Hydrogen Gas Inhalation Attenuates Acute Impulse Noise Trauma: A Preclinical In Vivo Study

Objective: Molecular hydrogen (H2) has shown therapeutic potential in several oxidative stress-related conditions in humans, is well-tolerated, and is easily administered via inhalation.The aim of this preclinical in vivo study was to investigate whether impulse noise trauma can be prevented by H2 when inhaled immediately after impulse noise exposure. Methods: Guinea pigs (n = 26) were subjected to impulse noise (n = 400; 156 dB SPL; 0.33/s; n = 11; the Noise group), to impulse noise immediately followed by H2 inhalation (2 mol%; 500 ml/min; 1 hour; n = 10; the Noise + H2 group), or to H2 inhalation (n = 5; the H2 group). The acoustically evoked ABR threshold at 3.15, 6.30, 12.5, 20.0, and 30.0 kHz was assessed before and 4 days after impulse noise and/or H2 exposure. The cochleae were harvested after the final ABR assessment for quantification of hair cells. Results: Noise exposure caused ABR threshold elevations at all frequencies (median 35, 35, 30, 35, and 35 dB SPL, the Noise group; 20, 25, 10, 13, and 20 dB SPL, the Noise + H2 group; P < .05) but significantly less so in the Noise + H2 group (P < .05). Outer hair cell (OHC) loss was in the apical, mid, and basal regions 8.8%, 53%, and 14% in the Noise group and 3.5%, 22%, and 1.2% in the Noise + H2 group. The corresponding inner hair cell (IHC) loss was 0.1%, 14%, and 3.5% in the Noise group and 0%, 2.8%, and 0% in the Noise + H2 group. The difference between the groups was significant in the basal region for OHCs (P = .003) and apical (P = .033) and basal (P = .048) regions for IHCs. Conclusions: Acute acoustic trauma can be reduced by H2 when inhaled immediately after impulse noise exposure. Keywords: auditory brainstem response; auditory hair cell; guinea pig; hearing loss; molecular hydrogen; noise-induced; otoprotection.

Inhalation of Molecular Hydrogen, a Rescue Treatment for Noise-Induced Hearing Loss

Noise exposure is the most important external factor causing acquired hearing loss in humans, and it is strongly associated with the production of reactive oxygen species (ROS) in the cochlea. Several studies reported that the administration of various compounds with antioxidant effects can treat oxidative stress-induced hearing loss. However, traditional systemic drug administration to the human inner ear is problematic and has not been successful in a clinical setting. Thus, there is an urgent need to develop rescue treatment for patients with acute acoustic injuries. Hydrogen gas has antioxidant effects, rapid distribution, and distributes systemically after inhalation.The purpose of this study was to determine the protective efficacy of a single dose of molecular hydrogen (H2) on cochlear structures. Guinea pigs were divided into six groups and sacrificed immediately after or at 1 or 2 weeks. The animals were exposed to broadband noise for 2 h directly followed by 1-h inhalation of 2% H2 or room air. Electrophysiological hearing thresholds using frequency-specific auditory brainstem response (ABR) were measured prior to noise exposure and before sacrifice. ABR thresholds were significantly lower in H2-treated animals at 2 weeks after exposure, with significant preservation of outer hair cells in the entire cochlea. Quantification of synaptophysin immunoreactivity revealed that H2 inhalation protected the cochlear inner hair cell synaptic structures containing synaptophysin. The inflammatory response was greater in the stria vascularis, showing increased Iba1 due to H2 inhalation.Repeated administration of H2 inhalation may further improve the therapeutic effect. This animal model does not reproduce conditions in humans, highlighting the need for additional real-life studies in humans.

Saturated hydrogen saline protects against noise–induced hearing loss

Objective: To study effects of saturated hydrogen saline in preventing noise–induced hearing loss. Methods: Fifteen guinea pigs were randomly divided into 3 groups (5 each), group one was for control, group two was treated with normal saline and group three was treated with saturated hydrogen saline, which was given intraperitoneally at 1 hour before noise exposure at 1 ml/100 g. One hundred rounds of impulse noise (157 dB SPL peak) were delivered as noise exposure. Immediately after exposure to impulse noise and on Days 1, 2, 4 and 8 following exposure, auditory brainstem response (ABR) thresholds were measured. Outer hair cell morphological changes and succinate dehydrogenase (SDH) activity were examined on Day 8 post–exposure. Results: Immediately after noise exposure, ABR thresholds in saturated hydrogen saline treated animals were lower than the non–treated animals (P < 0.05). Microscopy showed little SDH staining, cell swelling and irregular cell arrangement in the non–treated or normal saline treated animals. Whereas in the saturated hydrogen saline treated animals, there was deep SDH staining with significantly reduced cell loss and more regular cellular arrangement compared to the other two groups. The surviving cells counts was 45.17 ± 12.15 for non–treated animals, 44.50 ± 10.02 for normal saline treated animals and,116.50±2.38 for animals treated with saturated hydrogen saline. While the count was similar between non–treated and normal saline treated animals, it was significantly higher in saturated hydrogen saline treated animals (P < 0.05). Conclusions: Intraperitoneal injection of saturated hydrogen saline appears to protect the cochlea against noise–induced damage.

Hydrogen in drinking water attenuates noise-induced hearing loss in guinea pigs

It has been shown that molecular hydrogen acts as a therapeutic and preventive antioxidant by selectively reducing the hydroxyl radical, the most cytotoxic of the reactive oxygen species. In the present study, we tested the hypothesis that acoustic damage in guinea pigs can be attenuated by the consumption of molecular hydrogen. Guinea pigs received normal water or hydrogen-rich water for 14 days before they were exposed to 115 dB SPL 4-kHz octave band noise for 3h. Animals in each group underwent measurements for auditory brainstem response (ABR) or distortion-product otoacoustic emissions (DPOAEs) before the treatment (baseline) and immediately, 1, 3, 7, and 14 days after noise exposure. The ABR thresholds at 2 and 4 kHz were significantly better on post-noise days 1, 3, and 14 in hydrogen-treated animals when compared to the normal water-treated controls. Compared to the controls, the hydrogen-treated animals showed greater amplitude of DPOAE input/output growth functions during the recovery process, with statistical significance detected on post-noise days 3 and 7. These findings suggest that hydrogen can facilitate the recovery of hair cell function and attenuate noise-induced temporary hearing loss.

The effects of methane and hydrogen gases produced by enteric bacteria on ileal motility and colonic transit time

Background: Gases produced by intestinal flora may modulate intestinal motor function in healthy individuals as well as those with functional bowel disease. Methane, produced by enteric bacteria in the human gut, is associated with slowed intestinal transit and constipation. The effects of hydrogen, another main gas produced by bacterial fermentation in the gut, on small bowel and colonic motor function remains unrecognized. Therefore, we set out to investigate whether intestinal gases including methane and hydrogen could influence the small bowel motility and colonic transit. Methods: Guinea pig ileum was placed in the peristaltic bath with tension transducers attached to measure velocity and amplitude of peristaltic contraction before and after the infusion of control, hydrogen, and methane gases. Also, changes in the intraluminal pressures were monitored before and after the gas infusions. Key results: Methane decreased peristaltic velocity and increased contraction amplitude significantly of guinea pig ileum (P < 0.05). The AUC of intraluminal pressure was significantly increased with methane in guinea pig ileum (P < 0.05). In a second experiment, guinea pig colon was placed in the peristaltic bath to measure transit time before and after control, hydrogen, methane, and methane-hydrogen mixture gas infusions. Hydrogen shortened colonic transit time by 47% in the proximal colon, and by 10% in the distal colon, when compared with baselines (P < 0.05). Conclusions & inferences: Methane delayed ileal peristaltic conduction velocity by augmenting contractility. Hydrogen shortened colonic transit, and that effect was more prominent in the proximal colon than distal colon.

Hydrogen-rich saline alleviates experimental noise-induced hearing loss in guinea pigs

To examine the efficiency of hydrogen-rich saline in the treatment of intensive noise-induced cochlear injury. Forty guinea pigs were assigned to one of four groups: HS+NOISE (i.p. injection hydrogen-rich saline), NS+NOISE (i.p. injection normal saline), NOISE ALONE (noise control), and NO TREATMENT (normal control) groups. The HS+NOISE, NS+NOISE, and NOISE ALONE groups were exposed to intensive noise (4 h at 115 dB SPL noise of 4000±100 Hz). The auditory brainstem response (ABR) was used to examine the hearing threshold in each group. Distortion product otoacoustic emission (DPOAE) was used to examine outer hair cell function. We also examined cochlear morphology to evaluate inner and outer hair cell trauma induced by noise exposure. Hydrogen-rich saline was administered twice daily for 6 days (2.5 ml/kg, i.p.) 24 h after noise exposure. Baseline ABR thresholds and DPOAE values were normal in all groups at the measured frequencies (2, 4, 8, and 16 kHz) before noise exposure. The ABR threshold shift was 50-55 dB across the frequencies tested, and average DPOAE declined in the NOISE ALONE, NS+NOISE, and HS+NOISE groups 24 h after noise exposure. However, the changes in cochlear parameters were different between groups. The HS+NOISE group showed a significantly decreased ABR threshold value as compared with the NS+NOISE or NOISE ALONE group (P<0.01) on day 7. The mean DPOAE recovered to some extent in the three noise exposure groups, but at most frequencies the HS+NOISE group showed significantly increased DPOAE on day 7 as compared with the NS+NOISE group or NOISE ALONE group (P<0.01). Surface Corti organ preparations stained with succinate dehydrogenase (SDH) showed that most outer hair cells (OHCs) were still dropsical and a few were missing 7 days after noise exposure in the NS+NOISE group. Only a few OHCs were slightly dropsical in the HS+NOISE group. The numbers of missing hair cells 7 days after noise exposure were significantly greater in the NOISE ONLY and NS+NOISE groups than the HS+NOISE group (P<0.01). Hydrogen-rich saline can alleviate experimental noise-induced hearing loss in guinea pigs, partially by preventing the death of cochlear hair cells after intensive noise exposure.

Hydrogen-rich saline protects retina against glutamate-induced excitotoxic injury in guinea pig

Molecular hydrogen (H(2)) is an efficient antioxidant that can selectively reduce hydroxyl radicals and inhibit oxidative stress-induced injuries. We investigated the protective effects and mechanism of hydrogen-rich saline in a glutamate-induced retinal injury model. Retinal excitotoxicity was induced in healthy guinea pigs by injecting glutamate into the vitreous cavity. After 30 min, hydrogen-rich saline was injected into the vitreous cavity, the peritoneal cavity or both. Seven days later, the retinal stress response was evaluated by examining the stress biomarkers, inducible nitric-oxide synthase (iNOS) and glucose-regulated protein 78 (GRP78). The impaired glutamate uptake was assessed by the expression of the excitatory amino acid transporter 1(EAAT-1). The retinal histopathological changes were investigated, focusing on the thicknesses of the entire retina and its inner layer, the number of cells in the retinal ganglion cell layer (GCL) and the ultrastructure of the retinal ganglion cells (RGCs) and glial cells. Compared with the glutamate-induced injury group, the hydrogen-rich saline treatment reduced the loss of cells in the GCL and thinning of the retina and attenuated cellular morphological damage. These improvements were greatest in animals that received H(2) injections into both the vitreous and the peritoneal cavities. The hydrogen-rich saline also inhibited the expression of glial fibrillary acidic protein (GFAP) in Müller cells, CD11b in microglia, and iNOS and GRP78 in glial cells. Moreover, the hydrogen-rich saline increased the expression of EAAT-1. In conclusion, the administration of hydrogen-rich saline through the intravitreal or/and intraperitoneal routes could reduce the retinal excitotoxic injury and promote retinal recovery. This result likely occurs by inhibiting the activation of glial cells, decreasing the production of the iNOS and GRP78 and promoting glutamate clearance.

Inhaled hydrogen gas therapy for prevention of noise-induced hearing loss through reducing reactive oxygen species

Reactive oxygen species (ROS) that form in the inner ear play an important role in noise-induced hearing loss (NIHL). Recent studies have revealed that molecular hydrogen (H2) has great potential for reducing ROS. In this study, we examined the potential of hydrogen gas to protect against NIHL. We tested this hypothesis in guinea pigs with 0.5%, 1.0% and 1.5% H2 inhalation in air for 5h a day after noise exposure, for five consecutive days. All animals underwent measurements for auditory brainstem response after the noise exposure; the results revealed that there was a better improvement in the threshold shift for the 1.0% and 1.5% H2-treated groups than the non-treated group. Furthermore, outer hair cell (OHC) loss was examined 7 days after noise exposure. A significantly higher survival rate of OHCs was observed in the 1.0% and 1.5% H2-treated group as compared to that of the non-treated group in the basal turn. Immunohistochemical analyses for 8-hydroxy-2′-deoxyguanosine (8-OHdG) were performed to examine the amount of oxidative DNA damage. While strong immunoreactivities against 8-OHdG were observed of the non-treated group, the H2-treated group showed decreased immunoreactivity for 8-OHdG. These findings strongly suggest that inhaled hydrogen gas protects against NIHL.

Hydrogen-Saturated Saline Protects Intensive Narrow Band Noise-Induced Hearing Loss in Guinea Pigs through an Antioxidant Effect

The purpose of the current study was to evaluate hydrogen-saturated saline protecting intensive narrow band noise-induced hearing loss. Guinea pigs were divided into three groups: hydrogen-saturated saline; normal saline; and control. For saline administration, the guinea pigs were given daily abdominal injections (1 ml/100 g) 3 days before and 1 h before narrow band noise exposure (2.5-3.5 kHz 130 dB SPL, 1 h). The guinea pigs in the control group received no treatment. The hearing function was assessed by the auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) recording. The changes of free radicals in the cochlea before noise exposure, and immediately and 7 days after noise exposure were also examined. By Scanning electron microscopy and succinate dehydrogenase staining, we found that pre-treatment with hydrogen-saturated saline significantly reduced noise-induced hair cell damage and hearing loss. We also found that the malondialdehyde, lipid peroxidation, and hydroxyl levels were significantly lower in the hydrogen-saturated saline group after noise trauma, indicating that hydrogen-saturated saline can decrease the amount of harmful free radicals caused by noise trauma. Our findings suggest that hydrogen-saturated saline is effective in preventing intensive narrow band noise-induced hearing loss through the antioxidant effect.

Changes in IL-4 and IL-13 expression in allergic-rhinitis treated with hydrogen-rich saline in guinea-pig model

Background: Medical gas hydrogen (H2) has a special role in airway inflammation; however, the effect of H2 on allergic rhinitis (AR) remains unclear. This study explored the possible roles of H2 on the pathogenesis of AR and observed the influences of H2 on cytokines IL-4 and IL-13. Methods: An AR guinea pig model was established by nasal ovalbumin sensitisation. Eighteen guinea pigs were divided into three groups, namely, saline control, AR-sensitised, and hydrogen-rich saline (HRS)-treated groups, with each group having six guinea pigs. The frequencies of sneezing and scratching were recorded. The IgE level and cytokine (IL-4 and IL-13) levels in the serum were measured. The expression levels of IL-4 and IL-13 mRNA and protein in the nasal mucosa were also determined by real-time reverse transcriptase-polymerase chain reaction and Western blot. We also observed the infiltration of cytokine (IL-4 and IL-13) in nasal mucosa by immunofluorescence. Results: The frequencies of sneezing and scratching, as well as the levels of IgE, IL-4, and IL-13, in the serum were higher in the AR group than in the control group (p<0.01), whereas all these parameters were decreased significantly after HRS treatment (p<0.05). The expression levels of IL-4 and IL-13 mRNA and protein in the nasal mucosa were also lower in guinea pigs treated with HRS than those in the AR group (p<0.05). Conclusions: HRS could affect anti-inflammation in AR and decreased the expression of IL-4 and IL-13.