Hydrogen gas ameliorates oxidative stress in early brain injury after subarachnoid hemorrhage in rats

Abstract

Objective: Hydrogen gas has been demonstrated to neutralize free radicals and reduce oxidative stress recently. Our objective was to determine the therapeutic effect of H2 inhalation and its antioxidative activity on early brain injury after subarachnoid hemorrhage.

Design: Controlled in vivo laboratory study.

Setting: Animal research laboratory.

Subjects: One hundred thirty-seven adult male Sprague-Dawley rats weighing 280-350 g.

Interventions: Subarachnoid hemorrhage was induced by endovascular perforation method in rats. Subarachnoid hemorrhage rats were treated with 2.9% hydrogen gas inhaled for 2 hrs after perforation. At 24 and 72 hrs, mortality, body weight, neurologic deficits, and brain water content were assessed. Blood-brain barrier permeability and apoptosis were also measured at 24 hrs. To investigate the antioxidative activity of hydrogen gas, the expression of malondialdehyde, nitrotyrosine, and 8-hydroxyguanosine, which are oxidative markers of lipid, protein, and DNA damage, respectively, were measured at 24 hrs.

Measurements and main results: Hydrogen gas significantly alleviated brain edema and blood-brain barrier disruption, reduced apoptosis, and improved neurologic function at 24 hrs but not 72 hrs after subarachnoid hemorrhage. These effects were associated with the amelioration of oxidative injury of lipid, protein, and DNA.

Conclusions: Hydrogen gas could exert its neuroprotective effect against early brain injury after subarachnoid hemorrhage by its antioxidative activity.

Figure 1

A, Mortality was 15.3% and 9.8% in the subarachnoid hemorrhage (SAH) group and SAH + hydrogen (H₂) group (p > .05, Fisher’s exact test). B, Similar severity of SAH bleeding was obtained in the SAH group (n = 27) and SAH + H₂ group (n = 31, p > .05). C, Hydrogen gas (SAH + H₂) alleviated neurologic dysfunction (p < .05 vs. SAH) at 24 hrs, whereas the improvement of neurobehavioral score at 72 hrs was not significant (p > .05 vs. SAH). D, This graph shows the body weight loss after SAH. No significant improvement was detected between the SAH group and SAH + H₂ group at either 24 (p > .05) or 72 hrs (p > .05). At 24 hrs, sham: n = 27, SAH: n = 27, SAH + H₂: n = 31; at 72 hrs, n = 6 in all groups. *p < .05 vs. sham, †p < .05 vs. SAH.

Figure 2

Hydrogen treatment decreased brain water content significantly in the right hemisphere, left hemisphere, and brain stem at 24 hrs after subarachnoid hemorrhage (SAH) (A) (p < .05, sham: n = 6, SAH: n = 6, SAH + hydrogen (H₂): n = 10) but not at 72 hrs (B) in any brain regions (p > .05, n = 6) after SAH. C, Extravasated Evans blue in all brain regions was observed in the SAH group (p < .05) and H₂ treatment decreased Evans blue extravasation in both hemispheres (p < .05), but not in the cerebellum and brain stem (p > .05) at 24 hrs after SAH (n = 6). *p < .05 vs. sham, ^†p < .05 vs. SAH.

Figure 3

Nissl staining was performed on brain sections obtained from the sham, subarachnoid hemorrhage (SAH), and hydrogen (H₂) treatment groups. Neurons in the cerebral cortex at 24 hrs after SAH (n = 3) are shown in upper panels. Intact neurons could be seen in sham animals. Arrows indicate normal neurons (A1). Numerous foci of neuronal damage were present in the cerebral cortex in SAH animals. Arrows indicate deformed neurons (A2). Hydrogen treatment prevented the appearance of injured neurons and more positive stained cells and better cellular shape were observed (A3). The bottom panels show the results of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining after 24 hrs after SAH (n = 3). No positive staining was detected in the sham group (B1). SAH rats presented massive terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-positive cells in the cerebral cortex (B2), whereas evident reduction of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-positive cells could be seen in the SAH + hydrogen group (B3). Arrows indicate cells that are positive. Scale bars represent 100 μm.

Figure 4

A, subarachnoid hemorrhage (SAH) increased malondialdehyde (MDA) concentration in right hemispheres (p < .05), whereas hydrogen (H₂) treatment significantly suppressed the production of malondialdehyde (p < .05) at 24 hrs after SAH (n = 6). B, SAH increased the expression of nitrotyrosine at 24 hrs after SAH, and this increase was alleviated by hydrogen treatment (n = 6, p < .05). *p < .05 vs. sham, ^†p < .05 vs. SAH.

Figure 5

A1–A3, 8 hydroxyguanosine (8-OHG) immunostaining in the cerebral cortex (n = 3) at 24 hrs after subarachnoid hemorrhage (SAH). No immunoreactivity of 8-OHG was observed in the sham group (A1). Stronger oxidative injures were detected in SAH rats (A2) compared with those given hydrogen (H₂) treatment (A3). The bottom three panels show the double fluorescent labeling to display the localization of 8-OHG in SAH rats (n = 3). 8-OHG-positive cells (B1, C1, D1); neuronal nuclei (NeuN), CD34, and glial fibrillary acidic protein-positive (GFAP)-positive cells that represented neurons (B2), endothelial cells (C2), and astrocytes (D2), respectively. Colocalization of 8-OHG with neurons (B3) and endothelial cells (C3) were revealed by merged pictures. However, 8-OHG rarely displayed in astrocytes (D3). Arrows indicate cells that are positive. Scale bars represent 100 μm.