4. Discussion and Conclusions
Targeting MOPs in the peripheral nervous system represents a novel approach to the clinical management of acute and chronic pain. Critical to this practice, however, is a thorough understanding of these receptors’ effects on ongoing pain-induced behavior and opioid analgesia. The selective deletion of MOPs in the DRG of primary sensory neurons enabled us to evaluate the functional contribution of these receptors to acute nociceptive, inflammatory, and neuropathic pain. By examining behavioral responses to noxious stimuli and the effects of exogenous opioid agonists, we found that the absence of MOPs in primary sensory neurons (1) does not alter the behavioral response to acute nociceptive stimuli or the development of inflammation-induced thermal hyperalgesia, (2) exacerbates nerve injury-induced mechanical allodynia, (3) abolishes the analgesic effect of DALDA, and (4) attenuates the analgesic effect of morphine.
In naïve Oprm1 cKO and WT mice, MOP expression in intestine and periaqueductal gray was similar, as was paw withdrawal response to mechanical and thermal stimuli. Prior research has shown that MOP immunoreactivity in the gastrointestinal tract of rodents is confined primarily to the myenteric plexus and that MOP myenteric neurons are most numerous in the small intestine, followed by the stomach and proximal colon (Sternini, Patierno, Selmer & Kirchgessner, 2004). Intestinal MOP expression was unaltered in ourOprm1 cKO animals. Global expression of MOP in the spinal cord (ventral and dorsal regions) did not decrease; however, MOP immunoreactivity was significantly reduced in the superficial laminae. This finding supports previous research carried out with Nav1.8Cre/+::Oprm1fl/flanimals (Severino et al., 2018). As behavioral response and sensitivity to acute noxious stimuli were unaffected by the absence of peripheral MOPs, our findings suggest that endogenous tone at peripheral MOPs does not modulate response to acute noxious stimuli. These results are consistent with previous studies showing comparable baseline nociceptive sensitivity in Nav1.8Cre/+::Oprm1fl/fl, TPRV1Cre/+::Oprm1fl/fl, AdvillinCre/+::Oprm1fl/fl, and control Oprm1fl/fl animals (Corder et al., 2017; Martinez-Navarro et al., 2020; Severino et al., 2018; Sun, Chen, Chen & Pan, 2019; Weibel et al., 2013)
By examining motor coordination, ataxia, and balance with the rotarod test, we showed that the absence of MOPs in primary sensory neurons does not affect gross motor function or ambulation under naïve conditions. However, our findings from the open field exploration assay are worth noting. Both male and female MOP cKO mice exhibited significantly reduced spontaneous exploration and increased thigmotaxis (the tendency to remain close to walls), thereby mimicking behavior consistent with enhanced anxiety (Seibenhener & Wooten, 2015). Previously, mice deficient in Preproenkephalin , anOprm1 pre-cursor gene, were found to display similar behavior, including increased anxiety and offensive aggressiveness (Konig et al., 1996).
Under naïve conditions, the Oprm1 cKO mice, like AdvillinCre/+::Oprm1fl/fl mice (Sun, Chen, Chen & Pan, 2019), did not show opioid-induced analgesia during acute nociceptive stimuli. Although TRPV1- and Nav1.8-expressing DRG neurons have widespread distribution and overlap, neither TPRV1Cre/+::Oprm1fl/fl nor Nav1.8Cre/+::Oprm1fl/fl mice showed a change in morphine-induced analgesia for acute pain, despite reduced MOP expression in the DRG. These results may be due in part to the different behavioral assays used to evaluate pain response (Corder et al., 2017; Weibel et al., 2013). Our observations suggest that an opioid-induced analgesic response to thermal and mechanical nociception may require MOP activation across multiple subpopulations of primary sensory neurons. As previously reported, MOPs present in TRPV1-expressing neurons are primarily associated with opioid-induced hyperalgesia (i.e., opposing opioid analgesia), as the opioid analgesic effect is significantly enhanced when TRPV1-expressing neurons are removed (Chen & Pan, 2006; Chen, Prunean, Pan, Welker & Pan, 2007). Therefore, deletion of MOPs in TRPV1-expressing neurons may reveal that opioid-induced analgesia is a result of MOP activation in different subpopulations of DRG neurons.
In the CFA-induced pain model, Oprm1 cKO and WT animals developed comparable paw edema and thermal hyperalgesia, suggesting that MOPs in primary sensory neurons do not affect the inflammatory process or the subsequent development of inflammatory hyperalgesia. Of note, studies with Nav1.8Cre/flMOP animals showed that CFA-induced mechanical allodynia was comparable between groups until 1-week post-CFA. From 2 weeks post-CFA, MOP Nav1.8 cKOs exhibited enhanced sensitivity compared to their control counterparts (Severino et al., 2018).
In the periphery, endogenous mechanisms counteracting pain and inflammation arise from interaction between leukocyte-derived opioid peptides and opioid receptors on peripheral endings of primary afferent neurons, as well as anti-inflammatory cytokines (Celik et al., 2016; Labuz, Mousa, Schäfer, Stein & Machelska, 2007; Rittner et al., 2006; Shaqura, Zollner, Mousa, Stein & Schafer, 2004; Stein & Machelska, 2011; Stein, Schafer & Machelska, 2003). Our observations suggest that the peripheral sensitization of nociceptors by inflammatory mediators such as prostaglandins, bradykinin, and other cytokines may be independent of the influence of neuronal MOP-mediated modulatory mechanisms.
We observed that Oprm1 cKO animals developed robust ipsilateral and contralateral mechanical allodynia after peripheral nerve injury, whereas WT mice exhibited only ipsilateral allodynia. This finding is consistent with our earlier studies, which showed similar bilateral mechanical allodynia after spinal nerve injury in global MOP knockout mice and in WT mice administered naloxone (Mansikka, Zhao, Sheth, Sora, Uhl & Raja, 2004). Previous studies have also shown a bilateral increase in MOP binding in spinal dorsal horn of rats with unilateral chronic constriction injury-induced neuropathy (Stevens, Kajander, Bennett & Seybold, 1991).
It is unclear what signaling mechanisms link the two sides of the body and mediate an effect on contralateral nonlesioned structures in the presence of peripheral neuropathy. Potential mechanisms of contralateral change have been differentiated based on CNS and peripheral nervous system signaling (Koltzenburg, Wall & McMahon, 1999). In the periphery, it has been postulated that bilateral changes are mediated by circulating factors. This pathway would suggest that, after unilateral axotomy, breakdown products from the damaged nerve or denervated tissue may circulate in the bloodstream and induce changes in contralateral neuronal populations. Alternatively, contralateral effects may be governed by transmedian sprouting, possibly in the spinal cord. Thus, in midline structures and other tissues receiving bilateral inputs, unilateral nerve lesions might result in collateral sprouting of nearby and contralateral neurons. Such sprouting may also cause other changes in these contralateral neurons. Although additional studies are required to fully clarify the relationship between peripheral neuropathy and bilateral nociceptive signaling, our findings indicate that MOPs in primary sensory neurons play a critical role in the development of tonic bilateral inhibition of pain after nerve injury.
Previous investigations have shown that the analgesic effect of morphine on CFA-induced hypersensitivity is reduced in Nav1.8Cre/+::Oprm1fl/flmice (Weibel et al., 2013). Similarly, we showed that the effects of morphine on inflammatory and neuropathic pain behaviors were attenuated in Oprm1 cKO mice, indicating that systemic opioids act in part via peripheral MOPs on sensory neurons. These data provide substantial evidence that MOPs in primary sensory neurons play a critical role in systemic opioid analgesia under conditions of inflammatory and neuropathic pain. Of note, one study (Sun, Chen, Chen & Pan, 2019) reported that morphine was completely unable to induce analgesia in Oprm1cKO animals under inflammatory pain conditions. Though they observed a slight increase in paw withdrawal threshold, it was not statistically significant. The differences in our findings may be due to the different animal models used. Nonetheless, these results are in line with previously reported genetic and pharmacological data (Labuz, Mousa, Schäfer, Stein & Machelska, 2007; Weibel et al., 2013), which suggest that peripheral MOPs mediate mechanisms for certain doses of morphine. Moreover, our study is consistent with previous reports showing that peripheral nerve injury markedly reduces MOP expression in the DRG and represses the analgesic effect of systemically administered opioids via epigenetic mechanisms involving G9a and neuron-restrictive silence factor (Uchida, Sasaki, Ma & Ueda, 2010; Xu et al., 2020).
Studies involving transgenic mouse models are commonly used to ascertain the role of distinct receptor mechanisms in pain signaling. We acknowledge that the evidence put forth by our Pirt-Cre+/-::Oprm1fl/fl mice must be interpreted with caution, on account of potential confounding and/or compensatory factors. Deleting the Oprm1 gene in primary sensory neurons may lead to changes in the expression of other genes or nonspecific factors, thereby affecting the behavior of our transgenic animals. Moreover, the deletion of MOP expression in DRG neurons was dependent on the expression and efficiency of Cre. It is important to note that not all MOP-expressing DRG neurons express Cre and therefore were not under control of the Pirt promoter.
In summary, we assessed the functional role of MOPs in inflammatory and neuropathic pain using a new MOP-flox-Pirt-Cre mouse model that lacked these receptors in all primary sensory neurons. We found that these peripheral MOPs mediated all of the analgesia induced by the peripheral opioid DALDA in inflammatory and neuropathic pain states and attenuated the analgesia induced by morphine. These findings enhance our current understanding of the functional significance of peripheral MOPs and can aid in the development of potent new therapeutic agents that are devoid of the deleterious side effects associated with systemic opioids that cross the blood-brain barrier.