Antinociceptive Response Research Articles

Orexin A induced antinociception in the ventral tegmental area involves D1 and D2 receptors in the nucleus accumbens

Previous studies have shown that there are functional interactions among the lateral hypothalamus (LH), ventral tegmental area (VTA) and the nucleus accumbens (NAc), implicating pain modulation in the central nervous system. It has been shown that the LH–VTA orexinergic projecting neurons play an important role in mediating the suppression of nociception in animal models. However, little is known about the function of intra-VTA orexin receptors and involvement of D1/D2 receptors within the NAc in this antinociception. In the present study, we investigated the effect of direct administration of orexin A into the VTA, and examined the role of intra-accumbal dopamine receptors in tail-flick test as a model of acute nociceptive responses in rats. Adult male Wistar rats were unilaterally implanted by two separate cannulae into the VTA and NAc. The results showed that intra-VTA orexin A (0.055, 0.55, 5.5 and 55ng/0.3μl saline) could induce antinociception in a dose-dependent manner. In two separated supergroups, rats received intra-accumbal infusions of D1 and D2 receptor antagonists, SCH-23390 and sulpiride (0.125, 0.25, 1 and 4μg/rat) prior to intra-VTA orexin A (5.5ng/rat) administration respectively. Antinociceptive responses of drugs are represented as maximal possible effect in 5, 15, 30, 45 and 60min after their administrations. Our findings showed that intra-accumbal SCH-23390 and sulpiride dose-dependently prevented intra-VTA orexin-induced antinociception. Nevertheless, this effect is more potent in animals that received D2 receptor antagonist. It is supposed that orexin A can induce the antinociception through activation of orexinergic receptors which activate the dopaminergic inputs to the NAc in rats.

Synergistic effect of the interaction between curcumin and diclofenac on the formalin test in rats

The association of non-steroidal anti-inflammatory drugs with certain plant extracts can increase antinociceptive activity, permitting the use of lower doses and thus limiting side effects. Therefore, the aim objective of the current study was to examine the effects of curcumin on the nociception and pharmacokinetics of diclofenac in rats. Antinociception was assessed using the formalin test. Diluted formalin was injected subcutaneously into the dorsal surface of the right hind paw. Nociceptive behavior was quantified as the number of flinches of the injected paw during 60min after injection, and a reduction in formalin-induced flinching was interpreted as an antinociceptive response. Rats were treated with oral diclofenac (1–31mg/kg), curcumin (3.1–100mg/kg) or the diclofenac–curcumin combination (2.4–38.4mg/kg). To determine the possibility of a pharmacokinetic interaction, the oral bioavailability of diclofenac (10mg/kg) was studied in presence and the absence of curcumin (31mg/kg). Diclofenac, curcumin, or diclofenac–curcumin combination produced an antinociceptive effect on the formalin test. ED30 values were estimated for the individual drugs, and an isobologram was constructed. The derived theoretical ED30 for the antinociceptive effect (19.2mg/kg) was significantly different from the observed experimental ED30 value (9.8mg/kg); hence, the interaction between diclofenac and curcumin that mediates the antinociceptive effect was synergistic. Notwithstanding, the interaction does not appear to involve pharmacokinetic mechanisms, as oral curcumin failed to produce any significant alteration in oral diclofenac bioavailability. Data suggest that the diclofenac–curcumin combination can interact at the systemic level and may have therapeutic advantages for the clinical treatment of inflammatory pain.

Inflammatory nociception responses do not vary with age, but diminish with the pain history.

Some of the relevant factors that must be considered when dealing with old age include its growing numbers in the general population and pain contention in this age group. In this sense, it is important to study whether antinociceptive responses change with age. To elucidate this point, persistent pain in animals is the preferred model. In addition, the response to inflammatory pain in the same individual must be explored along its lifetime. Male Wistar rats were infiltrated with carrageenan (50 μl intraplantar) and tested 3 h and 24 h after injection using thermal (plantar test) and mechanociceptive tests (von Frey). The rats were divided into the following groups: (a) young rats infiltrated for the first time at 12 weeks of age and re-infiltrated at 15 and 17 weeks; (b) adult rats infiltrated for the first time at 28 weeks of age and re-infiltrated at 44 and 56 weeks; and (c) old rats infiltrated for the first time at 56 weeks of age and re-infiltrated at 72 weeks. The rats tested for the first time at 12 and 56 weeks of age showed hyperalgesia due to carrageenan infiltration at 3 h and 24 h after injection. This result showed that old rats maintain the same antialgesic response due to inflammation. However, when the injection was repeated in the three age groups, the latency to the thermal and mechanociceptive responses at 3 h is increased when compared to animals exposed for the first time to inflammation. The response to thermal and mechanociception in old rats is the same as in young animals as long as the nociceptive stimulus is not repeated. The repetition of the stimulus produces changes compatible with desensitization of the response and evidences the significance of algesic stimulus repetition in the same individual rather than the age of the individual.

Fluorescent visualization of oxytocin in the hypothalamo-neurohypophysial system.

Oxytocin (OXT) is well known for its ability to the milk ejection reflex and uterine contraction. It is also involved in several other behaviors, such as anti-nociception, anxiety, feeding, social recognition, and stress responses. OXT is synthesized in the magnocellular neurosecretory cells (MNCs) in the hypothalamic paraventricular (PVN) and the supraoptic nuclei (SON) that terminate their axons in the posterior pituitary (PP). We generated transgenic rats that express the OXT and fluorescent protein fusion gene in order to visualize OXT in the hypothalamo-neurohypophysial system (HNS). In these transgenic rats, fluorescent proteins were observed in the MNCs and axon terminals in the PP. This transgenic rat is a new tool to study the physiological role of OXT in the HNS.

Interaction between the dopaminergic and opioidergic systems in dorsal hippocampus in modulation of formalin-induced orofacial pain in rats

The hippocampus is a region of the brain that serves several functions. The dopaminergic system acts through D1- and D2-like receptors to interfere in pain modulation and the opioid receptors play major roles in analgesic processes and there are obvious overlaps between these two systems. The present study investigated the interaction between the opioidergic and dopaminergic systems in the dorsal hippocampus (CA1) region for formalin-induced orofacial pain. Two guide cannulae were stereotaxically implanted in the CA1 region and morphine (0.5, 1, 2 and 4μg/0.5μl saline) and naloxone (0.3, 1 and 3μg/0.5μl saline) were used as the opioid receptor agonist and antagonist, respectively. SKF-38393 (1μg/0.5μl saline) was used as a D1-like receptor agonist, quinpirole (2μg/0.5μl saline) as a D2-like receptor agonist, SCH-23390 (0.5μg/0.5μl saline) as a D1-like receptor antagonist and sulpiride (3μg/0.5μl DMSO) as a D2-like receptor antagonist. To induce orofacial pain, 50μl of 1% formalin was subcutaneously injected into the left side of the upper lip. Our results showed that different doses of morphine significantly reduced orofacial pain in both phases induced by formalin. Naloxone (1 and 3μg) reversed morphine induced analgesia in CA1. SKF-38393 and quinpirole with naloxone (1μg) significantly decreased formalin-induced orofacial pain in both phases. SCH-23390 had no effect on the antinociceptive response of morphine in both phases of orofacial pain. Sulpiride reversed the antinociceptive effects of morphine only in the first phase, but this result was not significant. Our findings suggest that there is cross-talk between the opioidergic and dopaminergic systems. Opioidergic neurons also exerted antinociceptive effects by modulation of the dopaminergic system in the CA1 region of the brain.

Baclofen and 2-hydroxysaclofen modify acute hypolocomotive and antinociceptive effects of nicotine

The aim of the present study was to evaluate the possible involvement of GABAB receptors in nicotine-induced hypolocomotion and antinociceptive effects in mice. Animals were exposed to nicotine only once. Acute nicotine hydrogen tartrate salt (3mg/kg; subcutaneous, s.c.) administration induced hypolocomotion and antinociceptive responses in the tail-immersion and the hot-plate tests. The effects of pretreatment with either the GABAB receptor agonist baclofen (1, 2 and 3mg/kg; intraperitoneal, i.p.) or GABAB receptor antagonist 2-hydroxysaclofen (0.25, 0.5 and 1mg/kg; i.p.) were evaluated on these behavioral nicotine responses. The GABAB receptor agonist, baclofen (3mg/kg, i.p.) abolished nicotine-induced antinociceptive effects in the tail-immersion and the hot-plate tests, but did not modify nicotine-induced hypolocomotion. In addition, the GABAB receptor antagonist, 2-hydroxysaclofen (1mg/kg, i.p.) increased nicotine-induced antinociceptive effects in the tail-immersion and the hot-plate tests, and abolished nicotine-induced hypolocomotion. The present results shed light that the GABAB receptor has an important role in mediating specific acute nicotine responses such as hypolocomotion and antinociception in mice.

Toll-like receptor 4 mutant and null mice retain morphine-induced tolerance, hyperalgesia, and physical dependence.

The innate immune system modulates opioid-induced effects within the central nervous system and one target that has received considerable attention is the toll-like receptor 4 (TLR4). Here, we examined the contribution of TLR4 in the development of morphine tolerance, hyperalgesia, and physical dependence in two inbred mouse strains: C3H/HeJ mice which have a dominant negative point mutation in the Tlr4 gene rendering the receptor non-functional, and B10ScNJ mice which are TLR4 null mutants. We found that neither acute antinociceptive response to a single dose of morphine, nor the development of analgesic tolerance to repeated morphine treatment, was affected by TLR4 genotype. Likewise, opioid induced hyperalgesia and opioid physical dependence (assessed by naloxone precipitated withdrawal) were not altered in TLR4 mutant or null mice. We also examined the behavioural consequence of two stereoisomers of naloxone: (−) naloxone, an opioid receptor antagonist, and (+) naloxone, a purported antagonist of TLR4. Both stereoisomers of naloxone suppressed opioid induced hyperalgesia in wild-type control, TLR4 mutant, and TLR4 null mice. Collectively, our data suggest that TLR4 is not required for opioid-induced analgesic tolerance, hyperalgesia, or physical dependence.

A lectin from the green seaweed Caulerpa cupressoides reduces mechanical hyper-nociception and inflammation in the rat temporomandibular joint during zymosan-induced arthritis.

Seaweed lectins have been widely investigated as anti-nociceptive and anti-inflammatory agents. This study analyzed the anti-nociceptive and anti-inflammatory responses of a lectin from the green seaweed Caulerpa cupressoides (CcL) on zymosan-induced arthritis of the rat temporomandibular joint (TMJ). Rats received i.v. CcL 30 min prior to injection of zymosan (2mg/art.) or 0.9% saline into the left TMJ. Mechanical hyper-nociception was measured by the electronic von Frey method at baseline and 4h after zymosan injection. Animals were euthanized 6h after zymosan injection and the synovial fluid was collected for leukocyte counting and myeloperoxidase activity assessment. Other animals were treated with ZnPP-IX (3mg/kg; s.c.), a specific heme oxygenase-1 pathway inhibitor, and naloxone (10 μg/art.), a nonselective opioid receptor antagonist. TMJ tissues were excised to perform histopathological and immunohistochemistry analyses. CcL (0.1, 1 or 10mg/kg) significantly reduced zymosan-induced hyper-nociception (81, 83 and 89.5%, respectively) and inhibited the leukocyte influx (77.3, 80.7 and 98.5%, respectively) compared with the zymosan-only group, as confirmed by myeloperoxidase activity; however, treatment with naloxone or ZnPP-IX did not revert the effects of CcL (10mg/kg), suggesting that the naloxone-sensitive opioid and heme oxygenase-1 pathways are not involved. CcL also reduced the leukocyte influx and the expression of IL-1β and TNF-α in the TMJ, based on histopathological and immunohistochemistry analyses, respectively. Therefore, CcL reduces TMJ hyper-nociception and inflammation with a mechanism that is partially dependent on TNF-α and IL-1β inhibition. CcL reveals a potentially valuable alternative tool for future studies of TMJ disorders.

Why do male mice spit soluble enzymes that hydrolyze extracellular nucleotides? Focus on “Prostatic acid phosphatase is the main acid phosphatase with 5′-ectonucleotidase activity in the male mouse saliva and regulates salivation”

the 5′-ectonucleotidases found on the surface of most mammalian cells and in blood serum serve as a source of adenosine derived from extracellular nucleotide degradation (11). The 5′-ectonucleotidases are phosphatases whose substrate is extracellular adenosine 5′-monophosphate (AMP) generated from adenosine 5′-tri- and diphosphates (ATP and ADP) released from injured tissues to stimulate cellular responses via P2 purinergic receptors (7). A recent study by Quintero et al. (10) investigated prostatic acid phosphatase (PAP), a 5′-ectonucleotidase that plays a role in the development of adenocarcinoma of the prostate gland. These authors showed that PAP exists as two alternative splice variants, a transmembrane prostatic acid phosphatase (TMPAP) and secreted PAP (sPAP). TMPAP and sPAP are distinguished functionally from other 5′-nucleotidases through inhibition by l−(+)−tartrate, ability to cleave phosphomonoesters besides AMP, and activity over a wide pH range (i.e., 3–8). TMPAP activation inhibits endocytosis/exocytosis through interaction with snapin (10), a soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE)-associated protein (5) that negatively regulates secretory vesicle docking and fusion with plasma membranes (Fig. 1). In addition, adenosine generated by PAP is an agonist for G protein-coupled A1 adenosine receptors (A1R) that counteract the nociceptive (i.e., pain-producing) effects of ATP (4) occurring through activation of P2X receptor ATP-gated ion channels (2; Fig. 1). However, little information is available concerning physiological roles of sPAP in biological fluids. Fig. 1. Prostatic acid phosphatase (PAP) signaling in salivary glands and saliva. In this issue of American Journal of Physiology-Cell Physiology, Araujo et al. (1) attempt to understand the role of TMPAP and sPAP in a secretory organ, the submandibular gland (SMG), which secretes fluid, proteins, and other factors into saliva, thereby bathing the oral cavity with antimicrobial and digestive agents. Using PAP−/− mice, the authors evaluate effects of deletion of this 5′-ectonucleotidase on SMG function. Comparisons between PAP−/− and wild-type SMG indicate that TMPAP is primarily expressed in granular convoluted tubule (GCT) cells of the duct but not acinar cells. Remarkably, deletion of PAP increases α-adrenergic and muscarinic cholinergic receptor-mediated saliva secretion and enhances expression of genes promoting inflammation and proliferation, particularly interferon-dependent proinflammatory genes. The authors suggest that this increase in saliva secretion relates to loss of negative control by TMPAP of exocytosis (Fig. 1). PAP deletion also increases B and T cell infiltration of SMG and levels of miR-146a that have been associated with prevention of tissue damage. Together with observations of proliferating acinar cells after PAP deletion, these results strongly suggest that SMG homeostasis in PAP−/− mice is maintained by an immune response without salivary gland degeneration seen with immune cell infiltration of SMG in the autoimmune disease Sjogren's syndrome (SS; 3, 8). Another important finding is that sPAP is only present in male mouse saliva, which correlates with reduced levels of GCT cells in female versus male SMG and is consistent with sexual dimorphism in rodent salivary glands (6). The authors suggest that mouse sPAP plays a male-specific function in which licking of wounds (perhaps caused by fighting) deposits sPAP-containing saliva that induces an anti-nociceptive response in wounded tissue by participating in the generation of adenosine from released nucleotides to activate A1 receptors (Fig. 1). Since SS has a female predominance in rodents and humans (3, 8), it would be interesting to determine whether PAP in males plays a protective role in SS by promoting gland homeostasis and delay in the onset of salivary gland dysfunction and degenerative immune cell infiltration. Another question remaining is whether extracellular nucleotides are released into the oral cavity due to tissue injury of oral epithelium caused by inflammation, exposure to toxins, SS, and radiation-induced damage occurring as a side effect of cancer therapy. Once released, degradation of extracellular nucleotides by ectonucleotide diphosphohydrolases (eNTPDases), ectoATPases, and ectoADPases would generate AMP whereupon 5′-ectonucleotidases including PAP would yield adenosine that could either be taken up by nucleoside transporters in cells and salvaged or activate P1 adenosine receptors (7, 9, 11). Further study is needed to investigate whether sPAP in saliva plays a role in the regulation of purinergic signaling in the oral cavity under physiological and pathological conditions.

Fentanyl produces an anti-hyperalgesic effect through the suppression of sodium channels in mice with painful diabetic neuropathy

Diabetic neuropathy is one of the most frequent complications of diabetes mellitus. Therefore, the present study was designed to investigate the anti-hyperalgesic mechanism of fentanyl in a mouse model of streptozotocin-induced diabetic neuropathy. The antinociceptive response was assessed by recording the latency in a tail-flick test. The tail-flick latency in diabetic mice was significantly shorter than that in non-diabetic mice. Fentanyl, at doses of 3 and 10μg/kg, s.c., produced a dose-dependent increase in the tail-flick latencies in diabetic mice. While fentanyl (3μg/kg, s.c.) did not produce a significant inhibition of the tail-flick response in non-diabetic mice, it significantly prolonged the tail-flick latency in diabetic mice to the same level as the baseline latency in non-diabetic mice. Although pretreatment with naloxone (3mg/kg, s.c.) completely antagonized fentanyl-induced antinociception in non-diabetic mice, it had no effect on the antinociceptive effect of fentanyl in diabetic mice. Pretreatment with either of the voltage-gated sodium channel openers fenvarelarte and veratridine practically abolished the antinociceptive effects of fentanyl in diabetic mice. However, neither fenvarelate nor veratridine affected the antinociceptive effect of fentanyl in non-diabetic mice. These results suggest that the anti-hyperalgesic effect of fentanyl is mediated through the blockade of sodium channels in diabetic mice, whereas opioid receptors mediate the antinociceptive effect of fentanyl in non-diabetic mice.

The role of GABA in hyperbaric oxygen‐induced acute antinociception in mice (659.16)

Earlier we reported that mice responded with an acute antinociceptive effect during an 11‐min treatment with HBO2 at 3.5 atmospheres absolute (ATA) (Ohgami et al., NeuroReport 20:1325, 2009). We endeavored to explore the possible role of GABA in this HBO2‐induced effect by pretreating different groups of male NIH Swiss mice, 20‐30 g, intracerebroventricularly (i.c.v.) or intrathecally (i.t.) with various drugs that influenced GABA neurotransmission. The pretreatment drugs were the GABAA antagonist SR 95531, the GABAB antagonist CGP 35348 and the GABA neuronal uptake‐inhibitor (±)‐nipecotic acid. Antinociceptive responsiveness to HBO2 was assessed using the acetic acid‐induced abdominal constriction test conducted in a small animal hyperbaric chamber. HBO2 at 3.5 ATA evoked a strong antinociceptive effect that was antagonized in dose‐related manner by i.c.v.‐ or i.t.‐administered SR 95531. I.c.v. and i.t. pretreatment with CGP 35348 or nipecotic acid potentiated HBO2‐induced antinociception at higher doses. These data strongly suggest that GABA is involved in the acute antinociceptive response of mice to HBO2.Grant Funding Source: Supported by NIH Grant AT‐007222 and the Allen I. White Distinguished Professorship.

Loss of Morphine Reward and Dependence in Mice Lacking G Protein–Coupled Receptor Kinase 5

The clinical benefits of opioid drugs are counteracted by the development of tolerance and addiction. We provide in vivo evidence for the involvement of G protein-coupled receptor kinases (GRKs) in opioid dependence in addition to their roles in agonist-selective mu-opioid receptor (MOR) phosphorylation. In vivo MOR phosphorylation was examined by immunoprecipitation and nanoflow liquid chromatography-tandem mass spectrometry analysis. Using the hot-plate and conditioned place preference test, we investigated opioid-related antinociception and reward effects in mice lacking GRK3 or GRK5. Etonitazene and fentanyl stimulated the in vivo phosphorylation of multiple carboxyl-terminal phosphate acceptor sites, including threonine 370, serine 375, and threonine 379, which was predominantly mediated by GRK3. By contrast, morphine promoted a selective phosphorylation of serine 375 that was predominantly mediated by GRK5. In contrast to GRK3 knockout mice, GRK5 knockout mice exhibited reduced antinociceptive responses after morphine administration and developed morphine tolerance similar to wild-type mice but fewer signs of physical dependence. Also, morphine was ineffective in inducing conditioned place preference in GRK5 knockout mice, whereas cocaine conditioned place preference was retained. However, the reward properties of morphine were evident in knock-in mice expressing a phosphorylation-deficient S375A mutation of the MOR. These findings show for the first time that MOR phosphorylation is regulated by agonist-selective recruitment of distinct GRK isoforms that influence different opioid-related behaviors. Modulation of GRK5 function could serve as a new approach for preventing addiction to opioids, while maintaining the analgesic properties of opioid drugs at an effective level.

Antinociceptive effect of certain dimethoxy flavones in mice

The aim of the present study was to evaluate the antinociceptive action of certain dimethoxy flavones (DMF, (7,2׳-dimethoxy flavone, 7,3׳-dimethoxy flavone, 7,4׳-dimethoxy flavone and 7,8,-dimethoxy flavone) and the possible mechanisms involved. The antinociceptive effect of dimethoxy flavones was investigated in mice employing acetic acid-induced abdominal writhings, formalin-induced nociception and hot water tail immersion assay procedures. To identify the possible mechanisms involved in the antinociceptive action of these compounds, acetic acid-induced abdominal constriction assay alone was employed. Mice were pretreated with naloxone, yohimbine, ondansetron, haloperidol, bicuculline or glibenclamide before dimethoxy flavone treatment to identify the role of opioid, adrenergic, 5HT3-serotonergic, dopaminergic, gamma-amino butyric acid (GABA) receptor or potassium channels, respectively. The investigated dimethoxy flavones produced a significant reduction in the number of abdominal constrictions in acetic acid assay. A dose dependent decrease in paw-licking response time was evident in both the early and late phases of formalin induced nociception. A significant increase in reaction time was also evident after treatment with various dimethoxy flavones in hot water tail immersion assay. Pretreatment with naloxone, ondansetron or glibenclamide significantly attenuated the antinociceptive effect of all the four dimethoxy flavones. Yohimbine pretreatment attenuated the antinociceptive response of 7,3׳-dimethoxy flavone, 7,4׳-dimethoxy flavone and 7,8-dimethoxy flavone. Pretreatment with haloperidol potentiated the antinociceptive response of all the tested dimethoxy flavones. The antinociceptive effect of 7,2׳-dimethoxy flavone and 7,3׳-dimethoxy flavone was annulled by bicuculline pretreatment. The results of the present study reveal the antinociceptive effect of dimethoxy flavones involving multiple pathways.

Isobolographic Analysis of the Antinociceptive Interaction between Ursolic Acid and Diclofenac or Tramadol in Mice

It is considered that natural products used in folk medicine can potentiate the effect of drugs. The aim of this study was to evaluate the pharmacological interaction between ursolic acid, a triterpene isolated from herbal medicines to treat pain, and the analgesics diclofenac or tramadol. Individual dose-response curves of the antinociceptive effect of these compounds were built to calculate the ED50, as well as the pharmacological interaction, by using isobolographic analysis. All treatments decreased significantly and in a dose-dependent manner the writhing behavior with ED50 values of 103.50 ± 19.66, 20.54 ± 6.05, and 9.60 ± 1.69 mg/kg, for ursolic acid, diclofenac, and tramadol, respectively. An isobolographic analysis allowed the characterization of the pharmacological interaction produced by a fixed ratio combination of 1 : 1 and 1 : 3 of equi-effective doses of these compounds. Theoretical antinociceptive ED50 values of ursolic acid-diclofenac were 62.12 ± 10.28 and 41.43 ± 6.69 mg/kg, respectively, not statistically different from those obtained experimentally (44.52 ± 5.25 and 44.89 ± 49.05 mg/kg, respectively), reporting an additive interaction. Theoretical antinociceptive ED50 values of ursolic acid-tramadol (56.56 ± 9.87 and 33.08 ± 5.07 mg/kg, respectively) were significantly lower than those observed experimentally (138.36 ± 49.05 and 67.34 ± 18.98 mg/kg, respectively) reporting antagonism in this interaction. Antinociceptive response obtained from isobolograms in the writhing test was corroborated by using formalin test in mice. Adverse effects such as gastric damage in the ursolic acid-diclofenac combination did not increase in an additive form similarly as with antinociception. Conversely, sedative response was significantly increased in the ursolic acid-tramadol combination. As observed in the formalin test, the antagonism on the antinociceptive response between ursolic acid and tramadol (1 : 1) was not reverted in the presence of the opioid antagonist naltrexone (1 mg/kg, i. p.). These results provide evidence for a differential pharmacological interaction, in which ursolic acid does not interfere with the antinociceptive effect of diclofenac but antagonizes that obtained with tramadol in an independent opioid mechanism. Therefore, medicinal plants containing abundant presence of ursolic acid may also modify efficacy in the alternative combinations for the pain therapy.

Diabetes-Causing Gene, Kruppel-Like Factor 11, Modulates the Antinociceptive Response of Chronic Ethanol Intake

Alcohol (EtOH [ethanol]) is an antinociceptive agent, working in part, by reducing sensitivity to painful stimuli. The transcription factor Kruppel-like factor 11 (KLF11), a human diabetes-causing gene that also regulates the neurotransmitter metabolic enzymes monoamine oxidase (MAO), has recently been identified as an EtOH-inducible gene. However, its role in antinociception remains unknown. Consequently, we investigated the function of KLF11 in chronic EtOH-induced antinociception using a genetically engineered knockout mouse model. Wild-type (Klf11(+/+) ) and KLF11 knockout (Klf11(-/-) ) mice were fed a liquid diet containing EtOH for 28days with increasing amounts of EtOH from 0% up to a final concentration of 6.4%, representing a final diet containing 36% of calories primarily from EtOH. Control mice from both genotypes were fed liquid diet without EtOH for 28days. The EtOH-induced antinociceptive effect was determined using the tail-flick test before and after EtOH exposure (on day 29). In addition, the enzyme activity and mRNA levels of MAO A and MAO B were measured by real-time RT-PCR and enzyme assays, respectively. EtOH produced an antinociceptive response to thermal pain in Klf11(+/+) mice, as expected. In contrast, deletion of KLF11 in the Klf11(-/-) mice abolished the EtOH-induced antinociceptive effect. The mRNA and protein levels of KLF11 were significantly increased in the brain prefrontal cortex of Klf11(+/+) mice exposed to EtOH compared with control Klf11(+/+) mice. Furthermore, MAO enzyme activities were affected differently in Klf11 wild-type versus Klf11 knockout mice exposed to chronic EtOH. Chronic EtOH intake significantly increased MAO B activity in Klf11(+/+) mice. The data show KLF11 modulation of EtOH-induced antinociception. The KLF11-targeted MAO B enzyme may contribute more significantly to EtOH-induced antinociception. Thus, this study revealed a new role for the KLF11 gene in the mechanisms underlying the antinociceptive effects of chronic EtOH exposure.

The acute antinociceptive effect of hyperbaric oxygen is not accompanied by an increase in markers of oxidative stress

AimsExposure to hyperbaric oxygen (HBO2) causes an antinociceptive response in mice. However, breathing oxygen (O2) at an elevated pressure can potentially cause oxygen toxicity. The aim of this study was to identify the determinants of HBO2 antinociception and the toxicity profile of HBO2. Main methodsMale NIH Swiss mice were assessed for acute antinociceptive responsiveness under room air or 100% O2 at 1.0 or 3.5 atmospheres absolute (ATA), using the acetic acid-induced abdominal constriction test. For the oxygen toxicity test, mice were exposed to 3.5 ATA oxygen for 11min, 60min, and 60min daily for 2days (120min) or 60min daily for 4days (240min), then assessed by analyzing the levels of two oxidative stress markers, MDA (malondialdehyde) and protein carbonyl in brain, spinal cord and lung. Key findingsOnly the combination of 100% O2 and 3.5 ATA caused significant antinociception. The antinociceptive effect of 100% O2 was pressure-dependent up to 3.5 ATA. In the oxygen toxicity test, mice exposed to HBO2 for different time intervals had levels of brain, spinal cord and lung MDA and protein carbonyl that were comparable to that of control animals exposed to room air. SignificanceTreatment with 100% O2 evokes a pressure-dependent antinociceptive effect. Since there was no significant increase in levels of the oxidative stress markers in the tested tissues, it is concluded that HBO2 at 3.5 ATA produces antinociception in the absence of oxidative stress in mice.

Involvement of D1/D2 dopamine receptors within the nucleus accumbens and ventral tegmental area in the development of sensitization to antinociceptive effect of morphine

The nucleus accumbens (NAc) and the ventral tegmental area (VTA) are two major areas for the mesolimbic dopaminergic system which are strongly involved in the development of behavioral sensitization. In the present study, we investigated the role of D1/D2 dopaminergic receptors within the NAc or VTA in response to sensitization to morphine by the tail-flick test as a model of acute pain. Sensitization was induced by subcutaneous (SC) injection of morphine (5mg/kg), once daily for three days followed by 5days free of drug. After the sensitization period, antinociceptive responses induced by an ineffective dose of morphine (1mg/kg; SC) were obtained by the tail-flick test, and represented as maximal possible effect (%MPE). In experimental groups, D1 and D2 receptor antagonists, SCH-23390 and sulpiride (0.25, 1 and 4μg/rat), were separately microinjected into the NAc or VTA, 10min before morphine administration during the sensitization period, respectively. Results showed that injection of morphine during the sensitization period (development of sensitization) increased %MPE of the ineffective dose of morphine from 2.43±1.4% in naive to 47.75±4.01% in sensitized animals (P<0.001). Unilateral microinjections of different doses of the D1/D2 receptor antagonists, SCH-23390 and sulpiride, into the NAc dose-dependently decreased %MPEs in morphine-sensitized animals. Nonetheless, %MPEs were only affected by intra-VTA administration of SCH-23390 in morphine-sensitized animals (P<0.05). Our findings suggest that both the D1/D2 dopamine receptors in the NAc and the D1 receptors in the VTA may be of more important in the development of sensitization to morphine in rats.

Reduced antinociceptive responsiveness to hyperbaric oxygen in opioid‐tolerant mice

Hyperbaric oxygen (HBO2 ) therapy can produce analgesia in patients experiencing various conditions of chronic pain. Previously, we reported that naloxone antagonized the acute antinociceptive effect of both brief (11 min) and longer (60 min) HBO2 treatments. This implied a possible role for opioid receptors in the antinociceptive effects of HBO2 . The aim of this study was to determine whether mice previously rendered as tolerant to opioid agonists would exhibit a reduced antinociceptive responsiveness to HBO2 . Male NIH Swiss mice were given repeated injections of the opioid agonists morphine, fentanyl or (-)-U50488H over 4 days to induce tolerance at their respective opioid receptors. Mice receiving saline according to a similar injection schedule served as a vehicle control group. On day 5, 15 h after the last injection, mice received either an antinociceptive challenge dose of the opioid agonists or a 30-min HBO2 exposure at 3.5 atmosphere absolute. The antinociception was then assessed by the 0.6% acetic acid-induced abdominal constriction test. The results showed that mice rendered as tolerant to morphine, fentanyl or (-)-U50488H pretreatment all exhibited reduced antinociceptive responsiveness to themselves and HBO2 . These results demonstrated that both μ- and κ-opioid receptors are involved in mediation of the acute antinociceptive response to HBO2 .

Hyperbaric Oxygen Treatment Produces an Antinociceptive Response Phase and Inhibits Astrocyte Activation and Inflammatory Response in a Rat Model of Neuropathic Pain

Hyperbaric oxygen (HBO) treatment has been proven to be a promising candidate for protection of the nervous system after acute injury in animal models of neuropathic pain. The purposes of this study were to examine the antinociceptive response phase induced by HBO treatment in a model of neuropathic pain and to determine the dependence of the treatment's mechanism of alleviating neuropathic pain on the inhibition of spinal astrocyte activation. Neuropathic pain was induced in rats by chronic constriction injury of the sciatic nerve. Mechanical threshold and thermal latency were tested preoperatively and for 1week postoperatively, four times daily at fixed time points. Methane dicarboxylic aldehyde (MDA) and superoxide dismutase (SOD) parameters were used as indices of oxidative stress response and tested before and after the treatment. The inflammatory cytokines interleukin (IL)-1β and IL-10 were assayed in the sciatic nerve were with enzyme-linked immunoassay. Glial fibrillary acidic protein activation in the spinal cord was evaluated immunohistochemically. The rats exhibited temporary allodynia immediately after HBO treatment completion. This transient allodynia was closely associated with changes in MDA and SOD levels. A single HBO treatment caused a short-acting antinociceptive response phase. Repetitive HBO treatment led to a long-acting antinociceptive response phase and inhibited astrocyte activation. These results indicated that HBO treatment played a dual role in the aggravation and alleviation of neuropathic pain, though the aggravated pain effect (transient allodynia) was far less pronounced than the antinociceptive phase. Astrocyte inhibition and anti-inflammation may contribute to the antinociceptive effect of HBO treatment after nerve injury.

Vitamin D supplementation attenuated carageenan-induced paw edema and formalin-induced nociception in indomethacin-treated rats

Background: This work evaluated the effect of vitamin D supplementation on carrageenan-induced paw edema and formalin-induced nociception in indomethacin treated rats. Methods: Rats were grouped into six (A – F) of 5 each. Groups (C – F) were pretreated for 14 days with vitamin D; 250, 500, 1000 and 1000 iu/day respectively. On day 15, they were treated as follows: group A (10 ml/kg distilled water), B; (10 mg/kg indomethacin), C – E; (10 mg/kg indomethacin) F (1000 iu/day vitamin D alone). One hour later 50 1% carrageenan or 25 5% formalin was injected into the sub-plantal surface of left hind paw. For carageenan-induced paw edema, paw sizes were measured 1 h later, then every hour for 5h. For formalin-induced nociception, number of paw licking/lifting was counted for each rat from 0 – 5 min (first phase) and 25 – 30 min (second phase) post-formalin injection. Results: Paw sizes were significantly (P<0.05) lower from 2 – 5 h post-carageenan injection in the 250 iu/day vitamin D + 10 mg/kg indomethacin group when compared with both controls. In the formalin-induced nociception, the 250 iu/day vitamin D + 10 mg/kg indomethacin, 500 iu/day vitamin D + 10 mg/kg indomethacin and the 1000 iu/day vitamin D alone groups produced significantly (p<0.05) lower number of paw lifting when compared with both controls in both phases. Conclusion: These findings suggest that vitamin D exerts antiinflammatory and antinociceptive responses that could be relevant for clinical application.