Role of peripheral sensory neuron mu-opioid receptors in nociceptive, inflammatory, and neuropathic pain

Abstract

Background and objective: The role of peripheral mu-opioid receptors (MOPs) in chronic pain conditions is not well understood. Here, we used a combination of mouse genetics, behavioral assays, and pharmacologic interventions to investigate the contribution of primary afferent MOPs to nociceptive, inflammatory, and neuropathic pain, as well as to opioid analgesia.

Methods: We generated conditional knockout mice in which MOPs were selectively deleted in primary sensory neurons. Inflammatory and neuropathic pain states were induced in mutant and control wild-type mice and their behavioral responses to noxious stimuli were compared. Gross motor function was also evaluated. Immunohistochemistry was used to assess MOP expression in the dorsal root ganglia, periaqueductal gray, and small intestine. The effects of MOP agonists DALDA (dermorphin [D-Arg2, Lys4] (1-4) amide) and morphine were evaluated in pain behavior assays, and their effects on neuronal physiology in the dorsal root ganglia were evaluated in whole-cell patch-clamp recordings.

Results: Conditional MOP knockouts and control mice exhibited similar behavioral responses to acute nociceptive stimuli and developed similar inflammation-induced hypersensitivity. Unilateral nerve injury in animals lacking peripheral MOPs induced enhanced, bilateral mechanical allodynia. Subcutaneously administered DALDA was unable to decrease the hypersensitivity induced by inflammation and nerve injury in MOP knockout animals, and morphine's antinociceptive effects were significantly attenuated in the absence of peripheral MOPs.

Conclusion: MOPs in primary sensory neurons contribute to the modulation of neuropathic pain behavior and opioid analgesia. Our observations highlight the clinical potential of peripherally acting opioid agonists in the management of inflammatory and neuropathic pain.

Keywords: analgesics; chronic pain; drug-related side effects and adverse reactions; neuralgia; opioid; pain management.

Figure 1

Summary of methods. Image indicates the genetic make-up of mice used for this study. CFA, complete Freund’s adjuvant; cKO, conditional knockout; DALDA, dermorphin [D-Arg2, Lys4] (1–4) amide; MOP, mu-opioid receptor; SNI_t, modified spared nerve injury; WT, wild-type.

Figure 2

Oprm1 cKO selectively deletes MOPs in DRG neurons. Representative western blot images (A) and quantification (B) of MOP protein levels in the DRG, SC, small intestine, and PAG (n=4–7 mice per group). The amount of MOP protein (50 and 55 kDa) was normalized to that of GAPDH (37 kDa) in the same sample, and the mean MOP level in WT mice was considered to be 1. Data are shown as mean±SEM. Protein levels were evaluated with Welch’s t-test. Immunofluorescence-stained images and quantification of MOP immunoreactivity in lumbar DRG (C, D) and spinal cord dorsal horn (E, F) in male Pirt-Cre^+/- Oprm1^fl/fl (Oprm1 cKO) and WT mice (n=2–3 mice per group, 2–3 DRG sections per mouse). Arrow direction indicates dorsal side. Scale bar for DRG: 20 μm; scale bar for spinal cord: 50 μm. *P<0.05. cKO, conditional knockout; DRG, dorsal root ganglia; MOP, mu-opioid receptor; PAG, periaqueductal gray; SC, spinal cord; WT, wild-type.

Figure 3

DALDA and morphine reduce the mean amplitude of HVA-I_Ca in cultured small-diameter wild-type DRG neurons. (A) Representative traces of HVA-I_Ca in response to bath application of 1 μM DALDA (upper panel, n=4; black trace, pre-DALDA mean HVA-I_Ca from 0 to 60 s; gray trace, post-DALDA mean HVA-I_Ca from 120 to 145 s) and bath application of 1 μM morphine (lower panel, n=3; black trace, premorphine mean HVA-I_Ca from 0 to 60s; gray trace, postmorphine mean HVA-I_Ca from 120 to 145 s). Scale bars: 200 pA, 10 ms. (B) Upper panel: a single bath application of 1 μM DALDA produced a mean reduction in HVA-I_Ca magnitude. Lower panel: a single bath application of 1 μM morphine produced a mean reduction in HVA-I_Ca magnitude. Horizontal black bars indicate the duration of drug exposure. (C) Upper panel: bath application of 1 μM DALDA significantly reduced HVA-I_Ca (t₍₁₄₆₎=11.84, unpaired t-test). Lower panel: bath application of 1 μM morphine significantly reduced HVA-I_Ca (t₍₁₀₆₎=16.75, unpaired t-test). Subscript indicates degrees of freedom in unpaired t-test. *P<0.05. DALDA, dermorphin [D-Arg2, Lys4] (1–4) amide; DRG, dorsal root ganglia; HVA-I_Ca, high-voltage-activated calcium.

Figure 4

Absence of mu-opioid receptors in primary sensory neurons does not affect behavioral response to acute pain. Pirt-Cre^+/- Oprm1^{fl/ fl} (Oprm1 cKO,gray bars) and wild-type mice (black bars) were tested for (A) paw withdrawal latency in response to radiant heat stimulus, (B) response latency to hot plate, (C) paw withdrawal frequency in response to low-force (0.07 g) and high-force (0.4 g) von Frey monofilaments, and (D) latency to fall during the rotarod test (n=10, with 5 males and 5 females). Data are shown as mean±SEM and were compared using Welch’s t-test.

Figure 5

Systemic DALDA and morphine do not inhibit responses to acute nociceptive stimuli in Oprm1 cKO mice. Wild-type and Oprm1 cKO (Pirt-Cre^+/- Oprm1^fl/fl) mice were injected subcutaneously with 5 mg/kg DALDA or morphine. Time course of the effects of DALDA (A, B) and morphine (C, D) on the paw withdrawal latency in response to radiant heat stimuli and the paw withdrawal frequency in response to high-force (0.4 g) von Frey filaments (n=10, with 5 males and 5 females). Data are shown as mean±SEM. *P<0.05 compared with predrug (within the same genotype). #P<0.05 compared with wild-type (same time point) by repeated-measures two-way analysis of variance with Bonferroni’s post-hoc test. cKO, conditional knockout; DALDA, Dermorphin [D-Arg2, Lys4] (1–4) amide.

Figure 6

Absence of peripheral MOPs abolishes the analgesic effect of systemic DALDA and attenuates the analgesic effect of systemic morphine under inflammatory pain conditions. Wild-type and Oprm1 cKO (Pirt-Cre^+/- Oprm1^fl/fl) mice were administered 5 mg/kg DALDA or morphine subcutaneously 24 hours after CFA injection. Time course of the effects of DALDA (A) and morphine (B) on paw withdrawal latency in response to radiant heat stimuli (n=10, with 5 males and 5 females). Wild-type and Oprm1 cKO mice received a 20 μL subcutaneous injection of 1 mg/mL CFA in one hind paw. Time course of the effects on thermal hyperalgesia (C) and paw edema (D). Data are shown as mean±SEM. *P<0.05 compared with predrug or pre-CFA (within the same genotype). #P<0.05 compared with wild-type (same time point) by repeated-measures two-way analysis of variance with Bonferroni’s post-hoc test. CFA, complete Freund's adjuvant; cKO, conditional knockout; DALDA, dermorphin [D-Arg2, Lys4] (1–4) amide; MOPs, mu-opioid receptors.

Figure 7

Nerve injury induces bilateral mechanical allodynia in the absence of peripheral MOPs. Time course of the effects of SNI_t-induced neuropathic pain on ipsilateral and contralateral paw withdrawal frequency in response to (A) low-force (0.07 g) and (B) high-force (0.4 g) von Frey monofilaments in Pirt-Cre^+/- Oprm1^fl/fl (Oprm1 cKO) and wild-type mice (n=10, with 5 males and 5 females). Data are shown as mean±SEM. *P<0.05 (ipsilateral) and †P<0.05 (contralateral) compared with presurgery (within the same genotype). #P<0.05 (ipsilateral) and ‡P<0.05 (contralateral) compared with wild-type (same time point) by repeated-measures two-way analysis of variance with Bonferroni’s post-hoc test. cKO, conditional knockout; MOPs, mu-opioid receptors; SNI_t, modified spared nerve injury.

Figure 8

Absence of peripheral MOPs abolishes the analgesic effect produced by systemic DALDA and attenuates the analgesic effect produced by systemic morphine in mice with neuropathic pain. Wild-type and Oprm1 cKO (Pirt-Cre^+/- Oprm1^fl/fl) mice were administered 5 mg/kg DALDA or morphine by subcutaneous injection. Time course of the effects of DALDA (A, B) and morphine (C, D) on paw withdrawal frequency in response to low-force (0.07 g) and high-force (0.4 g) von Frey monofilaments (n=10, with 5 males and 5 females). Data are shown as mean±SEM. *P<0.05 (ipsilateral) and †P<0.05 (contralateral) compared with predrug (within the same genotype). #P<0.05 (ipsilateral) and ‡P<0.05 (contralateral) compared with WT (same time point) by repeated-measures two-way analysis of variance with Bonferroni’s post-hoc test. cKO, conditional knockout; DALDA, dermorphin [D-Arg2, Lys4] (1–4) amide; MOPs, mu-opioid receptors.