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By Mireia Carcolé, Sami Kummer, Leonor Gonçalves, Daniel Zamanillo, Manuel Merlos, Anthony H. Dickenson, Begoña Fernández-Pastor, David Cabañero, and Rafael Maldonado

Excerpt from the article published in British Journal of Pharmacology, 23 July 2019; 176: 3939– 3955. DOI: https://doi.org/10.1111/bph.14794

Abstract

Background and Purpose: Osteoarthritic pain is a chronic disabling condition lacking effective treatment. Continuous use of opioid drugs during osteoarthritic pain induces tolerance and may result in dose escalation and abuse. Sigma-1 (σ1) receptors, a chaperone expressed in key areas for pain control, modulates μ-opioid receptor activity and represents a promising target to tackle these problems. The present study investigates the efficacy of the σ1 receptor antagonist E-52862 to inhibit pain sensitization, morphine tolerance, and associated electrophysiological and molecular changes in a murine model of osteoarthritic pain.

Experimental Approach: Mice received an intra-knee injection of monoiodoacetate followed by 14-day treatment with E-52862, morphine, or vehicle, and mechanical sensitivity was assessed before and after the daily doses.

Key Results: Monoiodoacetate-injected mice developed persistent mechanical hypersensitivity, which was dose-dependently inhibited by E-52862. Mechanical thresholds assessed before the daily E-52862 dose showed gradual recovery, reaching complete restoration by the end of the treatment. When repeated treatment started 15 days after knee injury, E-52862 produced enhanced short-term analgesia, but recovery to baseline threshold was slower. Both a σ1 receptor agonist and a μ receptor antagonist blocked the analgesic effects of E-52862. An acute, sub-effective dose of E-52862 restored morphine analgesia in opioid-tolerant mice. Moreover, E-52862 abolished spinal sensitization in osteoarthritic mice and inhibited pain-related molecular changes.

Conclusion and Implications: These findings show dual effects of σ1 receptor antagonism alleviating both short- and long-lasting antinociception during chronic osteoarthritis pain. They identify E-52862 as a promising pharmacological agent to treat chronic pain and avoid opioid tolerance.

1 INTRODUCTION

Osteoarthritis is the most frequent chronic musculoskeletal pain condition (Breivik, Collett, Ventafridda, Cohen, & Gallacher, 2006), characterized by progressive destruction of articular cartilage (Sutton et al., 2009; Zhang, Ren, & Dubner, 2013). Pain is the major symptom of osteoarthritis and the reason for presentation of patients to clinical services. However, available therapeutic approaches do not control the progression of the disease and do not provide satisfactory analgesia (Wieland, Michaelis, Kirschbaum, & Rudolphi, 2005). Opioids are potent analgesics widely used for severe pain management. However, prolonged administration has been associated with tolerance, abuse liability, and hyperalgesia (Vowles et al., 2015). Long-term opioid prescriptions increased importantly over the last decade, mainly in the United States, becoming a public health problem with devastating consequences including overdose-related deaths (Lyden & Binswanger, 2019). Therefore, there is an urgent need to develop safer therapeutic alternatives for chronic pain.

The sigma-1 (σ1) receptor is a ligand-regulated chaperone located mainly in the endoplasmic reticulum (Alonso et al., 2000). It has been proposed as an amplifier of signal transduction cascades that modulate a variety of receptors and ion channels (Su, Hayashi, Maurice, Buch, & Ruoho, 2010; Zamanillo, Romero, Merlos, & Vela, 2013). The σ1 receptors are expressed in areas of the nervous system crucial for pain transmission such as the dorsal root ganglia (DRG), dorsal horn, and periaqueductal grey (Alonso et al., 2000; Bangaru et al., 2013). Behavioural studies showed their involvement in models of neuropathic and inflammatory pain (Romero, Merlos, & Vela, 2016).

Osteoarthritis involves inflammatory and neuropathic pain mechanisms (Ivanavicius et al., 2007; Wylde, Hewlett, Learmonth, & Dieppe, 2011) at distinct points of the disease (Arendt-Nielsen et al., 2010), but the role of σ1 receptors in these different stages has not been yet investigated. Moreover, functional interactions have been reported between σ1 receptors and μ-opioid receptors (Kim et al., 2010). Indeed, σ1 receptor antagonists enhance opioid-induced analgesia in rodent models of acute nociception and inflammation (Chien & Pasternak, 1995; Montilla-García et al., 2019; Vidal-Torres et al., 2013) and did not potentiate opioid-induced side effects such as tolerance or physical dependence (Vidal-Torres et al., 2013), which could represent an important advantage for long-term opioid treatment. However, it is not known if σ1 receptors modulate opioid analgesia and tolerance during osteoarthritis chronic pain. Interestingly, σ1 receptors are up-regulated and redistributed from the endoplasmic reticulum to other subcellular locations under cellular stress (Hayashi & Su, 2007; Zamanillo et al., 2013), which is associated to chronic pain and morphine tolerance (Inceoglu et al., 2015; Liu et al., 2018). Hence, characterizing the role of σ1 receptors in osteoarthritic pain and opioid tolerance could provide insights into improving the available therapeutic strategies.

This study investigated the role of σ1 receptors in the monoiodoacetate (MIA) mouse model of osteoarthritic pain and its participation on opioid tolerance. We assessed the antinociceptive effects of acute and chronic treatment with the σ1 receptor antagonist E-52862 (also named S1RA [Romero et al., 2012] and MR309 [Castany, Gris, Vela, Verdú, & Boadas-Vaello, 2018]) and evaluated its effects in vivo on spinal electrophysiological recordings performed in animals with osteoarthritic pain. Involvement of μ opioid receptorsin the analgesic effects of E-52862 and its participation on morphine tolerance were also investigated. Pain and morphine-associated biochemical alterations were also investigated in spinal cord and DRG of mice with osteoarthritic pain.

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3 RESULTS

3.1 The σ1 receptor antagonist E-52862 produces acute antinociception and a gradual normalization of mechanical sensitivity during osteoarthritic pain

To assess the therapeutic potential of E-52862 in pain due to chronic osteoarthritis, we evaluated its effects on mechanical allodynia in mice subjected to joint pain induced by MIA. Mice received vehicle or different i.p. E-52862 doses (5–10–20 mg·kg⁻¹) twice a day during 14 days, starting the first day after MIA or sham injection. Mechanical thresholds were measured before (PRE values) and 30 min after (POST) the first daily administration (Figure 1a). Baseline mechanical thresholds were similar in all groups, and sham animals did not show nociceptive changes during the experiment, regardless of the treatment (Figure 1b,g). Intra-knee injection of MIA led to a marked decrease of withdrawal thresholds to mechanical stimuli when compared with sham. Mechanical allodynia was shown from the first day after MIA until the end of the experiment in vehicle-treated mice (Figure 1b,f). Acute E-52862 doses of 5 and 10 mg·kg⁻¹ were sub-effective, whereas 20 mg·kg⁻¹ clearly alleviated MIA-induced hypersensitivity, demonstrating dose-dependent analgesic effects of E-52862 after single administration (POST values compared with PRE values at Day 1; Figure 1c,e). Interestingly, E-52862 also induced a gradual recovery of the mechanical thresholds measured before its daily administration. This recovery was significant after the seventh day of treatment for all doses tested (Figure 1c,e). Hence, the sustained recovery of mechanical thresholds was independent of the E-52862 doses tested.

To evaluate the persistence of E-52862-induced antiallodynic effects, we continued the evaluation of mechanical thresholds after ending the repeated treatment (wash-out period; Figure 1f,g). Mice previously treated with vehicle or E-52862 (20 mg·kg⁻¹, 14 days) were evaluated for 15 additional days after ending the treatment. The reduction of mechanical allodynia induced by the 14-day treatment was maintained for 9 days after interrupting E-52862 administration (Figure 1g). Therefore, the blockade of σ1 receptors had sustained antiallodynic effects that lasted for several days after treatment discontinuation.

3.2 The analgesic efficacy of acute or repeated treatments with E-52862 depends on the stage of the osteoarthritic pain sensitization

Treatment with the σ1 receptor antagonist showed acute and long-lasting efficacy inhibiting osteoarthritic pain at stages in which inflammatory and neuroplastic changes may have not been fully developed. Thus, we wanted to assess its analgesic efficacy once these changes were already present. We compared the efficacy of acute and chronic E-52862 treatments starting 1 (Early) or 15 (Late) days after MIA injection (Figure 2a). MIA-injected mice showed similar decrease of mechanical thresholds 1 and 15 days after MIA (Figures 1b and 2b). We observed a significant shift to the left of the dose–response curve of E-52862 measured 15 days after MIA injection, when compared with the curve assessed on Day 1 (Figure 2c). This was reflected in lower median effective dose (ED₅₀) of the σ1 receptor antagonist 15 days after MIA (ED₅₀ = 7.024 mg·kg⁻¹) than on Day 1 (ED₅₀ = 14.10 mg·kg⁻¹). Thus, the dose needed to induce pain relief on Day 15 was half of the dose required on Day 1.

Comparison between early and late repeated treatments with E-52862 (5 and 10 mg·kg⁻¹) revealed that the time period in which the σ1 receptor antagonist was applied had a significant effect on the gradual recovery of mechanical thresholds. MIA-induced sensitization was stable in vehicle-treated mice up to 28 days after intra-knee injection (Figure 2b). Mice receiving the late chronic E-52862 treatment also showed the sustained restoration of mechanical sensitivity observed in the previous treatment schedule (Figure 2d,f), but this improvement was slower compared to the treatment starting at Day 1, as reflected on reduced slopes of the time-course curves of mechanical allodynia (Figure 2e,g). Therefore, late repeated treatments with E-52862 required a longer duration of treatment to restore mechanical thresholds than that for early treatments, even if single administrations at late time points showed higher acute antinociceptive effects.

3.3 Spinal administration of the σ1 receptor antagonist reduces evoked firing frequency of spinal neurons from mice with osteoarthritic pain

To distinguish whether E-52862 could modulate central sensitization in the spinal cord of mice with osteoarthritic pain (Harvey & Dickenson, 2009), in vivo electrophysiological recordings were performed in WDR neurons of lamina V of the dorsal horn. These neurons respond to mechanical and thermal stimuli, including punctate stimulation, dynamic brush, static pressure, and heat (Figure 3a). In this exploratory experiment, evoked responses to punctate mechanical stimuli were facilitated in osteoarthritic mice (Figure 3b). However, firing frequency evoked by the other stimuli was similar in MIA and sham animals (Figure 3c). Application of 90 μg of E-52862 to the exposed dorsal horn of mice with osteoarthritis significantly reduced firing frequency in response to mechanical and thermal stimuli 30 min after administration (Figure 3d,e). Application of 180 μg induced earlier inhibitory effects that were evident 10 and 30 min after E-52862 (Figure 3f,g), indicating dose-dependent drug effects. Hence, MIA induced central sensitization characterized by increased firing frequency of spinal WDR neurons that was normalized by the pharmacological blockade of σ1 receptors. This effect is associated with the short-term antiallodynic effects of E-52862 observed in mice with osteoarthritic pain.

3.4 σ1 receptors and μ receptors show reciprocal modulation in chronic osteoarthritic pain

The σ1 receptors have been shown to modulate opioid tolerance during acute nociception (Chien & Pasternak, 1995; Mei & Pasternak, 2002), but this effect has not been investigated under chronic pain conditions. To address this question, we induced opioid tolerance in mice with osteoarthritic pain using a chronic treatment with 2.5 mg·kg⁻¹ morphine administered twice a day for 14 days. Afterwards, mice received acute morphine (2.5 mg·kg⁻¹), E-52862 (5–20 mg·kg⁻¹), or combinations of both, and mechanical sensitivity was assessed (Figure 4a). Osteoarthritic mice developed tolerance to the antinociceptive effects of morphine, revealed by the absence of an opioid response at the end of the repeated treatment (Figure 4b,d). This tolerance persisted for at least 9 days, as morphine antinociception was still absent 17, 20, and 23 days after MIA (Figure 4b). In this context, a single sub-effective dose of E-52862 (5 mg·kg⁻¹), combined with morphine, restored opioid analgesia (Days 17 and 23, Figure 4c). This restorative effect was not observed when morphine was administered alone 3 days after the first morphine-E-52862 combination, suggesting that may be a transient effect (Day 20; Figure 4c). To assess the effect of higher doses of the σ1 receptor antagonist, we co-administered E-52862 20 mg·kg⁻¹ with morphine. As expected, this combination produced complete restoration of mechanosensitivity to baseline levels (Days 17 and 23, Figure 4d). Interestingly, this dose induced a long-lasting recovery, revealed by increased mechanical thresholds when morphine was given alone 3 days after the first morphine-E-52862 combination (Day 20, Figure 4d). This was not a residual effect of the earlier E-52862 dose, because the mechanical sensitivity assessed before the administration of morphine showed regular nociceptive sensitization (Figure 4d). Therefore, these results suggest that σ1 receptors participate in morphine tolerance during chronic osteoarthritic pain and its antagonism can restore opioid analgesia.

The σ1 receptor antagonist showed efficacy increasing the antinociceptive effect of morphine. However, it is not known whether μ receptors contribute to the analgesic effects of σ1 receptor ligands. Thus, we evaluated the involvement of σ1 receptors and μ receptors on the analgesic efficacy of E-52862. Mice with osteoarthritic pain were treated with vehicle or E-52862 for 13 days and on Day 14 received acute doses of E-52862 (20 mg·kg⁻¹), naloxone (1 mg·kg⁻¹), the σ1 receptor agonist PRE-084 (32 mg·kg⁻¹), or combinations of these drugs (Figure 5a). MIA-injected mice receiving vehicle showed reduced mechanical thresholds before drug administration (PRE values; Figure 5b). Acute injection of E-52862 decreased mechanical allodynia, whereas acute naloxone or PRE-084 did not induce significant responses. As expected, PRE-084 reduced the acute antiallodynic effect of the σ1 receptor antagonist (Figure 5b). Interestingly, co-administration of E-52862 with naloxone revealed a trend towards reduced analgesic effects of the σ1 receptor antagonist (Figure 5b), suggesting μ receptors also contribute to these acute responses. We also investigated whether μ receptors participate in the sustained recovery of mechanical thresholds induced by repeated E-52862 (PRE values; Figure 5c). In these conditions, both naloxone and PRE-084 injections decreased the mechanical thresholds (Figure 5c). Hence, the results indicate that the effects of E-52862 were selective for the σ1 receptors and a possible participation of μ receptors on the acute and sustained effects of E-52862 was noted.

An additional experiment was conducted to assess whether chronic stimulation of μ receptors could in turn modulate E-52862-induced antinociception. Different E-52862 doses were administered to mice with osteoarthritic pain after chronic morphine or vehicle (Figure 5a,d). Interestingly, we observed a significant shift to the right of the E-52862 dose–response curve when the drug was administered to morphine-tolerant mice. The σ1 receptor antagonist produced higher antinociceptive effects when mice were not exposed to morphine (ED₅₀ = 7.028 mg·kg⁻¹) than when mice were chronically treated with the opioid (ED₅₀ = 17.77 mg·kg⁻¹). Thus, the antinociceptive effect of E-52862 was 2.5 times lower when μ receptors were desensitized after repeated morphine. Hence, the results suggest bidirectional modulation of σ1 receptors and μ receptors during chronic osteoarthritic pain.

3.5 Molecular changes associated with chronic osteoarthritic pain are reversed by repeated treatment with the σ1 receptor antagonist E-52862

The σ1 receptor antagonist induced acute and long-lasting analgesic effects in mice with osteoarthritis pain, in contrast with morphine, which was devoid of antinociceptive effects after 14-day repeated administration. To compare the effects of these drugs at the molecular level, expression of markers associated with chronic pain was assessed in DRG and spinal cord samples of sham- or MIA-injected mice treated with vehicle, morphine, or E-52862 (Figure 6). One day after MIA, BDNF and NPY were over-expressed in the DRG (Figure 6b,c). This over-expression was reduced by acute morphine administration, but not by acute E-52862 (Figure 6b,c). This suggests peripheral effects of acute morphine treatments at early time points of the intra-knee injury. At spinal level, MIA-injected mice also showed early over-expression of TNF-α (Figure 6d), whereas IL-1β and NPY were unchanged (Figure 6e,f). No effect of the treatments was observed on these spinal markers at this early time point (Figure 6d,f).

After the 14-day treatment with vehicle, BDNF and TNF-α were still increased in osteoarthritic mice (Figure 6b,d), whereas NPY levels in the DRG showed a marked over-expression (Figure 6c). At this time point, spinal IL-1β and NPY levels were also enhanced (Figure 6e,f). Thus, additional molecular changes were established at later stages of osteoarthritic pain. Continuous blockade of σ1 receptors normalized the over-expression of all these markers (Figure 6b,f), whereas chronic morphine further increased BDNF, IL-1β, and spinal NPY expression (Figure 6b,e,f). Thus, the σ1 receptor antagonist disrupted the expression of pain signalling-related molecules after chronic treatment, whereas repeated morphine promoted the expression of neuroinflammatory mediators.

To further investigate the neuroinflammatory mechanisms underlying the long-term effects of E-52862 and morphine, we also analysed protein levels of the macrophage and microglial marker Iba-1 in DRG and spinal cord (Figure 7a). We did not observe significant differences in DRG (Figure 7b). In contrast, increased spinal Iba-1 levels were detected in osteoarthritic mice and after repeated morphine (Figure 7c). Interestingly, E-52862 reversed the enhancement of Iba-1 expression associated with osteoarthritic pain, suggesting a role for σ1 receptors in modulating glial reactivity. As microgliosis is associated with glutamateexcitotoxicity on neurons (Takeuchi, 2013), we decided to explore the spinal levels of glutamate receptors. We observed that chronic morphine significantly increased the levels of GluA2 AMPA receptor subunit (Figure 7d), whereas MIA enhanced phosphorylation of the NMDA receptor subunits GluN1 and GluN2B and the expression of metabotropic glutamate mGlu₅ receptor (Figure 7e,g). While the increases in phospho-GluN1 and phospho-GluN2B were independent of the treatment, the results showed that over-expression of mGlu₅receptors was completely reversed by E-52862 (Figure 7g), revealing a novel effect of σ1 receptor antagonism in modulating glutamatergic signalling.

Overall, acute administration of morphine decreased expression of peripheral nociceptive markers, but chronic administration aggravated the pain-related molecular changes. On the contrary, E-52862 did not affect the expression of these markers after acute administration, but repeated treatment abolished central and peripheral long-term alterations associated with chronic osteoarthritic pain.

4 DISCUSSION

This work reveals that the selective σ1 receptor antagonist E-52862 has short- and long-term analgesic effects and reverses morphine tolerance in mice subjected to a model of chronic osteoarthritic pain. Our findings indicate dose-dependent, short-term antinociception observed 30 min after E-52862 and sustained recovery of the mechanical thresholds when the drug was repeatedly given. Importantly, electrophysiological recordings revealed that E-52862 inhibited central sensitization of spinal WDR neurons in osteoarthritic mice. These effects partly involved μ receptors and σ1 receptor antagonism reversed morphine tolerance during osteoarthritic pain, demnstrating a crosstalk between σ1 receptors and the opioid system. Biochemical assays identified common alterations of neuroinflammatory markers and glutamatergic signalling associated with chronic pain and repeated opioid exposure, both specifically inhibited by E-52862.

Acute E-52862 induced short-term inhibition of MIA-induced hypersensitivity, confirming the analgesic efficacy of the σ1 receptor antagonist observed in different pain models (Romero et al., 2016). E-52862 or vehicle treatments did not induce significant changes in mechanical sensitivity of sham mice, although minor variations associated with the intra-knee saline injections were occasionally observed. Thus, normal mechanosensitivity remains intact following σ1 receptor antagonism. In agreement, it was proposed that σ1 receptors do not have a primary role in physiological conditions (Su et al., 2010; Zamanillo et al., 2013). The repeated treatment with E-52862 also produced a gradual recovery of sensitivity, which was observed with all the doses tested and maintained after interrupting the treatment. Previous studies in mice investigated acute antinociceptive effects of E-52862 (González-Cano, Merlos, Baeyens, & Cendán, 2013; Gris et al., 2014; Romero et al., 2012) and its effects during chronic administration without testing mechanical allodynia before the daily dose of the compound (Bura, Guegan, Zamanillo, Vela, & Maldonado, 2013; Romero et al., 2012). Our work shows long-lasting restorative effects of the σ1 receptor antagonist, suggesting an additional benefit for long-term chronic pain treatments.

E-52862 showed different efficacy depending on the stage of the osteoarthritic pain sensitization. It was proposed that the initial mechanical hyperalgesia in osteoarthritis is caused by inflammatory processes, while later stages involve neuropathic mechanisms (Thakur, Rahman, Hobbs, Dickenson, & Bennett, 2012). In agreement, rat models showed an increase of the nerve injury marker ATF-3 in the DRG between 8 and 14 days after MIA (Ivanavicius et al., 2007; Orita et al., 2011), and our mice increased expression of neuroinflammatory markers 15 days after intra-knee injury. The doses of E-52862 needed to exert acute antiallodynic effect 15 days after MIA were lower than on 1 day post-injection. Thus, acute effects of E-52862 were more prominent once the neuropathic component of osteoarthritic pain was established. Such intra-model differences agree with previous results showing higher efficacy of E-52862 after neuropathic injuries than during inflammatory pain (Gris et al., 2014; Romero et al., 2012). The long-lasting alleviation of pain required longer exposure to the σ1 receptor antagonist when persistent neuroinflammatory changes were present, although E-52862 was still able to restore normal sensitivity. Thus, the σ1 receptor antagonist showed higher short-term analgesic efficacy when pain had become chronic, while preserving its long-term effects on chronic pain sensitization.

In vivo electrophysiological recordings showed facilitation of the firing frequency of spinal WDR neurons after MIA, revealing central sensitization in mice with osteoarthritic pain. Our result in CD1 mice agrees with previous work using C57Bl/6 mice, which revealed similar MIA-induced responses to mechanical stimuli (Harvey & Dickenson, 2009). Furthermore, the lack of increased firing in response to thermal stimuli is in agreement with previous behavioural studies showing that MIA-injected mice do not exhibit consistent heat hyperalgesia (La Porta, Bura, Aracil-Fernández, Manzanares, & Maldonado, 2013). Interestingly, we found that intrathecal application of E-52862 reduced these MIA-facilitated responses. This is in line with previous ex vivo electrophysiological studies showing that E-52862 inhibited wind-up responses elicited after repeated nociceptor stimulation (Romero et al., 2012). In agreement, spinal cords of σ1 receptor knockout mice exhibited reduced wind-up responses compared to wild-type mice (de la Puente et al., 2009). Therefore, our experiments demonstrate that spinal central sensitization associated with osteoarthritis pain in live animals was reversed by the σ1 receptor antagonist.

It has been suggested that σ1 receptor agonists induce phosphorylation of μ receptors, a process involved in opioid tolerance (Rodríguez-Muñoz et al., 2015; Rodríguez-Muñoz, Cortés-Montero, Pozo-Rodrigálvarez, Sánchez-Blázquez, & Garzón-Niño, 2015). We found modulation, by σ1 receptors, of opioid analgesia during chronic osteoarthritic pain, showing that a single sub-effective dose of E-52862 co-administered with morphine restored opioid antinociception. Earlier preclinical research had shown this modulatory role in the absence of chronic pain, using σ1 receptor knockout mice and σ1 receptor antagonists (Chien & Pasternak, 1994; Sánchez-Fernández et al., 2013; Sánchez-Fernández et al., 2014). Moreover, E-52862 demonstrated efficacy restoring morphine analgesia in tolerant animals with acute nociceptive and inflammatory pain (Montilla-García et al., 2019; Rodríguez-Muñoz, Sánchez-Blázquez, et al., 2015; Vidal-Torres et al., 2013). Surprisingly, there were no previous studies assessing the role of σ1 receptors in modulation of opioid analgesia under conditions of chronic pain. Our data suggest that σ1 receptor antagonists could be efficient not only alleviating pain by themselves but also restoring opioid analgesia in tolerant individuals. Taking into account that opioid tolerance drives dose escalation and abuse, and E-52862 did not produce tolerance development during this long-term treatment and it is void of reinforcing effects in animals without pain (Bura et al., 2013), the σ1 receptor antagonist could represent a valuable alternative for chronic pain treatment. Considering the lifespan of mice (2.5 years) and humans (80 years), the length of the treatment used in this study could be compared to the duration of chronic pain treatments that produce adverse events in patients.

We observed inhibition of the acute and sustained effects of E-52862 after administration of the σ1 receptor agonist PRE-084. Previous studies using pharmacological and genetic approaches demonstrated that acute analgesic effects of E-52862 are selectively mediated by σ1 receptors (Gris et al., 2014; Sánchez-Fernández et al., 2014). Our work also showed that long-term restoration of mechanical thresholds is also σ1 receptor-dependent. Interestingly, naloxone also diminished the acute and sustained antinociceptive effects of E-52862 revealing opioid participation. In addition, morphine-tolerant mice showed decreased E-52862 efficacy. antagonism of σ1 receptors facilitates σ1 receptor- μ receptor binding and protects μ receptors from phosphorylation, thus preserving their activity. Such phosphorylation is enhanced after persistent stimulation of μ receptors (Rodríguez-Muñoz, Cortés-Montero, et al., 2015, Rodríguez-Muñoz, Sánchez-Blázquez, et al., 2015). As both naloxone and μ receptor desensitization after chronic morphine decreased the antinociceptive effects of E-52862, it can be concluded that part of the analgesic effects of the σ1 receptor antagonist rely on the enhancement of endogenous μ receptor activity.

We investigated pain-related molecular alterations involved in morphine and E-52862 analgesia during osteoarthritis. One day after MIA, BDNF and NPY over-expression was observed in the DRG and TNF-α expression increased in the spinal cord. Previous studies reported similar changes after nerve injuries, including immediate increases in BDNF and TNF-α expression that persisted for long-term periods (Ohtori, Takahashi, Moriya, & Myers, 2004; Uchida, Matsushita, & Ueda, 2013). Several researchers also showed prominent NPY up-regulation in nerve-injured primary afferents (Benoliel, Eliav, & Iadarola, 2001; Son et al., 2007). This synthesis de novo could represent an adaptive response to nociceptive sensitization (Munglani et al., 1995). While acute morphine inhibited pain-related over-expression of BDNF and NPY, a single E-52862 administration did not provoke such effects, suggesting that its acute effects do not rely on these molecules.

Later stages of osteoarthritic pain involved pronounced increases of IL-1β and NPY levels in spinal cord or DRG, accompanied by enhancement of microglial marker Iba1. In addition, the enhanced levels of BDNF and TNF-α were maintained in DRG and spinal cord respectively. Thus, the intra-knee injury induced persistent changes in neuroinflammatory mediators possibly contributing to the osteoarthritic phenotype. These pain-related changes were reported to increase glutamate release and stimulate the glutamatergic system (Takeuchi, 2013; Vaz, Lérias, Parreira, Diógenes, & Sebastião, 2015). In agreement, osteoarthritic mice showed over-expression of mGlu₅ receptors, which is associated with increased glutamate levels in the nervous system (Wang, Wang, Zhong, Li, & Cong, 2012). As previously described, chronic morphine further increased neuroinflammation and glutamatergic signalling (Cabañero et al., 2013; Johnston et al., 2004), characterized by exacerbated BDNF, IL-1β, spinal microgliosis, and AMPA receptor expression. Interestingly, increased BDNF/TrkB signalling contributes to chronic pain by eliciting microglial reactivity and glutamate release (Zhou et al., 2011). At the same time, IL-1β and TNF-α are mainly released by activated microglia, which also liberate BDNF after chronic morphine (Takayama & Ueda, 2005). Thus, repeated morphine contributed to an overall increase of spinal neuroinflammation. In sharp contrast, chronic E-52862 normalized the expression of BDNF and proinflammatory cytokines. These findings agree with previous studies revealing potentiation of BDNF effects after over-expression of σ1 receptors (Yagasaki et al., 2006) or chronic σ1 receptor activation (Kikuchi-Utsumi & Nakaki, 2008). Likewise, recent experiments showed normalization of TNF-α and IL-1β expression in spinal cord-injured σ1 receptor knockout mice (Castany et al., 2018). E-52862 also reduced MIA-induced microgliosis, consistent with the effects of the σ1 receptor antagonist BD1047 attenuating spinal microgliosis in a model of bone cancer pain (Zhu et al., 2015) and with the high levels of σ1 receptors reported in microglia (Gekker et al., 2006). Interestingly, E-52862 was effective in preventing the increased microglial density induced by MIA in supraspinal structures (Carcolé et al., 2019). In addition, over-expression of mGlu₅ receptors was completely abolished by E-52862. Hence, contrary to chronic morphine, repeated treatment with the σ1 receptor antagonist normalized the expression of neuroinflammatory mediators and glutamate receptors involved in chronic osteoarthritic pain.

In summary, the present study shows a dual effect of the σ1 receptor antagonist E-52862 alleviating pain in our model of osteoarthritis. On the one hand, the σ1 receptor antagonist reduced acute mechanical allodynia, involving inhibition of spinal sensitization without modification of pain-related molecular alterations. On the other hand, repeated E-52862 exposure induced gradual recovery of the mechanical thresholds in osteoarthritic mice without inducing tolerance. This effect was associated with inhibition of biochemical changes related to osteoarthritic pain and opioid tolerance. Such alterations involve neuroinflammatory mediators, microglial reactivity and glutamatergic signalling, which could constitute a common pathway by which the σ1 receptor antagonist provided relief of chronic pain and restoration of opioid analgesia in tolerant individuals. Hence, the σ1 receptor antagonist could dampen deleterious side effects of opioid prescription drugs, which has reached now dramatic consequences in the United States (Lyden & Binswanger, 2019), and represents a promising alternative to opioids in chronic pain conditions requiring long-term treatment with analgesics.