Grape seed-derived procyanidins decreases neuropathic pain and nerve regeneration by suppression of toll-like receptor 4-myeloid differentiation factor-88 signaling.

Background: Recent studies have shown that peripheral nerve regeneration process is closely related to neuropathic pain. Toll-like receptor 4 (TLR4) signaling was involved in different types of pain and nerve regeneration. TLR4 induced the recruitment of myeloid differentiation factor-88 adaptor protein (MyD88) and NF-κB-depended transcriptional process in sensory neurons and glial cells, which produced multiple cytokines and promoted the induction and persistence of pain. Our study aimed to investigate procyanidins's effect on pain and nerve regeneration via TLR4-Myd88 signaling. Methods: Spinal nerve ligation (SNL) model was established to measure the analgesic effect of procyanidins. Anatomical measurement of peripheral nerve regeneration was measured by microscopy and growth associated protein 43 (GAP43) staining. Western blotting and/or immunofluorescent staining were utilized to detect TLR4, myeloid differentiation factor-88 adaptor protein (MyD88), ionized calcium-binding adapter molecule 1 (IBA1) and nuclear factor kappa-B-p65 (NF-κB-p65) expression, as well as the activation of astrocyte and microglia. The antagonist of TLR4 (LPS-RS-Ultra, LRU) were intrathecally administrated to assess the behavioral effects of blocking TLR4 signaling on pain and nerve regeneration. Result: Procyanidins reduced mechanical allodynia, thermal hyperalgesia and significantly suppressed the number of nerve fibers regenerated and the degree of myelination in SNL model. Compared with sham group, TLR4, MyD88, IBA1 and phosphorylation of NF-κB-p65 were upregulated in SNL rats which were reversed by procyanidins administration. Additionally, procyanidins also suppressed activation of spinal astrocytes and glial cells. Conclusion: Suppression of TLR4-MyD88 signaling contributes to the alleviation of neuropathic pain and reduction of nerve regeneration by procyanidins.

Combined antiallodynic effects of Neurotropin®-tramadol and Neurotropin®-mirogabalin in rats with L5-spinal nerve ligation.

We aimed to examine the efficacy of combination therapies of Neurotropin® with tramadol and Neurotropin with mirogabalin for neuropathic pain management. A neuropathic pain model (L5 spinal nerve ligation model: L5-SNL) using male Wistar rats was generated through tight ligation of the left fifth lumbar nerve using silk sutures. Mechanical allodynia was assessed using the 50% paw withdrawal threshold. The combined antiallodynic effects were evaluated using isobolographic analyses. Small intestinal transit was evaluated using the charcoal meal test, and motor coordination using the rota-rod test. Neurotropin (50-200 NU/kg, p.o.), tramadol (7.5-60 mg/kg, p.o.), and mirogabalin (3-30 mg/kg, p.o.) showed a dose-dependent antiallodynic effect in L5-SNL rats. The combined antiallodynic effects of Neurotropin and tramadol were additive or synergistic, whereas those of Neurotropin and mirogabalin were additive. Neurotropin (100-400 NU/kg, p.o.) did not affect the small intestinal transit, whereas tramadol (30-100 mg/kg, p.o.) significantly inhibited it. Neurotropin (100-400 NU/kg, p.o.) did not affect the walking time, whereas mirogabalin (10-100 mg/kg, p.o.) significantly decreased it. Neurotropin dose-dependently ameliorated mechanical allodynia in rats, and combination therapy with Neurotropin-tramadol or Neurotropin-mirogabalin may alleviate neuropathic pain without aggravating the adverse effects of tramadol and mirogabalin.

Electroacupuncture may alleviate neuropathic pain via suppressing P2X7R expression.

Neuropathic pain is a severe problem that is difficult to treat clinically. Reducing abnormal remodeling of dendritic spines/synapses and increasing the anti-inflammatory effects in the spinal cord dorsal horn are potential methods to treat this disease. Previous studies have reported that electroacupuncture (EA) could increase the pain threshold after peripheral nerve injury. However, the underlying mechanism is unclear. P2X7 receptors (P2X7R) mediate the activation of microglia and participate in the occurrence and development of neuropathic pain. We hypothesized that the effects of EA on relieving pain may be related to the downregulation of the P2X7R. Spinal nerve ligation (SNL) rats were used as a model in this experiment, and 2'(3')-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a P2X7R agonist. We found that EA treatment decreased dendritic spine density, inhibited synaptic reconstruction and reduced inflammatory response, which is consistent with the decrease in P2X7R expression as well as the improved neurobehavioral performance. In contrast to the beneficial effects of EA, BzATP enhanced abnormal remodeling of dendritic spines/synapses and inflammation. Furthermore, the EA-mediated positive effects were reversed by BzATP, which is consistent with the increased P2X7R expression. These findings indicated that EA improves neuropathic pain by reducing abnormal dendritic spine/synaptic reconstruction and inflammation via suppressing P2X7R expression.

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.

Microglial Activation of GLP-1R Signaling in Neuropathic Pain Promotes Gene Expression Adaption Involved in Inflammatory Responses.

Background: Neuropathic pain is a common chronic pain, which is related to hypersensitivity to stimulus and greatly affects the quality of life of patients. Maladaptive gene changes and molecular signaling underlie the sensitization of nociceptive pathways. We previously found that the activation of microglial glucagon-like peptide 1 receptor (GLP-1R) could potently relieve formalin-, bone cancer-, peripheral nerve injury-, and diabetes-induced pain hypersensitivity. So far, little is known about how the gene profile changes upon the activation of GLP-1R signaling in the pathophysiology of neuropathic pain. Methods: Spinal nerve ligation (SNL) was performed to induce neuropathic pain in rats. Mechanical allodynia was assessed using von Frey filaments. The expression of IL-10, ß-endorphin, and µ-opioid receptor (MOR) was examined by real-time quantitative polymerase chain reaction (qPCR) and whole-cell recording. Measurements of cellular excitability of the substantia gelatinosa (SG) neurons by whole-cell recording were carried out. R packages of differential gene expression analysis based on the negative binomial distribution (DESeq2) and weighted correlation network analysis (WGCNA) were used to analyze differential gene expression and the correlated modules among GLP-1R clusters in neuropathic pain. Results: The GLP-1R agonist, exenatide, has an antiallodynic effect on neuropathic pain, which could be reversed by intrathecal injections of the microglial inhibitor minocycline. Furthermore, differential gene expression analysis (WGCNA) indicated that intrathecal injections of exenatide could reverse the abnormal expression of 591 genes in the spinal dorsal horn induced by nerve injury. WGCNA revealed 58 modules with a close relationship between the microglial GLP-1R pathway and features of nerve injuries, including pain, ligation, paw withdrawal latency (PWL), and anxiety. The brown module was identified as the highest correlated module, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that inflammatory responses were most correlated with PWL. To further unravel the changes of hyperalgesia-related neuronal electrophysiological activity mediated by microglia GLP-1 receptors, whole-cell recording identified that MOR agonism stimulated a robust outward current in the sham groups compared with the spinal nerve ligation (SNL) groups. This inhibitory effect on the SNL group was more sensitive than that of the sham group after bath application of ß-endorphin. Conclusions: Our results further confirmed that the GLP-1R pathway is involved in alleviating pain hypersensitivity mediated by spinal microglia activation, and inflammatory responses were the most correlated pathway associated with PWL changes in response to exenatide treatment. We found that the identification of gene regulation in response to GLP-1R activation is an effective strategy for identifying new therapeutic targets for neuropathic pain. Investigation for the activation of spinal microglial GLP-1R which might ameliorate inflammatory responses through gene expression and structural changes is providing a potential biomarker in pain management.

Evaluation of the Antinociceptive, Antiallodynic, Antihyperalgesic and Anti-Inflammatory Effect of Polyalthic Acid.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are very commonly used, but their adverse effects warrant investigating new therapeutic alternatives. Polyalthic acid, a labdane-type diterpenoid, is known to produce gastroprotection, tracheal smooth muscle relaxation, and antitumoral, antiparasitic and antibacterial activity. This study aimed to evaluate the antinociceptive, antiallodynic, antihyperalgesic and anti-inflammatory effect of polyalthic acid on rats. Moreover, the effectiveness of treating hyperalgesia with a combination of polyalthic acid and naproxen was analyzed, as well as the type of drug-drug interaction involved. Nociception was examined by injecting 1% formalin into the right hind paw and thermal hyperalgesia and inflammation by injecting a 1% carrageenan solution into the left hind paw of rats. Allodynia was assessed on an L5/L6 spinal nerve ligation model. Polyalthic acid generated significant antinociceptive (56-320 mg/kg), antiallodynic (100-562 mg/kg), and antihyperalgesic and anti-inflammatory (10-178 mg/kg) effects. Antinociception mechanisms were explored by pretreating the rats with naltrexone, ODQ and methiothepin, finding the effect blocked by the former two compounds, which indicates the participation of opioid receptors and guanylate cyclase. An isobolographic analysis suggests synergism between polyalthic acid and naproxen in the combined treatment of hyperalgesia.

Novel RET agonist for the treatment of experimental neuropathies.

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) alleviate symptoms of experimental neuropathy, protect and stimulate regeneration of sensory neurons in animal models of neuropathic pain, and restore their functional activity. However, clinical development of GFL proteins is complicated by their poor pharmacokinetic properties and multiple effects mediated by several receptors. Previously, we have identified a small molecule that selectively activates the major signal transduction unit of the GFL receptor complex, receptor tyrosine kinase RET, as an alternative to GFLs, for the treatment of neuropathic pain. We then introduced a series of chemical changes to improve the biological activity of these compounds and tested an optimized compound named BT44 in a panel of biological assays. BT44 efficiently and selectively stimulated the GFL receptor RET and activated the intracellular mitogene-activated protein kinase/extracellular signal-regulated kinase pathway in immortalized cells. In cultured sensory neurons, BT44 stimulated neurite outgrowth with an efficacy comparable to that of GFLs. BT44 alleviated mechanical hypersensitivity in surgery- and diabetes-induced rat models of neuropathic pain. In addition, BT44 normalized, to a certain degree, the expression of nociception-related neuronal markers which were altered by spinal nerve ligation, the neuropathy model used in this study. Our results suggest that the GFL mimetic BT44 is a promising new lead for the development of novel disease-modifying agents for the treatment of neuropathy and neuropathic pain.

Supraspinal Opioid Circuits Differentially Modulate Spinal Neuronal Responses in Neuropathic Rats.

BACKGROUND: The anterior cingulate cortex and central nucleus of the amygdala connect widely with brainstem nuclei involved in descending modulation, including the rostral ventromedial medulla. Endogenous opioids in these circuits participate in pain modulation. The hypothesis was that a differential opioidergic role for the brain nuclei listed in regulation of spinal neuronal responses because separable effects on pain behaviors in awake animals were previously observed. METHODS: This study utilized in vivo electrophysiology to determine the effects of morphine microinjection into the anterior cingulate cortex, right or left central nucleus of the amygdala, or the rostral ventromedial medulla on spinal wide dynamic range neuronal responses in isoflurane-anesthetized, male Sprague-Dawley rats. Ongoing activity in the ventrobasal thalamus was also measured. In total, 33 spinal nerve ligated and 26 control age- and weight-matched control rats were used. RESULTS: Brainstem morphine reduced neuronal firing to 60-g von Frey stimulation in control rats (to 65 ± 12% of control response (means ± 95% CI), P < 0.001) with a greater inhibition in neuropathic rats (to 53 ± 17% of control response, P < 0.001). Contrasting anterior cingulate cortex morphine had only marginal modulatory effects on spinal neuronal responses with limited variance in effect between control and neuropathic rats. The inhibitory effects of morphine in the central nucleus of the amygdala were dependent on pain state and laterality; only right-side morphine reduced neuronal firing to 60-g stimulation in neuropathic rats (to 65 ± 14% of control response, P = 0.001). In addition, in neuropathic rats elevated ongoing neuronal activity in the ventral posterolateral thalamus was not inhibited by anterior cingulate cortex morphine, in contrast to evoked responses. CONCLUSIONS: Cumulatively the data support opioid modulation of evoked responses predominately through a lateralized output from the right amygdala, as well as from the brainstem that is enhanced in injured conditions. Minimal modulation of dorsal horn responses was observed after anterior cingulate cortex opioid administration regardless of injury state.

Paravertebral Nerve Block With Liposomal Bupivacaine for Pain Control Following Video-Assisted Thoracoscopic Surgery and Thoracotomy.

BACKGROUND: Some surgeons have adopted the use of video-assisted thoracoscopic surgery (VATS) or robotic surgery to perform resections for lung cancer. VATS is associated with less pain and a decrease in pulmonary complications compared with open thoracotomies. Long-acting liposomal bupivacaine (LB) intercostal nerve blocks are reported to provide superior pain relief compared with epidural catheters in the first 3 d after a thoracotomy. This study examined whether LB improves pain after VATS and if it provides effective analgesia after a thoracotomy. MATERIALS AND METHODS: A retrospective review was performed on 151 consecutive patients undergoing a VATS or thoracotomy who received paravertebral nerve blocks. VATS patients received paravertebral nerve blocks with LB (VATS-LB) or 0.25% bupivacaine with epinephrine (BE; VATS-BE). Thoracotomy patients received paravertebral nerve blocks via LB injections. Pain scores, narcotic utilization, complications, and hospital length of stay were examined. RESULTS: Fifty patients underwent a VATS-LB, 53 underwent a VATS-BE, and 32 underwent a thoracotomy. Thoracotomy and VATS-LB patients had pain scores lower than VATS-BE patients in the first 48 h after surgery (P < 0.004). Opioid use was not significantly different between the thoracotomy and VATS-LB patients throughout the first 2 wk postoperatively. CONCLUSIONS: LB paravertebral blocks significantly improve postoperative pain in comparison with 0.25% BE blocks in VATS patients. LB paravertebral blocks also provide effective analgesia in patients undergoing thoracotomies.

NT-3 Promotes Oligodendrocyte Proliferation and Nerve Function Recovery After Spinal Cord Injury by Inhibiting Autophagy Pathway.

BACKGROUND: Spinal cord injury (SCI) is a serious medical problem, leading to lifelong disability and increasing the health burden worldwide. Traditional treatments have limited effects on neuronal function recovery. Previous studies showed that neurotrophin-3 (NT-3) promoted oligodendrocyte survival and improved neuronal functional recovery after SCI. However, the mechanism by which NT-3 promotes oligodendrocyte survival after SCI remains unclear, which limits its application. MATERIALS AND METHODS: A total of 75 female Sprague-Dawley rats were randomly divided into three groups: the NS group, NT-3 group, and NT-3 + rapamycin group. After successful modeling, the spinal cord specimens were taken at the corresponding time points. Western blot was used to detect autophagy-related proteins and Olig1 protein expression and combined with pathology, immunohistochemistry, flow cytometry, and other methods to detect the proliferation of oligodendrocytes after NT-3 application. RESULTS: NT-3 was found to significantly promote the recovery of motor function by Basso-Beattie-Bresnahan scores analysis in the rat SCI model. Furthermore, intraspinal administration of NT-3 could downregulate the expression of Beclin-1 in oligodendrocytes, indicating that NT-3 could inhibit excessive autophagy of oligodendrocytes after SCI. The effects of NT-3 on oligodendrocyte survival could be blocked by an autophagy activator rapamycin. CONCLUSIONS: This study found that NT-3 could promote the recovery of motor function after SCI in rats. The underlying reason may be that NT-3 inhibits the expression of autophagy proteins in oligodendrocytes and promotes oligodendrocyte proliferation. This study provided evidence for the future clinical application of NT-3 in SCI patients.

CX3CR1-Targeted PLGA Nanoparticles Reduce Microglia Activation and Pain Behavior in Rats with Spinal Nerve Ligation.

Activation of CX3CR1 in microglia plays an important role in the development of neuropathic pain. Here, we investigated whether neuropathic pain could be attenuated in spinal nerve ligation (SNL)-induced rats by reducing microglial activation through the use of poly(D,L-lactic-co-glycolic acid) (PLGA)-encapsulated CX3CR1 small-interfering RNA (siRNA) nanoparticles. After confirming the efficacy and specificity of CX3CR1 siRNA, as evidenced by its anti-inflammatory effects in lipopolysaccharide-stimulated BV2 cells in vitro, PLGA-encapsulated CX3CR1 siRNA nanoparticles were synthesized by sonication using the conventional double emulsion (W/O/W) method and administered intrathecally into SNL rats. CX3CR1 siRNA-treated rats exhibited significant reductions in the activation of microglia in the spinal dorsal horn and a downregulation of proinflammatory mediators, as well as a significant attenuation of mechanical allodynia. These data indicate that the PLGA-encapsulated CX3CR1 siRNA nanoparticles effectively reduce neuropathic pain in SNL-induced rats by reducing microglial activity and the expression of proinflammatory mediators. Therefore, we believe that PLGA-encapsulated CX3CR1 siRNA nanoparticles represent a valuable new treatment option for neuropathic pain.

Suberoylanilide Hydroxamic Acid Triggers Autophagy by Influencing the mTOR Pathway in the Spinal Dorsal Horn in a Rat Neuropathic Pain Model.

Histone acetylation levels can be upregulated by treating cells with histone deacetylase inhibitors (HDACIs), which can induce autophagy. Autophagy flux in the spinal cord of rats following the left fifth lumber spinal nerve ligation (SNL) is involved in the progression of neuropathic pain. Suberoylanilide hydroxamic acid (SAHA), one of the HDACIs can interfere with the epigenetic process of histone acetylation, which has been shown to ease neuropathic pain. Recent research suggest that SAHA can stimulate autophagy via the mammalian target of rapamycin (mTOR) pathway in some types of cancer cells. However, little is known about the role of SAHA and autophagy in neuropathic pain after nerve injury. In the present study, we aim to investigate autophagy flux and the role of the mTOR pathway on spinal cells autophagy activation in neuropathic pain induced by SNL in rats that received SAHA treatment. Autophagy-related proteins and mTOR or its active form were assessed by using western blot, immunohistochemistry, double immunofluorescence staining and transmission electron microscopy (TEM). We found that SAHA decreased the paw mechanical withdrawal threshold (PMWT) of the lower compared with SNL. Autophagy flux was mainly disrupted in the astrocytes and neuronal cells of the spinal cord dorsal horn on postsurgical day 28 and was reversed by daily intrathecal injection of SAHA (n = 100 nmol/day or n = 200 nmol/day). SAHA also decreased mTOR and phosphorylated mTOR (p-mTOR) expression, especially p-mTOR expression in astrocytes and neuronal cells of the spinal dorsal horn. These results suggest that SAHA attenuates neuropathic pain and contributes to autophagy flux in astrocytes and neuronal cells of the spinal dorsal horn via the mTOR signaling pathway.

Programmed intermittent bolus infusion versus continuous infusion of 0.2% levobupivacaine after ultrasound-guided thoracic paravertebral block for video-assisted thoracoscopic surgery: A randomised controlled trial.

BACKGROUND: The analgesic benefits of programmed intermittent bolus infusion for thoracic paravertebral block remain unknown. OBJECTIVE: The aim of this study was to compare the analgesia from intermittent bolus infusion with that of a continuous infusion after thoracic paravertebral block. DESIGN: A randomised controlled study. SETTING: A single centre between December 2016 and November 2017. Seventy patients scheduled for video-assisted thoracoscopic surgery were included in the study. INTERVENTION(S): Patients were randomly assigned to receive 0.2% levobupivacaine via continuous infusion (5 ml h, continuous group) or programmed intermittent bolus infusion (15 ml every 3 h, bolus group) after an initial 15-ml bolus injection of 0.2% levobupivacaine. MAIN OUTCOME MEASURES: The main outcome was the amount of rescue fentanyl (per kg of body weight) consumed within 24 h after surgery. Secondary outcomes were postoperative pain scores, plasma levobupivacaine concentrations and the number of dermatomes anaesthetised. RESULTS: There was no significant difference between the continuous and bolus groups in the postoperative consumption of fentanyl (median [interquartile range] 5.5 [4 to 9.5] µg kg versus 6 [3.5 to 9] µg kg respectively, P = 0.45) and postoperative pain scores within 24 h. At 20 h after initiating the infusions, there was no statistically significant difference between the two groups in terms of the plasma levobupivacaine concentration. The number of dermatomes anaesthetised to pinprick and cold testing was significantly greater in the bolus group. CONCLUSION: Our findings suggest that postoperative pain and opioid usage are similar with either programmed intermittent bolus infusion or continuous infusion after thoracic paravertebral block. Programmed intermittent bolus infusion provides a wider sensory blockade and could benefit patients requiring a wider extent of anaesthesia. TRIAL REGISTRATION: UMIN Clinical Trials Registry (UMIN-CTR; URL: http://umin.ac.jp/ctr/, ID: UMIN000023378).

Modulation of SUR1 KATP Channel Subunit Activity in the Peripheral Nervous System Reduces Mechanical Hyperalgesia after Nerve Injury in Mice.

The ATP-sensitive K+ channel (KATP) is involved in hypersensitivity during chronic pain and is presumed to be a downstream target of mu opioid receptors. Multiple subtypes of KATP channels exist in the peripheral and central nervous system and their activity may be inversely correlated to chronic pain phenotypes in rodents. In this study, we investigated the different KATP channel subunits that could be involved in neuropathic pain in mice. In chronic pain models utilizing spinal nerve ligation, SUR1 and Kir6.2 subunits were found to be significantly downregulated in dorsal root ganglia and the spinal cord. Local or intrathecal administration of SUR1-KATP channel subtype agonists resulted in analgesia after spinal nerve ligation but not SUR2 agonists. In ex-vivo nerve recordings, administration of the SUR1 agonist diazoxide to peripheral nerve terminals decreased mechanically evoked potentials. Genetic knockdown of SUR1 through an associated adenoviral strategy resulted in mechanical hyperalgesia but not thermal hyperalgesia compared to control mice. Behavioral data from neuropathic mice indicate that local reductions in SUR1-subtype KATP channel activity can exacerbate neuropathic pain symptoms. Since neuropathic pain is of major clinical relevance, potassium channels present a target for analgesic therapies, especially since they are expressed in nociceptors and could play an essential role in regulating the excitability of neurons involved in pain-transmission.

Biomarker Analysis of Orally Dosed, Dual Active, Matrix Metalloproteinase (MMP)-2 and MMP-9 Inhibitor, AQU-118, in the Spinal Nerve Ligation (SNL) Rat Model of Neuropathic Pain.

There is an unmet medical need for the development of non-addicting pain therapeutics with enhanced efficacy and tolerability. The current study examined the effects of AQU-118, an orally active inhibitor of metalloproteinase-2 (MMP-2) and MMP-9, in the spinal nerve ligation (SNL) rat model of neuropathic pain. Mechanical allodynia and the levels of various biomarkers were examined within the dorsal root ganglion (DRG) before and after oral dosing with AQU-118. The rats that received the SNL surgery exhibited significant mechanical allodynia as compared to sham controls. Animals received either vehicle, positive control (gabapentin), or AQU-118. After SNL surgery, the dorsal root ganglion (DRG) of those rats dosed with vehicle had elevated messenger RNA (mRNA) expression levels for MMP-2, IL1-ß & IL-6 and elevated protein levels for caspase-3 while exhibiting decreased protein levels for myelin basic protein (MBP) & active IL-ß as compared to sham controls. Rats orally dosed with AQU-118 exhibited significantly reduced mechanical allodynia and decreased levels of caspase-3 in the DRG as compared to vehicle controls. Results demonstrate that oral dosing with the dual active, MMP-2/-9 inhibitor, AQU-118, attenuated mechanical allodynia while at the same time significantly reduced the levels of caspase-3 in the DRG.

Involvement of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin/peroxisome proliferator-activated receptor γ pathway for induction and maintenance of neuropathic pain.

Peripheral nerve injury induces neuropathic pain, which is characterized by the tactile allodynia and thermal hyperalgesia. N-type voltage-dependent Ca2+ channel (VDCC) plays pivotal roles in the development of neuropathic pain, since mice lacking Cav2.2, the pore-forming subunit of N-type VDCC, show greatly reduced symptoms of both tactile allodynia and thermal hyperalgesia. Our study on gene expression profiles of the wild-type and N-type VDCC knockout (KO) spinal cord and several pain-related brain regions after spinal nerve ligation (SNL) injury revealed altered expression of genes encoding catalytic subunits of phosphatidylinositol-3 kinase (PI3K). PI3K/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling is considered to be very important for cancer development and drugs targeting the molecules in this pathway have been tested in oncology trials. In the present study, we have tested whether the changes in expression of molecules in this pathway in mice having spinal nerve injury are causally related to neuropathic pain. Our results suggest that spinal nerve injury induces activation of N-type VDCC and the following Ca2+ entry through this channel may change the expression of genes encoding PI3K catalytic subunits (p110α and p110γ), Akt, retinoid X receptor α (RXRα) and RXRγ. Furthermore, the blockers of the molecules in this pathway are found to be effective in reducing neuropathic pain both at the spinal and at the supraspinal levels. Thus, the activation of PI3K/Akt/mTOR/peroxisome proliferator activated receptor gamma (PPARγ) pathway would be a hallmark of the induction and maintenance of neuropathic pain.

Cross-talk inhibition between 5-HT2B and 5-HT7 receptors in phrenic motor facilitation via NADPH oxidase and PKA.

Intermittent spinal serotonin receptor activation elicits phrenic motor facilitation (pMF), a form of spinal respiratory motor plasticity. Episodic activation of either serotonin type 2 (5-HT2) or type 7 (5-HT7) receptors elicits pMF, although they do so via distinct cellular mechanisms known as the Q (5-HT2) and S (5-HT7) pathways to pMF. When coactivated, these pathways interact via mutual cross-talk inhibition. Although we have a rudimentary understanding of mechanisms mediating cross-talk interactions between spinal 5-HT2 subtype A (5-HT2A) and 5-HT7 receptor activation, we do not know if similar interactions exist between 5-HT2 subtype B (5-HT2B) and 5-HT7 receptors. We confirmed that either spinal 5-HT2B or 5-HT7 receptor activation alone elicits pMF and tested the hypotheses that 1) concurrent activation of both receptors suppresses pMF due to cross-talk inhibition; 2) 5-HT7 receptor inhibition of 5-HT2B receptor-induced pMF requires protein kinase A (PKA) activity; and 3) 5-HT2B receptor inhibition of 5-HT7 receptor-induced pMF requires NADPH oxidase (NOX) activity. Selective 5-HT2B and 5-HT7 receptor agonists were administered intrathecally at C4 (3 injections, 5-min intervals) to anesthetized, paralyzed, and ventilated rats. Whereas integrated phrenic nerve burst amplitude increased after selective spinal 5-HT2B or 5-HT7 receptor activation alone (i.e., pMF), pMF was no longer observed with concurrent 5-HT2B and 5-HT7 receptor agonist administration. With concurrent receptor activation, pMF was rescued by inhibiting either NOX or PKA activity, demonstrating their roles in cross-talk inhibition between these pathways to pMF. This report demonstrates cross-talk inhibition between 5-HT2B- and 5-HT7 receptor-induced pMF and that NOX and PKA activity are necessary for that cross-talk inhibition.

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.

Plasticity and Function of Spinal Oxytocin and Vasopressin Signaling during Recovery from Surgery with Nerve Injury.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Recovery from pain after surgery is faster after cesarean delivery than after other abdominal procedures. The authors hypothesized that recovery in rats after surgery could be reversed by antagonism of spinal oxytocin or vasopressin receptors, that there may be a sex difference, and that spinal oxytocin innervation could change after surgery. METHODS: Male and female rats underwent partial spinal nerve ligation surgery. Effects of nonselective and selective oxytocin and vasopressin 1A receptor antagonists on mechanical hypersensitivity during partial recovery were assessed (n = 8 to 14/group). Oxytocin immunoreactivity in the dorsal horn of the spinal cord (n = 7 to 8/group) and messenger RNA (mRNA) expression for oxytocin-binding receptors in dorsal root ganglia and spinal cord (n = 8/group) were measured. RESULTS: Intrathecal injection of oxytocin and vasopressin receptor antagonists were similarly effective at reducing withdrawal threshold (in all experiments from 22 [19, 26] median [first quartile, third quartile]) g to 8.3 [6.4, 12] g after injection) in both sexes, while having no or minimal effects in animals without surgery. Oxytocin fiber immunoreactivity was 3- to 5-fold greater in lumbar than other regions of the spinal cord and was increased more than 2-fold in lumbar cord ipsilateral to surgery. Injury was also associated with a 6.5-fold increase in oxytocin receptor and a 2-fold increase in vasopressin 1A receptor messenger RNA expression in the L4 dorsal root ganglion ipsilateral to surgery. CONCLUSIONS: These findings suggest that the capacity for oxytocin signaling in the spinal cord increases after surgery and that spinal oxytocin signaling plays ongoing roles in both sexes in recovery from mechanical hypersensitivity after surgery with known nerve injury.

An imidazole based H-Phe-Phe-NH2 peptidomimetic with anti-allodynic effect in spared nerve injury mice.

The dipeptide amide H-Phe-Phe-NH2 (1) that previously was identified as a ligand for the substance P 1-7 (SP1-7) binding site exerts intriguing results in animal models of neuropathic pain after central but not after peripheral administration. The dipeptide 1 is derived from stepwise modifications of the anti-nociceptive heptapeptide SP1-7 and the tetrapeptide endomorphin-2 that is also binding to the SP1-7 site. We herein report a strong anti-allodynic effect of a new H-Phe-Phe-NH2 peptidomimetic (4) comprising an imidazole ring as a bioisosteric element, in the spare nerve injury (SNI) mice model after peripheral administration. Peptidomimetic 4 was stable in plasma, displayed a fair membrane permeability and a favorable neurotoxic profile. Moreover, the effective dose (ED50) of 4 was superior as compared to gabapentin and morphine that are used in clinic.