• Yuan Hong, AnWen Shao, Jianfeng Wang, Sheng Chen, HaiJian Wu, Devin W McBride, Qun Wu, XueJun Sun, and JianMin Zhang.
• Department of Neurosurgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
• Plos One. 2014 Jan 1;9(4):e96212.

BackgroundsEarly brain injury (EBI) plays a key role in the pathogenesis of subarachnoid hemorrhage (SAH). Neuronal apoptosis is involved in the pathological process of EBI. Hydrogen can inhibit neuronal apoptosis and attenuate EBI following SAH. However, the molecular mechanism underlying hydrogen-mediated anti-apoptotic effects in SAH has not been elucidated. In the present study, we aimed to evaluate whether hydrogen alleviates EBI after SAH, specifically neuronal apoptosis, partially via the Akt/GSK3β signaling pathway.MethodsSprague-Dawley rats (n = 85) were randomly divided into the following groups: sham group (n = 17), SAH group (n = 17), SAH + saline group (n = 17), SAH + hydrogen-rich saline (HS) group (n = 17) and SAH + HS + Ly294002 (n = 17) group. HS or an equal volume of physiological saline was administered immediately after surgery and repeated 8 hours later. The PI3K inhibitor, Ly294002, was applied to manipulate the proposed pathway. Neurological score and SAH grade were assessed at 24 hours after SAH. Western blot was used for the quantification of Akt, pAkt, GSK3β, pGSK3β, Bcl-2, Bax and cleaved caspase-3 proteins. Neuronal apoptosis was identified by double staining of terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining and NeuN, and quantified by apoptosis index. Immunohistochemistry and immunofluorescent double-labeling staining was performed to clarify the relationships between neuronal apoptosis and pAkt or pGSK3β.ResultsHS significantly reduced neuronal apoptosis and improved neurological function at 24 hours after SAH. The levels of pAkt and pGSK3β, mainly expressed in neurons, were markedly up-regulated. Additionally, Bcl-2 was significantly increased while Bax and cleaved caspase-3 was decreased by HS treatment. Double staining of pAkt and TUNEL showed few colocalization of pAkt-positive cells and TUNEL-positive cells. The inhibitor of PI3K, Ly294002, suppressed the beneficial effects of HS.ConclusionsHS could attenuate neuronal apoptosis in EBI and improve the neurofunctional outcome after SAH, partially via the Akt/GSK3β pathway.

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