Objectives: The metabolic pathophysiology of ischemic stroke remains poorly understood. We performed a comparative omics analysis to identify the effects of cerebral ischemia on metabolism of cerebral cortex in the rat model with middle cerebral artery occlusion (MCAO).
Methods: MCAO was induced with suture occlusion technique. After the desired period of MCAO (30, 60, 120min), the rats were sacrificed. Tissue samples from the ischemic lesion were collected. Sham operated rats were treated in the same way except MCAO. Water-soluble metabolites were extracted and measured by gas-chromatography/mass-spectrometry (GC/MS). The obtained data were analyzed using multivariate statistics to explore metabolic pathways involved in ischemia. The associated metabolic enzymes were investigated with real-time polymerase chain reaction (RT-PCR) and Immunoblot analysis.
Results: Metabolic profiling by GC/MS analysis showed clear separation between the ischemia and control group (Figure A, B). The decrease of fructose 6-phosphate and ribulose 5-phosphate suggested enhancement of the pentose phosphate pathway (PPP) during cerebral ischemia. Transcriptional profiling by microarray hybridization indicated that Toll-like receptor and mitogen-activated protein kinase (MAPK) signaling pathway were upregulated during cerebral ischemia. In relation to PPP, upregulation of heat shock protein 27 (HSP27) was observed in the MAPK signaling pathway and was confirmed through RT-PCR. Immunoblotting showed a slight increase in HSP27 protein expression and a marked increase in HSP27 phosphorylation (FigureC). Corresponding upregulation of glucose 6-phosphate dehydrogenase (G6PD) activity and an increase in the NADPH/NAD+ ratio were also observed (Figure D, E).
Conclusions: G6PD activation via ischemia-induced HSP27 phosphorylation may be part of an endogenous antioxidant neuroprotection mechanism during the earliest stages of ischemia.