MECHANISMS OF ISCHEMIC PRECONDITIONING IN THE RAT RETINA in VIVO

Abstract

S241 INTRODUCTION: The intact in vivo rat retina provides an excellent model for examining the functional consequences of ischemia and the endogenous mechanisms of neuroprotection in the CNS [1]. We showed previously that ischemic preconditioning (IPC) completely prevents functional and histological damage after severe ischemia in this setting [1]. Based on its critical role in retinal ischemia [2,3], adenosine was hypothesized as a possible mediator. In addition, because inhibition of protein synthesis prevents protection induced by IPC [1], we studied the protein profiles of the retina after IPC to identify molecular factors that may be responsible. METHODS: IPC (5 min of ischemia) was induced 24 h before 60 min of ischemia produced by ligation of the central retinal artery in anesthetized rats. The electroretinogram (ERG) was measured to assess retinal function for up to 7 days later. Results were analyzed using ANOVA with P < 0.05 considered significant. Before or immediately after IPC, rats received i.p. injections of the specific adenosine A1 receptor antagonist DPCPX (0.45-4.5 mg/kg) Protein expression was studied in other groups subjected to the same time sequence of IPC and ischemia, but without A1 receptor blockade. RESULTS: DPCPX attenuated preconditioning protection in a dose-dependent manner (Figure 1). Protein gel electrophoresis identified a number of proteins in the 70-100 kDa range in preconditioned retinas. Among these, we suspected heat shock protein (HSP70). Western blotting (Figure 2) using a monoclonal antibody to both constitutive (HSPc, arrowhead) and inducible HSP (HSPi, small arrow) revealed increased expression of both at 72 h after preconditioning and ischemia.Figure 1: DPCPX blocks IPC protection. X-axis: Post-ischemic followup time. Y-axis: ERG b wave recovery. * = P < 0.05 vs vehicle.Figure 2: HSP70 protein is induced by IPCDISCUSSION: We showed a critical role for the adenosine A1 receptor in the powerful and exciting functional protection afforded by IPC. Heat shock proteins may contribute to protection as well through as yet unknown mechanisms. Grant support: NIH EY10343 to Dr Roth