Paw Pressure Threshold Research Articles

New Insights into the Opioid Analgesic Profile of cis-(-)-N-Normetazocine-derived Ligands.

In this work, we report on the in vitro and in vivo pharmacological properties of LP1 analogs to complete the series of structural modifications aimed to generate compounds with improved analgesia. To do that, the phenyl ring in the N-substituent of our lead compound LP1 was replaced by an electron-rich or electron-deficient ring and linked through a propanamide or butyramide spacer at the basic nitrogen of the (-)-cis-N-normetazocine skeleton. In radioligand binding assays, compounds 3 and 7 were found to display nanomolar binding affinity for the μ opioid receptor (MOR) (Ki = 5.96 ± 0.08 nM and 1.49 ± 0.24 nM, respectively). In the mouse vas deferens (MVD) assay, compound 3 showed an antagonist effect against DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin), a highly selective MOR prototype agonist, whereas compound 7 produced naloxone reversible effect at MOR. Moreover, compound 7, as potent as LP1 and DAMGO at MOR, was able to reduce thermal and inflammatory pain assessed by the mouse tail-flick test and rat paw pressure thresholds (PPTs) measured by a Randall-Selitto test.

Analgesic properties of newly synthesized N pyrrolyl hydrazide hydrazones

Purpose: To screen a series of newly synthesized N-pyrrolyl hydrazide hydrazones for analgesic activity via Paw-pressure (PP) test and hot plate test (HPT).
 Methods: The compounds newly synthesized through the classical Paal-Knor cyclization, N-pyrrolyl hydrazide-hydrazones were administered intraperitoneally at a dose of 20 mg/kg. Paw pressure and hot plate tests were applied to assess the analgesic properties. In addition, stress-induced analgesia with naloxone as a non-selective opioid receptor antagonist was performed.
 Results: The compound (DI-5g), containing an izatine carbonyl fragment, was the most promising. It presented the highest paw pressure threshold (25 AU at 30th min) by exceeding the analgesic activity of the referent metamizole (23 AU at 30th min). The relative effect from the hot plate test was consistent with the paw pressure results. Opioid receptors were involved in the analgesic activities of N-pyrrolyl hydrazide-hydrazones.
 Conclusion: The N-pyrrolyl carboxylic acids are synthesized and identified as new compounds and their hydrazide-hydrazone derivatives as promising leads for future design and synthesis of drugs with possibly prolonged analgesic activity.

The Acute Antiallodynic Effect of Tolperisone in Rat Neuropathic Pain and Evaluation of Its Mechanism of Action.

Current treatment approaches to manage neuropathic pain have a slow onset and their use is largely hampered by side-effects, thus there is a significant need for finding new medications. Tolperisone, a centrally acting muscle relaxant with a favorable side effect profile, has been reported to affect ion channels, which are targets for current first-line medications in neuropathic pain. Our aim was to explore its antinociceptive potency in rats developing neuropathic pain evoked by partial sciatic nerve ligation and the mechanisms involved. Acute oral tolperisone restores both the decreased paw pressure threshold and the elevated glutamate level in cerebrospinal fluid in neuropathic rats. These effects were comparable to those of pregabalin, a first-line medication in neuropathy. Tolperisone also inhibits release of glutamate from rat brain synaptosomes primarily by blockade of voltage-dependent sodium channels, although inhibition of calcium channels may also be involved at higher concentrations. However, pregabalin fails to affect glutamate release under our present conditions, indicating a different mechanism of action. These results lay the foundation of the avenue for repurposing tolperisone as an analgesic drug to relieve neuropathic pain.

Mu opioid receptor (MOR) agonist‐induced antihyperalgesic effects following spared nerve injury in male and female rats

In most preclinical models of neuropathic pain, hypersensitivity to pain often resolves after a few weeks. However, spared nerve injury (SNI) produces a persistent pain state that lasts at least 18 months. The goal of the current study was to evaluate antihyperalgesic effects of mu‐opioid receptor (MOR) agonists varying in potency and efficacy in the SNI model. We hypothesized that the MOR agonists, fentanyl, morphine, and nalbuphine, would produce antihyperalgesic effects in both male and female rats. Mechanical hypersensitivity was evaluated by measuring responses to increasing pressure (g) applied to each paw (paw pressure test or Randall‐Selitto test) with a maximum cut‐off of 300 g. Using a within‐subjects design, responses were evaluated before and after SNI or sham surgery in male and female rats, and cumulative dose effects curves for fentanyl (0.01‐0.1 mg/kg), morphine (0.3‐10 mg/kg), and nalbuphine (0.3‐10 mg/kg) were determined. Both male and female sham rats demonstrated an initial hypersensitivity on their ipsilateral paw following surgery that dissipated within 7 d. Following SNI surgery, paw pressure thresholds on the injured paw were lower as compared with the pre‐surgical response and with the contralateral paw for at least 20 wks in male and female rats. In male rats, fentanyl, morphine, and nalbuphine dose‐dependently alleviated SNI‐induced hypersensitivity, with EC50 values of 0.03 (± 0.007), 3.9 (± 0.8), and 5.3 (± 0.8) mg/kg, respectively. In female rats, fentanyl, morphine, and nalbuphine also alleviated SNI‐induced hypersensitivity in a dose‐dependent manner, with EC50 values of 0.01 (± 0.002), 4.1 (± 0.8), and 5.5 (± 0.8) mg/kg, respectively. Naltrexone (0.3 mg/kg) produced rightward shifts in the fentanyl and morphine dose effect curves. Interestingly, fentanyl and morphine also increased paw pressure thresholds in the uninjured, contralateral paws and sham‐treated ipsilateral paws in male and female rats. In contrast, gabapentin produced anti‐hyperalgesic effects in both male and female rats at 180 mg/kg as demonstrated by a return to pre‐surgical, paw pressure thresholds without altering thresholds in the non‐injured paw. These results demonstrate MOR agonists produce dose‐dependent increases in paw pressure thresholds following SNI surgery in both male and female rats. As expected, MOR agonists demonstrated a rank order in potency with fentanyl > morphine > nalbuphine in both sexes, and only fentanyl was slightly more potent in females. Fentanyl and morphine also increased paw pressure thresholds in non‐injured paws, suggesting that large doses inhibited responses to mechanical stimulation perhaps due to sedation and/or behavioral suppression. Overall, these findings demonstrate that MOR agonists produce antihyperalgesic effects in male and female rats potentially with narrow therapeutic indices. Future studies will investigate other behavioral effects of MOR agonists, such as reinforcing and interoceptive effects, in this chronic neuropathic pain model.

Aldosterone Synthase in Peripheral Sensory Neurons Contributes to Mechanical Hypersensitivity during Local Inflammation in Rats.

Recent emerging evidence suggests that extra-adrenal synthesis of aldosterone occurs (e.g., within the failing heart and in certain brain areas). In this study, the authors investigated evidence for a local endogenous aldosterone production through its key processing enzyme aldosterone synthase within peripheral nociceptive neurons. In male Wistar rats (n = 5 to 8 per group) with Freund's complete adjuvant hind paw inflammation, the authors examined aldosterone, aldosterone synthase, and mineralocorticoid receptor expression in peripheral sensory neurons using quantitative reverse transcriptase-polymerase chain reaction, Western blot, immunohistochemistry, and immunoprecipitation. Moreover, the authors explored the nociceptive behavioral changes after selective mineralocorticoid receptor antagonist, canrenoate-K, or specific aldosterone synthase inhibitor application. In rats with Freund's complete adjuvant-induced hind paw inflammation subcutaneous and intrathecal application of mineralocorticoid receptor antagonist, canrenoate-K, rapidly and dose-dependently attenuated nociceptive behavior (94 and 48% reduction in mean paw pressure thresholds, respectively), suggesting a tonic activation of neuronal mineralocorticoid receptors by an endogenous ligand. Indeed, aldosterone immunoreactivity was abundant in peptidergic nociceptive neurons of dorsal root ganglia and colocalized predominantly with its processing enzyme aldosterone synthase and mineralocorticoid receptors. Moreover, aldosterone and its synthesizing enzyme were significantly upregulated in peripheral sensory neurons under inflammatory conditions. The membrane mineralocorticoid receptor consistently coimmunoprecipitated with endogenous aldosterone, confirming a functional link between mineralocorticoid receptors and its endogenous ligand. Importantly, inhibition of endogenous aldosterone production in peripheral sensory neurons by a specific aldosterone synthase inhibitor attenuated nociceptive behavior after hind paw inflammation (a 32% reduction in paw pressure thresholds; inflammation, 47 ± 2 [mean ± SD] vs. inflammation + aldosterone synthase inhibitor, 62 ± 2). Local production of aldosterone by its processing enzyme aldosterone synthase within peripheral sensory neurons contributes to ongoing mechanical hypersensitivity during local inflammation via intrinsic activation of neuronal mineralocorticoid receptors.

The antidepressant effects of rosiglitazone on rats with depression induced by neuropathic pain

A growing number of studies reported that rosiglitazone (a PPARgamma agonist) could ameliorate the painful state and prevent stress-induced depression. However, whether rosiglitazone can prevent pain-induced depression is unclear. This study aimed to explore the antidepressant effects of rosiglitazone in L5 spinal nerve transection (SNT) induced neuropathic pain rats. In addition, AMPK inhibitor (Compound C) and autophagic antagonist (3-methyladenine, 3-MA) were applied to investigate the underlying therapeutic mechanisms. L5 SNT-induced neuropathic pain symptoms and depressive like-behaviors were detected by paw pressure threshold test (PPT), open-field test (OFT), forced swimming test (FST), tail suspension test (TST), sucrose preference test (SPT). Rosiglitazone could ameliorate L5 SNT-induced neuropathic pain symptoms and depressive like-behaviors and the effect could be reversed by Compound C or 3-MA. Compared with the sham group, the levels of BDNF, AMPK, Beclin-1 and LC3B in rats hippocampus significantly decreased in L5 SNT group. On the contrary, rosiglitazone administration significantly up-regulated the levels of AMPK, BDNF, Beclin-1 and LC3B in rats hippocampus. Compared with sham group, the levels of TNF-α, IL-1β, superoxide dismutase (SOD) and malondialdehyde (MDA) in rat hippocampus significantly increased in L5 SNT group. Besides, rosiglitazone administration significantly decreased the levels of TNF-α, IL-1β, SOD and MDA in hippocampus. Compared with rosiglitazone group, 3-MA administration, but not Compound C administration, significantly increased the levels of TNF-α, IL-1β, SOD and MDA in hippocampus. In conclusion, rosiglitazone can counteract down-regulation of AMPK and BDNF induced by L5 SNT rats in hippocampus, and activate autophagic pathway. These effects may contribute to the antidepressant effect of rosiglitazone on the rats with depression induced by L5 SNT.

Preclinical assessment of utility of M6S for multimodal acute and chronic pain treatment in diabetic neuropathy

AimsPrevious reports from our laboratory have established that morphine-6-O-sulfate (M6S) is a mixed μ/δ opioid receptor (OR) agonist and a potential improved alternative to morphine for treatment of chronic multimodal pain in non-diabetic rats. This study extends the antinociceptive effects of M6S and morphine in STZ-induced diabetic rats. Materials and methodsEffects of morphine and M6S were studied across a range of pain modalities, using hot plate threshold (HPT), pinprick sensitivity threshold (PST) and paw pressure threshold (PPT) tests. Key findingsAcutely, M6S was 3- to 5-fold more potent and 2- to 3-fold more efficacious than morphine in HPT and PST tests. No differences in analgesic drug potency/efficacy were detected in the PPT test. After 7–9days of chronic treatment, tolerance developed to the antinociceptive effects of morphine, but not to M6S, in all three pain tests. Furthermore, morphine-tolerant rats were not cross-tolerant to M6S. The selective δ-OR antagonist, naltrindole, blocked M6S-induced antinociception by 62±3% in the HPT test, 93±5% in the PST test, and 30±17% in the PPT test when examined acutely. SignificanceThese studies provide additional confirmation for the mixed μ/δ activity of M6S and demonstrate potential improved clinical utility for dual μ/δ agonists relative to morphine in treatment of diabetic neuropathy across multiple pain domains.

Evaluation of Analgesia, Tolerance, and the Mechanism of Action of Morphine-6-O-Sulfate Across Multiple Pain Modalities in Sprague-Dawley Rats.

Morphine-6-O-sulfate (M6S) is a mixed μ/δ-opioid receptor (OR) agonist and potential alternative to morphine for treatment of chronic multimodal pain. To provide more support for this hypothesis, the antinociceptive effects of M6S and morphine were compared in tests that access a range of pain modalities, including hot plate threshold (HPT), pinprick sensitivity threshold (PST) and paw pressure threshold tests. Acutely, M6S was 2- to 3-fold more potent than morphine in HPT and PST tests, specifically, derived from best-fit analysis of dose-response relationships of morphine/M6S half-effective dose (ED50) ratios (lower, upper 95% confidence interval [CI]) were 2.8 (2.0-5.8) in HPT and 2.2 (2.1, 2.4) in PST tests. No differences in analgesic drug potencies were detected in the PPT test (morphine/M6S ED50 ratio 1.2 (95% CI, 0.8-1.4). After 7 to 9 days of chronic treatment, tolerance developed to the antinociceptive effects of morphine, but not to M6S, in all 3 pain tests. Morphine-tolerant rats were not crosstolerant to M6S. The antinociceptive effects of M6S were not sensitive to κ-OR antagonists. However, the δ-OR antagonist, naltrindole, blocked M6S-induced antinociception by 55% ± 4% (95% CI, 39-75) in the HPT test, 94% ± 4% (95% CI, 84-105) in the PST test, and 5% ± 17% (95% CI, -47 to 59) or 51% ± 14% (95% CI, 14-84; 6 rats per each group) in the paw pressure threshold test when examined acutely or after 7 days of chronic treatment, respectively. Activity via δ-ORs thus appears to be an important determinant of M6S action. M6S also exhibited favorable antinociceptive and tolerance profiles compared with morphine in 3 different antinociceptive assays, indicating that M6S may serve as a useful alternative for rotation in morphine-tolerant subjects.

A Novel Approach for the Control of Inflammatory Pain: Prostaglandin E2 Complexation by Randomly Methylated β-Cyclodextrins.

Inhibitors of cyclooxygenase, which block the formation of prostaglandin (PG) E2, are the standard treatment of inflammatory pain. These drugs, however, have serious gastrointestinal, renal, and cardiovascular side effects that limit their clinical use. Cyclodextrins are neutral glucose oligomers that form a hydrophilic outer and a hydrophobic interior cavity used to carry hydrophilic substances. Methyl-β-cyclodextrins are used currently in several drugs as enhancers and also to deliver PGs. We therefore hypothesized that randomly methylated β-cyclodextrins (RAMEB) could be used for pain treatment. An in silico screening for important inflammatory mediators (eg, PGE2, substance P, bradykinin, and calcitonin gene-related peptide) was performed to predict the probability of these molecules binding to RAMEB. Thereafter, a comprehensive in vitro study investigated the complexation affinity of the best target toward RAMEB or its RAMEB-fraction L (FL) using capillary electrophoresis.Wistar rats were injected intraplantarly with complete Freund's adjuvant (CFA) for 96 hours to induce inflammatory hyperalgesia. Subsequently, rats were treated intraplantarly or intravenously either with RAMEB or RAMEB FL and compared with the respective controls. Parecoxib was used as positive control. Mechanical (paw pressure threshold, PPT) and thermal (paw withdrawal latency) nociceptive thresholds were determined before injection and at the indicated time points thereafter. Paw tissue was collected after treatments, and PGE2 and PGD2 contents were measured. Analysis of variance was used for data analysis followed by appropriate post hoc comparisons. In silico screening indicated that PGE2, with the highest affinity, was the best candidate for RAMEB binding. Likewise, in capillary electrophoresis experiments, RAMEB had a high affinity to form inclusion complexes with the PGE2 (stability constant [K], 360 1/M; 95% confidence interval [C]: 347.58-372.42 M). Local treatment with RAMEB alleviated CFA-induced mechanical (PPT: 76.25 g; 95% CI: 56.24-96.25 g) and thermal hyperalgesia (PPT: 8.50 seconds; 95% CI: 6.76-10.23 seconds). Moreover, a systemic administration of RAMEB decreased CFA-induced mechanical (PPT: 126.66 g; 95% CI: 114.54-138.77 g) and thermal hyperalgesia (paw withdrawal latency: 11.47 seconds; 95% CI: 9.26-13.68 seconds). RAMEB FL resulted in greater in vitro PGE2-binding capacity and decreased PG content as well as hyperalgesia in vivo to a similar extent. Motor activity of the rats was not altered by RAMEB or RAMEB FL. Capture of PGs by cyclodextrins could be a novel and innovative tool for the treatment of inflammatory pain and bypassing some unwanted side effects of cyclooxygenase inhibitors.

The efficacy of Dynorphin fragments at the κ, μ and δ opioid receptor in transfected HEK cells and in an animal model of unilateral peripheral inflammation

BackgroundDynorphin 1–17 is an endogenous peptide that is released at sites of inflammation by leukocytes, binding preferentially to κ-opioid receptors (KOP) to mediate nociception. We have previously shown that dynorphin 1–17 is rapidly biotransformed to smaller peptide fragments in inflamed tissue homogenate. This study aimed to determine the efficacy and potency of selected dynorphin fragments produced in an inflamed environment at the KOP, μ and δ-opioid receptors (MOP and DOP respectively) and in a model of inflammatory pain. Functional activity of Dynorphin 1–17 and fragments (1–6, 1–7 and 1–9) were screened over a range of concentrations against forskolin stimulated human embryonic kidney 293 (HEK) cells stably transfected with one of KOP, MOP or DOP. The analgesic activity of dynorphin 1–7 in a unilateral model of inflammatory pain was subsequently tested. Rats received unilateral intraplantar injections of Freund’s Complete Adjuvant to induce inflammation. After six days rats received either dynorphin 1–7, 1–17 or the selective KOP agonist U50488H and mechanical allodynia determined. Dynorphin 1–7 and 1–9 displayed the greatest activity across all receptor subtypes, while dynorphin 1–7, 1–9 and 1–17 displaying a potent activation of both KOP and DOP evidenced by cAMP inihibition. Administration of dynorphin 1–7 and U50488H, but not dynorphin 1–17 resulted in a significant increase in paw pressure threshold at an equimolar dose suggesting the small peptide dynorphin 1–7 mediates analgesia. These results show that dynorphin fragments produced in an inflamed tissue homogenate have changed activity at the opioid receptors and that dynorphin 1–7 mediates analgesia.

Effect of pioglitazone on neuropathic pain and spinal expression of TLR-4 and cytokines.

The molecular mechanisms underlying neuropathic pain have yet to be elucidated. The present study aimed to examine the modulation of neuroimmune activation in the spinal cord by the synthetic peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist, pioglitazone (Pio), in a rat model of neuropathic pain induced by chronic constriction injury (CCI). Rats were randomly assigned into four groups: Sham surgery with vehicle, chronic constriction injury with vehicle or Pio (10 mg/kg), and chronic constriction injury with Pio and a PPAR-γ antagonist GW9662 (2 mg/kg). Pio or vehicle was administered 1 h prior to the surgery and continued daily until day 7 post-surgery. Paw pressure threshold was measured prior to surgery and on days 0, 1, 3 and 7 post-surgery. Microglia activation markers macrophage antigen complex-1, the mRNA expression levels of tumor necrosis factor α and interleukin-1β, and the mRNA expression levels of toll like receptor (TLR-4) in the lumbar spinal cord were determined. Administration of Pio resulted in the prominent attenuation of mechanical hyperalgesia. In addition, Pio was able to significantly inhibit neuroimmune activation characterized by glial activation, the production of cytokines and expression levels of TLR-4. Concurrent administration of a PPAR-γ antagonist, GW9662, reversed the effects of Pio. The antihyperalgesic effect of administration of Pio in rats receiving CCI may, in part, be attributed to the inhibition of neuroimmune activation associated with the sustaining of neuropathic pain.

The electroacupuncture-induced analgesic effect mediated by 5-HT1, 5-HT3 receptor and muscarinic cholinergic receptors in rat model of collagenase-induced osteoarthritis.

BackgroundOsteoarthritis (OA) is an degenerative disease characterized by chronic joint pain. Complementary and alternative treatment such as acupuncture have been utilized to alleviate pain. The objective of this study was to investigate the analgesic mechanisms of electroacupuncture (EA) in the collagenase-induced osteoarthritis (CIOA) rat model.MethodsFour weeks after inducing CIOA by injecting collagenase solution into the left knee of 5-week-old male Sprague-Dawley rats, 2 Hz and 100 Hz EA on Zusanli (ST 36) was performed. The analgesic effect of EA was evaluated by the tail flick latency (TFL) and paw pressure threshold (PPT) tests. To investigate the analgesic mechanism, serotonergic and muscarinic cholinergic receptor agonists and antagonists were injected 20 min prior to EA and the resultant changes were evaluated by the TFL and PPT tests.ResultsEA on Zusanli (ST 36) demonstrated an analgesic effect in the CIOA rat model. The 2 Hz EA treatment showed a significantly greater analgesic effect than the 100 Hz treatment. The analgesic effect of 2 Hz EA was not strengthened by 5-HT1, 5-HT2, 5-HT3, and muscarinic cholinergic receptor agonist pretreatment, was blocked by 5-HT1, 5-HT3, and muscarinic cholinergic receptor antagonist pretreatment, but not blocked by 5-HT2 receptor antagonist pretreatment.ConclusionsIn the CIOA rat model, EA on Zusanli (ST 36) exhibited analgesic effects, and 2 Hz EA resulted in a significantly greater analgesic effect than 100 Hz EA. The analgesic effect of 2 Hz EA was reduced by pretreatment of 5-HT1 receptor, 5-HT3 receptor and muscarinic cholinergic receptor antagonists.

Topical Application of a Novel Oxycodone Gel Formulation (Tocopheryl Phosphate Mixture) in a Rat Model of Peripheral Inflammatory Pain Produces Localized Pain Relief Without Significant Systemic Exposure

This study was designed to assess the analgesic efficacy and systemic exposure of oxycodone administered topically in a novel tocopheryl phosphate mixture (TPM) gel formulation, to the inflamed hindpaws in a rat model of inflammatory pain. Unilateral hindpaw inflammation was induced in male Sprague-Dawley rats by intraplantar (i.pl.) injection of Freund's complete adjuvant (FCA). Mechanical hyperalgesia and hindpaw inflammation were assessed by measuring paw pressure thresholds and hindpaw volume, respectively, just prior to i.pl. FCA and again 5-6 days later. The analgesic effects of oxycodone administered topically (1 mg in TPM gel) or by i.pl. injection (50 μg), were assessed. Systemic oxycodone exposure was assessed over an 8-h postdosing interval following topical application. Skin permeation of oxycodone from the gel formulation was assessed in vitro using Franz diffusion cells. Oxycodone administered topically or by i.pl. injection produced significant (p < 0.05) analgesia in the inflamed hindpaws. Systemic oxycodone exposure was insignificant after topical dosing. The in vitro cumulative skin permeation of oxycodone was linearly related to the amount applied. Topical TPM/oxycodone gel formulations have the potential to alleviate moderate to severe inflammatory pain conditions with minimal systemic exposure, thereby avoiding central nervous system (CNS)-mediated adverse effects associated with oral administration of opioid analgesics.

Peripheral antinociceptive efficacy and potency of a novel opioid compound 14-O-MeM6SU in comparison to known peptide and non-peptide opioid agonists in a rat model of inflammatory pain

This study compared the peripheral analgesic effects of a novel opioid agonist 14-O-methylmorphine-6-O-sulfate (14-O-MeM6SU), to that of non-peptide (morphine, fentanyl) and peptide opioid agonists (Met-enkephalin; met-ENK and β-endorphin; β-END) in a model of localized inflammatory pain evoked by intraplantar (i.pl.) Freund's complete adjuvant (FCA). Nociceptive responses to local opioid agonists were measured by pressure paw-withdrawal procedures. In addition, the antinociceptive efficacy and potency of these test compounds in vivo was compared to that in vitro using the rat vas deferens (RVD) bioassay. Intraplantar 14-O-MeM6SU (0.32–2.53nmol/rat), morphine (14.95–112.15nmol/rat), fentanyl (0.19–2.36nmol/rat), met-ENK (0.10–10nmol/rat) and β-END (0.77–5.00nmol/rat) dose dependently increased paw pressure thresholds exclusively in inflamed hindpaws. At higher doses analgesic effects were also seen in noninflamed paws for 14-O-MeM6SU, morphine and fentanyl but not for met-ENK or β-END. The maximal possible local analgesic effect (%) measured in inflamed paws was 50.6±2.7, 18.23±1.78, 37.44±2.17, 36.00±1.43, and 40.69±0.91 for 14-O-MeM6SU, morphine, fentanyl, met-ENK and β-END, respectively. Interestingly, i.pl. administered opioid peptides met-ENK and β-END displayed a peripheral analgesic ceiling effect. This local antinociception was antagonized by co-administered opioid antagonist naloxone-methiodide (NAL-M). Similar to the analgesic testing, the RVD showed the following efficacy order of the test compounds: 14-O-MeM6SU>β-END>fentanyl>met-ENK⪢morphine. Taken together, 14-O-MeM6SU was more potent than morphine, fentanyl and met-ENK and β-END and displayed superiority in the maximum antinociceptive effects. The superiority of local antinociceptive effects of 14-O-MeM6SU might be due to both pharmacodynamic and pharmacokinetic factors.

Long‐term antinociception by electroacupuncture is mediated via peripheral opioid receptors in free‐moving rats with inflammatory hyperalgesia

Electroacupuncture (EA) has been widely accepted and applied as an important acupuncture-related technique for acupuncture analgesia (AA) research. The involvement of opioid peptides and receptors in acute AA has been shown via pre-EA application of opioid receptor/peptide antagonists. In this study, we intended to reproducibly institute acupoint position and needling excluding influences from anaesthesia or restrainers on rats with complete Freund's adjuvant (CFA) hind paw inflammatory pain, as well as to explore opioid-dependency and anti-inflammatory effects in sustained acupuncture analgesia. Accurate position and needling approach on acupoint GB30 was modelled by computer-based three-dimensional (3D) images and followed by an optimal EA treatment protocol (100 Hz, 2-3 mA, 20 min) at 0 and 24 h post-CFA in conscious free-moving rats. Opioid receptor antagonists, naloxone (NLX) and naltrindole (NTI) were applied intraplantarly post-EA at late phase (96 h) of CFA. Nociceptive thresholds were assessed by paw pressure threshold (Randall-Sellito) or paw withdrawal latency (Hargreaves), and anti-inflammatory effects were evaluated by measurement of plantar temperature and paw volume. EA elicited significant sustained mechanical and thermal antinociception up to 144 h. Mechanical antinociception of EA was suppressed by peripheral intraplantar application of NLX and NTI. EA also reduced paw temperature and volume during the same time frame indicating anti-inflammatory effects. By employing a reproducible EA treatment model on GB30 in free-moving rats, we demonstrated the involvement of peripheral opioid receptors mediated EA-induced long-term antinociception. Future studies should examine the specific neuroimmunological connection of EA-induced sustained antinociception in inflammation.

Spinal neuroimmune activation inhibited by repeated administration of pioglitazone in rats after L5 spinal nerve transection

Neuroimmune activation contributes to the generation and maintenance of neuropathic pain after peripheral nerve injury. Peroxisome proliferator activated receptor gamma (PPAR-γ) agonists have potential neuroprotection. The current study aimed to determine the effects of a PPAR-γ agonist pioglitazone on mechanical hyperalgesia and neuroimmune activation in a rat model of neuropathic pain induced by L5 spinal nerve transection (SNT). Thirty-two rats were equally randomized into 4 groups: sham operation with vehicle; L5 SNT with vehicle or pioglitazone; or L5 SNT with pioglitazone and a PPAR-γ antagonist GW9662. Pioglitazone or vehicle was administered 1h before operation and continued daily to day 14 after operation. The paw pressure threshold (PPT) was measured before operation and on days 3, 7, 14 after operation. Glial fibrillary acidic protein (GFAP) expression, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 levels, and nuclear factor-kappa B (NF-κB) activity in the lumbar spinal cord were determined on day 14 after operation. The results displayed pioglitazone improved the mechanical hyperalgesia, and attenuated the astrocyte and NF-κB activation and the inflammatory cytokine upregulation in nerve-injured rats, which might be reversed by GW9662. In conclusion, pioglitazone ameliorates the mechanical hyperalgesia induced by L5 SNT via inhibiting the spinal neuroimmune activation in rats, suggesting spinal PPAR-γ signaling pathway may be involved in the pathogenesis of mechanical hyperalgesia.

Relative contributions of peripheral versus supraspinal or spinal opioid receptors to the antinociception of systemic opioids

The contribution of supraspinal, spinal or peripheral mu-opioid receptors (MORs) to the overall antinociception of systemic centrally penetrating versus peripherally restricted opioids has not been thoroughly investigated. Therefore, we examined paw pressure thresholds in Wistar rats with complete Freund's adjuvant hindpaw inflammation following different doses of intraplantar (i.pl.) as well as intravenous (i.v.) fentanyl (6.25-50 μg/kg), morphine (1-7.5 mg/kg) or loperamide (1-7.5 mg/kg). Antagonism of the i.v. mu-opioid agonists by intracerebroventricular (i.c.v.), intrathecal (i.t.) or i.pl. naloxone-methiodide (NLXM) revealed the relative contributions of supraspinal, spinal and peripheral MOR to the overall antinociceptive effects. In parallel, the MOR density at these three levels of pain transmission was assessed by radioligand binding. Antinociceptive effects of i.v. fentanyl and morphine, but not of the peripherally restricted loperamide were two- to threefold greater and longer lasting compared with their i.pl. administration. I.c.v. but not i.pl. NLXM significantly antagonized fentanyl's and morphine's antinociception by 70-80%, whereas i.t. NLXM reduced it by 20-30%. In contrast, antinociception of i.v. loperamide was abolished by i.pl. but not by i.c.v. or i.t. NLXM. In parallel, a respective 32- and sixfold higher MOR density in supraspinal and spinal versus peripheral sensory neurons was detected. In conclusion, in comparison with supraspinal and spinal opioid receptors, peripheral opioid receptors do not significantly contribute to the antinociception of systemic fentanyl and morphine during inflammatory pain. Antinociception of their i.v. administration was superior over both i.v and i.pl. loperamide, acting exclusively via peripheral MOR. These findings may guide the future development of novel peripherally restricted opioids.

Effects of clonidine on bilateral pain behaviors and inflammatory response in rats under the state of neuropathic pain

This study was conducted to investigate the effects of clonidine on bilateral pain behaviors and inflammatory responses in neuropathic pain induced by partial sciatic nerve ligation (PSNL), and to better understand whether the antinociception of clonidine was related to α2-adrenoceptor mechanisms. Rats were divided randomly into five groups: sham-operation with saline, i.p.; PSNL with clonidine (0.2mg/kg) or saline, i.p.; PSNL with yohimbine (2mg/kg) followed by clonidine (0.2mg/kg), i.p.; and PSNL with naloxone (0.3mg/kg) followed by clonidine (0.2mg/kg), i.p. On post-operative days 1, 3, 7, 14, and 21, both ipsilateral and contralateral pain behaviors were measured. In rats receiving antagonists, bilateral behavioral changes were measured on day 14. Bilateral paw pressure threshold and paw withdrawal latencies were measured, and the extent of glial activation was dertermined by measuring macrophage antigen complex-1 (Mac-1) and glial fibrillary acidic protein (GFAP). Additionally, the levels of tumor necrosis factor α (TNF-α) and interleukin (IL)-6 were determined. PSNL induced bilateral behavioral hyperalgesia, with the ipsilateral level displaying a higher extent of behavior changes than the contralateral side. In addition, the glial activation markers and cytokine production were augmented bilaterally. Clonidine caused significant attenuation of bilateral mechanical allodynia and thermal hyperalgesia, accompanied by inhibition of glial activation and the expression of cytokines. The effects of clonidine were blocked by the α2-adrenoceptor antagonist yohimbine and partially reversed by the μ-opioid receptor antagonist naloxone. These data suggest that the bilateral antinoceptive effects of clonidine might mediate through immunomodulation by acting on α2-adrenoceptor in rats undergoing neuropathic pain.

P38 Mitogen–activated Protein Kinase Activation by Nerve Growth Factor in Primary Sensory Neurons Upregulates μ-Opioid Receptors to Enhance Opioid Responsiveness Toward Better Pain Control

Sensory neuron opioid receptors are targets for spinal, epidural, and peripheral opioid application. Although local nerve growth factor (NGF) has been identified as a mediator of sensory neuron μ-opioid receptor (MOR) up-regulation, the signaling pathways involved have not been yet identified. Wistar rats were treated with intraplantar vehicle, Freund's complete adjuvant, NGF, NGF plus intrathecal p38 mitogen-activated protein kinase (MAPK) inhibitors, or NGF plus extracellular signal-regulated kinase-1/2 MAPK inhibitors. After 4 days of treatment, paw pressure thresholds of an intraplantar full (fentanyl) or partial (buprenorphine) opioid agonist were determined by algesiometry. Tissue samples from rat dorsal root ganglia were subjected to radiolabeled ligand binding, Western blot analysis, and confocal immunofluorescence. Exogenous and endogenous NGF resulting from Freund's complete adjuvant inflammation produced significant potentiation and enhanced efficacy in fentanyl- and buprenorphine-induced dose-dependent antinociception, respectively. Furthermore, in the ipsilateral dorsal root ganglia, NGF produced a significant increase in MOR binding sites, proteins, and immunoreactive neurons. In parallel, phosphorylated p38-MAPK protein, the number of phosphorylated p38-MAPK immunoreactive neurons expressing MOR in dorsal root ganglia, and the peripherally directed axonal transport of MOR significantly increased. Finally, NGF-induced effects occurring in dorsal root ganglia, on axonal transport, and on the potentiation or enhanced efficacy of opioid antinociception were abrogated by inhibition of p38, but not extracellular signal-regulated kinase-1/2, MAPK. Local NGF through activation of the p38-MAPK pathway leads to adaptive changes in sensory neuron MOR toward enhanced susceptibility to local opioids. This effect may act as a counter-regulatory response to p38-MAPK-induced pain (e.g., inflammatory pain) to facilitate opioid-mediated antinociception.

Involvement of peripheral opioid receptors in electroacupuncture analgesia for carrageenan-induced hyperalgesia

Acupuncture is widely used to relieve pain; however, the mechanism underlying electroacupuncture analgesia (EAA) during inflammatory pain is unclear. We investigated whether endogenous peripheral opioid receptors participated in EAA during hyperalgesia elicited by carrageenan-induced inflammation. Moreover, we investigated which subtype of opioid receptor was involved in EAA. Carrageenan was subcutaneously administered by intraplanter (i.pl.) injection into the left hind paw. Nociceptive thresholds were measured using the paw pressure threshold (PPT). Rats received 3 Hz electroacupuncture (EA) for 1 h after carrageenan injection. The nonselective peripheral opioid receptor antagonist, naloxone methiodide, was administered by i.pl. injection of the inflamed paw 5 min before EA. Also, animals received i.pl. or intravenous (i.v.) injection of selective antagonists against μ(D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-ThrNH2, CTOP), δ(naltrindole, NTI), or κ (nor-Binaltorphimine, nor-BNI) opioid receptors 1 h before EA. PPT decreased significantly 3 h after carrageenan injection. EA resulted in significant increases of PPT, moreover, PPT elevations persisted for 9 h after carrageenan injection. PPT elevations produced by EA were antagonized by local i.pl. injection of naloxone methiodide at 3 and 5 h after cessation of EA. NTI, nor-BNI and CTOP blocked EAA from immediately, 1 h, and 3 h after EA cessation, respectively. The EAA in the inflamed paw could not be blocked by i.v. injection of NTI, nor-BNI and CTOP. These findings suggest that peripheral μ, δ and κ receptors on peripheral nerve terminals are activated by EA, although there is a time difference among these activations.