G-CSF ameliorates neuronal apoptosis through GSK-3β inhibition in neonatal hypoxia-ischemia in rats

Granulocyte-colony stimulating factor (G-CSF), a growth factor, has known neuroprotective effects in a variety of experimental brain injury models. Herein we show that G-CSF administration attenuates neuronal apoptosis after neonatal hypoxia-ischemia (HI) via glycogen synthase kinase-3 beta (GSK-3 beta) inhibition. Ten day old Sprague-Dawley rat pups (n = 157) were subjected to unilateral carotid artery ligation followed by 2.5 h of hypoxia or sham surgery. HI animals received control siRNA, GSK-3 beta siRNA (4 mu L/pup), G-CSF (50 mu g/kg), G-CSF combined with 0.1 or 0.4 nM G-CSF receptor (G-CSFR) siRNA, phosphatidylinositol 3-kinase (PI3K) inhibitor Wortmannin (86 ng/pup), or DMSO (vehicle for Wortmannin). Pups were euthanized 48 h post-HI to quantify brain infarct volume. G-CSFR, activated Akt (p-Akt), activated GSK-3 beta (p-GSK-3 beta), Cleaved Caspase-3 (CC3), Bcl-2, and Bax were quantified using Western blot analysis and the localizations of each was visualized via immunofluorescence staining. Neuronal cell death was determined using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Our results showed p-GSK-3 beta increased after HI until its peak at 48 h post-ictus, and both GSK-3 beta siRNA and G-CSF administration reduced p-GSK-3 beta expression, as well as infarct volume. p-GSK-3 beta and CC3 were generally co-localized in neurons. Furthermore, G-CSF increased p-Akt expression and the Bcl-2/Bax ratio and also decreased p-GSK-3 beta and CO expression levels in the ipsilateral hemisphere, which were all reversed by G-CSFR siRNA, Wortmannin, and GSK-3 beta siRNA. In conclusion, G-CSF attenuated caspase activation and reduced brain injury by inhibiting GSK-3 beta activity after experimental HI in rat pups. This neuroprotective effect was abolished by both G-CSFR siRNA and Wortmannin. (C) 2014 Elsevier Inc. All rights reserved.