No abstract

Preconditioning and postconditioning are mild ischemic exposures before or after severe injurious ischemia, respectively, that elicit endogenous neuroprotective responses. Molecular mechanisms of neuroprotection through preconditioning and postconditioning are not completely understood. Here we optimized the in vitro oxygen and glucose deprivation (OGD) models of preconditioning and postconditioning in primary cortical neuron cultures that allow the studies of the corresponding molecular mechanisms of neuroprotection. We found that the cortical cells preconditioned with a single 45-min OGD treatment administered 24 h prior to injurious 2 h OGD were robustly protected after both 3 h and 16 h of reperfusion. For the postconditioning treatment, we found that three cycles of 15 min OGD followed by 15 min reperfusion, applied immediately after injurious 2 h OGD and prior to complete reperfusion, resulted in effective neuroprotection at both 3 h and 16 h of reperfusion. Using real-time RT-PCR arrays focused on genes of the apoptosis and PI3K-Akt pathways, we found that injurious OGD mainly induced apoptosis-related and repressed PI3K-Akt pathway-related genes after either 3 h or 16 h of reperfusion. Preconditioning treatment resulted in the activation of both pro-survival and anti-apoptotic pathways after 3 h of reperfusion and mainly anti-apoptotic pathway after 16 h of reperfusion. In contrast, the activation of PI3K-Akt pathway mainly contributed to the neuroprotective effect by the postconditioning treatment after 3 h of reperfusion, but differential gene expression likely contributed minimally, if at all, to the neuroprotection observed after 16 h of reperfusion. Among the novel markers of neuroprotection, Nol3 gene upregulation was observed after 3 h of reperfusion following either preconditioning or postconditioning treatments and after 16 h of reperfusion following preconditioning treatment.

Mild ischaemic exposures before or after severe injurious ischaemia that elicit neuroprotective responses are referred to as preconditioning and post-conditioning. The corresponding molecular mechanisms of neuroprotection are not completely understood. Identification of the genes and associated pathways of corresponding neuroprotection would provide insight into neuronal survival, potential therapeutic approaches and assessments of therapies for stroke. The objectives of this study were to use global gene expression approach to infer the molecular mechanisms in pre- and post-conditioning-derived neuroprotection in cortical neurons following oxygen and glucose deprivation (OGD) in vitro and then to apply these findings to predict corresponding functional pathways. To this end, microarray analysis was applied to rat cortical neurons with or without the pre- and post-conditioning treatments at 3-h post-reperfusion, and differentially expressed transcripts were subjected to statistical, hierarchical clustering and pathway analyses. The expression patterns of 3,431 genes altered under all conditions of ischaemia (with and without pre- or post-conditioning). We identified 1,595 genes that were commonly regulated within both the pre- and post-conditioning treatments. Cluster analysis revealed that transcription profiles clustered tightly within controls, non-conditioned OGD and neuroprotected groups. Two clusters defining neuroprotective conditions associated with up- and downregulated genes were evident. The five most upregulated genes within the neuroprotective clusters were Tagln, Nes, Ptrf, Vim and Adamts9, and the five most downregulated genes were Slc7a3, Bex1, Brunol4, Nrxn3 and Cpne4. Pathway analysis revealed that the intracellular and second messenger signalling pathways in addition to cell death were predominantly associated with downregulated pre- and post-conditioning associated genes, suggesting that modulation of cell death and signal transduction pathways plays a role in the neuroprotection. A high degree of similarity in the pathways associated with the differentially expressed genes in the pre- and post-conditioning treatments suggests that similar molecular mechanisms may mediate their neuroprotective effects.