Evaluation of the effect of enriched hydrogen saline solution on distant organ (lung) damage in skeletal muscle ischemia reperfusion in rats.

INTRODUCTION: Ischemia-reperfusion (IR) injury is a major concern that frequently occurs during vascular surgeries. Hydrogen-rich saline (HRS) solution exhibits antioxidant and anti-inflammatory properties. This study aimed to examine the effects of HRS applied before ischemia in the lungs of rats using a lower extremity IR model. MATERIAL AND METHODS: After approval was obtained from the ethics committee, 18 male Wistar albino rats weighing 250-280 g were randomly divided into three groups: control (C), IR and IR-HRS. In the IR and IR-HRS groups, an atraumatic microvascular clamp was used to clamp the infrarenal abdominal aorta, and skeletal muscle ischemia was induced. After 120 min, the clamp was removed, and reperfusion was achieved for 120 min. In the IR-HRS group, HRS was administered intraperitoneally 30 min before the procedure. Lung tissue samples were examined under a light microscope and stained with hematoxylin-eosin (H&E). Malondialdehyde (MDA) levels, total sulfhydryl (SH) levels, and histopathological parameters were evaluated in the tissue samples. RESULTS: MDA and total SH levels were significantly higher in the IR group than in the control group (p < 0.0001 and p = 0.001, respectively). MDA and total SH levels were significantly lower in the IR-HRS group than in the IR group (p < 0.0001 and p = 0.013, respectively). A histopathological examination revealed that neutrophil infiltration/aggregation, alveolar wall thickness, and total lung injury score were significantly higher in the IR group than in the control group (p < 0.0001, p = 0.001, and p < 0.0001, respectively). Similarly, alveolar wall thickness and total lung injury scores were significantly higher in the IR-HRS group than in the control group (p = 0.009 and p = 0.004, respectively). A statistically significant decrease was observed in neutrophil infiltration/aggregation and total lung injury scores in the IR-HRS group compared to those in the IR group (p = 0.023 and p = 0.022, respectively). CONCLUSION: HRS at a dose of 20 mg/kg, administered intraperitoneally 30 min before ischemia in rats, reduced lipid peroxidation and oxidative stress, while also reducing IR damage in lung histopathology. We believe that HRS administered to rats prior to IR exerts a lung-protective effect.

Effect of hydrogen-rich saline on melanopsin after acute blue light-induced retinal damage in rats.

Excessive exposure to blue light can cause retinal damage. Hydrogen-rich saline (HRS), one of the hydrogen therapies, has been demonstrated to be effective in eye photodamage, but the effect on the expression of melanopsin in intrinsically photosensitive retinal ganglion cells (ipRGCs) is unknown. In this study, we used a rat model of light-induced retinal injury to observe the expression of melanopsin after HRS treatment and to determine the effect of HRS on retinal ganglion cell protection. Adult SD rats were exposed to blue light (48 h) and treated with HRS for 0, 3, 7, and 14 days. Real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB) were performed to find the expression of genes and proteins, respectively. The function of retinal ipRGCs was measured by pattern-evoked electroretinography (pERG). The number and morphological changes of melanopsin-positive ganglion cells in the retina were observed by immunofluorescence (IF). Acute blue light exposure caused a decrease in ipRGC function, decreased expression of melanopsin protein and the melanopsin-positive RGCs, and diminished immunoreactivity in dendrites. However, over time, melanopsin showed a tendency to self-recovery, with an increase in melanopsin protein expression and the number of melanopsin-positive RGCs, with incomplete recovery of function within two weeks. HRS treatment accelerated the recovery process, with a significant increase in melanopsin expression and the number of melanopsin-positive RGCs, and an improvement in the pERG waveform within two weeks.

Protective effects of hydrogen rich saline solution in rats with experimental myocardial ischemia reperfusion injury.

Aim: The aim of our study is to show whether the administration of hydrogen-rich saline solution (HRSS) intraperitoneally before left main coronary artery (LAD) ischemia protects the myocardium against ischemia-reperfusion (IR) injury. Materials and methods: After ethics committee approval, 24 Wistar Albino rats were divided into 4 groups, 6 rats in each group. For experimental IR, myocardial ischemia was performed by LAD ligation. Left thoracotomy was performed without ischemia in the Control group (Group C). Left thoracotomy was performed without myocardial ischemia to the rats in the HRSS group, and HRSS was given intraperitoneally (ip) at a rate of 10 ml/kg throughout the procedure. In the MIR-HRSS group, a single dose of 10 ml/kg HRSS was administered 5 min before reperfusion. Histopathological and biochemical parameters were compared in myocardial tissue samples taken at the end of the reperfusion period. Results: When the groups were compared among themselves in terms of TOS and TAS levels, there was a significant difference between the groups (p = 0.006, p = 0.002). The severity of cardiomyocyte degeneration was significantly greater in MIR group than that in the control and HRSS groups (p = 0.002 and p = 0.001, respectively), as well as severity score of cardiomyocyte degeneration was higher in MIR-HRSS group compared with HRSS group (p = 0.035). Conclusion: Our study shows that HRSS is protective in IR injury, with the application of HRSS 5 min before reperfusion, interstitial edema severity, subendocardial haemorrhage are reduced, and oxidant status parameters are increased, while antioxidant status parameters are decreased. We believe that when it is supported by other studies, the protective effects of HRSS on IR damage will be shown in detail and its indications will be expanded.

Hydrogen-rich saline alleviates cardiomyocyte apoptosis by reducing expression of calpain1 via miR-124-3p.

AIMS: Molecular hydrogen has been exhibited a protective function in heart diseases. Our previous study demonstrated that hydrogen-rich saline (HRS) could scavenge free radicals selectively and alleviate the inflammatory response in the myocardial ischaemia/reperfusion (I/R) injury, but the underlying mechanism has not been fully clarified. METHODS AND RESULTS: Adult (10 weeks) C57BL/6 male mice and neonatal rat cardiomyocytes were used to establish I/R and hypoxia/reoxygenation (H/R) injury models. I/R and H/R models were treated with HRS to classify the mechanisms of cardioproctective function. In this study, we found that miR-124-3p was significantly decreased in both I/R and H/R models, while it was partially ameliorated by HRS pretreatment. HRS treatment also alleviated ischaemia-induced apoptotic cell death and increased cell viability during I/R process, whereas silencing expression of miR-124-3p abolished this protective effect. In addition, we identified calpain1 as a direct target of miR-124-3p, and up-regulation of miR-124-3 produced both activity and expression of calpain1. It was also found that compared with the HRS group, overexpression of calpain1 increased caspase-3 activities, promoted cleaved-caspase3 and Bax protein expressions, and correspondingly decreased Bcl-2, further reducing cell viability. These results illustrated that calpain1 overexpression attenuated protective effect of HRS on cardiomyocytes in H/R model. CONCLUSIONS: The present study showed a protective effect of HRS on I/R injury, which may be associated with miR-124-3p-calpain1 signalling pathway.

Hydrogen-Rich Saline-A Novel Neuroprotective Agent in a Mouse Model of Experimental Cerebral Ischemia via the ROS-NLRP3 Inflammasome Signaling Pathway In Vivo and In Vitro.

BACKGROUND: Our previous research revealed that inflammation plays an important role in the pathophysiology of cerebral ischemia. The function of the NOD-like receptor protein 3 (NLRP3) inflammasome is to activate the inflammatory process. Recent findings suggest that reactive oxygen species (ROS) are essential secondary messengers that activate the NLRP3 inflammasome. Hydrogen-rich saline (HS) has attracted attention for its anti-inflammatory properties. However, the protective effect and possible mechanism of HSin brain ischemia have not been well elucidated. METHODS: To test the therapeutic effect of HS, we established a mouse model of distal middle cerebral artery occlusion (dMCAO) and an in vitro model of BV2 cells induced by lipopolysaccharide (LPS). The ROS scavenger N-acetylcysteine (NAC) was used to investigate the underlying mechanisms of HS. RESULTS: HS significantly improved neurological function, reduced infarct volume, and increased cerebral blood flow in a dMCAO mouse model. ROS, NLRP3, Caspase-1, and IL-1ß expression increased after cerebral ischemia, and this was reversed by HS treatment. In BV2 cells, the application of NAC further demonstrated that HS could effectively inhibit the expression of the ROS-activated NLRP3 inflammasome. CONCLUSIONS: HS, as a novel therapeutic option, could exert protect the brain by inhibiting the activation of the ROS-NLRP3 signaling pathway after cerebral ischemia.

Hyperbaric oxygen combined with hydrogen-rich saline protects against acute lung injury.

Background: This study sought to investigate therapeutic effects of hydrogen-rich saline (HRS) combined with hyperbaric oxygen (HBO2) in an experimental rat model of acute lung injury (ALI). Method: Forty male Sprague-Dawley rats were randomly divided into sham, LPS, LPS + HBO2, LPS + HRS, and LPS + HBO2 + HRS groups. After an intratracheal injection of LPS-induced ALI, the rats were given a single-agent HBO2 or HRS or HBO2 + HRS treatment. The treatments were continued for three days in this experimental rat model of ALI. At the end of experiment, the lung pathological, inflammatory factors, and cell apoptosis in the pulmonary tissue were detected by Tunel method and cell apoptosis rate was calculated accordingly. Results: In the groups treated with HBO2 + HRS, pulmonary pathological data, wet-dry weight ratio, and inflammatory factors of pulmonary tissues and alveolar lavage fluid were significantly superior to those of the sham group (p≺0.05). Cell apoptosis detection revealed that no single agent treatment of HRS or HBO2, or combination treatment, could alleviate all cell apoptosis. HRS combined with HBO2 treatment was superior to single treatment (p≺0.05). Conclusion: HRS or HBO2 single treatment could decrease inflammatory cytokines release in lung tissue, reduce the accumulation of oxidative products and alleviate apoptosis of pulmonary cells, then lead to positive therapeutic effects on ALI induced by LPS. Furthermore, HBO2 combined with HRS treatment presented a synergy effect on cell apoptosis decrease and a decline in inflammatory cytokine release and related inflammatory product generation, compared with a single treatment.

Protective Effect of Hydrogen-Rich Saline on Spinal Cord Damage in Rats.

The anti-inflammatory and anti-apoptotic effects of molecular hydrogen, delivered as hydrogen-rich saline (HRS), on spinal cord injury was investigated. Four-month-old male Sprague Dawley rats (n = 24) were classified into four groups: (1) control-laminectomy only at T7-T10; (2) spinal injury-dura left intact, Tator and Rivlin clip compression model applied to the spinal cord for 1 min, no treatment given; (3) HRS group-applied intraperitoneally (i.p.) for seven days; and (4) spinal injury-HRS administered i.p. for seven days after laminectomy at T7-T10 level, leaving the dura intact and applying the Tator and Rivlin clip compression model to the spinal cord for 1 min. Levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were measured in blood taken at day seven from all groups, and hematoxylin-eosin (H & E) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) were used to stain the tissue samples. IL-6 and TNF-α levels were significantly lower in the group treated with HRS following the spinal cord injury compared to the group whose spinal cord was damaged. A decrease in apoptosis was also observed. The anti-inflammatory and anti-apoptotic effect of IL-6 may be a clinically useful adjuvant therapy after spinal cord injury.

Hydrogen-Rich Saline Attenuates Chronic Allodynia after Bone Fractures via Reducing Spinal CXCL1/CXCR2-Mediated Iron Accumulation in Mice.

PURPOSE: Neuroinflammation often initiates iron overload in the pathogenesis of neurological disorders. Chemokine-driven neuroinflammation is required for central sensitization and chronic allodynia following fractures, but specific molecular modulations are elusive. This present study explored whether hydrogen-rich saline, as one potent anti-inflammatory pharmaceutical, could alleviate fracture-caused allodynia by suppressing chemokine CXCL1 expression and iron overload. METHODS: A mouse model of tibial fracture with intramedullary pinning was employed for establishing chronic allodynia. Three applications of hydrogen-rich saline (1, 5 or 10 mL/kg) were administrated intraperitoneally on a daily basis from days 4 to 6 following fractures. Spinal CXCL1 and its receptor CXCR2 levels, transferrin receptor 1 (TfR1) expression and iron concentration were examined. Recombinant CXCL1, a selective CXCR2 antagonist and an iron chelator were used for verification of mechanisms. RESULTS: Repetitive injections of hydrogen-rich saline (5 and 10 mL/kg but not 1 mL/kg) prevent fracture-caused mechanical allodynia and cold allodynia in a dose-dependent manner. Single exposure to hydrogen-rich saline (10 mL/kg) on day 14 after orthopedic surgeries controls the established persistent fracture allodynia. Furthermore, hydrogen-rich saline therapy reduces spinal CXCL1/CXCR2 over-expression and TfR1-mediated iron accumulation in fracture mice. Spinal CXCR2 antagonism impairs allodynia and iron overload following fracture surgery. Intrathecal delivery of recombinant CXCL1 induces acute allodynia and spinal iron overload, which is reversed by hydrogen-rich saline. Moreover, iron chelation alleviates exogenous CXCL1-induced acute pain behaviors. CONCLUSIONS: These findings identify that hydrogen-rich saline confers protection against fracture-caused chronic allodynia via spinal down-modulation of CXCL1-dependent TfR1-mediated iron accumulation in mice.

Protective Effects of Hydrogen-Rich Saline on Experimental Intestinal Volvulus in Rats.

BACKGROUND: Intestinal volvulus can cause morbidity and mortality. Surgical reduction, on the other hand, could result in ischemia-reperfusion (I/R) injury. Hydrogen rich saline solution (HRSS neutralizes free radicals in the body. This study aimed to investigate the effects of HRSS in I/R injury in experimental intestinal volvulus in rats. METHODS: Thirty rats were randomly allocated into 5 groups. All procedures were done under general anesthesia and sterile conditions in each animal. Five ml/kg of saline and HRSS were administered intraperitoneally (ip) in Sham (Group 1) and HRSS (Group 2) groups, respectively. Groups 3, 4, and 5 constituted the study groups in which volvulus was created in a 5-cm- long ileal segment 2 cm proximal to the ileocecal valve. After 2 hours the volvuli were reduced and following 2 hours of reperfusion, these segments were removed. In volvulus-I/R group (Group 3) no additional procedure was done. HRSS was administered shortly before reperfusion (reduction of the volvulus) in Treatment I (Group 4) and 1 h before experimental volvulus in Treatment II (Group 5) groups. Blood and intestinal tissue samples were obtained from all rats at the 4th hour. Both tissue and blood total oxidant (TOS) and antioxidant status (TAS) levels were determined and tissue histomorphologies were studied. Oxidative stress indices (TOS ÷ TAS) (OSI) were calculated. RESULTS: Tissue TOS and OSI levels and histomorphological injury scores were statistically lower in treatment groups than I/R group, whereas blood TOS and OSI levels were similar between the groups. CONCLUSIONS: This study provides biochemical and histomorphological evidence that HRSS prevents intestinal damage in I/R injury caused by volvulus.

Microbial hydrogen economy alleviates colitis by reprogramming colonocyte metabolism and reinforcing intestinal barrier.

With the rapid development and high therapeutic efficiency and biosafety of gas-involving theranostics, hydrogen medicine has been particularly outstanding because hydrogen gas (H2), a microbial-derived gas, has potent anti-oxidative, anti-apoptotic, and anti-inflammatory activities in many disease models. Studies have suggested that H2-enriched saline/water alleviates colitis in murine models; however, the underlying mechanism remains poorly understood. Despite evidence demonstrating the importance of the microbial hydrogen economy, which reflects the balance between H2-producing (hydrogenogenic) and H2-utilizing (hydrogenotrophic) microbes in maintaining colonic mucosal ecosystems, minimal efforts have been exerted to manipulate relevant H2-microbe interactions for colonic health. Consistent with previous studies, we found that administration of hydrogen-rich saline (HS) ameliorated dextran sulfate sodium-induced acute colitis in a mouse model. Furthermore, we demonstrated that HS administration can increase the abundance of intestinal-specific short-chain fatty acid (SCFA)-producing bacteria and SCFA production, thereby activating the intracellular butyrate sensor peroxisome proliferator-activated receptor γ signaling and decreasing the epithelial expression of Nos2, consequently promoting the recovery of the colonic anaerobic environment. Our results also indicated that HS administration ameliorated disrupted intestinal barrier functions by modulating specific mucosa-associated mucolytic bacteria, leading to substantial inhibition of opportunistic pathogenic Escherichia coli expansion as well as a significant increase in the expression of interepithelial tight junction proteins and a decrease in intestinal barrier permeability in mice with colitis. Exogenous H2 reprograms colonocyte metabolism by regulating the H2-gut microbiota-SCFAs axis and strengthens the intestinal barrier by modulating specific mucosa-associated mucolytic bacteria, wherein improved microbial hydrogen economy alleviates colitis.

Hydrogen-rich saline alleviates early brain injury through inhibition of necroptosis and neuroinflammation via the ROS/HO-1 signaling pathway after traumatic brain injury.

Traumatic brain injury (TBI) has been recognized as a serious public health issue and a key contributor to disability and death, with a huge economic burden worldwide. Hydrogen, which is a slight and specific cytotoxic oxygen radical scavenger, has been demonstrated to ameliorate early brain injury (EBI) through reactive oxygen species (ROS), oxidative stress injury, apoptosis and necroptosis. Necroptosis refers to a type of programmed cell death process that has a vital function in neuronal cell death following TBI. The specific function of necroptosis in hydrogen-mediated neuroprotection after TBI, however, has yet to be determined. The present study aimed to examine the neuroprotective effects and possible molecular basis that underly hydrogen-rich saline in TBI-stimulated EBI by examining neural necroptosis in the C57BL/6 mouse model. The brain water content, neurological score, neuroinflammatory cytokines (NF-κΒ, TNF-α, IL-6 and IL-1ß) and ROS were evaluated using flow cytometry. Malondialdehyde, superoxide dismutase (SOD) and glutathione (GSH) levels were evaluated using a biochemical kit. Receptor-interacting protein kinase (RIP)1, RIP3, Nrf2 and Heme oxygenase-1 (HO-1) were evaluated using western blotting. mRNA of Nrf2 and HO-1 were evaluated using quantitative PCR. Neuronal death was evaluated by TUNEL staining. The outcomes illustrated that hydrogen-rich saline treatment considerably enhanced the neurological score, increased neuronal survival, decreased the levels of serum MDA and brain ROS, increased the levels of serum GSH and SOD. In addition the protein expression levels of RIP1 and RIP3 and the cytokines NF-κB, TNF-α, IL-1ß and IL-6 were downregulated compared with the TBI group, which demonstrated that hydrogen-rich saline-induced inhibition of necroptosis and neuroinflammation ameliorated neuronal death following TBI. The neuroprotective capacity of hydrogen-rich saline was demonstrated to be partly dependent on the ROS/heme oxygenase-1 signaling pathway. Taken together, the findings of the present study indicated that hydrogen-rich saline enhanced neurological outcomes in mice and minimized neuronal death by inducing protective effects against neural necroptosis as well as neuroinflammation.

Hydrogen-rich saline regulates the polarization and apoptosis of alveolar macrophages and attenuates lung injury via suppression of autophagy in septic rats.

BACKGROUND: Hydrogen-rich saline (HRS) has a protective effect on sepsis-induced lung injury. However, the underlying mechanisms are still unclear. Polarization and apoptosis of macrophages are essential factors in the pathogenesis of acute lung injury (ALI). Moreover, autophagy is involved in the regulation of both macrophage polarization and apoptosis. Therefore, this study investigated the ability of HRS to attenuate ALI through regulation of the polarization and apoptosis of alveolar macrophages (AMs) during sepsis by modulating autophagy. METHODS: Male Sprague-Dawley (SD) rats were used to prepare the sepsis-induced lung injury animal model. Rat lung tissue was harvested after lipopolysaccharide (LPS) treatment, in the presence or absence of HRS, and the AMs were analyzed for changes in polarization, apoptosis, and autophagy. The rat AM cell line NR8383 was used to examine these processes in vitro using Western blot analysis, flow cytometry, and transmission electron microscopy. RESULTS: LPS-induced ALI in rats was associated with an increase in autophagy, apoptosis, and M1 polarization but a decrease in M2 polarization in AMs. These effects were reversed by administration of HRS. Inhibition of AM autophagy with 3-methyladenine (3-MA) decreased apoptosis and M1 polarization and increased M2 polarization, paralleling the effects of HRS. CONCLUSIONS: HRS could attenuate ALI in septic rats through regulation of AM polarization and a reduction in apoptosis by suppressing autophagy. This may represent a potential novel therapeutic target for the treatment of ALI caused by sepsis.

Hydrogen-saturated saline mediated neuroprotection through autophagy via PI3K/AKT/mTOR pathway in early and medium stages of rotenone-induced Parkinson's disease rats.

Some cardiovascular symptoms in the early stage of Parkinson's disease (PD) were related to degeneration of the rostral ventrolateral medulla (RVLM) catecholaminergic neurons. To date, little is known about the effects of hydrogen water on early stage of PD. Here, protective actions of hydrogen-saturated saline (HS) on rotenone-induced PD rats, as well as its underlying mechanisms were investigated. HS was used to treat PD rats at three general stages; early, medium and late, which were represented by rotenone induced rats for 0, 7 and 14 days. HS treatment significantly alleviated the cardiovascular and motor symptoms in rotenone-induced PD rats, improved the survival number of RVLM catecholaminergic neurons and nigral dopamine neurons only in early and medium stages of PD rats. Decreased levels of reactive oxygen species (ROS) and alpha-synuclein (α-Syn), transformation of microtubule associated protein 1 light chain 3 (LC3)-I/II and degradation of sequestosome 1 (p62) were detected, as well as increased expression level of autophagy related protein 5 (ATG5) and B-cell lymphoma-2 interacting protein 1 (Beclin-1) in the RVLM and substantia nigra (SN) after HS treatment in early and medium stages of PD rats. In addition, phosphorylation levels of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) and mammalian rapamycin target protein (mTOR) decreased after HS treatment in early and medium stages of PD rats. The results suggested that HS treatment exerted beneficial effects in early and medium stages before motor impairments emerged but not in the late stage of rotenone-induced PD rats. It exerted neuroprotection with RVLM catecholaminergic neurons and nigral dopamine neurons, mediated in part by decreasing levels of ROS and α-Syn through increasing autophagy machinery which were partly via inhibiting PI3K-Akt-mTOR pathway.

Peritoneal lavage with hydrogen-rich saline can be an effective and practical procedure for acute peritonitis.

PURPOSE: Acute peritonitis has remained a fatal disease despite of recent advances in care and treatment, including antibiotic and anticoagulant treatments. The cause of death is mostly sepsis-induced multiple organ failure. Oxidative stress can play an important role in this situation, but antioxidant therapy to capture any excessive reactive oxygen species has not yet been fully established. METHODS: Two experiments were performed. In the first experiment, we confirmed the effects of peritoneal lavage with hydrogen-rich saline (HRS) after a cecal ligation and puncture (CLP) operation in rats. In the second experiment, the changes in the hemodynamic state following this procedure were observed in a porcine model of abdominal sepsis to evaluate its safety and utility. RESULTS: Peritoneal lavage with HRS significantly improved the survival after CLP in rats, and it ameliorated the levels of sepsis-induced organ failure. Moreover, it showed anti-inflammatory and anti-apoptosis as well as antioxidant effects. The second experiment demonstrated the potential safety and feasibility of this procedure in a large animal model. CONCLUSION: This procedure can improve survival after sepsis through mitigating the sepsis-induced organ failure by inhibiting oxidative stress, apoptosis, and inflammatory pathways. Peritoneal lavage with HRS may therefore be an effective, safe, and practical therapy for patients with acute peritonitis.

Protective effects of hydrogen rich saline solution on ventral penile mathieu type flap with penile tourniquet application in rats.

INTRODUCTION: Penile tourniquet (Pt) application aims to work in a bloodless field in penile surgery. When the tourniquet is released, reperfusion injury occurs with the resumption of blood flow. Molecular hydrogen can easily attach to biomembranes and enter cytosol, mitochondria and other organelles of the cell and convert the formed OH- to H2O to prevent cell and tissue damage. AIM: We investigated the effects of hydrogen rich saline solution (HRSS) on penile Mathieu type flap tissue with Pt application in rats. STUDY DESIGN: Thirty-six Wistar-albino male rats were randomly divided into six groups. No operations were performed in the Sham group. Ventral penile Mathieu type flap was prepared and Pt was applied to the root of the penis with a plastic band in other groups. Pt was applied 10 and 30 min in the PT1° and PT³° groups. HRSS was injected intraperitoneally (ip) 5 ml/kg just before Pt was released in the HRSS1° and HRSS³° groups. In the HRSSB group, HRSS was injected 1 h before 10 min of Pt application. At the 4th hour of experiments the rats were sacrificed and tissue samples were taken for biochemical and histopathological studies. Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), myeloperoxidase (MPO), malondialdehyde (MDA) and glutathione (GSH) levels were determined in the penile tissue. The results were analyzed with one-way ANOVA and Pearson's Chi-Squared test. RESULTS: Tissue MDA, MPO, IL-6 and TNF-α values were significantly lower in all HRSS groups compared to PT1° and PT³° groups. Tissue GSH levels of HRSS groups were higher compared to PT groups. Histopathologically, inflammation was found to be higher in PT groups compared to HRSS groups. Interestingly, in the HRSSB group with HRSS administration prior to Pt, the damage was less in grade, but not statistically different than the other HRSS groups (p > 0.05). DISCUSSION: In previous studies, damage in histopathological examinations after Pt could only be demonstrated long after tourniquet applications such as 24 h and with longer duration of Pt such as 30 min. Structural changes in different Pt application times could be demonstrated at 60 min by electron microscopy and 48 h by light microscopy. In this study, the histopathological effect of Pt application could be demonstrated at the 4th hour after release and HRSS was observed to reduce the damage histopathologically as well as biochemically with its anti-inflammatory and antioxidant effects. It was observed that administration of HRSS either before or following Pt did not cause an alteration statistically. CONCLUSION: HRSS reduces tissue oxidative stress and inflammation on the flap tissue and has a protective effect in Pt applied to the hypospadias model created with a penile flap.

Hydrogen-rich saline reduces tissue injury and improves skin flap survival on a rat hindlimb degloving injury model.

BACKGROUND: Degloving injuries represent a challenge in plastic surgery. The aim of this study is to acknowledge the protective effects of hydrogen-rich saline (HRS) solution on a rat hindlimb degloved skin flap. METHODS: Twenty-one Sprague-Dawley rats were divided into three groups (control, saline and HRS). Degloving injury model was established, and flaps were sutured back following 5 min of ischemia. The control group did not receive any treatment. The saline group received intraperitoneal physiological saline (10 ml/kg) and the HRS group received intraperitoneal HRS solution (10 ml/kg) postoperatively and daily for 5 days after the operation. Skin samples were obtained for histological, immunohistochemical and biochemical evaluations. RESULTS: Inflammation was lower in the HRS compared with saline (p = 0.02) and control (p = 0.004) groups. Edema was lower in the HRS compared with saline (p = 0.02) and control (p = 0.001) groups. Malondialdehyde (MDA) level was lower in the HRS than the control group (p = 0.01). Total antioxidant level was higher in the HRS compared with saline (p = 0.009) and control (p = 0.03) groups. Total oxidant level was lower in the HRS than the control group (p = 0.02). Oxidative stress index was lower in the HRS compared with saline (p = 0.001) and control (p = 0.0001) groups`. Vascular proliferation was higher in the HRS compared with the control group (p = 0.01). CONCLUSION: Repeated HRS injections after trauma increased the viability of skin flap in rat degloving injury model by decreasing local tissue injury, due to its antioxidant, anti-inflammatory and angiogenic effects.

Molecular Hydrogen Application in Stroke: Bench to Bedside.

Stroke is a major cause of mortality and morbidity worldwide. Effective treatments are limited. Molecular hydrogen is emerging as a novel medical gas with therapeutic potential for various neurological diseases, including stroke. We reviewed the experimental and clinical findings of the effects of molecular hydrogen therapy in stroke patients and models. The underlying neuroprotective mechanisms against stroke pathology were also discussed.

Hydrogen Gas as an Exotic Performance-Enhancing Agent: Challenges and Opportunities.

BACKGROUND: Hydrogen gas (H2) has entered the world of experimental therapeutics approximately four and a half decades ago. Over the years, this simple molecule appears to drive more scientific attention, perhaps due to a dualism of H2 affirmative features demonstrated in numerous in vitro, animal and human studies on one side, and still puzzling mechanism(s) of its biological activity on the other. Up to this point, H2 was scrutinized for more than 170 different disease models and pathologies, and many research groups across the world have lately started to dynamically investigate its conceivable performance-enhancing potential. METHODS: We outlined here the studies indexed in leading research databases (PubMed, Web of Science, SCOPUS, JSTORE) that explored the effects of hydrogen on exercise performance, and also addressed important restraints, open questions, and windows of opportunities for forthcoming research and possible H2 enactment in exercise physiology. About two dozen trials have been identified in this domain, with most of the trials published during the past 5 years, while drinking hydrogen-rich water recognized as the most convenient method to deliver H2 in both animal and human studies. RESULTS: Either administered as an inhalational gas, enteral hydrogen-rich water, or intravenous hydrogen-rich saline, H2 seems to favorably affect various exercise performance outcomes and biomarkers of exercise-associated fatigue, inflammation, and oxidative stress. Not all studies have shown corroborative effects, and it appears that the gold-standard protocol for applying H2 in the field of exercise science does not exist at the moment, with studies markedly differ in the dose of H2 administered, the duration of treatment, and the source of hydrogen. CONCLUSION: H2 is a newfangled and rather effective performance-enhancing agent, yet its promising ergogenic potency has to be further validated and characterized in more well-controlled, appropriately sampled and longterm mechanistic trials. Also, appropriate regulation of hydrogen utilization in sport as an exotic medical gas may require distinctive legislative actions of relevant regulatory agencies in the future.

Hydrogen-rich saline mitigates pressure overload-induced cardiac hypertrophy and atrial fibrillation in rats via the JAK-STAT signalling pathway.

OBJECTIVE: To investigate if hydrogen-rich saline (HRS), which has been shown to have antioxidant and anti-inflammatory properties, could mitigate cardiac remodelling and reduce the incidence of atrial fibrillation (AF) in the rat model of cardiac hypertrophy. METHODS: Pressure overload was induced in rats by abdominal aortic constriction (AAC). The animals were separated into four groups: sham; AAC group; AAC plus low dose HRS (LHRS); AAC plus high dose HRS (HHRS). The sham and AAC groups received normal saline intraperitoneally and the LHRS and HHRS groups received 3 or 6 ml/kg HRS daily for six weeks, respectively. In vitro research was also performed using cardiotrophin-1 (CT-1)-induced hypertrophy of cultured neonatal rat cardiomyocytes. RESULTS: Cardiac hypertrophy was successfully induced by AAC and low and high dose HRS mitigated the pressure overload as shown by lower heart and atrial weights in these treatment groups. AF incidence and duration of the HRS groups were also significantly lower in the HRS groups compared with the AAC group. Atrial fibrosis was also reduced in the HRS groups and the JAK-STAT signalling pathway was down-regulated. In vitro experiments showed that hydrogen-rich medium mitigated the CT-1-induced cardiomyocyte hypertrophy with a similar effect as the JAK specific antagonists AG490. CONCLUSIONS: HRS was found to mitigate cardiac hypertrophy induced by pressure overload in rats and reduce atrial fibrosis and AF which was possibly achieved via inhibition of the JAK-STAT signalling pathway.

Hydrogen-rich saline protects intestinal epithelial tight junction barrier in rats with intestinal ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress-induced apoptosis pathway.

AIM: To investigate the effects of hydrogen-rich saline (HRS) on intestinal epithelial tight junction (TJ) barrier in rats with intestinal ischemia-reperfusion injury (IIRI). MATERIALS AND METHODS: Thirty-two healthy male Sprague-Dawley (SD) rats were randomly divided into four groups (n = 8 each): Sham group, I/R group, HRS group and 4-PBA group. After 45 min of ischemia and 6 h of reperfusion, the rats were sacrificed to collect serum and ileum for detection. Hematoxylin and eosin (H&E) staining was used to observe the morphology of small intestine. The serum expression levels of intestinal fatty acid binding protein (IFABP), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) were determined by enzyme linked immunosorbent assay (ELISA). Imunohistochemistry, immunofluorescence and Western blot were used to detect key proteins in intestinal epithelial TJs, ERS, and ERS-induced apoptosis, including occludin, zonula occludens-1 (ZO-1), glucose-regulated protein 78 (GRP78), X-box binding protein-1 (XBP1), C/EBP-homologous protein (CHOP) and caspase-3. Data was presented as mean ±â€¯SEM and compared using one-way ANOVA. A p-value <0.05 was considered significant. RESULTS: Compared with rats in the I/R group, the Chiu score of ileum damage in the HRS group and 4-PBA group were lower. The levels of serum IFABP, TNF-α, and IL-1ß were statistically significant among the groups. Increased expression of TJ proteins occludin and ZO-1 by reducing various parameters of ERS and ERS-induced apoptosis evidenced by down-regulation of the protein levels of GRP78, XBP1, CHOP and caspase-3 were shown in the HRS and 4-PBA groups. CONCLUSION: HRS had potential protective effects on intestinal barrier in IIRI rats. This study suggested that inhibition of excessive ERS and ERS-induced apoptosis by HRS may reduce intestinal epithelial cells damage and maintain the integrity of intestinal epithelial TJ barrier in rats with IIRI.