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

Exp Neurol. 2015 Jan;263:141-9. doi: 10.1016/j.expneurol.2014.10.004. Epub 2014 Oct 18.

Abstract

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-3β (GSK-3β) inhibition. Ten day old Sprague-Dawley rat pups (n=157) were subjected to unilateral carotid artery ligation followed by 2.5h of hypoxia or sham surgery. HI animals received control siRNA, GSK-3β siRNA (4 μL/pup), G-CSF (50 μg/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-3β (p-GSK-3β), 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-3β increased after HI until its peak at 48 h post-ictus, and both GSK-3β siRNA and G-CSF administration reduced p-GSK-3β expression, as well as infarct volume. p-GSK-3β 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-3β and CC3 expression levels in the ipsilateral hemisphere, which were all reversed by G-CSFR siRNA, Wortmannin, and GSK-3β siRNA. In conclusion, G-CSF attenuated caspase activation and reduced brain injury by inhibiting GSK-3β activity after experimental HI in rat pups. This neuroprotective effect was abolished by both G-CSFR siRNA and Wortmannin.

Keywords: Apoptosis; GSK-3β; Granulocyte-colony stimulating factor (G-CSF); Hypoxia–ischemia (HI); Neonatal; PI3K.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Animals, Newborn
  • Apoptosis / drug effects*
  • Apoptosis / physiology
  • Blotting, Western
  • Disease Models, Animal
  • Fluorescent Antibody Technique
  • Glycogen Synthase Kinase 3 / metabolism*
  • Glycogen Synthase Kinase 3 beta
  • Granulocyte Colony-Stimulating Factor / pharmacology*
  • Hypoxia-Ischemia, Brain / metabolism*
  • Hypoxia-Ischemia, Brain / pathology
  • In Situ Nick-End Labeling
  • Neurons / drug effects
  • Neurons / metabolism
  • Neurons / pathology
  • Neuroprotective Agents / pharmacology*
  • RNA, Small Interfering
  • Rats
  • Rats, Sprague-Dawley
  • Transfection

Substances

  • Neuroprotective Agents
  • RNA, Small Interfering
  • Granulocyte Colony-Stimulating Factor
  • Glycogen Synthase Kinase 3 beta
  • Gsk3b protein, rat
  • Glycogen Synthase Kinase 3