Thermal Paw Withdrawal Threshold Research Articles

Distinct antinociceptive and conditioned behavioral effects are produced by individual cannabinoids and a cannabis-derived mixture

Cannabinoids have been proposed as therapeutics for pain mitigation. Therefore, the antihyperalgesic effects of a proprietary cannabis-derived mixture, Non-Euphoric Phytocannabinoid Elixir #14 (NEPE14), were examined in a persistent Complete Freund's Adjuvant (CFA)-induced model of inflammatory pain. The acute antinociceptive and operant behavioral effects of NEPE14 were then compared with single cannabinoid preparations of Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-THC, the synthetic cannabinoid (−)-CP 55,940 (CP), and cannabidiol (CBD). The THC isomers and CP were also administered with cannabinoid-type-1 receptor (CB1R) antagonist, AM251, and NEPE14 was administered in combination with THC or CP. To induce inflammation, CFA or saline was administered into the paw of male and female Wistar rats. After injections, mechanical hypersensitivity was assessed with Von Frey filaments, and thermal hyperalgesia with a thermal probe. Nine Sprague Dawley rats were also trained to respond under a fixed-ratio 30 schedule for food reinforcers during a 60-min session. Response rates were recorded during the session and warm-water tail-withdrawal latency post session. In CFA-administered rats, mechanical and thermal paw-withdrawal thresholds significantly decreased compared to vehicle, indicating hyperalgesia. Both i.p. (6.6–20.7 ml/kg) and o.m. (30–300 μL) NEPE14 significantly reduced the mechanical and thermal hyperalgesia. In contrast, neither NEPE14 (3.7–20.7 mL/kg i.p., 100–1000 μL o.m.) nor CBD (10–100 mg/kg) significantly decreased response rates or increased tail-withdrawal latency. Acute Δ9-THC, Δ8-THC (1–5.6 mg/kg), and CP (0.032–0.18 mg/kg) significantly and dose-dependently decreased overall response rate and increased tail-withdrawal latency compared to vehicle. AM251 significantly antagonized the rate-decreasing effects of THC, and CP, as well as the antinociceptive effects of CP. Combinations of NEPE14 with Δ9-THC or CP were not significantly different from these cannabinoids alone. In summary, while NEPE14 significantly reduced CFA-induced hyperalgesia, it was more similar to CBD than Δ9-THC, Δ8-THC, and CP for significantly reducing thermal nociception and disrupting conditioned behavior.

Evaluating the impact of age and inflammatory duration on behavioral assessments of nociception

Pain is a prevalent issue for elderly individuals. Unfortunately, it remains unclear how acute and chronic pain differs as a function of age, and surprisingly, there is even disagreement on how the sensory and affective dimensions of pain change with age. Therefore, the current investigation evaluated such age differences with behavioral methodology using a preclinical model of arthritis. The primary factors of interest were age and chronicity of pain using behavioral assessments designed to measure sensory and affective dimensions of pain processing. Mechanical and thermal paw withdrawal thresholds demonstrated unique outcomes associated with sensory processing across age. The processing of pain affect measured by the Place Escape/Avoidance Paradigm (PEAP testing) also demonstrated age related effects. Overall, younger animals appeared more sensitive to nociceptive stimuli than older animals. However, the results from the current study suggest that chronicity of pain can be impactful for how older animals process pain related affect and avoidance. The finding of unique patterns of pain across age and duration of pain highlights the clinical literature. Future research should aim to elucidate mechanisms for affective processing of chronic pain in older subjects.

Effect of curcumin on lidocaine-induced nerve injury in rats: the role of Akt and ERK1/2 signaling pathway

Objective To evaluate the effect of curcumin on lidocaine-induced nerve injury and the role of protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway in rats. Methods Eighty SPF male Sprague-Dawley rats, aged 8-10 weeks, weighing 220-250 g, were divided into 8 groups (n=10 each) using a random number table method: control group(group C), sham operation group(group S), lidocaine group (group L), dimethyl sulfoxide (DMSO) group, low-dose curcumin group (group CL), high-dose curcumin group (group CH), curcumin plus ERK inhibitor PD98059 group (group P) and curcumin plus Akt inhibitor MK-2206 group (group M). The model of nerve injury was established by injecting 2% lidocaine 10 μl via the catheter in anesthetized rats in the other six groups except for C and S groups.Drugs were injected through a microcatheter for 14 consecutive days starting from 3rd day after IT catheterization as follows: DMSO 10 μl, curcumin 100 μg/10 μl and curcumin 500 μg/10 μl were injected once a day in D, CL and CH groups, respectively; curcumin 500 μg/10 μl was injected once a day, and D98059 10 μg and MK-2206 12 μg (in 5 μl DMSO) were intraperitoneally injected once a week in P and M groups, respectively.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal threshold (TWL) on the operated side were measured on 1 day before IT catheterization, before administration on 3rd day after IT catheterization, and on 14th day after administration.The spinal cord was removed on 1st day after the end of administration for determination of the expression of ERK1/2, phosphorylated ERK1/2 (p-ERK1/2), Akt, phosphorylated Akt (p-Akt), c-fos, Bcl-2 and Bax (by Western blot) and expression of ERK1/2 and Akt mRNA (by real-time polymerase chain reaction). Results Compared with group C, the MWT was significantly decreased, the TWL was shortened, the expression of ERK1/2 protein and mRNA, p-ERK1/2, c-fos, Akt protein and mRNA, p-Akt and Bcl-2 was down-regulated, and the expression of Bax was up-regulated in group L (P<0.05). Compared with group L, the MWT was significantly increased, and the TWL was prolonged in CL, CH, P and M groups, the expression of p-ERK1/2, c-fos, p-Akt and Bcl-2 was up-regulated, and the expression of Bax was down-regulated in CL and CH groups, the expression of p-Akt and Bcl-2 was up-regulated, and the expression of Bax was down-regulated in group P, and the the expression of p-ERK1/2 and c-fos was up-regulated in group M (P<0.05). Compared with group CH, the MWT was significantly decreased, and the TWL was shortened in P and M groups, the expression of p-ERK1/2 and c-fos was down-regulated in group P, and the expression of p-Akt and Bcl-2 was down-regulated, and the expression of Bax was up-regulated in group M (P<0.05). Conclusion Curcumin can reduce lidocaine-induced nerve injury, and the mechanism may be partly related to enhancing the activity of Akt and ERK1/2 signaling pathway in rats. Key words: Curcumin; Lidocaine; Drug-related side effects and adverse reactions; MAP kinase signaling system

Effect of dexmedetomidine on postoperative pain in rats with preoperative sleep deprivation

Objective To evaluate the effect of dexmedetomidine on postoperative pain in rats with preoperative sleep deprivation. Methods Fifty healthy adult male Sprague-Dawley rats, aged 12-14 weeks, weighing 200-300 g, were divided into 5 groups (n=10 each) using a random number table: control group (group C), sleep deprivation group (group SD), incisional pain group (group IP), sleep deprivation plus incisional pain group (group SD+ IP) and sleep deprivation plus incisional pain plus dexmedetomidine group (group SD+ IP+ DEX). Sleep deprivation was induced by the flower pot technique, and then the incisional pain model was carried out on first day after completion of sleep deprivation.Dexmedetomidine 50 μg/kg was intraperitoneally injected for 3 consecutive days before establishing the model of incisional pain in group SD+ IP+ DEX, and the equal volume of normal saline was given in the other groups.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal threshold (TWT) were measured before operation or at 1 day before sleep deprivation and after operation or at 12, 24 and 72 h after sleep deprivation.Blood samples were collected and spinal cord tissues were removed after the end of behavior test for determination of serum corticosterone concentrations (by enzyme-linked immunosorbent assay and content of 5-hydroxytryptamine (5-HT) in spinal dorsal horns (by high-performance liquid chromatography). Results Compared with group C, the MWT and TWT were significantly decreased, and the serum corticosterone concentrations and content of 5-HT in spinal dorsal horns were increased in the other 4 groups (P 0.05). Compared with group SD+ IP, the MWT and TWT were significantly increased, and the serum corticosterone concentrations and content of 5-HT in spinal dorsal horns were decreased in group SD+ IP+ DEX (P<0.05). Conclusion Dexmedetomidine can reduce postoperative pain in rats with preoperative sleep deprivation, and the mechanism may be related to inhibiting stress responses and levels of 5-HT in spinal dorsal horns. Key words: Dexmedetomidine; Sleep deprivation; Pain, postoperative

Role of calcium/calmodulin-dependent kinase II alpha in central nucleus of amygdale in fentanyl-induced hyperalgesia in rats: the relationship with mEPSCs

Objective To evaluate the role of calcium/calmodulin-dependent kinase Ⅱ alpha(CaMKⅡα)in the central nucleus of the amygdale(CeA)in fentanyl-induced hyperalgesia in rats and the relationship with miniature excitatory postsynaptic currents(mEPSCs). Methods Thirty-two male Sprague-Dawley rats, weighing 50-80 g, in which the CeA was successfully cannulated, were randomly divided into 4 groups(n=8 each)using a random number table: control 1 group(group C1), fentanyl-induced hyperalgesia 1 group(group FIH1), KN92 group, and KN93 group.Normal saline was injected subcutaneously, and dimethyl sulfoxide(DMSO)was given into the amygdale in group C1.In group FIH1, fentanyl was injected subcutaneously(60 μg/kg per time, 4 times in total, 15-min interval, cumulative dose of 240 μg/kg)to establish the model of hyperalgesia.In KN92 and KN93 groups, KN92 and KN93 10 nmol were given into the CeA after establishing the model.The mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal threshold(TWT)were measured at 6 and 7 h after fentanyl or normal saline injection.Another 12 Sprague-Dawley rats were selected and randomly divided into either control 2 group(group C2)or fentanyl-induced hyperalgesia 2 group(group FIH2)using a random number table with 6 rats in each group.The brains were removed and sliced 12 h later, and the frequency and amplitude of mEPSCs were recorded.KN93 10 nmol was then added to the artificial cerebral spinal fluid, and the frequency and amplitude of mEPSCs were recorded by whole cell patch-clamp technique. Results Compared with group C1, the MWT and TWT were significantly decreased at 6 h after fentanyl or normal saline injection in FIH1, KN92 and KN93 groups, and at 7 h after fentanyl or normal saline injection in FIH and KN92 groups(P 0.05). Compared with group C2, the frequency and amplitude of mEPSCs were significantly increased before administration of KN93(P 0.05). Compared with the value before KN93 administration, no significant change was found in the frequency and amplitude of mEPSCs after administration of KN93 in group C2(P>0.05), and the frequency and amplitude of mEPSCs were significantly decreased after administration of KN93 in group FIH2(P<0.05). Conclusion Activation of CaMKⅡα in the CeA enhances synaptic excitation in neurons, which is involved in fentanyl-induced hyperalgesia in rats. Key words: Calcium-calmodulin-dependent protein kinases; Fentanyl; Hyperalgesia; Excitatory postsynaptic potentials; Amygdala

The thermal probe test: A novel behavioral assay to quantify thermal paw withdrawal thresholds in mice

ABSTRACTRodent models are frequently used to improve our understanding of the molecular mechanisms of pain and to develop novel analgesics. Robust behavioral assays that quantify nociceptive responses to different sensory modalities, such has heat, are therefore needed. Here, we describe a novel behavioral assay to quantify thermal paw withdrawal thresholds in mice, called the thermal probe test, and compared it with other methods commonly used to measure heat thresholds, namely the Hargreaves test and the dynamic and conventional hot plate tests. In the thermal probe test, a slightly rounded 2.5 mm diameter metal probe that heats on contact at a rate of 2.5°C/sec, is applied to the plantar surface of the hind paw in mice at a starting temperature of ∼37°C, and the temperature at which a withdrawal response occurs, designated as the paw withdrawal temperature, is automatically recorded. The thermal probe test is effective at quantifying thermal allodynia in carrageenan-induced inflammation (paw withdrawal temperature 3 h: contralateral, 50.3 ± 0.6°C; ipsilateral, 43.1 ± 1.0°C), burns injury (paw withdrawal temperature 3 d: contralateral, 50.8 ± 0.5°C; ipsilateral, 43.2 ± 0.6°C) and after topical capsaicin (paw withdrawal temperature: vehicle control, 49.7 ± 0.6°C; capsaicin, 44.8 ± 1.2°C), giving comparable results to the Hargreaves test. In addition, the thermal probe test can detect opioid mediated analgesia in carrageenan-induced inflammation. Therefore the thermal probe test is a novel behavioral assay effective for quantifying thermal allodynia and analgesia in mouse models of pain.

CaMKII Phosphorylation in Primary Somatosensory Cortical Neurons is Involved in the Inhibition of Remifentanil-induced Hyperalgesia by Lidocaine in Male Sprague-Dawley Rats.

Previous clinical studies have shown that lidocaine can alleviate severe postoperative pain after remifentanil-based anesthesia. Experimental studies have also demonstrated that lidocaine can inhibit remifentanil-induced hyperalgesia, yet the mechanism remains unknown. The present study explored the role of the primary somatosensory (S1) cortex in remifentanil-induced hyperalgesia as well as its inhibition by lidocaine through evaluation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylation and protein expression levels in S1 cortical neurons. Male Sprague-Dawley rats were randomly allocated to the following 3 groups: remifentanil only (R), lidocaine only (L), and remifentanil+lidocaine (RL). Experimentally naive animals were used as controls for immunoblotting and immunofluorescence evaluations. Via intravenous tail vein administration (24 G catheter), the animals received remifentanil at 2.4 μg/kg/min, lidocaine at 200 μg/kg/min, and remifentanil at 2.4 μg/kg/min plus lidocaine at 200 μg/kg/min for 2 hours. Paw withdrawal threshold (PWT) values for both mechanical and thermal hyperalgesia, along with immunoblotting and immunofluorescence, were used to measure remifentanil-induced hyperalgesia and changes in CaMKII phosphorylation (P-CaMKII) and total protein expression (T-CaMKII). There was a significant decrease in the PWT for mechanical stimulation at 0.5 and 2 hours after discontinuing infusion in groups R and RL (P<0.05, n=10 per group). However, there were no differences in thermal PWT in any group at any time period when compared with that of baseline. There was also a significant increase of P-CaMKII (not T-CaMKII) in S1 cortical neurons of group R (not L and RL groups) at 0 to 2 hours after discontinuing infusion when compared with that of the corresponding control group (P<0.05, n=6 per group) as determined by immunoblotting and immunofluorescence microscopy. These results suggested that the phosphorylation of CaMKII in S1 cortical neurons increases significantly during the process of remifentanil-induced hyperalgesia. The increase of CaMKII phosphorylation could be inhibited by systemic application of lidocaine. This inhibition may play a role in the antihyperalgesia effects of lidocaine.

Disruption of Spinal Noradrenergic Activation Delays Recovery of Acute Incision-Induced Hypersensitivity and Increases Spinal Glial Activation in the Rat

Results of clinical studies suggest that descending inhibitory controls from the brainstem are important for speeding recovery from pain after surgery. We examined the effects of destroying spinally projecting noradrenergic neurons via intrathecally administered antibody to dopamine β-hydroxylase conjugated to saporin (DβH-saporin) on recovery in an acute incisional pain model. Mechanical and thermal paw withdrawal thresholds and nonevoked spontaneous guarding scores were tested for several weeks postoperatively and analyzed using mixed effects growth curve modeling. DβH-saporin treatment resulted in a significant prolongation in the duration of mechanical and to a lesser degree thermal hypersensitivity in the ipsilateral paw of incised rats but did not increase the duration of spontaneous guarding. DβH-saporin treatment was also associated with increased microglial and astrocyte activation in the ipsilateral spinal cord 21 days after incision compared with immunoglobulin G-saporin treated controls. Chronic intrathecal administration of the α2 adrenergic receptor antagonist atipamezole (50–200 μg/d) produced similar effects. These data suggest that spinally projecting noradrenergic pathways and spinal α2 adrenergic receptor activation are important for speeding recovery from hypersensitivity after surgical incision possibly by reducing spinal glial activation. Interventions that augment the noradrenergic system might be important to speed recovery from pain after surgery. PerspectiveEndogenous descending spinal noradrenergic activation promotes resolution of incision-induced hypersensitivity and inhibits spinal microglial and astrocyte activation in part through α2 adrenergic receptors.

Role of extracellular signal-regulated kinase in central amygdala on fentanyl-induced hyperalgesia in rats

Objective To investigate the role of extracellular signal-regulated kinase1/2(ERK1/2)in the central amygdala on fentanyl-induced hyperalgesia in rats. Methods Thirty-two male Sprague-Dawley rats, weighing 60–100 g, were randomly divided into 4 groups(n=8 each)using a random number table: control group(group C), fentanyl-induced hyperalgesia group(group H), U0124 group(group U1), and U0126 group(group U2). A catheter was implanted in the central amygdale.In group C, normal saline was injected subcutaneously, and 6.5 h later dimethyl sulfoxide(DMSO)was injected via the catheter.In group H, fentanyl was injected subcutaneously to induce hyperalgesia, and 6.5 h later DMSO was injected via the catheter.In group U1, hyperalgesia was induced, and 6.5 h later ERK1 inhibitor U0124 1.5 nmol was injected via the catheter.In group U2, hyperalgesia was induced, and 6.5 h later ERK1/2 inhibitor U0126 1.5 nmol was injected via the catheter.Mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal threshold(TWT)were measured before fentanyl injection, at 6.5 h after injection, and at 30 min after DMSO or U0124/U0126 administration via the catheter(T0-2). After the last measurement of pain threshold, the rats were sacrificed, and the amygdala tissues were sampled for detection of the expression of phosphorylated ERK1/2(p-ERK1/2)by Western blot in groups C and H. Results Compared with group C, the MWT and TWT were significantly decreased at T1, 2in H and U1 groups, and at T1in group U2(P 0.05). Compared with group H, the MWT and TWT were significantly increased at T2 in group U2(P 0.05). Conclusion ERK2 activation in the central amygdala is involved in the development of fentanyl-induced hyperalgesia in rats. Key words: Extracellular signal-regulated kinase(ERK); Fentanyl; Hyperalgesia; Amygdala

Resveratrol attenuates morphine antinociceptive tolerance via SIRT1 regulation in the rat spinal cord

In recent years, researchers have begun to pay more attention to the role of Sirtuin 1 (SIRT1, a class III histone deacetylase) in pain. However, little research has been conducted examining the involvement of SIRT1 in chronic morphine tolerance. The aim of this study was to investigate the role of spinal SIRT1 and acetyl-histone H3(Ac-H3) in chronic morphine tolerance in rats. Chronic morphine tolerance was induced by twice-daily intrathecal (i.t.) injections of morphine (10μg) for 6 days. Control rats received normal saline (NS). Resveratrol (Res, a SIRT1 stimulant, 30μg i.t.) or dimethyl sulfoxide (DMSO, 10μl i.t.) was then injected on days 7–13. The thermal paw withdrawal threshold was assessed to determine the analgesic effects of morphine (10μg). qRT-PCR, western blotting and immunohistochemistry were used to detect the expression of SIRT1 and global Ac-H3. Administration of morphine for 6 days induced a stabilized antinociceptive tolerance, down-regulated SIRT1 expression and up-regulated Ac-H3 expression in the spinal dorsal horn. Resveratrol treatment from day 7 to 13 increased SIRT1 expression, suppressed global Ac-H3 expression compared to the morphine tolerance (MT) group, and significantly reversed morphine antinociceptive tolerance. These results suggest that resveratrol reversed morphine tolerance by upregulating the expression of SIRT1 in the spinal dorsal horn. SIRT1 and global Ac-H3 in the spinal cord may play an important role in the mechanisms of chronic morphine tolerance.

Gene knockdown with lentiviral vector‐mediated intrathecal RNA interference of protein kinase C gamma reverses chronic morphine tolerance in rats

Although morphine is a widely used opioid analgesic, morphine tolerance (MT) has limited the use of the drug because it creates the necessity for high doses. Protein kinase C (PKC), especially the PKCγ isoform, is considered to play a key role in the development of MT. Because RNA interference provides a powerful method for the investigation of gene function, and lentiviral delivery systems have been approved for human use, this present study examined rats tolerant to morphine to determine whether an intrathecal injection of a lentiviral vector of PKCγ short hairpin RNA (LV-shPKCγ) down-regulated the expression of the PKCγ gene and reversed MT. MT was induced by intrathecal morphine (10 µg b.i.d.) for six consecutive days. A lentiviral-mediated short hairpin RNA (shRNA) system was synthesized to deliver the PKCγ shRNAs to the spinal cord of the rats with MT. Mechanical and thermal paw withdrawal threshold were assessed to determine the analgesic effects of morphine. Expression of PKCγ mRNA and protein was determined by reverse transcriptase-polymerase chain reaction and western blotting analysis, respectively. The chronic administration of morphine induced a stabilized analgesic tolerance. A single injection of LV-shPKCγ significantly reversed morphine antinociceptive tolerance. Compared to the control group, PKCγ mRNA and protein levels were dramatically down-regulated in the LV-shPKCγ group. A single injection of LV-shPKCγ reversed MT by reducing the expression of PKCγ in the spinal cord. These findings indicate that the use of LV-shPKCγ might be a potential strategy for therapy in MT.

Activation of the Neuronal Extracellular Signal-Regulated Kinase 2 in the Spinal Cord Dorsal Horn Is Required for Complete Freund's Adjuvant-Induced Pain Hypersensitivity

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 signaling in the spinal cord dorsal horn (SCDH) has been implicated in injury-induced pain hypersensitivity. Available ERK pathway inhibitors cannot distinguish between ERK1 and ERK2, nor can they differentially target the expression of neuronal or glial ERK1/2. We selectively inhibited the expression of ERK2 in neurons of the adult mouse SCDH by use of an ERK2 small interfering RNA (siRNA) delivered by a neurotropic adenoassociated viral vector. In situ hybridization revealed a siRNA vector-induced decrease in ERK2 mRNA in the ipsilateral SCDH. Immunohistochemistry showed a decreased neuronal phospho-ERK1/2 (pERK1/2), and Western blot analysis revealed that both ERK2 expression and phosphorylation were reduced by the siRNA vector. In contrast, basal ERK1 expression was not affected, although pERK1 was slightly increased. The siRNA vector-induced knockdown of ERK2 expression in the SCDH did not alter the baseline mechanical or thermal paw withdrawal thresholds. Hindpaw intraplantar injection of complete Freund's adjuvant (CFA) produced peripheral inflammation, mechanical allodynia, and thermal hyperalgesia in vector control animals that persisted for at least 96 h. It also caused an increase in SCDH ERK1 and ERK2 levels at 96 h and pERK1 and pERK2 levels at 1 and 96 h. The ERK2 siRNA vector prevented changes in ERK1, ERK2, and pERK2. In addition, the siRNA vector protected the animals from developing mechanical allodynia and thermal hyperalgesia throughout the 96 h after CFA. These findings indicate that ERK2 in the SCDH neurons is critical for the development of inflammatory pain hypersensitivity.

Mirtazapine and its enantiomers differentially modulate acute thermal nociception in rats

The antidepressant mirtazapine is an optically active drug and currently marketed as a racemic compound consisting of its S(+) and R(−)-enantiomers in a 50:50 mixture. As stereochemistry of antidepressants has become increasingly important to consider for the relevance of their analgesic properties, we investigated the effect of (+/−)-mirtazapine and its enantiomers in an animal model of acute thermal nociception. Wistar rats were injected intrathecal with either (+/−)-mirtazapine, R(−)-mirtazapine, S(+)-mirtazapine from 1 to 0.001 mg/kg and vehicle (0.9% NaCl), respectively. The effects on thermal paw withdrawal thresholds were monitored using the Hargreaves test. (+/−)-Mirtazapine exerted pro- and antinociceptive effects in acute thermal nociception, whereas R(−)-mirtazapine showed solely antinociceptive and S(+)-mirtazapine pronociceptive properties. These results clearly demonstrate a differential effect of (+/−)-mirtazapine and its enantiomers on nociception. As R(−)-mirtazapine exerts the antinociceptive activity of the racemic mixture it may be a putative candidate for an enantioselective use as analgesic.