Inhibition of Hepatic Ischemic Reperfusion Injury Using Saline Exposed to Electron Discharge in a Rat Model

Abstract

The pathogenesis of ischemia/reperfusion (I/R) injury in surgical trauma, organ transplantations, and brain and myocardial infarctions is attributable to excessive oxidative stress caused by reactive oxygen species and their metabolites. We prepared a physiological saline solution treated with simple exposure to a microampere current with electron discharge onto the surface; this treatment increased reduction potential and caused proton reduction. We examined the reductive potency of the electron-treated solution (ETS) on hepatocellular I/R injury in a rat model. When the ETS was administered in four doses at 0, 3, 10, and 20 min after reperfusion, severe hepatocellular injury was suppressed, as revealed by a decrease in serum alanine aminotransferase levels and histopathological improvement of liver damage. Since a preparation of hydrogen gas-dissolved saline solution (HDS) was shown to be capable of suppressing I/R injury, the possible involvement of dissolved hydrogen gas in the effectiveness of ETS was examined. When HDS was treated by degasification, the aforementioned effectiveness was decreased. In contrast, the same treatment did not alter the effectiveness of ETS. These results suggest that the antioxidative efficacy of ETS is not attributable to dissolved hydrogen gas but presumably to the molecule(s) produced from the stepwise reduction of protons in the solution.

INTRODUCTION

The antioxidative network of the body is well developed to detoxify oxidative stresses occurring during normal physiological reactions. However, the network becomes unbalanced when acute and excessive oxidative stresses are induced by ischemia/reperfusion (I/R) in clinical settings, many types of surgeries performed under hypoxia, organ transplantations, and myocardial and brain infarctions, frequently leading to serious tissue damage and life-threatening events and disabilities (4,10,18,21).

Oxidative stress is mitigated following the consumption of high levels of endogenous antioxidants such as glutathione (GSH) and vitamins, as they scavenge reactive oxygen species (ROS), hydroxyl radicals, lipid peroxyl radicals, and peroxynitrite and their peroxide metabolites.

To alleviate I/R-induced tissue damage, the current detoxification strategy against pathogenic oxidative stress includes primarily enhancing the antioxidative network potential of the body by administration of radical scavenger compounds. Previous approaches with antioxidants such as glutathione (16,20), α-tocopherol (12), α-lipoic acid (2), and edaravone (7) demonstrated a reduction in liver damage with lower increases in plasma levels of alanine aminotransferase (ALT) in hepatic I/R injury model rats. Moreover, Ohta and colleagues (3,6,14) reported the therapeutic benefits of hydrogen gas inhalation in myocardial, cerebral, and hepatic I/R injuries in rat models. The detoxification efficacy of the gas was marked, and the scavenger activity was selective for cytotoxic hydroxyl radicals, but not for other ROS, superoxide anion radicals, nitric oxide, or hydrogen peroxide.

In this study, we investigate whether electron-treated physiological saline solution (ETS), which is capable of exhibiting increased reductive potential of the solution as a result of greater proton reduction capacity and oxidation and reduction potentials (ORPs), suppresses hepatocellular I/R damage in rat models and examine the possible role of dissolved hydrogen gas in the reductive potential of ETS.

MATERIALS AND METHODS

Preparation of ETS

The treatment of physiological saline solution with electron discharge was performed by electron absorption apparatus as illustrated in Figure 1A. A 0.45-Φ stainless sawing (Clover Mfg. Co. Ltd., Osaka, Japan) needle connected to a negative output terminal of direct current (DC) power supply (PMC18-2, Kikusui Electronics Corp., Yokohama, Japan) was positioned 10 mm above the solution surface. An acrylic cylinder cell with an 18-mm internal diameter and 22-mm height was covered on the bottom of the cylinder cell with an electron-transferable hydrophobic membrane filter (FP-100, Sumitomo Electric Fine Polymer, Osaka, Japan) and placed on a metal electrode connected to the ground terminal.

Figure 1.

The preparation of ETS and suppression of hepatic I/R injury. (A) Schematic diagram of electron absorption apparatus in vitro. Physiological saline solution was added to the cylinder. The electron discharge at microampere current was applied on the surface of the solution. Black arrows indicate the direction of electron release. (B) A single administration of electron-treated physiological saline solution (ETS) attenuated the injury. Partial hepatic ischemia was maintained for 60 min. A 1.5-ml sample of ETS (○), twofold diluted ETS (•), or normal saline (▵) (control) was administered from the caudal vein 1 min before reperfusion (n = 6 per group). Blood was collected from the subclavian vein 1, 3, 7, and 24 h after hepatic reperfusion for alanine aminotransferase (ALT) measurements. Each point represents the mean ± standard error of the mean (SEM) of six cases. *Statistically significant difference (p < 0.05) from the control group (normal saline). I/R, ischemia/reperfusion.

Five milliliters of physiological saline solution (Otsuka Pharma, Tokyo, Japan) was added to the cylinder. The initiation of electron exposure onto the surface of a physiological saline solution was carried out by supplying 10 µA of DC using a high-voltage (−5,000 V) circuit (TA-5111-1, JET Cham Enterprise Co. Ltd., Taipei, Taiwan) connected to a 12-V DC power source. The current measured in the “cylinder cell-ground” circuit was monitored and controlled by changing the power supply.

Changes in pH and ORP values were monitored using a pH and ORP meter (DKK-TOA, Tokyo, Japan), respectively.

In theory, the ETS preparation system differed from the system used for electrolysis of a saline solution because the needle does not make contact with the solution (8).

Rat Hepatic I/R Injury Model

Sprague–Dawley male rats (232–310 g) were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan). They were anesthetized with isoflurane gas. A midline laparotomy incision was performed, and an atraumatic clip was used to interrupt the blood supply to the left lateral and median lobes of the liver to achieve 70% liver ischemia (1). After 60 or 70 min of partial hepatic ischemia, the clip was removed to initiate hepatic reperfusion. Afterwards, hematoxylin and eosin (H&E)-stained specimens were microscopically reviewed. Serum ALT levels were determined using an automated analyzer (DRI-CHEM 3500i, FUJIFILM, Tokyo, Japan).

Hydrogen Gas-Dissolved Physiological Saline Solution (HDS)

We used commercially available high-concentration H₂-dissolved water (1,500 ppb, ORP value −600 mV, Global Balance Co., Japan). Physiological osmosis was adjusted by adding 36% NaCl solution (Sigma, Tokyo, Japan).

Degasification Treatment for HDS

Light degasification treatment of the HDS was carried out using a vacuum pump G-25S (Ulvac, Kanagawa, Japan) for 15 s. The vacuum pressure was 20–30 mbar, as determined using Data logger (Pico VACQ, TMI-Orion, Reston, VA, USA).

Statistical Analysis

Statistical significance was determined by a one-way ANOVA and Fisher’s protected least significant difference (PLSD) test using the StatView software program, version 5.0 for Macintosh (SAS Institute, Cary, NC, USA).

Animal Ethics

All animal experiments were performed in accordance with the institutional animal ethics guidelines of the National Center for Child Health and Development.

RESULTS AND DISCUSSION

The method used for the preparation of ETS in the present study was different from the principle of electrolysis for a saline solution. The surface of the solution was exposed to discharged electrons at a microampere current released from the stainless steel needle without making contact with the solution (Fig. 1A). By contrast, in electrolysis (8), when the needle is dipped into the solution, the dilute NaCl solution is dissociated into acidic electrolyzed water (pH 2–3; ORP > 1,100 mV) that produces chlorine derivatives, such as chloride gas and hypochlorous acid, with an active chlorine content of 10–90 ppm and basic electrolyzed water (pH 10–13; ORP −800 to −900 mV) that produces H₂. The method used for ETS preparation did not produce such dramatic changes in the saline solution.

Notably, one of the physicochemical changes in the ETS was a decrease in H⁺ concentration. In this study, we have obtained an ETS with an increase in pH of 0.36 ± 0.04 (from 6.55 ± 0.06) over 60 min with exposure to a 10-µA current. This suggests that the decrease in H⁺ concentration in the solution was due to the reduction in H⁺ followed by production of atomic hydrogen, hydrogen anion, H₃O⁻, or hydrogen gas by stepwise proton reduction. Another change in the ETS was with respect to ORP values, which decreased by 84 ± 6 mV (from 366 ± 2 mV), indicating an increase in the reductive environment with increased electron concentration (8,15).

To further investigate the reducing potential of the ETS, we induced hepatic I/R injury in a rat model using 70% partial ischemia; this method is sensitive and widely used to induce acute, massive oxidative stress in vivo (11). The serum ALT values in the normal saline-administered control group increased exponentially from 1 h after reperfusion, peaked at 8,179 ± 916 U/L after 3 h, then gradually decreased over 24 h (Fig. 1B). When 1.5 ml of undiluted ETS was administered to the rats via the caudal vein 1 min before reperfusion, the inhibitory effect on the I/R injury at 3 h caused a 32.4% decrease in the ALT value, which was 5,529 ± 744 U/L (p = 0.002). When twofold diluted ETS with normal saline was administered, the inhibitory effect was attenuated with a 10.6% decrease in the ALT value, which was 7,316 ± 599 U/L (p = 0.296) (Fig. 1B). Furthermore, 1 ml of undiluted ETS administered in four doses at 0, 3, 10, and 20 min after reperfusion markedly decreased ALT values by 41.9% (to 5,691 ± 1,041 U/L) compared to the control value of 9,793 ± 911 U/L at 3 h (p = 0.005) (Fig. 2). In addition, when the ETS was separately treated with a light degasification, the decrease in ALT was either unaffected or slightly enhanced. The decrease in ALT values correlated with improved histopathological findings of H&E-stained hepatocellular injury, including degenerated hepatocytes, zonal cytoplasmic vacuolization, and hemorrhage in whole sections (Fig. 3). Hence, the ETS could suppress the acute severe oxidative stress induced in the hepatic I/R injury model.

Figure 2.

Effects of multiple dose administration (1 ml) on hepatic I/R injury with the ETS. Partial hepatic ischemia was maintained for 70 min. A 1-ml sample of ETS was administered via the penile vein in four doses at 0, 3, 10, and 20 min after reperfusion. Original ETS (n = 7), degassed ETS (n = 8), and normal saline (control, n = 8). Data are presented as the mean ± SEM.

Figure 3.

Histopathological examination. ETS was administered to rats with induced hepatic I/R injury as described in Figure 2.

We further examined the stability of the antioxidative activity of the ETS. When the ETS was kept for 24 h at room temperature, the ORP values of the ETS remained as low as 302 ± 2 mV (n = 3) without significant change compared to fresh preparations (300 ± 5 mV, n = 3). However, the ETS left for 24 h at room temperature could no longer suppress I/R injury, thus indicating that the active reducing ingredients present in the ETS are not stable (Fig. 4).

Figure 4.

Effect of 24-h storage of ETS at room temperature (n = 8) and control saline solution (n = 8) on hepatic I/R injury. Blood was collected 3 h after hepatic reperfusion for the ALT measurements. Data are presented as the mean ± SEM.

Ohta and colleagues (3,6,14) reported that hydrogen gas inhalation protected against brain, hepatic, and myocardial I/R injuries in animal models; therefore, we used the I/R injury model to examine the possible involvement of dissolved hydrogen gas in the efficacy of the ETS to suppress I/R injury. We prepared an artificial HDS using a commercially available, high concentration of hydrogen gas-dissolved water (1,500 ppb, ORP value −600 mV) adjusted for physiological osmosis with high concentration of NaCl solution. We administered the HDS to the rats with hepatic I/R injury. Marked suppression of injury was observed with four doses of the HDS solution at 0, 3, 10, and 20 min after reperfusion, showing a 41.2% decrease in ALT values from 6,133 ± 1,058 U/L to 3,603 ± 446 U/L (p = 0.017) (Fig. 5), similar to the effectiveness of the ETS (Fig. 2). However, pretreatment of the HDS with light degasification using a vacuum pump for 15 s attenuated the efficacy to 23.9%, rendering an ALT value of 4,670 ± 341 U/L, with loss of statistical significance (p = 0.150) as shown in Figure 5. In contrast, the effectiveness of ETS treated with the same light degasification was either unaffected or slightly enhanced as shown in Figure 2. These results indicate that suppression of I/R injury with the ETS was not attributable to the dissolved hydrogen gas, even though small amounts of hydrogen gas were produced in the solution.

Figure 5.

Effect of hydrogen-dissolved saline solution on hepatic I/R injury. Partial hepatic ischemia was maintained for 70 min. Hydrogen-dissolved saline (HDS; 1.5 ml) with (n = 8) or without (n = 8) degasification treatment was administrated via the penile vein 0, 3, 10, and 20 min after reperfusion. Blood was collected after 3 h of hepatic reperfusion for ALT measurements. Each column represents the mean ± SEM of eight cases.

Alternatively, we speculate that considering the possible hydrogenation potency of the ETS, this solution might stimulate the reactivation of extra- and intracellular levels of oxidized antioxidants, GSH, and vitamins. Levels of these antioxidants are lowered under excessive oxidative stress and with the massive production of peroxide metabolites induced by I/R treatment (5,19). In addition, IV administration of GSH suppressed hepatic I/R injury, resulting in smaller increases in plasma ALT levels (16,20). Thus, one of the contributions of ETS to the alleviation of tissue injury might be attributed to the synthesis of antioxidants, GSH, and vitamins directly or indirectly via increased availability of a mediator ion (hydrogen anion or hydride ion), which is usually produced at the expense of the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH), to form NADP⁺ and the hydride ion by NADPH-dependent GSH reductase (14). This interesting hypothesis should be examined in future studies.

At this stage, physicochemical studies on the antioxidative molecular species induced in the ETS are required. However, candidates for the detoxification of excessively produced cytotoxic free radicals generated by I/R treatment were presumably the added electron and increased negativity in molecule(s) such as atomic hydrogen (17), hydrogen anion, H₃O⁻, or the cluster assembly, not the dissolved hydrogen gas.

Finally, it is important that the antioxidative potency of the ETS can be transferable to a biological system as an antioxidant. The medical applications of this procedure, such as an infusion system and continuous flow device to treat surgical trauma, organ preservation, and brain and myocardial infarctions, should be beneficial.