Rodent Models Of Neuropathic Pain Research Articles

PGC‐1α activation ameliorates cancer-induced bone pain via inhibiting apoptosis of GABAergic interneurons

Cancer-induced bone pain (CIBP) stands out as one of the most challenging issues in clinical practice due to its intricate and not fully elucidated pathophysiological mechanisms. Existing evidence has pointed toward the significance of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) down-regulation in contributing to pain behaviors in various rodent models of neuropathic pain. In our current study, we aimed to investigate the role of PGC-1α in CIBP. Our results unveiled a reduction in PGC-1α expression within the spinal cord of CIBP rats, particularly in GABAergic interneurons. Notably, intrathecal administration of the PGC-1α activator ZLN005 suppressed the loss of spinal GABAergic interneurons. This suppression was achieved by inhibiting caspase-3-mediated apoptosis, ultimately leading to the alleviation of mechanical allodynia in CIBP rats. Further exploration into the mechanism revealed that PGC-1α activation played a pivotal role in mitigating ATP depletion and reactive oxygen species accumulation linked to mitochondrial dysfunction. This was achieved through the restoration of mitochondrial biogenesis and the activation of the SIRT3-SOD2 pathway. Impressively, the observed effects were prominently reversed upon the application of SR18292, a specific PGC-1α inhibitor. In conclusion, our findings strongly suggest that PGC-1α activation acts as a potent inhibitor of apoptosis in spinal GABAergic interneurons. This inhibition is mediated by the improvement of mitochondrial function, facilitated in part through the enhancement of mitochondrial biogenesis and the activation of the SIRT3-SOD2 pathway. The results of our study shed light on potential therapeutic avenues for addressing CIBP.

Transient Receptor Potential Channels: Multiple Modulators of Peripheral Neuropathic Pain in Several Rodent Models.

Neuropathic pain, a prevalent chronic condition in clinical settings, has attracted widespread societal attention. This condition is characterized by a persistent pain state accompanied by affective and cognitive disruptions, significantly impacting patients' quality of life. However, current clinical therapies fall short of addressing its complexity. Thus, exploring the underlying molecular mechanism of neuropathic pain and identifying potential targets for intervention is highly warranted. The transient receptor potential (TRP) receptors, a class of widely distributed channel proteins, in the nervous system, play a crucial role in sensory signaling, cellular calcium regulation, and developmental influences. TRP ion channels are also responsible for various sensory responses including heat, cold, pain, and stress. This review highlights recent advances in understanding TRPs in various rodent models of neuropathic pain, aiming to uncover potential therapeutic targets for clinical management.

Ninety-Hz Spinal Cord Stimulation–Induced Analgesia Is Dependent on Active Charge Balance and Is Nonlinearly Related to Amplitude: A Sham-Controlled Behavioral Study in a Rodent Model of Chronic Neuropathic Pain

BackgroundNinety-Hz active-recharge spinal cord stimulation (SCS) applied at below sensory-threshold intensity, as used with fast-acting subperception therapy spinal cord stimulation, has been shown clinically to produce significant analgesia, but additional characterization is required to better understand the therapy. This preclinical study investigates the behavioral effect of multiple 90-Hz SCS variants in a rodent model of neuropathic pain, focusing on charge balance and the relationship between 90-Hz efficacy and stimulation intensity. Materials and MethodsRats (n = 24) received a unilateral partial sciatic nerve ligation to induce neuropathic pain and were implanted with a quadripolar lead at T13. Mechanical hypersensitivity was assessed before, during, and after 60 minutes of SCS. After a prescreen with 50-Hz SCS 67% motor threshold ([MT], the positive control), rats underwent a randomized-crossover study including sham SCS and several 90-Hz SCS paradigms (at 40% MT or 60% MT, either using active or pseudopassive recharge) (experiment 1, n = 16). A second, identical experiment (experiment 2) was performed to supplement data with 90-Hz SCS at 20% and 80% MT (experiment 2, n = 8). ResultsExperiment 1: At 40% MT, 90-Hz active-recharge SCS produced a significantly larger recovery to baseline than did 90-Hz pseudopassive SCS at both tested intensities and sham SCS. Experiment 2: Only the 90-Hz SCS active recharge at 40% MT and 50-Hz SCS positive control caused mean recovery to baseline that was statistically better than that of sham SCS. ConclusionsThe degree to which 90-Hz SCS reduced mechanical hypersensitivity during stimulation depended on the nature of charge balance, with 90-Hz active-recharge SCS generating better responses than did 90-Hz pseudopassive recharge SCS. In addition, our findings suggest that the amplitude of 90-Hz active-recharge SCS must be carefully configured for efficacy.

Evaluation of trans- and cis-4-[18F]Fluorogabapentin for Brain PET Imaging.

Gabapentin, a selective ligand for the α2δ subunit of voltage-dependent calcium channels, is an anticonvulsant medication used in the treatment of neuropathic pain, epilepsy, and other neurological conditions. We recently described two radiofluorinated derivatives of gabapentin (trans-4-[18F]fluorogabapentin, [18F]tGBP4F, and cis-4-[18F]fluorogabapentin, [18F]cGBP4F) and showed that these compounds accumulate in the injured nerves in a rodent model of neuropathic pain. Given the use of gabapentin in brain diseases, here we investigate whether these radiofluorinated derivatives of gabapentin can be used for imaging α2δ receptors in the brain. Specifically, we developed automated radiosynthesis methods for [18F]tGBP4F and [18F]cGBP4F and conducted dynamic PET imaging in adult rhesus macaques with and without preadministration of pharmacological doses of gabapentin. Both radiotracers showed very high metabolic stability, negligible plasma protein binding, and slow accumulation in the brain. [18F]tGBP4F, the isomer with higher binding affinity, showed low brain uptake and could not be displaced, whereas [18F]cGBP4F showed moderate brain uptake and could be partially displaced. Kinetic modeling of brain regional time-activity curves using a metabolite-corrected arterial input function shows that a one-tissue compartment model accurately fits the data. Graphical analysis using Logan or multilinear analysis 1 produced similar results as compartmental modeling, indicating robust quantification. This study advances our understanding of how gabapentinoids work and provides an important advancement toward imaging α2δ receptors in the brain.

Melatonin Improves Mitochondrial Dysfunction and Attenuates Neuropathic Pain by Regulating SIRT1 in Dorsal Root Ganglions

Neuropathic pain is a debilitating chronic pain condition and is refractory to the currently available treatments. Emerging evidence suggests that melatonin exerts analgesic effects in rodent models of neuropathic pain. Nevertheless, the exact underlying mechanisms of the analgesic effects of melatonin on neuropathic pain are largely unknown. Here, we observed that spinal nerve ligation (SNL) in rats L5 and L6 induced an obvious decrease in the 50% paw withdrawal threshold (PWT) and paw withdrawal latency (PWL), indicating the induction of mechanical allodynia and the hyperalgesia, and melatonin prevented the genesis and maintenance of mechanical allodynia and the hyperalgesia. Notably, the inhibitory action of melatonin on SNL-induced mechanical allodynia and heat hypersensitivity was inhibited by a SIRT1 inhibitor (EX527). Melatonin treatment increased the expression of neuronal sirtuin1 (SIRT1) in DRGs following nerve injury. Furthermore, melatonin treatment restored the injury-dependent decrease in mitochondrial membrane potential and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and reduced the injury-dependent increase in hydrogen peroxide and 8-hydroxy-2-deoxyguanosine (8-OHdG), which was inhibited by EX527. In addition, we found that EX527 impeded the inhibitory effects of melatonin on the SNL-induced increased expression of cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β). In conclusion, the above data demonstrated that melatonin alleviated mechanical allodynia and hyperalgesia induced by peripheral nerve injury via SIRT1 activation. Melatonin resolved mitochondrial dysfunction—oxidative stress-dependent and neuroinflammation mechanisms that were driven by SIRT1 after nerve injury.

Identification and targeting of a unique NaV1.7 domain driving chronic pain

Small molecules directly targeting the voltage-gated sodium channel (VGSC) NaV1.7 have not been clinically successful. We reported that preventing the addition of a small ubiquitin-like modifier onto the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 function and was antinociceptive in rodent models of neuropathic pain. Here, we discovered a CRMP2 regulatory sequence (CRS) unique to NaV1.7 that is essential for this regulatory coupling. CRMP2 preferentially bound to the NaV1.7 CRS over other NaV isoforms. Substitution of the NaV1.7 CRS with the homologous domains from the other eight VGSC isoforms decreased NaV1.7 currents. A cell-penetrant decoy peptide corresponding to the NaV1.7-CRS reduced NaV1.7 currents and trafficking, decreased presynaptic NaV1.7 expression, reduced spinal CGRP release, and reversed nerve injury-induced mechanical allodynia. Importantly, the NaV1.7-CRS peptide did not produce motor impairment, nor did it alter physiological pain sensation, which is essential for survival. As a proof-of-concept for a NaV1.7 -targeted gene therapy, we packaged a plasmid encoding the NaV1.7-CRS in an AAV virus. Treatment with this virus reduced NaV1.7 function in both rodent and rhesus macaque sensory neurons. This gene therapy reversed and prevented mechanical allodynia in a model of nerve injury and reversed mechanical and cold allodynia in a model of chemotherapy-induced peripheral neuropathy. These findings support the conclusion that the CRS domain is a targetable region for the treatment of chronic neuropathic pain.

Noradrenergic Input from Nucleus of the Solitary Tract Regulates Parabrachial Activity in Mice.

The parabrachial complex (PB) is critically involved in aversive processes, and chronic pain is associated with amplified activity of PB neurons in rodent models of neuropathic pain. Here, we demonstrate that catecholaminergic input from the caudal nucleus of the solitary tract (cNTScat), a stress responsive region that integrates interoceptive and exteroceptive signals, causes amplification of PB activity and their sensory afferents. We used a virally mediated expression of a norepinephrine (NE) sensor, NE2h, fiber photometry, and extracellular recordings in anesthetized mice to show that noxious mechanical and thermal stimuli activate cNTS neurons. These stimuli also produce prolonged NE transients in PB that far outlast the noxious stimuli. Similar NE transients can be evoked by focal electrical stimulation of cNTS, a region that contains the noradrenergic A2 cell group that projects densely on PB. In vitro, optical stimulation of cNTScat terminals depolarized PB neurons and caused a prolonged increase the frequency of excitatory synaptic activity. A dual opsin approach showed that sensory afferents from the caudal spinal trigeminal nucleus are potentiated by cNTScat terminal activation. This potentiation was coupled with a decrease in the paired pulse ratio (PPR), consistent with an cNTScat-mediated increase in the probability of release at SpVc synapses. Together, these data suggest that A2 neurons of the cNTS generate long lasting NE transients in PB which increase excitability and potentiate responses of PB neurons to sensory inputs. These reveal a mechanism through which stressors from multiple modalities may potentiate the aversiveness of nociceptive stimuli.

Loss of astrocytic A1 adenosine receptor is involved in a chemotherapeutic agent-induced rodent model of neuropathic pain.

Oxaliplatin is a commonly used platinum chemotherapy drug, whereas peripheral neurotoxicity is a widely observed adverse reaction lacking a satisfactory therapeutic strategy. Different adenosine receptors underlying the common neuropathic phenotype play different roles through varied pathophysiological mechanisms. In this study, we investigated the role of adenosine receptor A1 (A1R) in oxaliplatin-induced neuropathic pain and its potential use in an effective therapeutic strategy. We established an oxaliplatin-induced neuropathic pain model simulating the mode of chemotherapy administration and observed the related neuropathic behavioral phenotype and implicated mechanisms. Five weekly injections of oxaliplatin for 2 weeks induced a severe and persistent neuropathic pain phenotype in mice. A1R expression in the spinal dorsal horn decreased during this process. Pharmacological intervention against A1R verified its importance in this process. Mechanistically, the loss of A1R expression was mainly attributed to its decreased expression in astrocytes. Consistent with the pharmacological results, the oxaliplatin-induced neuropathic pain phenotype was blocked by specific therapeutic interventions of A1R in astrocytes via lentiviral vectors, and the expression of glutamate metabolism-related proteins was upregulated. Neuropathic pain can be alleviated by pharmacological or astrocytic interventions via this pathway. These data reveal a specific adenosine receptor signaling pathway involved in oxaliplatin-induced peripheral neuropathic pain, which is related to the suppression of the astrocyte A1R signaling pathway. This may provide new opportunities for the treatment and management of neuropathic pain observed during oxaliplatin chemotherapy.

Inhibition of NKCC1 in spinal dorsal horn and dorsal root ganglion results in alleviation of neuropathic pain in rats with spinal cord contusion.

Previous studies have confirmed the relationship between chloride homeostasis and pain. However, the role of sodium potassium chloride co-transporter isoform 1 (NKCC1) in dorsal horn and dorsal root ganglion neurons (DRGs) in spinal cord injury (SCI)-induced neuropathic pain (NP) remains inconclusive. Therefore, we aimed to explore whether suppression of NKCC1 in the spinal cord and DRGs alleviate the NP of adult rats with thoracic spinal cord contusion. Thirty adult female Sprague-Dawley rats (8week-old, weighing 250-280g) were randomly divided into three groups with ten animals in each group (sham, SCI, and bumetanide groups). The paw withdrawal mechanical threshold and paw withdrawal thermal latency were recorded before injury (baseline) and on post-injury days 14, 21, 28, and 35. At the end of experiment, western blotting (WB) analysis, quantitative real-time Polymerase Chain Reaction (PCR) and immunofluorescence were performed to quantify NKCC1 expression. Our results revealed that NKCC1 protein expression in the spinal cord and DRGs was significantly up-regulated in rats with SCI. Intraperitoneal treatment of bumetanide (an NKCC1 inhibitor) reversed the expression of NKCC1 in the dorsal horn and DRGs and ameliorated mechanical ectopic pain and thermal hypersensitivities in the SCI rats. Our study demonstrated the occurrence of NKCC1 overexpression in the spinal cord and DRGs in a rodent model of NP and indicated that changes in the peripheral nervous system also play a major role in promoting pain sensitization after SCI.

Identification and characterisation of lipids that are positive allosteric modulators of glycine receptors.

Glycine receptors are ligand-gated ion channels that mediate fast inhibitory neurotransmission within the nervous system. They have recently emerged as targets for chronic pain therapies due to their role in nociceptive signalling. N-arachidonyl glycine is an endogenous lipid that modulates glycine receptors, however its mechanism of action remains unclear. We have identified a series of N-acyl amino acids that positively modulate glycine receptors. From this, a second series of lipids containing a phenylene-moiety within the lipid tail were synthesised. This improved their efficacy by up to 6-fold, and increased the potency of receptor activation by over 10-fold. (8-2-octylphneyl)octanoyl)glycine (OPOG) had the greatest activity - potentiating the glycine EC5 by 1500% with an EC50 of 664 nM at the α1 receptor. OPOG was also found to be analgesic and produces a dose-dependent reversal of allodynia in rodent models of neuropathic pain. The second aim of this study was to identify lipid binding sites on the glycine receptor. Molecular dynamic simulations of OPOG at the α1 receptor were conducted and indicated consistent binding within a cavity at the intracellular portion of transmembrane domains (TM) 1 and 4. This site has previously been suggested to bind neurosteroids. A cryoEM structure of OPOG bound to the zebrafish glycine receptor was also obtained and identified binding to an inter-subunit cavity between the extracellular portions of TM1(-), TM2(+) and TM3(+). This cavity has previously been shown to bind the positive allosteric modulator ivermectin. Mutagenesis of the residues identified within the two sites was indicated potentiation occurs primarily through the neurosteroid binding site involving aromatic and positively charged residues. Further studies are required to explore binding within the ivermectin cavity and its relative contribution to lipid modulation.

Reversal of Peripheral Neuropathic Pain by the Small-Molecule Natural Product Narirutin via Block of Nav1.7 Voltage-Gated Sodium Channel

Neuropathic pain is a refractory chronic disease affecting millions of people worldwide. Given that present painkillers have poor efficacy or severe side effects, developing novel analgesics is badly needed. The multiplex structure of active ingredients isolated from natural products provides a new source for phytochemical compound synthesis. Here, we identified a natural product, Narirutin, a flavonoid compound isolated from the Citrus unshiu, showing antinociceptive effects in rodent models of neuropathic pain. Using calcium imaging, whole-cell electrophysiology, western blotting, and immunofluorescence, we uncovered a molecular target for Narirutin's antinociceptive actions. We found that Narirutin (i) inhibits Veratridine-triggered nociceptor activities in L4-L6 rat dorsal root ganglion (DRG) neurons, (ii) blocks voltage-gated sodium (NaV) channels subtype 1.7 in both small-diameter DRG nociceptive neurons and human embryonic kidney (HEK) 293 cell line, (iii) does not affect tetrodotoxin-resistant (TTX-R) NaV channels, and (iv) blunts the upregulation of Nav1.7 in calcitonin gene-related peptide (CGRP)-labeled DRG sensory neurons after spared nerve injury (SNI) surgery. Identifying Nav1.7 as a molecular target of Narirutin may further clarify the analgesic mechanism of natural flavonoid compounds and provide an optimal idea to produce novel selective and efficient analgesic drugs.

Discovery of pyrazole-1-carboxamide derivatives as novel Gi-biased μ-opioid receptor agonists.

μ-Opioid receptor (MOR) Gi-biased agonists with no recruitment of β-arrestin were introduced as a new analgesic strategy to overcome the conventional undesirable side effects of opioid receptor-targeted drugs, such as tolerance, addiction, respiratory depression, and constipation. For the development of novel Gi-biased MOR agonists, the design, synthesis, and structure-activity relationship(SAR) analysis of the aminopyrazole core skeleton were conducted according to the current SAR data of PZM21 (2a) and its derivatives. New derivatives were biologically evaluated for their agonistic effects on cyclic adenosine monophosphate(cAMP) levels for the Gi pathway and β-arrestin recruitment in MOR/κ-opioid receptor/δ opioid receptor. An optimized selective Gi-biased agonist, Compound 17a, was discovered with potent cAMP inhibitory activities, with a 50% efficacy concentration valueof 87.1 nM and no activity in the MOR β-arrestin pathway and other subtypes. The in vivo pain relief efficacy of Compound 17a was confirmed in a dose-dependent manner with spinal nerve ligation and cisplatin-induced peripheral neuropathy rodent neuropathic pain models.

O-13: INHIBITION OF THE ENDOCANNABINOID REGULATING ENZYME MONOACYLGLYCEROL LIPASE AMELIORATES THE HYPER-NOCICEPTIVE PHENOTYPE IN THE BERKELEY MOUSE MODEL OF SICKLE CELL DISEASE

Purpose: Sickle Cell Disease (SCD) is a major cause of morbidity and mortality worldwide, and affects more than 100,000 people in the US. Recurrent episodes of acute, vaso-occlusive pain crisis, a hallmark of the disease, lead to chronic pain for many patients, though the mechanisms of this transition are poorly understood. Although opioids remain the standard of care to treat SCD chronic pain, their myriad adverse side effects (e.g., constipation, respiratory depression, abuse liability, dependence) as well as the fact that SCD chronic pain requires prolonged opioid treatment that results in tolerance, severely limit their therapeutic utility. Thus, a pressing need exists to identify effective non-opioid analgesic strategies to reduce SCD chronic pain. Humanized mouse models of SCD, such as the Berkeley (BERK) model, provide a useful tool to investigate disease pathophysiology and evaluate novel therapeutic strategies. Dorsal Root Ganglion (DRG) neurons are peripheral sensory neurons essential for the transmission of nociceptive afferents to the CNS. In rodent models of neuropathic pain, harvested DRG neurons show hyperexcitability. Inhibitors of the major degradative enzyme of 2-arachidonoylglycerol, monoacylglycerol lipase (MAGL), reduce nociceptive behavior in neuropathic and inflammatory preclinical models of pain through cannabinoid receptor-dependent and -independent mechanisms. As MAGL inhibitors have yet to be tested in BERK mice, here we test whether this approach will ameliorate the hyper-nociceptive phenotype of HbSS-BERK mice. Materials and methods: Male and female HbSS-BERK (sickle) and HbAA-BERK (control) mice were used as subjects for these experiments. Nociceptive behaviors were assessed using the von Frey and Hot Plate tests. Motor-functional behavior was assessed using the Grip Strength and Nesting assays. Neuronal hyperexcitability was assessed using whole cell patch clamp electrophysiology of L4-S1 DRG neurons. Data were analyzed as two- and three-way ANOVAs followed by Tukey post-hoc analysis when appropriate (p < 0.05 considered significant). Results: HbSS-BERK mice displayed profound mechanical, thermal and deep tissue/musculoskeletal hypersensitivity. HbSS-BERK mice also exhibited deficits in nest-building behavior. Patch clamp studies revealed that DRG neurons harvested from HbSS-BERK mice displayed extremely hyperexcitability. MJN-110 dose-dependently reduced mechanical allodynia and thermal hyperalgesia, and ameliorated deficits in grip strength in HbSS-BERK mice. Importantly, seven days of daily injections of MJN-110 (5 mg/kg) continued to ameliorate the hyper-nociceptive phenotype of HbSS-BERK mice, which did not undergo tolerance. Moreover, DRG harvested from the MJN-treated HbSS-BERK mice showed similar DRG neuronal activity as seen in control mice. Conclusion: These findings validate that HbSS-BERK mice show hypersensitive responses to mechanical and heat stimuli, and exhibit motor-functional deficits in grip strength and nest building behavior. Additionally, this work demonstrates hyperexcitability of sensory neurons from the lower lumbar region of the spine of HbSS-BERK mice. Finally, the observations that MJN110 reduces these hyper-nociceptive behaviors and ameliorates neuronal hyperexcitability in HbSS-BERK mice suggest that MAGL inhibition represents a viable strategy to reduce chronic pain related to sickle cell disease. The authors do not declare any conflict of interest

Discovery of (S)-1-((2',6-Bis(difluoromethyl)-[2,4'-bipyridin]-5-yl)oxy)-2,4-dimethylpentan-2-amine (BMS-986176/LX-9211): A Highly Selective, CNS Penetrable, and Orally Active Adaptor Protein-2 Associated Kinase 1 Inhibitor in Clinical Trials for the Treatment of Neuropathic Pain.

Recent mouse knockout studies identified adapter protein-2 associated kinase 1 (AAK1) as a viable target for treating neuropathic pain. Potent small-molecule inhibitors of AAK1 have been identified and show efficacy in various rodent pain models. (S)-1-((2',6-Bis(difluoromethyl)-[2,4'-bipyridin]-5-yl)oxy)-2,4-dimethylpentan-2-amine (BMS-986176/LX-9211) (34) was identified as a highly selective, CNS penetrant, potent AAK1 inhibitor from a novel class of bi(hetero)aryl ethers. BMS-986176/LX9211 (34) showed excellent efficacy in two rodent neuropathic pain models and excellent central nervous system (CNS) penetration and target engagement at the spinal cord with an average brain to plasma ratio of 20 in rat. The compound exhibited favorable physicochemical and pharmacokinetic properties, had an acceptable preclinical toxicity profile, and was chosen for clinical trials. BMS-986176/LX9211 (34) completed phase I trials with good human pharmacokinetics and minimum adverse events and is currently in phase II clinical trials for diabetic peripheral neuropathic pain (ClinicalTrials.gov identifier: NCT04455633) and postherpetic neuralgia (ClinicalTrials.gov identifier: NCT04662281).

Neuronal allodynic mechanisms of Slc7a5 (LAT1) in the spared nerve injury rodent model of neuropathic pain.

High-impact chronic pain is suffered by 1 in 5 patients in the USA and globally. Effective, non-addictive, non-opioid therapeutics are urgently needed for the treatment of chronic pain. Slc7a5 (Lat1), also known as system L-neutral amino acid transporter, is involved in a number of physiological processes related to inflammation. Transcriptomics studies have shown that Slc7a5 and its binding partner Slc3a2 are expressed in neurons of the dorsal root ganglia (DRG) and spinal dorsal horn, which are critical to the initiation and maintenance of nociception and pathophysiology of chronic pain. In addition, Slc7a5 is a transporter for the first-line anti-allodynic gabapentinoid drugs and binds to ion channels implicated in nociception and chronic pain including the voltage-gated sodium channel Nav1.7 and the voltage-gated potassium channels Kv1.1 and Kv1.2. We found that blocking Slc7a5 with intrathecal administration of the drug JPH203 alleviated allodynia in the spared nerve injury (SNI) rodent model of neuropathic pain. Western blot and immunohistochemistry studies revealed an increase in Slc7a5 protein levels in the spinal cord and DRGs of SNI mice compared to control mice. Using whole-cell current-clamp electrophysiology, we observed that JPH203 treatment reduced excitability of small-diameter (< 30µm) DRG neurons from SNI mice, in agreement with its behavioral effects. Voltage-clamp recordings from JPH203-treated naïve rat DRGs identified an effect on tetrodotoxin-resistant (TTX-R) sodium currents. Altogether, these results demonstrate that Slc7a5 is dysregulated in chronic neuropathic pain and can be targeted to provide relief of hypersensitivity.

Differential Expression of Long Non-Coding RNAs and Their Role in Rodent Neuropathic Pain Models.

Neuropathic pain, which is accompanied by an unpleasant sensation, affects the patient’s quality of life severely. Considering the complexity of the neuropathic pain, there are huge unmet medical needs for it while current effective therapeutics remain far from satisfactory. Accordingly, exploration of mechanisms of neuropathic pain could provide new therapeutic insights. While numerous researches have pointed out the contribution of sensory neuron-immune cell interactions, other mechanisms of action, such as long non-coding RNAs (lncRNAs), also could contribute to the neuropathic pain observed in vivo. LncRNAs have more than 200 nucleotides and were originally considered as transcriptional byproducts. However, recent studies have suggested that lncRNAs played a significant role in gene regulation and disease pathogenesis. A substantial number of long non-coding RNAs were expressed differentially in neuropathic pain models. Besides, therapies targeting specific lncRNAs can significantly ameliorate the development of neuropathic pain, which reveals the contribution of lncRNAs in the generation and maintenance of neuropathic pain and provides a new therapeutic strategy. The primary purpose of this review is to introduce recent studies of lncRNAs on different neuropathic pain models.

Pilot study on the effects of low intensity focused ultrasound in a swine model of neuropathic pain.

The authors' laboratory has previously demonstrated beneficial effects of noninvasive low intensity focused ultrasound (liFUS), targeted at the dorsal root ganglion (DRG), for reducing allodynia in rodent neuropathic pain models. However, in rats the DRG is 5 mm below the skin when approached laterally, while in humans the DRG is typically 5-8 cm deep. Here, using a modified liFUS probe, the authors demonstrated the feasibility of using external liFUS for modulation of antinociceptive responses in neuropathic swine. Two cohorts of swine underwent a common peroneal nerve injury (CPNI) to induce neuropathic pain. In the first cohort, pigs (14 kg) were iteratively tested to determine treatment parameters. liFUS penetration to the L5 DRG was verified by using a thermocouple to monitor tissue temperature changes and by measuring nerve conduction velocity (NCV) at the corresponding common peroneal nerve (CPN). Pain behaviors were monitored before and after treatment. DRG was evaluated for tissue damage postmortem. Based on data from the first cohort, a treatment algorithm was developed, parameter predictions were verified, and neuropathic pain was significantly modified in a second cohort of larger swine (20 kg). The authors performed a dose-response curve analysis in 14-kg CPNI swine. Specifically, after confirming that the liFUS probe could reach 5 cm in ex vivo tissue experiments, the authors tested liFUS in 14-kg CPNI swine. The mean ± SEM DRG depth was 3.79 ± 0.09 cm in this initial cohort. The parameters were determined and then extrapolated to larger animals (20 kg), and predictions were verified. Tissue temperature elevations at the treatment site did not exceed 2°C, and the expected increases in the CPN NCV were observed. liFUS treatment eliminated pain guarding in all animals for the duration of follow-up (up to 1 month) and improved allodynia for 5 days postprocedure. No evidence of histological damage was seen using Fluoro-Jade and H&E staining. The results demonstrate that a 5-cm depth can be reached with external liFUS and alters pain behavior and allodynia in a large-animal model of neuropathic pain.

PCC-0105002, a novel small molecule inhibitor of PSD95-nNOS protein-protein interactions, attenuates neuropathic pain and corrects motor disorder associated with neuropathic pain model

In view of postsynaptic density 95kDA (PSD95) tethers neuronal NO synthase (nNOS) to N-methyl-d-aspartate receptor (NMDAR), the PSD95-nNOS complex represents a therapeutic target of neuropathic pain. This study therefore sought to explore the ability of PCC-0105002, a novel PSD95-nNOS small molecule inhibitor, to alter pain sensitivity in rodent neuropathic pain models. Firstly, the IC50 of PCC-0105002 for PSD95 and NOS1 binding activity was determined using an Alpha Screen assay kit. Then, we examined the effects of PCC-0105002 in the mouse formalin test and in the rat spinal nerve ligation (SNL) model, and explored the ability of PCC-0105002 to mediate analgesia and to effect motor coordination in a rota-rod test. Moreover, the mechanisms whereby PCC-0105002 mediates analgesia was explored via western blotting, Golgi staining, and co-immunoprecipitation experiments in dorsal horn. The outcomes indicated that PCC-0105002 exhibited dose-dependent attenuation of phase II pain-associated behaviors in the formalin test. The result indicated that PCC-0105002 disrupted the PSD95-nNOS interaction with IC50 of 1.408 μM. In the SNL model, PCC-0105002 suppressed mechanical allodynia, thermal hyperalgesia, and abnormal dorsal horn wide dynamic range neuron discharge. PCC-0105002 mediated an analgesic effect comparable to that of MK-801, while it was better able to enhance motor coordination as compared with MK-801. Moreover, PCC-0105002 altered signaling downstream of NMDAR and thus functionally and structurally attenuating synaptic plasticity through respective regulation of the NR2B/GluR1/CaMKIIα and Rac1/RhoA pathways. These findings suggest that the novel PSD95-nNOS inhibitor PCC-0105002 is an effective agent for alleviating neuropathic pain, and that it produces fewer motor coordination-associated side effects than do NMDAR antagonists.

Novel Functionalized Amino Acids as Inhibitors of GABA Transporters with Analgesic Activity.

Neuropathic pain resistance to pharmacotherapy has encouraged researchers to develop effective therapies for its treatment. γ-Aminobutyric acid (GABA) transporters 1 and 4 (mGAT1 and mGAT4) have been increasingly recognized as promising drug targets for neuropathic pain (NP) associated with imbalances in inhibitory neurotransmission. In this context, we designed and synthesized new functionalized amino acids as inhibitors of GABA uptake and assessed their activities toward all four mouse GAT subtypes (mGAT1–4). According to the obtained results, compounds 2RS,4RS-39c (pIC50 (mGAT4) = 5.36), 50a (pIC50 (mGAT2) = 5.43), and 56a (with moderate subtype selectivity that favored mGAT4, pIC50 (mGAT4) = 5.04) were of particular interest and were therefore evaluated for their cytotoxic and hepatotoxic effects. In a set of in vivo experiments, both compounds 50a and 56a showed antinociceptive properties in three rodent models of NP, namely, chemotherapy-induced neuropathic pain models (the oxaliplatin model and the paclitaxel model) and the diabetic neuropathic pain model induced by streptozotocin; however compound 56a demonstrated predominant activity. Since impaired motor coordination is also observed in neuropathic pain conditions, we have pointed out that none of the test compounds induced motor deficits in the rotarod test.

Single-chain Fragment variable antibody targeting cholecystokinin-B receptor for pain reduction

The cholecystokinin B receptor and its neuropeptide ligand are upregulated in chronic neuropathic pain models. Single-chain Fragment variable antibodies were generated as preferred non-opioid targeting therapy blocking the cholecystokinin B receptor to inhibit chronic neuropathic pain models in vivo and in vitro. Engineered antibodies of this type feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their smaller size. More importantly, single-chain Fragment variable antibodies have promising biotherapeutic applications for both nervous and immune systems, now recognized as interactive in chronic pain. A mouse single-chain Fragment variable antibody library recognizing a fifteen amino acid extracellular peptide fragment of the cholecystokinin B receptor was generated from immunized spleens. Ribosome display, a powerful cell-free technology, was applied for recombinant antibody selection. Antibodies with higher affinity, stability, solubility, and binding specificity for cholecystokinin B not A receptor were selected and optimized for in vivo and in vitro efficacy. A single dose of the lead candidate reduced mechanical and cold hypersensitivity in two rodent models of neuropathic pain for at least seven weeks. Continuing efficacy was evident with either intraperitoneal or intranasal dosing. Likewise, the lead single-chain Fragment variable antibody totally prevented development of anxiety- and depression-like behaviors and cognitive deficits typical in the models. Reduction of neuronal firing frequency was evident in trigeminal ganglia primary neuronal cultures treated in vitro with the cholecystokinin B receptor antibody. Immunofluorescent staining intensity in the trigeminal neuron primary cultures was significantly reduced incrementally after overnight binding with increasingly higher dilutions of the single-chain Fragment variable antibody. While it is reported that single-chain Fragment variable antibodies are removed systemically within 2–6 h, Western blot evidence indicates the His-tag marker remained after 7 weeks in the trigeminal ganglia and in the dorsolateral medulla, providing evidence of brain and ganglia penetrance known to be compromised in overactivated states. This project showcases the in vivo efficacy of our lead single-chain Fragment variable antibody indicating its potential for development as a non-opioid, non-addictive therapeutic intervention for chronic pain. Importantly, studies by others have indicated treatments with cholecystokinin B receptor antagonists suppress maintenance and reactivation of morphine dependence in place preference tests while lowering tolerance and dose requirements. Our future studies remain to address these potential benefits that may accompany the cholecystokinin B receptor biological therapy. Both chronic sciatic and orofacial pain can be unrelenting and excruciating, reducing quality of life as well as diminishing physical and mental function. An effective non-opiate, non-addictive therapy with potential to reduce chronic neuropathic pain long term is greatly needed.