antioxidants Article Hydrogen Sulfide Interacting with Cannabinoid 2 Receptors during Sciatic Nerve Injury-Induced Neuropathic Pain Xue Bai 1,2 , Gerard Batallé 1,2 , Ignacio Martínez-Martel 1,2 and Olga Pol 1,2, * 1 Grup de Neurofarmacologia Molecular, Institut d’Investigació Biomèdica Sant Pau (IIB Sant Pau), 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: Hydrogen sulfide (H2 S) donors make opioids more effective in inhibiting nociception during inflammatory and neuropathic pain. We examined whether the analgesic, anxiolytic and/or antidepressant actions of the cannabinoid 2 receptor (CB2R) agonist, JWH-133, might be improved by pretreatment with H2 S donors, DADS and GYY4137 in mice with sciatic nerve injury-provoked neuropathy (CCI). The reversion of the antinociceptive effects of these treatments with the CB2R antagonist, AM630, and the regulatory actions of H2 S in the phosphorylation of NF-κB inhibitor alpha (IKBα) and in the brain-derived neurotrophic factor (BDNF), CB2R, Nrf2 and heme oxygenase 1 (HO-1) levels in prefrontal cortex (PFC), ventral hippocampus (vHIP) and periaqueductal gray matter (PAG), were examined. Data showed that the analgesic effects of JWH-133, systemically and locally administered, were improved by the DADS or GYY4137 pretreatment. The co-treatment of GYY4137 with JWH-133 also stopped anxiodepressive-like activities that concur with neuropathy. Our data likewise showed that both H2 S donors normalized the inflammatory (p-IKBα), neurotrophic (BDNF) variations caused by CCI, increased the expression of CB2R and activated the Nrf2/HO-1 antioxidant pathway in PFC, v-HIP and/or PAG of animals with neuropathic pain. In addition, the blockade of the analgesia produced by high doses of DADS and GYY4137 with AM630 indicated the contribution of the endocannabinoid system in the effects of H2 S during neuropathic pain, thus supporting the positive interaction between H2 S and CB2R. Therefore, this study demonstrates the potential use of Citation: Bai, X.; Batallé, G.; CB2R agonists combined with H2 S donors as a possible treatment for peripheral nerve injury-caused Martínez-Martel, I.; Pol, O. Hydrogen neuropathic pain and the associated emotional disturbances. Sulfide Interacting with Cannabinoid 2 Receptors during Sciatic Nerve Keywords: analgesia; anxiety; BDNF; cannabinoids; cannabinoid 2 receptors; depression; hydrogen Injury-Induced Neuropathic Pain. sulfide; neuropathic pain; oxidative stress Antioxidants 2023, 12, 1179. https:// doi.org/10.3390/antiox12061179 Academic Editor: Rosanna Di Paola 1. Introduction Received: 1 May 2023 Revised: 22 May 2023 Neuropathic pain is a chronic disease with clinical features such as allodynia, hyper- Accepted: 25 May 2023 algesia, paresthesia and spontaneous pain, among others. Patients with peripheral nerve Published: 30 May 2023 injury causing neuropathic pain experience high rates of comorbidities, such as depression and anxiety, which can persist for a long time and have a negative impact on patients’ lives [1,2]. Most pharmacological treatments used for the management of peripheral neuropathic Copyright: © 2023 by the authors. pain, including the use of opioids, pregabalin and gabapentin, are ineffective and have Licensee MDPI, Basel, Switzerland. serious side effects that limited their therapeutic use [3–5]. Therefore, there is a need to This article is an open access article identify new therapies for the management of neuropathic pain and its accompanying distributed under the terms and affective disorders, with few side effects. conditions of the Creative Commons The therapeutic potential of the cannabinoid system in different neurological diseases Attribution (CC BY) license (https:// has been widely demonstrated [6]. There are two types of cannabinoid receptors: type 1 creativecommons.org/licenses/by/ (CB1R) and 2 (CB2R) [7,8]. CB1Rs are mostly found in the central nervous system (CNS) [9], 4.0/). Antioxidants 2023, 12, 1179. https://doi.org/10.3390/antiox12061179 https://www.mdpi.com/journal/antioxidants Antioxidants 2023, 12, 1179 2 of 19 and CB2Rs are found in the microglia and immune cells, although they were also detected in the spinal cord and dorsal root ganglia [10]. More recent research further revealed that CB2Rs were also distributed in the hippocampus (HIP), cortex and cerebellum [11]. Interestingly, since CB2R agonists do not cause psychiatric disorders like CB1R ag- onists, their therapeutic role in pain has been extensively investigated [12–14]. Various studies showed that selective agonists of CB2R protected neurons from apoptosis [15] and reduced neuroinflammation after spinal cord injury [16]. Moreover, although some findings reported that CB2R agonists could induce anxiety-like behaviors and that the overexpres- sion of CB2R produced depressive-like behaviors in mice [17,18], the antidepressant and anxiolytic effects of CB2R agonists in animals suffering diabetic neuropathy, inflammatory or osteoarthritis pain have also been proven [12,13,19]. Nevertheless, the possible effects of CB2R agonists in mood disorders associated with the chronic constriction of sciatic nerve (CCI)-provoked neuropathic pain have not been fully investigated. Previous research demonstrated that the repetitive administration of two hydro- gen sulfide (H2 S) donors, DADS, diallyl disulfide and/or GYY4137, morpholin-4-ium 4-methoxyphenyl (morpholino) phosphinodithioate dichloromethane complex, alleviated neuropathic pain provoked by CCI [20], as well as of neuropathy caused by chemother- apy and osteoarthritic pain besides their accompanying anxiety- and depressive-like behaviors [21,22]. On the other hand, these two H2 S donors augmented the analgesic effects of opioids and those produced by carbon monoxide (CO) donors or a heme oxyge- nase 1 (HO-1) inducer in mice with neuropathic and joint pain [23,24]. However, whether treatment with DADS and/or GYY4137 could potentiate the analgesic actions of CB2R agonists, its effects on the emotional disorders and/or regulate the brain levels of CB2R in animals with CCI-induced neuropathic pain remains unknown, being the main objectives of this study. Oxidative stress and central sensitization are inextricably linked to the development of neuropathic pain and its accompanying mood disorders [25,26]. These associated emo- tional disorders are also linked with neuroinflammation and high levels of proinflammatory targets such as the nuclear factor κB (NF-κB), of which the activation induces the phospho- rylation of kappa-B inhibitor (p-IKBα) in the CNS and PNS [27,28]. Moreover, exogenous melatonin and irisin alleviated neuropathic pain-associated affective disorders and ethanol- induced behavioral deficits by suppressing the NF-κB activation pathway [29,30]. Brain-derived neurotrophic factor (BDNF) is important for controlling the neuroplastic- ity and neurogenesis in the CNS (cortex, HIP and subventricular zone) [31] and peripheral nervous system (PNS), for example, in the dorsal root ganglia [32]. Thus, previous studies have shown that stress, injury or inflammation could induce the upregulation of BDNF in the PNS [33], and the down- and upregulation in specific brain areas, such as the HIP and periaqueductal gray matter (PAG), respectively [34,35]. Nonetheless, the effects of H2 S in the brain expression of BDNF of animals with affective-like behaviors linked with chronic pain are not fully known. Hence, in male mice with anxiodepressive-like behaviors accompanying CCI-incited neuropathic pain, we examined: (1) the result of the combined administration of DADS and GYY4137 with JWH-133 in modulating the nociceptive responses; (2) the reversion of the analgesics properties of JWH-133, DADS and GYY4137 with AM630 (a CB2R antagonist); (3) the anxiolytic and/or antidepressant actions of JWH-133 alone and co-administered with GYY4137; (4) the effects of H2 S in the expression of p-IKBα, BDNF, CB2R, NRF2 and HO-1 in the PFC, ventral HIP (vHIP) and PAG, areas deeply involved in the control of nociception and emotions [36–39]. 2. Materials and Method 2.1. Animals We used male C57BL/6 mice from Envigo Laboratories (Barcelona, Spain) to carry out the experiments. The mice (age, 5–6 weeks; weight, 21–26 g) were kept under stan- dard conditions of light/dark (12/12 h), temperature (22 ± 1 ◦ C), and relative humidity Antioxidants 2023, 12, 1179 3 of 19 (55 ± 10%), with free access to water and food. The experiments were performed be- tween 9:00 a.m. and 5:00 p.m., after a week of acclimatization and according to the ethical guidelines of the European Commission’s directive (2010/63/EC) and the Spanish Law (RD 53/2013), and were approved by the local Committee of Animal Use and Care of the Autonomous University of Barcelona (ethical code 9863). Maximal efforts to reduce the number and suffering of animals were accomplished. 2.2. Neuropathic Pain Induction CCI was utilized for inducing neuropathic pain. Under isoflurane anesthesia condi- tions (3% induction, 2.5% maintenance), after the biceps femoris and the gluteus superfi- cialis were separated by blunt dissection, three ligatures (4/0 silk) across the sciatic nerve were performed. The same process, excluding nerve ligation, was employed in the control mice (sham). 2.3. Mechanical Allodynia Mechanical allodynia was assessed by evaluating the hind paw withdrawal reaction to the von Frey filament stimulation, from 0.4 to 3.5 g (North Coast Medical, Inc., San Jose, CA, USA). The mice were positioned in Plexiglas tubes (20 cm high × 9 cm diameter) with a wire grid bottom. Using the up–down paradigm [40], a filament of 0.4 g was applied first, and depending on the animal’s reaction, the strength of the next filament was increased or reduced. The threshold of the response was estimated applying an Excel program (Microsoft Iberia SRL, Barcelona, Spain) that incorporated curve-fitting of the data. 2.4. Thermal Hyperalgesia Thermal hyperalgesia was evaluated by measuring the paw withdrawal latency in response to a radiant heat using the plantar test (Ugo Basile, Varese, Italy) [41]. The mice were arranged in Plexiglas tubes (20 cm high × 9 cm diameter) and positioned on a glass surface. The heat source sited on the plantar surface of the hind paws was activated with a light beam intensity until the paw withdrawal. The paw withdrawal latencies were obtained from the mean of the three separate trials. 2.5. Cold Allodynia Cold allodynia was determined by recording the amount of elevation of each hind paw of the mice exposed to the cold plate (4 ± 0.5 ◦ C) (Ugo Basile, Italy) for 5 min. In all paradigms, both the ipsilateral and contralateral paws were assessed. 2.6. Anxiety-like Behaviors We used the elevated plus maze (EPM), an X-shaped structure with 2 open and 2 closed arms, to evaluate the anxiety-like behaviors. All arms were 5 cm wide and 35 cm long, and the closed ones had 15 cm high walls. At the beginning of the test, the mouse was placed in the central area of the maze, always looking at the same open arm, and its behavior was recorded with a digital camera for 5 min. Both the number of entries into the open and closed arms were recorded, as well as the percentage of time spent in the open arms. 2.7. Depressive-like Behaviors The tail suspension test (TST) and the forced swimming test (FST) were used to evaluate depressive-like behaviors. In the TST, an adhesive tape, attached 1 cm from the end of the tail, was used to suspend the animals from a firm structure placed 35 cm from the ground. The animals were recorded for 8 min and their immobility time during the last 6 min was quantified. In the FST, the mice were placed individually inside a plexiglass tube (25 cm high × 10 cm diameter) containing water at 24 ± 1 ◦ C, up to a height of 10 cm. The mice were recorded for 6 min and their immobility time was measured for the last 4 min. Antioxidants 2023, 12, 1179 4 of 19 All these tests were performed by researchers blinded to the experimental conditions and the animals, between 9 and 10 weeks old, were familiarized with the testing room for 1 h before the start of the experiment. 2.8. Western Blot The animals were euthanized by a cervical dislocation at 28 days after surgery (CCI or sham). The brain, obtained by performing a craniotomy, was dissected using a brain matrix. The PFC, HIP and PAG areas were identified following the Paxinos and Franklin’s stereotaxic coordinates [42]. Additionally, vHIP was separated from the dorsal HIP using the methodology described by [43]. The specific areas were extracted by cutting with a clean scalpel or a punch, pushing the metal gently, but firmly, into the tissue. Rocking it back and forth allowed for making the cut and harvesting the region of interest, which was then placed into labeled pre-chilled Eppendorf tubes and stored at –80 ◦ C until further use. The samples from two animals were pooled into one experimental sample to obtain sufficient protein levels to perform Western blot analysis. The expression of p-IKBα, BDNF, CB2R, NRF2 and HO-1 were evaluated. The tissues were sonicated in a cold lysis buffer, the RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA). After 1 h of dissolution (4 ◦ C), the crude homogenate was sonicated (10 s) and centrifuged at 700× g (20 min) at 4 ◦ C. After that, 60 µg of the total protein combined with 4x Laemmli loading buffer was loaded onto a 4% stacking/12% separating sodium dodecyl sulfate polyacrylamide gel. The electrophoretic transfer of the proteins onto polyvinylidene fluoride membranes for 120 min was successfully performed. The membranes were blocked with phosphate-buffered saline (PBS; Sigma-Aldrich, MO, USA) containing 5% nonfat dry milk, Tris-buffered saline with Tween 20 containing 5% bovine serum albumin (BSA; Sigma-Aldrich, MO, USA) or 5% nonfat dry milk and PBS with Tween 20 containing 5% BSA (75 min). The membranes were incubated with rabbit primary antibodies anti-p-IKBα (1:150), BDNF (1:150) and NRF2 (1:150) from Abcam (Cambridge, UK); IKBα (1:150) from Cell Signaling Technology (Danvers, MA, USA); HO-1 (1:100) from Enzo Life Sciences (New York, NY, USA); CB2R (1:150) from Cayman Chemical Company (Ann Arbor, MI, USA); and anti-glyceraldehyde-3- phosphate dehydrogenase (GAPDH, 1:5000) from Merck (Billerica, MA, USA) as a loading control, at 4 ◦ C overnight. The blots were incubated at room temperature for 1 h with secondary polyclonal antibodies conjugated to horseradish peroxidase (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The ECL kit (GE, Healthcare, Little Chalfont, Buckinghamshire, UK) was used to detect the proteins, and the Image-J program (National Institutes of Health, Bethesda, MD, USA) for densitometric analysis. 2.9. Experiments Our objective is to find a treatment that inhibits not only the allodynia and hyperalge- sia provoked by nerve injury, but also the anxiodepressive-like behaviors accompanying neuropathic pain. Therefore, considering that nerve injury leads to sensorial hypersensi- tivity from the onset of injury, whereas the associated anxiodepressive-like behaviors are only obvious after several weeks (4–6 weeks) of injury [44], all these experiments were performed at 28 days after CCI. Firstly, baseline responses were established using the following test sequence: von Frey filaments, plantar and cold plate tests. After baseline measurements, neuropathic pain was induced by CCI, and the animals were tested again at day 28 after surgery using the same sequence as previously. To examine the impact of H2 S in the pain reliever actions of JWH-133, a CB2R agonist [6] in neuropathic pain, the antinociceptive effects of the systemic co-administration of small doses of DADS (3.5 mg/kg) or GYY4137 (0.7 mg/kg) with JWH-133, 2 mg/kg, injected intraperitoneally and 5 µg subplantarly, were assessed (n = 8 animals/group). The impact of the intraperitoneal co-injection of 0.7 mg/kg GYY4137 with 2 mg/kg JWH-133 in the anxiodepressive-like behaviors accompanying neuropathic pain were also determined in parallel within different groups of animals (n = 8 animals per group). These Antioxidants 2023, 12, 1179 5 of 19 experiments were performed at day 28 after CCI induction. The doses of JWH-133 were chosen from the dose-response curves of this study and those of the DADS and GYY4137 from preceding works [23]. In all cases, the animals were treated with DADS and GYY4137 15 min prior to JWH-133 injection, and the tests were accomplished after 45 min. The reversal of the painkilling effects produced by 50 mg/kg or 150 µg JWH-133, 30 mg/kg DADS or 24 mg/kg GYY4137 with 3 mg/kg or 90 µg AM630, administered intraperitoneally or subplantarly, were also tested. The JWH-133 and H2 S donors were administered 15 min prior to AM630, and the tests were performed 45 min afterward (n = 8 mice/group). The doses of DADS, GYY4137 and AM630 were chosen in accordance with previous findings [23]. Lastly, using the samples of animals that have undergone functional tests, the effects of DADS or GYY4137 on the protein levels of p-IKBα, BDNF, CB2R, NRF2 and HO-1 in the PFC, v-HIP and PAG were evaluated. The controls were sham-operated mice administered with vehicle. In these experiments, 4 samples/group were evaluated based on the sample size analysis performed with the data obtained in a pilot experiment, accepting a risk of α = 0.05 and β = 0.2 in a two-tailed test. 2.10. Drugs Both GYY4137 and DADS from Sigma-Aldrich (St. Louis, MO, USA) were dissolved in 0.9% saline, and given intraperitoneally (10 mL/kg) 1 h before testing [21]. JWH-133 (Sigma-Aldrich, St. Louis, MO, USA) dissolved in a saline solution with 1% Tween 80 (Sigma-Aldrich, St. Louis, MO, USA), and AM630 (LabClinics (Barcelona, Spain) dissolved in a mixed solution containing 90% saline, 5% DMSO and 5% Tween 80, were both injected in a volume of 10 mL/kg (intraperitoneal) or 30 µL (subplantar) [45,46]. All drugs were freshly prepared, and for each group treated with a drug, the respective control group received the same volume of the corresponding vehicle. 2.11. Statistical Analyses We used the Prism 8.0 (Graphpad, La Jolla, CA, USA) and the SPSS (version 28, IBM, Madrid, Spain) programs for the statistical analysis. The normal distribution of the data was determined using the Kolmogorov–Smirnov test, and the sample size was calculated using GRANMO program. A two-way analysis of variance (ANOVA) was used to evaluate the antinociceptive actions of JWH-133 and the anxiolytic and antidepressant effects of JWH-133 alone and with GYY4137. A one-way ANOVA followed by a Tukey test was employed to see the effects of DADS and GYY4137 alone or with JWH-133 or AM630, as well as the impact of H2 S donors on protein levels. The results are presented as the mean values ± standard error of the mean (SEM). A p value < 0.05 was considered significant. 3. Results 3.1. The Antinociceptive Effects of JWH-133 during Neuropathic Pain Our results showed that JWH-133, given intraperitoneally (1–50 mg/kg) and subplan- tarly (5–150 µg) dose-dependently inhibited the allodynia and hyperalgesia generated by CCI (Figure 1). For all tests, the two-way ANOVA demonstrated the significant effects of the dose and group, and their interaction (p < 0.001). Nerve injury reduced the threshold of paw withdrawal to a mechanical stimulus in comparison to sham-operated mice treated with the vehicle (p < 0.001, one-way ANOVA) (Figure 1A,D). Mechanical allodynia was progressively reduced with the administration of 1, 2 and 3 mg/kg or 5 and 10 µg of JWH-133, and was totally inhibited with 5, 10, 20 and 50 mg/kg or 30, 50 and 150 µg of this drug, given intraperitoneally or subplantarly (p < 0.001; one-way ANOVA vs. CCI vehicle-treated mice). Nerve injury reduced the threshold of paw withdrawal to a mechanical stimulus in comparison to sham-operated mice treated with the vehicle (p < 0.001, one-way ANOVA) (Figure 1A,D). Mechanical allodynia was progressively reduced with the administration of 1, 2 and 3 mg/kg or 5 and 10 µg of JWH-133, and was totally inhibited with 5, 10, 20 and Antioxidants 2023, 12, 1179 50 mg/kg or 30, 50 and 150 µg of this drug, given intraperitoneally or subplantarly6(p < of 19 0.001; one-way ANOVA vs. CCI vehicle-treated mice). Figure 1. Effects of the intraperitoneal and subplantar administration of JWH-133 on the allodynia Figure 1. Effects ofgenerated and hyperalgesia the intraperitoneal and subplantar by CCI. Effects administration of several doses of JWH-133 ofintraperitoneally JWH-133 on the (A–C) allodynia and and hyperalgesia generated by CCI. Effects of several doses of JWH-133 intraperitoneally (A–C) and subplantarly (D–F) administered on the mechanical allodynia (A,D), thermal hyperalgesia (B,E) and subplantarly (D–F) administered on the mechanical allodynia (A,D), thermal hyperalgesia (B,E) and cold allodynia (C,F) caused by CCI in the ipsilateral paws. The effects of JWH-133 or vehicle (VEHI) cold allodynia (C,F) caused by CCI in the ipsilateral paws. The effects of JWH-133 or vehicle (VEHI) inthe in theipsilateral ipsilateralpaws pawsofofsham-operated sham-operatedanimals animals and and those those of of thethe vehicle vehicle in the in the ipsilateral ipsilateral paws paws of of CCI mice were also represented. For each assay, the symbols denote significant changes CCI mice were also represented. For each assay, the symbols denote significant changes versus, * versus, * SHAM–VEHI, SHAM–VEHI, ++ SHAM–JWH-133 SHAM–JWH-133 andand# #CCI–JWH-133 CCI–JWH-133(p(p<<0.05;0.05;one-way one-wayANOVA ANOVAfollowed followedbyby Tukeytest). Tukey test). Results Resultsare arepresented presented as as the the mean values ±±SEM; mean values SEM;nn==8 8animals/group. animals/group. Nerveinjury Nerve injuryalso alsosignificantly significantlydecreased decreasedthe thethreshold thresholdfor for evoking evoking paw paw withdrawal withdrawal to a thermal stimulus (p < 0.001; one-way ANOVA) (Figure 1B,E). Thermal hyperalgesia to a thermal stimulus (p < 0.001; one-way ANOVA) (Figure 1B,E). Thermal hyperalgesia was gradually reduced with the administration of 1 to 50 mg/kg or 5 to 150 µg of JWH-133 (p < 0.001; one-way ANOVA vs. CCI vehicle-treated mice). The increased paw lift number provoked by the cold thermal stimulation was detected in CCI mice administered with the vehicle (p < 0.001; one-way ANOVA vs. sham-operated mice) (Figure 1C,F). Thermal allodynia was also gradually decreased with the administra- tion of 1, 2, 3, 5 and 10 mg/kg or 5, 10 and 30 µg of JWH-133 and was entirely inhibited with 20 and 50 mg/kg or 50 and 150 µg of this drug injected intraperitoneally or subplantarly (p < 0.001; one-way ANOVA vs. CCI vehicle-treated mice). JWH-133 or vehicle had no effect on the ipsilateral paws of sham-operated mice (Figure 1), nor the contralateral paws of sham-operated or CCI mice. 3.2. The Analgesic Effects of JWH-133 in Animals with Neuropathic Pain Co-Treated with DADS or GYY4137 The antiallodynic and antihyperalgesic activities produced by the systemic injection of 3.5 mg/kg or 0.7 mg/kg of DADS or GYY4137 mixed with JWH-133, intraperitoneally (2 mg/kg) or subplantarly (5 µg), were evaluated (Figure 2). of 3.5 mg/kg or 0.7 mg/kg of DADS or GYY4137 mixed with JWH-133, intraperitoneally (2 mg/kg) or subplantarly (5 µg), were evaluated (Figure 2). The administration of DADS or GYY4137 significantly improved the mechanical (Fig- ure 2A,D) and cold antiallodynic effects (Figure 2C,F), in addition to the thermal antihy- peralgesic effects (Figure 2B,E) made by JWH-133, systemically or locally injected, as re- Antioxidants 2023, 12, 1179 lated with the effects produced by the vehicle, JWH-133, DADS or GYY4137 given alone 7 of 19 (one-way ANOVA, p < 0.001). Figure2.2.The Figure Theanalgesic analgesicactions actionsofofJWH-133 JWH-133co-administered co-administered with with DADS DADS oror GYY4137 GYY4137 inin CCI CCI mice. mice. Effectsofofthe Effects theco-administration co-administrationofof3.5 3.5mg/kg mg/kg of of DADS DADS oror 0.70.7 mg/kg mg/kg of GYY4137 of GYY4137 withwith 2 mg/kg 2 mg/kg or 5or 5 of µg of JWH-133, µgJWH-133, injectedinjected intraperitoneally intraperitoneally or subplantarly, or subplantarly, on the allodynia on the mechanical mechanical allodynia (A,D), ther- mal hyperalgesia (A,D), (B,E) and cold thermal hyperalgesia allodynia (B,E) (C,F) and cold caused(C,F) allodynia by CCI in theby caused ipsilateral CCI in thepaws are displayed. ipsilateral paws are displayed. The actions of these drugs injected alone are also presented. For each assay, the symbols denote significant changes versus * SHAM–VEHI–VEHI, + CCI–VEHI–VEHI, # CCI–VEHI–JWH-133 and & CCI–DADS–VEHI or CCI–GYY4137–VEHI (p < 0.05; a one-way ANOVA, followed by Tukey test). VEHI (vehicle). The results are presented as the mean values ± SEM; n = 8 animals/group. The administration of DADS or GYY4137 significantly improved the mechanical (Figure 2A,D) and cold antiallodynic effects (Figure 2C,F), in addition to the thermal antihyperalgesic effects (Figure 2B,E) made by JWH-133, systemically or locally injected, as related with the effects produced by the vehicle, JWH-133, DADS or GYY4137 given alone (one-way ANOVA, p < 0.001). DADS or GYY4137, administered separately and combined with JWH-133, intraperi- toneally or subplantarly, had no impact on the contralateral paws of CCI- or sham-operated mice, nor on the ipsilateral paws of sham-operated animals. 3.3. Reversion of the Antinociceptive Actions of JWH-133, DADS and GYY4137 with AM630 The intraperitoneal (3 mg/kg) and subplantar (90 µg) administration of the CB2R antagonist AM630 reversed the mechanical (Figure 3A,D) and cold antiallodynic actions (Figure 3C,F), as well as thermal antihyperalgesic activities (Figure 3B,E) produced by JWH- 133 injected intraperitoneally (50 mg/kg) or subplantarly (150 µg), respectively (one-way ANOVA, p < 0.001). 3.3. Reversion of the Antinociceptive Actions of JWH-133, DADS and GYY4137 with AM630 The intraperitoneal (3 mg/kg) and subplantar (90 µg) administration of the CB2R an- tagonist AM630 reversed the mechanical (Figure 3A,D) and cold antiallodynic actions (Figure 3C,F), as well as thermal antihyperalgesic activities (Figure 3B,E) produced by Antioxidants 2023, 12, 1179 JWH-133 injected intraperitoneally (50 mg/kg) or subplantarly (150 µg), respectively (one- 8 of 19 way ANOVA, p < 0.001). Figure 3. Reversal of the antiallodynic and antihyperalgesic effects of JWH-133 with AM630 in CCI Figure 3. Reversal of the antiallodynic and antihyperalgesic effects of JWH-133 with AM630 in CCI mice. mice. Effects Effects of of AM630 AM630 injectedintraperitoneally injected intraperitoneally (3 (3mg/kg) mg/kg)ororsubplantarly subplantarly (90(90 on on µg)µg) the the inhibition inhibition of the mechanical allodynia (A,D), thermal hyperalgesia (B,E) and cold allodynia of the mechanical allodynia (A,D), thermal hyperalgesia (B,E) and cold allodynia (C,F) produced(C,F) produced by by the intraperitoneal (50 mg/kg) or subplantar (150 µg) administration of JWH-133 in the intraperitoneal (50 mg/kg) or subplantar (150 µg) administration of JWH-133 in the ipsilateral the ipsilateral pawspaws of CCI of CCI mice. mice. For For eachassay each assayand andadministration administration route, route,the thesymbols symbols denote significant denote significantchanges changes versus versus * *SHAM–VEHI–VEHI, SHAM–VEHI–VEHI, + +CCI–VEHI–VEHI, CCI–VEHI–VEHI, # CCI–VEHI–AM630 # CCI–VEHI–AM630 and & CCI–JWH-133–AM630 and & CCI–JWH-133– (p < 0.05; AM630 (p