Microglia Activation and Polarization Associated with Retinal Inflammation and Degeneration in rd1 mouse model

Abstract

Purpose : Microglia has been recognized to be a special type of immune cells in retina. However, the role and mechanism of microglia in retinal degeneration disease are poorly understood. This study was to explore the activation and phenotype of microglia in rd1 mice, a widely used animal model of retinitis pigmentosa.

Methods : Rd1 (Pde6b^rd1/rd1) mice were used for study with wild-type C57BL/6 mice of the same ages as controls. Retinal degeneration condition were observed by the fundus imaging, ERG and OCT. Eyeballs and retina of rd1 mice at different postnatal days (P7, P14, P21, P28, P56 and P180) were collected and prepared for cryosections, whole mount, retinal cells and cell lysate. The microglia maker Iba-1, activation marker CD68 and CX3CR1, and phenotype markers CD86, CD206 and CD16/32, as well as pro-inflammatory cytokines were evaluated by immunofluorescent staining, flow cytometry, RT-qPCR, ELISA and Western blot analysis.

Results : The retinal degeneration was observed at early age of P7, developed progressively with thinner retina at P14 and P21, and lost most photoreceptors at P28 in rd1 mice when compared with C57 mice. The expression of CD68 and CX3CR1 increased significantly with ages at P7, P14, P21 and P28 in rd1 retinae, indicating microglia activation at the acute stage. Morphologically, activated microglia was characterized by round shape with short dendrites in rd1 mice. The number of microglia increased significantly by ages with the peak levels at p14. Flow cytometry revealed that the percentage of CD86⁺CD206^- microglia (M1 phenotype) increased markedly during P14 to P28 in rd1 retinae compared to C57 mice. The CD16/32⁺ (M1 marker) microglia was immune-localized mainly in ONL at P14 while in INL at P28, suggesting potential migration to the degeneration area. Furthermore, RT-qPCR revealed the higher expression of pro-inflammatory cytokines (TNF-a, IL-6 and CCL2) in rd1 mice than C57 mice.

Conclusions : Our findings uncover a novel mechanism that microglia is activated and polarized to M1 phenotype that is associated with stimulated pro-inflammatory cytokines during retinal degenerative process in rd1 mice. This study sheds lights on pathogenesis of retinal regeneration disease, and may provide novel targets for therapeutic potential.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.