Neuropathic pain is a debilitating chronic pain condition resulting from a lesion or disease of the somatosensory system (Colloca et al., 2017). The prevalence of neuropathic pain in the general population has been estimated in the range of 6.9–10% (van Hecke et al., 2014), and it is expected to rise in the future (Colloca et al., 2017). Despite the enormous efforts devoted to both clinical and preclinical research, neuropathic pain treatment remains an unmet clinical need (Finnerup et al., 2015).

The sigma-1 (σ₁) receptor is a unique ligand-operated chaperone expressed at high levels in several key pain control areas in both the peripheral and central nervous system, where it interacts with different receptors and ion channels to modulate them (Su et al., 2016; Sánchez-Fernández et al., 2017). The pharmacology of σ₁ receptors has been deeply studied, and there are currently selective σ₁ agonists (such as PRE-084) and antagonists (such as S1RA), to study σ₁ receptor function (Sánchez-Fernández et al., 2017). Substantial evidence points to a prominent role for these receptors in neuropathic pain of diverse etiology (Merlos et al., 2017; Sánchez-Fernández et al., 2017), and shows that pain-like behaviors are attenuated in σ₁-knockout (KO) mice (de la Puente et al., 2009; Nieto et al., 2012) and in wild-type (WT) animals treated with σ₁ receptor antagonists (Roh et al., 2008; Nieto et al., 2012; Romero et al., 2012; Gris et al., 2016; Kang et al., 2016). The mechanisms involved in the antineuropathic effects of σ₁ inhibition are only partially known and have been well studied in the central nervous system, specifically in the dorsal spinal cord, where these receptors control central sensitization (reviewed in Sánchez-Fernández et al., 2017).

There are differences between the pathophysiological mechanisms and responses to drug treatment for neuropathic pain induced by different types of injury to the peripheral nerves (Aley and Levine, 2002; Baron et al., 2010; Hershman et al., 2014; Finnerup et al., 2015). In particular, different neuroplastic changes (Casals-Díaz et al., 2009) and gene expression profiles (Griffin et al., 2007; Costigan et al., 2010) have been reported after denervation or constriction/ligation of the sciatic nerve. Surgical interventions inevitably results in nerve transection, and as a consequence, significant number of patients experience neuropathic pain (Borsook et al., 2013). However, all studies to date on the role of σ₁ receptors in neuropathic pain after mechanical injury to peripheral nerves has focused on models of sciatic nerve constriction/ligation (Roh et al., 2008; de la Puente et al., 2009; Espinosa-Juárez et al., 2017a); thus the role of σ₁ receptors in neuropathic pain induced by nerve transection has never been explored. Transection of the tibial and common peroneal branches of the sciatic nerve results in persistent neuropathic pain in rodents, manifested by marked hypersensitivity in the territory of the intact sural branch. Hence, this neuropathic pain model is termed the spared nerve injury (SNI) model (Decosterd and Woolf, 2000).

In view of these antecedents, the first goal of the present study was to test whether the inhibition of σ₁ receptors alleviated the painful hypersensitivity associated with SNI-induced neuropathic pain. This was investigated by comparing SNI-induced neuropathic hypersensitivity in WT and σ₁-KO mice, and by testing the effects, in animals with neuropathy, of the acute and repeated administration of the selective σ₁ antagonist S1RA, which is currently under clinical development for the treatment of neuropathic pain (Abadias et al., 2013; Bruna et al., 2018).

Opioid receptors have been described as part of the interactome of σ₁ receptors. This is relevant since σ₁ receptors physically interact with opioid receptors restraining their functioning (reviewed in Sánchez-Fernández et al., 2017), so that σ₁ receptor inhibition enhances analgesia induced by opioid drugs in nociceptive pain at both central (Mei and Pasternak, 2002) and peripheral sites (Sánchez-Fernández et al., 2013; Sánchez-Fernández et al., 2014; Prezzavento et al., 2017), and can increase the antihyperalgesic effects of endogenous opioid peptides (EOPs) produced naturally by immune cells that accumulate at the inflamed site to relieve inflammatory pain (Tejada et al., 2017). During neuropathic pain there is a prominent recruitment of immune cells harboring EOPs at both peripheral and central sites (reviewed in Ref. Tejada et al., 2018). However, whether σ₁ receptors modulate endogenous opioid analgesia in neuropathic pain remains completely unknown. Therefore, the second goal of this study was to evaluate the possible contribution of the endogenous opioid system to the antineuropathic effects induced by S1RA in the mouse model of SNI.

The main findings of the present study are that: 1) pharmacological antagonism or genetic inactivation of σ₁ receptors reduces neuropathic pain induced by peripheral nerve transection (SNI model); 2) the ameliorative effects on SNI-induced hypersensitivity to mechanical and heat stimuli (but not to cold stimuli) produced by σ₁ receptor antagonism are mediated by modulation of the endogenous opioid system; and 3) repeated treatment with S1RA induces prolonged ameliorative effects which lasted longer than the presence of the drug in the organism.

Basal sensitivity to mechanical, cold, and heat stimulation in mice lacking σ₁ receptors, in the absence of nerve injury, did not differ from that in WT mice. This is in agreement with previous studies (de la Puente et al., 2009; Nieto et al., 2012) and suggests that the basic mechanisms of nociceptive transduction are intact in mice lacking σ₁ receptors. We showed that WT mice after SNI surgery developed mechanical, cold, and heat hypersensitivities with time courses similar to those previously reported in mice (Bourquin et al., 2006; Guida et al., 2015; Liu et al., 2015; Cobos et al., 2018). In addition, the extent of the sensory hypersensitivity was similar between female and male WT mice. In mice lacking σ₁ receptors from either sex, however, SNI surgery induced a very different pattern of painful hypersensitivity: these mutant mice did not develop cold allodynia and showed significantly less mechanical allodynia, whereas they developed heat hyperalgesia normally. This is consistent with previous work in other neuropathic pain models, which found that σ₁-KO mice had significantly reduced mechanical and cold allodynia induced by chemotherapy (Nieto et al., 2012) or by partial sciatic nerve ligation (de la Puente et al., 2009); the latter study also found that heat hyperalgesia developed normally (de la Puente et al., 2009). However, it was recently reported that σ₁-KO mice showed, in addition to the reduction in mechanical allodynia, a significant attenuation of heat hypersensitivity induced by spinal cord injury (Castany et al., 2018) or diabetic neuropathy (Wang et al., 2018). Taken together, studies with σ₁-KO mice suggest that the role of σ₁ receptors during neuropathic pain depends on the sensory modality explored and the type of injury.

The acute pharmacological antagonism of σ₁ receptor with S1RA administration, once the painful neuropathy was fully established in WT mice, significantly (although partially) attenuated mechanical allodynia and fully reversed cold hypersensitivity induced by SNI. In addition, and in contrast to our findings with σ₁-KO mice, acute S1RA administration also abolished SNI-induced heat hyperalgesia in female or male WT mice. Previous studies in other neuropathic pain models reported that the systemic acute administration of σ₁ antagonists (including S1RA) reversed not only neuropathic cold and mechanical allodynia (Nieto et al., 2012; Romero et al., 2012; Gris et al., 2016; Espinosa-Juárez et al., 2017b; Paniagua et al., 2017; Castany et al., 2018; Wang et al., 2018) but also heat hyperalgesia (Díaz et al., 2012; Romero et al., 2012; Paniagua et al., 2017; Castany et al., 2018; Wang et al., 2018), findings which are also in clear agreement with our results. Therefore, there is an apparent divergence between the reduction in heat hyperalgesia in S1RA-treated WT mice, and the normal development of heat hypersensitivity in σ₁-KO mice after SNI. This inconsistency cannot be explained by a nonspecific off-target effect of S1RA, because here we show that the acute ameliorative effects induced by S1RA in all three sensory modalities explored in the present study were abolished by the selective σ₁ agonist PRE-084, which did not show any effects “per se,” as previously described in a different neuropathic pain model (Espinosa-Juárez et al., 2017b). In addition, the administration of S1RA to σ₁-KO mice had no effect on SNI-induced mechanical and heat hypersensitivity. Therefore, these results argue in favor of a σ₁-mediated action induced by this drug. In fact, it is known that S1RA has exquisite selectivity for σ₁ receptors (Romero et al., 2012). The discrepancy between the effect of genetic and pharmacological inhibition of σ₁ receptors on heat hypersensitivity was also found in other studies of neuropathic pain (de la Puente et al., 2009; Romero et al., 2012) and during inflammatory pain (Tejada et al., 2014). In addition, conflicting results between σ₁ knockout and pharmacological antagonism have been reported in the modulation of opioid-induced analgesia in nociceptive heat pain (Vidal-Torres et al., 2013). Studies of pain mechanisms that used the genetic and pharmacological inhibition of other receptors have also obtained contradictory results (Petrus et al., 2007; Bonin et al., 2011), which have been attributed to compensatory mechanisms developed by mutant mice. Therefore, this issue appears to be a general concern in experiments with knockout animals. Taking into account these antecedents, we suggest that σ₁-KO mice develop these purported compensatory mechanisms in heat pain pathways but not in other pain pathways in which the knockout replicated the effects of σ₁ antagonists.

After peripheral nerve injury, neuroinflammatory processes occur with the recruitment of myriad immune cells at the site of injury (Treutlein et al., 2018) and in the dorsal root ganglia (Kwon et al., 2013), where the somas of peripheral sensory neurons are located. These immune cells release a variety of inflammatory mediators that contribute to neuropathic pain, but they also produce EOPs which have analgesic potential (reviewed in Refs. Ji et al., 2014; Stein, 2016; Tejada et al., 2018). In our experimental conditions, opioid antagonism caused by the administration of naloxone or its peripherally restricted analog naloxone methiodide did not exacerbate pain hypersensitivity in any sensory modality explored, suggesting that the tonic endogenous activity of the opioid system is limited in SNI mice. Importantly, the ameliorative effects induced by S1RA in SNI-induced mechanical and heat hypersensitivity were reversed by both naloxone and naloxone methiodide. These results suggest that σ₁ inhibition ameliorates SNI-induced mechanical and heat hypersensitivity through a mechanism dependent on peripherally produced EOPs, whose actions are tonically limited by σ₁ receptors. This dependence on the peripheral opioid system of the effects induced by S1RA on mechanical and heat hypersensitivity was seen in both female and male mice, indicating a lack of sexual dimorphism in these effects. It was recently reported that σ₁ antagonism produced opioid-dependent antihyperalgesic effects during inflammation by enhancing the action of EOPs released by immune cells that accumulate at the inflamed site (Tejada et al., 2017). Interestingly, σ₁ receptors are expressed in the somas of all peripheral sensory neurons (Mavlyutov et al., 2016; Montilla-García et al., 2018), at a much higher density than in central pain-related areas (Sánchez-Fernández et al., 2014). In light of these antecedents, it can be hypothesized that our results for the effects of S1RA on neuropathic mechanical and heat hypersensitivity may also be attributable to the enhancement, by σ₁ antagonism, of the peripheral antinociceptive actions of EOPs produced by immune cells. Further research is guaranteed to determine the exact EOP/EOPs involved in the opioid-dependent effects induced by σ₁ antagonism during neuropathic pain.

It has been reported that peripheral immune cells do not contribute equally to every modality of sensory hypersensitivity after peripheral nerve injury. In fact, peripheral macrophages and T-cells promote both mechanical allodynia and heat hyperalgesia (Kobayashi et al., 2015; Cobos et al., 2018), whereas their influence in cold allodynia is very limited, which suggest that it is due to neuronal mechanisms rather than to neuroimmune interactions (Cobos et al., 2018). Here we show that the effects of S1RA on cold allodynia in either female or male mice are independent of opioid activation, which is consistent with the known inhibitory effects of S1RA on neuronal sensitization (Romero et al., 2012; Paniagua et al., 2017). In this connection it was previously reported that the effects of σ₁ antagonists in other pain models such as capsaicin-induced secondary hypersensitivity (Entrena et al., 2009) or formalin-induced pain (Tejada et al., 2017) were not sensitive to naloxone treatment. Taking into account the wide variety of protein partners (other than opioid receptors) that benefit from the chaperoning actions of σ₁ receptors (reviewed in Refs. Su et al., 2016; Sánchez-Fernández et al., 2017), it is not surprising that multiple opioid and nonopioid mechanisms simultaneously participate on the ameliorative effects of σ₁ antagonism.

We also found that whereas morphine only partially reversed mechanical allodynia, it was able to fully suppress heat and cold hypersensitivity induced by SNI—effects which resemble those induced by S1RA. The effects of morphine on mechanical and heat hypersensitivity, but not on cold allodynia, were sensitive to the peripheral opioid antagonist naloxone methiodide. These results suggest that peripheral opioid effects are markedly weaker in cold allodynia than in mechanical or heat hypersensitivity, and are consistent with the absence of peripherally mediated opioid effects on the inhibition of cold allodynia induced by S1RA.

We also tested the effects of the repeated administration of S1RA, which was administered preemptively before surgery and subsequently administered twice a day during the next 9 days. Plasma levels of S1RA in mice after repeated treatment with this drug were similar to or lower than the levels of this drug in humans after daily oral S1RA treatment at therapeutic doses (Abadias et al., 2013; Bruna et al., 2018). We found that the sustained administration of S1RA induced prolonged ameliorative effects on the hypersensitivity to mechanical, heat, and cold stimuli, without any evidence of tolerance to the antineuropathic effects during the evaluation period. This may be relevant given that some effects of S1RA, as shown in the present study, involve the opioid system, and it is well known that sustained opioid treatment induced analgesic tolerance (Morgan and Christie, 2011). Therefore, although we show that the effects of σ₁ antagonism are partially mediated by the opioid system, this does not necessarily imply the development of analgesic tolerance.

We evaluated the effects of the repeated administration of S1RA starting before neuropathic pain was established, and found that it had marked effects on mechanical, heat, and cold hypersensitivity. However, when the same dose of S1RA was acutely administered once neuropathic pain was fully established, its effects were limited and observed only in cold allodynia. Therefore, S1RA showed higher efficacy after repeated treatment than after a single treatment. It is unlikely that the greater effects induced by repeated treatment of S1RA were due to drug accumulation, since we previously showed that repeated treatment with the same protocol as in the present study did not result in increased levels of S1RA with time (Romero et al., 2012). In addition, here we show that 12 h after treatment was discontinued, there were no appreciable levels of S1RA in either plasma or brain tissue, indicating the complete elimination of this compound between doses. Interestingly, although no S1RA remained in the organism 12 h after its last administration, drug effects were still significantly evident in all three sensory modalities. Our results are consistent with previous studies in which the repeated administration of σ₁ antagonists (including S1RA) consistently induced a long-lasting reduction of the development of mechanical, cold, and heat hypersensitivity in models of neuropathic pain of different etiologies (Nieto et al., 2012; Gris et al., 2016; Paniagua et al., 2017). It is unclear whether these prolonged effects induced by the repeated treatment with S1RA might be due to the production of an active metabolite not detected in our determinations. However, it is known that σ₁ receptors can influence gene transcription, which might account for the long-lasting effects observed (Tsai et al., 2015). In fact, repeated treatment with the σ₁ agonist PRE-084, which is chemically unrelated to S1RA, had long-lasting proallodynic effects (Entrena et al., 2016). Taken together, these results suggest that the repeated treatment with σ₁ ligands might have sustained pharmacodynamic effects, not restricted to S1RA and its possible active metabolites, although further experiments are needed to clarify this issue. Regardless of the precise mechanism, our data suggest that repeated treatment with S1RA may have potential therapeutic utility in the context of neuropathies induced by nerve transection during surgery, when the precise time of nerve injury can be anticipated and preventive treatment can be given.

In summary, this study demonstrates that σ₁ antagonism may be a potentially effective therapeutic tool to inhibit neuropathic pain induced by peripheral nerve transection. In addition, our findings support the notion that σ₁ antagonism induces both opioid-dependent and -independent effects during neuropathic pain.

EJC and FRN designed research; IB-C, GP, SY, DC, and FRN performed research; IB-C, SY, FRN, and JMB analyzed data; IB-C, JMB, EJC, and FRN wrote the paper. All authors read and approved the final version of the manuscript.

IB-C was supported by an FPU grant from the Spanish Ministry of Education, Culture, and Sports. This study was partially supported by the Spanish Ministry of Economy and Competitiveness (MINECO, grant SAF2016-80540-R), the Junta de Andalucía (grant CTS 109), and funding from Esteve and the European Regional Development Fund (ERDF). This research was done in partial fulfillment of the requirements for the doctoral thesis of IB-C.

SY was employed by Esteve.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The authors declare that this study received funding from Esteve. This company had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.