Conflicting findings on the effectiveness of hydrogen therapy for ameliorating vascular leakage in a 5-day post hypoxic-ischemic survival piglet model

Neonatal hypoxic-ischemic encephalopathy (HIE) is a major cause of morbidity and mortality in newborns in both high- and low-income countries. The important determinants of its pathophysiology are neural cells and vascular components. In neonatal HIE, increased vascular permeability due to damage to the blood-brain barrier is associated with seizures and poor outcomes in both translational and clinical studies. In our previous studies, hydrogen gas (H2) improved the neurological outcome of HIE and ameliorated the cell death. In this study, we used albumin immunohistochemistry to assess if H2 inhalation effectively reduced the cerebral vascular leakage. Of 33 piglets subjected to a hypoxic-ischemic insult, 26 piglets were ultimately analyzed. After the insult, the piglets were grouped into normothermia (NT), H2 ventilation (H2), therapeutic hypothermia (TH), and H2 combined with TH (H2-TH) groups. The ratio of albumin stained to unstained areas was analyzed and found to be lower in the H2 group than in the other groups, although the difference was not statistically significant. In this study, H2 therapy did not significantly improve albumin leakage despite the histological images suggesting signs of improvement. Further investigations are warranted to study the efficacy of H2 gas for vascular leakage in neonatal HIE.

Molecular hydrogen: prospective treatment strategy of kidney damage after cardiac surgery

Cardiac surgery-associated acute kidney injury (CS-AKI) is a common postoperative complication, mostly due increasing oxidative stress. Recently, molecular hydrogen (H2 gas), has also been applied to cardiac surgery due to its ability to reduce oxidative stress. We evaluated the potential effect of H2 application on the kidney in an in vivo model of simulated heart transplantation. Pigs underwent cardiac surgery with 3 hours while connected to extracorporeal circulation (ECC) and subsequent 60 minutes of spontaneous reperfusion of the heart. We used two experimental groups: T – pigs after transplantation, TH – pigs after transplantation treated with 4% H2 mixed with air during inhalation of anesthesia and throughout oxygenation of blood in ECC. The levels of creatinine, urea and phosphorus were measured in plasma. Renal tissue samples were analyzed by Western blot method for protein levels of Nrf2, Keap-1, and SOD1. After cardiac surgery, selected plasma biomarkers were elevated. However, H2 therapy was followed by the normalization of all these parameters. Our results suggest activation of Nrf2/Keap1 pathway as well as increased SOD1 protein expression in the group treated with H2. The administration of H2 had a protective effect on the kidneys of pigs after cardiac surgery, especially in terms of normalization of plasma biomarkers to control levels.

Impact of hydrogen gas inhalation during therapeutic hypothermia on cerebral hemodynamics and oxygenation in the asphyxiated piglet

We previously reported the neuroprotective potential of combined hydrogen (H2) gas ventilation therapy and therapeutic hypothermia (TH) by assessing the short-term neurological outcomes and histological findings of 5-day neonatal hypoxic-ischemic (HI) encephalopathy piglets. However, the effects of H2 gas on cerebral circulation and oxygen metabolism and on prognosis were unknown. Here, we used near-infrared time-resolved spectroscopy to compare combined H2 gas ventilation and TH with TH alone. Piglets were divided into three groups: HI insult with normothermia (NT, n = 10), HI insult with hypothermia (TH, 33.5 ± 0.5 °C, n = 8), and HI insult with hypothermia plus H2 ventilation (TH + H2, 2.1-2.7%, n = 8). H2 ventilation and TH were administered and the cerebral blood volume (CBV) and cerebral hemoglobin oxygen saturation (ScO2) were recorded for 24 h after the insult. CBV was significantly higher at 24 h after the insult in the TH + H2 group than in the other groups. ScO2 was significantly lower throughout the 24 h after the insult in the TH + H2 group than in the NT group. In conclusion, combined H2 gas ventilation and TH increased CBV and decreased ScO2, which may reflect elevated cerebral blood flow to meet greater oxygen demand for the surviving neurons, compared with TH alone.

Development of a novel porcine ischemia/reperfusion model inducing different ischemia times in bilateral kidneys-effects of hydrogen gas inhalation

Background: Acute kidney injury and its central pathology, renal ischemia reperfusion injury (IRI), have been studied in many animal models. Although renal IRI has been induced in pig models in many ways, simultaneous bilateral ischemia or unilateral ischemia along with contralateral nephrectomy models only provide data on the renal response to a single ischemia time. Moreover, it has been reported that prolonged renal ischemia time in pigs for 120 min or more can cause irreversible renal damage and increase animal mortality. Methods: We developed a model that induces prolonged ischemia time and subsequent reperfusion injury without threatening the lives of pigs by subjecting the left and right kidneys to ischemia for 120 and 60 min, respectively. Using this novel model, we investigated whether hydrogen gas inhalation could alleviate renal IRI. Results: All animals (n=4) survived until the end of the observation period of 3 months in this model. Evaluation of the left and right kidneys immediately before and after IRI could be performed separately by blood sampling from each renal vein and renal biopsy during surgery, although the results of peripheral blood sampling during the follow-up were the mixed results of bilateral kidneys. The release of degraded DNA from the kidneys immediately after IRI and subsequent renal fibrosis at 3 months increased in response to ischemia time. Although the effect of hydrogen gas on pathological findings was not obvious, the release of degraded DNA from the kidney, an acute marker of IRI, appeared to be suppressed. Conclusions: We have developed a novel model in which IRI of different ischemia times is induced in the bilateral kidney that provides two-fold information and allows for safe long-term observation experiments in pigs. Using this model, hydrogen gas inhalation appeared to reduce acute renal IRI, although the effect was not statistically significant. Keywords: Animal model; chronic kidney disease; hydrogen gas; ischemia reperfusion injury (IRI); porcine.

Pharmacokinetics of hydrogen after ingesting a hydrogen-rich solution: A study in pigs

Drinking hydrogen (H2)-rich water is a common way to consume H2. Although many studies have shown efficacy of drinking H2-rich water in neuropsychiatric and endocrine metabolic disorders, their authenticity has been questioned because none examined the associated pharmacokinetics of H2. Therefore, we performed the first study to investigate the pharmacokinetics of H2 in pigs given an H2-rich glucose solution with the aim to extrapolate the findings to humans. We inserted blood collection catheters into the jejunal and portal veins, suprahepatic inferior vena cava, and carotid artery of 4 female pigs aged 8 weeks. Then, within 2 min we infused 500 ml of either H2-rich or H2-free glucose solution into the jejunum via a percutaneous gastrostomy tube and measured changes in H2 concentration in venous and arterial blood over 120 min. After infusion of the H2-rich glucose solution, H2 concentration in the portal vein peaked at 0.05 mg/L and remained at more than 0.016 mg/L (H2 saturation level, 1%) after 1 h; it also increased after infusion of H2-free glucose solution but remained below 0.001 mg/L (H2 saturation level, 0.06%). We assume that H2 was subsequently metabolized in the liver or eliminated via the lungs because it was not detected in the carotid artery. In conclusion, drinking highly concentrated H2-rich solution within a short time is a good way to increase H2 concentration in portal blood and supply H2 to the liver.

Pharmacokinetics of a single inhalation of hydrogen gas in pigs

The benefits of inhaling hydrogen gas (H2) have been widely reported but its pharmacokinetics have not yet been sufficiently analyzed. We developed a new experimental system in pigs to closely evaluate the process by which H2 is absorbed in the lungs, enters the bloodstream, and is distributed, metabolized, and excreted. We inserted and secured catheters into the carotid artery (CA), portal vein (PV), and supra-hepatic inferior vena cava (IVC) to allow repeated blood sampling and performed bilateral thoracotomy to collapse the lungs. Then, using a hydrogen-absorbing alloy canister, we filled the lungs to the maximum inspiratory level with 100% H2. The pig was maintained for 30 seconds without resuming breathing, as if they were holding their breath. We collected blood from the three intravascular catheters after 0, 3, 10, 30, and 60 minutes and measured H2 concentration by gas chromatography. H2 concentration in the CA peaked immediately after breath holding; 3 min later, it dropped to 1/40 of the peak value. Peak H2 concentrations in the PV and IVC were 40% and 14% of that in the CA, respectively. However, H2 concentration decay in the PV and IVC (half-life: 310 s and 350 s, respectively) was slower than in the CA (half-life: 92 s). At 10 min, H2 concentration was significantly higher in venous blood than in arterial blood. At 60 min, H2 was detected in the portal blood at a concentration of 6.9-53 nL/mL higher than at steady state, and in the SVC 14-29 nL/mL higher than at steady state. In contrast, H2 concentration in the CA decreased to steady state levels. This is the first report showing that inhaled H2 is transported to the whole body by advection diffusion and metabolized dynamically.

Low-Flow Nasal Cannula Hydrogen Therapy

Background: Molecular hydrogen (H2) is a biologically active gas that is widely used in the healthcare sector. In recent years, on-site H2 gas generators, which produce high-purity H2 by water electrolysis, have begun to be introduced in hospitals, clinics, beauty salons, and fitness clubs because of their ease of use. In general, these generators produce H2 at a low-flow rate, so physicians are concerned that an effective blood concentration of H2 may not be ensured when the gas is delivered through a nasal cannula. Therefore, this study aimed to evaluate blood concentrations of H2 delivered from an H2 gas generator via a nasal cannula. Methods: We administered 100% H2, produced by an H2 gas generator, at a low-flow rate of 250 mL/min via a nasal cannula to three spontaneously breathing micro miniature pigs. An oxygen mask was placed over the nasal cannula to administer oxygen while minimizing H2 leakage, and a catheter was inserted into the carotid artery to monitor the arterial blood H2 concentration. Results: During the first hour of H2 inhalation, the mean (standard error (SE)) H2 concentrations and saturations in the arterial blood of the three pigs were 1,560 (413) nL/mL and 8.85% (2.34%); 1,190 (102) nL/mL and 6.74% (0.58%); and 1,740 (181) nL/mL and 9.88% (1.03%), respectively. These values are comparable to the concentration one would expect if 100% of the H2 released from the H2 gas generator is taken up by the body. Conclusions: Inhalation of 100% H2 produced by an H2 gas generator, even at low-flow rates, can increase blood H2 concentrations to levels that previous non-clinical and clinical studies demonstrated to be therapeutically effective. The combination of a nasal cannula and an oxygen mask is a convenient way to reduce H2 leakage while maintaining oxygenation.

Effectiveness of Acidic Oxidative Potential Water in Preventing Bacterial Infection in Islet Transplantation

At a number of points in the current procedures of islet isolation and islet culture after the harvesting of donor pancreata, microorganisms could potentially infect the islet preparation. Furthermore, the use of islets from multiple donors can compound the risks of contamination of individual recipients. Acidic oxidative potential water (also termed electrolyzed strong acid solution, function water, or acqua oxidation water), which was developed in Japan, is a strong acid formed on the anode in the electrolysis of water containing a small amount of sodium chloride. It has these physical properties: pH, from 2.3 to 2.7; oxidative-reduction potential, from 1,000 to 1,100 mV; dissolved chlorine, from 30 to 40 ppm; and dissolved oxygen, from 10 to 30 ppm. Because of these properties, acidic oxidative potential water has strong bactericidal effects on all bacteria including methicillin-resistant Staphylococcus aureus (MRSA), viruses including HIV, HBV, HCV, CMV, and fungi as a result of the action of the active oxygen and active chlorine that it contains. We conducted this study to evaluate the effect of acidic oxidative potential water irrigation on bacterial contamination on the harvesting of porcine pancreata from slaughterhouses for islet xenotransplantation by counting the number of pancreatic surface bacteria using the Dip-slide method, and on the results of islet culture; and to evaluate the direct effect on isolated islets when it is used to prevent bacterial contamination by the static incubation test and by morphological examination. Direct irrigation of the pancreas by acidic oxidative potential water was found to be very effective in preventing bacterial contamination, but direct irrigation of isolated islets slightly decreased their viability and function.

On the Physiology of Hydrogen Diving and Its Implication for Hydrogen Biochemical Decompression

Biochemical decompression, a novel approach for decreasing decompression sickness (DCS) risk by increasing the tissue washout rate of the inert gas, was tested in pigs during simulated H2 dives. Since there is only limited physiological data on the use of H2 as a diving gas, direct calorimetry and respirometry were used to determine whether physiological responses to hyperbaric H2 and He are different in guinea pigs. The data suggested that responses in hyperbaric heliox and hydrox cannot be explained solely by the thermal properties of the two gas mixtures. To increase the washout rate of H2, a H2-metabolizing microbe (Methanobrevibacter smithii) was tested that converts H2 to H2O and CH4. Using pigs (Sus scrofa) comparisons were made between untreated controls, saline-injected controls, and animals injected with M. smithii into the large intestine. To simulate a H2 dive, pigs were placed in a dry hyperbaric chamber and compressed to different pressures (22.3-25.7 atm) for times of 30-1440 min. Subsequently, pigs were decompressed to 11 atm at varying rates (0.45-1.80 atm • min-1), and observed for severe symptoms of DCS for 1 h. Chamber gases (O2, N2, He, H2, CH4) were monitored using gas chromatography throughout the dive. Release of CH4 in untreated pigs indicated that H2 was being metabolized by native intestinal microbes and results indicated that native H2-metabolizing microbes may provide some protection against DCS following hyperbaric H2 exposure. M. smithii injection further enhanced CH4 output and lowered DCS incidence. A probabilistic model estimated the effect of H2-metabolism on the probability of DCS, P(DCS), after hyperbaric H2 exposure. The data set included varying compression and decompression sequences for controls and animals with intestinal injections of H2 metabolizing microbes. The model showed that increasing total activity of M. smithii injected into the animals reduced P(DCS). Reducing the tissue concentration of the inert gas significantly reduced the risk of DCS in pigs, further supporting the hypothesis that DCS is primarily caused by elevated tissue inert gas tension. The data provide promising evidence for the development of biochemical decompression as an aid to human diving.

Hydrogen is neuroprotective and preserves cerebrovascular reactivity in asphyxiated newborn pigs

Hydrogen (H2) has been reported to neutralize toxic reactive oxygen species. Oxidative stress is an important mechanism of neuronal damage after perinatal asphyxia. We examined whether 2.1% H2-supplemented room air (H2-RA) ventilation would preserve cerebrovascular reactivity (CR) and brain morphology after asphyxia/reventilation (A/R) in newborn pigs. Anesthetized, ventilated piglets were assigned to one of the following groups: A/R with RA or H2-RA ventilation (A/R-RA and A/R-H2-RA; n = 8 and 7, respectively) and respective time control groups (n = 9 and 7). Asphyxia was induced by suspending ventilation for 10 min, followed by reventilation with the respective gases for 4 h. After euthanasia, the brains were processed for neuropathological examination. Pial arteriolar diameter changes to graded hypercapnia (5-10% CO2 inhalation), and NMDA (10(-4) M) were determined using the closed cranial window/intravital microscopy before and 1 h after asphyxia. Neuropathology revealed that H2-RA ventilation significantly reduced neuronal injury induced by A/R in virtually all examined brain regions including the cerebral cortex, the hippocampus, basal ganglia, cerebellum, and the brainstem. Furthermore, H2-RA ventilation significantly increased CR to hypercapnia after A/R (% vasodilation was 23 ± 4% versus 41 ± 9%, p < 0.05). H2-RA ventilation did not affect reactive oxygen species-dependent CR to NMDA. In summary, H2-RA could be a promising approach to reduce the neurologic deficits after perinatal asphyxia.