Role of PKC zeta in microglia/ macrophages trafficking during diabetic retinopathy in Goto Kakizaki rats

Abstract

Purpose: : Diabetic retinopathy is associated with ocular inflammation leading to retinal barriers breakdown. We investigated the molecular mechanisms involved in microglia/macrophages trafficking in the retina and the role of protein kinase C zeta (PKCζ) in this process.

Methods: : We used Goto Kakizaki (GK) rats, a model for spontaneous type 2 diabetes. The study was performed on 3 stages: 2, 5 and 12 months of hyperglycemia. To demonstrate that PKCζ activity is involved in trafficking, control and diabetic rats were given an intravitreal injection of PKCζ inhibitor. Immunostaining on retina flatmounts and cryosections were performed to determine the activation and migration of microglia/macrophages in retina and the modification of the retinal pigment epithelium (RPE).

Results: : We showed an increase of activated microglia/macrophage cells in the retina with an accumulation in subretinal space and RPE alteration, after 12 months of hyperglycemia. Surprisingly, after 5 months of diabetes, we observed F-actin remodeling, with a central actin ring forming a "pore" in some RPE cells, without alteration of their junctions. ICAM-1, CAV-1 and PKCζ were localized in the pore structure. RPE pores were quantified in diabetic rats at 2, 5, and 12 months of hyperglycemia and in age-matched controls The number of pores significantly increased from 5 to 12 months, in diabetic rats and significantly decreased at 12 months, while pore number increased with age in control rats.The intravitreous injection of PKC zeta inhibitor in 12 months old diabetic rats impaired microglia cells migration in the outer retina. Inhibitor supressed the expression of iNOS and induced a change in microglia morphology showing a resting dendritic shape.

Conclusions: : We show for the first time that a physiological transcellular pathway takes place through RPE cells and contributes to microglia/macrophages retinal trafficking. Chronic hyperglycemia causes alteration of this pathway and subsequent subretinal accumulation of activated microglia/macrophages that may participate to the pathogenesis of diabetic retinopathy. We have shown that inhibition of PKCζ activity impaired microglia/ macrophage cells migration in the outer retina. Furthermore, inhibition of PKCζ activation significantly reduced pore formation in RPE from treated diabetic and control rats compared to RPE from untreated diabetic and control rats.

Keywords: diabetic retinopathy • retinal pigment epithelium • microglia