR.

Results: : Lectin stained, flat-mounted retinas from ω-3 or ω-6 fed mice with oxygen-induced retinopathy were assessed for severity of vaso-obliteration (VO) and neovascularization (NV). As previously shown both VO and NV were more severe in ω-6 fed mice (%VO: 19.0 ± 1.0; %NV: 10.2 ± 0.7) than in the ω-3 fed counterparts (%VO: 14.7 ± 1.5, p < 0.05; %NV: 2.9 ± 0.5, p < 0.001). Up-regulation of activated macrophage markers, pro-inflammatory mediators, angiogenic mediators, as well as adhesion markers required for microglia recruitment existed in retinas of ω-6 fed mice compared to their ω-3 fed counterparts.

Conclusions: : These findings suggest that up-regulation of adhesion markers in ω-6 fed mice may be responsible for recruiting inflammatory cells and activated macrophages, which in turn stimulate increased angiogenic signals resulting in more severe neovascularization.

Keywords: microglia • retina • gene microarray