Hindpaw Withdrawal Latency Research Articles

Biological Evaluation of Bioactive Compounds of Selaginella plana in Treating Diabetic Neuropathy

Introduction: The study is aimed to assess the plant bioactive compounds of Selaginella plana in treating diabetic neuropathy. The plant components were assessed using ethanol as the solvent. Method: A phytochemical screening was conducted to ascertain the existence of secondary metabolites in the plant extracts. The efficacy of plant extract against neuropathic pain was determined using plantar test and Von Frey filament stimulation. Biochemical tests were performed to determine the level of SGOT, SGPT, ALP, HDL, CHO, LDL, Creatinine and Urea. The percentage yield of ethanol and aqueous extract was 19.22 g and 15.76 g, respectively. Result: The ethanolic extract showed presence of most of the phytoconstituents like alkaloids, flavonoids, carbohydrates, tannins, phytosterols, glycosides, proteins, and gum. Thus, ethanolic extract was used for further studies. In contrast to disease induced rats, rats given the extract chronically had a considerably shorter hind paw withdrawal latency. In STZ-induced diabetic rats, the mechanical withdrawal threshold dramatically reduced mechanical allodynia after 7 days by a dose of 400 mg/kg. Conclusion: The major findings of the study demonstrated that STZ-induced diabetic rats administered leaf extract had reduced neuropathic pain. It can be concluded that S. plana may be used as an alternative drug in the treatment of diabetic neuropathy.

Therapeutic effects of electroacupuncture at different sites on rats with neuropathic pain via the spinal Nrf2/HO-1 pathway: 大鼠脊髓Nrf2/HO-1通路参与不同部位电针调节神经性疼痛效应的机制探讨

ObjectiveThis study aimed to assess the effects of electroacupuncture (EA) at the contralateral, ipsilateral, or bilateral “Zusanli (ST36)” and “Yanglingquan(GB34)” on neuropathic pain caused by chronic contractile injury (CCI) and to explore the role of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in the effects of EA. MethodsMale Sprague-Dawley rats were subjected to the CCI model to induce neuropathic pain. A total of 45 rats were randomly divided into five groups (n = 9): sham, CCI, EA-Co (CCI + EA at contralateral acupoints), EA-Ip (CCI + EA at ipsilateral acupoints), and EA-Bi (CCI + EA at bilateral acupoints). The rats received EA treatment on day 8 after CCI, once every alternate day, for a total of eight times. The time courses of mechanical pain threshold (MWT), hind paw withdrawal latency (HWL), and sciatic functional index (SFI) were determined. The expression levels of 8-hydroxydeoxyguanosine (8-OHdG), glutathione (GSH), superoxide dismutase (SOD) activity, interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor factor-alpha (TNF-α) in the spinal cord were measured. The distribution of Nrf2, its expression of Nrf2 in both the cytosol and nucleus, and the protein levels of its downstream target genes, NQO1 and HO-1, were detected via double immunofluorescence staining and western blotting, respectively. ResultsFollowing CCI, both MWT and HWL in the CCI group significantly decreased from day 14 after surgery (P < 0.001). EA treatment exhibited significant antinociceptive effects induced by CCI by increasing the MWT and HWL values, especially bilateral EA (P < 0.05). The SFI of the CCI group was significantly lower than that of the sham group (P < 0.001). Only bilateral EA improved the SFI scores compared to the CCI group (P < 0.05). 8-OHdG levels in the spinal cord of the CCI group were significantly higher than those in the sham group (P < 0.05), whereas GSH levels and SOD activity in the spinal cord of the CCI group were significantly lower than those in the sham group (P < 0.001 and P < 0.01, respectively). Bilateral EA administration significantly downregulated 8-OHdG levels (P < 0.01) and upregulated GSH levels and SOD activity in the spinal cord (P < 0.01). CCI significantly enhanced the production of IL-1β, IL-6, and TNF-α in the spinal cord compared with that in the sham group (all P < 0.001). Meanwhile, the effects of EA were also accompanied by markedly decreased expression of IL-1β and IL-6 in the spinal cord (P < 0.05). TNF-α levels were only decreased in the EA-Ip and EA-Bi groups compared with those in the CCI group (P < 0.001). Confocal microscopy revealed that Nrf2 was mainly localized in the neurons of the spinal cord. Notably, EA treatment enhanced nuclear translocation of Nrf2 in neurons. CCI significantly decreased the production of Nrf2, HO-1, and NQO1 in the spinal cord compared to the sham group (P < 0.001), and bilateral EA up-regulated the protein levels of Nrf2 and its target genes HO-1 and NQO1(all P < 0.001). ConclusionOur results suggest that bilateral EA is an optimal therapeutic strategy for neuropathic pain. The effects of EA on neuropathic pain may be mediated by the restoration of the Nrf2 pathway in the spinal cord.

Melatonin Relieves Paclitaxel-Induced Neuropathic Pain by Regulating pNEK2-Dependent Epigenetic Pathways in DRG Neurons.

The neurohormone melatonin (MLT) demonstrates promising potential in ameliorating neuropathic pain induced by paclitaxel (PTX) chemotherapy. However, little is known about its protective effect on dorsal root ganglion (DRG) neurons in neuropathic pain resulting from the chemotherapeutic drug PTX. Here, PTX-treated rats revealed that intrathecal administration of MLT dose-dependently elevated hind paw withdrawal thresholds and latency, indicating that MLT significantly reversed PTX-induced neuropathic pain. Mechanistically, the analgesic effects of MLT were found to be mediated via melatonin receptor 2 (MT2), as pretreatment with an MT2 receptor antagonist inhibited these effects. Moreover, intrathecal MLT injection reversed the pNEK2-dependent epigenetic program induced by PTX. All of the effects caused by MLT were blocked by pretreatment with an MT2 receptor-selective antagonist, 4P-PDOT. Remarkably, multiple MLT administered during PTX treatment (PTX+MLTs) exhibited not only rapid but also lasting reversal of allodynia/hyperalgesia compared to single-bolus MLT administered after PTX treatment (PTX+MLT). In addition, PTX+MLTs exhibited greater efficacy in reversing PTX-induced alterations in pRSK2, pNEK2, JMJD3, H3K27me3, and TRPV1 expression and interaction in DRG neurons than PTX+MLT. These results indicated that MLT administered during PTX treatment reduced the incidence and/or severity of neuropathy and had a better inhibitory effect on the pNEK2-dependent epigenetic program compared to MLT administered after PTX treatment. In conclusion, MLT/MT2 is a promising therapy for the treatment of pNEK2-dependent painful neuropathy resulting from PTX treatment. MLT administered during PTX chemotherapy may be more effective in the prevention or reduction of PTX-induced neuropathy and maintaining quality.

Calcium/calmodulin-dependent protein kinase II is involved in the transmission and regulation of nociception in naïve and morphine-tolerant rat nucleus accumbens.

Synaptic plasticity contributes to nociceptive signal transmission and modulation, with calcium/calmodulin-dependent protein kinase II (CaMK II) playing a fundamental role in neural plasticity. This research was conducted to investigate the role of CaMK II in the transmission and regulation of nociceptive information within the nucleus accumbens (NAc) of naïve and morphine-tolerant rats. Randall Selitto and hot-plate tests were utilized to measure the hindpaw withdrawal latencies (HWLs) in response to noxious mechanical and thermal stimuli. To induce chronic morphine tolerance, rats received intraperitoneal morphine injection twice per day for seven days. CaMK II expression and activity were assessed using western blotting. Intra-NAc microinjection of autocamtide-2-related inhibitory peptide (AIP) induced an increase in HWLs in naïve rats in response to noxious thermal and mechanical stimuli. Moreover, the expression of the phosphorylated CaMK II (p-CaMK II) was significantly decreased as determined by western blotting. Chronic intraperitoneal injection of morphine resulted in significant morphine tolerance in rats on Day 7, and an increase of p-CaMK II expression in NAc in morphine-tolerant rats was observed. Furthermore, intra-NAc administration of AIP elicited significant antinociceptive responses in morphine-tolerant rats. In addition, compared with naïve rats, AIP induced stronger thermal antinociceptive effects of the same dose in rats exhibiting morphine tolerance. This study shows that CaMK II in the NAc is involved in the transmission and regulation of nociception in naïve and morphine-tolerant rats.

Hangover headache and its behavioral changes in rats.

The present study aims to establish and evaluate a rat model for hangover headaches caused by alcoholic drinks. Chronic migraine (CM) model rats were divided into 3 groups, and intragastrically administered alcoholic drinks (sample A, B, or C) to simulate hangover headache attacks. The withdrawal threshold for the hind paw/face and the thermal latency of hind paw withdrawal were detected after 24 hr. Serum was collected from the periorbital venous plexus of rats in each group, and enzymatic immunoassays were used to determine the serum levels of calcitonin gene-related peptide (CGRP), substance P (SP), and nitric oxide (NO). Compared with the control group, the mechanical hind paw pain threshold was significantly lower in rats administered Samples A and B after 24 hr; however, no significant difference was observed across groups for the thermal pain threshold. The mechanical threshold for periorbital pain was only significantly reduced in rats administered Sample A. Immunoassays further indicated that serum levels of SP in the group administered Sample A were significantly higher than those in the control group; the serum levels of NO and CGRP were significantly higher in the group of rats receiving Sample B. We successfully developed an effective and safe rat model for investigating alcohol drink induced hangover headaches. This model could be used to investigate the mechanisms associated with hangover headaches for the development of novel and promising candidates for the future treatment or prophylaxis of hangover headaches.

Modulation of Pain-Associated Behaviors in Sickle Mice Using Low-Intensity Transcranial Focused Ultrasound

Pain is a major comorbidity of sickle cell disease (SCD), which is the most genetic diathesis affecting mostly African Americans in the United States. Opioids are used to reduce pain, however the increased risk of drug abuse, overdose, and addiction has led to an urgent need to develop non-pharmacologic approaches to treat pain safely and effectively. Low-intensity transcranial focused ultrasound (tFUS) is a non-invasive brain stimulation method featured with high precision and deep brain penetration, which holds great promise in managing pain. Here, we report how tFUS modulates pain-related behaviors in wild-type mice and humanized sickle mice by targeting specific brain regions involved in the processing and perception of pain. We used wild-type mice (C57BL/6J) and humanized transgenic BERK mice on a mixed genetic background (FVB/N, 129, DBA/2, C57BL/6, and Black Swiss) murine α and β globin knockouts (α−/−, β−/−) with sickle mice expressing >99% human sickle hemoglobin S (HbS), demonstrating severe SCD. Mice were generated in-house by cross-breeding homozygous (SS) males with hemizygous (AS) females. All studies in mice were approved by IACUC of Carnegie Mellon University and University of California at Irvine, and complied with NIH guidelines. The tFUS was applied to hindlimb of the primary somatosensory cortex (S1HL) at the left hemisphere of the brain, and the effect of tFUS stimulation was further investigated by comparing with control groups such as sham treatment (applying tFUS to a vicinity region of S1HL) and negative control (not applying tFUS while keeping the same experimental procedures). A modified hot-plate behavior test was used to assess unilateral effects of tFUS by comparing withdrawal responses of contralateral (right) and ipsilateral (left) hind paws. The nociceptive responses to noxious heat stimuli were blindly assessed and analyzed in terms of ∆hind paw withdrawal latency (∆hPWL) (s) (= contralateral hPWL- ipsilateral hPWL) before and after specific treatment. We observed a marked reduction in sensitivity to noxious heat stimuli in tFUS-treated male and female wild-type mice. Compared to the baseline value, tFUS stimulation with lower pulse repetition frequency (PRF) (i.e., 40 Hz) at left S1HL led to a significant increase in the averaged ∆hPWL (***p<0.001, N=8 male wild-type mice for 3 min after tFUS; *p<0.05, N=8 for 8 min after tFUS; **p<0.01, N=8 female wild-type mice for 3 min after tFUS; *p<0.05, N=8 for 8 min after tFUS; Friedman test), but not with the sham treatment (p>0.05, N=8 male and female wild-type mice for 3 and 8 min after sham treatment; Friedman test). Next, we assessed whether tFUS could modulate heat hyperalgesia in female sickle mice. Fig. 1 shows that tFUS stimulation with PRF of 40 Hz at left S1HL resulted in a significant change of ∆hPWL in the sickle mice compared to pre-stimulation baseline (**p<0.01, N=10 for 3 and 8 min after tFUS; Friedman test). This effect was specific to tFUS stimulation as a remarkable difference of averaged ∆hPWL between the sham treatment/negative control and pre-sham/negative control baseline was not observed (p>0.05, N=10 for 3 and 8 min after sham treatment and negative control; Friedman test), which suggests that tFUS stimulation with a lower PRF at S1HL can attenuate heat hyperalgesia in the sickle mice. In addition, when tFUS with higher PRF (i.e., 3 kHz) was applied at left S1HL, we found a negative averaged value of ∆hPWL based on the decreased contralateral (right) hind paw latency compared to latency of ipsilateral (left) hind paw, which is significantly different from the behavioral responses to heat stimuli with the PRF of 40 Hz (###p<0.001, N=10 for 3 min after tFUS with PRF of 40 and 3 kHz; ####p<0.0001, N=10 for 8 min after tFUS with PRF of 40 and 3 kHz; Kruskal-Wallis test). Since, tFUS stimulation with a higher PRF leads to exacerbation of heat hyperalgesia in the sickle mice, it is critical to optimize the level of PRF to achieve an analgesic effect. We show that tFUS stimulation can be effective in non-invasively attenuating heat pain sensitivity in wild-type mice and heat hyperalgesia in humanized sickle mice at a lower PRF, but exacerbating at a higher PRF by stimulating pre-identified pain-processing brain circuit. These observations have a translational potential for establishing an effective, non-addictive, non-pharmacological, non-invasive device-based neuromodulation technique for managing sickle cell pain. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Bone marrow mesenchymal stem cell-derived exosomes shuttling miR-150-5p alleviates mechanical allodynia in rats by targeting NOTCH2 in microglia

BackgroundThis study probes into the function and mechanism of bone marrow mesenchymal stem cell (BMSC)-derived exosomes loaded with miR-150-5p in mechanical allodynia.MethodsBMSCs were infected with miR-150-5p inhibition lentiviruses to obtain exosomes with low miR-150-5p expression. A L5 spinal nerve ligation (SNL) model was established in rats where exosomes, NOTCH2 overexpression/inhibition plasmids, or microglial cells were intrathecally administered. Hind paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) of rats were measured. TUNEL staining was used to measure the apoptotic rate in rat spinal dorsal horn (SDH), ELISA to evaluate pro-inflammatory factor levels, and RT-qPCR, western blotting, and immunohistochemistry to detect miR-150-5p and NOTCH2 expression. Immunofluorescence was used for localizing exosomes and NOTCH2 and detecting the expression of OX42, a maker for microglia. Dual luciferase reporter and RNA pull down assays were performed to validate the putative binding between miR-150-5p and NOTCH2.ResultsNOTCH2 expressed at a high level and miR-150-5p was downregulated in SDH of SNL rats. Exosomes injected were localized in rat SDH. BMSC-exosomes or NOTCH2 downregulation increased PWT and PWL of SNL rats and reduced apoptosis and inflammation in SDH. In contrast, NOTCH2 overexpression aggravated mechanical allodynia and SDH injury. Moreover, inhibiting miR-150-5p in BMSC-exosomes offset the therapeutic effects of BMSC-exosomes. Microglia activation induced mechanical allodynia in wild rats, while intrathecal injection of microglial cells incubated with BMSC-exosomes showed alleviated mechanical allodynia in SNL rats. NOTCH2 was targeted by miR-150-5p.ConclusionBMSC-derived exosomal miR-150-5p alleviates mechanical allodynia by targeting NOTCH2 in microglial cells.

L-cysteine methyl ester overcomes the deleterious effects of morphine on ventilatory parameters and arterial blood-gas chemistry in unanesthetized rats.

We are developing a series of thiolesters that produce an immediate and sustained reversal of the deleterious effects of opioids, such as morphine and fentanyl, on ventilation without diminishing the antinociceptive effects of these opioids. We report here the effects of systemic injections of L-cysteine methyl ester (L-CYSme) on morphine-induced changes in ventilatory parameters, arterial-blood gas (ABG) chemistry (pH, pCO2, pO2, sO2), Alveolar-arterial (A-a) gradient (i.e., the index of alveolar gas-exchange within the lungs), and antinociception in unanesthetized Sprague Dawley rats. The administration of morphine (10 mg/kg, IV) produced a series of deleterious effects on ventilatory parameters, including sustained decreases in tidal volume, minute ventilation, inspiratory drive and peak inspiratory flow that were accompanied by a sustained increase in end inspiratory pause. A single injection of L-CYSme (500 μmol/kg, IV) produced a rapid and long-lasting reversal of the deleterious effects of morphine on ventilatory parameters, and a second injection of L-CYSme (500 μmol/kg, IV) elicited pronounced increases in ventilatory parameters, such as minute ventilation, to values well above pre-morphine levels. L-CYSme (250 or 500 μmol/kg, IV) also produced an immediate and sustained reversal of the deleterious effects of morphine (10 mg/kg, IV) on arterial blood pH, pCO2, pO2, sO2 and A-a gradient, whereas L-cysteine (500 μmol/kg, IV) itself was inactive. L-CYSme (500 μmol/kg, IV) did not appear to modulate the sedative effects of morphine as measured by righting reflex times, but did diminish the duration, however, not the magnitude of the antinociceptive actions of morphine (5 or 10 mg/kg, IV) as determined in tail-flick latency and hindpaw-withdrawal latency assays. These findings provide evidence that L-CYSme can powerfully overcome the deleterious effects of morphine on breathing and gas-exchange in Sprague Dawley rats while not affecting the sedative or early stage antinociceptive effects of the opioid. The mechanisms by which L-CYSme interferes with the OR-induced signaling pathways that mediate the deleterious effects of morphine on ventilatory performance, and by which L-CYSme diminishes the late stage antinociceptive action of morphine remain to be determined.

Protective effects of anandamide against cisplatin-induced peripheral neuropathy in rats

Cisplatin (CIS) is an effective antineoplastic agent used in the treatment of several cancer types. Peripheral neuropathy is a major dose-limiting side-effect in CIS therapy. Cannabinoids may alleviate this painful side effect. This study investigated the analgesic effects of anandamide (AN) on CIS-induced peripheral neuropathy, in vitro effects of AN in CIS neurotoxicity, and the contribution of nitric oxide (NO) in this effect. This is an experimental animal study. Primary dorsal root ganglion (DRG) cultures were prepared from one-day-old rats for in vitro investigations. DRG cells were incubated with CIS (100–300 M), and AN (10, 50, 100, and 500 μM) was administered with the submaximal concentration of CIS. Female Sprague Dawley rats were divided into control, CIS, CIS+AN, CIS+AN+L-NG-nitro arginine methyl ester (LNAME). CIS was administered 3 mg/kg i.p once weekly for 5 weeks. AN (1 mg/kg i.p) or in combination with 10 mg/kg i.p LNAME was administrated 30 min before CIS injection. Mechanical allodynia, thermal hyperalgesia, and tail clip tests were performed. After intracardiac perfusion, sciatic nerves (SN), and DRGs were isolated and semi-thin sections were stained with toluidine blue and investigated histologically. SPSS v. 21.0 and Sigma STAT 3.5 were used for statistical analysis. One/two way ANOVA, Kruskal–Wallis, and Wilcoxon signed ranks tests were used. A p-value of 0.05 was accepted as significant. CIS caused significant mechanical allodynia. AN and AN+LNAME significantly increased hind paw withdrawal latency in mechanical allodynia test. The degenerated axons significantly increased in CIS group, while decreased in AN group. The frequency of larger neurons seemed to be higher in CIS+AN group. AN may be a therapeutic alternative for the treatment of CIS-induced peripheral neuropathy. However, its central adverse effects must be considered.

The activation of galanin receptor 2 plays an antinociceptive effect in nucleus accumbens of rats with neuropathic pain

Our previous research has shown that galanin plays an antinociceptive effect via binding to galanin receptors (GalRs) in nucleus accumbens (NAc). This study focused on the involvement of GalR2 in galanin-induced antinociceptive effect in NAc of neuropathic pain rats. The chronic constriction injury of sciatic nerve (CCI) was used to mimic neuropathic pain model. The hind paw withdrawal latency (HWL) to thermal stimulation and hind paw withdrawal threshold (HWT) to mechanical stimulation were measured as the indicators of pain threshold. The results showed that 14 and 28 days after CCI, the expression of GalR2 was up-regulated in bilateral NAc of rats, and intra-NAc injection of GalR2 antagonist M871 reversed galanin-induced increases in HWL and HWT of CCI rats. Furthermore, intra-NAc injection of GalR2 agonist M1145 induced increases in HWL and HWT at day 14 and day 28 after CCI, which could also be reversed by M871. Finally, we found that M1145-induced antinociceptive effect in NAc of CCI rats was stronger than that in intact rats. These results imply that the GalR2 is activated in the NAc from day 14 to day 28 after CCI and GalR2 is involved in the galanin-induced antinociceptive effect in NAc of CCI rats.

Galanin Receptor 2 Is Involved in Galanin-Induced Analgesic Effect by Activating PKC and CaMKII in the Nucleus Accumbens of Inflammatory Pain Rats.

It has been reported that galanin has an analgesic effect via activating galanin receptors (GALRs). This study focused on the involvement of GALR2 in the galanin-induced analgesic effect and its signaling mechanism in the nucleus accumbens (NAc) of inflammatory rats. Animal models were established through injecting carrageenan into the plantar of rats’ left hind paw. The results showed that GALR2 antagonist M871 weakened partially the galanin-induced increases in hind paw withdrawal latency (HWL) to thermal stimulation and hind paw withdrawal threshold (HWT) to mechanical stimulation in NAc of inflammatory rats. Moreover, the GALR2 agonist M1145 prolonged the HWL and HWT, while M871 blocked the M1145-induced increases in HWL and HWT. Western blotting showed that the phosphorylation of calcium/calmodulin-dependent protein kinase II (p-CaMKII) and protein kinase C (p-PKC) in NAc were upregulated after carrageenan injection, while p-PKC and p-CaMKII were downregulated after intra-NAc administration of M871. Furthermore, the CaMKII inhibitor KN93 and PKC inhibitor GO6983 attenuated M1145-induced increases in HWL and HWT in NAc of rats with inflammatory pain. These results prove that GALR2 is involved in the galanin-induced analgesic effect by activating CaMKII and PKC in NAc of inflammatory pain rats, implying that GALR2 agonists probably are potent therapeutic options for inflammatory pain.

Antinociceptive effects of rotigotine-loaded microspheres and its synergistic interactions with analgesics in inflammatory pain in rats

Rotigotine-loaded microspheres (RoMS) are sustained-release formulations with prolonged anti-Parkinson's effects. Given that pain is a non-motor symptom of Parkinson's disease, this study investigated the antinociceptive effects of RoMS and their synergistic effects with analgesics on inflammatory pain. A model of inflammatory pain was prepared by intraplantarly injecting male Sprague-Dawley rats with carrageenan. The antinociceptive effects of RoMS, acetaminophen, and tramadol, both alone and in combination, were evaluated using the hind paw withdrawal latency in the hot plate test and Randall–Selitto test. The rotigotine concentrations in serum and tissues were assayed using ultra-performance liquid chromatography–tandem mass spectrometry. Isobolographic analysis was performed to evaluate the nature of the interactions of RoMS with acetaminophen or tramadol. The results showed that hind paw withdrawal latency to thermal and mechanical stimuli was significantly increased on day 3 and 7 after administered RoMS. Rotigotine could be detected in serum and tissues 3 and 7 days after an intramuscular injection of RoMS. However, the rotigotine concentration fell the detection limit of the assay on day 14 after administration. RoMS produced synergistic antinociceptive effects in the inflammatory pain model when RoMS is combined with acetaminophen or tramadol. These findings suggest that RoMS can relieve inflammatory pain in rats. Furthermore, the combination of RoMS with acetaminophen or tramadol produces synergistic antinociception, which may be clinically worthy because combination therapies may reduce the drug doses required for antinociception.

The Effect of Rotigotine Extended-Release Microspheres Alone or With Celecoxib on the Inflammatory Pain.

Clinical trials of rotigotine extended-release microspheres (RTGT-MS), which provides a sustained release of rotigotine for near 2 weeks in vivo, have been conducted in the treatment of Parkinson’s disease (PD). This study was to investigate the analgesic effect of RTGT-MS, and to know whether RTGT-MS have synergistic interaction with non-steroidal anti-inflammatory drug, celecoxib. The inflammatory pain model of rats was prepared by carrageenan-induced paw edema. The thermal and mechanical stimuli were applied and the hindpaw withdrawal latency (HWL) response was evaluated. Treatment with RTGT-MS increased the HWL in a dose-dependent manner. The ED50 of RTGT-MS was 24.68 ± 1.02 mg/kg. Isobolographic analysis shows that the combination of RTGT-MS and celecoxib resulted in a synergistic antinociceptive effect. Further results demonstrated that antinociceptive effect of RTGT-MS was accompanied with that PKA, cAMP, COX-2, and PGE2 levels were decreased. Chlorpromazine, a dopamine receptor blocker, not only weakened the analgesic effect of RTGT-MS, but also increased the levels of cAMP, PKA, COX-2, and PGE2. These findings provide a rationale for the combination of RTGT-MS and celecoxib in the treatment of PD, which may reduce the dose of celecoxib, thereby lowering the incidence of adverse effects and improving the pain management in PD patients.

Thermal Hyperalgesia and Mechanical Allodynia Elicited by Histamine and Non-histaminergic Itch Mediators: Respective Involvement of TRPV1 and TRPA1

Acute itch is elicited by histamine, as well as non-histaminergic itch mediators including chloroquine, BAM8-22 and Ser-Leu-Ile-Gly-Arg-Leu (SLIGRL). When injected intradermally, histamine binds to histamine H1 and H4 receptors that activate transient receptor potential vanilloid 1 (TRPV1) to depolarize pruriceptors. Chloroquine, BAM8-22, and SLIGRL, respectively, bind to Mas-related G-protein-coupled receptors MrgprA3, MrgprC11, and MrgprC11/PAR2 that in turn activate transient receptor potential ankyrin 1 (TRPA1). In this study we tested if histamine, chloroquine, BAM8-22 and SLIGRL elicit thermal hyperalgesia and mechanical allodynia in adult male mice. We measured the latency of hindpaw withdrawal from a noxious heat stimulus, and the threshold for hindpaw withdrawal from a von Frey mechanical stimulus. Intraplantar injection of histamine resulted in significant thermal hyperalgesia (p < 0.001) and mechanical allodynia (p < 0.001) ipsilaterally that persisted for 1 h. Pretreatment with the TRPV1 antagonist AMG-517 (10 or 20 μg), but not the TRPA1 antagonist HC-030031 (50 or 100 μg), significantly attenuated the magnitude and time course of thermal hyperalgesia and mechanical allodynia elicited by histamine (p < 0.001 for both), indicating that these effects are mediated by TRPV1. In contrast, pretreatment with the TRPA1 antagonist significantly reduced thermal hyperalgesia and mechanical allodynia elicited by chloroquine (p < 0.001 for both ), BAM-822 (p < 0.01, p < 0.001, respectively) and SLGRL (p < 0.05, p < 0.001, respectively), indicating that effects elicited by these non-histaminergic itch mediators require TRPA1. TRPV1 and TRPA1 channel inhibitors thus may have potential use in reducing hyperalgesia and allodynia associated with histaminergic and non-histaminergic itch, respectively.

Pharmacopuncture of Bauhinia variegata Nanoemulsion Formulation against Diabetic Peripheral Neuropathic Pain.

ObjectivesThe objective of the study was to prepare Bauhinia variegata loaded nanoemulsion(formulation and determine the efficacy of herbal drug formulation against diabetic peripheral neuropathic pain through acupuncture technique.MethodsNine different ba tches of nanoemulsion (NE1 NE9) of BVN was prepared by varying the Smix ratio and the concentration of oil. BVN was characterized to determine particle size, shape, zeta potential, polydispersity index, optical transmittance, drug release profile and stora ge stability.The optimized formulation was subjected to plantar test, behavioral tests of neuropathic pain and Von Frey filament stimulation test. Diabetes was induced by intraperitoneal injection of freshly prepared solution of Streptozotocin (60 mg/kg) to the experimental rats. Animals were made diabetic divided into four groups, Group I was untreated normal control group, Group II was diabetic control group, Group III was Bauhinia variegata extract ( treated group (100 mg/kg/day, p.o) and Group IV was BVN treated groups (100 mg/kg/day, p.o) acute and chronically.ResultsThe prepared B. variegata loaded nanoemulsion was nanosized (124 nm), spherical, uniform and stable over the period of 180 days with no change in physiochemical properties. The bl ood glucose and body weight of animals was normalizing after four weeks of treatment that was significant with BVN in comparison to diabetic control group. The chronic administration of BVN significantly (P<0.001) decreased hind paw withdrawal latency an d attenuated mechanical allodynia as compared with diabetic rats.ConclusionThus, BVN may be an effective drug formulation against diabetic peripheral neuropathic pain.

Role of mu-opioid receptor in nociceptive modulation in anterior cingulate cortex of rats.

Lots of studies have demonstrated that anterior cingulate cortex plays important roles in the pain perception and pain modulation. The present study explored the role of mu-opioid receptor in nociceptive modulation in anterior cingulate cortex of rats with neuropathic pain. Neuropathic pain model was set up by chronic constriction injury of the left sciatic nerve of rats. The hindpaw withdrawal latency to thermal and mechanical stimulation, by hot plate and Randall Selitto Test respectively, was used to evaluate the rat’s responses to noxious stimulation. Results showed that intra-anterior cingulate cortex injection of morphine could induce the antinociception dose-dependently. By intra-anterior cingulate cortex injection of opioid receptor antagonist, the morphine-induced antinociception could be attenuated by naloxone, as well as much significantly by the selective mu-opioid receptor antagonist β-funaltrexamine, indicating that mu-opioid receptor is involved in the morphine-induced antinociception in anterior cingulate cortex of rats with neuropathic pain. The morphine-induced antinociception was much more decreased in rats with neuropathic pain than that in normal rats, and there was a significant decrease in mu-opioid receptor messenger RNA levels in anterior cingulate cortex of rats with neuropathic pain, indicating that there may be a down-regulation in mu-opioid receptor expression in anterior cingulate cortex of rats with neuropathic pain. To further confirm the role of mu-opioid receptor in morphine-induced antinociception in anterior cingulate cortex, normal rats were received intra-anterior cingulate cortex administration of small interfering RNA targeting mu-opioid receptor and it was found that there was a down-regulation in mu-opioid receptor messenger RNA levels, as well as a down-regulation in mu-opioid receptor expression in anterior cingulate cortex tested by real-time polymerase chain reaction and western blotting. Furthermore, the morphine-induced antinociceptive effect decreased significantly in rats with small interfering RNA targeting mu-opioid receptor, which indicated that knockdown mu-opioid receptor in anterior cingulate cortex could also attenuate morphine-induced antinociceptive effect. These results strongly suggest that mu-opioid receptor plays a significant role in nociceptive modulation in anterior cingulate cortex of rats.

Galanin plays a role in antinociception via binding to galanin receptors in the nucleus accumbens of rats with neuropathic pain

Galanin and galanin receptors (GalRs) play important roles in the transmission and modulation of nociceptive information. Our previous research has shown that the expression of GalR1 is upregulated and that GalR1 activation in the nucleus accumbens (NAc) of rats with neuropathic pain has an antinociceptive effect. However, the antinociceptive effect of NAc galanin in neuralgia remains unclear. The present study aimed to explore the antinociceptive effect induced by galanin in rats with neuropathic pain and the underlying mechanism. The results showed that the intra-NAc injection of galanin induced a dose-dependent increase in hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation in mononeuropathic rats and that this effect was stronger than that in intact rats. The intra-NAc injection of the non-selective GalR antagonist galantide reduced HWL in the rats with neuropathic pain, but there was no influence of galantide on HWL in intact rats. Moreover, galanin expression in the NAc was upregulated after sciatic nerve ligation. All of these results demonstrate that galanin plays a role in antinociception via binding to GalRs in the NAc of rats and that endogenous galanin is involved in the antinociception after peripheral nerve injury.

Galanin receptor 1 plays an antinociceptive effect via inhibiting PKA activation in the nucleus accumbens of rats with neuropathic pain.

Galanin and galanin receptors (GalRs) have been reported to be involved in the transmission and modulation of nociceptive information in the central nervous system (CNS). However, the underlying mechanism of the antinociception of GalRs in neuropathic pain remains unclear. This study investigated the antinociception induced by galanin receptor 1 (GalR1) via protein kinase A (PKA) signaling pathway in the nucleus accumbens (NAc) of rats with neuropathic pain. A mononeuropathy model was replicated by ligation of the left sciatic nerve, following which the expression of phospho-PKA (p-PKA) in the NAc were markedly up-regulated at 14(th) and 28(th) day after ligation of sciatic nerve, and p-PKA expression was down-regulated by intra-NAc injection of GalR1 agonist M617, but the GalR1 antagonist M35 did not have an effect. We also found that M35 in the NAc blocked the M617-induced increase in the hind paw withdrawal latencies (HWLs) of rats with mononeuropathy, but M35 alone had no effect on HWLs, and PKA inhibitor H-89 attenuated the M617-induced an increase in the HWLs. These results suggested that GalR1 induced an antinociception via inhibiting PKA activation, implying that GalR agonists may be potential and potent therapeutic options to treat chronic neuropathic pain.

Electroacupuncture exerts an anti-migraine effect via modulation of the 5-HT7 receptor in the conscious rat.

Acupuncture has been recommended as an alternative therapy for migraine. Emerging evidence suggests that the 5-HT7 receptor (5-HT7R) plays a significant facilitatory role in descending modulation in migraine pathophysiology, and that activation of 5-HT7R in the descending pathway is involved in migraine central sensitisation. To investigate the ability of electroacupuncture (EA) to ameliorate central sensitisation via modulation of 5-HT7R in the descending pain pathways using a rat model of migraine induced by repetitive dural electrical stimulation (DES). 64 male Sprague-Dawley rats were randomly divided into four groups: Normal group; DES group (receiving dural electrical stimulation only); DES+GB20 group (DES model group treated with EA at GB20); and DES+Sham group (DES model group treated with EA at a non-traditional (sham) acupuncture point). The presence of cutaneous allodynia was determined by measuring facial and hind-paw withdrawal latencies to electronic von-Frey. The expression of 5-HT7R in the descending pathways (periaqueductal grey, raphe magnus nucleus, and trigeminal nucleus caudalis) was assessed using immunofluorescence and Western blotting. Facial and hind-paw withdrawal thresholds were significantly increased in the DES+GB20 group compared with the untreated DES group. The expression of 5-HT7R was significantly decreased in the DES+GB20 group compared with the DES group (one-way analysis of variance (ANOVA), P<0.05). No significant differences in behaviour or expression were found between the rats in the DES+Sham group and the untreated DES group (one-way ANOVA, P>0.05). EA at GB20 may ameliorate central sensitisation in migraine by inhibiting the activation of 5-HT7 receptors in the descending pain pathway in a rat model of migraine.

Heroin-based crack induces hyperalgesia through β-arrestin 2 redistribution and phosphorylation of Erk1/2 and JNK in the periaqueductal gray area

Continuous use of crack induces hyperalgesia which is related to drug tolerance. Despite cumulative evidence based on the growth rate of crack abuse, no serious study has been focused on the mechanisms of crack-induced hyperalgesia. This study aimed to elucidate whether extracellular signal-regulated kinases (Erk1/2)/β-arrestin pathways are involved in the crack-induced hyperalgesia. Fifty adult male Wistar rats were randomly divided into five groups: normal saline (NS), crack (0.9 mg/kg/day), heroin (1 mg/kg/day), crack + barbadin (100 μM), and heroin + barbadin groups, which received their intraperitoneal (i.p) treatments for four weeks. The thermal sensitivity was assessed using the hot-plate test. Moreover, phosphorylation of the Erk1/2 and JNK, as well as expression of protein kinase C-alpha (PKC-α), Mu-receptor (MOR), and β-arrestin 2 were determined in the whole lysate and membrane fraction using immunoblotting assay in the periaqueductal gray (PAG) area. The results demonstrated that chronic administration of crack and heroin significantly decreased hind-paw withdrawal latency compared to the NS group. Furthermore, crack as well as heroin administration increased phosphorylated Erk1/2 and JNK in the PAG. In addition, membrane β-arrestin 2 and PKC-α were significantly increased in the crack and heroin-received groups, while membrane MOR expression was decreased in the PAG. Nevertheless, co-administration of barbadin, an inhibitor of β-arrestin, and crack or heroin reversed all these changes. Our findings may partially confirm the role of β-arrestin 2 and PKC rearrangements, Erk1/2 and JNK phosphorylation in crack-induced hyperalgesia and provide potential therapeutic targets to attenuate crack-induced hyperalgesia.