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. Author manuscript; available in PMC: 2021 Oct 15.
Published in final edited form as: Neuroscience. 2020 Aug 25;446:102–112. doi: 10.1016/j.neuroscience.2020.08.022

Topical application of loperamide/oxymorphindole, mu and delta opioid receptor agonists, reduces sensitization of C-fiber nociceptors that possess NaV1.8

Megan L Uhelski 1, Daniel Bruce 2, Rebecca Speltz 1,2, George L Wilcox 2,3,4, Donald A Simone 1
PMCID: PMC7532699  NIHMSID: NIHMS1629426  PMID: 32858141

Abstract

It was recently shown that local injection, systemic administration or topical application of the peripherally-restricted mu-opioid receptor (MOR) agonist loperamide (Lo) and the delta-opioid receptor (DOR) agonist oxymorphindole (OMI) synergized to produce highly potent anti-hyperalgesia that was dependent on both MOR and DOR located in the periphery. We assessed peripheral mechanisms by which this Lo/OMI combination produces analgesia in mice expressing the light-sensitive protein channelrhodopsin2 (ChR2) in neurons that express NaV1.8 voltage-gated sodium channels. These mice (NaV1.8-ChR2+) enabled us to selectively target and record electrophysiological activity from these neurons (the majority of which are nociceptive) using blue light stimulation of the hind paw. We assessed the effect of Lo/OMI on nociceptor activity in both naïve mice and mice treated with complete Freund’s adjuvant (CFA) to induce chronic inflammation of the hind paw. Teased fiber recording of tibial nerve fibers innervating the plantar hind paw revealed that the Lo/OMI combination reduced responses to light stimulation in naïve mice and attenuated spontaneous activity as well as responses to light and mechanical stimuli in CFA-treated mice. These results show that Lo/OMI reduces activity of C-fiber nociceptors that express NaV1.8 and corroborate recent behavioral studies demonstrating the potent analgesic effects of this drug combination. Because of its peripheral site of action, Lo/OMI might produce effective analgesia without the side effects associated with activation of opioid receptors in the central nervous system.

Keywords: hyperalgesia, nociceptor sensitization, antinocicepton, electrophysiology, opioids

INTRODUCTION

Opioids are widely used for relief of chronic pain but are associated with a variety of serious side effects including constipation, sedation, pruritus, tolerance, dependence, addiction, and respiratory depression that can lead to death (Benyamin et al., 2008). Many of these side effects are the consequence of activation of opioid receptors in the central nervous system (Bates et al., 2004). However, opioid receptors, including mu- and delta-opioid receptors (MOR and DOR, respectively), are also expressed in the periphery by nociceptive dorsal root ganglion neurons (Werz et al., 1983; Obara et al., 2009) and on the peripheral terminals of nociceptive afferent fibers (Parsons et al., 1990; Coggeshall et al., 1997). MORs are highly expressed in both myelinated and unmyelinated peptidergic DRG neurons (Minami et al., 1995). DORs are expressed in a small proportion of small-sized DRG neurons that tend to be non-peptidergic C-fibers (Minami et al., 1995; Scherrer et al., 2009; Bardoni et al., 2014; Francois & Scherrer, 2017). Co-expression has been demonstrated in a subset of peptidergic nociceptive DRG neurons; however, the proportion of neurons identified as expressing both MOR and DOR has not been consistent across studies (Erbs et al., 2015; Scherrer et al., 2009; Wang et al., 2010; Rau et al., 2005). Activation of MOR and DOR in the periphery reduced hyperalgesia produced by inflammation (Stein, 1995; Zhou et al., 1998; Pacheco et al., 2005) and nerve injury (Truong et al., 2003; Kabli & Cahill, 2007), and decreased sensitization of nociceptors (Andreev et al., 1994; Cook & Nickerson, 2005). Thus, targeting multiple peripheral opioid receptor subtypes may produce analgesic efficacy without the side effects associated with activation of these receptors in the central nervous system.

Recent studies have shown that co-activation of certain G protein-coupled receptors (GPCRs) in the central and peripheral terminals of nociceptors frequently resulted in analgesic synergy, allowing 10–100-fold reduction in doses, increased analgesic efficacy and reduced side effect profiles (Janson & Stein, 2003, Stein & Lang, 2009). Identifying drug pairs that yield potent analgesic synergy and understanding the underlying cellular signaling mechanisms is one approach to improve therapeutic outcomes by maximizing analgesia while minimizing drug doses and side effects. For example, several behavioral studies have shown that analgesia following co- administration of MOR and DOR agonists was greater than that following the individual agonists (Sutters et al., 1990; Miaskowski et al., 1992; Joseph & Levine, 2010; Schramm & Honda, 2010; Schuster et al., 2015). Wilcox and colleagues (Bruce et al., 2019) recently showed that intraplantar injection or topical application of the peripherally-restricted MOR agonist loperamide (Lo) and the DOR agonist oxymorphindole (OMI)(Portoghese et al., 1988) synergized to produce potent antinociception to heat and mechanical stimuli in behavioral studies. In naïve mice, the ED50 value for intraplantar administration of the Lo/OMI combination was 10 times lower than the theoretical additive ED50 value. In mice with inflammation of the hind paw produced by complete Freund’s adjuvant (CFA), Lo/OMI produced both thermal and mechanical anti-hyperalgesia with 100-fold higher potency. Moreover, whereas topical application of Lo or OMI in inflamed mice exhibited similar antinociceptive potencies in behavioral studies, combined application was approximately 50 times more potent. Thus, peripheral application of Lo/OMI synergized to produce potent antinociception in behavioral studies through peripheral mechanisms.

In the present study we further characterized the effect of topical application of Lo/OMI on hyperalgesia by evaluating the responses of nociceptors under normal conditions and following inflammation. We used NaV1.8-ChR2+ mice that co-express the light-sensitive protein channelrhodopsin2 (ChR2) and the voltage-gated sodium channel NaV1.8. Using this approach, we recently characterized NaV1.8-containing primary afferent fibers in vivo and the majority (>70%) of recorded fibers were polymodal nociceptors (Uhelski et al., 2017a).

EXPERIMENTAL PROCEDURES

Subjects

Thirty-eight adult (>6 weeks old) mice of both sexes (n = 20 male, n = 18 female) were utilized in the current study. NaV1.8-ChR2+ mice were created as described previously (Stirling et al., 2005; Daou et al., 2013) using homozygous NaV1.8-Cre mice crossed with Ai32 mice carrying the ChR2(H134R)-EYFP gene in the Gt(ROSA)26Sor locus (Madisen et al., 2012) that is separated from its CAG promoter by a loxP-flanked transcription STOP cassette to enable Cre-dependent expression (Jackson Laboratory). Mice were housed in cages of 3–4 on a 12 h light/dark cycle and had free access to food and water. All protocols and procedures were approved by the University of Minnesota Institutional Animal Care and Use Committee and were conducted according to the guidelines established by the International Association for the Study of Pain (Zimmerman, 1983).

Mechanical pain sensitivity

Naïve mice were placed inside individual glass containers on a wire mesh platform and allowed to acclimate for at least 20 min. A single von Frey monofilament (bending force of 3.9 mN) was applied 10 times for 1–2 s/trial at 5–10 s intervals to random locations of the plantar surface of each hind paw and the frequency of withdrawal responses was determined. Vigorous paw withdrawals were counted and a withdrawal response frequency calculated for each hind paw. Mice were tested for 3 consecutive days prior to receiving CFA to acclimate to the testing environment. Withdrawal response frequencies were obtained just prior to the electrophysiology experiments (3–17 d after CFA injection) to assess the degree of mechanical hyperalgesia.

Intraplantar injection of CFA

Mice were placed in an induction chamber and anesthetized with isoflurane (2–3%). Anesthetized mice were moved to the procedure area and placed in prone position with a nosecone delivering 1–3% isoflurane throughout the procedure. CFA (50:50 emulsion with saline) was injected under the skin of the left plantar hind paw in a volume of 20 μL using a 30 gauge needle. Excess liquid was removed with an alcohol wipe and the mouse placed in an empty cage with a clean paper towel to recover from anesthesia. Mice were monitored until they were fully alert and active, and then returned to the home cage.

Electrophysiology studies

The surgical procedure for electrophysiological recording from the tibial nerve has been described earlier (Cain et al., 2001; Uhelski et al., 2017a). Mice were anesthetized using inhaled isoflurane (2–3% induction, 1–2% maintenance). The level of anesthesia was evaluated by pinching the right hind paw or tail and/or testing for corneal reflexes. Once anesthetized, the hair around the left hind leg was removed and an incision was made in the skin over the gastrocnemius muscle, which was dissected and removed to access the tibial nerve. The skin was sutured to a stainless steel ring (1.3 cm inner diameter) to form a pool that was filled with mineral oil. Dental impression material (COE-FLEX, GC America) was applied to the skin and around the ring to prevent mineral oil from leaking out of the pool and to stabilize the hind paw. After the impression material had cured (~10 min), the nerve was gently dissected from surrounding tissue and placed on a small mirror platform for dissection. The epineurium was removed allowing small bundles of fibers to be cut proximally, teased into fine filaments using fine forceps, and placed on a silver wire recording electrode. Action potentials from individual fibers were filtered below 1 and above 3,000 Hz, amplified by 10,000, audiomonitored, visualized on an oscilloscope, and stored on a PC using Spike 2.0 software (CED, Cambridge, UK). Nociceptors were initially identified by mild pinching and/or applying pressure to the glabrous skin of the hind paw. Calibrated von Frey monofilaments were used to identify the precise location of the receptive field (RF), which was marked on the skin with a felt-tip pen. In some cases, blue light (470 nm LED, Plexon) was used as a search stimulus and was applied to the skin for a few seconds at a time.

Conduction velocity (CV) of individual fibers was determined by electrically stimulating the RF with two fine pin electrodes that were inserted under the skin outside the RF. Beginning with a voltage below threshold, electrical pulses (200 μs) were delivered every 2 s until the response threshold was reached, and the conduction latency was calculated using a stimulus of 1.5 times the threshold value and the CV determined by dividing the conduction distance (distance from RF to recording electrode) by the latency of the action potential. Fibers with CV of 1.2 m/s or less were classified as C-fibers, while those with CV between 1.2 m/s and 10 m/s were classified as Aδ-fibers and those with CV > 10 m/s were classified as Aβ-fibers. C-fiber nociceptors responding to blue light stimulation (i.e., NaV1.8+ fibers) were preferentially studied.

Electrophysiological responses of nociceptors

Once a nociceptor was identified, the rate of spontaneous activity was determined for a period of two minutes before any testing. Mechanical response thresholds (mN) were obtained using calibrated von Frey monofilaments. The RF was stimulated multiple times with a single filament, and if no response was elicited, the next higher force (or lower force if there was a response) was applied. Response threshold was defined as the lowest force eliciting a response on 50% or more of the trials.

Responses evoked by suprathreshold mechanical stimuli were determined using a single suprathreshold von Frey monofilament that delivered a force of 147 mN. This monofilament was applied three times, each for 5 s with an inter-stimulus interval of 60 s, and the evoked response was defined as the mean number of impulses from the three trials.

Nociceptors were assessed for response to blue light stimulation by positioning the fiber optic cable ~1 mm above the RF using a micromanipulator. A blue light stimulus of 16 mW/mm2 was delivered for 5 s and the number of evoked impulses was recorded.

A Peltier device (contact area 25 mm2) was used to deliver heat stimuli to the skin. The probe was maintained at a base temperature of 32°C and heat stimuli from 34°C to 50°C were delivered in ascending order of 2°C. Each stimulus was applied for 5 s every 60 s. We were unable to apply heat to some fibers due to the location of their RF.

Topical application of loperamide/oxymorphindole

Loperamide hydrochloride was obtained from Sigma and oxymorphindole hydrochloride was obtained from Dr. Philip Portoghese’ lab. Stock solutions of loperamide/oxymorphindole (Lo/OMI) were prepared in 0.9% saline with 5% dimethyl sulfoxide (DMSO) and 5% Cremaphor; dilution to experimental concentrations with saline resulted in DMSO and Cremaphor concentrations <1%. Solutions for topical application were further diluted with 95% ethanol to a 50% ethanol solution. Naïve mice were given vehicle (50% ethanol in saline) or Lo/OMI at concentrations of 0.5, 5 and 50 μM applied to the RF. For CFA-treated mice, vehicle (50% ethanol) followed by Lo/OMI in concentrations of 0.05, 0.5, and 5 μM were applied to the RF. These concentrations were ten-fold lower than those used in the naïve mice and were comparable to those used by Wilcox and colleagues (Bruce et al., 2019) and were based on behavioral data indicating higher potency under inflammatory conditions. Also based on these prior behavioral studies, the combination had equimolar concentrations of the two agents, i.e. a 1:1 concentration ratio, such that the 5 μM combination, for example, contained 5 μM loperamide and 5 μM oxymorphindole. Vehicle and Lo/OMI were applied to the skin using a 3 mm diameter piece of filter paper which was fully submerged in solution just prior to administration. The filter paper was then placed directly on the skin over the RF for 5 min and removed before testing. An approximate 20 min washout period was used between consecutive treatments.

Experimental design

We determined the effects of vehicle and Lo/OMI on spontaneous activity (SA) and on responses evoked by mechanical, thermal, and blue light stimuli for up to 30 min after treatment. Mechanical thresholds and responses evoked by mechanical, heat and cold stimuli were determined before application of any drug, and at 15 min after application of vehicle and the various concentrations of Lo/OMI which were given in ascending order of concentration. In naïve mice, responses evoked by blue light stimulation were obtained before and every 5 min for 15 min after vehicle or Lo/OMI application (0.5, 5, and 50 μM in that order). Vehicle was applied 3 times at 20–30 min intervals, and Lo/OMI was applied three times at 20–30 min intervals beginning with 0.5 μM, then 5 μM, and 50 μM. Pilot studies were conducted to ensure that the vehicle itself did not alter response characteristics and to determine the appropriate length of the washout period for Lo/OMI (data not shown). CFA-treated mice were given vehicle followed by 0.05, 0.5, and 5 μM Lo/OMI at 20–30 min intervals. Responses were assessed at 15 min after each application.

Statistical Analyses

All data are expressed as mean ± SEM or median [1st quartile, 3rd quartile]. For naïve mice, the percent change in blue light responses (derived from the number of impulses during a 5 s stimulation) was analyzed for each treatment condition (vehicle vs 0.5, 5, and 50 μM Lo/OMI) at 5, 10 and 15 min post-application using repeated measures analyses of variance (ANOVA) with drug condition as the between-subjects factor and time as the within-subjects factor. Mechanical thresholds (mN) were converted to a log scale in order to allow for parametric testing. Mean mechanical response thresholds (in mN) at baseline were compared using the Mann-Whitney U test, and percent change scores were calculated from thresholds (in mN) in order to compare vehicle-treated C-fibers to those treated with 0.5, 5, and 50 μM Lo/OMI using repeated measures ANOVA using group (vehicle or Lo/OMI) as the between subjects factor and treatment (1st, 2nd and 3rd application) as the within-subjects factor. Baseline responses to a 5-s application of a 147 mN von Frey (expressed as the number of impulses) were compared using an independent t-test. The percent change in responses to a 147 mN stimulus (derived from the number of impulses during the 5 s stimulation) relative to baseline was analyzed using repeated measures ANOVA with drug condition (vehicle or Lo/OMI) as the between subjects factor and treatment (1st, 2nd and 3rd application) as the within-subjects factor. Heat thresholds (°C) were analyzed using repeated measures ANOVA with drug condition (Lo/OMI or vehicle) as the between-subjects factor and treatment (baseline, 1st, 2nd and 3rd application) as the within-subjects factor. The cumulative number of impulses evoked by all heat stimuli were converted to percent change scores relative to baseline values and analyzed using repeated measures ANOVA with drug condition (Lo/OMI or vehicle) as the between-subjects factor and treatment (1st, 2nd and 3rd application) as the within-subjects factor. Fisher’s LSD post-hoc tests were used to determine where differences were significant.

For CFA-treated mice, mechanical paw withdrawal frequencies (expressed as the number of positive withdrawal responses out of 10 stimulations) were analyzed for the left (treated) hind paw using a paired t-test. Baseline response characteristics were compared between naïve and CFA-treated mice using independent t-tests (spontaneous activity, blue light responses, responses to 147 mN stimulus) and Mann-Whitney U tests (mechanical response thresholds).

Spontaneous activity (expressed as the frequency of spontaneous impulses over a 2 min time period) was analyzed using repeated measures ANOVA with treatment as the within-subjects factor to compare SA before treatment and after vehicle, 0.05, 0.5, and 5 μM Lo/OMI. The percent change in responses evoked by blue light (derived from the number of impulses produced by 5-s stimulation) was analyzed using repeated measures ANOVA with treatment as the within-subjects factor in order to compare vehicle, 0.05, 0.5, and 5 μM Lo/OMI. Mechanical thresholds (mN) were converted to a log scale in order to allow for parametric testing. Log-transformed thresholds were analyzed using repeated measures ANOVA with treatment as the within-subjects factor in order to compare vehicle, 0.05, 0.5, and 5 μM Lo/OMI. The number of impulses evoked by the 147 mN stimulus was analyzed using repeated measures ANOVA with treatment as the within-subjects factor in order to compare vehicle, 0.05, 0.5, and 5 μM Lo/OMI. Post-hoc comparisons were made using Fisher’s LSD tests.

RESULTS

Topical Lo/OMI reduced excitability of C-fiber nociceptors in naïve mice

In naïve mice (n = 30), recordings were made from 39 C-fibers (mean CV: 0.55± 0.03 m/s). Two separate groups of mice received either three concentrations of Lo/OMI (0.5 μM, 5.0 μM and 50 μM) or three consecutive treatments with vehicle (50% ethanol solution). Since only 3 fibers exhibited low levels of ongoing SA prior to vehicle or drug application (mean rate 0.03 ± 0.01 Hz), SA was not studied in this condition.

Topical application of Lo/OMI (n = 7) (Fig. 1) dose-dependently reduced the number of impulses evoked by blue light at 5, 10, and 15 min after application of 0.5 μM, 5 μM, and 50 μM (Fig. 1A–C). Responses evoked by blue light were unaltered following repeated applications of vehicle (n = 8). Representative traces for each treatment group are presented in Fig. 1 D–E. The percent change in mechanical response thresholds was significantly higher for all concentrations of Lo/OMI relative to vehicle (Fig. 2A). Percent changes in the responses to the 147 mN von Frey monofilament were lower at all Lo/OMI concentrations (n = 7; Fig. 2B) relative to vehicle treatment (n = 8). Heat thresholds (Fig. 3A) increased relative to baseline after Lo/OMI (n = 5), but did not differ from vehicle-treated (n = 5) fibers, which increased after the first vehicle treatment only. Cumulative responses to heat (Fig. 3B) did decrease at all three concentrations of Lo/OMI, but did not change after vehicle treatments. An example of the effects of Lo/OMI on responses of a single C-fiber nociceptor to heat stimulation is shown in Fig. 3C.

Figure 1.

Figure 1.

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Dose-dependent decreases in responses evoked by blue light following topical application of Lo/OMI (n = 7). The number of evoked impulses were reduced following doses of (A) 0.5 μM (F1,14 = 19.2, p<.001), (B) 5 μM (F1,14 = 64.6, p<.001), and (C) 50 μM (F1,14 = 62.5, p<.001) relative to vehicle treatment (n = 8). Data are shown as mean (±SEM) percent change from baseline. (D) Representative example of raw traces from a vehicle-treated C-fiber showing the baseline response to a 5-s blue light stimulus (upper left) and responses following the first, second and third applications of vehicle (right). Conduction latency (lower left) consists of three overlapping traces to show consistency. Left arrow shows the onset of the electrical stimulus and the right arrow indicates the action potential of the fiber of interest. (E) Representative example of raw traces from an Lo/OMI-treated C-fiber showing the baseline response to a 5-s blue light stimulus (upper left) and responses following application of 0.5, 5 and 50 μM (right). Conduction latency (lower left) as described above. Left arrow shows the onset of the electrical stimulus and the right arrow indicates the action potential of the fiber of interest. *p≤.01, **p<.005, ***p<.001 vs Vehicle. L/O: loperamide/oxymorphindole.

Figure 2.

Figure 2.

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Lo/OMI decreased responses to mechanical stimuli. (A) While baseline response thresholds did not differ between fibers treated with Lo/OMI (21.0±5.1 mN, n = 7) and vehicle (15.7±4.0 mN, n = 11; Mann-Whitney U = 28.0, n1 = 11, n2 = 7, n.s.), Lo/OMI increased mechanical response thresholds relative to vehicle treatment as shown by mean (±SEM) percent changes from baseline (group: F1,16 = 13.8, p<.005; dose: F2,32 = 3.2, n.s.; group x dose: F2,32 = 0.7, n.s.). (B) Baseline responses to a 5-s application of 147 mN von Frey monofilament did not differ between vehicle (80.3±19.6 impulses, n = 6) and L/O treatment groups (64.5±9.4 impulses, n = 7; t11 = 0.8, n.s.). Responses evoked by 147 mN decreased at 15 min after application of 0.5, 5, and 50 μM Lo/OMI relative to vehicle treatment (group: F1,11= 19.7, p<.005; treatment: F2,22 = 1.9, n.s.; group x treatment: F2,22 = 1.9, n.s.) as shown by mean (±SEM) percent changes from baseline. Application of 50 μM Lo/OMI caused a greater percent decrease than 0.5 μM and 5 μM. *p<.05 vs Vehicle; **p<.01 vs Vehicle; ***p<.005 vs Vehicle; #p<.05 vs 0.5 μM Lo/OMI; ##p<.005 vs 5 μM Lo/OMI. L/O: loperamide/oxymorphindole

Figure 3.

Figure 3.

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Lo/OMI decreased responses to heat. (A) Mean (±SEM) heat response thresholds of nociceptors increased after topical application of Lo/OMI (n = 5) but did not differ from those following vehicle (n = 5; group: F1,8 = 0.3, n.s.; treatment: F3,24 = 4.8, p<.01; group x treatment: F3,24 = 1.0, n.s.). There was an increase in heat thresholds after the first vehicle treatment only. (B) Cumulative responses to heat decreased at all concentrations 15 min after the application of Lo/OMI (group: F1,8 = 26.9, p<.005; treatment: F2,16 = 3.3, n.s.; group x treatment: F2,16 = 0.1, n.s.). Data are expressed as mean (±SEM) percent change from baseline response. (C) Representative example of raw traces from a C-fiber showing responses to heat stimuli before and after treatment with loperamide/oxymorphindole (L/O). **p<.01, ***p<.005 vs Vehicle; #p<.05, ##p<.005, ###p<.001 vs baseline.

Lo/OMI decreased activity of nociceptors in inflamed skin

A total of 8 mice were injected in the left hind paw with CFA prior to electrophysiological recording (12.0 ± 1.6 days post-injection, range 3–17 days). Mechanical paw withdrawal frequencies (6.5 ± 0.4) increased after CFA treatment in comparison to baseline values (0.8 ± 0.4, t7 = 9.7, p<.001). We recorded from 8 C-fibers (mean CV: 0.61 ± 0.07 m/s) from CFA-treated mice which received four treatments: vehicle (50% ethanol), 0.05 μM, 0.5 μM, and 5 μM Lo/OMI. Based on the results in naïve mice, spontaneous activity and responses to mechanical and blue light stimuli were assessed at 15 min following application. A 20–30 min washout period was used between consecutive treatments.

The mean rate of baseline spontaneous activity in C-fibers from CFA-treated mice was 0.17 ± 0.04 Hz (vs 0.00 ± 0.00 Hz in naïve mice, t36 = 8.6, p<.001). Overall, there was no evidence of sensitization to mechanical stimuli based on baseline response thresholds (13.7[5.9,39.2] mN in naïve mice vs 19.6[10.8,19.6] mN in CFA-treated mice, Mann-Whitney U = 115.0, n1 = 31, n2 = 8, n.s.) or responses to 147 mN von Frey stimulation (61.6 ± 9.3 impulses in naïve mice vs 40.8 ± 9.4 impulses in CFA-treated mice, t20 = 1.1, n.s.). Responses to blue light stimulation also did not differ between the two groups (11.7 ± 2.1 impulses in naïve mice vs 10.0 ± 2.8 impulses in CFA-treated mice, t27 = 0.4, n.s.).

Spontaneous activity did not change after vehicle treatment but deceased in a dose-dependent manner following Lo/OMI treatments (n = 7; Fig. 4A). A representative raw trace showing the decrease in spontaneous activity following Lo/OMI treatment is presented in Fig. 4B. Lo/OMI also dose-dependently decreased cumulative responses evoked by blue light, while vehicle had no effect (n = 6; Fig. 5). Mechanical response thresholds did not change following vehicle, 0.05 or 0.5 μM Lo/OMI, but did increase following application of 5 μM Lo/OMI (n = 6; Fig. 6A). Responses evoked by a 147 mN von Frey monofilament decreased following Lo/OMI concentration of 0.5 μM and 5 μM Lo/OMI, but not vehicle or 0.05 μM Lo/OMI (n = 5; Fig. 6B).

Figure 4.

Figure 4.

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Effect of Lo/OMI on spontaneous activity. Lo/OMI decreased spontaneous activity of nociceptors (n = 7; F4,24 = 18.5, p<.001) in CFA-inflamed hind paws. (A) Mean (±SEM) discharge rates (impulses/sec) prior to any drug application (baseline) and at 15 min after application of vehicle and different concentrations of Lo/OMI. Spontaneous activity decreased after all concentration of Lo/OMI but not after vehicle. (B) Raw traces from a C-fiber showing spontaneous activity before and after treatment with vehicle and loperamide/oxymorphindole (L/O). **p≤.005 vs baseline.

Figure 5.

Figure 5.

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Effect of Lo/OMI on cumulative responses evoked by blue light following inflammation. Data show mean (±SEM) percent change from baseline in cumulative responses to blue light from nociceptors (n = 6; F3,15 = 14.2, p<.001) in CFA-inflamed hind paws 15 min following application of loperamide/oxymorphindole (L/O) at all concentrations tested when compared to vehicle, which did not change from baseline. *p<.05, **p<.01,***p<.001 vs vehicle.

Figure 6.

Figure 6.

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Lo/OMI increased mechanical response thresholds and decreased responses to mechanical stimuli following inflammation. (A) Mean (±SEM) log-transformed mechanical thresholds of nociceptors (n = 6; 5.0 μM; F4,20 = 4.8, p<.01) in CFA-inflamed hind paws did not differ from baseline 15 min after vehicle, 0.05 μM Lo/OMI or 0.5 μM Lo/OMI, but did increase after 5 μM Lo/OMI. (B) Mean (±SEM) number of impulses evoked by 147 mN did not change after treatment with vehicle or 0.05 μM Lo/OMI, but decreased 15 min after application of 0.5 and 5 μM Lo/OMI (n = 5; F4,16 = 11.5, p<.001). *p<.05 vs baseline. L/O: loperamide/oxymorphindole.

DISCUSSION

Selective activation of peripheral opioid receptors can provide pain relief without the burden of side effects normally associated with opioid use (Bates et al., 2004). It was shown recently that co-activation of peripheral mu- and delta-opioid receptors produced synergistic behavioral antinociception that enhanced analgesic potency with reduced side effects (Bruce et al., 2019). We applied a topical treatment of the 1:1 combination of the mu- and delta-opioid receptor agonists loperamide and oxymorphindole (Lo/OMI) to the receptive fields of C-fiber nociceptors and recorded their response characteristics. We used mice that co-express the light-sensitive channelrhodopsin2 in neurons expressing NaV1.8 voltage-gated sodium channels (NaV1.8-ChR2+, Stirling et al., 2005; Daou et al., 2013; Madisen et al., 2012). Using these mice, we were able to selectively activate nociceptors in the periphery with blue light stimulation of the skin of the plantar hind paw. Blue light activates nociceptors without delivering noxious, potentially damaging stimuli to the skin and does not sensitize nociceptors during brief applications (Uhelski et al., 2017a). Using blue light in naïve mice, we were able to determine that the time course of the Lo/OMI effect on nociceptor responses had a rapid onset, occurring within 5 min of topical treatment, and a short duration, ending within 30 min of application. We found that the peak effect for reducing nociceptor activity was at 15 min following application, and therefore determined our drug effects on additional evoked responses at this time point. Mice that were pre-treated with CFA were tested only at 15 min after drug treatment. Although topical application of loperamide and oxymorphindole in behavioral studies produced potent antinociception that was synergistic (Bruce et al., 2019), we do not know if the decrease in responses of nociceptors following topical application of Lo/OMI in the present study was due to synergy since effects of these individual compounds were not tested. Rather, were we were interested in determining the time course and dose-resonse relationships of Lo/OMI on excitability of C-fiber nociceptors that possessed NaV1.8. Loperamide has previously been shown to inhibit high-voltage-gated L-type calcium channels and hyperpolarization-activated cyclic nucleotide–gated (HCN) channels in DRG neurons, but electrophysiological studies with oxymorphindole are lacking (Hagiwara et al., 2003; Vasilyev et al., 2007).

In naïve mice, topical Lo/OMI consistently attenuated responses of C-fiber nociceptors to blue light, mechanical, and heat stimuli. Based on earlier behavioral studies (Bruce et al., 2019), we selected a range of Lo/OMI concentrations for CFA-treated mice that was ten-fold lower than those found to be efficacious in naïve mice (0.05, 0.5, and 5 μM vs 0.5, 5, and 50 μM). Even at ten-fold lower concentrations, Lo/OMI had a robust effect in reducing evoked responses of nociceptors. Spontaneous activity, a hallmark of sensitization, decreased considerably after application of Lo/OMI and was nearly eliminated at the highest concentration. Responses of nociceptors evoked by blue light stimulation decreased at all three concentrations, whereas responses to mechanical stimuli were reduced only by the higher concentrations.

Although CFA increased spontaneous activity in C-fiber nociceptors, we did not observe any changes in responses evoked by blue light or mechanical stimuli in nociceptors with spontaneous activity. Earlier studies of nociceptor recordings from CFA-treated rats also exhibited increased spontaneous activity without sensitization to mechanical stimuli (Andrew & Greenspan, 1999). In those studies, there was no change in mechanical response thresholds or responses evoked by a 1 mm2 probe, but responses were increased when mechanical stimuli were applied using a 0.1 mm2 probe. The 147 mN von Frey monofilament used in the present study has a diameter of 0.48 mm, suggesting that the physical attributes of the stimulus could impact whether responses of C-fibers differ between naïve and CFA-treated mice. Further, these studies occurred within 24 hours of the CFA injection, during the peak of acute inflammation, whereas our studies took place at least 72 hours after the injection when hyperalgesia is still present but swelling and overt signs of inflammation are lessened. Thus, while many nociceptors were still spontaneously active at this time, fewer may have been sensitized to mechanical stimuli. However, Lo/OMI was still able to decrease evoked responses of spontaneously active nociceptors, suggesting that Lo/OMI is capable of reducing sensitization. The use of a different experimental pain model where nociceptor sensitization includes increased responses to mechanical and thermal stimuli, such as chemotherapy-induced neuropathy or sickle cell disease (Uhelski et al., 2015, 2017b), would be ideal for future studies with Lo/OMI. Whether or not the responses of nociceptors to blue light would be impacted under a different experimental pain model has yet to be determined.

Earlier behavioral studies suggested that the combination of MOR and DOR agonists synergized when given intrathecally or into the skin (Sutters et al., 1990; Miaskowski et al., 1992; Joseph & Levine, 2010; Schramm & Honda, 2010; Schuster et al., 2015). The exact mechanisms by which Lo/OMI synergized to produce potent behavioral antinociception and reduced nociceptor activity are unclear. However, since this drug combination was applied topically to the skin, it is likely that the effect of Lo/OMI occurred via opioid receptors located on nociceptor terminals in the epidermis or on non-neuronal cells such as keratinocytes (Bigliardi et al., 1998, 2009; Leong et al., 2017).

In recent behavioral studies using intraplantar administration of Lo/OMI (Bruce et al., 2019), the analgesic synergy was dependent on MOR and DOR since selective antagonists for either receptor subtype greatly reduced the maximum analgesic effect. This outcome also suggests that the two opioid receptors co-localize in nociceptors. The synergism in those studies was also dependent on the epsilon isoform of protein kinase C, an intracellular signaling molecule present in 95% of primary afferent fibers (Schuster et al., 2015). Since analgesia following intrathecal opioids is dependent on activation of opioid receptors on the central terminals of primary afferent fibers (Yaksh, 1997), one possibility is that MOR and DOR are co-expressed on both central and peripheral terminals of nociceptors. Co-localization of MOR and DOR has been identified in DRG neurons; however, the observed proportion of neurons co-expressing both receptors has not been consistent (Erbs et al., 2015; Scherrer et al., 2009; Wang et al., 2010; Rau et al., 2005). Furthermore, we are unaware of studies that have assessed the degree of co-localization following CFA-induced inflammation; it is possible that inflammation-induced upregulation of opioid receptor expression (Ji et al., 1995; Mousa, 2003; Patwardhan et al., 2005; Pettinger et al., 2013; Puehler et al., 2004) could increase the degree of co-localization and thereby enhance the analgesic effect of peripherally administered opioids (Stein, 2018).

Co-expression of receptors allows for unique receptor interactions, including the formation of heterodimers (Ferre et al., 2014; Gurevich and Gurevich, 2008). Furthermore, ligand pharmacology, receptor function, signal transduction, and cellular trafficking can be altered by receptor dimerization (Hiller et al., 2013). With respect to opioid receptors, it has been shown that they can form heteromers with a number of different GPCRs and this can be modulated in pathological states (Costantino et al., 2012; Gomes et al., 2013). The existence of MOR-DOR heteromers is supported by the development of bivalent ligands whereby the pharmacophores are tethered with specific spacer lengths, which is directly related to analgesic potency (Daniels et al., 2005).

In conclusion, topical application of the 1:1 combination of loperamide and oxymorphindole produced rapid-onset decreases in responses of C-fiber nociceptors to blue light, mechanical, and heat stimuli in naïve mice, and reduced spontaneous activity and nociceptor responses to blue light and mechanical stimuli in CFA-inflamed mice at ten-fold lower concentrations as used for behavioral studies. This outcome is consistent with the hypothesis that nociceptor terminals located in the epidermis are hyperpolarized by Lo/OMI, preventing the initiation of action potentials upon activation of stimulus-gated cation channels such as TRP and ChR2. Opioid compounds designed to activate peripheral opioid receptors such as loperamide (Kopsky et al., 2019), and perhaps to target opioid receptor heteromers, may be a useful and effective alternative for pain management rather than traditional opioids that are currently hindered by unwanted side effects and the potential for abuse and addiction.

Highlights.

  • Topical application of a combination of loperamide (Lo) and oxymorphindole (OMI) produces potent anti-hyperalgesia.

  • We determined the effects of Lo/OMI combination on responses of C-fiber nociceptors that possess the Nav1.8 sodium channel.

  • Topical application of Lo/OMI decreased responses of C-fiber nociceptors evoked by blue light and mechanical stimulation.

ACKNOWLEDGEMENTS

The authors extend their thanks to Dr. Philippe Séguela of McGill University for providing homozygous NaV1.8-Cre mice to start the breeding colony for production of the NaV1.8-ChR2+ mice. We also thank Drs. Phil Portoghese and Eyup Akgün for providing the OMI. This work was supported by NIH grant CA241627 to DAS, the R.W. Goltz Professorship in Dermatology (GLW), and the University of Minnesota Academic Health Center (GLW).

Footnotes

CONFLICT OF INTEREST

G.L.W. and D.B. are inventors on a University of Minesota international patent application. The remaining authors declare no competing financial interest.

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