antioxidants Article The Recovery of Cognitive and Affective Deficiencies Linked with Chronic Osteoarthritis Pain and Implicated Pathways by Slow-Releasing Hydrogen Sulfide Treatment Gerard Batallé 1,2 , Xue Bai 1,2 , Enric Pouso-Vázquez 1,2 , Gerad Roch 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.batalle@e-campus.uab.cat (G.B.); xue.bai@e-campus.uab.cat (X.B.); enrique.pousovazquez@e-campus.uab.cat (E.P.-V.); gerard.roch@e-campus.uab.cat (G.R.); 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: Chronic osteoarthritis pain is accompanied by several comorbidities whose treatment has not been completely resolved. The anti-inflammatory, analgesic, and antidepressant effects of slow-releasing hydrogen sulfide (H2 S) donors during osteoarthritic pain have been shown, but their actions in the accompanying memory impairment and anxious-like behaviors have not yet been  demonstrated. Using female mice with chronic osteoarthritic pain, the effects of natural, diallyl  disulfide (DADS) or synthetic, morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate Citation: Batallé, G.; Bai, X.; dichloromethane complex (GYY4137) slow-releasing H2 S donors, on associated cognitive and grip Pouso-Vázquez, E.; Roch, G.; strength deficits and anxiodepressive-like behaviors, were assessed. Their effects on specific brain Rodríguez, L.; Pol, O. The Recovery areas implicated in the modulation of pain and emotional responses were also determined. Results of Cognitive and Affective demonstrated an improvement in memory and grip strength deficits, as well as in the anxious-like Deficiencies Linked with Chronic behaviors associated with chronic pain in GYY4137 and/or DADS treated mice. The painkiller and Osteoarthritis Pain and Implicated antidepressant properties of both treatments were also established. Treatment with DADS and/or Pathways by Slow-Releasing GYY4137 inhibited: oxidative stress in the amygdala; phosphoinositide 3-kinase overexpression in the Hydrogen Sulfide Treatment. amygdala, periaqueductal gray matter, and anterior cingulate cortex; protein kinase B activation in Antioxidants 2021, 10, 1632. https:// the amygdala and infralimbic cortex; up-regulation of inducible nitric oxide synthase in the amygdala, doi.org/10.3390/antiox10101632 periaqueductal gray matter and infralimbic cortex and apoptotic responses in the amygdala. These Academic Editor: Stanley Omaye results might explain the recovery of memory and grip strength and the inhibition of allodynia and associated anxiodepressive-like behaviors by these treatments. In conclusion, this study revealed Received: 30 August 2021 new properties of slow-releasing H2 S donors in cognitive impairment and affective disorders linked Accepted: 13 October 2021 with chronic osteoarthritis pain and their effects on the central nervous system. Published: 16 October 2021 Keywords: anxiety; apoptosis; cognitive deficits; depression; grip strength impairments; hydrogen Publisher’s Note: MDPI stays neutral sulfide; osteoarthritic pain; oxidative stress with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Osteoarthritis is a degenerative disorder and one of the most common forms of arthritis. It is also one of the most frequent sources of chronic pain, affecting more than Copyright: © 2021 by the authors. 100 million people worldwide. However, despite its high incidence in society, especially in Licensee MDPI, Basel, Switzerland. women (35%), its etiology and treatment have not been fully solved [1]. This article is an open access article Osteoarthritis is characterized by the progressive loss of articular cartilage, bone, and distributed under the terms and synovial remodeling, thus leading to subchondral bone alterations and inflammation, and conditions of the Creative Commons resulting in severe pain and loss of joint function [2,3]. In addition, and as a consequence Attribution (CC BY) license (https:// of the pain and suffering, patients also experience related cognitive and affective disorders, creativecommons.org/licenses/by/ including memory dysfunction and depressive-like and anxiety-like behaviors [4]. These 4.0/). Antioxidants 2021, 10, 1632. https://doi.org/10.3390/antiox10101632 https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, 1632 2 of 17 emotional comorbidities further potentiate hypersensitivity and a more rapid cartilage degradation and disability, thus contributing to a worse quality of life in patients [5]. Dif- ferent studies have suggested that the underlying mechanisms involved in chronic pain processes are also implicated in memory deficits, as seen in patients with fibromyalgia or severe pain who have exhibited relevant alterations in working memory [6–8]. Moreover, osteoarthritis and several emotional and cognitive deficits share a common pathophys- iology that involves the activation of the phosphatidylinositol 3 kinase (PI3K)-protein kinase B (AKT) pathway, oxidative stress, and increased proinflammatory responses [9–12]. The PI3K/p-AKT axis is involved in several cellular processes essential to maintaining homeostasis, including cell growth, cell survival, inflammation, and metabolism [13,14]. This pathway is also implicated in the progression of osteoarthritis pain, and its inhibition has been found to attenuate joint damage by restoring cartilage homeostasis, enhancing au- tophagy, and suppressing inflammation [10]. The PI3K/p-AKT pathway is also associated with negative mood disorders and may be an important target in the neuroprotection for depressive and anxiety syndromes [15,16]. Furthermore, the inactivation of this pathway has been found to additionally improve the cognitive alterations associated with chronic pain [17]. Several studies revealed that oxidative stress takes part in the pathogenesis of os- teoarthritis [18,19]. Therefore, oxidative stress as a result of high reactive oxygen species synthesis, modest antioxidant defenses and lipid-rich formation is a negative factor for cer- tain neuropsychological illnesses, such as alterations in learning and memory functions [11], anxiety [20], depression [21], and nociception [22]. Indeed, oxidative stress blockage can prevent the appearance of these behavioral syndromes, and the emotional and cognitive im- pairments have been manifested under low levels of antioxidant defenses, thus supporting the relationship between oxidative stress and emotional/memory deficits [11,23,24]. It has been demonstrated that in the progress of osteoarthritis, affected chondrocytes and synovial cells overproduce inflammatory mediators such as interleukin-1β and nitric oxide, which accelerate the degradation of cartilage [25]. Nitric oxide synthetized by the inducible nitric oxide synthase (NOS2) is one of the major proinflammatory and destruc- tive mediators in osteoarthritis [26]. Indeed, high levels of NOS2 have been found in the synovial membrane of individuals with osteoarthritis [27]. Moreover, experiments per- formed on animals have supported this idea by showing that NOS2 inhibition could be an attractive way to treat osteoarthritis [26]. Finally, the excess production of reactive oxygen species and nitric oxide has been linked with apoptotic responses during osteoarthritis [19]. Therefore, the up-regulation of 4-HNE, a marker of oxidative damage, ends up causing increased BAX levels, leading to cellular apoptosis [18]. In this study, we evaluated the expression of 4-HNE and BAX as markers of oxidative stress and apoptosis. Hydrogen sulfite (H2 S) is an endogenous gaseous neurotransmitter that takes part in several physiological processes in the central and peripheral nervous systems [28,29]. Accordingly, H2 S releasing compounds, especially slow-releasing compounds, have been explored as therapeutic agents in several diseases [30,31]. Numerous findings have demon- strated the beneficial anti-inflammatory and protective actions induced by the exogenous administration of H2 S in the cartilage and chondrocytes in cell cultures [32,33]. Other works have also proven the positive effects of the intra-articular administration of H2 S donors on rheumatic disease progression [34], cartilage destruction, and oxidative damage in rats [35]. The antinociceptive and antidepressant effects of the systemic administration of H2 S donors, such as isothiocyanates, in mice with knee osteoarthritis have also been shown [2,36]. Nevertheless, these compounds have failed to inhibit the anxiolytic-like behaviors concomitant with osteoarthritic or neuropathic pain [24,36]. Our objective is to find new treatments that can palliate the memory and grip strength impairments and the anxiodepressive-like behaviors accompanying chronic osteoarthritic pain, and to establish the principal signaling pathways implicated in these actions in specific areas of the central nervous system. Thus, we evaluated the effects of the systemic administration of two slow-releasing H2 S donors, a natural garlic bioactive component Antioxidants 2021, 10, 1632 3 of 17 (diallyl disulfide, DADS) and a synthetic compound (morpholin-4-ium 4-methoxyphenyl (morpholino) phosphinodithioate, GYY4137), on nociceptive, grip strength, and memory deficits, as well as on the anxiodepressive symptoms provoked by the intra-articular administration of monosodium acetate (MIA). To examine the probable pathways involved in these actions, we analyzed their effects on the amygdala, periaqueductal gray matter, infralimbic cortex, and anterior cingulate cortex, all of which are highly involved in the modulation of nociception, memory, and emotional disorders [37–40]. 2. Materials and Methods 2.1. Animals Experiments were performed with female C57BL/6 mice (21–26 g; 5–6 weeks old), acquired from Envigo Laboratories (Barcelona, Spain). Animals were accommodated under standard light/dark (12/12 h), temperature (22 ◦ C), and humidity (66%) requirements, with free access to food and water. Experiments were done following 7 days of acclimatization to the environmental conditions, performed between 9:00 a.m. and 5:00 p.m., and in compliance with the guidelines of the European Commission’s directive (2010/63/EC) and the Spanish Law (RD 53/2013) regulating animal research, and were approved by the local Committee of Animal Use and Care of the Autonomous University of Barcelona (ethical code: 9863). Every effort was made to reduce the amount and suffering of the animals used. 2.2. Induction of Osteoarthritis Pain Osteoarthritis pain was induced under isoflurane anesthesia conditions (3% induction, 2.5% maintenance) 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 injected. Control animals were injected with the same volume of SS. 2.3. Mechanical Allodynia Mechanical allodynia was evaluated by measuring the hind paw withdrawal response after stimulation with von Frey filaments of different bending forces (0.008–3.5 g). The animals were placed in Plexiglas tubes (20 cm high × 9 cm diameter) with a wire grid bottom, through which the filaments (North Coast Medical, Inc., San Jose, CA, USA) were applied by using the up–down paradigm [41]. The filament of 0.4 g was applied first, and the filament of 3.0 g was used as a cut-off. The strength of the next filament was increased or decreased depending on the animal’s response. The threshold of the response was calculated using an Excel program (Microsoft Iberia SRL, Barcelona, Spain), which includes curve fitting of the data. The animals were habituated to the environment for 1 h before the experiment. Both ipsilateral and contralateral paws were tested. 2.4. Measurement of Grip Strength We used a computerized grip strength meter (Model 47200, Ugo Basile, Varese, Italy) to measure grip strength according to the method reported by [42]. 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 both 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 employed as a reference for the following determinations. Antioxidants 2021, 10, 1632 4 of 17 2.5. Cognitive Behavior Object recognition memory was evaluated in a gray box (44 × 44 cm) with a non- reflecting base and four walls. This task consisted of 4 sessions of 10 min each (habituation, training, and test). On days 1 and 2, the mice were habituated to the empty box. On the 3rd day, for the training session, the mice were placed again in the box and 2 identical objects were presented. Twenty-four hours later, the mice were placed once again in the box, and one of the familiar objects was replaced by a novel object. The time exploring each of the 2 objects (novel and familiar) was filmed. The discrimination index ((time exploring the novel object–time exploring the familiar)/(time exploring novel + familiar) ∗ 100) was utilized as a measure of cognitive behavior, according to [43]. High values of discrimination represent good recognition memory. Mice were habituated to the testing room for 1 h before starting the evaluation, and the equipment was carefully cleaned between subjects. 2.6. Depressive Behavioral Tests The evaluation of the depressive-like behaviors was performed by using the forced swimming test (FST) and tail suspension test (TST), in which the duration of immobility of the animals was quantified according to the methods described by [44,45], respectively. In the FST, each mouse was placed in a transparent Plexiglas cylinder (25 cm × 10 cm) 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 the mice showed a sufficiently stable level of immobility. In the TST, the mice, isolated acoustically and visually, were suspended by the tail from a horizontal wooden bar (35 cm above the floor) using adhesive tape (1 cm from the tip of the tail). The immobility time in seconds was recorded for 6 min. In both tests, the mice were considered immobile when they remained completely quiet. 2.7. Anxiety Behavioral Tests The anxiety-like behavior was assessed by using the elevated plus maze (EPM) [46], which consists of an X-shape apparatus with four arms, each 5 cm wide and 35 cm long, with two being open and two being closed, with walls 15 cm high. It was elevated off the ground by 45 cm. The animal was placed in the center of the maze facing the open arms, and its behavior was recorded by a digital camera for 5 min. The number of entries into the open and closed arms and the percentage of time they stayed in the open arms were calculated for each animal. The mice were habituated to the testing room for 1 h before starting the evaluation, and the equipment was carefully cleaned between subjects. All these experiments were performed by experimenters blinded to the experi- mental conditions. 2.8. Western Blot Analysis Twenty-nine days after MIA or SS injection, the animals were euthanized by cervical dislocation and the contralateral amygdala, periaqueductal gray matter, infralimbic cortex and anterior cingulate cortex were quickly extracted, frozen, and maintained at −80 ◦ C until usage. In this study, we analyzed the expression of 4-HNE, PI3K, p-Akt, NOS2 and BAX. The sonication of tissues was made in cold lysis buffer RIPA Buffer (R0278; Sigma- Aldrich, MO, USA). After solubilization for 1 h at 4 ◦ C, crude homogenates were sonicated for 10 s and centrifuged at 700× g for 20 min at 4 ◦ C. The supernatant (60 µg of total protein) was mixed with 4 × Laemmli loading buffer and loaded onto 4% stacking/12% separating sodium dodecyl sulfate polyacrylamide gels. Proteins were electrophoretically transferred onto a polyvinylidene fluoride membrane for 120 min and blocked with phosphate-buffered saline (PBS; P-5493; Sigma-Aldrich, MO, USA) containing 5% nonfat dry milk, Tris-buffered saline with Tween 20 containing 5% bovine serum albumin (BSA; A-7906; Sigma-Aldrich, MO, USA) or 5% nonfat dry milk, and PBS with Tween 20 containing 5% BSA, for 1 h and 15 min. Then, the membranes were incubated with specific rabbit primary antibodies, the anti 4-HNE (1:200; ab46545, Abcam, Cambridge, UK), PI3K (1:150; ab109006, Abcam, Antioxidants 2021, 10, 1632 5 of 17 Cambridge, UK), phospho-Akt (1:150; 4060S, Cell Signaling Technology, Danvers, MA, USA), total Akt (1:250; 9272S, Cell Signaling Technology, Danvers, MA, USA), NOS2 (1:200; ab15323, Abcam, Cambridge, UK), BAX (1:250; 14796S, Cell Signaling Technology, Danvers, MA, USA) or β-actin (1:5000, ab8227, Abcam, Cambridge, UK) antibodies overnight at 4 ◦ C. Afterward, blots were incubated for 1 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare, Little Chalfont, UK), and proteins were detected with chemiluminescence reagents (ECL kit; GE Healthcare, Little Chalfont, UK). Densitometric analysis was done using the Image-J program (National Institutes of Health, Bethesda, MD, USA). 2.9. Experimental Procedures In the first experiments, we evaluated the effects of repetitive intraperitoneal admin- istration, twice daily, of 200 µmol/kg of DADS and 32 µmol/kg of GYY4137, over 3 and 4 consecutive days, on the cognitive and grip strength losses and mechanical allodynia caused by osteoarthritis (n = 6–8 animals for each group). The doses of DADS and GYY4137 were chosen in conformity with another research [47]. The effects of the same treatments on associated depressive- and anxiety-like behaviors were further assessed (n = 8 animals for each group). In all experiments, SS plus vehicle-treated mice were used as controls. MIA-injected animals treated with DADS, GYY4137 or vehicle (0.9% SS) were euth- anized by cervical dislocation, and the protein levels of 4-HNE, PI3K, p-Akt, NOS2, and BAX in the amygdala, periaqueductal gray matter, infralimbic cortex and anterior cingulate cortex were evaluated by western blot. In these experiments, SS-injected mice treated with vehicle were used as controls (n = 3–4 samples per group). 2.10. Drugs DADS and GYY4137 were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in SS. Both drugs were intraperitoneally administered 1 h before testing, in a final volume of 10 mL/kg, in accordance with our previous pilot studies and other work [47]. Both drugs were prepared before use, and for each group treated with a drug, the respective control group received the same volume of corresponding vehicle. 2.11. Statistical Analyses Data are expressed as the mean values ± standard error of the mean (SEM). We used the GraphPad software (version 8.0) for the statistical analysis. The effects of DADS and GYY4137 on the allodynia, anxiodepressive-like behaviors, grip strength, and memory deficits associated with osteoarthritis pain were analyzed by using a one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test. The effects of both treatments on the expression of several proteins in different brain areas were also analyzed by using a one-way ANOVA and the post hoc Student–Newman–Keuls test. A value of p < 0.05 was considered significant. 3. Results 3.1. Treatment with DADS and/or GYY4137 Inhibits Osteoarthritis-Induced Cognitive Impairment, Hind Limb Grip Strength Deficits and Mechanical Allodynia The effects of the repetitive intraperitoneal administration, twice daily, of 200 µmol/kg of DADS or 32 µmol/kg of GYY4137, over 3 and 4 consecutive days, on mechanical allody- nia and the grip strength and cognitive impairments caused by osteoarthritis were evaluated. Our data demonstrated the complete reversion of the mechanical allodynia (p < 0.0001; one-way ANOVA followed by the Student–Newman–Keuls test as compared with SS- vehicle treated mice) (Figure 1A) and grip strength impairments (p < 0.0001; one-way ANOVA followed by the Student–Newman–Keuls test as compared with SS-vehicle treated mice) (Figure 1B) provoked by the intra-articular injection of MIA in animals treated with were evaluated. Our data demonstrated the complete reversion of the mechanical allodynia (p < 0.0001; one-way ANOVA followed by the Student–Newman–Keuls test as compared with SS-vehicle treated mice) (Figure 1A) and grip strength impairments (p < 0.0001; one-way Antioxidants 2021, 10, 1632 ANOVA followed by the Student–Newman–Keuls test as compared with SS-vehicle 6 of 17 treated mice) (Figure 1B) provoked by the intra-articular injection of MIA in animals treated with DADS or GYY4137. Both treatments did not have any significant impact on the mechanical allodynia in the contralateral paws of either MIA- or SS-injected mice (data DADS or GYY4137. Both treatments did not have any significant impact on the mechanical not shown). allodynia in the contralateral paws of either MIA- or SS-injected mice (data not shown). Figure 1. Treatment with DADS and/or GYY4137 inhibited the mechanical allodynia, grip strength, and cognitive Figure 1. Treatment deficits induced by with DADS and/or monosodium GYY4137 acetate (MIA)inhibited theThe injection. mechanical allodynia, mechanical gripgrip allodynia, strength, andand strength, cognitive memory deficits deficits induced by monosodium acetate (MIA) injection. The mechanical allodynia, grip strength, and memory deficits induced by osteoarthritis were evaluated on day 29 after MIA or saline solution (SS) injection and treatment with DADS induced by osteoarthritis were evaluated on day 29 after MIA or saline solution (SS) injection and treatment with DADS (200 μg/kg, (200 µg/kg, twice daily for 3 days) or GYY4137 (32 µg/kg, twice daily for 4 days) in the von Frey filament strength (g) (A), twice daily for 3 days) or GYY4137 (32 μg/kg, twice daily for 4 days) in the von Frey filament strength (g) (A), grip strength grip strength (%) (B) and novel object recognition (discrimination index) (C) tests. A schematic representation of the novel (%) (B) and novel object recognition (discrimination index) (C) tests. A schematic representation of the novel object recog- object nition testrecognition test For is shown (D). is shown (D). *For each test, each test, denotes * denotes significant significant differences vs.differences SS-vehicle vs. SS-vehicle treated mice, #treated denotesmice, # denotes significant significant differences vs.differences MIA-injectedvs. mice MIA-injected mice treated with treated DADS, with and DADS, significant $ denotes and $ denotes significant differences differences vs.mice vs. MIA-injected MIA-injected treated mice with treated(pwith GYY4137 GYY4137 < 0.05; one-way (p