Involvement of IL-6R and GP130 in Retinal Ischemia-reperfusion in Rats

Abstract

Abstract: : Purpose: Previously, we demonstrated an upregulation of IL-6 mRNA after ischemia reperfusion (I/R) injury to the rat retina and that IL-6 had a neuroprotective effect. To characterize the targets of IL-6 action, we examined apoptosis after I/R injury and the spatial distribution of IL-6 receptor (IL-6R) and its associated protein, GP130. Methods: Cannulas, connected to a saline column that generated a pressure of 110 mmHg, were inserted into the anterior chambers of adult Lewis albino rats to generate retinal ischemia. After 1 hour, the cannulas were removed to restore retinal reperfusion. Groups of animals were euthanized at 0, 4, 12, 18 or 48 hours post reperfusion and their eyes were enucleated. The retinas of some eyes were dissected and homogenized for semiquantitative Real Time RT-PCR analysis of IL-6R (n=3) and GP130 (n=3). Other eyes were fixed, processed, embedded in paraffin and sectioned. Immunohistochemistry was performed using anti-IL-6R (n=3) and anti-GP130 (n=3) antibodies. Apoptosis was studied by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick end-labeling (TUNEL) method. Results: Semiquantitative Real Time RT-PCR analysis demonstrated that IL-6R mRNA was significantly upregulated at 12 hours (13.4 fold increase ± 2.4, p<0.001, Tukey’s test) when compared to normal and IL-6 mRNA returned to normal levels by 48 hours. In contrast, there were no significant changes in GP130 gene expression throughout this time course. IL-6R and GP130 protein localized to cells within the retinal ganglion cell layer and inner plexiform layer that resemble retinal ganglion or amacrine cells. In addition, these IL-6R and GP-130 positive cells did not colocalize with TUNEL positive cells. Conclusions: Upregulation of IL-6R mRNA and its protein may serve as an endogenous neuroprotective response by the retina to I/R injury.

Keywords: ischemia • cytokines/chemokines • neuroprotection