Neuroprotective effect of hydrogen-rich saline against neurologic damage and apoptosis in early brain injury following subarachnoid hemorrhage: possible role of the Akt/GSK3β signaling pathway.
BACKGROUND
Early 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.


METHODS
Sprague-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.


RESULTS
HS 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.


CONCLUSIONS
HS could attenuate neuronal apoptosis in EBI and improve the neurofunctional outcome after SAH, partially via the Akt/GSK3β pathway.
Department of Neurosurgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
Yuan
Y
Hong
Department of Neurosurgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
AnWen
A
Shao
Department of Neurosurgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China; Department of Neurosurgery, Taizhou Enze Medical Center (Group), Taizhou Hospital, Taizhou, China.
Jianfeng
J
Wang
Department of Neurosurgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
Sheng
S
Chen
Department of Neurosurgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
HaiJian
H
Wu
Department of Physiology and Pharmacology, Loma Linda University School of Medicine, Loma Linda, California, United States of America.
Devin W
DW
McBride
Department of Neurosurgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
Qun
Q
Wu
Department of Diving Medicine, The Second Military Medical University, Shanghai, China.
XueJun
X
Sun
Department of Neurosurgery, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
JianMin
J
Zhang
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D002540:Cerebral Cortex / Q000187:drug effects / Q000473:pathology; D002867:Chromones / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006859:Hydrogen; D008297:Male; D009025:Morpholines / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000473:pathology; D018696:Neuroprotective Agents / Q000494:pharmacology* / Q000627:therapeutic use; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011897:Random Allocation; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D012965:Sodium Chloride / Q000494:pharmacology* / Q000627:therapeutic use; D013345:Subarachnoid Hemorrhage / Q000473:pathology / Q000503:physiopathology / Q000517:prevention & control*
D051017:Apoptosis Regulatory Proteins; D002867:Chromones; D009025:Morpholines; D018696:Neuroprotective Agents; D000081082:Phosphoinositide-3 Kinase Inhibitors; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D012965:Sodium Chloride; D006859:Hydrogen; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3