Human birth tissue products as a regenerative medicine to inhibit post-surgical pain through multi-modal action.

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 and, more importantly, preventing its transition to a chronic state. In a mouse model of 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. Importantly, preemptive drug treatment also inhibited the transition of post-surgical pain to a prolonged state. This effect was achieved through direct inhibition of nociceptive dorsal root ganglion (DRG) neurons via CD44-dependent pathways, and indirect pain relief by attenuating immune cell recruitment. 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.

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.

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.

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.

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.

Oligomerization of MrgC11 and µ-opioid receptors in sensory neurons enhances morphine analgesia.

The µ-opioid receptor (MOR) agonist morphine is commonly used for pain management, but it has severe adverse effects and produces analgesic tolerance. Thus, alternative ways of stimulating MOR activity are needed. We found that MrgC11, a sensory neuron-specific G protein-coupled receptor, may form heteromeric complexes with MOR. Peptide-mediated activation of MrgC11 enhanced MOR recycling by inducing coendocytosis and sorting of MOR for membrane reinsertion. MrgC11 activation also inhibited the coupling of MOR to ß-arrestin-2 and enhanced the morphine-dependent inhibition of cAMP production. Intrathecal coadministration of a low dose of an MrgC agonist potentiated acute morphine analgesia and reduced chronic morphine tolerance in wild-type mice but not in Mrg-cluster knockout (Mrg KO) mice. BAM22, a bivalent agonist of MrgC and opioid receptors, enhanced the interaction between MrgC11 and MOR and produced stronger analgesia than did the individual monovalent agonists. Morphine-induced neuronal and pain inhibition was reduced in Mrg KO mice compared to that in wild-type mice. Our results uncover MrgC11-MOR interactions that lead to positive functional modulation of MOR. MrgC shares genetic homogeneity and functional similarity with human MrgX1. Thus, harnessing this positive modulation of MOR function by Mrg signaling may enhance morphine analgesia in a sensory neuron-specific fashion to limit central side effects.

Peripherally Acting µ-Opioid Receptor Agonists Attenuate Ongoing Pain-associated Behavior and Spontaneous Neuronal Activity after Nerve Injury in Rats.

BACKGROUND: Ongoing neuropathic pain is difficult to treat. The authors examined whether dermorphin [D-Arg2, Lys4] (1-4) amide, a peripherally acting µ-opioid receptor agonist, attenuates ongoing pain-associated manifestations after nerve injury in rats and mice. METHODS: Using conditioned place preference assay, the authors tested whether animals show a preference to the environment associated with drug treatment. Wide-dynamic range and dorsal root ganglion neuronal activities were measured by electrophysiology recording and calcium imaging. RESULTS: Nerve-injured animals stayed longer in dermorphin [D-Arg2, Lys4] (1-4) amide-paired chamber after conditioning than during preconditioning (rats: 402.4 ± 61.3 vs. 322.1 ± 45.0 s, 10 mg/kg, n = 9, P = 0.009; mice: 437.8 ± 59.4 vs. 351.3 ± 95.9 s, 2 mg/kg, n = 8, P = 0.047). Topical ganglionic application of dermorphin [D-Arg2, Lys4] (1-4) amide (5 µM, 1 µl, n = 5) reduced the numbers of small-diameter dorsal root ganglion neurons that showed spontaneous activity (1.1 ± 0.4 vs. 1.5 ± 0.3, P = 0.044) and that were activated by test stimulation (15.5 ± 5.5 vs. 28.2 ± 8.2, P = 0.009) after injury. In neuropathic rats, dermorphin [D-Arg2, Lys4] (1-4) amide (10 mg/kg, n = 8) decreased spontaneous firing rates in wide-dynamic range neurons to 53.2 ± 46.6% of predrug level, and methylnaltrexone (5 mg/kg, n = 9) blocked dermorphin [D-Arg2, Lys4] (1-4) amide-induced place preference and inhibition of wide-dynamic range neurons. Dermorphin [D-Arg2, Lys4] (1-4) amide increased paw withdrawal threshold (17.5 ± 2.2 g) from baseline (3.5 ± 0.7 g, 10 mg/kg, n = 8, P = 0.002) in nerve-injured rats, but the effect diminished after repeated administrations. CONCLUSIONS: Peripherally acting µ-opioids may attenuate ongoing pain-related behavior and its neurophysiologic correlates. Yet, repeated administrations cause antiallodynic tolerance.

Targeting human Mas-related G protein-coupled receptor X1 to inhibit persistent pain.

Human Mas-related G protein-coupled receptor X1 (MRGPRX1) is a promising target for pain inhibition, mainly because of its restricted expression in nociceptors within the peripheral nervous system. However, constrained by species differences across Mrgprs, drug candidates that activate MRGPRX1 do not activate rodent receptors, leaving no responsive animal model to test the effect on pain in vivo. Here, we generated a transgenic mouse line in which we replaced mouse Mrgprs with human MrgprX1 This humanized mouse allowed us to characterize an agonist [bovine adrenal medulla 8-22 (BAM8-22)] and a positive allosteric modulator (PAM), ML382, of MRGPRX1. Cellular studies suggested that ML382 enhances the ability of BAM8-22 to inhibit high-voltage-activated Ca2+ channels and attenuate spinal nociceptive transmission. Importantly, both BAM8-22 and ML382 effectively attenuated evoked, persistent, and spontaneous pain without causing obvious side effects. Notably, ML382 by itself attenuated both evoked pain hypersensitivity and spontaneous pain in MrgprX1 mice after nerve injury without acquiring coadministration of an exogenous agonist. Our findings suggest that humanized MrgprX1 mice provide a promising preclinical model and that activating MRGPRX1 is an effective way to treat persistent pain.

Activation of cannabinoid CB1 receptor contributes to suppression of spinal nociceptive transmission and inhibition of mechanical hypersensitivity by Aß-fiber stimulation.

Activation of Aß-fibers is an intrinsic feature of spinal cord stimulation (SCS) pain therapy. Cannabinoid receptor type 1 (CB1) is important to neuronal plasticity and pain modulation, but its role in SCS-induced pain inhibition remains unclear. In this study, we showed that CB1 receptors are expressed in both excitatory and inhibitory interneurons in substantia gelatinosa (SG). Patch-clamp recording of the evoked excitatory postsynaptic currents (eEPSCs) in mice after spinal nerve ligation (SNL) showed that electrical stimulation of Aß-fibers (Aß-ES) using clinical SCS-like parameters (50 Hz, 0.2 millisecond, 10 µA) induced prolonged depression of eEPSCs to C-fiber inputs in SG neurons. Pretreatment with CB1 receptor antagonist AM251 (2 µM) reduced the inhibition of C-eEPSCs by Aß-ES in both excitatory and inhibitory SG neurons. We further determined the net effect of Aß-ES on spinal nociceptive transmission in vivo by recording spinal local field potential in SNL rats. Epidural SCS (50 Hz, Aß-plateau, 5 minutes) attenuated C-fiber-evoked local field potential. This effect of SCS was partially reduced by spinal topical application of AM251 (25 µg, 50 µL), but not CB2 receptor antagonist AM630 (100 µg). Finally, intrathecal pretreatment with AM251 (50 µg, 15 µL) in SNL rats blocked the inhibition of behavioral mechanical hypersensitivity by SCS (50 Hz, 0.2 millisecond; 80% of motor threshold, 60 minutes). Our findings suggest that activation of spinal CB1 receptors may contribute to synaptic depression to high-threshold afferent inputs in SG neurons after Aß-ES and may be involved in SCS-induced inhibition of spinal nociceptive transmission after nerve injury.

Activation of Peripheral µ-opioid Receptors by Dermorphin [D-Arg2, Lys4] (1-4) Amide Leads to Modality-preferred Inhibition of Neuropathic Pain.

BACKGROUND: Opioids have long been regarded as the most effective drugs for the treatment of severe acute and chronic pain. Unfortunately, their therapeutic efficacy and clinical utility have been limited because of central and peripheral side effects. METHODS: To determine the therapeutic value of peripheral µ-opioid receptors as a target for neuropathic pain treatment, the authors examined the effects of dermorphin [D-Arg2, Lys4] (1-4) amide (DALDA), a hydrophilic, peripherally acting µ-opioid receptor agonist, in male and female rats with spinal nerve ligation-induced neuropathic pain. The authors also utilized behavioral, pharmacologic, electrophysiologic, and molecular biologic tools to characterize DALDA's possible mechanisms of action in male rats. RESULTS: DALDA, administered subcutaneously, had 70 times greater efficacy for inhibiting thermal (n = 8 to 11/group) than mechanical hypersensitivity (n = 6 to 8/group) in male rats. The pain inhibitory effects of DALDA on mechanical and heat hypersensitivity were abolished in animals pretreated with systemic methylnaltrexone (n = 7 to 9/group), a peripheral µ-opioid receptor antagonist. In the spinal wide-dynamic range neurons, systemic DALDA inhibited C-fiber-mediated, but not A-fiber-mediated, response in neuropathic male rats (n = 13). In primary sensory neurons, DALDA inhibited the capsaicin-induced [Ca2+] increase more than the ß-alanine-induced [Ca] increase (n = 300); capsaicin and ß-alanine activate subpopulations of neurons involved in the signaling of heat and mechanical pain, respectively. DALDA-treated rats (n = 5 to 8/group) did not exhibit motor deficits and locomotor impairment suggesting that it does not induce central side effects. CONCLUSIONS: These findings suggest that DALDA may represent a potential alternative to current opioid therapy for the treatment of neuropathic pain and is likely to be associated with minimal adverse effects.

Electrical stimulation of low-threshold afferent fibers induces a prolonged synaptic depression in lamina II dorsal horn neurons to high-threshold afferent inputs in mice.

Electrical stimulation of low-threshold Aß-fibers (Aß-ES) is used clinically to treat neuropathic pain conditions that are refractory to pharmacotherapy. However, it is unclear how Aß-ES modulates synaptic responses to high-threshold afferent inputs (C-, Aδ-fibers) in superficial dorsal horn. Substantia gelatinosa (SG) (lamina II) neurons are important for relaying and modulating converging spinal nociceptive inputs. We recorded C-fiber-evoked excitatory postsynaptic currents (eEPSCs) in spinal cord slices in response to paired-pulse test stimulation (500 µA, 0.1 millisecond, 400 milliseconds apart). We showed that 50-Hz and 1000-Hz, but not 4-Hz, Aß-ES (10 µA, 0.1 millisecond, 5 minutes) induced prolonged inhibition of C-fiber eEPSCs in SG neurons in naive mice. Furthermore, 50-Hz Aß-ES inhibited both monosynaptic and polysynaptic forms of C-fiber eEPSC in naive mice and mice that had undergone spinal nerve ligation (SNL). The paired-pulse ratio (amplitude second eEPSC/first eEPSC) increased only in naive mice after 50-Hz Aß-ES, suggesting that Aß-ES may inhibit SG neurons by different mechanisms under naive and nerve-injured conditions. Finally, 50-Hz Aß-ES inhibited both glutamatergic excitatory and GABAergic inhibitory interneurons, which were identified by fluorescence in vGlut2-Td and glutamic acid decarboxylase-green fluorescent protein transgenic mice after SNL. These findings show that activities in Aß-fibers lead to frequency-dependent depression of synaptic transmission in SG neurons in response to peripheral noxious inputs. However, 50-Hz Aß-ES failed to induce cell-type-selective inhibition in SG neurons. The physiologic implication of this novel form of synaptic depression for pain modulation by Aß-ES warrants further investigation.

Electrical stimulation of dorsal root entry zone attenuates wide-dynamic-range neuronal activity in rats.

OBJECTIVES: Recent clinical studies suggest that neurostimulation at the dorsal root entry zone (DREZ) may alleviate neuropathic pain. However, the mechanisms of action for this therapeutic effect are unclear. Here, we examined whether DREZ stimulation inhibits spinal wide-dynamic-range (WDR) neuronal activity in nerve-injured rats. MATERIALS AND METHODS: We conducted in vivo extracellular single-unit recordings of WDR neurons in rats after an L5 spinal nerve ligation (SNL) or sham surgery. We set bipolar electrical stimulation (50 Hz, 0.2 msec, 5 min) of the DREZ at the intensity that activated only Aα/ß-fibers by measuring the lowest current at which DREZ stimulation evoked a peak antidromic sciatic Aα/ß-compound action potential without inducing an Aδ/C-compound action potential (i.e., Ab1). RESULTS: The elevated spontaneous activity rate of WDR neurons in SNL rats (n = 25; data combined from post-SNL groups at days 14-16 [n = 15] and days 45-75 [n = 10]) was significantly decreased from the prestimulation level (p < 0.01) at 0-15 min and 30-45 min post-stimulation. In both sham-operated (n = 8) and nerve-injured rats, DREZ stimulation attenuated the C-component, but not the A-component, of the WDR neuronal response to graded intracutaneous electrical stimuli (0.1-10 mA, 2 msec) applied to the skin receptive field. Further, DREZ stimulation blocked windup (a form of brief neuronal sensitization) to repetitive noxious stimuli (0.5 Hz) at 0-15 min in all groups (p < 0.05). CONCLUSIONS: Attenuation of WDR neuronal activity may contribute to DREZ stimulation-induced analgesia. This finding supports the notion that DREZ may be a useful target for neuromodulatory control of pain.

Activation of MrgC receptor inhibits N-type calcium channels in small-diameter primary sensory neurons in mice.

Mas-related G-protein-coupled receptor subtype C (mouse MrgC11 and rat rMrgC), expressed specifically in small-diameter primary sensory neurons, may constitute a novel pain inhibitory mechanism. We have shown previously that intrathecal administration of MrgC-selective agonists can strongly attenuate persistent pain in various animal models. However, the underlying mechanisms for MrgC agonist-induced analgesia remain elusive. Here, we conducted patch-clamp recordings to test the effect of MrgC agonists on high-voltage-activated (HVA) calcium current in small-diameter dorsal root ganglion (DRG) neurons. Using pharmacological approaches, we show for the first time that an MrgC agonist (JHU58) selectively and dose-dependently inhibits N-type, but not L- or P/Q-type, HVA calcium channels in mouse DRG neurons. Activation of HVA calcium channels is important to neurotransmitter release and synaptic transmission. Patch-clamp recordings in spinal cord slices showed that JHU58 attenuated the evoked excitatory postsynaptic currents in substantia gelatinosa (SG) neurons in wild-type mice, but not in Mrg knockout mice, after peripheral nerve injury. These findings indicate that activation of endogenously expressed MrgC receptors at central terminals of primary sensory fibers may decrease peripheral excitatory inputs onto SG neurons. Together, these results suggest potential cellular and molecular mechanisms that may contribute to intrathecal MrgC agonist-induced analgesia. Because MrgC shares substantial genetic homogeneity with human MrgX1, our findings may suggest a rationale for developing intrathecally delivered MrgX1 receptor agonists to treat pathological pain in humans and provide critical insight regarding potential mechanisms that may underlie its analgesic effects.