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.