Loss of μ opioid receptor signaling in nociceptors, but not microglia, abrogates morphine tolerance without disrupting analgesia

Abstract

Opioid pain medications have detrimental side effects including analgesic tolerance and opioid-induced hyperalgesia (OIH). Tolerance and OIH counteract opioid analgesia and drive dose escalation. The cell types and receptors on which opioids act to initiate these maladaptive processes remain disputed, which has prevented the development of therapies to maximize and sustain opioid analgesic efficacy. We found that μ opioid receptors (MORs) expressed by primary afferent nociceptors initiate tolerance and OIH development. RNA sequencing and histological analysis revealed that MORs are expressed by nociceptors, but not by spinal microglia. Deletion of MORs specifically in nociceptors eliminated morphine tolerance, OIH and pronociceptive synaptic long-term potentiation without altering antinociception. Furthermore, we found that co-administration of methylnaltrexone bromide, a peripherally restricted MOR antagonist, was sufficient to abrogate morphine tolerance and OIH without diminishing antinociception in perioperative and chronic pain models. Collectively, our data support the idea that opioid agonists can be combined with peripheral MOR antagonists to limit analgesic tolerance and OIH.

Figure 1. MOR is required for morphine antinociceptive tolerance and OIH, but is not expressed by spinal microglia

(a–b) Behavioral indices of chronic morphine side effects: (a) analgesic tolerance (F_{3, 20} = 61.26, P < 0.0001) and (b) OIH (F_{3, 20} = 16.96, P < 0.0001) in control and MOR KO mice (n = 6 mice for all groups). (c,d) Densitometry analysis of anti-CD11b immunoreactivity in spinal cord dorsal horn to assess microglial activation in control and MOR KO mice (P = 0.0032). Scale bar = 500 µm. (e) in situ hybridization for Oprm1 mRNA in CX3CR1-eGFP mouse spinal cord. (f) Immunohistochemistry for MOR protein in CX3CR1-eGFP mouse spinal cord. (g) MOR-mCherry reporter mouse spinal cord immunostained for CD11b. Scale bars = 50 µm for panels e–f. (h) Example Wiggle plots for mapped reads of Oprm1 (red) in whole DRG (bottom plot) and in purified spinal microglia (top plot). Reads were registered for the partially overlapping microglial gene Ipcef1 (gray) but not Oprm1 exons 1–4 for microglia (top plot). In DRG, reads mapped to Oprm1 exons 1 through 4 with little to no reads for Ipcef1 (bottom plot). (i) Mapped reads for several cell types from RNA-seq transcriptome profiling of acutely purified spinal microglia in uninjured mice and in mice with Chronic Constriction Injury (CCI) of the sciatic nerve after 2 and 7 days. n = 2 independent sequencing experiments per group, consisting of pooled microglia from n = 15 mice for controls, n = 4 mice from CCI day 2 and n = 5 mice from CCI day 7. One-way ANOVA + Bonferroni (a, b) and Kruskal-Wallis (d).★ P < 0.05. Error bars are mean ± SEM. Overlaid points are individual subject scores. FPKM = Fragments Per Kilobase of transcript per Million mapped reads.

Figure 2. Conditional deletion of MOR from primary afferent nociceptors does not alter nociceptive behavior or reduce systemic morphine antinociception

(a) Trpv1^Cre mice were crossed with Oprm1^flox/flox mice. Exons 2 and 3 of the Oprm1 gene are flanked by loxP sites (triangles) and are excised in TRPV1 neurons expressing Cre recombinase. (b) PCR on genomic DNA showing excision of the floxed DNA fragment (363 bp). (c,d) in situ hybridization for Oprm1 mRNA and (e,f) anti-MOR immunostaining in MOR cKO mice compared to littermate controls (n = 3 per genotype). (g–j) MOR co-immunostaining with CGRP in spinal laminae I and II outer in (i) control and (j) MOR cKO mice. (k,l) MOR co-immunostaining with IB4 in spinal laminae I and II outer in (k) control and (l) MOR cKO mice. (m–o) Baseline nociceptive hypersensitivity or affective-motivational behavior to (m) von Frey filament mechanical stimulation of the hindpaw, (n) increasing water bath temperatures during tail immersion, or (o) increasing hotplate temperatures on the hindpaws between MOR cKOs (n = 15) and controls (n = 14). (p–r) Antinociception, resulting from an acute spinal morphine administration (1 µg, intrathecal) in MOR cKOs (n = 6) and controls (n = 9), in response to (p) von Frey mechanical stimulation (F_{1, 13} = 5.132, P = 0.0412), (q) 50 °C water tail immersion (F_{1, 13} = 12.53, P = 0.0036), and (r) 52.5 °C hotplate (F_{1, 13} = 5.827, P = 0.0313). (s–u) Antinociception, resulting from an acute systemic morphine administration (10 mg/kg, subcutaneous) in MOR cKO mice (n = 13) compared to controls (n = 17), when assessed by (s) von Frey mechanical filaments (F_{1, 28} = 18.46, P = 0.0002), (h) 50 °C water tail immersion (F_{1, 28} = 18.78, P < 0.0001), or (i) 52.5 °C hotplate (F_{1, 28} = 12.95, P < 0.0001). Repeated measures Two-way ANOVA + Bonferroni. ★ P < 0.05. Error bars are ± SEM. Overlaid points are individual subject scores. Scale bars = 50 µm, throughout. BL = baseline.

Figure 3. Conditional deletion of MOR from TRPV1 nociceptors prevents the onset of morphine antinociceptive tolerance and OIH

(a,d,g) Daily nociceptive behavior and opioid antinociception throughout a 10 day chronic morphine schedule (10 mg/kg, subcutaneous, once daily). Nociceptive behavior (pre-morphine BL timepoints only): von Frey: F_{1, 30} = 9.863, P = 0.004; tail immersion: F_{1, 30} = 0.7311; hotplate: F_{1, 30} = 0.2581, P = 0.0615. Antinociception (post-morphine +30 min timepoints only): von Frey: F_{1, 30} = 4.812, P = 0.0367; tail immersion: F_{1, 30} = 19.74, P < 0.0001; hotplate: F_{1, 30} = 17.23, P = 0.0004. (b,e,h) Antinociceptive tolerance: (left panels) Maximal possible effect (MPE) for morphine antinociception from the first administration (Day 1: +30 min) compared to the last administration (Day 10: +30 min) (von Frey: F_{1, 30} = 7.621, P = 0.0097; tail immersion: F_{1, 30} = 28.27, P < 0.0001; hotplate: F_{1, 30} = 12.27, P = 0.0015), and (right panels) the percent change for each subject. (c,f,i) OIH: (left panels) Percent change in the pre-morphine baseline nociceptive behaviors prior to the first administration (Day 1: BL) compared to the last (Day 10: BL) (von Frey: F_{1, 30} = 6.13, P < 0.001; tail immersion: F_{1, 30} = 23.08, P < 0.0001; hotplate: F_{1, 30} = 5.163, P = 0.0304), and (right panels) the percent change for each subject. Control, n = 19; MOR cKO, n = 13. BL = baseline. Student’s t Test, two-tailed (right panels of b,c,e,f,h,i). Repeated Measures Two-way ANOVA + Bonferroni (a,d,g and left panels of b,c,e,f,h,i). ★ P < 0.05. Error bars are mean ± SEM. Overlaid points are individual animal scores. BL = baseline.

Figure 4. Opioid-induced spinal long-term potentiation (LTP) is initiated by presynaptic MOR in nociceptors

(a,b) MOR cKO mice crossed with a mouse line expressing Channelrhodopsin2 (ChR2-eYFP) in a Cre-dependent manner produces expression of ChR2-eYFP in Trpv1^Cre+ DRG nociceptor cell bodies and central terminals in the spinal cord dorsal horn. Scale bars = 50 µm, throughout. (c,d) Blue light-evoked (473 nm, 1.0 mW/cm², 0.2 ms, 0.05 Hz) EPSCs recorded in laminae I and II outer spinal neurons in slices from MOR cKO and littermate controls. Numbered inset traces correspond to individual EPSCs before, during, and after wash-out of bath applied DAMGO (500 nM; 5 min duration). Scale bar = 100 pA, 10 ms. (c) DAMGO-induced depression of EPSC amplitude and rebound LTP after washout (Control + LTP; n = 8 / 15 neurons) in control slices. (d) MOR cKO spinal neurons do not show DAMGO-induced depression of light-evoked EPSCs or rebound LTP upon DAMGO washout (n = 9 / 9 neurons). Error bars are mean ± SEM.

Figure 5. Pharmacological blockade of peripheral MOR by methylnaltrexone bromide (MNB) dose-dependently prevents the onset of morphine antinociceptive tolerance and OIH

(a) Temporal raster plot of nociception-induced sensory-reflexive and affective-motivational behaviors in an inescapable noxious environment (enclosed 52.5 °C hotplate). Each row displays the behavioral profile over 45 s for an individual mouse given either saline (n = 17), morphine (n = 19), or morphine + MNB (n = 10), on the first treatment (Day 1) and after chronic treatment (Day 7). (b,c) Summary of reflexive paw flinches in panel a: (b) cumulative summation and AUC analysis for Day 1 and Day 7 trials (F_{2, 43} = 8.749, P = 0.0006), and (c) the dose-response effect of MNB on morphine antinociceptive tolerance displayed as the percent change in AUC between trial days. (d,e) Summary of all affective-motivational behaviors in panel a: (d) cumulative summation and AUC analysis for Day 1 and Day 7 trials (F_{2, 43} = 24.61, P < 0.0001), and (e) the dose-response effect of MNB on morphine tolerance. (f–k) Effect of MNB co-administration at multiple doses on acute morphine antinociception for (f) mechanically-induced nociceptive paw flinches (F_{1, 42} = 182.9, P < 0.0001), (g) noxious thermal-induced paw attending and guarding (F_{1, 39} = 279.9, P < 0.0001), (h,i) antinociceptive tolerance (reflexive hypersensitivity: F_{4, 42} = 3.54, P = 0.0141; affective-motivational: F_{4, 40} = 6.115, P = 0.0006), and (j,k) OIH (reflexive hypersensitivity: F_{4, 42} = 6.825, P = 0.0003; affective-motivational: F_{4, 40} = 3.619, P = 0.0131). MNB doses 0.0 – 1.0 mg/kg, n = 10; MNB 10 mg/kg, n = 7 for reflexive tests and n = 5 for affective-motivational tests. Best-fit lines were generated following non-linear regression analysis based on the % MPE for each mouse. One-way ANOVA + Bonferroni (left panels of b,d,f–k). ★ P < 0.05. Error bars are mean ± SEM. Overlaid points are individual animal scores. PID = post-injury day.

Figure 6. Combination therapy of morphine and MNB delivers long-lasting antinociception, without the onset of tolerance, during perioperative and chronic pain states

(a,d,g,j) Timecourse for (a,g) nociceptive hypersensitivity and (d,j) affective-motivational behaviors following an orthotrauma (tibia fracture and bone pinning), or a peripheral nerve injury (Chronic Constriction Injury, CCI), and the effect of chronic saline (n = 10 for fracture, n = 5 for CCI), 10 mg/kg morphine (n = 10 / injury model), or 10 mg/kg morphine + 10 mg/kg MNB (n = 10 / injury model) treatments. Treatments are first given on PID 7 and re-administered once daily until PID 14, with the exception that the Saline group is administered acute morphine only on PID 7 and PID 14. (b,e,h,k) Antinociceptive tolerance: (left panels) Maximal possible effect (MPE) for morphine antinociception from the first administration (PID 7: +30 min) compared to the last administration (PID 14: +30 min) (Fracture: von Frey, F2, 21 = 2.05, P = 0.0023; 55 °C drop, F2, 21 = 6.084, P = 0.0082. CCI: von Frey, F2, 27 = 13.8, P = 0.0009; Acetone drop, F2, 27 = 5.976, P = 0.0213), and (right panels) the percent change for each subject. (c,f,i,l) OIH: (left panels) Percent change in the pre-morphine baseline nociceptive behaviors prior to the first administration (PID 7: BL) compared to just to the last (PID 14: BL) (Fracture: von Frey, F_{2, 22} = 4.253, P = 0.0274; 55 °C drop, F_{2, 21} = 5.347, P = 0.0133. CCI: von Frey, F_{2, 27} = 0.05814, P = 0.9436; Acetone drop, F_{2, 27} = 0.4272, P = 0.6567), and (right panels) the percent change for each subject. (m,n) Schematic detailing the influence of opioid-induced nociceptor maladaptive potentiation over CNS analgesic circuits to initiate tolerance and OIH. Conditional MOR deletion or MNB blockade of MOR signaling in nociceptors abrogates this potentiation, thereby maintaining opioid analgesic efficacy and reducing OIH. Two-way ANOVA + Bonferroni (for panels c,f,i,l two separate ANOVAs were run for Pre-injury vs. PID7, and PID7 vs. PID14). Student’s t Test, two-tailed (right panels of b,c,e,f,h,i,k,l). ★ P < 0.05. Error bars are mean ± SEM. Overlaid points are individual animal scores. PID = post-injury day.