Real-time Hydrogen Monitoring in Rat TissuesScientific Research

INTRODUCTION

Hydrogen gas (H₂) is colorless and odorless at standard temperature and pressure. Due to its small molecular weight and hydrophobic properties, H₂ can easily permeate cell membranes and even enter cell organelles. H₂ has long been regarded as a biochemical inert gas. In 2007, Ohsawa et al.¹ demonstrated its selective antioxidant effects in a rat cerebral ischemia/reperfusion model. A large number of studies have since shown the therapeutic and preventive effects of H₂ in various animal disease models.²³⁴⁵ Meanwhile, some clinical investigations have also confirmed its beneficial effects on different diseases.⁶⁷ Various ways have been explored to administer H₂, mainly including inhaling H₂, drinking H₂-rich water, injecting H₂-rich saline, and direct incorporation of H₂ by diffusion (such as bath and eye-drops). Due to its bio-safety, countries and regions such as USA,⁸ Japan,⁹ Europe,¹⁰ and China¹¹ have recently allow to use H₂ as a food additive. At present, the therapeutic effects of H₂ have attracted increasing attention worldwide.¹²

Despite rapid advances in understanding the biological effects of H₂, the underlying mechanism is yet to be elucidated. In addition to the aforementioned hypothesis regarding selective scavenging of toxic free radicals, H₂ can exert its bio-functions by reducing inflammation and apoptosis events.¹³ A hypothesis based on the bio-enzyme basis of H₂ was recently proposed,¹⁴ and a new study showed that H₂-rich water could significantly increase the activity of pepsin and change the protein structure and dynamic properties.¹⁵

Until now, the existing molecular mechanism of the biological effect of H₂ has not been fully explained due to lack of solid pharmacokinetic data. In addition, to the best of our knowledge, only a few studies have explored its concentration and distribution after intake. A previous study¹⁶ determined H₂ concentrations in different rat tissues following the administration of H₂ via various routes. The study revealed variable dynamics of H₂ in various tissues over time and different H₂ concentrations in the same tissue with different methods of H₂ uptake. Another in vivo study¹⁷ monitored the sequential changes of H₂ concentrations in tissues over time with continuous inhalation of 3% H₂. However, conclusions of both studies were not entirely consistent. Moreover, previous studies were performed only after a single concentration of H₂ intake. The dose-response curve had not been illustrated. Therefore, more accurate and detailed studies are needed to acquire the exact pharmacokinetics of H₂.

This study pioneered a comprehensive and quantitative assessment of H₂ distributions within various tissues in vivo after different concentrations of H₂ being inhaled, and obtained the dose-response curve by real-time monitoring.

MATERIALS AND METHODS

Gas preparation

Different concentrations of H₂ (4%, 42%, 67%; v/v) were prepared using a lab-made gas mixing device (Figure 1A). H₂ and oxygen gas (O₂) from cylinders and air from a generator (LCA- 3, LICHEN-BX instrument technology Co., Ltd., Shanghai, China) with different flow rates were adjusted by flowmeters and mixed in a sealed box. The targeted concentration of H₂ was confirmed with a gas detector (XP-3140, New-cosmos Co. Ltd., Tokyo, Japan). Meanwhile, the O₂ concentration was kept at ~21% and verified with an O₂ detector (JR2000-02, Jingruibo Technology Co., Ltd., Beijing, China). The mixed gas was administered to a rat through a gas supply hood at a total flow rate of 3 L/min.

Animals and experimental design

Fifty 8-week-old specific-pathogen-free level male Sprague- Dawley rats weighing 180–210 g (Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China; SCXK (Lu) 20190003) were used in this study. All rats were maintained under standard conditions (21 ± 1°C; 12/12 hours light/dark cycle). Water and food were provided ad libitum. The experiments were approved by the Laboratory Animal Ethics Committee of Shandong First Medical University & Shandong Academy of Medical Sciences (approval No. 2020-1028) on March 18, 2020.

After overnight fasting, rats were sedated by intraperitoneal injection of 20% urethane (7 mL/kg, Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China). After losing consciousness, rats were put on a warming plate maintained at 38°C and then dissected to expose the target tissue with minimum incisions. The exposed tissue was covered with a layer of humid gauze with an opened small hole to maintain moisture in order to mimic the internal environment better, and then the microsensor tip was inserted into the tissue through the hole.

Seven tissues, including the left brain, median lobe of the liver, spleen, left kidney, thigh muscle (left hind gastrocnemius muscle), gonadal (visceral) white adipose tissue (WAT), and inguinal (subcutaneous) WAT (n = 3-6 per tissue), were monitored (Figure 1B). Only one target tissue was exposed at a time per rat.

H₂ concentration monitoring in vivo

The measuring device included a miniaturized Clark-type hydrogen microsensor with an internal reference electrode and a sensing anode (tip diameter 40–60 μm), a micromanipulator, and a microsensor multimeter (Unisense, Aarhus, Denmark). The signals of Clark-type sensors were controlled by target gas concentrations, sensor dimensions, temperature, and salinity.¹⁸ A standard curve was established by diluting the H₂-saturated phosphate-buffered saline at 38°C. The tip of the microsensor was inserted into the exposed tissue at a depth of ~1 mm below the tissue surface. At first, the air was administered to maintain a stable baseline. Next, a required concentration of H₂ was provided continuously until the H₂ concentration in the target tissue reached equilibrium. Then, the gas was replaced by pure air and monitoring was continued until the H₂ concentration returned to baseline. Predicted by Henry’s law for the solubility of a gas in a liquid,¹⁹ and the solubility of H₂ at a certain temperature and salinity,²⁰ the theoretical equilibrium concentrations (C_e) of 4%, 42%, and 67% H₂ in blood are about 28.5, 299.0, and 476.9 μM, respectively. The detection started before hydrogen inhalation. At the beginning, the rats were given air until reaching a stable baseline, then hydrogen was provided.

Statistical analysis

Statistical analyses were conducted using Origin v8.5 (Origin Lab Corporation, Northampton, MA, USA) and GraphPad Prism v8.0.1 (GraphPad Prism Software, Inc., La Jolla, CA, USA). An ordinary one-way analysis of variance and Tukey’s multiple comparison test were used to assess the significance of differences in H₂ concentrations between various tissues. P < 0.05 was considered statistically significant. Data are representative of at least three independent experiments, and expressed as the mean ± standard deviation (SD).

RESULTS

C_e of H₂ in different tissues

C_e of H₂ in different tissues after inhaling different concentrations of H₂ are shown in Figure 2. The figures display highest mean C_e in the brain, followed by inguinal WAT and kidney, and lowest in the thigh muscle regarding the three concentrations of H₂. Inter-organ comparisons revealed significant differences between the brain/thigh muscle and other tissues (P < 0.001 or P < 0.01) for different concentrations of H₂. For 4% H₂, C_e showed a significant difference between inguinal WAT and liver (P < 0.05). For 67% H₂, C_e showed statistically significant differences between inguinal WAT and liver, spleen, gonadal WAT, respectively (P < 0.05). C_e of H₂ in all tissues exhibited a dose-dependent increase corresponding to the concentration of inhaled H₂. C_e values after inhaling 42% and 67% H₂ were about 10.5- and 16.8-time greater than 4% H₂ in various tissues. The theoretical C_e of H₂ based on a blood flow model and Henry’s law for the solubility of gas are also shown in Figure 2.

H₂ saturation dynamics in different tissues

The H₂ dynamic curves for different tissues after inhaling various concentrations of H₂ are summarized in Figure 3A–F. In general, H₂ concentrations in the brain, liver, kidney, and spleen increased faster than in the thigh muscle, inguinal WAT, and gonadal WAT (Figure 3). For 4% H₂, the concentrations in the brain, liver, kidney, and spleen rose rapidly in a short time and then gradually leveled in about two minutes for the liver, kidney, spleen and three minutes for the brain (Figure 3A). However, the plots for the thigh muscle, inguinal WAT, and gonadal WAT exhibited a gradual increase after H₂ was inhaled and they needed much more time to reach the C_e (Figure 3D). The ascending order of different tissues reaching 50% and 90% saturation concentrations was spleen, liver, kidney, brain, gonadal WAT, thigh muscle, and inguinal WAT (Figure 3G and H). For 42% and 67% H₂, trends similar to those of 4% H₂ were observed (Figure 3B, C, E, and F) and the orders were same as 4% H₂ (Figure 3G and H). In the same tissue, similar time was needed to reach 50% C_e for different H₂ concentrations applied. Meanwhile, a dose-dependent relationship for the time to reach 90% C_e was observed (Figure 3G and H).

H₂ desaturation dynamics in different tissues

The H₂ concentrations in different tissues started decreasing after H₂ administration was withdrawn (Figure 4A–F). In the beginning, the brain, liver, kidney, and spleen exhibited a similar sharp drop right after H₂ withdrawal, and then plots decreased gradually until reached the baseline (Figure 4A–C). However, the curves of the thigh muscle, inguinal WAT, and gonadal WAT exhibited a more gradual decrease after H₂ withdrawal and took more time to reach the baseline (Figure 4D–F). In ascending order of the time needed for different tissues to reach 50% and 90% desaturation, the spleen and liver took the least time, followed by kidney, brain, thigh muscle, gonadal WAT, and inguinal WAT (Figure 4G and H). In the same tissue, the times to reach 50% and 90% desaturation concentrations exhibited a dose-dependent relationship. More time was required to reach baseline for higher H₂ concentrations.

DISCUSSION

As a small molecular gas, H₂ can diffuse into the target tissues without any hindrance, even while passing through the blood- brain barrier. Inhalation is a common method to administer H₂. When mixed with air, the explosive range of H₂ is 4–75% (v/v).²¹ Hence, inhalation of 2–4% H₂ gas is frequently used in medical researches.¹ The development of H₂ generator has recently led to an increasing number of studies using high H₂ concentrations. A H₂ generator produces a mixture of H₂ (67%) and O₂ (33%) by electrolyzing water. A commercial medical- grade H₂/O₂ ventilator has been shown to ameliorate different diseases in animal models²²²³ and patients.²⁴²⁵ However, an abnormal O₂ content may have an ambiguous effect on research results. Therefore, in some experiments, the H₂/O₂ mixture gas was diluted with nitrogen (N₂) to obtain 42% H₂ and maintain the same O₂ concentration (~21%) as in the atmosphere.²⁶ The distribution of H₂ in tissues after inhaling low concentrations of H₂ has been investigated.^16172728 However, how the specific molecular mechanisms of high concentrations of H₂ differ from that of low concentrations needs further investigation. In addition, the distribution or concentration differences of H₂in vivo after inhaling different concentrations of H₂ also need further exploration. In this study, we monitored the concentrations of H₂ in different tissues in rats after inhaling 4%, 42%, and 67% H₂.

In the beginning of inhalation, H₂ enters the arterial blood through the alveoli and is then taken into tissues by a pressure gradient until reaches equilibrium. In this study, H₂ in the brain reached the highest C_e, followed by inguinal WAT, kidney, gonadal WAT, spleen, and liver tissues. In the thigh muscle, the H₂ concentration was significantly lower than in other tissues. This is consistent with a previous report suggesting that the saturation concentration of H₂ in the thigh muscle is much lower than in the blood and myocardium of rats.²⁷ However, the distribution of H₂ concentrations in different tissues found in this study was contradictory to results in some other literatures. Yamamoto et al.¹⁷ reported that after inhalation of 3% H₂, the saturation concentration was highest in the liver and lowest in the kidney. Another study revealed that the H₂ concentrations in different tissues varied with different administration methods and H₂ inhalation resulted in the highest H₂ concentration in the muscle.¹⁶ The reasons for the contradictory results varied and may be attributed to the different detected locations and depths chosen to represent the whole organ, the different concentrations and durations for H₂ inhalation, or the methodological differences.

Intermittent and continuous measurements have been applied to determine the H₂ concentrations in tissues. An intermittent measurement requires taking samples at regular intervals. After homogenization, H₂ is released into an airtight tube and then the gas is collected and measured with gas chromatography.¹⁶ In contrast, continuous measurement leads to the ability to create a continuous concentration curve against time using a microsensor in vivo,¹⁷ which has been used in biomedical fields.²⁹³⁰ A continuous measurement was performed in this study to obtain a complete equilibrium curve.

The C_e of H₂ for 4%, 42%, and 67% H₂ at 0.9% salinity have been shown to be about 28.5, 299.0, and 476.9 μM, respectively.²⁰ A previous study revealed that the H₂ concentration in the blood was in accordance with the value predicted by Henry’s law for the solubility of a gas in liquid.²⁸ It would be interesting to compare the actual C_e with theoretical concentrations in tissues rather than blood. In this study, only the C_e of H₂ in the brain was consistent with the theoretical concentrations, whereas in other tissues the C_e of H₂ were lower, especially in the thigh muscle. This may result from the diffusion of H₂ into the surroundings. A previous study detected the H₂ concentrations in air samples from the surface of the skin after volunteers inhaled 4% H₂.³¹ The gaseous diffusion model and the blood flow model have been applied in theoretical models of H₂ distributions.¹⁷ As per the gaseous diffusion model based on the distance between the gas supply hood and each organ, the brain reached a higher H₂ concentration due to the short distance between the face and head. In the blood flow model, different tissues reached the same C_e of H₂ at different rates based on the blood flow.¹⁷ This study indicated that both of the models worked because highest C_e of H₂ was found in the brain, and the C_e of H₂ in most tissues approached the theoretical concentrations.

H₂ concentrations of different tissues showed different saturation and desaturation rates in this study. In general, spleen and liver tissues needed less time to reach 50% and 90% C_e, followed by the kidney and brain. In tissues of the thigh muscle, gonadal WAT, and inguinal WAT, the equilibrium rates were slower than other tissues. The blood flow of muscle and adipose tissue was slower than that of abdominal tissues.³²³³ This may lead to a slower equilibrium rate. Moreover, as a fat-soluble gas, H₂ accumulated in adipose tissues. This leads to a slower rate when approaching equilibrium compared with other tissues.³⁴ A previous report¹⁷ showed that the saturation time was significantly longer and the concentration increased more slowly in muscle than the other examined organs for 3% H₂. Another study revealed that for 2% H₂, the arterial H₂ concentration in rats reached a maximum level after 5 minutes, whereas the increasing rate of H₂ concentration was much slower in the center of the thigh muscle and it reached the maximum level after 30 minutes.²⁷

The blood flow rate in various tissues has been measured by the H₂ clearance method.³³³⁵ Therefore, the desaturation of H₂ occurs mainly through blood circulation to the lung and then is released out of the body.

An in vitro experiment has shown a dose-dependent relationship for H₂ in protecting cells from cell death and reacting with hydroxyl radicals.¹ In an in vivo experiment, inhalation of H₂ (1-4%) was applied for hepatic injury caused by ischemia-reperfusion, and 2-4% H₂ was found to work the best.³⁶ Another study²⁷ on myocardial ischemia-reperfusion injury revealed that inhalation of 0.5-2% H₂ significantly reduced infarct size in a dose-dependent manner with 2% H₂ providing the most prominent effects. In contrast, 4% H₂ inhalation did not show the alleviating effect.²⁷ These results indicated the importance of choosing the appropriate dosage of H₂ for various tissue injuries. Considering the different C_e, the optimal concentration of H₂ inhalation may vary from disease to disease. Moreover, given the different saturation rates in tissues, different inhalation times may be needed to achieve the desired effect for lesions in different tissues, especially in muscle and adipose tissue.

We used a microsensor tip inserted into the tissue at a depth of 1 mm below the organ surface. The concentrations of H₂ may vary between different locations and depths in a same tissue and it is needed to estimate in future studies. This study revealed the H₂ distribution in different tissues under anesthesia in rats. However, it is known that anesthesia results in the tendency for blood flow rates to decrease in most tissues.³³ To confirm these results, it would be of interest to measure the H₂ distribution in different tissues while the animal is in a conscious state. However, at present, this would be difficult to achieve because of the constraints of the current measurement methods. Pharmacokinetics of H₂in vivo varies with methods of administration and thus influence the biomedical effects. Other H₂ intake methods, such as drinking H₂-rich water, are also worth further exploration.

To summarize, the H₂ distribution in different tissues of rats during and after inhaling different concentrations of H₂ over time was investigated. The results provide a reference for H₂ dose selection for animal and clinical trials and promote the use of H₂ in clinical therapies.

Acknowledgements

The authors are grateful to technical instructors of Shanghai Weizai Technology Co., Ltd. for their help in the monitoring technology.