antioxidants Article A Novel Therapy for Cisplatin-Induced Allodynia and Dysfunctional and Emotional Impairments in Male and Female Mice Ignacio Martínez-Martel 1,2 and Olga Pol 1,2, * 1 Grup de Neurofarmacologia Molecular, Institut de Recerca Sant Pau, Sant Quintí 77-79, 08041 Barcelona, Spain 2 Grup de Neurofarmacologia Molecular, Institut de Neurociències, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain * Correspondence: opol@santpau.es; Tel.: +34-619-757-054 Abstract: Patients undergoing chemotherapy with cisplatin (CIS) develop neuropathy in addition to other symptoms such as, anxiety, depression, muscle wasting and body weight loss. This symptoma- tology greatly weakens patients and may even lead to adjournment of chemotherapy. The protecting actions of molecular hydrogen in many neurological illnesses have been described, but its effect on the functional and emotional deficiencies caused by CIS has not been assessed. In C57BL/6J male and female mice injected with CIS, we examined the impact of the prophylactic treatment with hydrogen-rich water (HRW) on: (i) the tactile and cold allodynia, (ii) the deficits of grip strength and weight loss, (iii) the anxiodepressive-like behaviors and (iv) the inflammatory and oxidative reactions incited by CIS in the dorsal root ganglia (DRG) and prefrontal cortex (PFC). The results demonstrate that the mechanical allodynia and the anxiodepressive-like comportment provoked by CIS were similarly manifested in both sexes, whereas the cold allodynia, grip strength deficits and body weight loss produced by this chemotherapeutic agent were greater in female mice. Nonetheless, the prophylactic treatment with HRW prevented the allodynia and the functional and emotional impairments resulting from CIS in both sexes. This treatment also inhibited the inflammatory and oxidative responses activated by CIS in the DRG and PFC in both sexes, which might explain the Citation: Martínez-Martel, I.; Pol, O. therapeutic actions of HRW in male and female mice. In conclusion, this study revealed the plausible A Novel Therapy for Cisplatin- use of HRW as a new therapy for the allodynia and physical and mental impairments linked with Induced Allodynia and CIS and its possible mechanism of action. Dysfunctional and Emotional Impairments in Male and Female Keywords: anxiety; chemotherapy-induced peripheral neuropathy; cisplatin; depression; Mice. Antioxidants 2023, 12, 2063. https://doi.org/10.3390/ hydrogen-rich water; inflammation; molecular hydrogen; oxidative stress antiox12122063 Academic Editor: Alessandra Napolitano 1. Introduction Received: 26 October 2023 Chemotherapy-associated neuropathic pain is a major unpleasant effect resulting Revised: 23 November 2023 from this treatment in cancer patients. Patients undergoing chemotherapy also develop Accepted: 28 November 2023 other signs, such as anxiety, depression, muscle wasting and cognitive deficits that have Published: 30 November 2023 a significant negative impact on the well-being and quality of life of patients and may even lead to the need for adjournment of the chemotherapy [1,2]. Furthermore, therapies to prevent or treat this type of pain such as anticonvulsant, antidepressant, and non- Copyright: © 2023 by the authors. steroidal anti-inflammatory drugs are not significantly effective and produce noteworthy Licensee MDPI, Basel, Switzerland. side effects [3,4]. Therefore, our objective is to find an effective and safe treatment for This article is an open access article alleviating the neuropathic pain related to chemotherapy, as well as the functional deficits distributed under the terms and and mood disorders that are associated with it. conditions of the Creative Commons Cisplatin (CIS), a platinum analogue discovered by Rosenberg et al. (1969) [5], remains Attribution (CC BY) license (https:// one of the most used chemotherapeutic agents to treat solid tumors located in different creativecommons.org/licenses/by/ organs, including the ovaries, testes, head, neck, breast and lung [6,7]. However, despite 4.0/). the demonstrable clinical advantages of CIS as an antineoplastic drug, this treatment entails Antioxidants 2023, 12, 2063. https://doi.org/10.3390/antiox12122063 https://www.mdpi.com/journal/antioxidants Antioxidants 2023, 12, 2063 2 of 18 neuropathic pain and affective deficits, as well as muscle wasting and body weight loss, which can persist for months and greatly weaken patients [8–10]. All the side effects caused by CIS chemotherapy are relevant since they are the main reasons for a clinical decline in cancer patients [10]. Thus, effective treatments dismissing CIS-caused adverse effects are necessary. It is accepted that gender differences exist in the pathology of neuropathic pain. Several clinical studies have demonstrated that women are more sensitive to pain than men [11,12]. Additionally, animal investigations have also reported that female rodents show a lower threshold for pain in different models [13]. Regarding the impact of sex in the chemotherapy-induced neuropathic pain provoked by CIS, contradictory results exist. Some studies revealed that the mechanical and/or cold allodynia caused by CIS are similarly displayed in male and female animals [4,14], whereas another showed sex discrepancies in the maintenance of mechanical allodynia [15]. Nevertheless, possible differences among sexes in the development of CIS-induced emotional disorders, muscle wasting and/or weight lose have been poorly investigated. Treatment with CIS activates the transcription factor NF-κB and the successive pro- inflammatory cytokines (TNF-α, IL-6 and IL-8), leading to neuronal inflammation [16,17]. These inflammatory mediators play an important role in muscle wasting [18] as well as in the development of the anxiodepressive-like behaviors [19]. It is also well documented that the dysregulation of the NLRP3 inflammasome leads to excessive production of pro- inflammatory cytokines, leading to severe inflammation and/or various diseases such chronic pain [20]. The release of reactive oxygen species (ROS) from the dysfunctional mitochondria is a key upstream action for inducing the activation of the NLRP3 inflam- masome [21]. Recent studies have demonstrated that NLRP3 activation drives paclitaxel-, vincristine- and oxaliplatin-induced peripheral neuropathy [22–24]. These works further revealed that the inhibition or deletion of NLRP3 prevents the development of neuro- pathic pain caused by vincristine and oxaliplatin without affecting the chemotherapy efficacy [22,23]. Nonetheless, the role played by this inflammasome in CIS-induced neu- ropathy is not completely known. Abundant evidence also indicates that the beginning and progression of chemotherapy- induced neuropathic pain are linked with oxidative stress [25]. Oxidative stress also participates in the CIS-mediated neuropathy through a dysregulation of the products of lipid peroxidation (thiobarbituric acid reactive substance (TBARS) or 4-hydroxy-2-nonenal (4-HNE)) and Nrf2 transcription factor levels, which protect against the free radical injury by regulating antioxidant enzymes, for example heme oxygenase-1 (HO-1), superoxide dismutase-1 (SOD-1) and catalase [26]. Therefore, a decreased catalase and SOD-1 activity in the dorsal root ganglia (DRG) [27] and prefrontal cortex (PFC) of CIS-injected mice [28] was demonstrated. In line with this, increased levels of TBARS were also observed in the PFC of CIS-injected mice [28]. Therefore, the maintenance of a balance between the inflam- matory and antioxidant pathways might be key for alleviating CIS-induced neuropathy and associated emotional disorders. It is acknowledged that CIS-based chemotherapy is also related to other side effects, including muscle wasting and body weight loss [29,30]. In line with this, decreased muscle strength and body weight are manifested in CIS-injected mice [31–33]. However, the impact of sex and the possible treatment of these physical dysfunctions produced by CIS requires further investigation. Molecular hydrogen (H2 ) produces strong anti-inflammatory and antioxidant actions that are predominantly attributed to its capacity to inhibit the synthesis of NF-κB and the subsequent release of pro-inflammatory factors [34,35], as well as by reducing ROS levels and increasing the expression of antioxidant enzymes such as SOD-1 and HO-1 [36]. Recent experiments further revealed that the therapeutic administration of hydrogen-rich water (HRW) has beneficial effects in animals with paclitaxel-induced neuropathic pain [37]. This treatment also reduced the nociceptive and emotive disturbances occasioned by nerve injury and persistent paw inflammation in male mice [38,39]. There is also evidence that Antioxidants 2023, 12, 2063 3 of 18 H2 exhibits antidepressant and/or anxiolytic properties in animals with traumatic brain injury [40] or chronic stress-induced depressive- and anxiety-like comportments [41] and further improves psychiatric illnesses in humans [42]. Nevertheless, it is not known whether the prophylactic treatment with HRW can avoid the CIS-caused mechanical and thermal allodynia and some physical disabilities. Moreover, given that the emotional deficits can have long-lasting effects and negatively modulate pain sensation [43,44], it is also crucial to assess whether HRW administration can effectively inhibit the anxiety- and depressive-like behaviors associated with CIS-induced neuropathy. Therefore, our study aims to investigate the potential beneficial effects of prophylactic treatment with HRW as a novel approach for controlling CIS-induced neuropathy, weight loss and limb grip strength, as well as the development of anxiodepressive-like behaviors. The impact of sex on these actions and the main pathways involved will also be studied. 2. Materials and Methods 2.1. Animals Male and female C57BL/6J mice at 10 weeks of age at the beginning of the experiments were used in this study. Mice acquired from Envigo Laboratories (Barcelona, Spain) were housed with a 12 h dark/light cycle, 22 ◦ C temperature and 66% humidity with food and water ad libitum. After 7 days of acclimatization to the housing conditions, the experimental procedure was conducted between 9:00 to 17:00 h in agreement with the guidelines of the European Commission’s directive (2010/63/EC) and the Spanish Law (RD 53/2013) regulating animal research; the experiments were approved by the local Committee of Animal Use and Care of the Autonomous University of Barcelona (CEEA-UAB-protocol # 4581). A total of 128 mice (64 male and 64 female) were used in this study. Mice were randomly assigned to experimental groups. Animals were housed in polypropylene cages with four male or female mice per cage in an enriched environment that included a carton hut and cellulose fragments. The experiments were performed by a researcher blinded to the experimental conditions. All efforts were made to minimize animal suffering and to reduce the number of animals used. 2.2. Experimental Design In different groups of male and female mice: (1) The development of tactile and cold allodynia, deficits of grip strength and body weight loss provoked by the intraperitoneal administration of CIS or VEH were evaluated on days 2, 4, 6, 9, 11, 13, 16, 18, 20, 23, 25, 27 and 30 after their first injection (n = 8 animals). (2) The anxiety- and/or depressive-like behaviors caused by CIS or VEH were evalu- ated at day 30 after their first injection (n = 8 animals). (3) The inhibitory actions of co-treatment with HRW (from day 0 to 30) on the tactile and cold allodynia, grip strength deficits and body weight loss caused by CIS or VEH were evaluated on days 2, 4, 6, 9, 11, 13, 16, 18, 20, 23, 25, 27 and 30 after the first injection of CIS or VEH (n = 8 animals). (4) The possible anxiolytic and antidepressant effects produced by the co-treatment with HRW were evaluated at day 30 after the first administration of CIS or VEH (n = 8 animals). (5) The effects of HRW on the protein levels of NLRP3, 4-HNE, HO-1 and SOD-1 in the DRG and PFC were evaluated at day 30 after the first administration of CIS or VEH using Western blot (n = 3 samples for group). The experimental design is represented in Figure 1. Antioxidants 2023, 12, 2063 4 of 18 Antioxidants 2023, 12, 2063 4 of 18 Figure 1. Schematic Figure 1. Schematic representation representation ofof the the experimental experimentaltimeline. timeline.VF, VF,von vonFrey; Frey;CP, CP,cold coldplate; plate;GS, GS, grip strength; grip strength; EPM, EPM, elevated elevated plus plus maze; maze; TST, TST,tail tailsuspension suspensiontest; test;FST, FST,forced forcedswimming swimmingtest; I.P., test; I.P., intraperitoneal; CIS, intraperitoneal; CIS, cisplatin; cisplatin; VEH, VEH, vehicle; vehicle; HRW, HRW, hydrogen-rich hydrogen-rich water. water. 2.3. 2.3. Drug Drug Administration Administration CIS CIS (Sigma Aldrich, (Sigma Aldrich, St. Louis, MO, St. Louis, MO, USA) USA)waswasdiluted dilutedininsterile sterilesaline saline(0.9% (0.9%NaCl) NaCl)andand given given intraperitoneally daily at a dosage of 2.8 mg/kg for 5 days, followed by 5 days ofof intraperitoneally daily at a dosage of 2.8 mg/kg for 5 days, followed by 5 days rest, rest, and and asas second second round round ofof 55 doses, doses, according accordingto toaamodified modifiedversion versionreported reportedinin[45,46]. [45,46]. The doses and administration regimen of CIS were chosen as they The doses and administration regimen of CIS were chosen as they were similar to those were similar to those used used in patients undergoing this type of chemotherapy [47]. HRW was prepared using a a in patients undergoing this type of chemotherapy [47]. HRW was prepared using hydrogen hydrogenwater watergenerator generatorfrom fromhydrogen hydrogen(Osmo-star (Osmo-star Soriano Soriano S.L., Alicante, S.L., Alicante,Spain) accord- Spain) ac- ing to Cheng et al. (2020) [48]. All drugs were newly prepared before cording to Cheng et al. (2020) [48]. All drugs were newly prepared before their admin- their administration. For each group istration. treated For each with group a drug, treated the arespective with drug, thecontrol group respective received control the received group same volume the of analogous vehicle (VEH). same volume of analogous vehicle (VEH). For For evaluating evaluating whether whether HRWHRW could could prevent preventthetheweight weightloss,loss,mechanical mechanicalallodynia, allodynia, cold allodynia and GS deficits induced by CIS, male and female mice cold allodynia and GS deficits induced by CIS, male and female mice were pretreated with were pretreated with HRW, HRW, intraperitoneally administered at a concentration of 0.3 mM, 1 h before testing inina a intraperitoneally administered at a concentration of 0.3 mM, 1 h before testing final final volume volume of of 10 10 mL/kg [38], followed mL/kg [38], followedby byCISCISor orvehicle vehicleintraperitoneally intraperitoneallyadministered administered 22 hh later. A similar protocol was used for assessing whether later. A similar protocol was used for assessing whether HRW could prevent HRW could prevent CIS- CIS-in- induced anxiety- duced anxiety- andand depressive-like depressive-like behaviors. behaviors. 2.4. Allodynia 2.4. Allodynia Mechanical allodynia was measured as the hind paw withdrawal response to von Frey Mechanical allodynia was measured as the hind paw withdrawal response to von hair stimulation using the up-and-down method as described previously [49]. Mice were Frey hair stimulation using the up-and-down method as described previously [49]. Mice placed in methacrylate cylinders (20 cm high/9 cm diameter) in a grid bottom by which were placed in methacrylate cylinders (20 cm high/9 cm diameter) in a grid bo om by a series of von Frey hairs (0.4 to 3 g; North Coast Medical, Inc., San Jose, CA, USA) were which a series of von Frey hairs (0.4 to 3 g; North Coast Medical, Inc., San Jose, CA, USA) perpendicularly applied to the plantar surface of each hind paw. A positive response was were perpendicularly applied to the plantar surface of each hind paw. A positive response defined as a clear paw withdrawal or shaking. Whenever a positive response happened, was defined as a clear paw withdrawal or shaking. Whenever a positive response hap- the next lower hair was applied, and when a negative response occurred, the next higher pened, the next lower hair was applied, and when a negative response occurred, the next hair was applied. higher hair was applied. For cold allodynia, we used a cold plate analgesiometer (Ugo Basile, Varese, Italy) at For cold allodynia, we used a cold plate analgesiometer (Ugo Basile, Varese, Italy) at 4 ± 0.5 ◦ C and the number of elevations of each hind paw was noted for 5 min. 4 ± 0.5 °C and the number of elevations of each hind paw was noted for 5 min. 2.5. Forelimb Grip Strength Test 2.5. Forelimb Grip Strength Test The grip strength was measured using a grip strength meter (Ugo Basile, Varese, Italy) The grip according strength to [50]. was measured The mice using were allowed to agrab gripthe strength meter metal bar (Ugo with Basile, both hind Varese, It- legs before aly) according to [50]. The mice were allowed to grab the metal bar with both the mouse’s tail was gently pulled back; the maximal force (g) was recorded automatically hind legs before by the mouse’s the machine. Thetail was tests gently were pulled back; performed three the maximal times for eachforce (g) was animal recorded at one-min auto- intervals before CIS or VEH injection. This value was considered as 100% grip strength andone- matically by the machine. The tests were performed three times for each animal at was min intervals before CIS or VEH injection. This value was considered used as a reference for subsequent determinations. The force value was used to reflectas 100% grip muscle weakness. Antioxidants 2023, 12, 2063 5 of 18 2.6. Anxiety and Depressive Conducts The elevated plus maze (EPM) with 4 arms, 2 closed by 15 cm high walls and 2 open, elevated at 45 cm from the floor was used to assess the anxiety-like behaviors [51]. Each arm was 5 cm wide and 35 cm long. Mice were placed in the center of the maze and permitted to explore it for 5 min. During this time, the animals were recorded with a digital camera and the number of entrances and proportion of time spent in the open arms were counted. The tail suspension test (TST) and the forced swimming test (FST) were used to assess the depressive-like behaviors. In the TST, mice were suspended at 35 cm from the floor by using adhesive tape to the tip of the tail and fixing it to a surface. The animals were filmed, and the immobility time was evaluated for 6 min [52]. In the FST, mice were put into transparent cylinders (height, 25 cm; radius, 10 cm) containing water (24 ± 2 ◦ C) up to a 10 cm depth for 6 min. The time that the animals spent immobile during the last 4 min was recorded [53]. Animals were familiarized with the testing room for 1 h before beginning the tests. 2.7. Western Blot For Western blot analysis, animals were euthanized by cervical dislocation at day 30 after the first injection of CIS or VEH. DRG from the ipsilateral lumbar section (L3 to L5) and PFC were extracted and preserved at −80 ◦ C. Tissues were sonicated with cold RIPA buffer (Sigma-Aldrich, MO, USA). After solubi- lization for 1 h at 4 ◦ C, the homogenizes were sonicated again for 10 s and centrifuged for 20 min at 700 g and 4 ◦ C. Samples (60 µg total protein) were separated on a sodium dodecyl sulfate polyacrylamide gel (12%), transferred to polyvinylidene fluoride membranes for 120 min and blocked with phosphate-buffered saline plus Tween 20 or Tris-buffered saline plus Tween 20 + 5% of nonfat dry milk or bovine serum albumin for 75 min. Membranes were incubated overnight at 4 ◦ C with specific primary antibodies (Table 1), followed by horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Little Chalfont, UK). Proteins were detected with chemiluminescence reagents (ECL kit; GE Healthcare, Little Chalfont, UK), and densitometric analysis was performed using the Image-J program (version 1.8.0; National Institutes of Health, Bethesda, MD, USA). Table 1. Primary antibodies used in this study. Antibody Dilution Purchased NLRP3 1:200 Adipogen Life Sciences, Epalinges, Switzerland 4-HNE 1:200 Abcam, Cambridge, UK HO-1 1:100 Enzo Life Science, New York, NY, USA SOD-1 1:150 Novus Biologic, Littleton, CO, USA GAPDH 1:5000 Merck, Billerica, MA, USA 2.8. Statistical Analyses The results are expressed as the mean values ± standard error of the mean (SEM). A three-way repeated measures ANOVA with time, sex and treatment as the variation factors was used to assess the actions of the co-treatment with HRW on CIS-provoked allodynia, grip strength deficits and body weight loss. A two-way ANOVA was used to assess the influence of the co-treatment with HRW on the emotional behaviors caused by CIS. A one-way ANOVA plus Tukey test was used to analyze the impact of HRW treatment on the expression of several proteins. We used SPSS (version 28, IBM, Madrid, Spain) and GraphPad Prism 9.0 software (La Jolla, CA, USA). A p < 0.05 was declared as significant. Antioxidants 2023, 12, 2063 6 of 18 Antioxidants 2023, 12, 2063 6 of 18 3. Results 3.1. The Prophylactic Treatment with HRW Inhibited the Mechanical and Cold Allodynia 3. Resultsby CIS in Male and Female Mice Generated 3.1. The Prophylactic Treatment with HRW Inhibited the Mechanical and Cold Allodynia On the Generated byevaluation CIS in Maleof andthe effects Female of HRW on the mechanical allodynia provoked by Mice CIS inOnboth hind paws of male and female the evaluation of the effects of HRW mice, themechanical on the three-way allodynia ANOVA repeated provoked measures by CIS test revealed in both a significant hind paws timefemale of male and effect mice, (p < 0.001), a significant the three-way ANOVA treatment repeatedeffect (p < test measures 0.001) and a significant time by treatment interaction (p < 0.001) in the von Frey test. revealed a significant time effect (p < 0.001), a significant treatment effect (p < 0.001) and a The results significant indicated time by treatment that the tactile interaction (p