Antinociceptive Response Research Articles

Pharmacological restoration of anti-nociceptive functions in the prefrontal cortex relieves chronic pain.

Chronic pain affects one in four adults, and effective non-sedating and non-addictive treatments are urgently needed. Chronic pain causes maladaptive changes in the cerebral cortex, which can lead to impaired endogenous nociceptive processing. However, it is not yet clear if drugs that restore endogenous cortical regulation could provide an effective therapeutic strategy for chronic pain. Here, we studied the nociceptive response of neurons in the prelimbic region of the prefrontal cortex (PL-PFC) in freely behaving rats using a spared nerve injury (SNI) model of chronic pain, and the impact of AMPAkines, a class of drugs that increase central glutamate signaling, on such response. We found that neurons in the PL-PFC increase their firing rates in response to noxious stimulations; chronic neuropathic pain, however, suppressed this important cortical pain response. Meanwhile, CX546, a well-known AMPAkine, restored the anti-nociceptive response of PL-PFC neurons in the chronic pain condition. In addition, both systemic administration and direct delivery of CX546 into the PL-PFC inhibited symptoms of chronic pain, whereas optogenetic inactivation of the PFC neurons or administration of AMPA receptor antagonists in the PL-PFC blocked the anti-nociceptive effects of CX546. These results indicate that restoration of the endogenous anti-nociceptive functions in the PL-PFC by pharmacological agents such as AMPAkines constitutes a successful strategy to treat chronic neuropathic pain.

Nitric oxide-mediated defensive and antinociceptive responses organised at the anterior hypothalamus of mice are modulated by glutamatergic inputs from area 24b of the cingulate cortex.

Previous studies suggested that Cg1 area of the cingulate cortex of rats controls glutamate-mediated fear-induced defensive behaviour and antinociception organised at the posterior hypothalamus. In turn, microinjection of the nitric oxide donor SIN-1 into the anterior hypothalamus of mice produced defensive behaviours and fear-induced antinociception. However, it remains unknown whether Cg1 also modulates the latter mechanisms in mice. The present study examined the influence of Cg1 on SIN1-evoked fear-induced defensive behaviour and antinociception organised at the anterior hypothalamus of mice. The fear-like behavioural and antinociceptive responses to the microinjection of SIN-1 (300 nmol) into the anterior hypothalamus were evaluated after the microinjection of either N-methyl-D-aspartic acid receptor agonist (0.1, 1 and 10 nmol) or physiological saline into the cingulate cortex of C57BL/6 male mice. In addition, neurotracing and immunohistochemistry were used to characterise Cg1-anterior hypothalamus glutamatergic pathways. The data showed that activation of Cg1 N-methyl-D-aspartic acid receptors increased escape while reducing freezing and antinociceptive responses to SIN-1 microinjections into the anterior hypothalamus. Anterograde neural tract tracer co-localised with VGLUT2-labelled fibres suggests these responses are mediated by glutamatergic synapses at the anterior hypothalamus. In contrast with previous studies showing that Cg1 facilitates both escape and antinociception to chemical stimulation of the posterior hypothalamus in rats, the present data suggest that Cg1 facilitates escape while inhibiting defensive antinociception produced by the microinjection of SIN-1 in the anterior hypothalamus of mice. Accordingly, Cg1 may have opposite effects on antinociceptive responses organised in the anterior and posterior hypothalamus of mice and rats, respectively.

The Sigma 2 receptor promotes and the Sigma 1 receptor inhibits mu-opioid receptor-mediated antinociception

The Sigma-1 receptor (σ1R) has emerged as an interesting pharmacological target because it inhibits analgesia mediated by mu-opioid receptors (MOR), and also facilitates the development of neuropathic pain. Based on these findings, the recent cloning of the Sigma-2 receptor (σ2R) led us to investigate its potential role as a regulator of opioid analgesia and of pain hypersensitivity in σ2R knockout mice. In contrast to σ1R deficient mice, σ2R knockout mice developed mechanical allodynia following establishment of chronic constriction injury-induced neuropathic pain, which was alleviated by the σ1R antagonist S1RA. The analgesic effects of morphine, [D-Ala, N-MePhe, Gly-ol]-encephalin (DAMGO) and β-endorphin increased in σ1R−/− mice and diminished in σ2R−/− mice. The analgesic effect of morphine was increased in σ2R−/− mice by treatment with S1RA. However, σ2R−/− mice and wild-type mice exhibited comparable antinociceptive responses to the delta receptor agonist [D-Pen2,5]-encephalin (DPDPE), the cannabinoid type 1 receptor agonist WIN55,212-2 and the α2-adrenergic receptor agonist clonidine. Therefore, while σR1 inhibits and σ2R facilitates MOR-mediated analgesia these receptors exchange their roles when regulating neuropathic pain perception. Our study may help identify new pharmacological targets for diminishing pain perception and improving opioid detoxification therapies.

Role of D1- and D2-like dopamine receptors within the dentate gyrus in antinociception induced by chemical stimulation of the lateral hypothalamus in an animal model of acute pain

The dentate gyrus (DG) as the main gateway of the hippocampal formation (HF) plays a crucial role in pain modulation. For this purpose, the HF receives dopaminergic inputs originated from the substantia nigra and the ventral tegmental area. It has previously been shown that the lateral hypothalamus (LH) stimulation produces antinociception via orexinergic projections of the LH to the DG region. So, given the presence of dopamine receptors in the DG and the undeniable role of the dopaminergic system in pain modulation, the current study was conducted to investigate the role of dopamine receptors located within the DG in the LH stimulation-induced pain modulation. Adult male Wistar rats weighing 220–250 g were unilaterally implanted with two separate cannulas into the LH and DG. Intra-DG administration of D1- or D2-like dopamine receptor antagonist (0.125, 0.25, 1, and 4 µg) was performed just 5 min before chemical stimulation of the LH by carbachol (250 nM). Nociceptive assay was done using the tail-flick apparatus immediately after the last microinjection. The results demonstrated that the administration of SCH23390 or Sulpiride into the DG decreased the intra-LH carbachol-induced antinociceptive responses and decreased the tail-flick latency times. The role of D2-like dopamine receptor of the DG was more prominent than that of D1-like dopamine receptor in antinociceptive response produced by the LH stimulation. It seems to be a complex neural circuitry in which the LH produces antinociceptive effects, in part, via dopamine receptors located in the DG region.

The antinociceptive mechanisms of melatonin: role of L-arginine/nitric oxide/cyclic GMP/KATP channel signaling pathway.

Pain is one of the most common medical challenges, reducing life quality. Despite the progression in pain management, it has remained a clinical challenge, which raises the need for investigating novel antinociceptive drugs with correspondence signaling pathways. Besides, the precise antinociceptive mechanisms of melatonin are not revealed. Accordingly, owing to the critical role of L-arginine/nitric oxide (NO)/cyclic GMP (cGMP)/KATP in the antinociceptive responses of various analgesics, the role of this signaling pathway is evaluated in the antinociceptive effects of melatonin. Male NMRI mice were intraperitoneally pretreated with the injection of L-arginine (NO precursor, 100 mg/kg), N(gamma)-nitro-L-arginine methyl ester [L-NAME, NO synthase (NOS) inhibitor, 30 mg/kg], S-nitroso-N-acetylpenicillamine (SNAP, NO donor, 1 mg/kg), sildenafil (phosphodiesterase inhibitor, 0.5 mg/kg), and glibenclamide (KATP channel blocker, 10 mg/kg) alone and before the administration of the most effective dose of melatonin amongst the intraperitoneal doses of 50, 100, and 150 mg/kg. The formalin test (2%, 25 µL, intra-plantarly) was done following the melatonin administration, then the nociceptive responses of mice were evaluated during the early phase for 5 min and the late phase for 15 min. The results showed that 100 mg/kg dose of melatonin carried out the most antinociceptive effects. While the antinociceptive effect of melatonin was increased by L-arginine, SNAP, and sildenafil, it was significantly reduced by L-NAME and glibenclamide in both phases of the formalin test, with no relation to the sedative effects of melatonin evaluated by the inclined plane test. In conclusion, the antinociceptive effect of melatonin is mediated through the L-arginine/NO/cGMP/KATP pathway.

Behavioural and molecular effects of cannabidiolic acid in mice

AimsCannabidiolic acid (CBDA) is one of the most abundant phytocannabinoid acids in the Cannabis sativa plant. It has been shown that it is able to exert some therapeutic effects such as antiemetic, anti-inflammatory, anxiolytic or antidepressant, although some of them remain under debate. In the present study we aim to assess the potential behavioural effects of CBDA as well as its modulation of neuroinflammatory markers in the prefrontal cortex (PFC). Main methodsThe effects of acute and repeated CBDA (0.001–1 mg/kg i.p.) treatments were evaluated on cognitive, emotional, motivational and nociceptive behaviours in male CD1 mice. For this, Y-maze and elevated plus maze paradigms, spontaneous locomotor activity, social interaction, hot-plate, formalin and tail suspension tests were used. We also studied the effects of CBDA on the rewarding responses of cocaine in the conditioned place preference (CPP) paradigm. Finally, PFC was dissected after acute and repeated CBDA treatments to evaluate inflammatory markers. Key findingsAcute CBDA treatment induced antinociceptive responses in the hot-plate test. A 10-day CBDA treatment reduced despair-like behaviour in the tail suspension test. CBDA did not alter the results of the remaining behavioural tests assayed, including cocaine-induced reward in the CPP. Regarding the biochemical analysis, repeated CBDA treatment diminished the level of peroxisome proliferator-activated receptor gamma (PPAR-γ) and increased that of interleukin-6 (IL-6) protein in PFC. SignificanceThese results show that CBDA has limited in vivo effects on the modulation of mice behaviour, supporting the current skepticism regarding its therapeutic potential.

Modulatory role of hippocampal dopamine receptors in antinociceptive responses induced by chemical stimulation of the lateral hypothalamus in an animal model of persistent inflammatory pain

The lateral hypothalamus (LH) plays a complicated role in the modulation of inflammatory pain. There are a number of connections between the LH and the hippocampus. This study evaluated the pain modulatory role of intra-CA1 dopamine receptors in LH chemical stimulation-induced antinociception in the formalin test (persistent inflammatory pain model). Vehicle control groups received saline or DMSO into the CA1 and saline into the LH. Carbachol control groups received carbachol (250 nM) into the LH 5 min after saline or DMSO injections into the CA1. In the treatment groups, intra-CA1 administration of SCH-23,390 or Sulpiride (D1- or D2-like dopamine receptor antagonists, respectively) was performed 5 min before carbachol injection. Formalin tests were done in all rats 5 min after the second injection. LH chemical stimulation-induced antinociception during both phases of the formalin test was alleviated by the intra-CA1 administration of dopamine receptor antagonists. The inhibitory effects of the D1 or D2-like dopamine receptor antagonist on LH chemical stimulation-induced analgesia was nearly the same in the both phases of formalin‐induced pain-related behaviors. The findings show that the LH-CA1 pathway contributes to the modulation of formalin-induced pain. Moreover, the results indicate that D1- and D2-like dopamine receptors in the CA1 participate in the LH chemical stimulation-induced antinociception.

Pain relief devoid of opioid side effects following central action of a silylated neurotensin analog

Neurotensin (NT) exerts naloxone-insensitive antinociceptive action through its binding to both NTS1 and NTS2 receptors and NT analogs provide stronger pain relief than morphine on a molecular basis. Here, we examined the analgesic/adverse effect profile of a new NT(8–13) derivative denoted JMV2009, in which the Pro10 residue was substituted by a silicon-containing unnatural amino acid silaproline. We first report the synthesis and in vitro characterization (receptor-binding affinity, functional activity and stability) of JMV2009. We next examined its analgesic activity in a battery of acute, tonic and chronic pain models. We finally evaluated its ability to induce adverse effects associated with chronic opioid use, such as constipation and analgesic tolerance or related to NTS1 activation, like hypothermia. In in vitro assays, JMV2009 exhibited high binding affinity for both NTS1 and NTS2, improved proteolytic resistance as well as agonistic activities similar to NT, inducing sustained activation of p42/p44 MAPK and receptor internalization. Intrathecal injection of JMV2009 produced dose-dependent antinociceptive responses in the tail-flick test and almost completely abolished the nociceptive-related behaviors induced by chemical somatic and visceral noxious stimuli. Likewise, increasing doses of JMV2009 significantly reduced tactile allodynia and weight bearing deficits in nerve-injured rats. Importantly, repeated agonist treatment did not result in the development of analgesic tolerance. Furthermore, JMV2009 did not cause constipation and was ineffective in inducing hypothermia. These findings suggest that NT drugs can act as an effective opioid-free medication for the management of pain or can serve as adjuvant analgesics to reduce the opioid adverse effects.

Role of Dopaminergic Receptors Within the Ventral Tegmental Area in Antinociception Induced by Chemical Stimulation of the Lateral Hypothalamus in an Animal Model of Orofacial Pain.

IntroductionThe ventral tegmental area (VTA), as one of the classical components of the brain reward circuitry, shares large neural networks with the pain processing system. We previously showed the role of VTA dopamine receptors in modulation of lateral hypothalamus (LH)-induced antinociception in acute pain conditions. However, considering the fact that the neural systems involved in the mediation of tonic pain are not the same as those that mediate phasic pain. In the present study, we aimed to examine the role of intra-VTA dopamine receptors in LH-induced antinociceptive responses during tonic orofacial pain conditions.MethodsMale Wistar rats weighing 230–250 g were implanted with two separate cannulae into the LH and VTA on the same side. Different solutions of carbachol (62.5, 125 and 250 nM), as a non-selective cholinergic receptor agonist that activates the LH projecting neurons, were microinjected into the LH. In the other groups, D1-like dopamine receptor antagonist, SCH-23390 (0.25, 1 and 4 µg/03 µL saline) or D2-like dopamine receptor antagonist, Sulpiride (0.25, 1 and 4 µg/0.3 µL DMSO 12%) were microinjected into VTA, 5 min prior intra-LH carbachol (250 nM), then subjected to orofacial formalin test. Intra-LH carbachol microinjection dose-dependently attenuated biphasic orofacial pain.ResultsIntra-VTA administration of SCH-23390 or Sulpiride dose-dependently decreased intra-LH carbachol-induced antinociception during both phases of orofacial formalin test with further effects in the late phase.DiscussionThe findings suggest that chemical stimulation of the LH by carbachol possibly activates the orexin projecting neurons and subsequently, the VTA dopaminergic neurons involved in the orofacial pain modulation. Detecting such neural circuitry offers an alternative approach in the development of more efficient therapies for such debilitating pain conditions.

Evaluation of Topical Anti-Inflammatory Effects of a Gel Formulation with Plantago Lanceolata, Achillea Millefolium, Aesculus Hippocastanum and Taxodium Distichum

Medicinal plants hold a significant place as alternative treatments available for inflammatory diseases, with many phytoconstituents being frequently tested in vitro for their biological activities. In the current study, we investigated the in vivo anti-inflammatory properties of a novel active gel formulation, combining Achillea millefolium and Taxodium distichum essential oils with extracts of Aesculus hippocastanum seeds and Plantago lanceolata leaves. The toxicity of the obtained extracts and volatile oils was determined using the invertebrate model based on Daphnia magna. Anti-inflammatory potential was evaluated by the plethysmometric method on Wistar rats, expressed as the inhibition of the inflammatory oedema (%IIO), while the antinociceptive response was determined on NMRI mice, according to the tail-flick latency method. The tested gel’s efficacy was similar to the 5% diclofenac standard (maximal %IIO of 42.01% vs. 48.70%, respectively), with the anti-inflammatory effect being observed sooner than for diclofenac. Our active gel also produced a significant prolongation of tail-flick latencies at both 60 and 120 min, comparable to diclofenac. Consequently, we can imply that the active constituents present in vivo anti-inflammatory properties, and the prepared gel may be suited for use as an alternative treatment of topical inflammatory conditions.

C57BL/6 substrain differences in formalin-induced pain-like behavioral responses

Substantial evidence from preclinical models of pain suggests that basal and noxious nociceptive sensitivity, as well as antinociceptive responses to drugs, show significant heritability. Individual differences to these responses have been observed across species from rodents to humans. The use of closely related C57BL/6 inbred mouse substrains can facilitate gene mapping of acute nociceptive behaviors in preclinical pain models. In this study, we investigated behavioral differences between C57BL/6 J (B6 J) and C57BL/6 N (B6 N) substrains in the formalin test, a widely used tonic inflammatory pain model, using a battery of pain-related phenotypes, including reflexive tests, nesting, voluntary wheel running, sucrose preference and anxiety-like behavior in the light/dark test at two different time points (1-h and 24-h). Our results show that these substrains did not differ in reflexive thermal and mechanical responses at the 1-h time point. However, B6 N substrain mice showed increased sensitivity to spontaneous pain-like behaviors. In addition, B6 N substrain continued to show higher levels of mechanical hypersensitivity compared to controls at 24-h. indicating that mechanical hypersensitivity is a more persistent pain-related phenotype induced by formalin. Finally, no sex differences were observed in our outcome measures. Our results provide a comprehensive behavioral testing paradigm in response to an inflammatory agent for future mouse genetic studies in pain.

Daily intermittent fasting in mice enhances morphine-induced antinociception while mitigating reward, tolerance, and constipation.

The opioid epidemic has plagued the United States with high levels of abuse and poor quality of life for chronic pain patients requiring continuous use of opioids. New drug discovery efforts have been implemented to mitigate this epidemic; however, new medications are still limited by low efficacy and/or high side effect and abuse potential. Intermittent fasting (IF) has recently been shown to improve a variety of pathological states, including stroke and neuroinflammation. Numerous animal and human studies have shown the benefits of IF in these disease states, but not in pain and opioid treatment. We thus subjected male and female CD-1 mice to 18-hour fasting intervals followed by 6-hour feed periods with standard chow for 1 week. Mice that underwent this diet displayed an enhanced antinociceptive response to morphine both in efficacy and duration using thermal tail-flick and postoperative paw incision pain models. While showing enhanced antinociception, IF mice also demonstrated no morphine reward and reduced tolerance and constipation. Seeking a mechanism for these improvements, we found that the mu-opioid receptor showed enhanced efficacy and reduced tolerance in the spinal cord and periaqueductal gray, respectively, from IF mice using a S-GTPγS coupling assay. These improvements in receptor function were not due to changes in mu-opioid receptor protein expression. These data suggest that a daily IF diet may improve the therapeutic index of acute and chronic opioid therapies for pain patients in the clinic, providing a novel tool to improve patient therapy and reduce potential abuse.

Glabridin Alleviates Inflammation and Nociception in Rodents by Activating BK<sub>Ca</sub> Channels and Reducing NO Levels

Inflammation, and the pain that accompanies it, is a natural response of the body. The licorice plant (Glycyrrhiza glabra) have demonstrated anti-inflammatory, anti-edematous, and anti-nociceptive effects of its extracts. The effective ingredient remains unidentified; however, one possibility is the unique isoflavone glabridin. The anti-nociceptive, and anti-inflammatory effects of glabridin and its possible mechanism with focus on the large conductance Ca2+-activated K+ (BKCa) channels and L-arginine-nitric oxide (NO) pathway were examined by using different tests. In order to determine the anti-edematous, anti-nociceptive, and anti-oxidative effects of glabradin, some tests such as the tail flick, hotplate, carrageenan-induced paw edema, air pouch, acetic-acid-induced writhing, formalin, and capsaicin tests, as well as toxicity and open field tests were made. Glabridin was administered to rats (n = 8) or mice (n = 8) for 3 d at 3 doses (10, 20, and 40 mg/kg). Glabridin inhibited cytokine production and showed an anti-nociceptive response via the activating of BKCa channels and downregulating NO level and partially transient receptor potential vanilloid-1 pathways. It also demonstrated anti-inflammatory effects by inhibiting cyclooxygenase (COX) activity, while showing no cytotoxicity. Glabridin, however, showed no anti-nociceptive effect in the neurogenic phase. Glabridin is a promising substance in terms of its anti-nociceptive and anti-inflammatory effects by disrupting peripheral NO production, inhibiting cyclic guanosine monophosphate (cGMP) activation and activating BKCa channels and its lack of acute and subacute toxic effects.

Neuroprotective and anti-inflammatory activity of DAT inhibitor R-phenylpiracetam in experimental models of inflammation in male mice.

R-phenylpiracetam (R-PhP, (4R)-2-(4-phenyl-2-oxopyrrolidin-1-yl)acetamide) is an optical isomer of phenotropil, a clinically-used nootropic drug that improves physical condition and cognition. Recently, R-PhP was shown to bind to the dopamine transporter (DAT). Since growing evidence suggests that dysfunction of the dopaminergic system is associated with persistent neuroinflammation, the aim of this study was to determine whether R-PhP, an inhibitor of DAT, has neuroprotective and anti-inflammatory effects in male mice. The pharmacokinetic profiles ofR-PhPin mouse plasma and its bioavailability in brain tissue were assessed. To study possible molecular mechanisms involved in the anti-inflammatory activity of R-PhP, target profiling was performed using radioligand binding and enzymatic activity assays. To clarify the neuroprotective and anti-inflammatory effects of R-PhP, we used a lipopolysaccharide (LPS)-induced endotoxaemia model characterized by reduced body temperature and overexpression of inflammatory genes in the brain. In addition, the antinociceptive and anti-inflammatory effects of R-PhP were tested using carrageenan-induced paw oedema and formalin-induced paw-licking tests. R-PhP (50mg/kg) reached the brain tissue 15min after intraperitoneal (ip) and peroral (po) injections. The maximal concentration of R-PhP in the brain tissues was 28µg/g and 18µg/g tissue after ip and po administration, respectively. In radioligand binding assays, DAT was the only significant molecular target found for R-PhP. A single ip injection of R-PhP significantly attenuated the LPS-induced body temperature reduction and the overexpression of inflammatory genes, such as tumour necrosis factor-α (TNF-α), interleukin 1 beta (IL-1β) and inducible nitric oxide synthase (iNOS). Seven-day po pretreatment with R-PhP dose-dependently reduced paw oedema and the antinociceptive response, as shown by the carrageenan-induced paw oedema test. In addition, R-PhP decreased the nociceptive response during the inflammatory phase in the formalin-induced paw-licking test. Our study showed that R-PhP possesses neuroprotective and anti-inflammatory effects, demonstrating the potential of DAT inhibitors as effective therapeutics.

Synergistic interaction between butorphanol and dexmedetomidine in antinociception

Opioid analgesics and the α2-adrenergic receptor (α2AR) agonists are found to produce synergistic antinociception when administered in combination. In this study interactions between butorphanol and dexmedetomidine were investigated in the thermal pain and autonomous locomotor activity. Butorphanol and dexmedetomidine were administered subcutaneously alone and in combination in a fixed-dose ratio (3:1) to assess the antinociceptive and sedative responses. Butorphanol produced antinociception in the hot-plate test via three major opioid receptor subtypes, i.e. MORs, KORs and DORs, while in the tail-immersion test the antinociception was produced by MORs and KORs, whereas dexmedetomidine exhibited antinociception by α2ARs in both tests. They exhibited dose- and time-dependent antinociception and inhibition of locomotor activity when administered alone, while their combination displayed enhanced therapeutic effects. Isobolographic analysis revealed that combined butorphanol and dexmedetomidine produced synergistic interactions in the hot-plate, tail-immersion and locomotor activity tests. Furthermore, the analgesic synergy was also approved to be modulated by MORs, KORs, DORs and α2ARs. Hence we concluded from this study that combined butorphanol and dexmedetomidine produced synergistic antinociception that may be helpful in facilitating clinical management of acute nociceptive pain.

A Role for Keratinocytes in Peripheral Kappa Opioid Receptor‐Mediated Antinociception

Peripherally‐expressed kappa opioid receptors (KOR) are a promising target to treat inflammatory pain that would be devoid of CNS related adverse effects. We have found that local injectionof KOR agonists into the rat hindpaw produces a profound antinociceptive response that is mediated by activation of G protein‐coupled inwardly‐rectifying potassium (GIRK) channels. We confirmed with immunohistochemistry that both KOR and GIRK2 channels are expressed in intraepidermal nerve fibers (IENFS), which propagate nociceptive signals from the periphery to the spinal cord. Interestingly, we also found that KOR and GIRK2 channels are expressed in keratinocytes. Keratinocytes make up the majority of cells in the epidermis and evidence suggests that they play a role in pain and sensory processing. A variety of nociceptive signaling receptors are expressed in keratinocytes, including transient receptor potential vanilloid channels 1 through 4 (TRPV1‐4), suggesting that keratinocytes can modulate and possibly initiate nociception in the epidermis. Here we sought to determine if expression of GIRK channels in keratinocytes plays a role in KOR‐mediated peripheral antinociception. We assessed the effect of local hindpaw administration of a GIRK2 shRNA expression plasmid fused to a HIV‐1 Tat peptide that facilitates transport into cells. Unlike IENFs, keratinocytes are capable of transcription, thus we used this method to ensure knockdown would occur in keratinocytes and not in IENFs. Rats were injected intraplantarly (i.pl., 50ul) with either GIRK2 shRNA or scrambled non‐effective (sNE) shRNA plasmid complex along with Fugene HD daily for two days. 48 hours after the second injection, the rats were injected i.pl. with ML297 (33ug), a direct GIRK channel activator, and paw withdrawal latencies (PWL) to a thermal stimulus were determined over a 20 min period. ML297 produced a robust (+5 sec above baseline) antinociceptive response in contralateral (non‐shRNA plasmid injected) paws and in hindpaws injected with the sNE shRNA plasmid. Theantinociceptive response to ML297 was lost completely in hindpaws injected with GIRK2 shRNA plasmid. We next determined if local knockdown of GIRK2 in keratinocytes altered the behavioral response to the KOR agonist, U50,488. Following administration of either GIRK2 shRNA or sNE shRNA plasmid, rats were injected with PGE2 (i.pl., 0.3ug) with or without U50,488 (0.1ug) and PWL to the heat stimulus was measured. The sNE shRNA plasmid did not alter the KOR agonist‐mediated reduction of PGE2‐evoked thermal allodynia, however, the GIRK2 shRNA completely blocked the KOR‐mediated response. PGE2‐evoked thermal allodynia in the absence of agonist was unaltered in hindpaws treated with either shRNA plasmid. These data suggest that GIRK channels expressed in keratinocytes play an essential role in peripheral KOR‐mediated antinociceptive signaling in the rat hindpaw.Support or Funding InformationThis work is supported by NIH/NIDA RO1 DA 038645.

Chronic Fluoxetine Impairs the Effects of 5-HT1A and 5-HT2C Receptors Activation in the PAG and Amygdala on Antinociception Induced by Aversive Situation in Mice.

Growing evidence suggests an important role of fluoxetine with serotonin 5-HT1A and 5-HT2C receptors in the modulation of emotion and nociception in brain areas such as the amygdala and periaqueductal gray (PAG). Acute fluoxetine impairs 5-HT2C (but not 5-HT1A) receptor activation in the amygdaloid complex. Given that fluoxetine produces its clinical therapeutic effects only when given chronically, this study investigated the effects of chronic treatment with fluoxetine on the effects produced by 5-HT1A or 5-HT2C receptors activation in the amygdala or PAG on fear-induced antinociception. We recorded the effects of chronic fluoxetine on serotonin and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) levels as well as serotonin turnover; 5-HT1A and 5-HT2C receptor protein levels in the amygdala and PAG. Also, we evaluated the effects of chronic fluoxetine combined with intra-amygdala or intra-PAG injection of MK-212 (a 5-HT2C agonist; 0.63 nmol) or 8-OH-DPAT (a 5-HT1A agonist; 10 nmol) on the antinociceptive response in mice confined in the open arm of the elevated plus-maze (EPM). Nociception was assessed with the writhing test induced by intraperitoneal injection of 0.6% acetic acid. Results showed that fluoxetine (20 mg/kg, s.c.) enhanced the open-arm induced antinociception (OAA) and reduced the number of writhes in mice confined in the enclosed arm, featuring an analgesic effect. In addition, fluoxetine increased the expression of 5-HT2C receptors and 5-HT levels whereas reduced its turnover in the amygdala. While fluoxetine did not change 5-HT and 5-HIAA levels, and its turnover in the PAG, it up-regulated 5HT1A and 5-HT2C receptors in this midbrain area. Chronic fluoxetine (5.0 mg/Kg, an intrinsically inactive dose on nociception) antagonized the enhancement of OAA produced by intra-amygdala or intra-PAG injection of MK-212. Fluoxetine also impaired the attenuation of OAA induced by intra-amygdala injection of 8-OH-DPAT and totally prevented OAA in mice that received intra-PAG 8-OH-DPAT. These results suggest that (i) 5-HT may facilitate nociception and intensify OAA, acting at amygdala 5-HT1A and 5-HT2C receptors, respectively, and (ii) fluoxetine modulates the OAA through activation of 5-HT2C receptors within the PAG. These findings indicate that chronic fluoxetine impairs the effects of 5-HT1A and 5-HT2C receptors activation in the amygdala and PAG on fear-induced antinociception in mice.

Role of hippocampal dopamine receptors in the antinociceptive responses induced by chemical stimulation of the lateral hypothalamus in animal model of acute pain

Dopamine is the predominant catecholamine neurotransmitter in the mammalian brain which has been shown to play a critical role in antinociceptive process. Previous studies have shown that the role of CA1 region of the hippocampus in antinociception induced by stimulation of the lateral hypothalamus (LH) through the dopaminergic system in tonic pain. In this study, we tried to assess the involvement of intra-hippocampal D1- and D2-like dopamine receptors in the LH stimulation-induced antinociception during the tail-flick test as an animal model of acute pain. Ninety-five male Wistar rats were unilaterally implanted with two separate cannulae into the LH and CA1. Animals received intra-CA1 infusion of SCH-23390 (0.25, 1 and 4 µg/rat), as a D1-like dopamine receptor antagonist and sulpiride (0.125, 0.25, 1 and 4 µg/rat), as a D2-like dopamine receptor antagonist, 2 min before intra-LH administration of carbachol (250 nM/rat). The antinociceptive effects of SCH-23390 and sulpiride were measured by using a tail-flick analgesiometer and represented as the maximal possible effect (%MPE). Also, the locomotion tracking apparatus was used to measure the locomotor activity of animals. Results showed that intra-CA1 administration of SCH-23390 or sulpiride could prevent the intra-LH carbachol-induced antinociception. This effect was a little more dominant after blocking the D2-like dopamine receptor in the CA1. These findings revealed that D1- and D2-like dopamine receptors within the CA1 play an important role in antinociceptive responses induced by chemical stimulation of the LH. It could be suggested that dopamine receptors in the CA1 were triggered by LH orexinergic projections.

Role of β-Caryophyllene in the Antinociceptive and Anti-Inflammatory Effects of Tagetes lucida Cav. Essential Oil.

Tagetes lucida Cav. (Asteraceae) is an ancient medicinal plant commonly used to alleviate pain. Nevertheless, scientific studies validating this property are lacking in the literature. Animal models of pain were used to evaluate the antinociceptive and anti-inflammatory activities of T. lucida essential oil (TLEO) and a bioactive metabolite. The chemical constitution and possible toxicity of the extract and the mechanism of action of β-caryophyllene were also explored. Temporal course curves and dose–response graphics were generated using TLEO (0.1–10 mg/kg or 3.16–31.62 mg/kg) and β-caryophyllene (3.16–10 mg/kg). Metamizole (80 mg/kg) and indomethacin (20 mg/kg) were used as reference drugs in the formalin assay and writhing test in rats and mice, respectively. The β-caryophyllene mechanism of action was explored in the presence of naloxone (1 mg/kg), flumazenil (10 mg/kg), WAY100635 (0.16 mg/kg), or nitro-l-arginine methyl ester (L-NAME) (20 mg/kg) in the formalin test in rats. GC/MS analysis demonstrated the presence of geranyl acetate (49.89%), geraniol (7.92%), and β-caryophyllene (6.27%). Significant and dose-dependent antinociceptive response was produced by TLEO and β-caryophyllene without the presence of gastric damage. In conclusion, β-caryophyllene was confirmed as a bioactive compound in the T. lucida analgesic properties by involving the participation of receptors like opioids, benzodiazepines, and Serotonin 1A receptor (5-HT1A), as well as nitric oxide.

CANNABINOID RECEPTORS ARE NOT INVOLVED IN ANTINOCICEPTION INDUCED BY SYSTEMIC DICLOFENAC IN MICE

Aims: It has been long suspected that the cannabinoid system participates in the antinociceptive effects of nonsteroidal anti-inflammatory drugs. We studied the possible effects of cannabinoid receptor antagonism on diclofenac-induced antinociception in the writhing test in mice. Methods: In our study, male BALB/c mice, weighing 20-30 g, were used. Writhing responses were produced by intraperitoneal injection of 0.6% acetic acid. Different doses of diclofenac (3, 10, 30 mg/kg, i.p.) were tested, then the influence of AM-251 (1 mg/kg, i.p.), a cannabinoid CB1 receptor antagonist and AM-630 (3 mg/kg, i.p.), a cannabinoid CB2 receptor antagonist on the antinociceptive effects of diclofenac was studied. Results: Diclofenac administration elicited a significant, dose-dependent antinociceptive response; however, neither the cannabinoid CB1 receptor antagonist AM-251 nor the cannabinoid CB2 receptor antagonist AM-630 had any influence on the antinociceptive effect of diclofenac. Conclusion: Iinhibition of cannabinoid receptors does not contribute to the antinociceptive action of systemic diclofenac. Further studies are needed to explain the antinociceptive mechanism of diclofenac. Keywords: AM-251, AM-630, antinociception, cannabinoid receptors, diclofenac