{"id":26787,"date":"2024-01-03T21:38:21","date_gmt":"2024-01-03T19:38:21","guid":{"rendered":"https:\/\/hho-bulgaria.com\/real-time-hydrogen-monitoring-in-rat-tissues\/"},"modified":"2024-02-05T04:30:33","modified_gmt":"2024-02-05T02:30:33","slug":"real-time-hydrogen-monitoring-in-rat-tissues","status":"publish","type":"post","link":"https:\/\/hho-bulgaria.com\/en\/real-time-hydrogen-monitoring-in-rat-tissues\/","title":{"rendered":"Real-time Hydrogen Monitoring in Rat Tissues"},"content":{"rendered":"
\n

INTRODUCTION<\/h2>\n

Hydrogen gas (H2<\/sub>) is colorless and odorless at standard temperature and pressure. Due to its small molecular weight and hydrophobic properties, H2<\/sub> can easily permeate cell membranes and even enter cell organelles. H2<\/sub> has long been regarded as a biochemical inert gas. In 2007, Ohsawa et al.1<\/a><\/sup> 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<\/sub> in various animal disease models.2<\/a><\/sup>3<\/a><\/sup>4<\/a><\/sup>5<\/a><\/sup> Meanwhile, some clinical investigations have also confirmed its beneficial effects on different diseases.6<\/a><\/sup>7<\/a><\/sup> Various ways have been explored to administer H2<\/sub>, mainly including inhaling H2<\/sub>, drinking H2<\/sub>-rich water, injecting H2<\/sub>-rich saline, and direct incorporation of H2<\/sub> by diffusion (such as bath and eye-drops). Due to its bio-safety, countries and regions such as USA,8<\/a><\/sup> Japan,9<\/a><\/sup> Europe,10<\/a><\/sup> and China11<\/a><\/sup> have recently allow to use H2<\/sub> as a food additive. At present, the therapeutic effects of H2<\/sub> have attracted increasing attention worldwide.12<\/a><\/sup><\/p>\n

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

Until now, the existing molecular mechanism of the biological effect of H2<\/sub> 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<\/a><\/sup> determined H2<\/sub> concentrations in different rat tissues following the administration of H2<\/sub> via various routes. The study revealed variable dynamics of H2<\/sub> in various tissues over time and different H2<\/sub> concentrations in the same tissue with different methods of H2<\/sub> uptake. Another in vivo<\/em> study17<\/a><\/sup> monitored the sequential changes of H2<\/sub> concentrations in tissues over time with continuous inhalation of 3% H2<\/sub>. However, conclusions of both studies were not entirely consistent. Moreover, previous studies were performed only after a single concentration of H2<\/sub> intake. The dose-response curve had not been illustrated. Therefore, more accurate and detailed studies are needed to acquire the exact pharmacokinetics of H2<\/sub>.<\/p>\n

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

MATERIALS AND METHODS<\/h2>\n

Gas preparation<\/h3>\n

Different concentrations of H2<\/sub> (4%, 42%, 67%; v\/v) were prepared using a lab-made gas mixing device (Figure 1A<\/a>). H2<\/sub> and oxygen gas (O2<\/sub>) 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<\/sub> was confirmed with a gas detector (XP-3140, New-cosmos Co. Ltd., Tokyo, Japan). Meanwhile, the O2<\/sub> concentration was kept at ~21% and verified with an O2<\/sub> 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.<\/p>\n

\n
\"F1-6\"<\/a>
Figure 1: <\/a><\/p>\n
Device for H2<\/sub> inhalation (A) and the seven representative tissues targeted for H2<\/sub> concentration measurements (B)<\/strong>.Note: H2<\/sub>: Hydrogen gas; O2<\/sub>: oxygen gas; WAT: white adipose tissue.<\/div>\n<\/figcaption><\/figure>\n<\/section>\n

H2<\/sub> concentration monitoring in vivo<\/em><\/h3>\n

The measuring device included a miniaturized Clark-type hydrogen microsensor with an internal reference electrode and a sensing anode (tip diameter 40\u201360 \u03bcm), 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><\/sup> A standard curve was established by diluting the H2<\/sub>-saturated phosphate-buffered saline at 38\u00b0C. 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<\/sub> was provided continuously until the H2<\/sub> concentration in the target tissue reached equilibrium. Then, the gas was replaced by pure air and monitoring was continued until the H2<\/sub> concentration returned to baseline. Predicted by Henry\u2019s law for the solubility of a gas in a liquid,19<\/a><\/sup> and the solubility of H2<\/sub> at a certain temperature and salinity,20<\/a><\/sup> the theoretical equilibrium concentrations (Ce<\/sub>) of 4%, 42%, and 67% H2<\/sub> in blood are about 28.5, 299.0, and 476.9 \u03bcM, respectively. The detection started before hydrogen inhalation. At the beginning, the rats were given air until reaching a stable baseline, then hydrogen was provided.<\/p>\n

Statistical analysis<\/h3>\n

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\u2019s multiple comparison test were used to assess the significance of differences in H2<\/sub> concentrations between various tissues. P<\/em> < 0.05 was considered statistically significant. Data are representative of at least three independent experiments, and expressed as the mean \u00b1 standard deviation (SD).<\/p>\n

RESULTS<\/h2>\n

Ce<\/sub> of H2<\/sub> in different tissues<\/h3>\n

Ce<\/sub> of H2<\/sub> in different tissues after inhaling different concentrations of H2<\/sub> are shown in Figure 2<\/a>. The figures display highest mean Ce<\/sub> in the brain, followed by inguinal WAT and kidney, and lowest in the thigh muscle regarding the three concentrations of H2<\/sub>. Inter-organ comparisons revealed significant differences between the brain\/thigh muscle and other tissues (P<\/em> < 0.001 or P<\/em> < 0.01) for different concentrations of H2<\/sub>. For 4% H2<\/sub>, Ce<\/sub> showed a significant difference between inguinal WAT and liver (P<\/em> < 0.05). For 67% H2<\/sub>, Ce<\/sub> showed statistically significant differences between inguinal WAT and liver, spleen, gonadal WAT, respectively (P<\/em> < 0.05). Ce<\/sub> of H2<\/sub> in all tissues exhibited a dose-dependent increase corresponding to the concentration of inhaled H2<\/sub>. Ce<\/sub> values after inhaling 42% and 67% H2<\/sub> were about 10.5- and 16.8-time greater than 4% H2<\/sub> in various tissues. The theoretical Ce<\/sub> of H2<\/sub> based on a blood flow model and Henry\u2019s law for the solubility of gas are also shown in Figure 2<\/a>.<\/p>\n

\n
\"F2-6\"<\/a>
Figure 2: <\/a><\/p>\n
Equilibrium H2<\/sub> concentrations in the brain, liver, spleen, kidney, thigh muscle, gonadal white adipose tissue (WAT), and inguinal WAT after inhaling different concentrations of H2<\/sub><\/strong>.Note: (A\u2013C) 4%, 42%, 67% H2<\/sub>. The dotted line represents the theoretical Ce<\/sub> of H2<\/sub>. Data are shown as the mean \u00b1 SD (n<\/em> = 3-6 rats per tissue). **P<\/em> < 0.01, ***P<\/em> < 0.001, vs<\/em>. brain; ##P<\/em> < 0.01, ###P<\/em> < 0.001, vs. thigh muscle; &P<\/em> < 0.05, vs<\/em>. inguinal WAT (one-way analysis of variance followed by Tukey\u2019s multiple comparison test). H2<\/sub>: Hydrogen gas.<\/div>\n<\/figcaption><\/figure>\n<\/section>\n

H2<\/sub> saturation dynamics in different tissues<\/h3>\n

The H2<\/sub> dynamic curves for different tissues after inhaling various concentrations of H2<\/sub> are summarized in Figure 3A<\/a>\u2013F<\/a>. In general, H2<\/sub> concentrations in the brain, liver, kidney, and spleen increased faster than in the thigh muscle, inguinal WAT, and gonadal WAT (Figure 3<\/a>). For 4% H2<\/sub>, 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<\/a>). However, the plots for the thigh muscle, inguinal WAT, and gonadal WAT exhibited a gradual increase after H2<\/sub> was inhaled and they needed much more time to reach the Ce<\/sub> (Figure 3D<\/a>). 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<\/a> and H<\/a>). For 42% and 67% H2<\/sub>, trends similar to those of 4% H2<\/sub> were observed (Figure 3B<\/a>, C<\/a>, E<\/a>, and F<\/a>) and the orders were same as 4% H2<\/sub> (Figure 3G<\/a> and H<\/a>). In the same tissue, similar time was needed to reach 50% Ce<\/sub> for different H2<\/sub> concentrations applied. Meanwhile, a dose-dependent relationship for the time to reach 90% Ce<\/sub> was observed (Figure 3G<\/a> and H<\/a>).<\/p>\n

\n
\"F3-6\"<\/a>
Figure 3: <\/a><\/p>\n
H2<\/sub> saturation dynamics in the brain, liver, spleen, kidney, thigh muscle, gonadal white adipose tissue (WAT), and inguinal WAT after inhaling different concentrations of H2<\/sub><\/strong>.Note: (A\u2013F) Changes of H2<\/sub> concentrations in the brain, liver, spleen, kidney, thigh muscle, gonadal white adipose tissue (WAT), and inguinal WAT during inhalation of 4% (A, D), 42% (B, E), and 67% (C, F) H2<\/sub>. (G, H) Time taken to reach 50% (G) and 90% (H) saturation. Data are shown as the mean \u00b1 SD (n<\/em> = 3-6 rats per tissue), and were analyzed by one-way analysis of variance followed by Tukey\u2019s multiple comparison test. H2<\/sub>: Hydrogen gas.<\/div>\n<\/figcaption><\/figure>\n<\/section>\n

H2<\/sub> desaturation dynamics in different tissues<\/h3>\n

The H2<\/sub> concentrations in different tissues started decreasing after H2<\/sub> administration was withdrawn (Figure 4A<\/a>\u2013F<\/a>). In the beginning, the brain, liver, kidney, and spleen exhibited a similar sharp drop right after H2<\/sub> withdrawal, and then plots decreased gradually until reached the baseline (Figure 4A<\/a>\u2013C<\/a>). However, the curves of the thigh muscle, inguinal WAT, and gonadal WAT exhibited a more gradual decrease after H2<\/sub> withdrawal and took more time to reach the baseline (Figure 4D<\/a>\u2013F<\/a>). 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<\/a> and H<\/a>). 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<\/sub> concentrations.<\/p>\n

\n
\"F4-6\"<\/a>
Figure 4: <\/a><\/p>\n
Hydrogen desaturation dynamics in the brain, liver, spleen, kidney, thigh muscle, gonadal white adipose tissue (WAT), and inguinal WAT after inhaling different concentrations of H2<\/sub><\/strong>.Note: (A\u2013F) Changes of H2<\/sub> concentrations in the brain, liver, spleen, kidney, thigh muscle, gonadal white adipose tissue (WAT), and inguinal WAT after ending inhalation of 4% (A, D), 42% (B, E), and 67% (C, F) H2<\/sub>. (G, h) Time taken to reach 50% (G) and 90% (H) desaturation. Data are shown as the mean \u00b1 SD (n<\/em> = 3-6 rats per tissue), and were analyzed by one-way analysis of variance followed by Tukey\u2019s multiple comparison test. H2<\/sub>: Hydrogen gas.<\/div>\n<\/figcaption><\/figure>\n<\/section>\n

DISCUSSION<\/h2>\n

As a small molecular gas, H2<\/sub> 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<\/sub>. When mixed with air, the explosive range of H2<\/sub> is 4\u201375% (v\/v).21<\/a><\/sup> Hence, inhalation of 2\u20134% H2<\/sub> gas is frequently used in medical researches.1<\/a><\/sup> The development of H2<\/sub> generator has recently led to an increasing number of studies using high H2<\/sub> concentrations. A H2<\/sub> generator produces a mixture of H2<\/sub> (67%) and O2<\/sub> (33%) by electrolyzing water. A commercial medical- grade H2<\/sub>\/O2<\/sub> ventilator has been shown to ameliorate different diseases in animal models22<\/a><\/sup>23<\/a><\/sup> and patients.24<\/a><\/sup>25<\/a><\/sup> However, an abnormal O2<\/sub> content may have an ambiguous effect on research results. Therefore, in some experiments, the H2<\/sub>\/O2<\/sub> mixture gas was diluted with nitrogen (N2<\/sub>) to obtain 42% H2<\/sub> and maintain the same O2<\/sub> concentration (~21%) as in the atmosphere.26<\/a><\/sup> The distribution of H2<\/sub> in tissues after inhaling low concentrations of H2<\/sub> has been investigated.16<\/a><\/sup>17<\/a><\/sup>27<\/a><\/sup>28<\/a><\/sup> However, how the specific molecular mechanisms of high concentrations of H2<\/sub> differ from that of low concentrations needs further investigation. In addition, the distribution or concentration differences of H2<\/sub>in vivo<\/em> after inhaling different concentrations of H2<\/sub> also need further exploration. In this study, we monitored the concentrations of H2<\/sub> in different tissues in rats after inhaling 4%, 42%, and 67% H2<\/sub>.<\/p>\n

In the beginning of inhalation, H2<\/sub> enters the arterial blood through the alveoli and is then taken into tissues by a pressure gradient until reaches equilibrium. In this study, H2<\/sub> in the brain reached the highest Ce<\/sub>, followed by inguinal WAT, kidney, gonadal WAT, spleen, and liver tissues. In the thigh muscle, the H2<\/sub> concentration was significantly lower than in other tissues. This is consistent with a previous report suggesting that the saturation concentration of H2<\/sub> in the thigh muscle is much lower than in the blood and myocardium of rats.27<\/a><\/sup> However, the distribution of H2<\/sub> concentrations in different tissues found in this study was contradictory to results in some other literatures. Yamamoto et al.17<\/a><\/sup> reported that after inhalation of 3% H2<\/sub>, the saturation concentration was highest in the liver and lowest in the kidney. Another study revealed that the H2<\/sub> concentrations in different tissues varied with different administration methods and H2<\/sub> inhalation resulted in the highest H2<\/sub> concentration in the muscle.16<\/a><\/sup> 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<\/sub> inhalation, or the methodological differences.<\/p>\n

Intermittent and continuous measurements have been applied to determine the H2<\/sub> concentrations in tissues. An intermittent measurement requires taking samples at regular intervals. After homogenization, H2<\/sub> is released into an airtight tube and then the gas is collected and measured with gas chromatography.16<\/a><\/sup> In contrast, continuous measurement leads to the ability to create a continuous concentration curve against time using a microsensor in vivo<\/em>,17<\/a><\/sup> which has been used in biomedical fields.29<\/a><\/sup>30<\/a><\/sup> A continuous measurement was performed in this study to obtain a complete equilibrium curve.<\/p>\n

The Ce<\/sub> of H2<\/sub> for 4%, 42%, and 67% H2<\/sub> at 0.9% salinity have been shown to be about 28.5, 299.0, and 476.9 \u03bcM, respectively.20<\/a><\/sup> A previous study revealed that the H2<\/sub> concentration in the blood was in accordance with the value predicted by Henry\u2019s law for the solubility of a gas in liquid.28<\/a><\/sup> It would be interesting to compare the actual Ce<\/sub> with theoretical concentrations in tissues rather than blood. In this study, only the Ce<\/sub> of H2<\/sub> in the brain was consistent with the theoretical concentrations, whereas in other tissues the Ce<\/sub> of H2<\/sub> were lower, especially in the thigh muscle. This may result from the diffusion of H2<\/sub> into the surroundings. A previous study detected the H2<\/sub> concentrations in air samples from the surface of the skin after volunteers inhaled 4% H2<\/sub>.31<\/a><\/sup> The gaseous diffusion model and the blood flow model have been applied in theoretical models of H2<\/sub> distributions.17<\/a><\/sup> As per the gaseous diffusion model based on the distance between the gas supply hood and each organ, the brain reached a higher H2<\/sub> concentration due to the short distance between the face and head. In the blood flow model, different tissues reached the same Ce<\/sub> of H2<\/sub> at different rates based on the blood flow.17<\/a><\/sup> This study indicated that both of the models worked because highest Ce<\/sub> of H2<\/sub> was found in the brain, and the Ce<\/sub> of H2<\/sub> in most tissues approached the theoretical concentrations.<\/p>\n

H2<\/sub> 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% Ce<\/sub>, 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.32<\/a><\/sup>33<\/a><\/sup> This may lead to a slower equilibrium rate. Moreover, as a fat-soluble gas, H2<\/sub> accumulated in adipose tissues. This leads to a slower rate when approaching equilibrium compared with other tissues.34<\/a><\/sup> A previous report17<\/a><\/sup> showed that the saturation time was significantly longer and the concentration increased more slowly in muscle than the other examined organs for 3% H2<\/sub>. Another study revealed that for 2% H2<\/sub>, the arterial H2<\/sub> concentration in rats reached a maximum level after 5 minutes, whereas the increasing rate of H2<\/sub> concentration was much slower in the center of the thigh muscle and it reached the maximum level after 30 minutes.27<\/a><\/sup><\/p>\n

The blood flow rate in various tissues has been measured by the H2<\/sub> clearance method.33<\/a><\/sup>35<\/a><\/sup> Therefore, the desaturation of H2<\/sub> occurs mainly through blood circulation to the lung and then is released out of the body.<\/p>\n

An in vitro<\/em> experiment has shown a dose-dependent relationship for H2<\/sub> in protecting cells from cell death and reacting with hydroxyl radicals.1<\/a><\/sup> In an in vivo<\/em> experiment, inhalation of H2<\/sub> (1-4%) was applied for hepatic injury caused by ischemia-reperfusion, and 2-4% H2<\/sub> was found to work the best.36<\/a><\/sup> Another study27<\/a><\/sup> on myocardial ischemia-reperfusion injury revealed that inhalation of 0.5-2% H2<\/sub> significantly reduced infarct size in a dose-dependent manner with 2% H2<\/sub> providing the most prominent effects. In contrast, 4% H2<\/sub> inhalation did not show the alleviating effect.27<\/a><\/sup> These results indicated the importance of choosing the appropriate dosage of H2<\/sub> for various tissue injuries. Considering the different Ce<\/sub>, the optimal concentration of H2<\/sub> 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.<\/p>\n

We used a microsensor tip inserted into the tissue at a depth of 1 mm below the organ surface. The concentrations of H2<\/sub> 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<\/sub> 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<\/a><\/sup> To confirm these results, it would be of interest to measure the H2<\/sub> 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 H2<\/sub>in vivo<\/em> varies with methods of administration and thus influence the biomedical effects. Other H2<\/sub> intake methods, such as drinking H2<\/sub>-rich water, are also worth further exploration.<\/p>\n

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

Acknowledgements<\/h3>\n

The authors are grateful to technical instructors of Shanghai Weizai Technology Co., Ltd. for their help in the monitoring technology.<\/p>\n<\/section>\n","protected":false},"excerpt":{"rendered":"

In vivo microelectrode monitoring of real-time hydrogen concentration in different tissues of rats after inhaling hydrogen gas<\/p>\n","protected":false},"author":1,"featured_media":17899,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[130],"tags":[],"disease":[862],"body-organ":[1022],"applications":[679],"test_subjects":[1517],"report-topic":[1309],"class_list":["post-26787","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-hydrogen-health","disease-hydrogen-biology-2","body-organ-whole-body-2","applications-inhalation-2","test_subjects-rat-2","report-topic-hydrogen-safety-2"],"acf":[],"yoast_head":"\nReal-time Hydrogen Monitoring in Rat Tissues<\/title>\n<meta name=\"description\" content=\"In vivo microelectrode monitoring of real-time hydrogen concentration in different tissues of rats after inhaling hydrogen gas\" \/>\n<meta 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Animals and experimental design<\/h3>\n

Fifty 8-week-old specific-pathogen-free level male Sprague- Dawley rats weighing 180\u2013210 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 \u00b1 1\u00b0C; 12\/12 hours light\/dark cycle). Water and food were provided ad libitum<\/em>. 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.<\/p>\n

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\u00b0C 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.<\/p>\n

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<\/em> = 3-6 per tissue), were monitored (Figure 1B<\/a>). Only one target tissue was exposed at a time per rat.<\/p>\n