Genomic analysis of ischemic preconditioning in adult rat hippocampal slice cultures

Abstract

Understanding endogenous mechanisms of neuroprotection may have important clinical applications. It is well established that brain tissue becomes more resistant to ischemic injury following a sublethal ischemic insult. This process, called ischemic preconditioning (IPC), can be induced in adult rat hippocampal slice cultures by a brief oxygen–glucose deprivation (OGD) [Hassen, G.W., Tian, D., Ding, D., Bergold, P.J., 2004. A new model of ischemic preconditioning using young adult hippocampal slice cultures. Brain Res. Brain Res. Protoc. 13, 135–143]. We have analyzed the changes in gene expression brought about by IPC in this model in order to understand the mechanisms involved. Total RNA was isolated at different time points following a brief OGD (3, 6 and 12 h) and used to probe genome-wide expression microarrays. Genes were identified that were significantly up- or down-regulated relative to controls. We placed genes that were differentially expressed into statistically significant groups based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO) terms. Genes involved in signal transduction, transcription, and oxidative phosphorylation are differentially expressed at each time point. The analysis demonstrates that alterations in signaling pathways (TGF-β, Wnt, MAPK, ErbB, Toll-like receptor, JAK-STAT, VEGF) consistently accompany IPC. RT-PCR was used to confirm that members of these signaling pathways are regulated as predicted by the microarray analysis. We verified that protein translation following OGD is necessary for IPC. We also found that blocking the NMDA receptor during OGD does not significantly inhibit IPC in this model or produce large changes in gene expression. Our data thus suggests that changes in signaling pathways and their down-stream targets play an important role in triggering endogenous neuroprotection.

Introduction

Brief exposure to sublethal ischemia can induce tolerance to a subsequent, otherwise lethal ischemic insult in many tissues including brain (Dirnagl et al., 2003, Gidday, 2006, Steiger and Hanggi, 2007). Understanding mechanisms of endogenous protection from cerebral ischemia may lead to novel treatments for brain injury, especially stroke. Ischemic tolerance or ischemic preconditioning (IPC) can be induced in adult rat hippocampal slice cultures by a brief period of oxygen and glucose deprivation (OGD) (Hassen et al., 2004, Pellegrini-Giampietro et al., 1999). The exact mechanisms of IPC are unknown, but multiple modifications have been hypothesized including alterations in signal transduction, hypoxia-inducible factors, and inflammatory mediators (Gidday, 2006). In order to explore these possibilities, we have analyzed changes in gene expression after IPC in adult rat hippocampal slice cultures and characterized these transcripts using genome-wide DNA microarrays. Adult rat hippocampal slice cultures are an attractive model because they are a stable, well-defined population of neurons and glial cells that maintain much of the normal connectivity and architecture of the brain.

This is the first study to fully characterize IPC-related changes in gene expression in adult rat hippocampal slice cultures with a genome-wide DNA microarray. DNA microarray analysis allowed us to identify differentially expressed genes following IPC. Using bioinformatics techniques, we were then able to categorize these genes into functional groups and known pathways. The purpose of this analysis is to gain insight into the underlying mechanisms of neuroprotection associated with IPC.