antioxidants Article The Beneficial Effects of Heme Oxygenase 1 and Hydrogen Sulfide Activation in the Management of Neuropathic Pain, Anxiety- and Depressive-like Effects of Paclitaxel in Mice Gerard Roch 1,2 , Gerard Batallé 1,2 , Xue Bai 1,2 , Enric Pouso-Vázquez 1,2 , Laura Rodríguez 1,2 and Olga Pol 1,2, * 1 Grup de Neurofarmacologia Molecular, Institut d’Investigació Biomèdica Sant Pau, Hospital de la Santa Creu i Sant Pau, 08041 Barcelona, Spain; gerard.roch@e-campus.uab.cat (G.R.); gerard.batalle@e-campus.uab.cat (G.B.); xue.bai@e-campus.uab.cat (X.B.); enrique.pousovazquez@e-campus.uab.cat (E.P.-V.); laura.rodriguezpe@e-campus.uab.cat (L.R.) 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: Chemotherapy-induced peripheral neuropathy constitutes an unresolved clinical problem that severely decreases the quality of the patient’s life. It is characterized by somatosensory alterations, including chronic pain, and a high risk of suffering mental disorders such as depression and anxiety. Unfortunately, an effective treatment for this neuropathology is yet to be found. We investigated the therapeutic potential of cobalt protoporphyrin IX (CoPP), a heme oxygenase 1 inducer, and morpholin-  4-ium 4-methoxyphenyl(morpholino) phosphinodithioate dichloromethane complex (GYY4137), a  slow hydrogen sulfide (H2 S) donor, in a preclinical model of paclitaxel (PTX)-induced peripheral Citation: Roch, G.; Batallé, G.; Bai, X.; neuropathy (PIPN) in mice. At three weeks after PTX injection, we evaluated the effects of the Pouso-Vázquez, E.; Rodríguez, L.; repetitive administration of 5 mg/kg of CoPP and 35 mg/kg of GYY4137 on PTX-induced nociceptive Pol, O. The Beneficial Effects of Heme symptoms (mechanical and cold allodynia) and on the associated emotional disturbances (anxiety- Oxygenase 1 and Hydrogen Sulfide and depressive-like behaviors). We also studied the mechanisms that could mediate their therapeutic Activation in the Management of properties by evaluating the expression of key proteins implicated in the development of nociception, Neuropathic Pain, Anxiety- and oxidative stress, microglial activation, and apoptosis in prefrontal cortex (PFC) and dorsal root ganglia Depressive-like Effects of Paclitaxel in Mice. Antioxidants 2022, 11, 122. (DRG) of mice with PIPN. Results demonstrate that CoPP and GYY4137 treatments inhibited both https://doi.org/10.3390/ the nociceptive symptomatology and the derived emotional alterations. These actions were mainly antiox11010122 mediated through potentiation of antioxidant responses and inhibiting oxidative stress in the DRG and/or PFC of mice with PIPN. Both treatments normalized some plasticity changes and apoptotic Academic Editor: reactions, and GYY4137 blocked microglial activation induced by PTX in PFC. In conclusion, this Alessandra Napolitano study proposes CoPP and GYY4137 as good candidates for treating neuropathic pain, anxiety- and Received: 2 December 2021 depressive-like effects of PTX. Accepted: 4 January 2022 Published: 6 January 2022 Keywords: anxiety; depression; heme oxygenase 1; hydrogen sulfide; chemotherapy-induced Publisher’s Note: MDPI stays neutral peripheral neuropathy; oxidative stress; paclitaxel with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction During the last several decades, the survival rates for most cancers have been increas- ing, an improvement that can be partially attributed to new and improved treatments [1]. Copyright: © 2022 by the authors. Unfortunately, the same tendency is not observable for many of the secondary effects of on- Licensee MDPI, Basel, Switzerland. cotherapies, which remain unresolved and affect a high number of patients. Chemotherapy- This article is an open access article distributed under the terms and induced peripheral neuropathy (CIPN) is a side effect that has significant impacts, both conditions of the Creative Commons on cancer survival and on the patient’s quality of life; it is a crucial limiting factor that can Attribution (CC BY) license (https:// lead to a change or reduction in the therapy or, in severe cases, to its total cessation [1]. creativecommons.org/licenses/by/ The usual CIPN symptomatology includes multiple somatosensory alterations, such 4.0/). as allodynia, hyperalgesia, and spontaneous pain such as burning spams [2]. Remarkably, Antioxidants 2022, 11, 122. https://doi.org/10.3390/antiox11010122 https://www.mdpi.com/journal/antioxidants Antioxidants 2022, 11, 122 2 of 19 different studies have also concluded that, if CIPN persists over time, this symptomatol- ogy greatly increases the risk of developing severe emotional disorders such as anxiety, depression, and insomnia. In some cases, these tendencies are maintained long after treat- ment cessation, reinforcing the conception of CIPN as a chronic disease [3]. Unfortunately, there are currently no effective pharmacological therapies for CIPN, and the efficacy of the existing therapies is only moderate [4]. In the last decade, innovative pharmacological approaches have been tested in animal trials with positive analgesic results, e.g., the administration of reactive oxygen species (ROS) scavengers [5,6], antioxidants such as oltipraz [7], or anti-inflammatory substances [8]. However, very few studies have investigated new possible effective treatments for the emotional disorders associated with CINP. Considering that mental illnesses are hard to treat once established and that they often persist after the original cause has been resolved, it is crucial to assess the possible anxiolytic and antidepressant effects of the analgesics to improve cancer patients’ mental health. Carbon monoxide (CO) is a gaseous neurotransmitter with relevant implications in pain processing [9,10]. Accordingly, CO-releasing molecules and heme oxygenase 1 (HO-1) enzyme inducers have been demonstrated to be effective analgesic options in inflammatory and neuropathic pain models [11–13]. Additionally, different studies indicate that these substances have anxiolytic effects [10–14]. Hydrogen sulfide (H2 S), another gaseous neurotransmitter, also plays an important role in nociception [15]. Numerous studies have reported the painkilling actions of this neurotransmitter when administering slow H2 S-releasing agents such as isothiocyanates or morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate dichloromethane complex (GYY4137) in animals with osteoarthritic or nerve-injury-induced neuropathic pain [16–18]. These treatments also improved the affective behaviors accompanying chronic osteoarthritic pain and neuropathic pain generated by nerve injury or associated with diabetes [17,19,20]. Paclitaxel (PTX) is a taxane chemotherapeutic agent widely used for the treatment of cancer, including ovarian, breast, lung, and pancreatic cancer [21]. It has one of the highest CIPN incidence rates and the CIPN may persist for a long time, and it is accompanied by multiple affective disorders that interfere with positive outcomes [22,23]. In this study, we used a preclinical model of PTX-induced peripheral neuropathy (PIPN) developed by [24], which reproduces the somatosensory clinical features such as the bilateral mechanical and cold allodynia, as well as the anxiodepressive-like behaviors present in humans [22]. In this model, we evaluated the effects induced by two well-recognized antioxidant com- pounds: cobalt protoporphyrin IX (CoPP), an inducer of the antioxidant enzyme HO-1, and GYY4137, a slow-release H2 S donor, which also exhibits important antioxidant properties in several diseases. The mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathways are activated after PTX injection and are actively involved in the establishment of PIPN [25,26]. In this study, we evaluated the effects of CoPP and GYY4137 on the protein levels of p-P38, p-ERK1/2, PI3K, and p-Akt in the prefrontal cortex (PFC) and dorsal root ganglia (DRG), two specific areas sited in the central (CNS) and peripheral (PNS) nervous system implicated in the modulation of nociception and emotional disorders [27,28]. Oxidative stress also plays a crucial role in the development of CIPN [28]. Previ- ous studies have shown an upregulation of several oxidative-stress markers and/or the downregulation of the nuclear factor erythroid-2-related factor 2 (Nrf2), HO-1, NAD(P)H quinone dehydrogenase 1 (NQO1) and superoxide dismutase 1 (SOD-1) axis in animals with CINP [28,29]. Therefore, the activation of Nrf2 and HO-1 improves CIPN symptoma- tology [11,12]. We analyzed the effects of CoPP and GYY4137 on the protein levels of the oxidative biomarkers 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) and of the antioxidant enzymes HO-1, NQO1, and SOD-1 in the PFC and DRG of PTX-injected mice. Antioxidants 2022, 11, x FOR PEER REVIEW 3 of 20 the antioxidant enzymes HO‐1, NQO1, and SOD‐1 in the PFC and DRG of PTX‐injected Antioxidants 2022, 11, 122 mice. 3 of 19 Microglia are also implicated in the establishment of CIPN [30] and play a crucial role in the development of emotional disorders accompanying chronic pain [31]. The pos‐ sible contribution of microglia to the effects of both treatments was also assessed. Microglia are also implicated in the establishment of CIPN [30] and play a crucial role in theFinally, becauseofthe development deregulation emotional in calcium disorders homeostasis accompanying andpain chronic in the antioxidant [31]. re‐ The possible sponse elements induced by PTX provoke apoptotic responses [32], the BAX contribution of microglia to the effects of both treatments was also assessed. expression in the PFC and Finally, DRG the because of PTX‐injected deregulationmice treatedhomeostasis in calcium with CoPP or GYY4137 and was also ana‐ in the antioxidant re- lyzed. sponse elements induced by PTX provoke apoptotic responses [32], the BAX expression in the PFC and DRG of PTX-injected mice treated with CoPP or GYY4137 was also analyzed. 2. Materials and Methods 2. Materials 2.1. Animals and Methods 2.1. Animals Male 5‐6‐week‐old C57BL/6 mice (21–25 g), acquired from Envigo Laboratories (Bar‐ Male celona, 5-6-week-old Spain), were usedC57BL/6 mice (21–25 for the experiments. g), were They acquired kept from withinEnvigo Laboratories a plastic cage under (Barcelona, Spain), were used for the experiments. They were kept controlled environmental conditions (a temperature of 22 °C, a humidity of 66%, within a plastic andcage a 12 under controlled environmental conditions ◦ h dark/light cycle), and an ad libitum food(aand temperature of 22 was water supply C, aprovided. humidity Aof 7‐day 66%, and ac‐ aclimatation 12 h dark/light periodcycle), to theand an ad conditions housing libitum food andplace took waterbefore supply was provided. starting A 7-day the experiments, acclimatation period to the which were conducted housing between 9:00conditions a.m. and took placeAnimals 5:00 p.m. before starting the experiments, were executed in strict which accordance with the guidelines of the European Commission’s directive (2010/63/EC)strict were conducted between 9:00 a.m. and 5:00 p.m. Animals were executed in and accordance the Spanishwith Law the (RDguidelines of the European 53/2013) regulating animalCommission’s research, and directive (2010/63/EC) the procedure was ap‐ and the by proved Spanish Law the local (RD 53/2013) Committee regulating of Animal Use andanimal Care research, and the procedure of the Autonomous University wasof approved by the local Committee of Animal Use and Care of the Autonomous Barcelona (ethical code is 9863). All efforts were made to minimize both the number of University of Barcelona animals used(ethical and theircode is 9863). All efforts were made to minimize both the number of suffering. animals used and their suffering. 2.2. PTX‐Derived Peripheral Neuropathy Induction 2.2. PTX-Derived Peripheral Neuropathy Induction Painful neuropathy was induced with PTX (Tocris Bioscience, Bristol, UK) intraperi‐ Painful neuropathy was induced with PTX (Tocris Bioscience, Bristol, UK) intraperi- toneally (i.p.) injected at 2 mg/kg, every other day, for four consecutive days (Figure 1), as toneally (i.p.) injected at 2 mg/kg, every other day, for four consecutive days (Figure 1), per a previous study [24]. PTX was dissolved in Cremophor EL (Sigma‐Aldrich, St. Louis, as per a previous study [24]. PTX was dissolved in Cremophor EL (Sigma-Aldrich, St. MO, USA)/ethanol/saline (SS, 0.9% NaCl) in a mixture of 1:1:18. The control group re‐ Louis, MO, USA)/ethanol/saline (SS, 0.9% NaCl) in a mixture of 1:1:18. The control group ceived an equal volume of vehicle solution in the same schedule. received an equal volume of vehicle solution in the same schedule. Figure 1. Schematic illustration of the design of the experiment, performed to determine whether the Figure 1. administration repetitive Schematic illustration of CoPPof(5the designorof mg/kg) the experiment, GYY4137 performed (35 mg/kg) to determine intraperitoneally whether injected over the repetitive administration of CoPP (5 mg/kg) or GYY4137 (35 mg/kg) intraperitoneally injected 5 and 4 consecutive days, respectively, can reverse the nociceptive responses caused by PTX and the over 5 andemotional associated 4 consecutive days, respectively, disorders. can reverse the nociceptive responses caused by PTX PTX: paclitaxel. and the associated emotional disorders. PTX: paclitaxel. 2.3. Nociceptive Tests 2.3. Nociceptive MechanicalTests allodynia was evaluated by measuring the hind paw withdrawal response Mechanical after the stimulationallodynia with the was evaluated von Frey by measuring (VF) filaments the bending of different hind paw withdrawal forces (0.008–3.5 re‐ g). sponse after Animals werethe stimulation placed with the von inside individual Frey (VF)cylinders methacrylate filaments (20ofcm high × bending different forces 9 cm in diame- ter; Servei Estació, (0.008–3.5 Barcelona, g). Animals Spain) with were placed a grid inside bottommethacrylate individual through which the filaments cylinders (North (20 cm high Coast Medical, Inc., San Jose, CA, USA) were perpendicularly applied in the central area of the hind paws when mice were resting by using the up–down paradigm. The VF hairs were pressed for 2–3 s, starting with a filament of 0.4 g, and the stiffness of the following hair to be used was decided based on the elicited response. The threshold of the response was Antioxidants 2022, 11, 122 4 of 19 calculated using an Excel program (Microsoft Iberia SRL, Barcelona, Spain) that included curve fitting of the data. A fast withdrawal, licking, or shaking of the paw was registered as a positive response. Cold allodynia was assessed by using a cold plate (CP) analgesiometer (Ugo Basile, Italy). The plate temperature was set at 4 ± 0.5 ◦ C, and the number of elevations for each hind paw was registered for 5 min. In all tests, animals were habituated to the environment for 1 h before the experiment. Both ipsilateral and contralateral paws were tested. 2.4. Emotional Behavior Tests The anxiety-like behavior was assessed by utilizing the elevated plus maze (EPM) [33] and the open file (OF) tests [34]. The EPM apparatus used in this test had 4 arms, each of them 5 cm wide and 35 cm long. Two of them were closed by 15 cm high walls, while the other two were open. The maze was suspended 45 cm above the floor. Mice were placed in the center of the structure, always facing the same open arm, and allowed to explore for 5 min. Their movements were recorded by a digital camera. We registered the number of entries into the open and closed arms, and the percentage of time spent in the open arms. In the OF test, animals were placed in the center of a 44 cm × 44 cm box enclosed by grey walls that were 30 cm high. They were allowed to freely explore this space for 5 min, and their behavior during this time was recorded by a digital camera. The number of squares crossed and entries into the central area and the amount of time spent in this central area were considered. The evaluation of the depressive-like behaviors was performed by using the tail suspension test (TST) and the forced swimming test (FST), by which the duration of immobility of the animals was quantified according to the methods described in [35,36]. In the TST, animals isolated acoustically and visually were suspended at 35 cm from the floor by applying adhesive tape to the tip of the tail and attaching it to an elevated surface. Their movements were recorded for 8 min with a digital camera. The first 2 min were considered habituation time and were not analyzed. The amount of time spent in total immobility during the following 6 min was registered. In the FST, animals were placed in methacrylate cylinders (25 cm high × 10 cm in diameter; Servei Estació, Barcelona, Spain) filled with tempered water (24 ± 2 ◦ C) up to a 10 cm depth. Mice were then carefully placed in the water and left in this environment for 6 min. Their activity was recorded by a digital camera. The first 2 min were considered habituation time and were not analyzed. The amount of time spent in immobility during the following 4 min was registered. In both tests, mice were considered immobile when they remained completely still. 2.5. Western Blot Analysis Mice were euthanized by cervical dislocation at day 21 post-injection (PTX or vehicle). Tissues from the PFC and DRG were immediately bilaterally extracted, frozen in dry ice, and stored at −80 ◦ C until use. Cell disruption was done in an ice-cold lysis RIPA buffer (Sigma-Aldrich) plus a 0.5% protease inhibitor cocktail (Sigma-Aldrich) and a 1% phosphatase inhibitor cocktail (Sigma-Aldrich). A two-step sonication process (20 kHz, 10 s cooldown in ice) was used, leaving the samples at 4 ◦ C for 1 h in the lysis buffer between the steps. Samples were then centrifuged for 20 min at 9500 rpm and at 4 ◦ C. The supernatant was subsequently collected, and its protein concentration was quantified through a BCA assay by a plate reader (BioTek Synergy HT, Winooski, VT, USA) and stored at −80 ◦ C until use. For the electrophoresis, 60 µg of total protein were mixed with a 4x Laemmli loading buffer and loaded into 4% stacking/12% separating sodium dodecyl sulfate polyacrylamide gels. Thereafter, proteins were transferred onto a polyvinylidene fluoride membrane by electrophoresis (120 min) and blocked for 75 min with one of the following solutions: phosphate-buffered saline (PBS) + 5% nonfat dry milk, PBS with Tween Antioxidants 2022, 11, 122 5 of 19 20 (PBST) + 5% bovine serum albumin (BSA), or Tris-buffered saline with Tween 20 (TBST) + 5% nonfat dry milk or 5% BSA. Membranes were then incubated overnight at 4 ◦ C with rabbit primary antibodies against the following: p-P38 (1:200), P38 (1:250), p-ERK 1/2 (1:250), ERK 1/2 (1:250), p-Akt (1:200), Akt (1:200), and BAX (1:150), which were obtained from Cell Signaling Technology (Danvers, MA, United States); 4-HNE (1:150), MDA (1:150), PI3K (1:150), and HO-1 (1:150), which were bought at Abcam (Cambridge, United Kingdom); CD11b/c (1:200) and SOD- 1 (1:150), which were acquired from Novus Biologic (Littleton, CO, USA); and NQO1 (1:250) and β-actin (1:5000), which were obtained from Merck (Billerica, MA, USA). β-actin was used as the loading control. Afterwards, blots were incubated at room temperature for 1 h with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (GE Healthcare, Little Chalfont, United Kingdom). Finally, chemiluminescence reagents (ECL kit; GE Healthcare, Little Chalfont, United Kingdom) were added, and photon emission was scanned using the Chemidoc MP imaging system (Bio Rad, Hercules, CA, USA). Band density was determined and compared with the Image-J software (version 1.34s, National Institutes of Health, Bethesda, MD, USA). 2.6. Experimental Procedures We evaluated the analgesic, anxiolytic, and/or antidepressant properties of CoPP and GYY4137 in PTX-injected mice. To this end, mice were injected daily for five consecutive days with 5 mg/kg CoPP or vehicle, at days 17, 18, 19, 20, and 21 after PTX injection. For GYY4137, mice received four daily injections of 35 mg/kg of this drug or vehicle at days 18, 19, 20, and 21 after PTX injection, as depicted in Figure 1. For both treatments, nociceptive tests (VF and CP) were carried out on the same days of drug injection, while the emotional behavior tests (EPM, OF, TST, and FST) were performed at the end of treatment, on day 21 after PTX injection. For each drug, nociceptive and emotional behaviors were tested in different groups of mice. All animals were sacrificed at day 21 after PTX injection (n = 6–8 animals per group). All these experiments were performed by experimenters blinded to the experimental conditions and the animals receiving the experimental treatment or vehicle were injected and tested at the same day under the same conditions. 2.7. Drugs CoPP, obtained from Frontier Scientific (Livchem GmbH & Co., Frankfurt, Germany), dissolved in dimethyl sulfoxide (1% in SS) was i.p. administered at 5 mg/kg daily for five consecutive days, 3 h before testing, in accordance with [14]. GYY4137, acquired from Sigma-Aldrich (St. Louis, MO, USA), dissolved in SS was i.p. administered at 35 mg/kg daily for four consecutive days, 1 h before testing, in accordance with [20]. Both compounds were injected in a final volume of 10 mL/kg. All drugs were freshly prepared before use. For each group treated with a drug, the respective control group received the same volume of the corresponding vehicle. 2.8. Statistical Analyses The statistical studies were performed with Prism 8.0 (Graphpad, La Jolla, CA, USA). The results are expressed as the mean values ± standard error of the mean (SEM). The two-way repeated measures ANOVA, with treatment and time as the variation factors, the one-way ANOVA, and the Student–Newman–Keuls (SNK) test were used to assess the effects of CoPP and GYY4137 administration on PTX-induced nociception. The effects of both treatments on the emotional behaviors were analyzed by using the one-way ANOVA with treatment as the variation factor, followed by the corresponding SNK post-hoc test. The differences in protein expression were also evaluated using the one-way ANOVA followed by the SNK test. A value of p < 0.05 was considered significant. the effects of CoPP and GYY4137 administration on PTX‐induced nociception. The effects of both treatments on the emotional behaviors were analyzed by using the one‐way ANOVA with treatment as the variation factor, followed by the corresponding SNK post‐ hoc test. The differences in protein expression were also evaluated using the one‐way ANOVA followed by the SNK test. A value of p < 0.05 was considered significant. Antioxidants 2022, 11, 122 6 of 19 3. Results 3.1. Treatment with CoPP Inhibited the Mechanical and Cold Allodynia Caused by PTX 3. Results In both paws, two‐way repeated‐measures ANOVA showed the significant effects of 3.1. Treatment with CoPP Inhibited the Mechanical and Cold Allodynia Caused by PTX the treatment, time, and their interaction (p < 0.001) for the mechanical and thermal allo‐ dynia.In both paws, two-way repeated-measures ANOVA showed the significant effects of the treatment, time, and Concerning thetheir interaction mechanical (p < 0.001) allodynia, thefor the mechanical administration of and thermal CoPP allodynia.re‐ progressively Concerning the mechanical allodynia, the administration of CoPP progressively duced the mechanical allodynia induced by PTX from days 1 to 3 of treatment and com‐ re- duced the mechanical allodynia induced by PTX from days 1 to 3 of treatment and com- pletely inhibited it at day 5 of treatment in both hind paws (p < 0.001; one way ANOVA pletely inhibited it at day 5 of treatment in both hind paws (p < 0.001; one way ANOVA and SNK test; Figure 2A,B). Similar results were obtained for cold allodynia, as CoPP and SNK test; Figure 2A,B). Similar results were obtained for cold allodynia, as CoPP treatment significantly decreased the number of times that mice elevated both hind paws treatment significantly decreased the number of times that mice elevated both hind paws in the cold plate from days 1 to 3 of treatment, until complete reversal was achieved at in the cold plate from days 1 to 3 of treatment, until complete reversal was achieved at day day 5 of treatment (p < 0.014, one‐way ANOVA followed by SNK tests; Figure 2C,D). In 5 of treatment (p < 0.014, one-way ANOVA followed by SNK tests; Figure 2C,D). In both both tests and paws, CoPP did not produce any effect in the vehicle‐injected mice. tests and paws, CoPP did not produce any effect in the vehicle-injected mice. Figure 2. Figure Effects of 2. Effects of CoPP CoPP onon PTX‐induced PTX-induced mechanical mechanical andand cold cold allodynia. allodynia. Mechanical Mechanical(A: (A:left leftpaws; paws; right paws) B: right B: paws) and and cold cold (C: (C: left left paws; paws; D: right paws) D: right paws) antiallodynic antiallodynic effects effects produced produced byby the the repeated repeated administration of 5 mg/kg administration mg/kgof ofCoPP CoPPororvehicle vehiclefor forfive five days days from from days days 1717 to to 21 21 after after PTX PTX injection. injection. In In all all panels, panels, for for each each day day and and treatment treatment evaluated, evaluated, ** indicates indicates significant significant differences differences vs. vs. the the vehicle– vehicle– vehicle‐treated vehicle-treated animals and ++ vs. vs. PTX–vehicle‐treated PTX–vehicle-treated animals animals (p (p <<0.05, 0.05,one‐way one-wayANOVA ANOVA followed followed by by the the SNK SNK test). test). Results Results are are shown shown asas mean mean values values ±±SEM;SEM;nn==66animals animalsperperexperimental experimentalgroup. group. 3.2. CoPP Treatment Prevented the Emotional Behavioral Alterations Associated with PIPN Regarding the anxiety-like behaviors, CoPP reversed both the decreased number of entries into the EPM’s open arms (p < 0.021, one-way ANOVA followed by the SNK test as compared with the vehicle–vehicle-treated mice; Figure 3A) and the decreased time spent in the central area of the OF in PTX-injected animals (p < 0.035, one-way ANOVA followed by the SNK test as compared with the vehicle–vehicle-treated mice; Figure 3F). as compared with the vehicle–vehicle‐treated mice; Figure 3A) and the decreased spent in the central area of the OF in PTX‐injected animals (p < 0.035, one‐way AN followed by the SNK test as compared with the vehicle–vehicle‐treated mice; Figur The percentage of time spent in the open arms (Figure 3B) and the number of e into the closed arms of the EPM (Figure 3C), as well as the number of entries in the c Antioxidants 2022, 11, 122 7 of 19 area (Figure 3E) and the number of squares crossed in the OF (Figure 3G) did not significant differences among the groups. Figure 3. Effects of treatment with CoPP on the emotional behavioral disorders associated with PTX-induced neuropathic pain. Effects of the repetitive administration of 5 mg/kg of CoPP or vehicle from days 17 to 21 after PTX or vehicle (VEHI) injection on the anxiety- (EPM and OF tests) and depressive-like behaviors (TST and FST) associated with CIPN. In the EPM test, the number of times that animals entered the open arms (A), the percentage of total time spent in open arms (B), and the number of entrances into the closed arms (C) are represented. In the OF test, the number of entries into the central area (E), the percentage of time spent within the central area (F), and the total number of squares crossed (G) are shown. Figures (D,H) display the time the animals spent in immobility (s) in the TST (D) and FST (H). In all panels, * indicates significant differences vs. the vehicle–vehicle-treated animals; &, vs. the vehicle–CoPP-treated animals; and $, vs. the animals treated with PTX plus CoPP (p < 0.05, one-way ANOVA followed by the SNK test). Data are expressed as mean values ± SEM; n = 8 animals per experimental group. induced neuropathic pain. Effects of the repetitive administration of 5 mg/kg of CoPP or vehicle from days 17 to 21 after PTX or vehicle (VEHI) injection on the anxiety‐ (EPM and OF tests) and depressive‐like behaviors (TST and FST) associated with CIPN. In the EPM test, the number of times that animals entered the open arms (A), the percentage of total time spent in open arms (B), and the number of entrances into the closed arms (C) are represented. In the OF test, the number of entries Antioxidants 2022, 11, 122 into the central area (E), the percentage of time spent within the central area (F), and the total num‐ 8 of 19 ber of squares crossed (G) are shown. Figures D and H display the time the animals spent in immo‐ bility (s) in the TST (D) and FST (H). In all panels, * indicates significant differences vs. the vehicle– vehicle‐treated animals; &, vs. the vehicle–CoPP‐treated animals; and $, vs. the animals treated with PTX plusTheCoPP percentage (p < 0.05,ofone‐way time spent in the ANOVA open arms followed (Figure by the 3B) Data SNK test). and the number ofasentries are expressed mean into the closed arms of the EPM (Figure 3C), values ± SEM; n = 8 animals per experimental group. as well as the number of entries in the central area (Figure 3E) and the number of squares crossed in the OF (Figure 3G) did not show significant differences The results among regarding thethe groups. depressive‐like behaviors associated with PTX‐induced The results regarding the depressive-like neuropathic pain also revealed that CoPP normalized behaviors associated the increasedwith PTX-induced immobility neu- time ob‐ ropathic pain also revealed that CoPP normalized the increased immobility served in PTX–vehicle‐treated animals in both the TST (p < 0.001, one‐way ANOVA fol‐ time observed in PTX–vehicle-treated lowed animals by the SNK test; Figure 3D)inand both the(pTST FST (p