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

PLoS One. 2014 Apr 24;9(4):e96212. doi: 10.1371/journal.pone.0096212. eCollection 2014.

Abstract

Backgrounds: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects*
  • Apoptosis Regulatory Proteins / metabolism
  • Cerebral Cortex / drug effects
  • Cerebral Cortex / pathology
  • Chromones / pharmacology
  • Glycogen Synthase Kinase 3 / metabolism*
  • Glycogen Synthase Kinase 3 beta
  • Hydrogen
  • Male
  • Morpholines / pharmacology
  • Neurons / drug effects
  • Neurons / pathology
  • Neuroprotective Agents / pharmacology*
  • Neuroprotective Agents / therapeutic use
  • Phosphoinositide-3 Kinase Inhibitors
  • Phosphorylation
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Random Allocation
  • Rats, Sprague-Dawley
  • Signal Transduction
  • Sodium Chloride / pharmacology*
  • Sodium Chloride / therapeutic use
  • Subarachnoid Hemorrhage / pathology
  • Subarachnoid Hemorrhage / physiopathology
  • Subarachnoid Hemorrhage / prevention & control*

Substances

  • Apoptosis Regulatory Proteins
  • Chromones
  • Morpholines
  • Neuroprotective Agents
  • Phosphoinositide-3 Kinase Inhibitors
  • 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
  • Sodium Chloride
  • Hydrogen
  • Glycogen Synthase Kinase 3 beta
  • Gsk3b protein, rat
  • Proto-Oncogene Proteins c-akt
  • Glycogen Synthase Kinase 3

Grants and funding

This project was funded by Grant 81171096 and 81371369 from National Natural Science Foundation of China; Grant 20120101120030 from Doctoral Program of the Ministry of Education; Grant 2013KYA088 from Health department of Zhejiang province. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.