Effect of Hyperglycemic and hypoxic stresses on the primary cultures of retinal neuron and glial populations: a model system to understand the role of glia in diabetic retinopathy

Abstract

Purpose : Retinal gliosis has been shown to be one of the key features in the DR eyes by experimental and immunohistological methods. A human cell culture model system mimicking retinal composition could help in understanding the initial mechanisms of glial activation during disease progression. The aim of present study was to establish a mixed primary culture of retinal glia and neurons from the cadaveric retina for studying their role under the diabetic conditions.

Methods : Primary cultures of retinal neurons and glia were established from human cadaveric retina. The cells from the initial passages (P0-P2) were characterized by immunofluorescence and semi-quantitative PCR. These cells were subjected to a calculated level of hypoxia and hyperglycemia by treating them with CoCl₂ and D- glucose. Live cell imaging was done with Fluo-4 AM Ca²⁺ dye to check the activation of cells under stress. RNA and proteins were extracted from the harvested cells after the stress treatment and analyzed for the expression of inflammatory and glial markers by quantitative PCR. Comparisons of GFAP and Iba -1 protein expression (n=60) and Kernel density estimations (n=160) to analyze the spike response of Ca²⁺ were performed in treated versus untreated cells.

Results : Different types of retinal neurons and glial cells in the mixed retinal culture showed positive expression for the specific markers (GFAP, GS, Nestin, Iba-1 and Vimentin). Increased expression of angiogenic and neurodegenerative cytokines were observed in treated cells compared to untreated by real time PCR. Live cell imaging with Flu-4 AM Ca²⁺dye clearly showed elevated Ca²⁺signal transduction in the treated cells. Kernel density estimation of Ca²⁺ stained cells further showed higher spiking response and augmented network activity in the treated culture compared to untreated. Compared to untreated cells, 3 and 1.6 fold increase in GFAP protein (n=60) and 4-fold and 2 -fold increase of Iba-1 expression was noted in cells exposed to hypoxia and hyperglycemia respectively.

Conclusions : Hyperactivation of the cultured cells under stress and marked changes in the glial markers confirmed the role of glia in DR progression. The preliminary findings of this study indicate that the proposed culture system can serve as a model for detailed evaluation on the role of glia in DR progression.

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