{'fraction_non_alphanumeric': 0.03173431734317343, 'fraction_numerical': 0.025169935909885414, 'mean_word_length': 4.734075723830735, 'pattern_counts': {'":': 0, '<': 5, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Accurate in vivo real-time determination of the hydrogen concentration in different tissues of mice after hydrogen inhalation

As an antioxidant, anti-inflammatory and anti-apoptotic agent, hydrogen (H2) shows a promising potential in basic and clinical research against various diseases owing to its safety and efficacy. However, knowledge involving its underlying mechanisms of action, dosage effects, and dose duration remains limited. Previously, the dynamics of H2 concentrations in different tissues of rats after exogenous H2 inhalation had been detected by our team. Here, sequential changes of H2 concentrations in different tissues of another most commonly used experimental rodent mice were monitored in real time with an electrochemical H2 gas sensor during continuous different concentrations of H2 inhalation targeting on five tissues including brain, liver, spleen, kidney, and gastrocnemius. The results showed that the H2 saturation concentrations varied among tissues significantly regardless of the concentration of H2 inhaled, and they were detected the highest in the kidney but the lowest in the gastrocnemius. Meantime, it required a significant longer time to saturate in the thigh muscle. By comparing the H2 saturation concentrations of mice and rats, we found that there were no differences detected in most tissues except the kidney and spleen. Both gas diffusion and bloodstream transport could help the H2 reach to most organs. The results provide data reference for dosage selection, dose duration determination to ensure optimal therapeutic effects of H2 for mice experiments.


Introduction
As the number one element in the periodic table, hydrogen has the simplest structure and the smallest molecular weight. It usually forms a diatomic molecule (H 2 ) with a low density and physical non-polarity, which enables it to disperse quickly and even penetrate the organelle membrane. Although H 2 is an inert gas, its role in the prevention and treatment of various diseases has created quite a stir since 2007, when Ohsawa's team reported its antioxidant effect in a rodent model of ischemia-reperfusion injury (Ohsawa et al., 2007). Given its safety and effectiveness, H 2 is always commanding attention with numerous animal studies and clinical trials. Many countries and regions including USA, Japan, Europe, and China have recognized H 2 as a food additive, despite it is not accepted globally . It is worth mentioning that H 2 has played an indelible role in the treatment of the latest outbreak of coronavirus disease 2019 (COVID-19) (Guan et al., 2020).
It must be said that hydrogen biomedicine has indeed mushroomed in recent decades, but two major problems are also hindering its further development. One is the undefined mechanism. Currently, the protective effects of H 2 are largely attributed to its antioxidant, anti-inflammatory, and anti-apoptotic properties (Liu et al., 2021;Russell et al., 2021). Although few reports have proposed the effects on bio-enzyme activity, protein structural properties, and gut microbiota/short-chain fatty acids axis (Cheng et al., 2020;Ma et al., 2020;Ge et al., 2022), yet the direct target is still being unidentified. The other is the undetermined dose-effect, which is seriously affected by the diffusivity of H 2 and the instability of H 2 donor. At present, exogenous H 2 can be ingested by organisms via various methods mainly including H 2 -rich water drinking, H 2 -rich saline injection and H 2 inhalation (Ge et al., 2017). In the processes of different administrations, the H 2 saturation concentration and the saturation rates in various tissues are considered as important reference for the optimal administration selection. As the most straightforward therapeutic method, H 2 inhalation has been widely used since the first sensational report in 2007 (Ohsawa et al., 2007). Several previous studies have reported changes of H 2 concentrations in tissues of rats after exogenous H 2 inhalation (Liu et al., 2014;Yamamoto et al., 2019). However, greater inaccuracy tends to occur in results of ex vivo detection attributed to the rapid diffusion of H 2 in tissues during samples processing even by the aid of glass containers (Liu et al., 2014). In addition, only single concentration of H 2 (3%) was inhaled and the H 2 distribution in different tissues may differ during and after other concentrations of H 2 administration (Yamamoto et al., 2019). Recently, our team monitored the real-time H 2 concentrations in various tissues of rats during three doses of exogenous H 2 (4%, 42%, 67%; v/v) were inhaled. In particular, the electrochemical H 2 sensor was adopted creatively during the experiments and the dose-dependent response was revealed for the first time .
Since both mice and rats are the most commonly used experimental rodents in basic research, we further measured the sequential changes of H 2 concentrations over time in different tissues after mice inhaled different concentrations of exogenous H 2 . Considering both the easy collection and blood flow of the tissues, five tissues including brain, liver, spleen, kidney, and gastrocnemius with relatively large blood flow were targeted here. We hoped that the results could provide fundamental data reference for dose-effect studies and the determination of H 2 inhalation duration in mouse experiments to ensure the optimum effects of H 2 .

Preparation of exogenous H 2
Here, mice inhaled three different concentrations of exogenous H 2 (4%, 42%, 67%; v/v) using our self-made device . The H 2 concentration of the mixed gas was determined using a H 2 detector (XP-3140, New Cosmos Electric Co., Ltd., Osaka, Japan). 4% H 2 was used in the sensational article published by Japanese scholars in 2007 (Ohsawa et al., 2007), and then the protective role of low concentration of H 2 was reported in many diseases. Meanwhile, commercial machines produced 67% H 2 have been applied widely in clinical practice. 42% H 2 was selected as the intermediate concentration based on literature review (Huang et al., 2019). So here, these three concentrations of H 2 were used, which covered low, medium, and high concentrations.

Operation in animal experiments
25 eight-week-old SPF-level C57BL/6J mice weighing about 20 g were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., Shandong Province, China. All mice were housed under standard conditions (22 AE 1 C; 12/12 h light/dark cycle) with water and food ad libitum for at least one-week acclimatization. Then the mice were anesthetized by an intraperitoneal injection of 20% urethane (1.4 g/kg of bodyweight, Shanghai Aladdin Biochemical Technology Co., Ltd., China). Complete anesthesia was confirmed by muscle relaxation, disappearance of the corneal reflex and smooth breathing of mice. Then, the animals were dissected on a heating plate with constant temperature of 38 C and then tissues to be measured were exposed. To minimize the damage to the mice and create a more realistic simulation of the in-vivo condition, the operative incision should be made as small as possible. After the mice were secured, tip of the microelectrode was then dipped into the exposed tissue. Here, five representative tissues including the brain, median lobe of the liver, spleen, kidney, and rear gastrocnemius muscle, were targeted ( Figure 1). All animal procedures were conducted following the guidelines of the laboratory animal ethics committee of Shandong First Medical University and Shandong Academy of Medical Sciences.

H 2 concentration measurement by electrochemical H 2 sensor
An electrochemical H 2 sensor (40-60 μm tip diameter) assembled on a micromanipulator and connected to a multimeter (Unisense, Aarhus, Denmark) was used to monitor the H 2 concentrations of different tissues. Before experiments, the H 2 sensor was calibrated, and a standard curve was established by diluting the H 2 -saturated phosphate-buffered saline at 38 C. For in vivo measurements, the needle sensor tip was placed less than 1 mm into the exposed tissue. Initially, a stable baseline was obtained by supplying only air. Then, the mice were continuously supplied with the required concentration of H 2 until the H 2 concentration in the measured tissue reached the plateau stage. After that, pure air was supplied and the decline curve of H 2 concentration was recorded until it returned to the baseline level again. At least three independent measurements were conducted for each tissue (n ! 3). The H 2 sensor was rinsed with 0.01M hydrochloric acid between animals to remove the substances on the sensor tip.

Statistical analysis
Data are expressed as the mean AE SD (standard deviation). One-way analysis of variance followed by least significant difference (LSD) post hoc test was used for the data conforming to normal distribution to compare the H 2 concentrations between multi-measurement sites. Otherwise, Kruskal-Wallis multiple tests were performed. The independent samples ttest was used for comparison between the two groups. The P < 0.05 was considered significant. All data were analyzed using SPSS version 26.0 (IBM, Armonk, NY, USA) and graphed using GraphPad Prism 8.0.1 (GraphPad Software Inc., La Jolla, CA).

Real-time curve of H 2 concentration in different tissues
The real-time concentrations of H 2 in five different tissues of mice after different concentrations of exogenous H 2 inhalation were displayed in Figures 2A-C. It could be seen that the H 2 concentrations in five tissues of mice increased significantly over time during the H 2 inhalation of any concentration. However, the rising rate and amplitude of H 2 concentration varied in different tissues even after the same concentration of exogenous H 2 inhalation. Specifically, the H 2 concentrations in the kidney and brain rose at a faster rate, allowing both tissues to reach the saturated H 2 concentrations in a short time. The same ascent courses became slower in the liver and spleen. While the H 2 concentration in the gastrocnemius increased the slowest, and a long time was needed to reach a lower equilibrium concentration. When exogenous H 2 inhalation was stopped, the H 2 concentrations in the tissues began to decrease over time. Similarly, the gastrocnemius needed more time to reach the baseline reading compared with the other tissues ( Figure 2D-F).

Saturation concentration of H 2 in different tissues
As shown in Figure 3, most of the H 2 saturation concentrations detected in various tissues were significantly different after the same concentration of H 2 inhalation. The H 2 saturation concentrations were highest in the kidney (28.058 AE 0.949 μmol/L, 382.183 AE 5.336 μmol/ L, 508.522 AE 23.260 μmol/L after 4%, 42% and 67% inhalation, respectively; the same below), followed by the brain (  . The saturation concentration of H 2 in the kidney, spleen, liver, brain, and gastrocnemius of mice after inhaling different concentrations of H 2 (4%, 42% and 67%). Data are shown as the mean AE SD (n ¼ 3-5 mice/tissue). The statistical analysis was performed using one-way ANOVA followed by LSD test. Different letters indicate significant differences at P < 0.05 among tissues. 225.351 AE 14.711 μmol/L), and lowest in the gastrocnemius (5.927 AE 0.532 μmol/L, 73.364 AE 6.440 μmol/L, 160.765 AE 10.245 μmol/L).
There was no significant difference of H 2 saturation concentration detected in the kidney and brain after the low and high concentrations of exogenous H 2 inhalation. Other inter-tissue comparisons all showed significant differences (P < 0.05). Attentively, the difference of H 2 saturation concentration detected in the same tissue was directly proportional to the difference of inhaled exogenous H 2 concentration, namely, the saturation values after inhaling 42% and 67% H 2 were about 12.3-and 17.8-time greater than that after 4% H 2 inhalation in various tissues except for the gastrocnemius with 67% (about 27.1time).

Discussion
In this study, the real-time H 2 concentrations in five tissues of mice including brain, kidney, liver, spleen, and gastrocnemius were monitored after continuously inhaling three different concentrations of H 2 (4%, 42% and 67%) using the Unisense H 2 microsensor. Unsurprisingly, the results showed that H 2 concentration curves over time and H 2 saturation concentrations were significantly different among various tissues of mice (Figures 2 and 3), but similar with that in the corresponding tissues of rats except kidney and spleen ( Figure 4).
Undoubtedly, the H 2 concentration is an important indicator for the quality evaluation of H 2 inhalation equipment and H 2 -rich products. Currently, several methods including gas chromatography, H 2 microelectrode detection and redox titration (methylene blue) were often used to determine H 2 concentration.
Gas chromatography is a standard method for gas analysis, by which the concentration of H 2 in gas can be directly analyzed. However, H 2 dissolved in liquid (water or blood) must be released before measurement due to the interference of solvent signals. As early as 2007, Ohsawa determined H 2 concentration in blood using gas chromatography. They found that H 2 concentration in arterial blood increased after exogenous H 2 inhalation, and was higher than that in artery blood, which suggested that H 2 had been incorporated into tissues (Ohsawa et al., 2007). Other studies have also reported the H 2 concentrations in blood and/or tissues after exogenous H 2 treatment in rat models through this method (Nagata et al., 2009;Cardinal et al., 2010;Sobue et al., 2015). Liu et al. (2014) determined and compared the H 2 concentrations in various homogenized rat organs after several routes and doses of H 2 administration using high-quality sensor gas chromatography. They revealed different peak H 2 concentrations following one-time H 2 administration and the value changes over time (Liu et al., 2014). In one word, gas chromatography is popular for gas concentration determination, but this technique cannot realize real-time detection, and in-vivo studies are not yet possible.
The Unisense H 2 microsensor is a miniaturized Clark-type hydrogen sensor for measuring partial pressure of H 2 . Driven by the external partial pressure, H 2 will pass through the sensor tip membrane and be oxidized at the platinum anode surface. The picoammeter converts the resulting oxidation current to a signal, and finally to H 2 concentration. Depending on its ultra-fine needle size tip, excellent response time, and high sensitivity, the H 2 sensor facilitates reliable and fast measurements, which makes it especially popular in some advanced applications such as longterm H 2 monitoring in tissues or water. In addition, the electrode analysis is relatively simple, given the data can be read directly in real time when Figure 4. Comparison of the saturation concentration of H 2 in the kidney, spleen, liver, brain, and gastrocnemius of mice and rats after inhaling different concentrations of H 2 (4%, 42% and 67%). Data are shown as the mean AE SD (n ¼ 3-5 mice/tissue). The statistical analysis was performed using the independent samples t-test. Different letters indicate significant differences of the saturation concentration of H 2 in the same tissue between the mice and rats (P < 0.05). putting the electrode in H 2 -containing water or target tissue. So, the microsensor is widely used in a series of studies to monitor the timecourse of H 2 level changes in blood and tissues (Hayashida et al., 2008;Sobue et al., 2015;Yamamoto et al., 2019;Liu et al., 2022). During the measurement process, the mouse skin opening should be as small as possible. It can be considered to put a moist gauze on the exposed tissue, and the normal saline can also be sprayed intermittently to avoid tissue drying, so as to better simulate the in vivo environment and obtain more accurate measurement data. Nevertheless, the electrode measurement is not completely precise due to the interference of bioelectrical signals, which has become a bottleneck restricting the analysis of H 2 concentration in vivo.
Here, the dynamic distribution of H 2 in different tissues of another commonly used experimental rodent mice was explored after three different concentrations of exogenous H 2 Inhalation. The results showed that the concentration of H 2 in tissues depended on the time, the concentration of exogenous H 2 intake as well as the characteristics of the tissue itself. After the exogenous H 2 was inhaled by mice, it diffused through the alveoli into the bloodstream, then dissolved, and was transported through the whole body in a blood flow-dependent manner (Ichihara et al., 2021) despite of the unclear receptor molecules and downstream effectors (Sano et al., 2020). Instead of being concentrated in the blood vessels, H 2 diffused out of the blood vessels during travelling (Sano et al., 2020). It has been verified by the breath analysis that 60% was excreted in the breath and the rest was consumed by the body (Shimouchi et al., 2012(Shimouchi et al., , 2013. The dissolved or diffused H 2 entered tissues by a pressure gradient. In this study, the H 2 saturation concentration was highest in the kidney of mice, and followed by the brain, liver, spleen and lowest in the gastrocnemius (Figures 2 and 3).
As the organ with the most abundant blood supply of the whole body, kidneys receive approximately 20%-25% of the cardiac output (Povstyan et al., 2011). They are normally blooded from renal arteries that anatomically originates from the abdominal aorta (Karada g et al., 2020). The dissolved H 2 was transported into the kidneys along with the bloodstream of systemic circulation. In view of the abundant blood supply of the kidneys, H 2 was saturated in a short time and the peak H 2 concentration was the highest under any concentration of H 2 inhalation. So H 2 in the kidneys was mainly transported in a blood flow-dependent manner after inhalation despite the gaseous diffusion could not be excluded. The results were not completely consistent with the reports of Yamamoto et al. (2019) and Liu et al. (2022), where the H 2 saturation concentration in the kidney was not higher than that in the brain and even more other organs (Yamamoto et al., 2019;Liu et al., 2022).
The H 2 saturation concentration in the brain is second to that in the kidney only. There were no significant differences detected in H 2 concentrations of the two tissues after inhaling low or high concentrations of H 2 . The cerebral blood mainly comes from the carotid arterial, and its blood flow accounts for about 15% of the total cardiac output (Yu et al., 2016). As of now, several studies have explored the H 2 concentration in arterial blood after H 2 inhalation. Hayashida et al. (2008) investigated the regional delivery of inhaled 2% H 2 by monitoring the time-course of H 2 level changes in blood and myocardium by the aid of a needle-shaped H 2 sensor, and found that H 2 inhalation could efficiently increase the H 2 concentration in the arterial blood and "at risk" area of myocardial infarction and then alleviate ischemia-reperfusion injury (Hayashida et al., 2008). Sano et al. (2020) investigated the kinetics of single-dose inhalation of H 2 in the body of pigs. The results showed that H 2 concentration of carotid artery reached a very high level immediately after inhalation, indicating that the inhaled H 2 entered in the brain efficiently (Sano et al., 2020). Liu et al. (2014) reported that inhalation of H 2 induced slightly higher H 2 concentration in the brain compared to the other modes of H 2 administration (Liu et al., 2014). Yamamoto et al. (2019) showed that H 2 concentration would increase more rapidly and saturate at a higher value in the brain due to the short distance between the gas supply hood and the tissue, which was attributed to the gaseous diffusion model based on the fact that H 2 can traverse the blood-brain barrier (Yamamoto et al., 2019). Fortunately, the results of Yamamoto's study, our previous research in rats inhaling 4% H 2 , and the current study were similar. So, we inferred that both gas diffusion and bloodstream transport contributed to the H 2 concentration in the brain considerably after the H 2 inhalation.
Liver, the largest internal organ, has extremely rich blood flow. Moreover, the liver has strong ability to accumulate H 2 after exogenous supplement due to the high affinity of hepatic glycogen to hydrogen molecule (Kamimura et al., 2011). The previous study has detected the highest peak H 2 concentration in the liver of rats after inhaling 3% H 2 (Yamamoto et al., 2019). However, this study and our previous research have not detected such high H 2 concentration in the liver, which may result from the operational differences . The alleviating effects of H 2 on liver diseases are unquestionable, which has also been evidenced in our own investigation . As the largest immune organ, spleen can store part of blood in the sinuses. Here, the lowest H 2 concentration was detected in the spleen of the abdominal organs. We speculated that inhalation may not be a suitable way to ingest H 2 for the spleen, which has ever been shown in the study of Liu et al. (2014) (Liu et al., 2014). Consistent with most other studies, thigh muscle required a longer time to saturate and the H 2 saturation concentration was the lowest (Hayashida et al., 2008;Yamamoto et al., 2019;Liu et al., 2022). Following the blood flow model, H 2 concentration would increase more gradually in the muscle than other organs due to lower blood flow (Yamamoto et al., 2019;Liu et al., 2022).
H 2 concentration in arterial blood is proportional to the partial pressure of H 2 in the inhaled gas mixture (Sano et al., 2020). Similarly, a dose-dependent response of H 2 concentrations in the same tissue of mice after inhaling different concentrations of H 2 was found, which was consistent with the results in tissues of rats . By comparing the H 2 saturation concentrations in the same tissue after the same concentration of exogenous H 2 inhalation between rats and mice in detail, we found no differences of H 2 saturation concentrations in the liver and brain between both animals, regardless of any H 2 concentration inhaled. However, for the kidney and spleen, the results were reversed, with significant differences in H 2 concentrations detected in both tissues (P < 0.05). The H 2 saturation concentrations detected in gastrocnemius of mice and rats were significantly different after low concentration of H 2 inhalation, but there was no difference after medium and high concentrations of H 2 inhalation (Figure 4). The differences of the results reflected an inevitable problem during the measurement, the varieties among animals. After the same concentration of exogenous H 2 inhalation, the H 2 concentration detected in the same tissue of different mice may vary. For example, the H 2 concentration in the liver of mice ranged from about 12 to 22 μmol/L after 4% H 2 inhalation, and only five similar H 2 concentration curves were obtained in eight detected mice. The situation has close relationships to the physical differences of mice and the practical operation of measurement. Practical considerations include periodic calibration of the sensor with H 2 standards and the membrane permeability of the H 2 microsensor, which may decrease the sensitivity over time (Zhao et al., 2016).
Unfortunately, there are also some shortcomings in current research. Firstly, only inhalation was adopted here, whereas the distribution of H 2 in the body and the saturation concentrations of tissues may differ using other administration methods including drinking and injection. Secondly, detections of H 2 concentrations in the immediate target tissues such as respiratory and cardiovascular systems (including arterial and venous blood) were lacking, which were important to explore whether the H 2 concentration in the lung was the highest or not after inhalation, and to support the hypothesis that H 2 diffuses while being carried in a blood flow-dependent manner instead of diffusion simply (Sano et al., 2020). When the microsensor penetrated the lung, it might affect animals' normal breathing. And what was the worse, bleeding would be caused when the heart and blood vessels were penetrated, resulting in the change of blood volume, which might affect the blood flow and the amount of blood entering the tissue further. In addition, the spilled blood could easily clog the sensor tip. Meanwhile, the contraction and expansion of the lung together with the constant beating of the heart could easily lead to the sensor tip bending or rupture. All the above conditions could interfere the accuracy of the measurement results. Based on the above statement, more advanced techniques and equipment were required to achieve precise in vivo measurement of gas concentration in tissues though H 2 sensing shows more promise as an effective means.
In conclusion, dynamics of H 2 concentrations over time in different tissues of mice exposed to continuous different concentrations of H 2 gas inhalation were investigated. Significantly various H 2 saturation concentrations among tissues were detected. The kidney and brain reached a higher H 2 saturation concentration while the gastrocnemius had the lowest and required a longer time to saturate. H 2 could reach to most organs not only by simply diffusion but also following transportation of the bloodstream. All the results provide a data reference for the dosage selection and the dose duration, and finally guarantee the optimal effects of H 2 .

Data availability statement
Data will be made available on request.