antioxidants Article The Inhibitory Effects of Slow-Releasing Hydrogen Sulfide Donors in the Mechanical Allodynia, Grip Strength Deficits, and Depressive-Like Behaviors Associated with Chronic Osteoarthritis Pain Gerard Batallé 1,2,† , Laura Cabarga 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.batalle@e-campus.uab.cat (G.B.); laura.cabarga@e-campus.uab.cat (L.C.) 2 Institut de Neurociències, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain * Correspondence: opol@santpau.es; Tel.: +34-619-757-054 † Both authors contributed equally to this work.  Received: 5 December 2019; Accepted: 26 December 2019; Published: 29 December 2019  Abstract: Osteoarthritis and its associated comorbidities are important clinical problems that have a negative impact on the quality of life, and its treatment remains unresolved. We investigated whether the systemic administration of slow-releasing hydrogen sulfide (H2 S) donors, allyl isothiocyanate (A-ITC) and phenyl isothiocyanate (P-ITC), alleviates chronic osteoarthritis pain and the associated emotional disorders. In C57BL/6 female mice with osteoarthritis pain induced by the intra-articular injection of monosodium iodoacetate, we evaluated the effects of repeated administration of A-ITC and P-ITC on the (i) mechanical allodynia and grip strength deficits; (ii) emotional conducts; and (iii) glial activity and expression of inducible nitric oxide synthase (NOS2), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and antioxidant enzymes (heme oxygenase 1, NAD(P)H:quinone oxidoreductase-1, glutathione S-transferase mu 1 and alpha 1) in the hippocampus. The administration of A-ITC and P-ITC inhibited the mechanical allodynia, the grip strength deficits, and the depressive-like behaviors accompanying osteoarthritis. Both treatments inhibited microglial activation, normalized the upregulation of NOS2 and PI3K/p-Akt, and maintained high levels of antioxidant/detoxificant enzymes in the hippocampus. Data suggest that treatment with low doses of slow-releasing H2 S donors might be an interesting strategy for the treatment of nociception, functional disability, and emotional disorders associated with osteoarthritis pain. Keywords: analgesia; anxiety; depression; grip strength; hydrogen sulfide donors; inflammation; microglia; osteoarthritis pain; oxidative stress 1. Introduction Osteoarthritis is one of the most prevalent diseases affecting more than 100 million people worldwide. Osteoarthritis is a chronic degenerative joint disorder characterized by the destruction of articular cartilage causing subchondral bone alterations, inflammation, and intense pain [1]. Chronic osteoarthritis pain, which is characterized by persistent pain with inflammatory and neuropathic components, causes a physical inability to perform daily tasks, difficulty walking, etc. [2]. It is also accompanied by affective disorders such as anxiety and depression, which further contribute to the impairment in the quality of life of patients and exert a negative influence on the perception of pain, creating a vicious circle that leads to unwanted results [3]. Antioxidants 2020, 9, 31; doi:10.3390/antiox9010031 www.mdpi.com/journal/antioxidants Antioxidants 2020, 9, 31 2 of 20 At present, the treatment of chronic osteoarthritis pain remains a challenge. Therapies to treat osteoarthritis pain are limited, with modest efficacy and important adverse effects. Furthermore, it is important to note that while some treatments may relieve pain, few are able to reduce the emotional disorders associated with chronic osteoarthritis pain. Therefore, an investigation of new strategies that effectively relieve chronic osteoarthritis pain and the associated comorbidities, such as anxiety and depression, is essential. Hydrogen sulfide (H2 S), together with nitric oxide and carbon monoxide, is an integral part of the triad of neurotransmitter gases with important physiological and pathophysiological functions. H2 S is widely distributed in the central and peripheral nervous system and plays an important role in redox balance, apoptosis, and inflammatory processes [4]. However, despite numerous studies revealing that the exogenous administration of H2 S exerts powerful anti-inflammatory and antioxidant actions [5], the role of H2 S in pain modulation is controversial. Thus, conflicting data suggest that H2 S can be both pronociceptive and antinociceptive, depending on the type of H2 S-releasing substances used [6]. That is, compounds that release large amounts of H2 S in a short time and in an uncontrolled manner can relieve pain, not alter it, or even increase it, depending on the dose, the route of administration, and the time of the evaluation of their effects [7–10]. In contrast, more recent studies show that the administration of substances that are capable of releasing H2 S slowly, simulating the conditions of H2 S release in vivo, in addition to exerting potent anti-inflammatory effects [11], are also able to relieve neuropathic pain induced by the administration of antineoplastic drugs in animals [12]. Nevertheless, the effect of the administration of low doses of two slow-releasing H2 S compounds, such as allyl isothiocyanate (A-ITC) and phenyl isothiocyanate (P-ITC), on the mechanical allodynia and grip strength deficits induced by chronic osteoarthritis in mice has not yet been studied. Several studies also show that the exogenous administration of H2 S exerts anxiolytic and antidepressant effects in different animal models of anxiety and depression [13], as well as in the anxiety-like behavior associated with diabetes [14], without affecting locomotor activity. Thus, these data suggest that treatment with slow-releasing H2 S compounds may be a good approximation for the treatment of the anxiety-like and depressive-like behaviors associated with chronic osteoarthritis pain. The mechanisms implicated in the development of osteoarthritis are not completely known. Inflammation and oxidative stress are closely integrated in the osteoarthritis pathology. The levels of several inflammatory mediators, such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα), are augmented in the joint tissue of osteoarthritis patients [15]. Moreover, IL-1β and TNFα also stimulate the synthesis of prostaglandins, nitric oxide, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), nuclear factor κB (NF-κB), and diverse mitogen-activated protein kinase (MAPK) signaling pathways further contributing to osteoarthritis pain [16]. Accordingly, the PI3K/Akt/NF-κB and MAPK signaling pathways are activated in the spinal cord and different brain areas, such as the hippocampus and/or prefrontal cortex, of animals with chronic pain [17,18], and their inhibition reduced the development of osteoarthritis and neuropathic pain [19,20]. Microglia are likewise implicated in the progression of chronic pain, and their activation in the supraspinal areas is in part responsible for the molecular neuroplasticity changes associated with chronic pain [21] as well as the emotional disorders accompanying it [3]. Thus, microglial activation has been demonstrated in the medial prefrontal cortex and the hippocampus of animals with depressive-like conducts associated with osteoarthritis or neuropathic pain [18,22] as well as in several brain regions of depressive patients suffering chronic pain [23]. Consequently, the depressive-like behaviors accompanying chronic pain diminished with the administration of microglial inhibitors [24,25]. Oxidative stress also plays an essential role in the pathogenesis of osteoarthritis. High levels of reactive oxygen species have been demonstrated in the synovial tissue, chondrocytes, and fibroblasts of the joint, thus promoting the structural and functional damage of the cartilage [26], which can also potentiate the inflammatory responses by activating the production of nitric oxide synthetized by inducible nitric oxide synthase (NOS2). Therefore, in addition to the well-known positive effects of anti-inflammatory agents, treatment with antioxidant compounds, for example, inducers Antioxidants 2020, 9, 31 3 of 20 of nuclear factor erythroid 2-related factor 2 (Nrf2) and/or other enzymes activated by Nrf2, such as heme oxygenase 1 (HO-1), also attenuates the development of osteoarthritis by regulating redox homeostasis [27,28]. Nevertheless, the effects of knee osteoarthritis and those of the systemic treatment with slow-releasing H2 S compounds in the endogenous antioxidant system in the central nervous system have not been evaluated. Lastly and taking account that women are more likely to suffer chronic pain than men and because most of the pain symptoms and disorders associated with chronic osteoarthritis pain are more prevalent in women [29], this study was performed in female mice. In female mice with chronic osteoarthritis pain induced by the intra-articular administration of monosodium iodoacetate (MIA), at 29 days after injection, we assessed the effects of the repeated administration of 4.4 µmol/kg A-ITC or 13.3 µmol/kg P-ITC on (1) the mechanical allodynia; (2) the grip strength deficits; (3) the emotional behaviors associated with chronic osteoarthritis pain; (4) the inflammatory and molecular changes induced by MIA in the central nervous system by evaluating the protein levels of CD11b/c (a microglial marker), glial fibrillary acidic protein (GFAP, an astroglial marker), NOS2, and PI3K/p-Akt; and (6) the expression of antioxidant and detoxificant proteins such as HO-1, NAD(P)H:quinone oxidoreductase-1 (NQO1), glutathione S-transferase mu 1 (GSTM1), and glutathione S-transferase alpha 1 (GSTA1) in the hippocampus. 2. Materials and Methods 2.1. Animals We used female mice of 6–8 weeks of age acquired from Envigo laboratories (Barcelona, Spain). All mice weighed between 21 and 25 g and were accommodated in a room with 12/12 h of light/dark and a controlled temperature of 22 ◦ C and humidity of 66%. The animals had free access to food and water, and experiments started at 7 days after acclimatization to the housing conditions. All the proposed experiments were conducted between 9:00 a.m. and 5:00 p.m. and were carried out in accordance with the guidelines of the European Commission’s directive (2010/63/EC) and the Spanish Law (RD 53/2013) regulating animal research, and they were approved by the local Committee of Animal Use and Care of the Autonomous University of Barcelona (number: 1325R5). Maximum efforts were made to minimize the number of animals used and their suffering. 2.2. Induction of Osteoarthritis Pain Osteoarthritis pain was induced in anesthetized mice with isoflurane (2%) by the intra-articular injection of MIA (Sigma-Aldrich, St. Louis, MO, USA). The right knee joint was shaved and flexed at a 90◦ angle, and 10 µl of MIA (15 mg/mL) dissolved in saline solution (NaCl 0.9%; SS) was intra-articularly injected with a 30-gauge needle. Control mice received the same volume of SS. 2.3. Mechanical Allodynia Mechanical allodynia was evaluated by measuring the hind paw withdrawal response to von Frey filament stimulation. For this purpose, mice were placed in methacrylate cylinders (20 cm high and 9 cm in diameter) on a lifted wire grid through which the von Frey filaments (North Coast Medical, Inc., San Jose, CA, USA) with a bending force range of 0.008 to 3.5 g were applied to each hind paw, using the up–down paradigm reported by Chaplan et al. [30]. The test was started with the 0.4 g filament, and the strength of the next filament was increased or decreased in accordance with the response. Finally, the threshold of the response was calculated from the sequence of filament strength using an Excel program (Microsoft Iberia SRL, Barcelona, Spain) that includes the adjustment of the data curve. Both ipsilateral and contralateral hind paws were assessed, and animals were habituated for 1 h before starting the test to allow appropriate behavioral immobility. Antioxidants 2020, 9, 31 4 of 20 2.4. Measurement of Grip Strength Grip strength was measured with a computerized grip strength meter (Model 47200, Ugo Basile, Varese, Italy) according to the method reported by [31]. To measure grip strength in the hind paws, the experimenter held the animal by the base of the tail, allowing the mice to grasp the metal bar of the grip strength meter with its hind paws. The metal bar was connected to a force transducer that automatically recorded the peak force of each measurement in grams. For each mouse, the grip strength of the hind limbs was measured in triplicate. To prevent the mice from gripping the metal bar with their forepaws during the test, the animals were first allowed to grasp a wire mesh cylinder with their forepaws. Baseline grip strength values were recorded for each mouse as the average of three determinations before the administration of MIA or SS. This value was considered 100% of grip strength and was used as a reference for the following determinations. 2.5. Measurement of Anxiety-Like Behaviors The anxiety-like behaviors were evaluated using the elevated plus maze (EPM) and the open field (OF) tests. The EPM is an apparatus with 4 arms 5 cm wide and 35 cm long, two of which are open and two closed with walls 15 cm high. The distance of the EPM to the ground is 45 cm. The animal was placed in the central square of the maze facing one of the open arms, and its behavior was recorded by a digital camera for 5 min, according to the method described by [32]. The number of entries in the open and closed arms, as well as the percentage of time spent in the open arms, was calculated for each animal. The OF test was also used to evaluate the anxiety-like behavior of animals according to the method used by [33]. Mice were placed on a 44 × 44 cm box with a gray nonreflecting base and walls, and their behavior was recorded by a digital camera for 5 min. At the beginning of the test, mice were placed in the center of the arena and were allowed to move freely around the maze and to explore the environment. The number of entries in the central area, the time spent in it, and the number of squares crossed was determined for each animal. 2.6. Measurement of Depressive-Like Behaviors The evaluation of the depressive-like behaviors was realized using the tail suspension test (TST) in accordance with the procedures described by [34] and the forced swimming test (FST) according to the method described by [35]. In the TST, each mouse was suspended 35 cm above the floor with an adhesive tape attached to the tip of its tail. The entire experiment was recorded with a digital camera, and the immobility time was measured over a period of 6 min. The mice were considered immobile when they remained completely quiet. In the FST, each mouse was placed in a transparent Plexiglas cylinder (25 cm high × 10 cm diameter) containing water to a depth of 10 cm at 24 ◦ C ± 0.1 ◦ C. Each animal was subjected to forced swimming for 6 min, and the total duration of immobility was measured during the last 4 min, when mice show a sufficiently stable level of immobility. All the behavioral experiments were executed by an experimenter blinded to the treatment applied. 2.7. Western Blot Analysis Animals were euthanized by cervical dislocation at 29 days after injection (MIA or SS). Tissues from the contralateral hippocampus were extracted quickly after killing, frozen, and maintained at −80◦ C until use. The protein levels of CD11b/c, GFAP, NOS2, PI3K, p-Akt, HO-1, NQO1, GSTM1, and GSTA1 were analyzed. The homogenization of the tissues was performed in ice-cold lysis buffer (50 mM Tris-Base, 150 nM NaCl, 1% NP-40, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 Triton X-100, 0.1% sodium dodecyl sulfate, 1 mM Na3VO4, 25 mM NaF, 0.5% protease inhibitor cocktail, 1% phosphatase inhibitor cocktail). All reagents were acquired from Sigma-Aldrich (St. Louis, Antioxidants 2020, 9, 31 5 of 20 MO, USA), except for NP-40, which was purchased from Calbiochem (Darmstadt, Germany). After solubilization of crude homogenate for 1 h at 4 ◦ C, it was sonicated for 10 s and centrifuged at 4 ◦ C for 15 min at 700× g. Then, the supernatants (60 µg of total protein) were mixed with 4 × Laemmli loading buffer and loaded onto 4% stacking/10% separating sodium dodecyl sulfate polyacrylamide gels. Proteins were electrophoretically transferred onto a polyvinylidene fluoride membrane for 120 min and blocked with phosphate-buffered saline plus 5% nonfat dry milk or Tris-buffered saline with Tween 20 plus 5% nonfat dry milk or 5% bovine serum albumin for 1 h and 15 min and then incubated with specific rabbit primary anti CD11b/c (1:200), GFAP (1:5000), GSTM1 (1:150), and GSTA1 (1:150) from Novus Biologic, Litton, CO, (USA); NOS2 (1:200) and PI3K (1:200) from Abcam, Cambridge (United Kingdom); p-Akt (1:200) and Akt (1:200) from Cell Signaling Technology, Danvers, MA (USA); HO-1 (1:150) from Enzo Life Sciences, Lausen (Switzerland); and NQO1 (1:250) from Sigma-Aldrich, St. Louis, MO (USA) antibodies overnight at 4 ◦ C. Blots were incubated for 1 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare, Little Chalfont, United Kingdom) to detect proteins, which were then visualized by chemiluminescence reagents (ECL kit; GE Healthcare, Little Chalfont, United Kingdom) and exposure to Kodak film. Densitometric analysis was done using Image-J program (National Institutes of Health, Bethesda, MD, USA). We used a rabbit anti glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:5000; Merck, Billerica, MA, USA) as a loading control. 2.8. Experimental Procedures In the first experiments, baseline responses for the von Frey filaments and grip strength were established. After that, osteoarthritis pain was induced, and animals were tested again on days 19, 20, 22, 25, 26, 27, and 29 after MIA injection. SS-injected mice were used as controls (six animals per group). In other groups of animals, we investigated the effects of the intraperitoneal daily administration of 4.4 µmol/kg A-ITC or vehicle from day 25 to day 29 after MIA or SS injection and the effects of the intraperitoneal daily administration of 13.3 µmol/kg P-ITC or vehicle from day 19 to day 29 after MIA or SS injection on the mechanical allodynia and the grip strength deficits induced by MIA (n = six animals per group). The effects of A-ITC or the vehicle were evaluated on days 26, 27, and 29 post-MIA or SS injection, while the effects of P-ITC or the vehicle were measured on days 20, 22, 25, and 29 post-MIA or SS injection at 30 min after drug or vehicle injection. We also evaluated the effects of the treatment with 4.4 µmol/kg A-ITC or vehicle during 4 consecutive days (25 to 29 after MIA or SS injection) and the effects of 13.3 µmol/kg P-ITC or vehicle administered during 10 consecutive days (19 to 29 after MIA or SS injection) on the anxiety- and depressive-like behaviors associated with chronic osteoarthritis pain at 29 days after MIA injection. The anxiety-like behaviors were evaluated in the EPM and OF tests and the depressive-like behaviors in the TST and FST (n = 10 animals per group). The involvement of Kv7 potassium channels in the inhibition of the allodynia, grip strength deficits, and depressive-like behaviors produced by the administration of 4.4 µmol/kg A-ITC or 13.3 µmol/kg P-ITC during 4 days or 10 consecutive days was also studied by evaluating the reversion of these effects with the administration of 8.0 µmol/kg of the selective Kv7 potassium channel blocker, XE-991 [36]. Finally, at day 29 after MIA injection and at 4 (A-ITC) or 10 days (P-ITC) of drug or vehicle administration, mice were euthanized by cervical dislocation, and the protein levels of CD11b/c, GFAP, NOS2, PI3K, p-Akt, HO-1, NQO1, GSTM1, and GSTA1 in the hippocampus were evaluated by Western blot. In these experiments, SS-vehicle-treated mice were used as controls (n = 4 samples per group). 2.9. Drugs A-ITC and P-ITC, obtained from Sigma-Aldrich (St. Louis, MO, USA) and XE-991, purchased in Tocris Bioscience (Ellisville, MO, USA) were dissolved in SS. All drugs were freshly prepared before use and intraperitoneally administered in a final volume of 10 mL/kg, 30 min, and 45 min before testing, Antioxidants 2020, 9, 31 6 of 20 in accordance with our preliminary studies and other work [12]. For each group treated with a drug, the respective control group received the same volume of vehicle. 2.10. Statistical Analyses All data are expressed as the mean values ± standard error of the mean (SEM). The statistical results indicate the F value, the degrees of freedom Fx,y, and the p value of the ANOVA. Statistical analysis was carried out using the SPSS program (version 13 for Windows, IBM, Madrid, Spain). We used the three-way repeated measures analysis of variance (ANOVA) with injection, treatment, and time as the factors of variation, followed by one-way ANOVA and the Student–Newman–Keuls test to evaluate the effects of the repetitive treatment with A-ITC and P-ITC and their corresponding vehicle on the mechanical allodynia and grip strength deficits induced by MIA. The effects of the repetitive treatment with A-ITC and P-ITC on the anxiety-like and depressive-like behaviors associated with osteoarthritis pain were assessed using a two-way ANOVA followed by the corresponding one-way ANOVA and the Student–Newman–Keuls test. The reversion of the antinociceptive and antidepressant effects of A-ITC and P-ITC with XE-991were evaluated using a one-way ANOVA and the Student–Newman–Keuls test. Variations in the protein levels were also analyzed with a one-way ANOVA followed by the Student–Newman–Keuls test. A value of p < 0.05 was considered significant. 3. Results 3.1. Treatment with A-ITC Reverses the Mechanical Allodynia and the Grip Strength Deficits Induced by the Intra-Articular Injection of MIA in Mice The three-way repeated measures ANOVA revealed significant effects among the injection (F1,5 = 211.56, p < 0.001), treatment (F1,5 = 50.67, p < 0.001), and time (F3,15 = 5.37, p < 0.010), and interactions between injection and treatment (F1,5 = 79.12, p < 0.001), injection and time (F 3,15 = 5.51, p < 0.009), treatment and time (F3,15 = 4.26, p < 0.023), and among the three factors (F3,15 = 3.41, p < 0.045) for the mechanical allodynia. These results confirmed that MIA injection reduces the threshold of the ipsilateral hind paw withdrawal to the von Frey filament stimulation from days 25 to 29 after MIA injection (p < 0.001, one-way ANOVA versus the corresponding SS-injected mice treated with vehicle; Figure 1A, Table 1). The administration of A-ITC decreased the mechanical allodynia induced by MIA after one day of treatment. Thus, the threshold of the ipsilateral paw withdrawal to a mechanical stimulus in MIA-injected mice treated with A-ITC was similar to that obtained in SS-injected animals treated with vehicle or A-ITC for one day (Figure 1A). The intraperitoneal administration of A-ITC or vehicle did not produce any significant effect on the mechanical allodynia in either the ipsilateral paw of the SS-injected mice (Figure 1A) or the contralateral paw of the MIA-injected or SS-injected animals (data not shown). In the grip strength test, the three-way repeated measures ANOVA also revealed significant effects of the injection (F1,5 = 55.69, p < 0.001) and time (F3,15 = 3.75, p < 0.034) and the interaction between injection and treatment (F1,5 = 21.42, p < 0.006). The decreased hind limb grip strength induced by MIA from days 25 to 29 after injection (p < 0.009, one-way ANOVA versus the corresponding SS-injected mice treated with vehicle; Figure 1B, Table 1) was inhibited by A-ITC treatment in a time-dependent manner. The grip strength deficits induced by MIA were completely reversed after 4 days of treatment with A-ITC (Figure 1B). Antioxidants 2019, 8, x FOR PEER REVIEW 7 of 20 induced by MIA from days 25 to 29 after injection (p < 0.009, one-way ANOVA versus the corresponding SS-injected mice treated with vehicle; Figure 1B, Table 1) was inhibited by A-ITC treatment 2020, Antioxidants in a 9, time-dependent 31 manner. The grip strength deficits induced by MIA were completely 7 of 20 reversed after 4 days of treatment with A-ITC (Figure 1B). Figure 1. Treatment with allyl isothiocyanate (A-ITC) reduces the mechanical allodynia and the grip strength1.deficits Figure Treatmentinduced with by the allyl intra-articular(A-ITC) isothiocyanate injection of MIA. reduces theThe development mechanical of (A)and allodynia mechanical the grip allodyniadeficits strength in the ipsilateral induced by pawtheand (B) grip strength intra-articular deficits injection in theThe of MIA. hinddevelopment paws of the MIA- of (A)ormechanical SS-injected mice treated allodynia inwith the A-ITC or vehicle ipsilateral paw forand4 consecutive days aredeficits (B) grip strength shown.inThetheeffects hindofpaws A-ITCofwere the evaluated MIA- or at days 26, 27, and 29 after MIA or SS injection. For each test and time evaluated, * denotes SS-injected mice treated with A-ITC or vehicle for 4 consecutive days are shown. The effects of A-ITC significant differences were vs. their evaluated respective at days 26, 27,SS-injected and 29 aftermice, MIAand or $SSdenotes significant injection. For eachdifferences vs. MIA-injected test and time evaluated, * mice treated with A-ITC (p < 0.05; one-way ANOVA followed by the Student–Newman–Keuls denotes significant differences vs. their respective SS-injected mice, and $ denotes significant test). The results are differences vs. shown as the mean MIA-injected treated±with mice values n = 6 (p SEM;A-ITC animals < 0.05;per experimental one-way ANOVA group. followed by the Student–Newman–Keuls Table test). TheANOVA’s 1. Summary of the one-way results are shown aswith performed the mean values the results ± SEM; for obtained n =the 6 animals per mechanical experimental group. allodynia and grip strength deficits at 0, 1, 2, and 4 days after the administration of allyl isothiocyanate (A-ITC) or vehicle in saline (SS) and monosodium iodoacetate (MIA)-injected mice. 3.2. Treatment with P-ITC Reverses the Mechanical Allodynia and the Grip Strength Deficits Induced by the Intra-Articular Injection of MIA in Mice Time of Treatment (days) For mechanical allodynia, 0the three-way repeated 1 2 measures ANOVA demonstrated4 significant Mechanical F effects of the injection (F1,5 3,20 = 25.15 F = 679.25, p < 0.001) = 18.27 F and treatment (F3,20 3,20 = 52.24 F 1,5 = 12.58, p < 0.016).= 17.98 3,20 A significant allodynia p < 0.001 p < 0.001 p < 0.001 p < 0.001 Grip F3,20 = 7.97 F3,20 = 8.26 F3,20 = 5.05 F3,20 = 7.09 strength p < 0.001 p < 0.001 p < 0.009 p < 0.002 Antioxidants 2020, 9, 31 8 of 20 3.2. Treatment with P-ITC Reverses the Mechanical Allodynia and the Grip Strength Deficits Induced by the Intra-Articular Injection of MIA in Mice For mechanical allodynia, the three-way repeated measures ANOVA demonstrated significant effects of the injection (F1,5 = 679.25, p < 0.001) and treatment (F1,5 = 12.58, p < 0.016). A significant Antioxidants 2019, 8, x FOR PEER REVIEW 8 of 20 interaction among injection and treatment (F1,5 = 6.59, p < 0.050), injection and time (F4.20 = 3.35, p < 0.030), and among interaction the three factors injection and(F4.20 = 3.54,(Fp1,5<= 0.024) treatment 6.59, p