Transcranial direct current stimulation enhances the protective effect of isoflurane preconditioning on cerebral ischemia/reperfusion injury: A new mechanism associated with the nuclear protein Akirin2

Aims: Ischemic stroke is a major cause of disability and mortality worldwide. Transcranial direct current stimulation (tDCS) and isoflurane (ISO) preconditioning exhibit neuroprotective properties. However, it remains unclear whether tDCS en-hances the protective effect of ISO preconditioning on ischemic stroke, and the un-derlying mechanisms are yet to be clarified.Method: A model of middle cerebral artery occlusion (MCAO), a rat ischemia-reper-fusion (I/R) injury model, and an in vitro oxygen-glucose deprivation/re-oxygenation (O/R) model of ischemic injury were developed. ISO preconditioning and tDCS were administered daily for 7 days before MCAO modeling. Triphenyltetrazolium chloride staining, modified neurological severity score, and hanging-wire test were conducted to assess infarct volume and neurological outcomes. Untargeted metabolomic ex-periments, adeno- associated virus, lentiviral vectors, and small interfering RNA tech-niques were used to explore the underlying mechanisms.Results: tDCS/DCS enhanced the protective effects of ISO pretreatment on I/R injury-induced brain damage. This was evidenced by reduced infarct volume and improved neurological outcomes in rats with MCAO, as well as decreased cortical neuronal death after O/R injury. Untargeted metabolomic experiments identified oxi-dative phosphorylation (OXPHOS) as a critical pathological process for ISO- mediated neuroprotection from I/R injury. The combination of tDCS/DCS with ISO precondi-tioning significantly inhibited I/R injury- induced OXPHOS. Mechanistically, Akirin2, a small nuclear protein that regulates cell proliferation and differentiation, was found to decrease in the cortex of rats with MCAO and in cortical primary neurons subjected to O/R injury. Akirin2 functions upstream of phosphatase and tensin homolog de-leted on chromosome 10 (PTEN). tDCS/DCS was able to further upregulate Akirin2 levels and activate the Akirin2/PTEN signaling pathway in vivo and in vitro, with ISO pretreatment alone, thereby contributing to the improvement of cerebral I/R injur y.Conclusion: tDCS treatment enhances the neuroprotective effects of ISO precondi-tioning on ischemic stroke by inhibiting oxidative stress and activating Akirin2-PTEN signaling pathway, highlighting potential of combination therapy in ischemic stroke.