Neuronal KATP channels mediate hypoxic preconditioning and reduce subsequent neonatal hypoxic–ischemic brain injury

Abstract

Neonatal hypoxic–ischemic brain injury and its related illness hypoxic–ischemic encephalopathy (HIE) are major causes of nervous system damage and neurological morbidity in children. Hypoxic preconditioning (HPC) is known to be neuroprotective in cerebral ischemic brain injury. KATP channels are involved in ischemic preconditioning in the heart; however the involvement of neuronal KATP channels in HPC in the brain has not been fully investigated. In this study, we investigated the role of HPC in hypoxia–ischemia (HI)-induced brain injury in postnatal seven-day-old (P7) CD1 mouse pups. Specifically, TTC (2,3,5-triphenyltetrazolium chloride) staining was used to assess the infarct volume, TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling) to detect apoptotic cells, Western blots to evaluate protein level, and patch-clamp recordings to measure KATP channel current activities. Behavioral tests were performed to assess the functional recovery after hypoxic–ischemic insults. We found that hypoxic preconditioning reduced infarct volume, decreased the number of TUNEL-positive cells, and improved neurobehavioral functional recovery in neonatal mice following hypoxic–ischemic insults. Pre-treatment with a KATP channel blocker, tolbutamide, inhibited hypoxic preconditioning-induced neuroprotection and augmented neurodegeneration following hypoxic–ischemic injury. Pre-treatment with a KATP channel opener, diazoxide, reduced infarct volume and mimicked hypoxic preconditioning-induced neuroprotection. Hypoxic preconditioning induced upregulation of the protein level of the Kir6.2 isoform and enhanced current activities of KATP channels. Hypoxic preconditioning restored the HI-reduced PKC and pAkt levels, and reduced caspase-3 level, while tolbutamide inhibited the effects of hypoxic preconditioning. We conclude that KATP channels are involved in hypoxic preconditioning-induced neuroprotection in neonatal hypoxic–ischemic brain injury. KATP channel openers may therefore have therapeutic effects in neonatal hypoxic–ischemic brain injury.