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. 2022 Dec 27;17(12):e0279410. doi: 10.1371/journal.pone.0279410

Pharmacokinetics of hydrogen administered intraperitoneally as hydrogen-rich saline and its effect on ischemic neuronal cell death in the brain in gerbils

Momoko Hirano 1, Kazuhisa Sugai 1, Masahiko Fujisawa 1, Eiji Kobayashi 2,3,4, Yoshinori Katsumata 2,3, Yoji Hakamata 1,2,*, Motoaki Sano 2,3,*
Editor: Alexander A Mongin5
PMCID: PMC9794077  PMID: 36574398

Abstract

Intraperitoneal administration of hydrogen (H2)-containing saline inhibited neuronal cell death in ischemic stroke in a number of animal models, but it is unknown whether H2 is absorbed from the abdominal cavity into the blood and reaches the brain. In this study, we investigated whether intraperitoneal administration of saline containing H2 inhibits neuronal cell death caused by cerebral ischemia and measured the concentration of H2 in the carotid artery and inferior vena cava (IVC). Gerbils were subjected to transient unilateral cerebral ischemia twice, and saline or H2-rich saline was administered intraperitoneally three or seven times every 12 hours. We evaluated the number of apoptotic cells in the hippocampus and cerebral cortex on day 3 and the number of viable neurons in the hippocampus and cerebral cortex on day 7. In addition, a single dose of saline or H2-rich saline was administered intraperitoneally, and blood H2 levels in the carotid artery and IVC were measured. On day 3 of ischemia/reperfusion, the number of neurons undergoing apoptosis in the cortex was significantly lower in the H2-rich saline group than in the saline group, and on day 7, the number of viable neurons in the hippocampus and cerebral cortex was significantly higher in the H2-rich saline group. Intraperitoneal administration of H2-rich saline resulted in large increases in H2 concentration in the IVC ranging from 0.00183 mg/L (0.114%) to 0.00725 mg/L (0.453%). In contrast, carotid H2 concentrations remained in the range of 0.00008 mg/L (0.0049%) to 0.00023 (0.0146%). On average, H2 concentrations in carotid artery were 0.04 times lower than in IVC. These results indicate that intraperitoneal administration of H2-rich saline significantly suppresses neuronal cell death after cerebral ischemia, even though H2 hardly reaches the brain.

Introduction

Molecular hydrogen (H2) is the most abundant molecule in the universe, but its medical value has long gone unnoticed. Its effectiveness in medicine was first studied in a rat cerebral infarction model [1]. Since then, its efficacy has been demonstrated in many pathologies, especially those involving ischemia-reperfusion injury [2–9].

For a long time, the effect of hydrogen in reducing ischemia-reperfusion injury was thought to be based on scavenging of reactive oxygen species [1]. However, other mechanisms also may be involved [7, 10].

Although the efficacy of molecular H2 has been reported extensively, its pharmacokinetics and pharmacodynamics after administration were first investigated only recently: We performed a study in which we measured the blood concentration of H2 in miniature pigs after inhalation of H2 [5, 11] and injection of H2 water into the intestinal tract [4]; the study showed that after a single inhalation of H2 gas, the H2 concentration in carotid arterial blood rises quickly but drops below the detection limit within a few minutes [12]. H2 in the blood appears to be rapidly excreted through exhalation. By administering 500 ml of H2-rich water through a tube inserted into the jejunum of miniature pigs, we confirmed that H2 was absorbed into the blood through the jejunal wall and flowed into the portal vein and hepatic superior inferior vena cava (IVC); however, almost all of it was expired and only a little reached the carotid artery [4].

To date, studies examining changes in the blood concentration of H2 have been conducted only in pigs. Furthermore, to the best of our knowledge, no studies have examined this topic in rodents. However, rodent experiments would be important because these animals are frequently used in basic medical research, including as disease models.

Rodents are often used for basic experiments on cerebral infarction, but the anatomy of their cerebral collateral blood vessels varies, e.g., at the circle of Willis; these differences are important because collateral hemodynamics have a significant influence on experimental generation of cerebral ischemia. Gerbils do not have an anastomosis between the carotid and vertebrobasilar arteries at the circle of Willis, so unilateral common carotid occlusion in the neck can easily create ipsilateral cerebral ischemia (S1 Fig) and they are often used in ischemic stroke research [13]. Therefore, previously we established a model of cerebral ischemia in gerbils in which the only required manipulation is transient occlusion of the unilateral common carotid artery in the neck; consequently, this model has low variability of data and enables reliable testing of drug efficacy [14–16].

In this study, H2-rich saline solution was administered intraperitoneally to gerbils to evaluate whether H2 inhibits ischemic neuronal cell death and to assess the pharmacokinetics of H2.

Materials and methods

2.1 Ethics statement

This study was approved by the Institutional Animal Care and Use Committee in Nippon Veterinary and Life Science University (No. 2021K-19). All animal experiments complied with the ARRIVE guidelines and were performed in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals.

2.2 Animals and experimental group

Male (n = 54) and female (n = 54) Mongolian gerbils (body weight [BW], 70–90 g) were used. All animals were bred in our laboratory, maintained in pathogen-free environments, and provided with food and water ad libitum. The carotid artery was clipped in 90 of the 108 gerbils, and 22 ischemia-positive animals were subsequently selected (see below). No cerebral ischemia was induced in the remaining gerbils (n = 17), and they were used for staining normal brain tissue and measuring blood H2 concentration.

No criteria were set for including or excluding animals, and no potential confounders were identified. The researchers were not blinded to group allocation, but the experiments and data analysis were conducted by different people to avoid potential bias.

2.3 Preparation of H2-rich saline

To prepare H2-rich saline, a polyethylene terephthalate bottle was filled with saline; then, H2 was pressurized with a DAYS hydrogen gas filling system (Doctors Man Co., Ltd., Japan) and filled into the bottle to a gauge pressure of 0.4 MPa, as described previously [4]. The bottle was shaken for 30 seconds to dissolve the H2, and then the lid was opened to reduce the pressure to atmospheric pressure. H2-rich saline (H2 concentration, 2.6 mg/L) was prepared immediately before each set of injections.

2.4 Cerebral ischemia model in gerbils and experimental protocol

Gerbils were anesthetized with 2% isoflurane, and the left carotid artery was exposed by a mid-neck incision and occluded with a vascular clip. Anesthesia was immediately stopped, and the animals were awakened. Each animal’s behavior was carefully observed in the open field for 10 minutes, during which time neurological signs and the stroke index [17] were recorded. Animals with a stroke index of 10 or greater (scale, 0–25) were classified as ischemia positive and used in experiments to test the effects of intraperitoneal administration of H2-rich saline. The ischemia-negative animals were not used in subsequent experiments. In the ischemia-positive animals, the clip was released after 10 minutes of occlusion to restore blood flow, and the occlusion was repeated after 5 hours. After the second occlusion, the animals were randomly divided into H2-rich and saline groups. Animals in each group received repeated intraperitoneal injection with 20 ml/kg BW of solution three or seven times at 12-hour intervals. We examined two administration frequencies with the aim of confirming the existence of a dose-response relationship (S2 Fig).

2.5 Assessment of cerebral neurons

To assess cerebral neurons, 15 of the ischemia-positive animals were selected and assigned to the H2-rich saline group (n = 7) or the saline group (n = 8).

At day 7 after ischemia-reperfusion, the animals were anesthetized by intraperitoneal injection of pentobarbital (Kyoritsu Seiyaku, Japan, 65 mg/kg BW). The transcardiac approach was used to perfuse brains with heparinized saline followed by 10% phosphate-buffered formalin. Then, brains were further fixed overnight in 10% phosphate-buffered formalin, embedded in paraffin and cut into 5-μm sections and stained with hematoxylin and eosin (HE). Each brain section was scanned with a NanoZoomer-SQ (Hamamatsu Photonics K.K., Japan) x 40 mode (0.23 μm/pixel), and images were analyzed by NDP view software (NDPver2.0, Hamamastu Photonix K.K., Japan) to measure the number of neurons in the hippocampal CA1 area and layer III and V of the cerebral cortex in each hemisphere (S3 Fig). The area of measurement in each hemisphere was defined as a 300-μm wide column between the interhemispheric and rhinal fissures, as described previously [14].

2.6 Terminal deoxynucleotidyl transferase dUTP nick end labeling apoptosis assay

Apoptosis is involved in neuronal death after ischemia-reperfusion injury [18, 19], so we investigated whether it occurred in our gerbil model of cerebral ischemia-reperfusion injury and whether it is inhibited by intraperitoneal administration of H2-rich saline.

To assess apoptosis, 7 of the ischemia-positive animals were selected and randomly assigned to the H2-rich saline group (n = 4) or the saline group (n = 3).

On day 3 after ischemia-reperfusion, animals were anesthetized by intraperitoneal injection of pentobarbital (65 mg/kg BW; Kyoritsu Siyaku, Japan,). Brain sections were prepared as described above. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed on paraffin-embedded sections with an ApopTag® in situ apoptosis detection kit (Sigma-Aldrich Co., USA) according to the manufacturer’s protocol. After staining, counterstaining of nuclei was performed with hematoxylin. The method of Hanyu et al. [14] was used to image a 300-μm wide area midway between the longitudinal cerebral column and the left and right rhinal fissure in cerebral cortex and a 300-μm wide area in the hippocampus with NanoZoomer-SQ. The number of TUNEL-positive cells in layer III and V of the cerebral cortex and hippocampus was measured with image processing software (NDPver2.0, Hamamastu Photonix K.K., Japan).

2.7 Measurement of blood H2 concentration

To determine whether the hydrogen in H2-rich saline reaches the brain after intraperitoneal injection, blood samples were taken from the IVC and common carotid artery, the main vessels connecting the injection site (intraperitoneal) and the injured brain. Gerbils were anesthetized with isoflurane and injected intraperitoneally with 20 mL/kg BW of H2-rich saline (n = 9) or saline (n = 6). After 5 minutes, blood was collected from the carotid artery, and after 10 minutes, from the IVC. The blood H2 concentration was measured as described previously [4]. Briefly, to measure blood H2 concentration, a needle was first inserted into the rubber lid of a 13.5-mL sealed vial, 1 mL of air was removed, and 1 mL of blood was injected. The rubber lid was immediately coated with wax to seal the injection hole. In such a sealed vial, H2 in the blood is released into the air phase; therefore, the H2 concentration in the blood was estimated by measuring the H2 gas concentration in the air in the vial with a gas chromatograph (TRIlyzer mBA-3000, Taiyo Co.). Calibration curves were prepared with H2 gas concentrations of 0 (nitrogen gas), 5, 50, and 130 ppm. Samples were taken from the carotid artery and IVC, and each sample was measured twice. At the same time, the H2 gas concentration was measured in the air in vials in which no blood was injected. The H2 concentration in the air was 0.5 ppm, i.e., above the quantification limit. Therefore, the value in the air was subtracted from each sample measurement.

2.8 Statistical analysis

The number of cerebral neurons is presented as the ratio of neurons in the left and right hemispheres. Other data are presented as mean ± standard error. Experimental groups were compared by one-way analysis of variance (ANOVA) followed by the Tukey-Kramer multiple comparison test. P values below 0.05 were considered as significant.

Results

All ischemia-positive animals were eligible for inclusion in the analyses.

3.1 Number of surviving neurons in hippocampal tissue after ischemia-reperfusion

Ischemia-reperfusion injury markedly reduced the number of surviving neurons in the hippocampal CA1 region compared with the contralateral healthy region (Fig 1A and 1B). Surviving neurons had large round nuclei and well-defined nucleoli. Dead neurons had shrunken nuclei and were stained darkly with hematoxylin. The ratio of hippocampal neurons on the ischemic side to the healthy contralateral side after three and seven doses was 0.058 ± 0.013 (n = 5) and 0.033 ± 0.013 (n = 3) in the saline group and 0.117 ± 0.040 (n = 4) and 0.149 ± 0.071 (n = 3) in the H2-rich saline group. In seven dose groups, significantly more hippocampal neurons survived in the H2-rich saline group than in the saline group.

Fig 1. Effect of intraperitoneal injection of H2-rich saline on ischemic injury in hippocampal region CA1 in gerbils.

Fig 1

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(A) Hematoxylin and eosin staining of ischemic CA1 and contralateral (healthy) regions. The image on the right is an enlarged view of the inset in the image on the left. A closed arrowhead (△) indicates a typical surviving neuron, and an open arrowhead (▲), a dead neuron. (B) Ratio of number of surviving neurons on ischemic side to number on contralateral (healthy) side. *P < 0.05, **P < 0.01. S, saline, H2, hydrogen-rich saline; × 3, three doses; × 7, seven doses. Number of animals in each group: H2-rich saline, n = 7 (three doses, n = 4; seven doses, n = 3); saline, n = 8 (three doses, n = 5; seven doses, n = 3).

3.2 Number of surviving neurons in cerebral cortex after ischemia-reperfusion

Ischemia-reperfusion injury markedly reduced the number of surviving neurons in layer III and V of the ischemic cerebral cortex compared with the contralateral healthy region (Fig 2A and 2B). The ratio of surviving neurons on the ischemic to the contralateral side in layer III of the cortex after three and seven doses was 0.371 ± 0.153 (n = 5) and 0.443 ± 0.009 (n = 3) in the saline group and 0.592 ± 0.037 (n = 4) and 0.687±0.024 (n = 3) in the H2-rich saline group (Fig 2C). The ratio of surviving neurons in the ischemic compared to the contralateral side in layer V of the cortex after 3 and 7 doses was 0.346 ± 0.087 (n = 5) and 0.404 ± 0.040 (n = 3) in the saline group and 0.585 ± 0.117 (n = 4) and 0.644 ± 0.072 (n = 3) in the H2-rich saline group (Fig 2D). No difference was seen between the three- and seven-dose H2-rich saline groups regarding the effectiveness of intraperitoneal H2-rich saline injection in preventing neuronal death in the cerebral cortex.

Fig 2. Effect of intraperitoneal injection of H2-rich saline on ischemic injury in gerbil cerebral cortex.

Fig 2

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(A) Images of ischemic and contralateral (healthy) cortex stained with hematoxylin and eosin. (B) Higher magnification image of the inset in the image of the ischemic cortex and contralateral (healthy) cortex in (A). A closed arrowhead (△) indicates a typical surviving neuron, and an open arrowhead (▲), a dead neuron. (C) Ratio of number of surviving neurons in layer III of the cortex on ischemic side to number on contralateral (healthy) side. (D) Ratio of number of surviving neurons in layer V of the cortex on ischemic side to number on contralateral (healthy) side. * P < 0.05, ** P < 0.01. S, saline, H2, hydrogen-rich saline; × 3, three doses; × 7, seven doses. Number of animals in each group: H2-rich saline, n = 7 (three doses, n = 4; seven doses, n = 3); and saline, n = 8 (three doses, n = 5; seven doses, n = 3).

3.3 Inhibition of neuronal apoptosis after ischemia-reperfusion

TUNEL-positive neurons were observed in the hippocampus on day 3 after ischemia-reperfusion (Fig 3A). We found no differences in the number of positive cells between the groups (Fig 3B). The positive neurons were presented in layer III and V of the ischemic cerebral cortex on day 3 after ischemia-reperfusion (Fig 4A and 4B). Significantly fewer TUNEL-positive cells in layer III were seen in the H2-rich saline group (n = 4) than in the saline group (n = 3), but the number of TUNEL-positive cells in layer V was not significantly different between the two groups (Fig 4C).

Fig 3. Effect of intraperitoneal injection of H2-rich saline on ischemic neuronal apoptosis in gerbil hippocampus.

Fig 3

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(A) Images of ischemic and (healthy) contralateral hippocampus stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). An arrowhead (▲) indicates a typical apoptosis cell (upper right). (B) Number of TUNEL-positive cells in ischemic hippocampus.

Fig 4. Effect of intraperitoneal injection of H2-rich saline on ischemic neuronal apoptosis in gerbil cerebral cortex.

Fig 4

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(A) Images of ischemic contralateral and (healthy) cortex stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). (B) Higher magnification image of the inset in the image of the ischemic cortex in (A). A typical apoptosis cell is indicated by an arrowhead (▲). (C) Number of TUNEL-positive cells in layers III and V of the cerebral cortex. * P < 0.05. S, saline, H2, hydrogen-rich saline; × 7, seven doses. Number of animals in each group: H2-rich saline, n = 4; saline, n = 3.

3.4 H2 concentration in the supra-hepatic IVC and carotid artery

The H2 concentrations were measured in blood samples from the carotid artery and IVC 5 and 10 minutes after intraperitoneal administration of saline or H2-rich saline (Fig 5, S1 Table). When saline was administered intraperitoneally, H2 was detected in the IVC (mean, 0.00024 mg/L [0.0226%]) but not in the carotid artery in 5 of the 6 samples.

Fig 5. H2 concentration in the inferior vena cava and carotid artery of gerbils after intraperitoneal administration of H2-rich saline and saline.

Fig 5

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The not detectable H2 concentration was the same as that of the blank and was calculated as zero in the statistical test. ** P < 0.01. A, carotid artery; H2, hydrogen-rich saline; S, saline; V, inferior vena cava; Number of animals in each group: H2-rich group, n = 9; saline group, n = 6.

When saline was administered intraperitoneally, H2 was detected in the IVC (mean, 0.00024 mg/L [0.0226%]) but not in the carotid artery in 5 of the 6 samples. After intraperitoneal administration of H2-rich saline, H2 concentrations increased significantly in the IVC (range, 0.00183 mg/L [0. 114%] to 0.00725 mg/L [0.453%]). In contrast, H2 concentrations in the carotid artery remained in the range of 0.00008 mg/L (0.0049%) to 0.00023 (0.0146%). On average, H2 concentrations in carotid artery were 0.04 times lower than in IVC.

Discussion

In the present study, we showed that repeated, brief cerebral ischemia reproducibly induces neuronal death in the ischemia-vulnerable hippocampus and layers III and V of the cerebral cortex and that intraperitoneal administration of H2-rich saline inhibits neuronal cell death in these brain regions. The number of surviving hippocampal neurons after ischemic recanalization was significantly higher after seven than after three doses of H2-rich saline, indicating a dose-response relationship.

Although several studies have examined the efficacy of intraperitoneal administration of H2-rich saline on ischemia-reperfusion injury in the brain [18, 20–23], this study is novel in that it examined the effects on individual neuronal death in the hippocampus and cerebral cortex, rather than on infarct size reduction, in an experimental gerbil system, a highly reliable cerebral ischemia model [15, 16].

The second novelty of this study is that it determined the pharmacokinetics of H2 when injected intraperitoneally as H2-rich saline. Specifically, blood was injected into a sealed vial, and the vaporized hydrogen was measured by gas chromatography.

The H2 concentration of each sample was expressed as a corrected value obtained after subtracting the H2 concentration in air [4, 11, 12]. The lower detection limit of the analyzer was 0.1 ppm, but the H2 value in air was 0.5 ppm, well above the quantitation limit. Therefore, each sample containing H2 from air and blood should have a value greater than 0.5 ppm, i.e., above the quantitation limit. If the analyzer shows the hydrogen concentration in a sample as 0.5 ppm, that sample contains only H2 from air and no H2 from blood; in this case, the measured value would be described as not detectable (n.d.).

After intraperitoneal administration of saline, a mean of 0.00024 mg/L (0.0226%) of H2 was detected in the IVC, but no H2 was detected in the carotid artery. The H2 in the IVC is thought to be derived from intestinal bacteria [24–28]. We assume that most of the H2 was expelled with exhaled air while passing through the lungs and therefore did not reach the carotid artery.

Intraperitoneal administration of H2-rich saline resulted in a large increase in H2 concentration in the IVC (from 0.00183 mg/L [0.114%] to 0.00725 mg/L [0.453%]). We suggest that H2 in intraperitoneally administered H2-rich saline was absorbed by the portal venous system and subsequently reached the IVC. Experiments in pigs have shown that two thirds of the H2 in the portal venous blood are absorbed in the liver and the remainder reaches the IVC [4]. From these results, it can be concluded that intraperitoneal administration of H2-rich saline is a good model that can mimic the condition of drinking H2-rich water.

Intraperitoneal administration of H2-rich saline resulted in only very slight increases (ranging from 0.008 mg/L [0.0049%] to 0.00023 [0.0146%]) in H2 concentrations in the carotid artery. Unlike in pigs, in gerbils frequent blood sampling is too invasive, so carotid artery H2 concentrations were measured only once, 10 minutes after intraperitoneal administration of H2-rich saline. Previously, when an H2-rich solution was administered to the jejunum of minipigs, we observed that the H2 concentration in the portal blood increased after 2 minutes and continued to rise to approximately the same level at 5, 10, 20, and 30 minutes. Therefore, it is unlikely that the timing of the peak carotid H2 concentration was missed.

H2 may have been absorbed from the injected H2-rich saline into the intraperitoneal blood and transported via the portal vein, liver, and IFC to the lungs, where it was excreted almost entirely during exhalation. These results are similar to those of our previous study in which hydrogen water was injected directly into the jejunal lumen of pigs [4].

In both clinical trials and animal studies, inhalation of H2 is regulated to achieve a hydrogen concentration of 1% to 4% in the inhaled air [29]. Currently, it is unclear whether the inhibitory effect of H2 on ischemic neuronal cell death observed in this study is explained by a minute amount of H2, about 0.01 times the amount inhaled, reaching the brain or by an indirect effect via an inter-organ communication [30]. Further studies are needed.

Supporting information

S1 Fig. Anatomical comparison of basilar artery in rat and gerbil.

In rats (left), the internal carotid artery system, vertebral artery system, and left and right medial artery systems are connected at the base of the brain to form the circle of Willis. Therefore, if one artery is occluded, blood can still flow to the brain via the other vessels. In the gerbil (right), the basilar arterial ring lacks anastomoses between the right and left internal carotid arteries and between the carotid and vertebral arteries, so occlusion of the common carotid artery can easily induce ipsilateral cerebral ischemia.

(TIF)

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S2 Fig. Experimental protocols.

(A) Experimental protocol-1. After two 10-minute occlusions of the left carotid artery, the animals were randomly divided into H2-rich saline and saline groups. Animals in each group received 3 or 7 intraperitoneal injections of 20 ml/kg body weight of each solution. Histological analysis was performed on days 3 and 7. (B) Experimental protocol-2. H2-rich saline or saline solution (20 ml/kg BW) was administered intraperitoneally once. Blood was drawn from the carotid artery after 5 minutes and from the IVC after 10 minutes, and H2 concentration was measured by gas chromatography.

(TIF)

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S3 Fig. Measurement site of the number of survived neurons in the brain of gerbils after transient cerebral ischemia.

Cerebral histology of the cerebrum of a gerbil coronally sectioned at the levels of the infundibulum and stained with hematoxylin and eosin. Neuronal cells were counted in the hippocampal CA1 region (areas inside the blue rectangles) and the central part of the cerebral cortex (areas inside the black rectangles, 300 μm wide) in the left and right cerebral hemispheres.

(TIF)

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S1 Table. H2 concentration in the inferior vena cava and carotid artery of gerbils after intraperitoneal administration of H2-rich saline.

H2 measurements are shown in concentration (mg/L) and saturation (%). Because H2 dissolves at 1.6 mg/L under 0.1 MPa, the H2 saturation of each sample was converted by assuming 1.6 mg/L as 100% saturation. IVC, inferior vena cava, CA carotid artery, n.d., no detected, i.p., intraperitoneal injection.

(TIF)

Click here for additional data file. (138.6KB, tif)

Acknowledgments

The authors thank Sou Hashimoto and Yasuyo Aoyama (Doctors Man Co., Ltd.), Shigeo Ohta (Japanese Molecular Hydrogen Promotion Association [JHyPA]), Suga Kato (JHyPA), and Mayumi Takeda (JHyPA) for technical assistance.

Data Availability

All relevant data are within the article and its Supporting Information files.

Funding Statement

This work was supported by grants from Doctors Man Co., Ltd. (M.S.). Sou Hashimoto (Doctors Man Co., Ltd.) provided us with the H2 filling device. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Alexander A Mongin

7 Sep 2022

PONE-D-22-22387Hydrogen indirectly inhibits ischemic neuronal cell death in the gerbil brain after intraperitoneal administration of hydrogen-rich salinePLOS ONE

Dear Dr. Sano,

Thank you for submitting your manuscript to PLOS ONE. Your study has been evaluated by two experts in the field. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The major concerns of two reviewers and requests of Academic Editor that fall in line with the PLOS ONE publication expectations are outlined below:

  • [1] (Academic Editor) Your publication does not adhere to PLOS ONE Data Sharing Policy, as your primary results have not been included in supplemental files or made available through open database depository (such as OSF, Figshare, or Dryad).  See additional requirements at https://journals.plos.org/plosone/s/data-availability.  If your resubmission does not meet this criterion (provides access to primary information included in all statistical analyses) , it will be rejected outright.

[2] One of the major points of your submission is not fully supported by experimental data.  Specifically, I refer to close-to-undetectable levels of hydrogen in carotid circulation (and by extension in the brain) and the speculation that such levels are too low to explain protective effects of systemic hydrogen delivery.  Both reviewers questioned reliability of this conclusion and sensitivity of hydrogen detection method.  Mors specifically:

- (Reviewer 2) Your baseline hydrogen measurement was done only once.  This is insufficient for making conclusions and statistical comparisons.   Please add additional baseline measurements and run statistics comparisons.  

- (Reviewer 1) Sensitivity of the method may not be sufficient to detect increases in the intracarotid hydrogen levels.  Please add additional discussion in the text.

- (Academic Editor)  Due to short half-life of hydrogen in arterial circulation (~90 seconds), it is not clear if the selected timing for the intraarterial hydrogen measurements truly reflect transients of this gas upon intraperitoneal delivery in gerbils.  Please additionally discuss this in the text.

  • [3] (Academic Editor) In IHC assays, do you discriminate reliably between pycnotic and healthy nuclei?  This distinction is not obvious in provided representative images.  In Fig. 1 and Fig 2, please include additional insets with high magnification allowing for better discrimination between healthy and dying cells and include markings indicating healthy and injured/dead cells.

  • [4] (Reviewer 1) TUNEL staining quantification was performed in context but not vulnerable hippocampus.  If the relevant IHC slides/scans are preserved, please add hippocampal quantification or explain why it is impossible.

[5] (Reviewer 1, Review 2, Academic Editor) Your speculation that hydrogen provides indirect protection in the brain by acting on parasympathetic and sympathetic innervation is solely a speculation, which has not been tested by the presented experiments.  For this reason, please exclude references to indirect mechanisms and gut-brain interactions from abstract, discussion, and concluding statements in the manuscript.

- For example, on lines 358-360 you write [quotation] “Although we were able to show that intraperitoneally administered H2-rich saline protects brain neurons indirectly via the gut-brain connection, i.e., an inter-organ network…”  This is an example of unacceptable statement that is not supported by experimental evidence in the manuscript.</li></ul>

Please submit your revised manuscript by Oct 22 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Alexander A. Mongin, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, using intraperitoneal injection of hydrogen saline, HE staining and Tunel staining, the authors found that hydrogen saline injection can reduce ischemia caused by many times in hippocampus and cortex neuron loss, reduce the amount of cortex cell apoptosis, determination of portal vein and hydrogen content of the internal carotid artery, portal vein hydrogen content increased, However, hydrogen in carotid artery had no detected. Based on these results, the authors concluded that intraperitoneal injection of hydrogen saline may have a protective effect on brain injury caused by cerebral ischemia through indirect effects, but the specific mechanism is not confirmed.

 This is interesting work, However there are some problems should be considered.

According to the results of brain histological staining, the author verified the protective effect of intraperitoneal injection of hydrogen saline on cerebral ischemia, but the evidence was relatively thin. Although there were similar studies on the protection of hydrogen water on cerebral ischemia in the past, it should be supported by more research basis to be more convincing. For example, behavioral studies to verify changes in protein levels of apoptosis.

As for hydrogen acting through indirect effects, the conclusion obtained in this study is not rigorous enough. 1. There are differences in the effect of different doses and times of administration, and the dose-effect relationship is more supportive of direct effect, but certainly cannot be used as evidence to negate indirect effect. 2. The authors could not put only hydrogen from the internal carotid artery as a basis, and believed that hydrogen did not act directly on the brain, but guessed that it was realized through indirect effects such as the brain-gut axis.

Hydrogen could not be measured, possibly because the sensitivity of the method was not high enough. The author can measure in the portal vein to hydrogen, hydrogen can be absorbed in the abdominal cavity and enter the blood circulation, considering that the hydrogen is insufficient evidence of animal cell metabolism, hydrogen in animals is the inertia of the mantra, hydrogen is released only when pulmonary circulation, but such release cannot be no residual, less is retained, Resulting in undetectable detection by insensitive assays. If very little hydrogen enters the brain still can produce protective effect against cerebral ischemia injury, in other words, small dose and large effect can indicate that the effect of hydrogen is very strong, of course, indirect effect cannot be ruled out. If an indirect effect is to be judged, research evidence of such an effect should be provided, rather than simply relying on insufficient and rigorous evidence to guess.

A previous study by the authors[ref] found that inhaled hydrogen could measure higher levels in the internal carotid artery. Intraperitoneal injection of hydrogen saline could not measure the amount of hydrogen in the internal carotid artery. Given that the same group found that hydrogen inhalation could measure sufficient amounts of hydrogen in the internal carotid artery, inhalation should be used as a control to observe differences in the protective effects of hydrogen entry into the brain against neuronal loss following ischemic injury. However, the manuscript does not make such a comparative study.

Tunel staining only has cortical data, not hippocampal results. Why is this? This study has cortex and hippocampus

HE staining, but Tunel staining only in the cortex, there are no staining in hippocampus, which is not readily understood.

M. Sano, G. Ichihara, Y. Katsumata, T. Hiraide, A. Hirai, M. Momoi, T. Tamura, S. Ohata, E. Kobayashi

Pharmacokinetics of a single inhalation of hydrogen gas in pigs

PLoS One, 15 (2020), Article e0234626

Ichihara G, Katsumata Y, Moriyama H, Kitakata H, Hirai A, Momoi M, Ko S, Shinya Y, Kinouchi K, Kobayashi E, Sano M. Pharmacokinetics of hydrogen after ingesting a hydrogen-rich solution: A study in pigs. Heliyon. 2021 Nov 11;7(11):e08359. doi: 10.1016/j.heliyon.2021.e08359. PMID: 34816046; PMCID: PMC8591508.

Reviewer #2: Methodologically, this is a solid manuscript with appropriate controls and methods used except for the H2 concentration data. The manuscript is logical and well written.

Major concerns:

1. The study is confirmative. The reported data just confirm previously published data but on a different rodent stroke model and no new scientific information is reported. The manuscript would benefit from addressing the mechanism for H2 protective effect.

2. Table 1 data (H2 concentration): n=1 for saline group is not sufficient; statistical analysis is not performed.

Minor suggestions:

1. Blood H2 concentration assay should be described in more details

**********

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2022 Dec 27;17(12):e0279410. doi: 10.1371/journal.pone.0279410.r002

Author response to Decision Letter 0

10 Nov 2022

Point-by-point response to the academic editor’s and reviewers’ comments

Thank you for your constructive comments and suggestions, which have helped to improve our manuscript. Our point-by-point responses are shown below:

Responses to the Academic Editor and Reviewers

#1 Academic Editor

Your publication does not adhere to PLOS ONE Data Sharing Policy, as your primary results have not been included in supplemental files or made available through open database depository

Author Response: In accordance with the PLOS ONE Data Sharing Policy, we have revised all bar graphs as bar graph + error bars + dot blots to clearly show individual data (Figure 1 to Figure 4). The results of the hydrogen concentration measurements are shown in Figure 5 as bar graph + error bars + dot blots and were statistically analyzed. These raw data are presented in the supplementary table.

#2 Reviewer 2 comment

Your baseline hydrogen measurement was done only once. This is insufficient for making conclusions and statistical comparisons. Please add additional baseline measurements and run statistics comparisons.

Author Response: To clarify the changes in blood hydrogen concentration after intraperitoneal administration of H2-rich saline, 3 new animals were added to the hydrogen group (total: 9 animals) and 5 animals to the saline group (total: 6 animals). The n.d. (not detectable) point was calculated as zero for statistical tests. The raw data of the hydrogen concentration measurements are shown in the supplementary table, and to clarify the experimental results, we created a new Figure 5 showing the results of the statistical tests.

#2 Reviewer 1 comment

Sensitivity of the method may not be sufficient to detect increases in the intracarotid hydrogen levels. Please add additional discussion in the text.

Author Response: To measure blood H2 concentration, we first inserted a needle into the rubber lid of a 13.5-mL sealed vial, extracted 1 mL of air, and injected 1 mL of blood. To prevent outgassing, we immediately applied wax to the rubber lid to seal the injection hole. In such a sealed vial, H2 in the blood is released into the air phase because the air in the vial contains almost no H2 gas, so after injection of blood, most of the H2 gas moves from the blood (liquid phase) into the air (gas phase). Therefore, examining the concentration of H2 gas in the air inside the vial enabled the H2 concentration in the blood to be estimated. Some of the air phase (0.2 mL, 0.4 mL, or 1 mL, depending on the H2 concentration) was collected from the vial, and the H2 concentration was measured by gas chromatography (TRIlyzer mBA-3000, Taiyo, Co., Ltd.). A calibration curve was obtained by using H2 gas at a concentration of 0 (nitrogen gas), 5, 50, and 130 parts per million (ppm). The detectable value of the analyzer is 0.1 ppm (i.e., the analyzer shows values of 0.1 ppm or higher), and the limit of quantitation is considered to be approximately 0.2 ppm.

Samples were collected from the carotid artery after 5 minutes and from the inferior vena cava after 10 minutes, and each sample was measured twice. At the same time, air was collected from the exact same location and injected into a blood-free vial, and the H2 gas concentration was measured. The value of H2 in the air was 0.5 ppm, i.e., above the limit of quantitation. The air value was subtracted from each measured sample value to obtain a corrected value. Thus, only samples with an H2 value above 0.5 ppm contained H2 also from blood.

In the revised manuscript, the Methods section describes in detail the method used for measuring hydrogen concentration. In the Discussion section, we also explain the sensitivity of the H2 concentration measurement.

#2 Academic Editor comment

Due to short half-life of hydrogen in arterial circulation (~90 seconds), it is not clear if the selected timing for the intraarterial hydrogen measurements truly reflect transients of this gas upon intraperitoneal delivery in gerbils. Please additionally discuss this in the text.

Author Response: Thank you for pointing out this important point. In a previous study, when we administered an H2-rich solution to the jejunum of minipigs, we observed that the H2 concentration in the portal blood was increased at 2 minutes and continued to rise to 2 minutes and then remained at approximately the same level at 5, 10, 20, and 30 minutes. Therefore, it is unlikely that the timing of the peak carotid H2 concentration was missed. In the revised manuscript, we now explain the above in Discussion section to justify the timing of the H2 concentration measurement.

#3 Academic Editor comment

In Fig. 1 and Fig 2, please include additional insets with high magnification allowing for better discrimination between healthy and dying cells and include markings indicating healthy and injured/dead cells.

Author Response: Thank you for this suggestion. We have added a high magnification image in Figure 1-4 and have marked healthy and dying cells in it so that the reader can distinguish between them.

#4 Reviewer 1 comment

TUNEL staining quantification was performed in context but not vulnerable hippocampus. If the relevant IHC slides/scans are preserved, please add hippocampal quantification or explain why it is impossible.

Author Response: We have followed the Reviewer's instructions and added the results of TUNEL staining of the hippocampus as Figure 3. The number of TUNEL-positive cells in the hippocampus was measured and compared between the two groups. At day 3 of ischemia-reperfusion injury, there was no significant decrease in the number of TUNEL-positive cells after intraperitoneal administration of H2-rich saline.

#5 Reviewer 1, Review 2, Academic Editor comments

Please exclude references to indirect mechanisms and gut-brain interactions from abstract, discussion, and concluding statements in the manuscript.

Author Response: As directed by Reviewer 1, Reviewer 2, and the Academic Editor, we have excluded statements on indirect mechanisms and gut-brain interactions, as well as the cited references. We have completely rewritten the Discussion.

#5 Reviewer 1 comment

If an indirect effect is to be judged, research evidence of such an effect should be provided, rather than simply relying on insufficient and rigorous evidence to guess.

Author Response: We completely agree with this comment. As directed by Reviewer 1, Reviewer 2, and the Academic Editor, we have excluded statements on indirect mechanisms and gut-brain interactions, as well as the cited references. Intraperitoneal administration has been the route of administration most frequently used for observing the effects on rodents of H2 in H2-rich saline. To the best of our knowledge, no studies have evaluated the pharmacokinetics of H2 administered by this route. We hope that our study will help to elucidate the mechanism of action of H2.

HE staining, but TUNEL staining only in the cortex, there are no staining in hippocampus, which is not readily understood.

Author Response: We have added the results of TUNEL staining of the hippocampus as Figure 3. The number of TUNEL-positive cells in the hippocampus was measured and compared between the two groups. At day 3 of ischemia-reperfusion injury, there was no significant decrease in the number of TUNEL-positive cells after intraperitoneal administration of H2-rich saline.

#5 Reviewer 2 comment

The study is confirmative. The reported data just confirm previously published data but on a different rodent stroke model and no new scientific information is reported. The manuscript would benefit from addressing the mechanism for H2 protective effect.

Author Response: Intraperitoneal administration has been widely used as a route of administration for observing the brain-protective effects in rodents of H2 in H2-rich saline. To our knowledge, this article is the first to report on the pharmacokinetics of H2 administered by this route of administration. In addition, this study is novel in that it examined the effects of intraperitoneal administration of H2-rich saline on individual neuronal death in the hippocampus and cerebral cortex—rather than reduction in the size of necrotic brain tissue—in a gerbil experimental system, which is a reliable model of cerebral ischemia.

Table 1 data (H2 concentration): n=1 for saline group is not sufficient; statistical analysis is not performed.

Author Response: We completely agree with this comment. To clarify the changes in blood hydrogen concentration after intraperitoneal administration of H2-rich saline, we added 3 new animals to the H2 group (total: 9 animals) and 5 to the saline group (total: 6 animals). The n.d. (not detectable) point was calculated as zero for statistical tests. The raw data of the hydrogen concentration measurements are shown in the supplementary table, and to clarify the experimental results, we created a new Figure 5 showing the results of the statistical tests.

Minor suggestions:

Blood H2 concentration assay should be described in more details.

Author Response: Thank you for your important comment. To measure blood H2 concentration, we first inserted a needle into the rubber lid of a 13.5-mL sealed vial, extracted 1 mL of air, and injected 1 mL of blood. To prevent outgassing, we immediately applied wax to the rubber lid to seal the injection hole. In such a sealed vial, H2 in the blood is released into the air phase because the air in the vial contains almost no H2 gas, so after injection of blood, most of the H2 gas moves from the blood (liquid phase) into the air (gas phase). Therefore, examining the concentration of H2 gas in the air inside the vial enabled the H2 concentration in the blood to be estimated. Some of the air phase (0.2 mL, 0.4 mL, or 1 mL, depending on the H2 concentration) was collected from the vial, and the H2 concentration was measured by gas chromatography (TRIlyzer mBA-3000, Taiyo, Co., Ltd.). A calibration curve was obtained by using H2 gas at a concentration of 0 (nitrogen gas), 5, 50, and 130 parts per million (ppm). The detectable value of the analyzer is 0.1 ppm (i.e., the analyzer shows values of 0.1 ppm or higher), and the limit of quantitation is considered to be approximately 0.2 ppm.

Samples were collected from the carotid artery after 5 minutes and from the inferior vena cava after 10 minutes, and each sample was measured twice. At the same time, air was collected from the exact same location and injected into a blood-free vial, and the H2 gas concentration was measured. The value of H2 in the air was 0.5 ppm, i.e., above the limit of quantitation. The air value was subtracted from each measured value to obtain a corrected value. Thus, only samples with a value above 0.5 ppm contained H2 also from blood.

In the revised manuscript, the Methods section describes in detail the method of measuring hydrogen concentration. In the Discussion section, we also explain the sensitivity of the H2 concentration measurement.

Attachment

Submitted filename: Response letter_PLOSONE_edited_20221104.docx

Click here for additional data file. (31.7KB, docx)

Decision Letter 1

Alexander A Mongin

7 Dec 2022

Pharmacokinetics of hydrogen administered intraperitoneally as hydrogen-rich saline and its effect on ischemic neuronal cell death in the brain in gerbils

PONE-D-22-22387R1

Dear Dr. Sano,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Alexander A. Mongin, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The author responded to the review comments very seriously, and there have been many improvements in the new manuscript, but there are still some problems that have not been clarified, and even new problems have emerged in the process of revision, requiring the author to give explanations.

The basic conclusion of this study is that adding hydrogen to the abdominal pathway can have a protective effect on brain injury, but the changes in hydrogen concentration detected by carotid artery cannot explain the protective effect of hydrogen entering the brain. This conclusion is very difficult to understand. In order to reach such a conclusion, that is, to rule out the direct effect of hydrogen, in addition to the problem of detecting the sensitivity of the hydrogen method, there should also be evidence of this indirect effect, or that hydrogen is likely to play an indirect role in the abdominal cavity through an indirect route. There are many possibilities for indirect effects. For example, it may affect the immune inflammatory response through phytonerves. It also works through the blood circulation by influencing the release of other biomolecules from the gut flora. However, it is better that these effects should be studied accordingly, otherwise there is still a possibility that the conclusion cannot be understood.

After hydrogen is ingested by the body, there are three metabolic directions: one is captured and fixed by local tissues; the other is transferred with blood circulation. In this study, hydrogen ingested through the abdomen will mainly regress to the heart with blood and then enter the pulmonary circulation, most of which may spread into the air. There is also some hydrogen circulating from the skin to the outside world. Third, it is metabolically consumed by body tissues. All three cases have been reported in academic studies. But either way, you can't completely metabolize hydrogen. Any metabolic pattern in the body probably follows the rule of inverse exponential decay, which is that as the concentration decreases, the metabolic efficiency decreases. In other words, hydrogen is unlikely to disappear from the body in a very short time. However, with any detection method, there is a problem of detection sensitivity. In this study, hydrogen could not be analyzed in the internal carotid artery, mainly due to the detection sensitivity. This study adopts gas chromatography, which is a classical hydrogen analysis method. But the sensitivity of this analysis is also very limited. For example, the concentration of air can only be analyzed above 0.5ppm. While the headspace method itself has an effect on the efficiency of releasing hydrogen from the liquid, which in this study is blood. This may be the reason why this study could not detect sufficient levels of hydrogen, and also the reason why the changes could not be analyzed.

Regarding the peak hydrogen concentration, the authors report that "when an H2-rich solution was applied to the jejunum of the mini pigs, we observed that the portal blood H2 concentration increased at 2 minutes, continued to rise to 2 minutes, and then remained at roughly the same level at 5,10,20, and 30 minutes." The total body volume of an animal is a significant factor affecting the gas diffusion. The gas conversion patterns of gerbils and pigs are quite different, and the results measured in pigs should not be used to invert the results in mice. Just as you can't classify people by the gas diffusion patterns in an elephant's body.

For the problem of insufficient sample size in the original experiment, the author revised the feedback saying that the sample size was increased, so as to meet the requirements of statistical analysis. However, this completely violates the basic principle of experimental design. Statistical analysis requires that samples should not be randomly added in the middle, because the principle of random grouping cannot be realized in this way. If the author must do so, he must make this modification clear in the manuscript.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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Acceptance letter

Alexander A Mongin

15 Dec 2022

PONE-D-22-22387R1

Pharmacokinetics of hydrogen administered intraperitoneally as hydrogen-rich saline and its effect on ischemic neuronal cell death in the brain in gerbils

Dear Dr. Sano:

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on behalf of

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Academic Editor

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Anatomical comparison of basilar artery in rat and gerbil.

    In rats (left), the internal carotid artery system, vertebral artery system, and left and right medial artery systems are connected at the base of the brain to form the circle of Willis. Therefore, if one artery is occluded, blood can still flow to the brain via the other vessels. In the gerbil (right), the basilar arterial ring lacks anastomoses between the right and left internal carotid arteries and between the carotid and vertebral arteries, so occlusion of the common carotid artery can easily induce ipsilateral cerebral ischemia.

    (TIF)

    Click here for additional data file. (144.8KB, tif)
    S2 Fig. Experimental protocols.

    (A) Experimental protocol-1. After two 10-minute occlusions of the left carotid artery, the animals were randomly divided into H2-rich saline and saline groups. Animals in each group received 3 or 7 intraperitoneal injections of 20 ml/kg body weight of each solution. Histological analysis was performed on days 3 and 7. (B) Experimental protocol-2. H2-rich saline or saline solution (20 ml/kg BW) was administered intraperitoneally once. Blood was drawn from the carotid artery after 5 minutes and from the IVC after 10 minutes, and H2 concentration was measured by gas chromatography.

    (TIF)

    Click here for additional data file. (53.4KB, tif)
    S3 Fig. Measurement site of the number of survived neurons in the brain of gerbils after transient cerebral ischemia.

    Cerebral histology of the cerebrum of a gerbil coronally sectioned at the levels of the infundibulum and stained with hematoxylin and eosin. Neuronal cells were counted in the hippocampal CA1 region (areas inside the blue rectangles) and the central part of the cerebral cortex (areas inside the black rectangles, 300 μm wide) in the left and right cerebral hemispheres.

    (TIF)

    Click here for additional data file. (263.4KB, tif)
    S1 Table. H2 concentration in the inferior vena cava and carotid artery of gerbils after intraperitoneal administration of H2-rich saline.

    H2 measurements are shown in concentration (mg/L) and saturation (%). Because H2 dissolves at 1.6 mg/L under 0.1 MPa, the H2 saturation of each sample was converted by assuming 1.6 mg/L as 100% saturation. IVC, inferior vena cava, CA carotid artery, n.d., no detected, i.p., intraperitoneal injection.

    (TIF)

    Click here for additional data file. (138.6KB, tif)
    Attachment

    Submitted filename: Response letter_PLOSONE_edited_20221104.docx

    Click here for additional data file. (31.7KB, docx)

    Data Availability Statement

    All relevant data are within the article and its Supporting Information files.

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