Paw Pressure Research Articles

Structural determinants of the hyperalgesic activity of myotoxic Lys49-phospholipase\xa0A2

BackgroundBothropstoxin-I (BthTx-I) is a Lys49-phospholipase A2 (Lys49-PLA2) from the venom of Bothrops jararacussu, which despite of the lack of catalytic activity induces myotoxicity, inflammation and pain. The C-terminal region of the Lys49-PLA2s is important for these effects; however, the amino acid residues that determine hyperalgesia and edema are unknown. The aim of this study was to characterize the structural determinants for the Lys49-PLA2-induced nociception and inflammation.MethodsScanning alanine mutagenesis in the active-site and C-terminal regions of BthTx-I has been used to study the structural determinants of toxin activities. The R118A mutant was employed as this substitution decreases PLA2 myotoxicity. In addition, K115A and K116A mutants – which contribute to decrease cytotoxicity – and the K122A mutant – which decreases both myotoxicity and cytotoxicity – were also used. The H48Q mutant – which does not interfere with membrane damage or myotoxic activity – was used to evaluate if the PLA2 catalytic site is relevant for the non-catalytic PLA2-induced pain and inflammation. Wistar male rats received intraplantar injections with mutant PLA2. Subsequently, hyperalgesia and edema were evaluated by the paw pressure test and by a plethysmometer. Native and recombinant BthTx-I were used as controls.ResultsNative and recombinant BthTx-I induced hyperalgesia and edema, which peaked at 2 h. The R118A mutant did not induce nociception or edema. The mutations K115A and K116A abolished hyperalgesia without interfering with edema. Finally, the K122A mutant did not induce hyperalgesia and presented a decreased inflammatory response.ConclusionsThe results obtained with the BthTx-I mutants suggest, for the first time, that there are distinct residues responsible for the hyperalgesia and edema induced by BthTx-I. In addition, we also showed that cytolytic activity is essential for the hyperalgesic effect but not for edematogenic activity, corroborating previous data showing that edema and hyperalgesia can occur in a non-dependent manner. Understanding the structure-activity relationship in BthTx-I has opened new possibilities to discover the target for PLA2-induced pain.

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 synthetic peptide PnPP-19 induces peripheral antinociception via activation of NO/cGMP/KATP pathway: Role of eNOS and nNOS

Backgroundand purpose: The peptide PnPP-19, derived from the spider toxin PnTx2-6 (renamed as δ-CNTX-Pn1c), potentiates erectile function by activating the nitrergic system. Since NO has been studied as an antinociceptive molecule and PnPP-19 is known to induce peripheral antinociception, we intended to evaluate whether PnPP-19 could induce peripheral antinociception through activation of this pathway. Experimental approachNociceptive thresholds were measured by paw pressure test. PGE2 (2 μg/paw) was administered intraplantarly together with PnPP-19 and inhibitors/blockers of NOS, guanylyl cyclase and KATP channels. The nitrite concentration was accessed by Griess test. The expression and phosphorylation of eNOS and nNOS were determined by western blot. Key resultsPnPP-19 (5, 10 and 20 μg/paw) induced peripheral antinociception in rats. Administration of NOS inhibitor (L-NOarg), selective nNOS inhibitor (L-NPA), guanylyl cyclase inhibitor (ODQ) and the blocker of KATP (glibenclamide) partially inhibited the antinociceptive effect of PnPP-19 (10 μg/paw). Tissue nitrite concentration increased after PnPP-19 (10 μg/paw) administration. Expression of eNOS and nNOS remained the same in all tested groups, however the phosphorylation of nNOS Ser852 (inactivation site) increased and phosphorylation of eNOS Ser1177 (activation site) decreased after PGE2 injection. Administration of PnPP-19 reverted this PGE2-induced effect. Conclusions and implicationsThe peripheral antinociceptive effect induced by PnPP-19 is resulting from activation of NO-cGMP-KATP pathway. Activation of eNOS and nNOS might be required for such effect. Our results suggest PnPP-19 as a new drug candidate to treat pain and reinforce the importance of nNOS and eNOS activation, as well as endogenous NO release, for induction of peripheral antinociception.

Peripheral Antinociception Induced by Aripiprazole Is Mediated by the Opioid System.

Background Aripiprazole is an antipsychotic drug used to treat schizophrenia and related disorders. Our previous study showed that this compound also induces antinociceptive effects. The present study aimed to assess the participation of the opioid system in this effect. Methods Male Swiss mice were submitted to paw pressure test and hyperalgesia was induced by intraplantar injection of prostaglandin E2 (PGE2, 2 μg). Aripiprazole was injected 10 min before the measurement. Naloxone, clocinnamox, naltrindole, nor-binaltorphimine, and bestatin were given 30 min before aripiprazole. Nociceptive thresholds were measured in the 3rd hour after PGE2 injection. Results Aripiprazole (100 μg/paw) injected locally into the right hind paw induced an antinociceptive effect that was blocked by naloxone (50 μg/paw), a nonselective opioid receptor antagonist. The role of μ-, δ-, and κ-opioid receptors was investigated using the selective antagonists, clocinnamox (40 μg/paw), naltrindole (15, 30, and 60 μg/paw), and nor-binaltorphimine (200 μg/paw), respectively. The data indicated that only the δ-opioid receptor antagonist inhibited the peripheral antinociception induced by aripiprazole. Bestatin (400 μg), an aminopeptidase-N inhibitor, significantly enhanced low-dose (25 μg/paw) aripiprazole-induced peripheral antinociception. Conclusion The results suggest the participation of the opioid system via δ-opioid receptor in the peripheral antinociceptive effect induced by aripiprazole.

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.

Antinociceptive effect of co-administered NMDA and histamine H4 receptor antagonists in a rat model of acute pain.

The histamine H4 receptor (H4R) has attracted a lot of attention in terms of its role in antinociception. The N-Methyl-d-aspartic acid (NMDA) receptor is a well-characterized participant in pain pathways. However, it's influence on H4R signaling is poorly understood. Thus, in the present study we investigated the effect of a selective H4R antagonist JNJ7777120 (25mg/kg) and a NMDA receptor antagonist MK-801 (0.1-10μg) on nociceptive thresholds in a rat acute pain model. The influence of intrathecally (it), intracerebroventricularly (icv) and intraplantarly (ipl) administered MK-801 co-injected with JNJ7777120 administered intraperitoneally (ip) was determined in the modified Randall-Selitto paw pressure, the tail flick and plantar tests. Both ip JNJ7777120 as well as MK-801 delivered it and ipl increased nociceptive pain thresholds. It and ipl pretreatment with MK-801 additively augmented JNJ7777120 mechanical antinociception. A weaker effect was also observed after it co-administration with MK-801 and JNJ7777120 in the tail flick test. Intracerebroventricular MK-801 failed to produce any effect nor did ipl MK-801 in the plantar test. The results show for the first time that the H4R and NMDA receptors play a significant role in antinociception in an acute non-inflammatory pain. Furthermore, the blockage of NMDA receptors at the peripheral and spinal, but not supraspinal sites produces a profound antinociceptive response upon simultaneous H4R antagonism. Thus, this study clearly demonstrates the relevance of NMDA and H4R receptors in the modulation of pain signals and that their role is not exclusively limited to their modulatory activity in inflammatory pain states.

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.

Peltatoside Isolated from Annona crassiflora Induces Peripheral Antinociception by Activation of the Cannabinoid System.

Peltatoside is a natural compound isolated from leaves of Annona crassiflora Mart., a plant widely used in folk medicine. This substance is an analogue of quercetin, a flavonoid extensively studied because of its diverse biological activities, including analgesic effects. Besides, a previous study suggested, by computer structure analyses, a possible quercetin-CB1 cannabinoid receptor interaction. Thus, the aim of this work was to assess the antinociceptive effect of peltatoside and analyze the cannabinoid system involvement in this action. The mouse paw pressure test was used and hyperalgesia was induced by intraplantar injection of carrageenan (200 µg/paw). All used drugs were administered by intraplantar administration in Swiss male mice (n = 6). Peltatoside (100 µg/paw) elicited a local inhibition of hyperalgesia. The peripheral antinociceptive action of peltatoside was antagonized by the CB1 cannabinoid antagonist AM251 (160 µg/paw), but not by CB2 cannabinoid antagonist AM630 (100 µg/paw). In order to assess the role of endocannabinoids in this peripheral antinociceptive effect, we used (i) [5Z,8Z,11Z,14Z]-5,8,11,14-eicosatetraenyl-methyl ester phosphonofluoridic acid, an inhibitor of anandamide amidase; (ii) JZL184, an inhibitor for monoacylglycerol lipase, the primary enzyme responsible for degrading the endocannabinoid 2-arachidonoylglycerol; and (iii) VDM11, an endocannabinoid reuptake inhibitor. MAFP, JZL184, and VDM11 did not induce antinociception, respectively, at the doses 0.5, 3.8, and 2.5 µg/paw, however, these three drugs were able to potentiate the peripheral antinociceptive effect of peltatoside at an intermediary dose (50 µg/paw). Our results suggest that this natural substance is capable of inducing analgesia through the activation of peripheral CB1 receptors, involving endocannabinoids in this process.

Neuroprotective Effect of Matrine in Mouse Model of Vincristine-Induced Neuropathic Pain.

Chemotherapy drugs such as vincristine (VCR) can cause neuropathic pain, and there is still lack of ideal strategy to treat it. The current study was designed to investigate effect of matrine (MT) on VCR-induced neuropathic pain in animal model. VCR (75μg/kg, i.p. for 10 consecutive days) was administered to induce painful neuropathy model in mice. MT (15, 30 and 60mg/kg, i.p.) and pregabalin (10mg/kg, i.p.) were administered for 11 consecutive days. Various tests were performed to assess the degree of pain at different days (1, 6, 11, 16, and 21). Von Frey hair, hot plate, cold-plate and paw pressure tests were conducted to assess the degree of mechanical allodynia, thermal hyperalgesia, cold allodynia and mechanical hyperalgesia in the hind paw respectively. The electrophysiological and histopathological changes were also analyzed. Furthermore, tissue malondialdehyde (MDA), total antioxidant capacity (T-AOC),superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total calcium (TCA), myeloperoxidase (MPO), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10) were measured to investigate possible involvement of MT in inflammation and oxidative stress. Administration of MT attenuated the VCR-induced behavioral alterations as well as electrophysiological and histopathological changes in a dose dependent manner. Further, MT also attenuated the VCR-induced oxidative stress (MDA, T-AOC, GSH-Px, SOD and TCA) and inflammation (MPO, TNF-α, IL-6 and IL-10). Taken together, MT ameliorated VCR-induced painful neuropathy, which might be attributed to neuroprotective effects by subsequent reduction in oxidative stress and anti-inflammatory actions.

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.

Involvement of high mobility group box 1 in the development and maintenance of chemotherapy-induced peripheral neuropathy in rats.

Given that high mobility group box 1 (HMGB1), a nuclear protein, once released to the extracellular space, promotes nociception, we asked if inactivation of HMGB1 prevents or reverses chemotherapy-induced painful neuropathy in rats and also examined possible involvement of Toll-like receptor 4 (TLR4) and the receptor for advanced glycation endproduct (RAGE), known as targets for HMGB1. Painful neuropathy was produced by repeated i.p. administration of paclitaxel or vincristine in rats. Nociceptive threshold was determined by the paw pressure method and/or von Frey test in the hindpaw. Tissue protein levels were determined by immunoblotting. Repeated i.p. administration of the anti-HMGB1-neutralizing antibody or recombinant human soluble thrombomodulin (rhsTM), known to inactivate HMGB1, prevented the development of hyperalgesia and/or allodynia induced by paclitaxel or vincristine in rats. A single i.p. or intraplantar (i.pl.) administration of the antibody or rhsTM reversed the chemotherapy-induced neuropathy. A single i.pl. administration of a TLR4 antagonist or low molecular weight heparin, known to inhibit RAGE, attenuated the hyperalgesia caused by i.pl. HMGB1 and also the chemotherapy-induced painful neuropathy. Paclitaxel or vincristine treatment significantly decreased protein levels of HMGB1 in the dorsal root ganglia, but not sciatic nerves. HMGB1 thus participates in both development and maintenance of chemotherapy-induced painful neuropathy, in part through RAGE and TLR4. HMGB1 inactivation is considered useful to prevent and treat the chemotherapy-induced painful neuropathy.

Compensatory load redistribution in Labrador retrievers when carrying different weights--a non-randomized prospective trial.

BackgroundRetrievers are dogs particularly bred to retrieve birds or other small game, for the retrieval, the dogs are typically sent to the place where the shot game has fallen or to search the field for the wounded but still live game in order to return them to the hunter as quickly as possible. Examples of game animals are pheasants, mallard ducks and rabbits. For training, dummies with a variety of weights are used to simulate the retrieval of various types of game. The aim of this non-randomized prospective study was to investigate if peak vertical force, vertical impulse and paw pressure contact area are increased in the forelimbs when carrying different weights, and if the symmetrical weight distribution between contralateral limb pairs is disturbed. Ten actively working Labrador retrievers were walked over a pressure plate with or without carrying 0.5, 2.0 and 4.0 kg dummies. The aim of this study was to determine if vertical ground reaction forces and paw pressure contact area are increased in the forelimbs when carrying different weights, and if symmetrical weight distribution is disturbed between contralateral limb pairs.ResultsPeak vertical force and vertical impulse were significantly increased in the forelimbs and decreased in the hindlimbs in all weight carrying conditions.ConclusionsThese results demonstrate the significant effects of carrying weight in the mouth on the ground reaction forces, which likely produce additional stress on the forelimb joints. Carry of game or a dummy is likely to alter the forelimb load distribution.

Liposomal Formulation to Increase Stability and Prolong Antineuropathic Activity of Verbascoside.

Verbascoside (acteoside) possesses various pharmacological properties for human health, including antioxidant, anti-inflammatory, and antineoplastic properties in addition to numerous wound healing and neuroprotective properties, with an excellent and well-known safety profile. However, its poor chemical stability, due to hydrolysis, limits its use in the clinic. To overcome these limitations, we prepared unilamellar liposomal formulations of verbascoside for parenteral administration.Two formulations were prepared: V-L1 and V-L2, where V-L2 contains phospholipid and cholesterol about 4 times higher than the V-L1 sample, and about 2 times higher than verbascoside. The mean particle size of the liposomes prepared was found to be around 120 nm with a polydispersity index < 0.2. Encapsulation efficacy resulted in 30 %. A total of 82.28 ± 1.79 % of verbascoside was released from the liposomes within 24 hours. Liposomes ameliorate the stability of verbascoside by preventing its hydrolysis.The optimized drug delivery formulation was tested in the paw pressure test in two animal models of neuropathic pain: a peripheral mononeuropathy was produced either by a chronic constriction injury of the sciatic nerve or by an intra-articular injection of sodium monoiodoacetate. The performance of the liposomal formulation was compared with that of the free drug.For evaluating the paw pressure test in chronic constriction injury rats, a liposomal formulation administered i. p. at the dosage of 100 mg/kg showed a longer lasting antihyperalgesic effect in comparison with a 100-mg/kg verbascoside saline solution, as well as in the sodium monoiodoacetate models. The effect appeared 15 min after administration and persisted for up to 60 min.

κ-Opioid receptor participates of NSAIDs peripheral antinociception

NSAIDs represent some of the most widely prescribed drugs for relief of short-term fever, pain and inflammation. The participation of the opioid system in the peripheral is poorly understood. The aim of this study was evaluate the role of opioid system in the peripheral antinociception by diclofenac and dipyrone. To test this hypothesis, opioid receptor antagonists were evaluated using the rat paw pressure test, in which pain sensitivity is increased by intraplantar injection of prostaglandin E2 (PGE2, 2μg). Diclofenac (20μg/paw) and Dipyrone (40μg/paw) administered locally into the right paw elicited an antinociceptive effect. It was used naloxone (50μg/paw), a non-selective opioid receptor antagonist, which antagonized peripheral antinociception induced by diclofenac and dipyrone. Selectively, it was evaluated the μ-, δ- and κ-opioid receptor antagonists, respectively, clocinnamox (40μg/paw), naltrindole (50μg/paw) and nor-binaltorphimine (20, 40 and 80μg/paw). Our data indicated that only the κ-opioid antagonist was capable to reverse the peripheral antinociception by NSAIDs. The present results provide evidence that the opioid system participated in the diclofenac and dipyrone-induced peripheral antinociception by indirect activation of κ-opioid receptor probable by release of endogenous opioids such as dynorphins.

Top-Down Effect of Direct Current Stimulation on the Nociceptive Response of Rats

Transcranial direct current stimulation (tDCS) is an emerging, noninvasive technique of neurostimulation for treating pain. However, the mechanisms and pathways involved in its analgesic effects are poorly understood. Therefore, we investigated the effects of direct current stimulation (DCS) on thermal and mechanical nociceptive thresholds and on the activation of the midbrain periaqueductal gray (PAG) and the dorsal horn of the spinal cord (DHSC) in rats; these central nervous system areas are associated with pain processing. Male Wistar rats underwent cathodal DCS of the motor cortex and, while still under stimulation, were evaluated using tail-flick and paw pressure nociceptive tests. Sham stimulation and naive rats were used as controls. We used a randomized design; the assays were not blinded to the experimenter. Immunoreactivity of the early growth response gene 1 (Egr-1), which is a marker of neuronal activation, was evaluated in the PAG and DHSC, and enkephalin immunoreactivity was evaluated in the DHSC. DCS did not change the thermal nociceptive threshold; however, it increased the mechanical nociceptive threshold of both hind paws compared with that of controls, characterizing a topographical effect. DCS decreased the Egr-1 labeling in the PAG and DHSC as well as the immunoreactivity of spinal enkephalin. Altogether, the data suggest that DCS disinhibits the midbrain descending analgesic pathway, consequently inhibiting spinal nociceptive neurons and causing an increase in the nociceptive threshold. This study reinforces the idea that the motor cortex participates in the neurocircuitry that is involved in analgesia and further clarifies the mechanisms of action of tDCS in pain treatment.

HISTOLOGICAL AND FUNCTIONAL STUDY OF LASER AND LATEX TREATMENTS ON CRUSHED SCIATIC NERVE

This study evaluated the effects of low‐level laser (LLL 15J/cm2, 780nm) associated or not to natural latex (F1 protein − 0,1%) on the crushed sciatic nerve (15 kgf, axonotmesis, left nerve). We used 36 male Wistar rats (200g) divided into 6 groups (n = 6): C ‐ control; E ‐ exposed nerve; I ‐injured nerve; L – LLL; F1 ‐ F1 protein; LF1 ‐ LLL and F1. The functional analysis of mechanical allodynia (von Frey), paw pressure (forceps test) and grip strength (Grip strength meter) was performed after 1, 7, 14, 28, 56 and 180 days of nerve injury. The histological analyses were assessed after 15, 57 and 181 days.Mechanical allodyniaThe non‐injured paw (right) did not differ in all groups studied. The left paw (injured) revealed a higher initial value in Groups I, LLL, LLL + P1 and P1 compared to groups C and E these values decreased in subsequent periods. After seven days the F1 values were very close to the C and E while this effect was observed in the other groups after 14 days. Group I had the highest value for this test after 180 days.Paw pressureThe right paw (non‐injured) showed similar values during the 180 days of the experiment. The left paw (injured) showed higher initial values in groups I, LLL, F1, LLL+ F1 compared to groups C and E. Baseline values reduction was observed after 7 days in the LLL group and after 14 days in the group LLL + F1. After 180 days all groups had similar values, with F1 group presenting the lowest value.Grip StrengthDuring 180 days there was an increase in the values in all groups. The initial values of groups C and E were higher compared to the other groups up to 56 days. At the end of the experiment (180 days) P1 and LLL + F1 groups showed the highest values for the test.Histological analysisGroups C and E showed normal characteristics. Injured groups (I, LLL, LLL + P1 and P1) reveal reduced number and size of nerve fibers, mainly after 15 and 56 days. In the course of time the injured nerve morphology improved with the P1 and LLL+P1 groups showing the best results among the treated groups. In the 180‐day period, P1 and LLL + P1 groups showed similar morphology to groups C and E. The functional and histological analyzes showed improvement after damage in all injured groups. The functional tests showed that the LLL was more effective in reducing pain (paw pressure) after 7 days, probably due to the anti‐inflammatory property of the laser. LLL + P1 treatment reduced pain after 14 days and improved of grip force at long‐term (180 days). F1 showed the best results for mechanical allodynia after 7 days, paw pressure after 180 days and grip force after 180 days. Histology showed promising results from the use of F1 with or without association with the LLL in accelerating nerve regeneration.Support or Funding InformationNerve injury, Low‐level laser, Latex protein, functional tests, morphologic analyses

Changes in Brainstem Protein Levels During the Development and Decay of Tolerance to Morphine: Focus on PKCγ

Our hypothesis is that heterologous tolerance following chronic treatment with morphine is an adaptive process mediated by alterations in one or more cellular signaling proteins. This lab has previously characterized a time course for the onset and decay of tolerance following subcutaneous (sc) injection by assessing tolerance using paw pressure testing and the response of the longitudinal muscle/myenteric plexus (LM/MP) to DAMGO and 2‐CADO after various schedules of treatment. Western blot protein analysis in LM/MP homogenates from those animals revealed no change in the number of μ, A1, or A2a receptors, indicating that the tolerance observed in that tissue cannot be explained by a decrease in receptor abundance. Because the brainstem is involved in the analgesic effect produced by morphine, we examined the abundance of several proteins in tissue homogenates obtained from the same animals used for the previous time course study. Western blot analysis of brainstem homogenates also revealed no change in the abundance of μ, A1, and A2a receptors at all time points examined. In addition, the α2b receptor was also largely unchanged. Previous studies have shown that the α3 subunit of the Na+/K+ ATPase decreased as tolerance develops and returned to baseline as tolerance decays in the LM/MP. We therefore determined the abundance of the α1, α3, and β1 subunits of the Na+/K+ ATPase. Unlike results obtained from the LM/MP, we observed no change in any of these subunits in the brainstem. We also examined PKCγ and PKCɛ and observed that PKCγ decreases in concentration as tolerance develops and that this change reverses as tolerance decays. PKCγ is not present in the LM/MP and PKCɛ does not change significantly there. These data indicate that PKCγ may be an important mediator for the development of tolerance in the brainstem and that the cellular mechanism by which tolerance develops may be different between the LM/MP and brainstem.

X‐irradiation and Simulated Microgravity affects Rat Weight, Food Intake, Insulin and Paw Pressure Pain Threshold

The aim of this study is to examine various physiological measurements in response to range of space‐like radiation doses in a NASA‐validated rodent model of simulated microgravity.MethodsFour groups of rats (6 rats per group), control (CON), irradiated (IR), irradiated plus hindlimb suspended (IR+HLS) and suspended (HLS), were used in the study and maintained for two consecutive weeks. IR and IR+HLS groups were exposed to five sessions of X‐ray irradiation (0.5 Gy each, at 3–4 days intervals; cumulative dose 1.2 Gy). Body weights, food and water intakes, pressure pain thresholds and random blood plasma insulin levels were measured.ResultsCompared to CON, weight gain was slowed by about 25% in IR animals and by 50% in HLS and IR+HLS groups. IR acutely decreased food consumption by 20%‐50% when compared to pre‐exposure levels. Effect of HLS on food consumption (15% decrease) was evident during first day of post‐suspension. HLS but not IR resulted in deep tissue hyperalgesia (45% decreases in PPT) and a 40%–60% decrease in plasma insulin levels.Summary and ConclusionsThese observations suggest that exposure to IR may contribute to space flight‐associated anorexia and weight loss. However exposure to microgravity and lower limb disuse appear to be associated with complications. These include insulinopenia, deep tissue hyperalgesia and the muscle atrophy in addition to weight loss and, from our previous studies, bone degenerative effects.Support or Funding InformationSupported by a grant from Arkansas Space Grant Consortium

Role of monocyte chemoattractant protein-1, stromal derived factor-1 and retinoic acid in pathophysiology of neuropathic pain in rats.

Chemokines have been recently recognized to play a role in chronic pain syndromes' pathophysiology. This study investigated the role of monocyte chemoattractant protein-1 (MCP-1), stromal cell derived factor-1 (SDF-1), and retinoic acid (RA) as targets for the therapeutic approach of neuropathic pain. A chronic constriction injury (CCI) model of neuropathic pain by unilateral ligation of left sciatic nerve was performed in adult female Wistar rats. The effects of doxycycline (Dox, 50 mg/kg/day i.p. for 7 days), single dose of bicyclam (5 mg/kg i.p.), RA (15 mg/kg/day i.p. for 7 days), and their combination(s) on behavioral tests of nociception (Von Frey filaments; paw pressure test) on days 0, 1, 3, 5, and 7 of operation were studied. Serum concentrations of MCP-1 and SDF-1 were measured by ELISA. Histological examination of the sciatic nerve was investigated. CCI of sciatic nerve significantly induced mechanical allodynia and hyperalgesia and an increase of MCP-1 and SDF-1 serum levels. Dox-treated groups (Dox, Dox+bicyclam, Dox+RA, Dox+bicyclam+RA) and bicyclam-treated groups (bicyclam, Dox+bicyclam, bicyclam+RA, Dox+bicyclam+RA) attenuated CCI-induced behavioral and biochemical changes. RA inhibited CCI-induced mechanical hyperalgesia but produced a time-dependent reversal of allodynia. Histological findings showed degenerative changes of sciatic nerve after CCI that were partially recovered in Dox-treated groups. These findings demonstrate an association between serum MCP-1 and SDF-1 concentrations and behavioral manifestations of neuropathic pain. RA administration decreased neuropathic pain (antihyperalgesic effect) but did not cause any improvement in sciatic nerve tissues, either alone or in combination with chemokine antagonists. Thus, chemokines may serve as potential targets for drug development in neuropathic pain treatment.

PnPP-19, a spider toxin peptide, induces peripheral antinociception through opioid and cannabinoid receptors and inhibition of neutral endopeptidase.

The synthetic peptide PnPP-19 has been studied as a new drug candidate to treat erectile dysfunction. However, PnTx2-6, the spider toxin from which the peptide was designed, induces hyperalgesia. Therefore, we intended to investigate the role of PnPP-19 in the nociceptive pathway. Nociceptive thresholds were measured by paw pressure test. PnPP-19 was administered intraplantarly alone or with selective cannabinoid or opioid receptor antagonists. The hydrolysis of PnPP-19 by neutral endopeptidase (NEP) (EC 3.4.24.11), an enzyme that cleaves enkephalin, was monitored by HPLC and the cleavage sites were deduced by LC-MS. Inhibition by PnPP-19 and Leu-enkephalin of NEP enzyme activity was determined spectrofluorimetrically. PnPP-19 (5, 10 and 20 μg per paw) induced peripheral antinociception in rats. Specific antagonists of μ opioid receptors (clocinnamox), δ opioid receptors (naltrindole) and CB1 receptors (AM251) partly inhibited the antinociceptive effect of PnPP-19. Inhibition of fatty acid amide hydrolase by MAFP or of anandamide uptake by VDM11 enhanced PnPP-19-induced antinociception. NEP cleaved PnPP-19 only after a long incubation, and Ki values of 35.6 ± 1.4 and 14.6 ± 0.44 μmol·L(-1) were determined for PnPP-19 and Leu-enkephalin respectively as inhibitors of NEP activity. Antinociception induced by PnPP-19 appears to involve the inhibition of NEP and activation of CB1, μ and δ opioid receptors. Our data provide a greater understanding of the antinociceptive effects of PnPP-19. This peptide could be useful as a new antinociceptive drug candidate.