title: G-CSF attenuates neuroinflammation and stabilizes the blood-brain barrier via the PI3K/Akt/GSK-3β signaling pathway following neonatal hypoxia-ischemia in rats.
abstract: OBJECTIVE
  Neonatal hypoxia occurs in approximately 60% of premature births and is associated with a multitude of neurological disorders. While various treatments have been developed, translating them from bench to bedside has been limited. We previously showed G-CSF administration was neuroprotective in a neonatal hypoxia-ischemia rat pup model, leading us to hypothesize that G-CSF inactivation of GSK-3β via the PI3K/Akt pathway may attenuate neuroinflammation and stabilize the blood-brain barrier (BBB).
  
  
  METHODS
  P10 Sprague-Dawley rat pups were subjected to unilateral carotid artery ligation followed by hypoxia for 2.5h. We assessed inflammation by measuring expression levels of IKKβ, NF-κB, TNF-α, IL-1β, IL-10, and IL-12 as well as neutrophil infiltration. BBB stabilization was evaluated by measuring Evans blue extravasation, and Western blot analysis of Claudin-3, Claudin-5, ICAM-1, and VCAM-1.
  
  
  RESULTS
  First, the time course study showed that p-β-catenin/β-catenin, IKKβ, and NF-κB expression levels peaked at 48h post-HI. The knockdown of GSK-3β with siRNA prevented the HI-induced increase of p-β-catenin/β-catenin, IKKβ, and NF-κB expression levels 48h after HI. G-CSF treatment reduced brain water content and neuroinflammation by downregulating IKKβ, NF-κB, TNF-α, IL-1β, and IL-12 and upregulating IL-10, thereby reducing neutrophil infiltration. Additionally, G-CSF stabilizes the BBB by downregulating VCAM-1 and ICAM-1, as well as upregulating Claudins 3 and 5 in endothelial cells. G-CSFR knockdown by siRNA and Akt inhibition by Wortmannin reversed G-CSF's neuroprotective effects.
  
  
  CONCLUSIONS
  We demonstrate G-CSF plays a pivotal role in attenuating neuroinflammation and BBB disruption following HI by inactivating GSK-3β through the PI3K/Akt pathway.
authors.affiliation: Departments of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, CA, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.affiliation: Departments of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, CA, USA.
authors.forename: Devin W
authors.initials: DW
authors.lastname: McBride
authors.affiliation: Departments of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, CA, USA.
authors.forename: Desislava
authors.initials: D
authors.lastname: Doycheva
authors.affiliation: Departments of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, CA, USA.
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Dixon
authors.affiliation: Departments of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, CA, USA.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Krafft
authors.affiliation: Departments of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, CA, USA; Department of Anesthesiology, School of Medicine, Loma Linda University, Loma Linda, CA, USA.
authors.forename: John H
authors.initials: JH
authors.lastname: Zhang
authors.affiliation: Departments of Physiology and Pharmacology, School of Medicine, Loma Linda University, Loma Linda, CA, USA. Electronic address: jtang@llu.edu.
authors.forename: Jiping
authors.initials: J
authors.lastname: Tang
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D000893:Anti-Inflammatory Agents / Q000627:therapeutic use*; D001812:Blood-Brain Barrier / Q000187:drug effects* / Q000503:physiopathology; D016207:Cytokines / Q000378:metabolism; D004195:Disease Models, Animal; D004660:Encephalitis / Q000188:drug therapy* / Q000209:etiology; D007839:Functional Laterality; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism; D016179:Granulocyte Colony-Stimulating Factor / Q000627:therapeutic use*; D020925:Hypoxia-Ischemia, Brain / Q000150:complications; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000627:therapeutic use; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D000893:Anti-Inflammatory Agents; D016207:Cytokines; D034741:RNA, Small Interfering; D016179:Granulocyte Colony-Stimulating Factor; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3
keywords: Blood–brain barrier (BBB); GSK-3β; Granulocyte-colony stimulating factor (G-CSF); Hypoxia-ischemia (HI); Neonatal; Neuroinflammation; PI3K