Human birth tissue products as a non-opioid medicine to inhibit post-surgical pain.

Pain after surgery causes significant suffering. Opioid analgesics cause severe side effects and accidental death. Therefore, there is an urgent need to develop non-opioid therapies for managing post-surgical pain. Local application of Clarix Flo (FLO), a human amniotic membrane (AM) product, attenuated established post-surgical pain hypersensitivity without exhibiting known side effects of opioid use in mice. This effect was achieved through direct inhibition of nociceptive dorsal root ganglion (DRG) neurons via CD44-dependent pathways. We further purified the major matrix component, the heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) from human AM that has greater purity and water solubility than FLO. HC-HA/PTX3 replicated FLO-induced neuronal and pain inhibition. Mechanistically, HC-HA/PTX3 induced cytoskeleton rearrangements to inhibit sodium current and high-voltage activated calcium current on nociceptive neurons, suggesting it is a key bioactive component mediating pain relief. Collectively, our findings highlight the potential of naturally derived biologics from human birth tissues as an effective non-opioid treatment for post-surgical pain and unravel the underlying mechanisms.

Peripherally restricted cannabinoid and mu-opioid receptor agonists synergistically attenuate neuropathic mechanical hypersensitivity in mice.

ABSTRACT: Many medications commonly used to treat neuropathic pain are associated with significant, dose-limiting adverse effects, including sedation, dizziness, and fatigue. These adverse effects are due to the activity of these medications within the central nervous system. The objective of this work was to investigate the interactions between peripherally restricted cannabinoid receptor and mu-opioid receptor (MOR) agonists on ongoing and evoked neuropathic pain behaviors in mouse models. RNAscope analysis of cannabinoid receptor type 1 (CB1R) and MOR mRNA demonstrated that the mRNA of both receptors is colocalized in both mouse and human dorsal root ganglion. Single-cell RNAseq of dorsal root ganglion from chronic constriction injury mice showed that the mRNA of both receptors (Cnr1 and Oprm1) is coexpressed across different neuron clusters. Myc-CB1R and FLAG-MOR were cotransfected into immortalized HEK-293T cells and were found to interact at a subcellular level. We also find that CB-13 (a peripherally restricted dual CB1R and cannabinoid receptor type 2 agonist) and DALDA (a peripherally restricted MOR agonist) both attenuate mechanical hypersensitivity in a murine model of neuropathic pain. Using isobolographic analysis, we demonstrate that when coadministered, these agents synergistically attenuate mechanical hypersensitivity. Importantly, combination dosing of these agents does not cause any detectable preferential behaviors or motor impairment. However, repeated dosing of these agents is associated with the development of tolerance to these drugs. Collectively, these findings suggest that leveraging synergistic pain inhibition between cannabinoid receptor and MOR agonists in peripheral sensory neurons may be worth examining in patients with neuropathic pain.

Chronic pain after spine surgery: Insights into pathogenesis, new treatment, and preventive therapy.

Chronic pain after spine surgery (CPSS) is often characterized by intractable low back pain and/or radiating leg pain, and has been reported in 8-40% of patients that received lumbar spine surgery. We conducted a literature search of PubMed, MEDLINE/OVID with a focus on studies about the etiology and treatments of CPSS and low back pain. Our aim was to provide a narrative review that would help us better understand the pathogenesis and current treatment options for CPSS. This knowledge will aid in the development of optimal strategies for managing postoperative pain symptoms and potentially curing the underlying etiologies. Firstly, we reviewed recent advances in the mechanistic study of CPSS, illustrated both structural (e.g., fibrosis and scaring) and non-structural factors (e.g., inflammation, neuronal sensitization, glial activation, psychological factor) causing CPSS, and highlighted those having not been given sufficient attention as the etiology of CPSS. Secondly, we summarized clinical evidence and therapeutic perspectives of CPSS. We also presented new insights about the treatments and etiology of CPSS, in order to raise awareness of medical staff in the identification and management of this complex painful disease. Finally, we discussed potential new targets for clinical interventions of CPSS and future perspectives of mechanistic and translational research. CPSS patients often have a mixed etiology. By reviewing recent findings, the authors advocate that clinicians shall comprehensively evaluate each case to formulate a patient-specific and multi-modal pain treatment, and importantly, consider an early intraoperative intervention that may decrease the risk or even prevent the onset of CPSS. Translational potential statement: CPSS remains difficult to treat. This review broadens our understanding of clinical therapies and underlying mechanisms of CPSS, and provides new insights which will aid in the development of novel mechanism-based therapies for not only managing the established pain symptoms but also preventing the development of CPSS.

The Utility of Peripherally Restricted Kappa-Opioid Receptor Agonists for Inhibiting Below-Level Pain After Spinal Cord Injury in Mice.

Pain after spinal cord injury (SCI) can be difficult to treat. Drugs that target the opioid receptor (OR) outside the central nervous system (CNS) have gained increasing interest in pain control owing to their low risk of central side effects. Asimadoline and ICI-204448 are believed to be peripherally restricted KOR agonists withlimited access to the CNS. This study examined whether they can attenuate pain hypersensitivity in mice subjected to a contusive T10 SCI. Subcutaneous (s.c.) injection of asimadoline (5, 20 mg/kg) and ICI-204448 (1, 10 mg/kg) inhibited heat hypersensitivity at both doses, but only attenuated mechanical hypersensitivity at the high dose. However, the high-dose asimadoline adversely affected animals' exploratory performance in SCI mice and caused aversion, suggesting CNS drug penetration. In contrast, high-dose ICI-204448 did not impair exploration and remained effective in reducing both mechanical and heat hypersensitivities after SCI. Accordingly, we chose to examine the potential peripheral neuronal mechanism for ICI-204448-induced pain inhibition by conducting in vivo calcium imaging of dorsal root ganglion (DRG) in Pirt-GCaMP6s+/- mice. High-dose ICI-204448 (10 mg/kg, s.c.) attenuated the increased fluorescence intensity of lumbar DRG neurons activated by a noxious pinch (400 g) stimulation in SCI mice. In conclusion, systemic administration of ICI-204448 achieved SCI pain inhibition at doses that did not induce notable side effects and attenuated DRG neuronal excitability which may partly contribute to its pain inhibition. These findings suggest that peripherally restricted KOR agonists may be useful for treating SCI pain, but the therapeutic window must be carefully examined.

Spinal Cord Stimulation Increases Chemoefficacy and Prevents Paclitaxel-Induced Pain via CX3CL1.

INTRODUCTION: Despite increasing utilization of spinal cord stimulation (SCS), its effects on chemoefficacy, cancer progression, and chemotherapy-induced peripheral neuropathy (CIPN) pain remain unclear. Up to 30% of adults who are cancer survivors may suffer from CIPN, and there are currently no effective preventative treatments. MATERIALS AND METHODS: Through a combination of bioluminescent imaging, behavioral, biochemical, and immunohistochemical approaches, we investigated the role of SCS and paclitaxel (PTX) on tumor growth and PTX-induced peripheral neuropathy (PIPN) pain development in T-cell-deficient male rats (Crl:NIH-Foxn1rnu) with xenograft human non-small cell lung cancer. We hypothesized that SCS can prevent CIPN pain and enhance chemoefficacy partially by modulating macrophages, fractalkine (CX3CL1), and inflammatory cytokines. RESULTS: We show that preemptive SCS enhanced the antitumor efficacy of PTX and prevented PIPN pain. Without SCS, rats with and without tumors developed robust PIPN pain-related mechanical hypersensitivity, but only those with tumors developed cold hypersensitivity, suggesting T-cell dependence for different PIPN pain modalities. SCS increased soluble CX3CL1 and macrophages and decreased neuronal and nonneuronal insoluble CX3CL1 expression and inflammation in dorsal root ganglia. CONCLUSION: Collectively, our findings suggest that preemptive SCS is a promising strategy to increase chemoefficacy and prevent PIPN pain via CX3CL1-macrophage modulation.

scRNA-sequencing reveals subtype-specific transcriptomic perturbations in DRG neurons of PirtEGFPf mice in neuropathic pain condition.

Functionally distinct subtypes/clusters of dorsal root ganglion (DRG) neurons may play different roles in nerve regeneration and pain. However, details about their transcriptomic changes under neuropathic pain conditions remain unclear. Chronic constriction injury (CCI) of the sciatic nerve represents a well-established model of neuropathic pain, and we conducted single-cell RNA-sequencing (scRNA-seq) to characterize subtype-specific perturbations of transcriptomes in lumbar DRG neurons on day 7 post-CCI. By using PirtEGFPf mice that selectively express an enhanced green fluorescent protein in DRG neurons, we established a highly efficient purification process to enrich neurons for scRNA-seq. We observed the emergence of four prominent CCI-induced clusters and a loss of marker genes in injured neurons. Importantly, a portion of injured neurons from several clusters were spared from injury-induced identity loss, suggesting subtype-specific transcriptomic changes in injured neurons. Moreover, uninjured neurons, which are necessary for mediating the evoked pain, also demonstrated cell-type-specific transcriptomic perturbations in these clusters, but not in others. Notably, male and female mice showed differential transcriptomic changes in multiple neuronal clusters after CCI, suggesting transcriptomic sexual dimorphism in DRG neurons after nerve injury. Using Fgf3 as a proof-of-principle, RNAscope study provided further evidence of increased Fgf3 in injured neurons after CCI, supporting scRNA-seq analysis, and calcium imaging study unraveled a functional role of Fgf3 in neuronal excitability. These findings may contribute to the identification of new target genes and the development of DRG neuron cell-type-specific therapies for optimizing neuropathic pain treatment and nerve regeneration.

BzATP Activates Satellite Glial Cells and Increases the Excitability of Dorsal Root Ganglia Neurons In Vivo.

The purinergic system plays an important role in pain transmission. Recent studies have suggested that activation of P2-purinergic receptors (P2Rs) may be involved in neuron-satellite glial cell (SGC) interactions in the dorsal root ganglia (DRG), but the details remain unclear. In DRG, P2X7R is selectively expressed in SGCs, which closely surround neurons, and is highly sensitive to 3'-O-(4-Benzoyl) benzoyl-ATP (BzATP). Using calcium imaging in intact mice to survey a large number of DRG neurons and SGCs, we examined how intra-ganglionic purinergic signaling initiated by BzATP affects neuronal activities in vivo. We developed GFAP-GCaMP6s and Pirt-GCaMP6s mice to express the genetically encoded calcium indicator GGCaM6s in SGCs and DRG neurons, respectively. The application of BzATP to the ganglion induced concentration-dependent activation of SGCs in GFAP-GCaMP6s mice. In Pirt-GCaMP6s mice, BzATP initially activated more large-size neurons than small-size ones. Both glial and neuronal responses to BzATP were blocked by A438079, a P2X7R-selective antagonist. Moreover, blockers to pannexin1 channels (probenecid) and P2X3R (A317491) also reduced the actions of BzATP, suggesting that P2X7R stimulation may induce the opening of pannexin1 channels, leading to paracrine ATP release, which could further excite neurons by acting on P2X3Rs. Importantly, BzATP increased the responses of small-size DRG neurons and wide-dynamic range spinal neurons to subsequent peripheral stimuli. Our findings suggest that intra-ganglionic purinergic signaling initiated by P2X7R activation could trigger SGC-neuron interaction in vivo and increase DRG neuron excitability.

Thieno[2,3-d]pyrimidine-Based Positive Allosteric Modulators of Human Mas-Related G Protein-Coupled Receptor X1 (MRGPRX1).

Mas-related G protein-coupled receptor X1 (MRGPRX1) is a human sensory neuron-specific receptor and potential target for the treatment of pain. Positive allosteric modulators (PAMs) of MRGPRX1 have the potential to preferentially activate the receptors at the central terminals of primary sensory neurons and minimize itch side effects caused by peripheral activation. Using a high-throughput screening (HTS) hit, a series of thieno[2,3-d]pyrimidine-based molecules were synthesized and evaluated as human MRGPRX1 PAMs in HEK293 cells stably transfected with human MrgprX1 gene. An iterative process to improve potency and metabolic stability led to the discovery of orally available 6-(tert-butyl)-5-(3,4-dichlorophenyl)-4-(2-(trifluoromethoxy)phenoxy)thieno[2,3-d]pyrimidine (1t), which can be distributed to the spinal cord, the presumed site of action, following oral administration. In a neuropathic pain model induced by sciatic nerve chronic constriction injury (CCI), compound 1t (100 mg/kg, po) reduced behavioral heat hypersensitivity in humanized MRGPRX1 mice, demonstrating the therapeutic potential of MRGPRX1 PAMs in treating neuropathic pain.

Purinergic signaling between neurons and satellite glial cells of mouse dorsal root ganglia modulates neuronal excitability in vivo.

ABSTRACT: Primary sensory neurons in dorsal root ganglia (DRG) are wrapped by satellite glial cells (SGCs), and neuron-SGC interaction may affect somatosensation, especially nociceptive transmission. P2-purinergic receptors (P2Rs) are key elements in the two-way interactions between DRG neurons and SGCs. However, because the cell types are in such close proximity, conventional approaches such as in vitro culture and electrophysiologic recordings are not adequate to investigate the physiologically relevant responses of these cells at a population level. Here, we performed in vivo calcium imaging to survey the activation of hundreds of DRG neurons in Pirt-GCaMP6s mice and to assess SGC activation in GFAP-GCaMP6s mice in situ. By combining pharmacologic and electrophysiologic techniques, we investigated how ganglionic purinergic signaling initiated by α,ß-methyleneadenosine 5'-triphosphate (α,ß-MeATP) modulates neuronal activity and excitability at a population level. We found that α,ß-MeATP induced robust activation of small neurons-likely nociceptors-through activation of P2X3R. Large neurons, which are likely non-nociceptive, were also activated by α,ß-MeATP, but with a delay. Blocking pannexin 1 channels attenuated the late phase response of DRG neurons, indicating that P2R stimulation may subsequently induce paracrine ATP release, which could further activate cells in the ganglion. Moreover, ganglionic α,ß-MeATP treatment in vivo sensitized small neurons and enhanced responses of spinal wide-dynamic-range neurons to subsequent C-fiber inputs, suggesting that modulation via ganglionic P2R signaling could significantly affect nociceptive neuron excitability and pain transmission. Therefore, targeting functional P2Rs within ganglia may represent an important new strategy for pain modulation.

Role of primary sensory neurone cannabinoid type-1 receptors in pain and the analgesic effects of the peripherally acting agonist CB-13 in mice.

BACKGROUND: Cannabinoid type-1 receptors (CB1Rs) are expressed in primary sensory neurones, but their role in pain modulation remains unclear. METHODS: We produced Pirt-CB1R conditional knockout (cKO) mice to delete CB1Rs in primary sensory neurones selectively, and used behavioural, pharmacological, and electrophysiological approaches to examine the influence of peripheral CB1R signalling on nociceptive and inflammatory pain. RESULTS: Conditional knockout of Pirt-CB1R did not alter mechanical or heat nociceptive thresholds, complete Freund adjuvant-induced inflammation, or heat hyperalgesia in vivo. The intrinsic membrane properties of small-diameter dorsal root ganglion neurones were also comparable between cKO and wild-type mice. Systemic administration of CB-13, a peripherally restricted CB1/CB2R dual agonist (5 mg kg-1), inhibited nociceptive pain and complete Freund adjuvant-induced inflammatory pain. These effects of CB-13 were diminished in Pirt-CB1R cKO mice. In small-diameter neurones from wild-type mice, CB-13 concentration-dependently inhibited high-voltage activated calcium current (HVA-ICa) and induced a rightward shift of the channel open probability curve. The effects of CB-13 were significantly attenuated by AM6545 (a CB1R antagonist) and Pirt-CB1R cKO. CONCLUSION: CB1R signalling in primary sensory neurones did not inhibit nociceptive or inflammatory pain, or the intrinsic excitability of nociceptive neurones. However, peripheral CB1Rs are important for the analgesic effects of systemically administered CB-13. In addition, HVA-ICa inhibition appears to be a key ionic mechanism for CB-13-induced pain inhibition. Thus, peripherally restricted CB1R agonists could have utility for pain treatment.

Astrocytes contribute to pain gating in the spinal cord.

Various pain therapies have been developed on the basis of the gate control theory of pain, which postulates that nonpainful sensory inputs mediated by large-diameter afferent fibers (Aß-fibers) can attenuate noxious signals relayed to the brain. To date, this theory has focused only on neuronal mechanisms. Here, we identified an unprecedented function of astrocytes in the gating of nociceptive signals transmitted by neurokinin 1 receptor­positive (NK1R+) projection neurons in the spinal cord. Electrical stimulation of peripheral Aß-fibers in naïve mice activated spinal astrocytes, which in turn induced long-term depression (LTD) in NK1R+ neurons and antinociception through activation of endogenous adenosinergic mechanisms. Suppression of astrocyte activation by pharmacologic, chemogenetic, and optogenetic manipulations blocked the induction of LTD in NK1R+ neurons and pain inhibition by Aß-fiber stimulation. Collectively, our study introduces astrocytes as an important component of pain gating by activation of Aß-fibers, which thus exert nonneuronal control of pain.

Calcium imaging in population of dorsal root ganglion neurons unravels novel mechanisms of visceral pain sensitization and referred somatic hypersensitivity.

ABSTRACT: Mechanisms of visceral pain sensitization and referred somatic hypersensitivity remain unclear. We conducted calcium imaging in Pirt-GCaMP6s mice to gauge responses of dorsal root ganglion (DRG) neurons to visceral and somatic stimulation in vivo. Intracolonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced colonic inflammation and increased the percentage of L6 DRG neurons that responded to colorectal distension above that of controls at day 7. Colorectal distension did not activate L4 DRG neurons. TNBS-treated mice exhibited more Evans blue extravasation than did control mice and developed mechanical hypersensitivity in low-back skin and hind paws, which are innervated by L6 and L4 DRG neurons, respectively, suggesting that colonic inflammation induced mechanical hypersensitivity in both homosegmental and heterosegmental somatic regions. Importantly, the percentage of L4 DRG neurons activated by hind paw pinch and brush stimulation and calcium responses of L6 DRG neurons to low-back brush stimulation were higher at day 7 after TNBS than those in control mice. Visceral irritation from intracolonic capsaicin instillation also increased Evans blue extravasation in hind paws and low-back skin and acutely increased the percentage of L4 DRG neurons responding to hind paw pinch and the response of L6 DRG neurons to low-back brush stimulation. These findings suggest that TNBS-induced colitis and capsaicin-induced visceral irritation may sensitize L6 DRG neurons to colorectal and somatic inputs and also increase the excitability of L4 DRG neurons that do not receive colorectal inputs. These changes may represent a potential peripheral neuronal mechanism for visceral pain sensitization and referred somatic hypersensitivity.

Ubiquitin-mediated receptor degradation contributes to development of tolerance to MrgC agonist-induced pain inhibition in neuropathic rats.

ABSTRACT: Agonists to subtype C of the Mas-related G-protein-coupled receptors (MrgC) induce pain inhibition after intrathecal (i.t.) administration in rodent models of nerve injury. Here, we investigated whether tolerance develops after repeated MrgC agonist treatments and examined the underlying mechanisms. In animal behavior studies conducted in male rats at 4 to 5 weeks after an L5 spinal nerve ligation (SNL), the ability of dipeptide MrgC agonist JHU58 (0.1 mM, 10 µL, i.t.) to inhibit mechanical and heat hypersensitivity decreased after 3 days of treatment with a tolerance-inducing dose (0.5 mM, 10 µL, i.t., twice/day). In HEK293T cells, acute treatment with JHU58 or BAM8-22 (a large peptide MrgC agonist) led to MrgC endocytosis from the cell membrane and later sorting to the membrane for reinsertion. However, chronic exposure to JHU58 increased the coupling of MrgC to ß-arrestin-2 and led to the ubiquitination and degradation of MrgC. Importantly, pretreatment with TAK-243 (0.2 mM, 5 µL, i.t.), a small-molecule inhibitor of the ubiquitin-activating enzyme, during tolerance induction attenuated the development of tolerance to JHU58-induced inhibition of mechanical and heat hypersensitivity in SNL rats. Interestingly, morphine analgesia was also decreased in SNL rats that had become tolerant to JHU58, suggesting a cross-tolerance. Furthermore, i.t. pretreatment with TAK-243, which reduced JHU58 tolerance, also attenuated the cross-tolerance to morphine analgesia. These findings suggest that tolerance can develop to MrgC agonist-induced pain inhibition after repeated i.t. administrations. This tolerance development to JHU58 may involve increased coupling of MrgC to ß-arrestin-2 and ubiquitin-mediated receptor degradation.

Spinal Cord Stimulation Attenuates Below-Level Mechanical Hypersensitivity in Rats After Thoracic Spinal Cord Injury.

OBJECTIVES: The burden of pain after spinal cord injury (SCI), which may occur above, at, or below injury level, is high worldwide. Spinal cord stimulation (SCS) is an important neuromodulation pain therapy, but its efficacy in SCI pain remains unclear. In SCI rats, we tested whether conventional SCS (50 Hz, 80% motor threshold [MoT]) and 1200 Hz, low-intensity SCS (40% MoT) inhibit hind paw mechanical hypersensitivity, and whether conventional SCS attenuates evoked responses of wide-dynamic range (WDR) neurons in lumbar spinal cord. MATERIALS AND METHODS: Male rats underwent a moderate contusive injury at the T9 vertebral level. Six to eight weeks later, SCS or sham stimulation (120 min, n = 10) was delivered through epidural miniature electrodes placed at upper-lumbar spinal cord, with using a crossover design. Mechanical hypersensitivity was examined in awake rats by measuring paw withdrawal threshold (PWT) to stimulation with von Frey filaments. WDR neurons were recorded with in vivo electrophysiologic methods in a separate study of anesthetized rats. RESULTS: Both conventional SCS and 1200 Hz SCS increased PWTs from prestimulation level in SCI rats, but the effects were modest and short-lived. Sham SCS was not effective. Conventional SCS (10 min) at an intensity that evokes the peak Aα/ß waveform of sciatic compound action potential did not inhibit WDR neuronal responses (n = 19) to graded or repeated electrical stimulation that induces windup. CONCLUSIONS: Conventional SCS and 1200 Hz, low-intensity SCS modestly attenuated below-level mechanical hypersensitivity after SCI. Inhibition of WDR neurons was not associated with pain inhibition from conventional SCS.

Spinal Cord Stimulation Enhances Microglial Activation in the Spinal Cord of Nerve-Injured Rats.

Microglia can modulate spinal nociceptive transmission. Yet, their role in spinal cord stimulation (SCS)-induced pain inhibition is unclear. Here, we examined how SCS affects microglial activation in the lumbar cord of rats with chronic constriction injury (CCI) of the sciatic nerve. Male rats received conventional SCS (50 Hz, 80% motor threshold, 180 min, 2 sessions/day) or sham stimulation on days 18-20 post-CCI. SCS transiently attenuated the mechanical hypersensitivity in the ipsilateral hind paw and increased OX-42 immunoreactivity in the bilateral dorsal horns. SCS also upregulated the mRNAs of M1-like markers, but not M2-like markers. Inducible NOS protein expression was increased, but brain-derived neurotrophic factor was decreased after SCS. Intrathecal minocycline (1 µg-100 µg), which inhibits microglial activation, dose-dependently attenuated the mechanical hypersensitivity. Pretreatment with low-dose minocycline (1 µg, 30 min) prolonged the SCS-induced pain inhibition. These findings suggest that conventional SCS may paradoxically increase spinal M1-like microglial activity and thereby compromise its own ability to inhibit pain.

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

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.

Activation of µ-δ opioid receptor heteromers inhibits neuropathic pain behavior in rodents.

Several reports support the idea that µ- and δ-opioid receptors (ORs) may exist as heterodimers in brain regions involved in pain signaling. The unique pharmacology of these heteromers may present a novel analgesic target. However, the role of µ-δ heteromers in sensory neurons involved in pain and opioid analgesia remains unclear, particularly during neuropathic pain. We examined the effects of spinal nerve injury on µ-δ heteromer expression in dorsal root ganglion (DRG) neurons and the effects of a µ-δ heteromer-targeting agonist, CYM51010, on neuropathic pain behavior in rats and mice. An L5 spinal nerve ligation (SNL) in rats significantly decreased µ-δ heteromer expression in L5 DRG but increased heteromer levels in uninjured L4 DRG. Importantly, in SNL rats, subcutaneous injection of CYM51010 inhibited mechanical hypersensitivity in a dose-related manner (EC50: 1.09 mg/kg) and also reversed heat hyperalgesia and attenuated ongoing pain (2 mg/kg, subcutaneously). HEK-293T cell surface-labeled with µ- and δ-ORs internalized both receptors after exposure to CYM51010. By contrast, in cells transfected with µ-OR alone, CYM51010 was significantly less effective at inducing receptor internalization. Electrophysiologic studies showed that CYM51010 inhibited the C-component and windup phenomenon in spinal wide dynamic range neurons of SNL rats. The pain inhibitory effects of CYM51010 persisted in morphine-tolerant rats but was markedly attenuated in µ-OR knockout mice. Our studies show that spinal nerve injury may increase µ-δ heterodimerization in uninjured DRG neurons, and that µ-δ heteromers may be a potential therapeutic target for relieving neuropathic pain, even under conditions of morphine tolerance.

Dermorphin [D-Arg2, Lys4] (1-4) amide inhibits below-level heat hypersensitivity in mice after contusive thoracic spinal cord injury.

Opioid use for chronic pain is limited by severe central adverse effects. We examined whether activating mu-opioid receptors (MORs) in the peripheral nervous system attenuates spinal cord injury (SCI) pain-like behavior in mice. We produced a contusive SCI at the T10 vertebral level and examined motor and sensory dysfunction for 6 weeks. At 6 weeks, we tested the effect of subcutaneous (s.c.) injection of dermorphin [D-Arg2, Lys4] (1-4) amide (DALDA), a peripherally acting MOR-preferring agonist, on mechanical and heat hypersensitivity. Basso mouse scale score was significantly decreased after SCI, and mice showed hypersensitivity to mechanical and heat stimulation at the hind paw beginning at 2 weeks, as indicated by increased paw withdrawal frequency to mechanical stimulation and decreased paw withdrawal latency to heat stimulation. In wild-type SCI mice, DALDA (1 mg/kg, s.c.) attenuated heat but not mechanical hypersensitivity. The effect was blocked by pretreatment with an intraperitoneal injection of methylnaltrexone (5 mg/kg), a peripherally restricted opioid receptor antagonist, and was also diminished in Pirt-MOR conditional knockout mice. DALDA did not adversely affect exploratory activity or induced preference to drug treatment in SCI mice. In vivo calcium imaging showed that DALDA (1, 10 mg/kg, s.c.) inhibited responses of small dorsal root ganglion neurons to noxious heat stimulation in Pirt-GCaMP6s mice after SCI. Western blot analysis showed upregulation of MOR in the lumbar spinal cord and sciatic nerves at 6 weeks after SCI. Our findings suggest that peripherally acting MOR agonist may inhibit heat hypersensitivity below the injury level with minimal adverse effects.

Adjacent intact nociceptive neurons drive the acute outburst of pain following peripheral axotomy.

Injury of peripheral nerves may quickly induce severe pain, but the mechanism remains obscure. We observed a rapid onset of spontaneous pain and evoked pain hypersensitivity after acute transection of the L5 spinal nerve (SNT) in awake rats. The outburst of pain was associated with a rapid development of spontaneous activities and hyperexcitability of nociceptive neurons in the adjacent uninjured L4 dorsal root ganglion (DRG), as revealed by both in vivo electrophysiological recording and high-throughput calcium imaging in vivo. Transection of the L4 dorsal root or intrathecal infusion of aminobutyrate aminotransferase inhibitor attenuated the spontaneous activity, suggesting that retrograde signals from the spinal cord may contribute to the sensitization of L4 DRG neurons after L5 SNT. Electrical stimulation of low-threshold afferents proximal to the axotomized L5 spinal nerve attenuated the spontaneous activities in L4 DRG and pain behavior. These findings suggest that peripheral axotomy may quickly induce hyperexcitability of uninjured nociceptors in the adjacent DRG that drives an outburst of pain.

Sex differences in gene regulation in the dorsal root ganglion after nerve injury.

BACKGROUND: Pain is a subjective experience derived from complex interactions among biological, environmental, and psychosocial pathways. Sex differences in pain sensitivity and chronic pain prevalence are well established. However, the molecular basis underlying these sex dimorphisms are poorly understood particularly with regard to the role of the peripheral nervous system. Here we sought to identify shared and distinct gene networks functioning in the peripheral nervous systems that may contribute to sex differences of pain in rats after nerve injury. RESULTS: We performed RNA-seq on dorsal root ganglia following chronic constriction injury of the sciatic nerve in male and female rats. Analysis from paired naive and injured tissues showed that 1513 genes were differentially expressed between sexes. Genes which facilitated synaptic transmission in naïve and injured females did not show increased expression in males. CONCLUSIONS: Appreciating sex-related gene expression differences and similarities in neuropathic pain models may help to improve the translational relevance to clinical populations and efficacy of clinical trials of this major health issue.