antioxidants Article Treatment with Hydrogen-Rich Water Improves the Nociceptive and Anxio-Depressive-like Behaviors Associated with Chronic Inflammatory Pain in Mice Santiago Coral-Pérez 1,2,† , Ignacio Martínez-Martel 1,2,† , Maria Martínez-Serrat 1,2,† , Gerard Batallé 1,2 , Xue Bai 1,2 , Christie R. A. Leite-Panissi 3, * and Olga Pol 1,2, * 1 Grup de Neurofarmacologia Molecular, Institut d’Investigació Biomèdica Sant Pau (IIB SANT PAU), Sant Quintí 77-79, 08041 Barcelona, Spain 2 Grup de Neurofarmacologia Molecular, Institut de Neurociències, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 3 Department of Psychology, Faculty of Philosophy Science and Letters of Ribeirão Preto, University of São Paulo, Ribeirão Preto 14040-901, SP, Brazil * Correspondence: christie@usp.br (C.R.A.L.-P.); opol@santpau.es (O.P.); Tel.: +34-619-757-054 (O.P.) † These authors contributed equally to this work. Abstract: Chronic inflammatory pain is manifested in many diseases. The potential use of molecular hydrogen (H2 ) as a new therapy for neurological disorders has been demonstrated. Recent studies prove its analgesic properties in animals with neuropathic pain, but the possible antinociceptive, antidepressant, and/or anxiolytic actions of H2 during persistent inflammatory pain have not been investigated. Therefore, using male mice with chronic inflammatory pain incited by the subplantar injection of complete Freud’s adjuvant (CFA), we assessed the actions of hydrogen-rich water (HRW) Citation: Coral-Pérez, S.; systemically administered on: (1) the nociceptive responses and affective disorders associated and Martínez-Martel, I.; Martínez-Serrat, (2) the oxidative (4-hydroxy-2-nonenal; 4-HNE), inflammatory (phosphorylated-NF-kB inhibitor M.; Batallé, G.; Bai, X.; Leite-Panissi, alpha; p-IKBα), and apoptotic (Bcl-2-like protein 4; BAX) changes provoked by CFA in the paws and C.R.A.; Pol, O. Treatment with amygdala. The role of the antioxidant system in the analgesia induced by HRW systemically and Hydrogen-Rich Water Improves the locally administered was also determined. Our results revealed that the intraperitoneal administra- Nociceptive and Anxio-Depressive- tion of HRW, besides reducing inflammatory pain, also inhibited the depressive- and anxiolytic-like like Behaviors Associated with behaviors associated and the over expression of 4-HNE, p-IKBα, and BAX in paws and amygdala. Chronic Inflammatory Pain in Mice. The contribution of the nuclear factor erythroid 2-related factor 2/heme oxygenase 1 and NAD(P)H: Antioxidants 2022, 11, 2153. https:// doi.org/10.3390/antiox11112153 quinone oxidoreductase 1 pathway in the analgesic activities of HRW, systemically or locally admin- istered, was also shown. These data revealed the analgesic, antidepressant, and anxiolytic actions of Academic Editor: Stanley Omaye HRW. The protective, anti-inflammatory, and antioxidant qualities of this treatment during inflamma- Received: 24 September 2022 tory pain were also demonstrated. Therefore, this study proposes the usage of HRW as a potential Accepted: 28 October 2022 therapy for chronic inflammatory pain and linked comorbidities. Published: 30 October 2022 Keywords: allodynia; anxiety; apoptosis; depression; hyperalgesia; inflammatory pain; molecular Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in hydrogen; oxidative stress published maps and institutional affil- iations. 1. Introduction Chronic pain is generally defined as a syndrome characterized by persistent suffering Copyright: © 2022 by the authors. that is prolonged for long periods and is challenging to alleviate/eradicate in clinical Licensee MDPI, Basel, Switzerland. practice [1]. Different comorbidities commonly accompany chronic pain, for example, This article is an open access article cognitive deficits, sleep difficulties, and affectations on the emotional mood, especially distributed under the terms and depressive and anxiety disorders [2,3]. conditions of the Creative Commons In several preclinical models of chronic pain, such as induced by the complete or Attribution (CC BY) license (https:// partial constriction of the sciatic nerve in the case of neuropathic pain [4–7], a close rela- creativecommons.org/licenses/by/ 4.0/). tionship between chronic pain and alterations in the affective states related with anxiety- Antioxidants 2022, 11, 2153. https://doi.org/10.3390/antiox11112153 https://www.mdpi.com/journal/antioxidants Antioxidants 2022, 11, 2153 2 of 16 and depressive-like behaviors has been found. In contrast, a possible relationship be- tween chronic pain generated by peripheral inflammation and the concurrent development of anxiety- and depressive-like behaviors has not been fully established. While some authors showed that chronic inflammatory pain is accompanied by depressive-like behav- iors [3,8,9], others only estimated the anxiety-like behaviors associated with inflammatory pain [2,10–12]. Thus, one of our objectives is to examine the plausible development of both affective deficits during chronic inflammatory pain generated by the subplantar injection of complete Freund’s adjuvant (CFA) in mice. Several studies demonstrated the participation of pro-inflammatory cytokines, for instance interleukins (IL), IL-1α, IL-1β, TNFα, and IL-6, in the progress of inflammatory pain [13,14]. Oxidative stress and the nuclear factor-κB (NF-κB) activated by the nuclear factor of kappa light polypeptide gene enhancer in the B-cells inhibitor, alpha (IKBα), are two notable factors that significantly contribute to the progress of inflammatory pain through regulating a large collection of genes involved in different processes of the inflam- matory replies [15–17]. Several studies further revealed the apoptotic reactions produced by inflammation in the peripheral and central nervous system (CNS) of animals with chronic inflammatory pain [18]. A solid relationship between oxidative stress and inflammation with depressive and/or anxiety disorders has been revealed; indeed, an increased expression of oxidative and inflammatory biomarkers in specific brain areas of depressive patients was demon- strated [19,20]. Consequently, a therapy for mood disorders which focuses on the use of antioxidants as an antidepressant and/or anxiolytic agents, avoiding the important side effects induced by classic antidepressants and/or anxiolytics, was proposed [20]. In accordance, the administration of nuclear factor erythroid 2-related factor 2 (Nrf2) tran- scription factor activators, such as sulforaphane or oltipraz, inhibited the depressive- and anxiety-like behaviors accompanying chronic neuropathic pain in mice [21,22], and TBE-31 and MCE-1 are two possible candidates for treating the inflammation-associated depressive behaviors [23]. In the last years, some researchers demonstrated the beneficial effects of molecular hydrogen (H2 ). Currently, numerous studies propose the use of H2 as a therapy, because of: a) its facility to cross the blood–brain barrier and penetrate cell membranes, b) the lack of evidence revealing undesired side effects or the development of tolerance induced by the repetitive treatment with H2 , and c) the antioxidant actions of this molecule against various oxidative-related diseases [24,25]. The antioxidant effects of H2 are predominantly adjudicated to its capacity to incite the translocation of the Nrf2 transcription factor from the cytosol to the nucleus, with the following transcription of several genes, such as superoxide dismutase (SOD), heme oxygenase 1 (HO-1), and NAD(P) H:quinone oxidoreductase 1 (NQO1) [25,26]. Other studies have also shown the anti-inflammatory responses induced by H2 through inhibiting pro-inflammatory cytokines, for instance, interleukins IL-1β, IL-4, IL-5, and IL-13, among others [27,28]. In the last years, the potential antidepressant and/or anxiolytic effects of H2 have also been shown in animals with unpredictable mild stress [29] and in healthy patients [30]. All these attributes suggested that H2 might be a good can- didate for treating chronic pain and the linked affective disorders. Consequently, several authors have revealed the painkiller properties of this gas in animals with neuropathic pain [31,32]. Even so, its possible antinociceptive, antidepressant, and/or anxiolytic effects during chronic inflammatory pain have not been completely established. In mice with chronic inflammatory pain provoked by CFA, our aims are to evaluate: (1) the antinociceptive effects of the intraperitoneal and/or subplantar administration of hydrogen-rich water (HRW), (2) the feasible antidepressant and/or anxiolytic effects of HRW during chronic inflammatory pain, (3) the main pathways that take part in the antinociceptive actions of HRW, and (4) the effects of HRW in the oxidative, inflammatory, and/or apoptotic reactions generated by CFA in the paws and amygdala, as key structures in regulating the nociceptive [33] and emotional responses [2]. Antioxidants 2022, 11, 2153 3 of 16 2. Materials and Method 2.1. Animals The experimentations were achieved with 6–8 week old male C57BL/6 mice (25–26 g) purchased at Envigo Laboratories (Barcelona, Spain), maintained under 12/12 h light/dark conditions in a space acclimatized to 22 ◦ C and a humidity of 66% until use. All experiments were carried out with the guidelines of the European Commission’s directive (2010/63/EC) and the Spanish Law (RD 53/2013) that regulated animal research and were authorized by the local Committee of Animal Use and Care of the Autonomous University of Barcelona (ethical code: 9863). All endeavors were performed to diminish the suffering and the number of animals used. 2.2. Generation of Inflammatory Pain To induce inflammatory pain, 30 µL of CFA (Sigma-Aldrich, St. Louis, MO, USA) was injected into the right hind paw via the subplantar method. For this process, animals were anesthetized with isoflurane (3% induction, 2% maintenance), applying the protocol used in a previous study [34]. Control animals were treated with the equal amount of saline (NaCl 0.9%; SS). 2.3. Mechanical Allodynia Mechanical allodynia was estimated by determining the hind limb withdrawal re- sponses after its stimulation with the von Frey filaments, for which the flexion forces were between 0.4 g to 3.0 g. Animals were sited in Plexiglas cylinders (25 cm × 10 cm) on a platform of a wire grid where the von Frey filaments (North Coast Medical, Inc., San Jose, CA, USA) were applied. The test began with the 0.4 g filament, and the strength of the next filaments enhanced or diminished depending on whether the animal removed, licked, or shook the paw or not. The hind limb withdrawal response was evaluated by applying an Excel program (Microsoft Iberia SRL, Barcelona, Spain), using the data sequence of five applications of filaments. 2.4. Thermal Hyperalgesia Thermal hyperalgesia was assessed by measuring the hind limb withdrawal latency response in seconds when the animal was exposed to heat in the plantar test (Ugo Basile, Varese, Italy). Animals were put in Plexiglas cylinders (25 cm × 10 cm) over a crystal superficies. The heat supply was placed below the paw of the animal and activated to light up until the animal reacted, or up to 12 s. Mean paw withdrawal latencies were taken from three separate measurements of hind limb withdrawal. In all tests, both hind paws were evaluated. Animals were familiarized to the von Frey and plantar environment for 1 h prior to the test to permit correct immobility behavior. 2.5. Depressive-like Behaviors The assessment of the depressive-like behaviors was accomplished at day 16 after CFA injection by using the tail suspension test (TST) and forced swimming test (FST), where the immobility of the animals was calculated in seconds. In the TST, animals were individually suspended by the tail from a platform, sited at 35 cm high from the ground, utilizing adhesive tape (1 cm). Animals were recorded by a digital camera and the immobility time was quantified over 6 min [5,35]. Concerning the FST, each animal was put in a Plexiglas tube (25 cm × 10 cm) that contained water to 10 cm of deepness at 24 ◦ C. Mice were subjected to forced swimming for 6 min, and the duration of immobility was quantified during the last 4 min, when mice showed an adequately stable level of immobility, in accordance with [36–38]. In both tests, mice were familiarized to the testing space for 1 h before starting the test. Antioxidants 2022, 11, 2153 4 of 16 2.6. Anxiety-like Behaviors The anxiety-like behaviors were tested by using the elevated plus maze (EPM) as it was described by Kraeuter et al. (2019) [39] and open field (OF) by Kraeuter et al. (2019) [40]. The EPM is an apparatus with four arms orientated on a cross shape, each one of 5 cm wide and 35 cm long, where two of the arms stay open and the other two are closed, with walls of 15 cm. The distance of the EPM to the ground is 45 cm. Mice were sited in the center of the maze facing one of the open arms, and their behavior was filmed for 5 min. The number of entries in the open and closed arms and the percentage of time spent in the open arms were calculated for each animal. In the OF test, mice were positioned in the arena of a box (44 cm × 44 cm) with a grey non-reflecting base and four walls (30 cm high), and the behavior was recorded for 5 min. Animals were permitted to move freely across the apparatus and explore the environment. The number of entries and percentage of time passed in the central area and the number of squares crossed were quantified. In both tests, animals were familiarized to the testing space for 1 h before starting the evaluation. 2.7. Experimental Procedures Since one of our goals was to examine the possible development of both affective deficits (depressive and anxious behaviors) associated with chronic inflammatory pain generated by the subplantar injection of CFA, the evaluation of mechanical allodynia and thermal hyperalgesia was performed between days 13 and 16 after CFA injection, and affective responses were assessed on day 16 after CFA injection. In the first procedure, SS- or CFA-injected mice were intraperitoneally administered with vehicle (VEH) or HRW (0.3 mM) at one time/day (HRW 1T) or two times/day (HRW 2T) over three consecutive days. Allodynia and hyperalgesia were tested prior to CFA injection (day 0), one day before treatment (day 13 after CFA injection) and in each day of treatment (days 14–16 after CFA-injection) (n = 6 animals for group). In separate groups of SS- or CFA-injected mice, the antiallodynic and anti-hyperalgesic effects induced by the subplantar administration of HRW (0.3 mM) or VEH, injected at 2T per day from days 14 to 16 after CFA injection, were also evaluated (n = 6 animals for group). In distinct groups of animals, the evaluation of depressive- and anxiety-like behaviors was performed at day 16 after CFA injection. The possible antidepressant and/or anxiolytic effects produced by the 2T daily intraperitoneal administration of HRW from days 14 to 16 after CFA injection were also evaluated in the TST/FST and EPM/OF tests, respectively (n = 8 animals for each group). Animals treated with VEH were used as controls. Animals were tested 1 h after HRW or VEH injection [41]. To examine the probable involvement of the Nrf2/HO-1 and NQO1 pathway in the analgesic actions of HRW, the effects of the intraperitoneal and subplantar administration of HRW or VEH, injected at 2T or 1T per day, in animals intraperitoneally or subplantarly co-treated with 25 mg/kg or 625 µg of ML385, a Nrf2 inhibitor [42], 10 mg/kg or 250 µg of tin protoporphyrin IX (SnPP), an HO-1 inhibitor [43], 10 mg/kg or 250 µg of dicoumarol, a NQO1 inhibitor [44], or VEH (DMSO 1% in SS) were assessed (n = 6 animals for group). The HRW concentration and doses of ML-385, SnPP and dicoumarol were chosen in line with earlier studies [41,45]. At 16 days after CFA injection, animals injected with HRW or VEH were euthanized by cervical dislocation, and the expression of 4-hydroxy-2-nonenal (4-HNE), Bcl-2-like protein 4 (BAX), and phosphorylated IKBα (p-IKBα) were assessed in the paws and amygdala tissues using Western blot. All experiments were performed by a researcher blinded to the experimental conditions. Antioxidants 2022, 11, 2153 5 of 16 2.8. Drugs HRW was prepared using a hydrogen water generator from Hydrogen (Osmostar Soriano S.L., Alicante, Spain), which uses the electrolysis method to break down H2 O into H2 . SnPP was obtained from Frontier Scientific (Livchem GmbH & Co., Frankfurt, Germany) and ML-385 and dicoumarol from Eurodiagnostico S.L (Madrid, Spain); all were dissolved in DMSO 1% in SS. All drugs were intraperitoneally and subplantarly injected in a volume of 10 mL/kg and 30 µL, respectively. HRW was administered 1 h before testing, while ML-385, SnPP, and dicoumarol were administered at 30 min before the tests, in accordance with earlier studies [41,45]. All drugs were newly prepared prior to their administration. For each group treated with a drug, the corresponding control group received the identical volume of corresponding VEH. 2.9. Western Blot Analysis Sixteen days following CFA or SS injection, animals were euthanized by cervical dislo- cation, and the ipsilateral subplantar tissue of the hind paws and contralateral amygdala were removed, frozen, and kept at −80 ◦ C until usage. We evaluated the levels of 4-HNE, p-IKBα, and BAX. Tissues were sonicated (amygdala) or homogenized (paw) in cold lysis buffer RIPA Buffer (Sigma-Aldrich, St. Louis, MO, USA). Next to the solubilisation for 1 h at 4 ◦ C, tissues were sonicated (10 s) and centrifuged at 700× g at 4 ◦ C (20 min). The supernatant (60 µg of total protein) mixed with Laemmli loading buffer was loaded onto 4% stacking/12% separating sodium dodecyl sulfate-polyacrylamide gels. Afterward, proteins were electrophoretically transferred on a polyvinylidene fluoride membrane for 120 min and blocked with Tris-buffered saline with Tween 20 and nonfat dry milk, bovine serum albumin (5%), or phosphate-buffered saline with Tween 20 and nonfat dry milk (5%) for 75 min. Then, membranes were incubated overnight at 4 ◦ C with specific primary rabbit antibodies against 4-HNE (1:100; Abcam, Cambridge, UK), p-IKBα (1:150; Abcam, Cam- bridge, UK), IKBα (1:100; Abcam, Cambridge, UK), BAX (1:150; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:5000; Merck, Billerica, MA, USA). After that, blots were incubated at room temperature (60 min) with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare, Little Chalfont, UK), proteins were detected by using chemiluminescence reagents (ECL kit; GE Healthcare, Little Chalfont, Bucking- hamshire, UK), and band density was evaluated by densitometry with the Image-J program (National Institutes of Health, Bethesda, MD, USA). 2.10. Statistical Analysis All results were represented as the mean values ± standard error of the mean (SEM). To evaluate the effects of inflammation, treatment, and time, and the interactions be- tween them, the three-way analysis of variance (ANOVA) with repeated measures was performed using the Statistical Package for Social Sciences (SPSS, Version 17.0, IL, USA). For each day, the effects produced by different therapies in the allodynia, hyperalgesia, and anxiety–depressive-like behaviors were analyzed with a one-way ANOVA and, subse- quently, a Student–Newman–Keuls test using the Graph Pad Prism program (version 8 for Windows). A value of p < 0.05 was considered significant. 3. Results 3.1. Effects of the Administration of HRW in the Mechanical Allodynia and Thermal Hyperalgesia Caused by CFA Initially, we assessed the effects of HRW injected intraperitoneally at 1T or 2T per day for 3 consecutive days on the allodynia and hyperalgesia caused by peripheral inflammation on days 14 to 16 after CFA injection. In both tests, the three-way repeated measures ANOVA displayed significant effects of inflammation, treatment, and time (p < 0.001) and interactions among inflammation × treatment, inflammation × time, treatment × time, as well as between inflammation × treatment × time (p < 0.001). Hyperalgesia Caused by CFA Initially, we assessed the effects of HRW injected intraperitoneally at 1T or 2T per day for 3 consecutive days on the allodynia and hyperalgesia caused by peripheral in- flammation on days 14 to 16 after CFA injection. In both tests, the three-way repeated Antioxidants 2022, 11, 2153 measures ANOVA displayed significant effects of inflammation, treatment, and time (p < 6 of 16 0.001) and interactions among inflammation × treatment, inflammation × time, treatment × time, as well as between inflammation × treatment × time (p < 0.001). Thus, Thus,thethe reduced reducedthreshold thresholdof of thethe ipsilateral hind ipsilateral paw hind paw withdrawal withdrawal to von Frey to von fila- Frey ments stimulus provoked by CFA at 13 days after its injection (p < 0.001, filaments stimulus provoked by CFA at 13 days after its injection (p < 0.001, one-wayone-way ANOVA vs.ANOVA SS-injected mice injected vs. SS-injected micewith VEH; injected Figure with VEH;1A) was1A) Figure fully was inhibited after 2after fully inhibited and23anddays of 3treatment with HRW days of treatment injected with HRW at 2T or 1T injected perorday, at 2T respectively. 1T per Moreover, day, respectively. the antiallo- Moreover, the dynic effects induced antiallodynic by the double effects induced by the administration of HRW double administration at twoatdays of HRW of treatment two days were of treatment higher were than higherthose thanproduced by thebysingle those produced administration the single administration(p <(p0.001, one-way < 0.001, one-way ANOVA). ANOVA). Figure Figure 1. 1. Effects Effects ofofthe theintraperitoneal intraperitonealinjection injection of of HRW HRW or or VEH VEHon onthe themechanical mechanicalallodynia allodyniaandand thermal hyperalgesia induced by paw inflammation. Effects of the repetitive administration of thermal hyperalgesia induced by paw inflammation. Effects of the repetitive administration of HRW HRW or VEH, or VEH, injected injected at 1Tat 1T2T and and per2T perover day, day,the over the decreased decreased von Freyvon Frey filaments filaments strength strength (g) (A) and(g) the(A) and the withdrawal latency (s) (B) generated by CFA in the ipsilateral hind paws. For each test, * withdrawal latency (s) (B) generated by CFA in the ipsilateral hind paws. For each test, * indicates indicates significant significant changeschanges vs. their vs. their respective respective SS-injectedSS-injected mice (SS +mice VEH,(SS + VEH, SS + HRW 1T,SS +orHRW 1T, or2T), SS + HRW SS + HRW 2T), + represents + represents significantsignificant differencesdifferences vs. CFA + VEHvs. CFA + VEH injected injected mice, and # mice, denotesand # denotes significant significant differences differences vs. CFA +vs.HRW CFA1T + HRW 1T (p (p < 0.05, on