G-CSF attenuates neuroinflammation and stabilizes the blood–brain barrier via the PI3K/Akt/GSK-3β signaling pathway following neonatal hypoxia-ischemia in rats

• G-CSF reduces neuroinflammation after HI via the G-CSFR/PI3K/Akt pathway.
• G-CSF stabilizes the BBB after HI via the G-CSFR/PI3K/Akt pathway.
• Inhibition of either PI3K or G-CSFR prevents the effects of G-CSF.
• GSK-3β knockdown by siRNA stabilizes the BBB and reduces inflammation.

ObjectiveNeonatal 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).MethodsP10 Sprague–Dawley rat pups were subjected to unilateral carotid artery ligation followed by hypoxia for 2.5 h. 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.Measurements and main resultsFirst, the time course study showed that p-β-catenin/β-catenin, IKKβ, and NF-κB expression levels peaked at 48 h 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 48 h 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.ConclusionsWe demonstrate G-CSF plays a pivotal role in attenuating neuroinflammation and BBB disruption following HI by inactivating GSK-3β through the PI3K/Akt pathway.