Hydrogen saline offers neuroprotection by reducing oxidative stress in a focal cerebral ischemia-reperfusion rat model

All experimental procedures and protocols used in this study were reviewed and approved by the Animal Care and Use Committee of the Second Military Medical University. Furthermore, all were in accordance with the Guide for the Care and Use of Laboratory Animals. A total of 228 male Sprague-Dawley rats weighing 250-280 g were used in the present study. The rats were housed at 22-24°C under a 12-h-light/12-h-dark cycle, with food and water available ad libitumthroughout the studies. Rats were randomly distributed into three groups, sham group (n = 52), MCAO group (n = 72) and MCAO plus hydrogen group (n = 104). Rats in the sham group only received intraperitoneal administration of normal saline and those in the MCAO group underwent MCAO followed by administration of normal saline at different time points (0, 3 or 6 h) after reperfusion onset. However, rats in the MCAO plus hydrogen group received MCAO and intraperitoneal treatment with hydrogen saline (1 ml/100 g body weight) at designed time points (0, 3 or 6 h after reperfusion onset). MCAO was produced by the filament model initially reported by Zea-Longa et al [13] with some modifications. After 90 min of right middle cerebral artery occlusion, the reperfusion of the MCA was initiated by removing the MCA occlusive filament. Rats were sacrificed at 12, 24, 72 h, and 7 days after reperfusion, and immunihistochemistry and detections of malondidehyd (MDA), anti-superoxide anion, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were performed.

Neurological function was assessed using a standard scoring system [14]: 0 = no apparent deficits, 1 = contralateral forelimb flexion, 2 = decreased grip of contralateral forelimb, 3 = contralateral circling if pulled by tail, 4 = spontaneous contralateral circling.

Infarct volume was determined by staining with 2, 3, 5-triphenyltetrazolium chloride (TTC, Sigma) as previously described [15]. The infarct and total hemispheric areas of each section, at intervals of 2-mm in thickness, were traced and analyzed using image analysis system (Image J software). The infarct ratio was calculated by dividing the infarct volume by the total volume of the sections.

The brains were obtained and right hemisphere was quickly separated. Brain samples were weighted with a precise electronic balance and dried in an oven at 100 °C for 48 h [16]. Then, the samples were re-weighed and the water content was determined according to the following formula: [(wet weight - dry weight)/wet weight] × 100%.

For Nissl staining, the 4-μm sections were hydrated in 1% toluidine blue at 50 °C for 20 min. After rinsing with double distilled water, they were dehydrated and mounted with permount. The cortex from each animal was captured and Imaging-Pro-Plus (LEIKA DMLB) was used to perform quantitative analysis of cell numbers.

Immunohistochemistry was performed on 20 μm-thick free-floating coronal sections, which were prepared as previously described [17]. After incubation in 3% hydrogen peroxide (H₂O₂) in PBS, the sections were incubated overnight at 4 °C with primary antibodies against 8-hydroxyl-2'-deoxyguanosine (8-OHdG, 100:1; America Alpha Diagnostic international, a marker for DNA damage), Nitrotyrosine (40:1; America upstate, a marker for nitration), bax (100:1; America Abcam) and bcl-2 (600:1; Americ Millipore). Sections were then treated with secondary antibodies (1:2000, Vectastain, Vector Laboratories). Immunoreactivity was visualized subsequently by the avidin-biotin complex method (Vectastain, Vector Laboratories) as described previously [17].

In each section, 6 visual fields (0.6 mm²) of cerebral cortex were randomly photographed. The number of staining cells in each field was counted at higher magnification (×200). Data were expressed as the number of cells per high-power field.

Lipid peroxidation levels were measured with the thiobarbituric acid (TBA) reaction. This method was used to obtain a spectrophotometric measurement of the color produced during the reaction of TBA with MDA at 535 nm. For this purpose, 2.5 ml of 100 g/l trichloroacetic acid solution was added to 0.5 ml of homogenate in centrifuge tube followed by heating in boiling water for 15 min. The mixture was allowed to cool to room temperature and centrifuged (Eppendorf, 5810R) at 1000 × g for 10 min. Then, 2 ml of supernatant was added to 1 ml of 6.7 g/l TBA solution in a test tube, followed by heating in boiling water for 15 min. The solution was then cooled and the absorbance was measured with a spectrophotometer (UV-WFZ75, Shanghai, China). TBARS levels were expressed as nmol/mg protein in the brain.

Brain samples from the cortex and hippocampus were taken from the impaired hemispheres of rats 24 h after hydrogen saline administration. The activity of caspase-3 was measured with caspase-3/CPP32 Fluorometric Assay Kit (BIOVISION Research Products 980, USA). Briefly, brain samples were homogenized in ice-cold lysis buffer and kept at 4 °C for 1 h. Brain homogenate was centrifuged at 12,000 g for 15 min at 4 °C. The supernatant was collected and stored at -80 °C for use. Protein concentration was measured using the Enhanced BCA Protein Assay Kit. A total of 50 μg of cell lysates were incubated in a 96-well plate with 2 × Reaction Buffer (50 μl). The reaction was started by adding 1 mM DEVD-APC substrate (5 μl). After incubation in dark at 37°C, the plate was read with a fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter.

The levels of IL-1β and TNFα of brain tissues were determined with solid phase sandwich ELISA kit (Invitrogen, USA) under a microplate reader (Stat Fax 3200) at 450 nm.

The hydrogen was administered 0, 3 and 6 after reperfusion to explore the preferable therapeutic regimen and brains were removed as 24 h after reperfusion followed by detection of infarction and brain edema. Hydrogen saline significantly reduced the infarct ratio when applied at 0 or 3 h after reperfusion, but that was only slightly reduced when injected at 6 h after reperfusion (Figure 1A-B). The mean brain water content of injured hemisphere was 77.67 ± 0.6% in the shame group, 84.04 ± 0.96% in the MCAO group and 78.82 ± 1.09% (0 h), 79.38 ± 0.44% (3 h), and 81.41 ± 1.02% (6 h) in the MCAO plus hydrogen group (P< 0.01) (Figure 1C). Based on these results, application of hydrogen 3 h after reperfusion was used in the following experiments.

The brain weight and neurological score were performed 24 after reperfusion. In the MCAO group, a marked body weight loss was observed when compared with sham group (P< 0.05). After hydrogen saline treatment at 3 h after reperfusion, the body weight loss was 23.93 ± 3.60 g in the MCAO group and 13.91 ± 3.64 g in the hydrogen treated group (P< 0.01), but marked body weight loss was also observed between the hydrogen treated group and the sham group (P< 0.05) (Figure 2A). Neurological scores were dramatically reduced in the MCAO group (P< 0.05 vs. sham), and hydrogen saline treatment at 3 h after reperfusion significantly improved the neurological function (P< 0.01 vs. MCAO group) even though neurological dysfunction was still observed (P< 0.05 vs. sham group) (Figure 2B).

Figure 3A showed the Nissl staining of injured cortex at 12, 24, 72 h and 7 days after reperfusion. Numerous neuronal cells in the ischemic core died or shrunk with enlarged intercellular space. The cells were much better preserved in the hydrogen treated group and the number of viable cells was markedly increased (Figure 3B) (P< 0.05 vs. MCAO group).

Oxidative DNA damage was determined by measuring the amount of 8-OHdG in the injured cortex 12, 24, 72 h and 7 days after reperfusion. Less positive staining was detected in the sham operation group. After 12 h of reperfusion, strong 8-OHdG positive staining was observed in the nuclei of neurons located in the ischemic cortex in the MCAO group. Hydrogen saline treatment significantly decreased the number of 8-OHdG-positive cells (P< 0.05 vs. MCAO group) (Figure 4A, B).

In the MCAO group, numerous TUNEL positive cells were observed and the injured cells were characterized by a round and shrunken morphology in the injured cortex at 12, 24, 72 h and 7 days after reperfusion. Hydrogen saline dramatically decreased the number of TUNEL-positive cells when applied at 3 h after reperfusion (P< 0.05) (Figure 5A, B).

Results showed the number of cells positive for Bcl-2 or Bax in sham group was lower than that in MCAO group (P < 0.05). However, when compared with MCAO group, hydrogen saline applied 3 h after reperfusion significantly increased the expression of Bcl-2 (Figure 6A, C), and decreased that of Bax at 24 h after reperfusion (Figure 6B, C) (P< 0.01 vs. MCAO group). Similarly, when compared with sham group, the activity of caspase-3 was increased in the ischemic cortex of MCAO group at 24 h after reperfusion, which was significantly reduced by hydrogen saline treatment at 3 h after reperfusion (P< 0.01 vs. MCAO group) (Figure 6D) which however was still higher than that in the sham group (P < 0.05 vs. sham).

The lipid peroxidation was presented as the MDA level at 12, 24, 72 h and 7 days after reperfusion. In the MCAO group, the MDA level in the ischemic cortex was increased which was significantly alleviated by hydrogen saline administration at 3 h after reperfusion (P< 0.05 vs. MCAO group) (Figure 7).

When compared with sham group, the levels of IL-1β and TNF-α were increased after MCAO and the increased levels of IL-1β and TNF-α lasted at least 7 days. After application of hydrogen saline at 3 h after reperfusion, the elevated levels of IL-1β and TNF-α were markedly decreased (P< 0.05 vs. MCAO group) (Figure 8).