To date, miRNA expression studies on cerebral ischemia in both human and animal models have focused mainly on acute phase of ischemic stroke. In this study, we present the roles played by microRNAs in the spontaneous recovery phases in cerebral ischemia using rodent stroke models. Brain tissues were harvested at different reperfusion time points ranging from 0–168 hrs after middle cerebral artery occlusion using homologous emboli. MiRNA and mRNA expression profiles were investigated by microarray followed by multiple statistical analysis. Candidate transcripts were also validated by quantitative RT-PCR. Three specific groups of miRNAs were observed among a total of 346 differentially expressed miRNAs. miRNAs, miR-21, -142-3p, -142-5p, and -146a displayed significant upregulation during stroke recovery (48 hrs to 168 hrs) compared with those during acute phases (0 hrs to 24 hrs). On the other hand, an opposite trend was observed in the expression of miR-196a/b/c, -224 and -324-3p. Interestingly, miR-206, -290, -291a-5p and -30c-1*, positively correlated with the infarct sizes, with an initial increase up to 24hrs followed by a gradual decrease from 48 hrs to 168 hrs (R = 0.95). Taken together with the expression levels of corresponding mRNA targets, we have also found that Hedgehog, Notch, Wnt and TGF-β signaling pathways could play significant roles in stroke recovery and especially in neuronal repair.

Hypoxia inducible factor-1α facilitates cellular adaptation to hypoxic conditions. Hence its tight regulation is crucial in hypoxia related diseases such as cerebral ischemia. Changes in hypoxia inducible factor-1α expression upon cerebral ischemia influence the expression of its downstream genes which eventually determines the extent of cellular damage. MicroRNAs are endogenous regulators of gene expression that have rapidly emerged as promising therapeutic targets in several diseases. In this study, we have identified miR-335 as a direct regulator of hypoxia inducible factor-1α and as a potential therapeutic target in cerebral ischemia. MiR-335 and hypoxia inducible factor-1α mRNA showed an inverse expression profile, both in vivo and in vitro ischemic conditions. Given the biphasic nature of hypoxia inducible factor-1α expression during cerebral ischemia, miR-335 mimic was found to reduce infarct volume in the early time (immediately after middle cerebral artery occlusion) of embolic stroke animal models while the miR-335 inhibitor appears to be beneficial at the late time of stroke (24 hrs after middle cerebral artery occlusion). Modulation of hypoxia inducible factor-1α expression by miR-335 also influenced the expression of crucial genes implicated in neurovascular permeability, cell death and maintenance of the blood brain barrier. These concerted effects, resulting in a reduction in infarct volume bring about a beneficial outcome in ischemic stroke.