Electronic Von Frey Anesthesiometer Research Articles

Nociceptive and histomorphometric evaluation of the effects of ozone therapy on the rat masseter muscle in a carrageenan model of myofascial pain

ObjectiveThis study evaluated the effects of intramuscular ozone therapy on nociception, inflammation, and tissue damage caused by the injection of carrageenan in the masseter muscle of rats. DesignRat masseter muscles were injected with saline or carrageenan. Seventy-seven adult male rats were divided into six groups: Sal, saline; Car, carrageenan; Ibup + Sal, ibuprofen and saline; Ibup + Car, ibuprofen and carrageenan; O3 + Sal, ozone and saline; and O3 + Car, ozone and carrageenan. The mixture of 5% ozone and 95% oxygen (20 µg/mL) was administered three times in the course of a week. Nociceptive responses in the masseter muscles were measured using a head withdrawal threshold, determined by an electronic von Frey anesthesiometer. The animals were euthanized one or eight days after the carrageenan injection, and the masseters were submitted to histological and histomorphometric analyses. ResultsMechanical allodynia and inflammation levels were reduced in the Ibup + Car group compared to the other groups. Myonecrosis was similar among carrageenan-treated groups. Picrosirius red stained sections showed more collagen fibers and more regenerating myofibers in the O3 + Car group compared to the other groups. Eight days after carrageenan injection, the O3 + Car group showed neutrophils close to the regenerating myofibers. ConclusionsIntramuscular ozone therapy did not alleviate mechanical allodynia, and it did not protect the masseter muscle against the deleterious effects produced by carrageenan, probably due to the mode of administration of this therapeutic agent.

β-Caryophyllene Inhibits Monoacylglycerol Lipase Activity and Increases 2-Arachidonoyl Glycerol Levels In Vivo: A New Mechanism of Endocannabinoid-Mediated Analgesia?

The mechanisms of β-caryophyllene (BCP)-induced analgesia are not well studied. Here, we tested the efficacy of BCP in an acute postsurgical pain model and evaluated its effect on the endocannabinoid system. Rats were treated with vehicle and 10, 25, 50, and 75 mg/kg BCP. Paw withdrawal responses to mechanical stimuli were evaluated using an electronic von Frey anesthesiometer. Endocannabinoids, including 2-arachidonoylglycerol (2-AG), were also evaluated in plasma and tissues using high-performance liquid chromatography-tandem mass spectrometry. Monoacylglycerol lipase (MAGL) activity was evaluated in vitro as well as ex vivo. We observed a dose-dependent and time-dependent alleviation of hyperalgesia in incised paws up to 85% of the baseline value at 30 minutes after administration of BCP. We also observed dose-dependent increases in the 2-AG levels of about threefold after administration of BCP as compared with vehicle controls. Incubations of spinal cord tissue homogenates from BCP-treated rats with isotope-labeled 2-arachidonoylglycerol-d8 revealed a reduced formation of the isotope-labeled MAGL product 2-AG-d8 as compared with vehicle controls, indicating MAGL enzyme inhibition. In vitro MAGL enzyme activity assessment using 2-AG as the substrate revealed an IC50 of 15.8 µM for MAGL inhibition using BCP. These data showed that BCP inhibits MAGL activity in vitro and in vivo, causing 2-AG levels to rise. Since the endocannabinoid 2-AG is a CB1 and CB2 receptor agonist, we propose that 2-AG-mediated cannabinoid receptor activation contributes to BCP's mechanism of analgesia. SIGNIFICANCE STATEMENT: β-Caryophyllene (BCP) consumption is relatively safe and is approved by the Food and Drug Administration as a flavoring agent, which can be used in cosmetic and food additives. BCP is a potent anti-inflammatory agent that showed substantial antihyperalgesic properties in this study of acute pain suggesting that BCP might be an alternative to opioids. This study shows an additive mechanism (monoacylglycerol lipase inhibition) by which BCP might indirectly alter CB1 and CB2 receptor activity and exhibit its pharmacological properties.

Optogenetic manipulations of CeA-CRF neurons modulate pain- and anxiety-like behaviors in neuropathic pain and control rats

The amygdala plays a critical role in the emotional-affective component of pain and pain modulation. The central nucleus of amygdala (CeA) serves major output functions and has been linked to pain-related behaviors. Corticotropin releasing factor (CRF) in the CeA has emerged as an important modulator of pain and affective disorders. Here we measured the effects of optogenetic manipulation of CeA-CRF neurons on pain-related behaviors in a rat neuropathic pain model and under control conditions. Emotional-affective behaviors (vocalizations), mechanosensitivity (electronic von Frey anesthesiometer and calibrated forceps), and anxiety-like behaviors (open field test and elevated plus maze) were assessed in adult rats 1 week and 4 weeks after spinal nerve ligation (SNL model) and sham surgery (control). For optogenetic silencing or activation of CRF neurons, a Cre-inducible viral vector encoding enhanced halorhodopsin (eNpHR3.0) or channelrhodopsin 2 (ChR2) was injected stereotaxically into the right CeA of transgenic Crh-Cre rats. Light of the appropriate wavelength (590 nm for eNpHR3.0; 473 nm for ChR2) was delivered into the CeA with an LED optic fiber. Optical silencing of CeA-CRF neurons decreased the emotional-affective responses in the acute and chronic phases of the neuropathic pain model but had anxiolytic effects only at the chronic stage and no effect on mechanosensitivity. Optogenetic activation of CeA-CRF neurons increased the emotional-affective responses and induced anxiety-like behaviors but had no effect on mechanosensitivity in control rats. The data show the critical contribution of CeA-CRF neurons to pain-related behaviors under normal conditions and beneficial effects of inhibiting CeA-CRF neurons in neuropathic pain.This article is part of the Special Issue on ‘Advances in mechanisms and therapeutic targets relevant to pain’.

Wound Infusion of 0.35% Levobupivacaine Reduces Mechanical Secondary Hyperalgesia and Opioid Consumption After Cesarean Delivery: A Prospective, Randomized, Triple-Blind, Placebo-Controlled Trial.

Some patients still report moderate-to-severe postoperative pain after cesarean delivery. Local anesthetic wound infusion improves acute pain and might act on peripheral and central sensitization mechanisms; however, no studies have proved this hypothesis. We evaluated the potential benefits of continuous wound infusion of levobupivacaine after cesarean delivery on secondary hyperalgesia (primary end point) and primary hyperalgesia, pain relief, persistent pain, and inflammatory and metabolic stress response. Healthy women scheduled for elective cesarean delivery participated in this prospective, randomized, triple-blind, placebo-controlled trial (NCT01458431). All patients received spinal anesthesia with 0.5% hyperbaric bupivacaine with fentanyl and a multiholed wound catheter placed under the fascia. Women were randomized to receive continuous wound infusion (0.35% levobupivacaine 7 mL/h for 48 hours; group L) or an equal volume of saline (group S). Secondary hyperalgesia to punctate mechanical stimuli was evaluated using dynamic tests, and primary hyperalgesia was evaluated using an electronic von Frey anesthesiometer; both were assessed at 24, 48, and 72 hours. The following variables were collected: intensity of postoperative parietal and visceral pain at rest and on movement rated on a visual analog scale >72 hours, time to first bolus of patient-controlled analgesia (PCA), cumulative dose of rescue morphine (PCA) and acetaminophen, ability to sleep and sleep quality, and patient satisfaction. Persistent postoperative pain was evaluated during a telephone interview at 1, 3, 6, and 12 months after surgery. C-reactive protein, acid glycoprotein, preprandial glucose, insulin, cortisol, prolactin, growth hormone, and interleukin-6 were measured before cesarean delivery and at 8, 24, and 48 hours. Adverse events and patient outcomes were recorded. Seventy women were included. In group L, the area of secondary hyperalgesia was significantly reduced (43.4 [18.5-80] vs 68.4 [39.0-136] cm2 and 45.1 [0.9-89.8] vs 67.3 [31.3-175] cm2 at 24 and 48 hours, respectively; group:time interaction P value < .001), the mechanical pain threshold was significantly higher at 24 hours (633 [441-802] vs 417 [300-572] g.mm-2; P = .001), and morphine consumption was significantly lower at 24 hours (4 [2-11] vs 11[6-23]; P = .003) compared with group S. Levobupivacaine had no effect on persistent postoperative pain at 1, 3, 6, and 12 months. Plasma insulin levels in the immediate postoperative period and at 8, 24, and 48 hours were significantly lower in group L (P < .001). There were no significant differences in other biochemical parameters of inflammatory and endocrine-metabolic response. Levobupivacaine wound infusion provides adequate analgesia and might be an effective antihyperalgesic adjunct.

Evaluation of the Efficacy, Biocompatibility, and Permeation of Bupivacaine-Loaded Poly(epsilon-caprolactone) Nano-Capsules as an Anesthetic.

In this study bupivacaine (BVC) was encapsulated in Nano-capsules of poly-ε-caprolactone (PCL) and its cytotoxicity in HaCaT (MTT) cells, its permeability in the oesophageal epithelium of pigs, as well as its anesthetic effect in the incision model of rat's hind paw (electronic von Frey anesthesiometer) were evaluated. BVC and epinephrine-associated bupivacaine (BVC-Epi) have been compared to BVC-Nano and it was demonstrated that BVC-Nano had high physicochemical properties and remained stable for 120 days; also, encapsulation of bupivacaine did not affect its toxicity to HaCaT cells, but epinephrine reduced its toxicity. Although both methods of combination with epinephrine and encapsulation in nanocapsules resulted in an extended time of anesthesia, the efficacy of epinephrine was more favorable. The permeation evaluation indicated that encapsulation increased both the permeability coefficient and the steady-state flux of bupivacaine across the esophageal epithelium. BVC permeation was enhanced by encapsulation into Nano-capsules, as a new novel therapeutic strategy, facilitating future research as a topical anesthetic.

PRG-1 relieves pain and depressive-like behaviors in rats of bone cancer pain by regulation of dendritic spine in hippocampus.

Rationale: Pain and depression, which tend to occur simultaneously and share some common neural circuits and neurotransmitters, are highly prevalent complication in patients with advanced cancer. Exploring the underlying mechanisms is the cornerstone to prevent the comorbidity of chronic pain and depression in cancer patients. Plasticity-related gene 1 (PRG-1) protein regulates synaptic plasticity and brain functional reorganization during neuronal development or after cerebral lesion. Purinergic P2X7 receptor has been proposed as a therapeutic target for various pain and neurological disorders like depression in rodents. In this study, we investigated the roles of PRG-1 in the hippocampus in the comorbidity of pain and depressive-like behaviors in rats with bone cancer pain (BCP).Methods: The bone cancer pain rat model was established by intra-tibial cell inoculation of SHZ-88 mammary gland carcinoma cells. The animal pain behaviors were assessed by measuring the thermal withdrawal latency values by using radiant heat stimulation and mechanical withdrawal threshold by using electronic von Frey anesthesiometer, and depressive-like behavior was assessed by sucrose preference test and forced swim test. Alterations in the expression levels of PRG-1 and P2X7 receptor in hippocampus were separately detected by using western blot, immunofluorescence and immunohistochemistry analysis. The effects of intra-hippocampal injection of FTY720 (a PRG-1/PP2A interaction activator), PRG-1 overexpression or intra-hippocampal injection of A438079 (a selective competitive P2X7 receptor antagonist) were also observed.Results: Carcinoma intra-tibia injection caused thermal hyperalgesia, mechanical allodynia and depressive-like behaviors in rats, and also induced the deactivation of neurons and dendritic spine structural anomalies in the hippocampus. Western blot, immunofluorescence and immunohistochemistry analysis showed an increased expression of PRG-1 and P2X7 receptor in the hippocampus of BCP rats. Intra-hippocampal injection of FTY720 or A438079 attenuated both pain and depressive-like behaviors. Furthermore, overexpression of PRG-1 in hippocampus has similar analgesic efficacy to FTY720. In addition, they rescued neuron deactivation and dendritic spine anomalies.Conclusion: The results suggest that both PRG-1 and P2X7 receptor in the hippocampus play important roles in the development of pain and depressive-like behaviors in bone cancer condition in rats by dendritic spine regulation via P2X7R/PRG-1/PP2A pathway.

Effect of long-term resistance exercise on masseter muscle mechanical hyperalgesia in rats

To investigate the effect of long-term resistance exercise of hindlimb on mechanical hyperalgesia of bilateral masseter muscle in rats with or without occlusal interference. Six-teen male Sprague-Dawley rats (220-250 g) were randomly divided into four groups: the naive control group, naive exercise group, occlusal interference control group, and occlusal interference exercise group. The rats in occlusal interference groups (occlusal interference control group and occlusal interference exercise group) obtained occlusal interference with 0.4 mm-thick crowns bonded to the right maxillary first molars. The rats in exercise groups (naive exercise group and occlusal interference exercise group) performed squat-type resistance exercises for 30 minutes, once a day, 5 days/week, lasting for 14 weeks. Resistance exercise was recorded every day. Mechanical withdrawal thresholds of bilateral masseter muscle were tested per week by use of modified electronic von-frey anesthesiometer. The rats were weighed per week. After the 14-week exercise, the muscle strength of the hindlimb was tested with a grip strength meter. Muscle (gastrocnemius and soleus) weight of bilateral hindlimb and length of bilateral fibula of the rats were obtained. The muscle-mass/body-mass ratios and muscle-mass/fibula-length ratios were calculated. Between the naive control group and naive exercise group, there was no significant difference in the mechanical withdrawal thresholds of bilateral masseter muscle for the 0-4 weeks (P>0.05). During the 5-14 weeks, the mechanical withdrawal thresholds of the rats in the naive exercise group were higher than those in the naive control group (P<0.05). Between the occlusal interference control group and occlusal interference exercise group, there was no significant difference in the mechanical withdrawal thresholds of bilateral masseter muscle for the 0-6 weeks (P>0.05). During the 7-14 weeks, the mechanical withdrawal thresholds of rats in the naive exercise group were higher than those in the occlusal interference control group (P<0.05). After the 14week exercise, the body mass of the rats in nonexercise group (the naive control group and occlusal interference control group) were larger than those in exercise group [(462±6) g vs. (418±14) g, P<0.05]. And the muscle strength of hindlimb of the rats in exercise group were bigger than those in non-exercise group [(6.75±0.13) N vs. (5.41±0.15) N, P<0.01]. long-term resistance exercise can increase mechanical withdrawal thresholds of the bilateral masseter muscle in rats with or without masseter muscle mechanical hyperalgesia.

Clinical observation of the effect of prophylaxis on allodynia in patients with migraine.

ObjectiveThe clinical characteristics of migraine with and without allodynia were compared to evaluate the risk factors of cutaneous allodynia in migraine. The effects of prophylactic therapy on allodynia in patients with migraine were assessed based on the change in pain threshold after therapy.Patients and methodsA total of 71 patients with migraine admitted to the Department of Neurology of Shandong Provincial Hospital were recruited in this study. The included patients were aged 18–70 years and did not present positive symptoms according to the nervous system examination. The variation in cutaneous allodynia was assessed for the role of classic prophylactic therapy in migraine-related allodynia, also termed as central sensitization (CS). Patients with migraine were randomized into two groups (topiramate and flunarizine groups), and the effect of drugs was evaluated by the change in cutaneous pain threshold between the two groups. Fifty-five patients were tested for pain threshold due to instrument failure. Pressure allodynia was measured with a force gage, and pricking was measured with Electronic von Frey Anesthesiometer. The pain threshold was measured every 3 months for 6 months. The variations in pain threshold after treatment were compared using t-test and χ2 test.ResultsAllodynia was seen in 70.4% of the patients; of these, 76.3% were females. Female gender, duration of illness, and frequency of migraine attacks per month were significantly associated with allodynia. The rate of allodynia and frequency of headache declined significantly, the number of patients with allodynia was reduced, and the pain threshold improved dramatically after treatment. The composite adverse events were low after treatment with topiramate and flunarizine.ConclusionAllodynia was observed, especially in females who experienced frequent migraine attacks for a prolonged period. Gender, duration of illness, and number of migraine attacks per month were the major predictors of allodynia. Topiramate and flunarizine administered orally for a short duration can effectively improve the pain threshold and CS of patients with migraine, and efficiently relieve allodynia.

Quantitative sensory testing with Electronic von Frey Anesthesiometer and von Frey filaments in nonpainful cats: a pilot study

ObjectiveMeasurement of sensory thresholds could represent a complementary tool to behavioural pain scores in cats. The aim of this study was to investigate the feasibility of quantitative sensory testing (QST) with the Electronic von Frey Anesthesiometer (EVF) and von Frey filaments (VFF) in healthy cats, and to assess the limits of agreement (LOA) between the two devices. Study designProspective clinical study. AnimalsA total of 15 client-owned healthy cats. MethodsTwo investigators (A and B) carried out the measurements independently. The EVF and the VFF were applied on the upper lip and at the level of the medial aspect of the stifle. A 1-hour interval was allowed between the sets of measurements taken by investigators A and B; each investigator repeated the entire session of measurements after 24 hours. The LOA between the EVF and the VFF were analysed with the intraclass correlation coefficient (ICC), and with the Bland Altman method. ResultsQST with both the EVF and the VFF was feasible in healthy cats; however, the willingness of the cats to cooperate was negatively affected by the repetition of the measurements on the second day. The presence of the cat owners seemed to facilitate the trial. There was a fair agreement between the EVF and the VFF (ICC = 0.49; 95% confidence interval: 0.13–0.70). Conclusionsand clinical relevance Our findings indicate that both EVF and VFF may be used for QST in cats. Further trials will be needed to verify the usefulness of QST with EVF and VFF in feline patients suffering from actual chronic pain.

Electroacupuncture Relieves Pain by Down-regulating Expression of Hippocampal High Mobility Group Protein 1 and Contents of TNF-α and IL-1 β in Rats with Chronic Neuropathic Pain

To observe the effect of electroacupuncture (EA) on expression of high mobility group protein 1 (HMGB 1) and related downstream effectors of proinflammatory cytokines in the hippocampus in chronic neuropathic pain rats, so as to investigate its mechanism underlying neuropathic pain relief. Male SD rats were randomized into sham, model, and EA groups, with 12 rats in each group. The neuropathic pain model was established by ligature of the left sciatic nerve to induce chronic constriction injury (CCI). EA was applied to bilateral "Zusanli"(ST 36) and "Yanglingquan"(GB 34) for 30 min, once daily for 7 days. The mechanical withdrawal threshold (WMT) was detected using an electronic von Frey anesthesiometer. The expression level of HMGB 1 in the hippocampus was determined using quantitative RT-PCR and Western blot, separately, and the contents of hippocampal TNF-α and IL-1 β were detected by ELISA. Compared with the sham group, the MWT values were markedly decreased on day 7, 10 and 14 after modeling in the model group (P<0.001). On day 10 and 14 after modeling, the MWT values were significantly up-regulated in the EA group relevant to those of the model group (P<0.05, P<0.01). The expression levels of HMGB1 mRNA and protein, and the contents of hippocampal TNF-α and IL-1 β were markedly increased in the model group relevant to the sham group (P<0.001), and significantly down-regulated in the EA group relevant to the model group (P<0.001, P<0.01, P<0.05). EA stimulation of ST 36-GB 34 can relieve pain in chronic neuropathic pain rats, which may be related to its actions in down-regulating the levels of HMGB 1 and its downstream proinflammatory cytokines TNF-α and IL-1 β in the hippocampus.

Recent and advanced animal models used in the Screening of analgesics and anti-inflammatory activity

Non-Steroidal anti-inflammatory drugs (NSAIDs) are consisting of three major anti-pyretic, anti-inflammatory and anti-analgesics properties. They have reduced the sensation of pain, body temperature, and inflammation. It is also used for the treatment of the long-term health problems like arthritis (rheumatoid arthritis, osteoarthritis, and lupus). NSAIDs highly protect the lining of the stomach and intestines from the damaging effects of acid promote blood clotting by activating blood platelets, and promote normal function of the kidney. Incompatible with the action of NSAIDs many different types of drugs and plant use for the treatment of the analgesic, inflammation and pyretic activity. Diclofenac inhibit the cyclooxygenase (COX-2) enzyme with the greater potency that it (COX-1). NSAIDs are generally used in the management of pain because of the integrated role of the COX pathway that is recognition of pyretic, inflammation and analgesic. Introduction to painful procedures and/or stressors during the early neonatal period can reprogram the underlying neurocircuitry involved in nociception and neuropathic pain perception. The reprogramming of these systems can result in an enduring elevation in sympathy towards mechanical and thermal stimuli. During adolescence, hind paw mechanical removal thresholds were evaluated using an electronic von Frey Anesthesiometer. Animals challenged neonatally with LPS (nLPS) had increased pain sensitivity on this measure which was related with decreased Oprm1 expression in the prefrontal cortex (PFC) and periaqueductal gray (PAG) of both male and female rats. There was no effect of inflammatory treatment on either anxiety or depressive-like behavior suggesting that affective functioning did not account for differences in mechanical pain sensitivity.

Activation of the P2X7 receptor in midbrain periaqueductal gray participates in the analgesic effect of tramadol in bone cancer pain rats.

BackgroundCancer pain is a well-known serious complication in metastatic or terminal cancer patients. Current pain management remains unsatisfactory. The activation of spinal and supraspinal P2X7 receptors plays a crucial role in the induction and maintenance mechanisms of various kinds of acute or chronic pain. The midbrain periaqueductal gray is a vital supraspinal site of the endogenous descending pain-modulating system. Tramadol is a synthetic, centrally acting analgesic agent that exhibits considerable efficacy in clinically relieving pain. The purpose of this study was to determine whether the activation of P2X7 receptor in the ventrolateral region of the periaqueductal gray (vlPAG) participates in the analgesic mechanisms of tramadol on bone cancer pain in rats. The bone cancer pain rat model was established by intratibial cell inoculation of SHZ-88 mammary gland carcinoma cells. The analgesic effects of different doses of tramadol (10, 20, and 40 mg/kg) were assessed by measuring the mechanical withdrawal threshold and thermal withdrawal latency values in rats by using an electronic von Frey anesthesiometer and radiant heat stimulation, respectively. Alterations in the number of P2X7 receptor-positive cells and P2X7 protein levels in vlPAG were separately detected by using immunohistochemistry and Western blot assay. The effect of intra-vlPAG injection of A-740003 (100 nmol), a selective competitive P2X7 receptor antagonist, on the analgesic effect of tramadol was also observed.ResultsThe expression of P2X7 receptor in the vlPAG on bone cancer pain rats was mildly elevated, and the tramadol (10, 20, and 40 mg/kg) dose dependently relieved pain-related behaviors in bone cancer pain rats and further upregulated the expression of P2X7 receptor in the vlPAG. The intra-vlPAG injection of A-740003 pretreatment partly but significantly antagonized the analgesic effect of tramadol on bone cancer pain rats.ConclusionsThe injection of tramadol can dose dependently elicit analgesic effect on bone cancer pain rats by promoting the expression of the P2X7 receptor in vlPAG.

Effects of tianeptine on symptoms of fibromyalgia via BDNF signaling in a fibromyalgia animal model.

Previous reports have suggested that physical and psychological stresses may trigger fibromyalgia (FM). Stress is an important risk factor in the development of depression and memory impairments. Antidepressants have been used to prevent stress-induced abnormal pain sensation. Among various antidepressants, tianeptine has been reported to be able to prevent neurodegeneration due to chronic stress and reverse decreases in hippocampal volume. To assess the possible effect of tianeptine on FM symptoms, we constructed a FM animal model induced by restraint stress with intermittent cold stress. All mice underwent nociceptive assays using electronic von Frey anesthesiometer and Hargreaves equipment. To assess the relationship between tianeptine and expression levels of brain-derived neurotrophic factor (BDNF), cAMP response element-binding protein (CREB), and phosphorylated cAMP response element-binding protein (p-CREB), western blotting and immunohistochemistry analyses were performed. In behavioral analysis, nociception tests showed that pain threshold was significantly decreased in the FM group compared to that in the control group. Western blot and immunohistochemical analyses of medial prefrontal cortex (mPFC) and hippocampus showed downregulation of BDNF and p-CREB proteins in the FM group compared to the control group. However, tianeptine recovered these changes in behavioral tests and protein level. Therefore, this FM animal model might be useful for investigating mechanisms linking BDNF-CREB pathway and pain. Our results suggest that tianeptine might potentially have therapeutic efficacy for FM.

CXCR4 signaling in macrophages contributes to periodontal mechanical hypersensitivity in Porphyromonas gingivalis-induced periodontitis in mice.

BackgroundPeriodontitis is an inflammatory disease accompanied by alveolar bone loss and progressive inflammation without pain. However, the potential contributors eliminating pain associated with gingival inflammation are unknown.Resultswe examined the involvement of CXC chemokine receptor type 4 (CXCR4) on the mechanical sensitivity of inflamed periodontal tissue, using a mouse model of periodontitis established by the ligation of the tooth cervix of a maxillary second molar and inoculation with Porphyromonas gingivalis (P. gingivalis). Infiltration of inflammatory cells into gingival tissue was not observed following the inoculation. Under light anesthesia, the mechanical head withdrawal threshold (MHWT) on the buccal gingiva was measured using an electronic von Frey anesthesiometer. No significant changes in MHWT were observed in the mice with P. gingivalis-induced periodontitis during the experimental period. Continuous administration of CXCR4 neutralizing antibody to the gingival tissue significantly decreased MHWT and increased the number of gingival CXCR4 immunoreactive macrophages in the periodontitis group. Nitric oxide metabolites in the gingival tissue were significantly increased after the inoculation of P. gingivalis and were reduced by gingival CXCR4 neutralization. Gingival L-arginine administration induced gingival mechanical allodynia in naive animals. Moreover, the decrease in MHWT after treatment with P. gingivalis and CXCR4 neutralization was partially reversed by nitric oxide synthase inhibition in the gingival tissue. Nuclear factor-kappa B was expressed in infiltrating macrophages after inoculation of P. gingivalis and administration of the nuclear factor-kappa B activator betulinic acid induced gingival mechanical allodynia in naive mice.ConclusionsThese findings suggest that CXCR4 signaling inhibits nitric oxide release from infiltrating macrophages and is involved in modulation of the mechanical sensitivity in the periodontal tissue in P. gingivalis-induced periodontitis.

Binge Ethanol Consumption Increases Inflammatory Pain Responses and Mechanical and Cold Sensitivity: Tigecycline Treatment Efficacy Shows Sex Differences.

BackgroundPhysicians have long reported that patients with chronic pain show higher tendencies for alcohol use disorder (AUD), and AUD patients appear to have higher pain sensitivities. The goal of this study was to test 2 hypotheses: (i) binge alcohol consumption increases inflammatory pain and mechanical and cold sensitivities; and (ii) tigecycline is an effective treatment for alcohol‐mediated‐increased pain behaviors and sensitivities. Both female and male mice were used to test the additional hypothesis that important sex differences in the ethanol (EtOH)‐related traits would be seen.Methods“Drinking in the Dark” (DID) alcohol consuming and nondrinking control, female and male, adult C57BL/6J mice were evaluated for inflammatory pain behaviors and for the presence of mechanical and cold sensitivities. Inflammatory pain was produced by intraplantar injection of formalin (10 μl, 2.5% in saline). For cold sensation, a 20 μl acetone drop was used. Mechanical withdrawal threshold was measured by an electronic von Frey anesthesiometer. Efficacy of tigecycline (80 mg/kg i.p.) to reduce DID‐related pain responses and sensitivity was tested.Results DID EtOH consumption increased inflammatory pain behavior, while it also produced sustained mechanical and cold sensitivities in both females and males. Tigecycline produced antinociceptive effects in males; a pro‐nociceptive effect was seen in females in the formalin test. Likewise, the drug reduced both mechanical and cold sensitivities in males, but females showed an increase in sensitivity in both tests.ConclusionsOur results demonstrated that binge drinking increases pain, touch, and thermal sensations in both sexes. In addition, we have identified sex‐specific effects of tigecycline on inflammatory pain, as well as mechanical and cold sensitivities. The development of tigecycline as an AUD pharmacotherapy may need consideration of its pro‐nociceptive action in females. Further studies are needed to investigate the mechanism underlying the sex‐specific differences in nociception.

Mechanical allodynia corresponds to Oprm1 downregulation within the descending pain network of male and female rats exposed to neonatal immune challenge

Exposure to painful procedures and/or stressors during the early neonatal period can reprogram the underlying neurocircuitry involved in nociception and neuropathic pain perception. The reprogramming of these systems can result in an enduring elevation in sensitivity towards mechanical and thermal stimuli. Recent evidence suggests that exposure to mild inflammatory mediators during the neonatal period can induce similar pain responses in both adolescent and adult rats. Therefore, we sought to profile changes in the expression of several genes across brain areas involved in the active modulation of nociception and neuropathic pain using a well-recognized model of neonatal inflammation. In the present study male and female Sprague-Dawley rats were administered either the inflammatory endotoxin lipopolysaccharide (LPS; 0.05mg/kg, i.p.) or saline (equivolume) on postnatal days (PND) 3 and 5. During adolescence, hind paw mechanical withdrawal thresholds were evaluated using an electronic von Frey anesthesiometer. Animals challenged neonatally with LPS (nLPS) had increased pain sensitivity on this measure which was associated with decreased Oprm1 expression in the prefrontal cortex (PFC) and periaqueductal gray (PAG) of both male and female rats. Although a ‘second hit’ with LPS in adolescence (aLPS) did not confer protection or reveal additional vulnerabilities, aLPS given to animals treated neonatally with saline was associated with increased pain sensitivity, but only in females. Interestingly, adolescent inflammatory challenge decreased Hcrt2 mRNA in the PAG and elevated Trpv1 in the PAG and PFC of both sexes. There was no effect of inflammatory treatment on either anxiety or depressive-like behavior suggesting that affective functioning did not account for differences in mechanical pain sensitivity. Finally, a preliminary investigation demonstrated that administration of a broad spectrum antibiotic cocktail attenuated the mechanical sensitivity that followed nLPS. Together, these data extend upon evidence that inflammation imparts long term changes in quality of life and pain responses via interference within the descending pain network. Moreover, they highlight a potential window of opportunity to target the microbiota-gut-brain axis and reverse pain processing disturbances following perinatal inflammation.

D-serine in the midbrain periaqueductal gray contributes to morphine tolerance in rats.

BackgroundThe N-methyl-D-aspartate subtype of glutamate receptor plays a critical role in morphine tolerance. D-serine, a co-agonist of N-methyl-D-aspartate receptor, participates in many physiological and pathophysiological processes via regulating N-methyl-D-aspartate receptor activation. The purinergic P2X7 receptor activation can induce the D-serine release in the central nervous system. This study aimed to investigate the role of the ventrolateral midbrain periaqueductal gray D-serine in the mechanism of morphine tolerance in rats. The development of morphine tolerance was induced in normal adult male Sprague–Dawley rats through subcutaneous injection of morphine (10 mg/kg). The analgesic effect of morphine (5 mg/kg, i.p.) was assessed by measuring mechanical withdrawal thresholds in rats with an electronic von Frey anesthesiometer. The D-serine concentration and serine racemase expression levels in the ventrolateral midbrain periaqueductal gray were evaluated through enzyme-linked immunosorbent assay and Western blot analysis, respectively. The effects of intra-ventrolateral midbrain periaqueductal gray injections of the D-serine degrading enzyme D-amino acid oxidase and antisense oligodeoxynucleotide targeting the P2X7 receptor on chronic morphine-treated rats were also explored.ResultsWe found that repeated morphine administrations decreased the antinociceptive potency of morphine evidenced by the percent changes in mechanical pain threshold in rats. By contrast, the D-serine contents and the expression levels of the serine racemase protein were upregulated in the ventrolateral midbrain periaqueductal gray in morphine-tolerant rats. The development of morphine tolerance was markedly alleviated by intra-ventrolateral midbrain periaqueductal gray injections of D-amino acid oxidase or antisense oligodeoxynucleotide targeting the P2X7 receptor.ConclusionsOur data indicate that the development of antinociceptive tolerance to morphine is partially mediated by ventrolateral midbrain periaqueductal gray D-serine content, and the activation of the ventrolateral midbrain periaqueductal gray P2X7 receptor is an essential prelude to D-serine release. These results suggest that a cascade involving P2X7 receptor–D-serine–N-methyl-D-aspartate receptor mediated signaling pathway in the supraspinal mechanism of morphine tolerance.

Targeting Putative Mu Opioid/Chemokine Receptor Type 5 Heteromers Potently Attenuates Nociception in a Murine Model of Sickle Cell Disease

Background: Sickle cell disease (SCD) is one of the world's most common inherited diseases, due to a point mutation in hemoglobin that leads to the polymerization of hemoglobin S, giving red blood cells (RBCs) their sickle shape. The hallmarks of SCD are hemolysis and vaso-occlusion. Vaso-occlusive crises (VOC) frequently occur in response to inflammatory and hypoxic conditions such as infections, surgery, etc. Pain is a major defining characteristic of SCD starting early in life and continuing throughout adulthood. VOC-induced pain can be chronic or episodic and unpredictable, requiring frequent hospitalizations and analgesics. Opioids, predominantly morphine, have been the mainstay treatment for pain management of SCD but pose a serious challenge attributed to the many adverse side effects, including tolerance, respiratory depression, sedation, nausea, constipation, pruritus, and dependence. Given that analgesics characteristically used to treat pain appear to be relatively ineffective in alleviating pain associated with SCD as well as display their own dose-limiting adverse effects, there exists a requisite need for superior pharmacological agents in its treatment. Here we reveal that the bivalent ligand MCC22, is highly effective as an analgesic in a murine model of SCD. MCC22 (MW=1255) contains both mu agonist and chemokine CCR5 antagonist pharmacophores that are linked through a 22-atom-spacer. It was designed specifically to target the mu opiate receptor (MOR)-CCR5 heteromer, as there is evidence for crosstalk between MOR and CCR5 in cultured cells that reduces the efficacy of opioid analgesics employed in SCD. Intrathecal (i.t.) MCC22 was potent in reducing the mechanical and heat hyperalgesia in Complete Freund's Adjuvant (CFA) and lipopolysaccharide (LPS) inflammatory pain assays. ED50s for the i.t. administration were 0.019 and 0.015 pmol/mouse, respectively. MCC22 when given intraperitoneally (i.p.) to LPS pretreated mice had an ED50 of 5.6 µmol/kg. We next investigated whether MCC22 would be effective in alleviating pain in SCD mice.Methods:Townes transgenic humanized mice expressing sickle hemoglobin (HbSS / HbAS) and normal human hemoglobin (HbAA) controls (4-12 weeks of age, 20-25g) were used in this study. Mice were characterized for hyperalgesia by quantifying cutaneous mechanical sensitivity of the hind paw and forelimb grip force. Mechanical sensitivity of the hind paw was evaluated by determining the frequency of withdrawal responses and paw withdrawal threshold. The frequency of paw withdrawal evoked by a standard von Frey monofilament with a bending force of 9.3 mN applied to the plantar surface of the hind paws was determined from 10 trials on each paw. Withdrawal threshold was determined using an electronic von Frey anesthesiometer pressed against the plantar surface with increasing force until withdrawal occurred. To assess deep tissue hyperalgesia, the tensile force of peak forelimb exertion was measured using a computerized grip force meter. To evaluate the analgesic effects of MCC22, paw withdrawal frequency was determined before and at various times after administration of 8.0 µmol/kg i.p.Results: Pain Characterization: HbSS sickle mice exhibited robust mechanical hyperalgesia as shown by a significant increase in paw withdrawal frequency compared to HbAS and HbAA controls (Figure1A, p<0.001). Paw withdrawal frequency in HbAS mice was also greater than those for HbAA mice (p<0.001), but less than HbSS mice. HbSS mice had lower mechanical withdrawal thresholds compared to both HbAS and HbAA mice (Figure 1B, p<0.001), while HbAS did not differ from HbAA mice. HbSS and HbAS mice also displayed lower forelimb grip force compared to HbAA mice (Figure 1C, p<0.001), although grip force in HbSS was lower than HbAS mice (p<0.001). Administration of MCC22 (8.0 µmol/kg i.p.) reduced hyperalgesia within 30 minutes as evidenced by a decrease in paw withdrawal frequency in both HbSS and HbAS mice (Figure 1D, p<0.001).Conclusions: Hyperalgesia was demonstrated in both HbAS and HbSS mice. MCC22 potently attenuated mechanical hyperalgesia in these SCD mice. The use of bivalent ligands that target heteromers involved in signaling pain may offer novel and effective treatments for pain in patients with SCD. We speculate that an analgesic with potential anti-inflammatory and mu-agonist activity may provide a novel approach to sickle pain. [Display omitted] DisclosuresBelcher:CSL Behring: Research Funding; Seattle Genetics: Research Funding; Biogen Idec: Research Funding. Vercellotti:Biogen Idec: Research Funding; CSL Behring: Research Funding; Cydan: Research Funding; Seattle Genetics: Research Funding.

Antiallodynic and antihyperalgesic effects of zerumbone on a mouse model of chronic constriction injury-induced neuropathic pain

Neuropathic pain is a chronic condition that is difficult to be treated. Current therapies available are either ineffective or non-specific thus requiring newer treatment approaches. In this study, we investigated the antiallodynic and antihyperalgesic effects of zerumbone, a bioactive sesquiterpene from Zingiber zerumbet in chronic constriction injury (CCI)-induced neuropathic pain animal model. Our findings showed that single and repeated dose of intra-peritoneal administration of zerumbone (5, 10, 50, 100mg/kg) significantly attenuated the CCI-induced neuropathic pain when evaluated using the electronic von Frey anesthesiometer, cold plate, Randall–Selitto analgesiometer and the Hargreaves plantar test. Zerumbone significantly alleviated tactile and cold allodynia as well as mechanical and thermal hyperalgesia. Our findings are in comparison to the positive control drugs thatused gabapentin (20mg/kgi.p.) and morphine (1mg/kgi.p.). Together, these results showed that the systemic administration of zerumbone produced marked antiallodynic and antihyperalgesic effects in the CCI-induced neuropathic pain in mice and may serve as a potential lead compound for further analysis.

Effects of habituation to the testing facility on mechanical nociceptive thresholds of pigs

Abstract Aims This study aimed to gain methodological knowledge about quantification of mechanical nociceptive thresholds in pigs stimulated on the hind legs. Methods Thirty-two castrated male pigs were used, weighing 50–60kg, 16 weeks of age and housed in standard pens (4.40m×4.40m) with ad libitum access to water and feed. Mechanical nociceptive testing (4 stimulations/pig) was done by an electronic von Frey anesthesiometer (IITC Life Science Inc., CA) with a rigid, hollow plastic tip (cut-off: 1000 gf) and an area of 0.3 mm2. Stimulations targeted caudal aspects of the metatarsus while the pigs were restrained in a cage measuring 35cm×40cm (length×width). During the last 3d before nociceptive testing, half of the pigs were habituated to the experimental set-up twice daily. Results Across the three days, the habituation procedure led to a reduction in time required to approach and release the pigs from the home pen (P&lt;0.05) and to increased acceptance of the confinement (P&lt;0.01). The habituated animals had a lower mechanical nociceptive threshold (median: 495gf (302–675) vs 745gf (479–1000); P&lt;0.05), and a decreased occurrence of censored observations (0 vs 25%; P&lt;0.05) compared to animals tested without habituation. Conclusions The present experiment focussed on methodological aspects of hand-held tests of mechanical nociceptive thresholds in pigs. The results show that the pigs responded to a 3-day habituation regime by increased ease of handling and tolerance of being confined in a test cage. In addition, habituation to the test environment and handling led to reduced mechanical nociceptive thresholds as well as a lowered proportion of censored observations. The current results reiterate the value of habituation in research involving animal behaviour. Further characterization of the methodology is needed to allow its application in the evaluation of clinical conditions in pigs.