Activation of the spinal sigma-1 receptor enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NR1 subunit in mice.

BACKGROUND AND PURPOSE: Previously we demonstrated that the spinal sigma-1 receptor (Sig-1 R) plays an important role in pain transmission, although the exact mechanism is still unclear. It has been suggested that Sig-1 R agonists increase glutamate-induced calcium influx through N-methyl-D-aspartate (NMDA) receptors. Despite data suggesting a link between Sig-1 Rs and NMDA receptors, there are no studies addressing whether Sig-1 R activation directly affects NMDA receptor sensitivity. EXPERIMENTAL APPROACH: We studied the effect of intrathecal (i.t.) administration of Sig-1 R agonists on protein kinase C (PKC) and protein kinase A (PKA) dependent phosphorylation of the NMDA receptor subunit NR1 (pNR1) as a marker of NMDA receptor sensitization. In addition, we examined whether this Sig-1 R mediated phosphorylation of NR1 plays an important role in sensory function using a model of NMDA-induced pain. KEY RESULTS: Both Western blot assays and image analysis of pNR1 immunohistochemical staining in the spinal cord indicated that i.t. injection of the Sig-1 R agonists, PRE-084 or carbetapentane dose dependently enhanced pNR1 expression in the murine dorsal horn. This increased pNR1 expression was significantly reduced by pretreatment with the specific Sig-1 R antagonist, BD-1047. In another set of experiments Sig-1 R agonists further potentiated NMDA-induced pain behaviour and pNR1 immunoreactivity and this was also reversed with BD-1047. CONCLUSIONS AND IMPLICATIONS: The results of this study suggest that the activation of spinal Sig-1 R enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NMDA receptor NR 1 subunit.

The anti-inflammatory effect of bee venom stimulation in a mouse air pouch model is mediated by adrenal medullary activity.

Cutaneous electrical or chemical stimulation can produce an anti-inflammatory effect, which is dependent on adrenal medullary-sympathetic activation. We have previously shown that peripheral injection of bee venom (BV) also produces a significant anti-inflammatory effect that is neurally mediated. In the present study, we examined whether this anti-inflammatory effect is also dependent on the adrenal gland using the mouse inflammatory air pouch model. Subcutaneous (s.c.) BV injection produced a marked suppression of leucocyte migration and tumour necrosis factor (TNF)-alpha concentration induced by zymosan injection into the air pouch. The role of the adrenal gland in this suppression was evaluated in adrenalectomized mice. Adrenalectomy significantly reversed the suppression of leucocyte migration and TNF-alpha elevation caused by BV. Serum concentrations of corticosteroid were increased in mice with zymosan-induced air-pouch inflammation and this increase was reduced by BV administration, suggesting that adrenal corticosteroid release is not involved in mediating the anti-inflammatory effects of BV. To test this hypothesis, the corticosteroid receptor antagonist (RU486) was administered and found not to affect the BV-induced inhibition of leucocyte migration. By contrast, pretreatment with the beta-adrenergic antagonist propranolol reversed the BV-induced inhibitory effect on leucocyte migration. These results suggest that the anti-inflammatory effect of s.c. BV administration is mediated in part by the release of catecholamines from the adrenal medulla.

Distribution of nociceptin-like immunoreactivity in the central nervous system of the Mongolian gerbil: an immunohistochemical study.

This study is designed to demonstrate the distribution of nociceptin, endogenous ORL1 receptor ligand, in the central nervous system of the Mongolian gerbil. To intensify the nociceptin-like immunoreactivity (NOC-LI), colchicine was administered into the lateral ventricle, at 48 h prior to the transcardiac perfusion. In the group without colchicine treatment, NOC-LI was observed in the fibres of the spinal dorsal horn, specifically in the superficial layers. However, the NOC-LI in the superficial layers disappeared after the administration of colchicine. In the brain, NOC-LI was prominent in the hypothalamus, hippocampus, cerebral peduncle, substantia nigra, dorsal raphe, periaqueductal grey, locus coeruleus and trapezoid nucleus. Colchicine treatment markedly intensified the NOC-LI in the somata of the central nervous system, whereas the untreated sections were too weak to observe and analyse. The distribution of NOC-LI provides informative data for studies of the neuronal circuit that nociceptin may be involved in.

The analgesic efficacy of bee venom acupuncture for knee osteoarthritis: a comparative study with needle acupuncture.

The aim of this investigation was to determine whether bee venom (BV) administered directly into an acupoint was a clinically effective and safe method for relieving the pain of patients with knee osteoarthritis (OA) as compared to traditional needle acupuncture. We evaluated the efficacy of BV acupuncture using both pain relief scores and computerized infrared thermography (IRT) following 4 weeks of BV acupuncture treatment. We observed that a significantly higher proportion of subjects receiving BV acupuncture reported substantial pain relief as compared with those receiving traditional needle acupuncture therapy. Furthermore, the IRT score was significantly improved and paralleled the level of pain relief.

Visceral antinociception produced by bee venom stimulation of the Zhongwan acupuncture point in mice: role of alpha(2) adrenoceptors.

The goal of the present study was to determine whether bee venom (BV) injection into the Zhongwan acupoint (CV12), compared to injection into a non-acupoint, produced antinociception in an acetic acid-induced visceral pain model. This was accomplished by injecting BV subcutaneously into the Zhongwan acupoint or into a non-acupoint 30 min before intraperitoneal injection of acetic acid in ICR mice. BV injection into the acupoint produced a dose dependent suppression of acetic acid-induced abdominal stretches and of acetic acid-induced Fos expression in the spinal cord and the nucleus tractus solitarii. In contrast BV injection into the non-acupoint only produced antinociception at the highest dose of BV tested. Naloxone pretreatment did not alter the antinociceptive effect of BV acupoint injection on the abdominal stretch reflex. On the other hand, pretreatment with the alpha 2-adrenoceptor antagonist, yohimbine completely blocked the antinociceptive effect of BV acupoint injection. These results imply that BV acupoint stimulation can produce visceral antinociception that is associated with activation of alpha 2-adrenoceptors, but not with naloxone-sensitive opioid receptors.

Antinociceptive effects of bee venom acupuncture (apipuncture) in rodent animal models: a comparative study of acupoint versus non-acupoint stimulation.

From a clinical perspective, the alternative forms of acupoint stimulation including electroacupuncture, moxibustion and acupressure appear to have more potent analgesic effects than manual needle acupuncture. Bee venom (BV) injection has also been reported to produce persistent nociceptive stimulation and to cause neuronal activation in the spinal cord. In previous study, we observed that BV stimulation into acupoint, namely BV acupuncture or Apipuncture, produced more potent anti-inflammatory and antinociceptive potency in rodent arthritis model as comparing with that of non-acupoint injection. Based on previous report, we decided to further investigate that BV injection into an acupoint produces antinociception as a result of its potent chemical stimulatory effect in both abdominal stretch assay and formalin test. Different doses of BV were injected into an acupoint or a non-acupoint 30 min prior to intraplantar formalin injection or intraperitoneal acetic acid injection. Using the abdominal stretch assay, we found that the high dose of BV (1:100 diluted in 20microl saline) produced a potent antinociceptive effect irrespective of the site of BV injection. In contrast the antinociceptive effect observed in both the writhing and formalin tests following administration of a low dose of BV (1:1000 diluted in 20microl saline) was significantly different between acupoint and non-acupoint sites. BV injection into an acupoint (Zhongwan, Cv. 12) was found to produce significantly greater antinociception than non-acupoint injection (10 mm from Zhongwan, Cv. 12) in the abdominal stretch assay. Similarly, in the formalin test, acupoint (Zusanli, St. 36) injection of BV produced more potent antinociception than non-acupoint injection (gluteal muscle). In contrast, BV injection into an arbitrary non-acupoint site on the back did not produce antinociception in either the writhing or formalin test. These results indicate that BV injection directly into an acupoint can produce a potent antinociceptive effect and suggest that this alternative form of acupoint stimulation (Apipuncture) may be a promising method for the relief of pain.

Bee venom pretreatment has both an antinociceptive and anti-inflammatory effect on carrageenan-induced inflammation.

Although the injection of bee venom (BV) has been reported to evoke tonic pain and hyperalgesia, there is conflicting evidence in the literature indicating that BV can also exert an anti-inflammatory and antinociceptive effects on inflammation. In this regard, BV has been traditionally used in Oriental medicine to relieve pain and to treat chronic inflammatory diseases such as rheumatoid arthritis. The present study was designed to test the hypothesis that BV induces acute nociception under normal conditions, but that it can serve as a potent anti-inflammatory and antinociceptive agent in a localized inflammatory state. The experiments were designed to evaluate the effect of BV pretreatment on carrageenan (CR)-induced acute paw edema and thermal hyperalgesia. In addition, spinal cord Fos expression induced by peripheral inflammation was quantitatively analyzed. In normal animals subcutaneous BV injection into the hindlimb was found to slightly increase Fos expression in the spinal cord without producing detectable nociceptive behaviors or hyperalgesia. In contrast pretreatment with BV (0.8 mg/kg) 30 min prior to CR injection suppressed both the paw edema and thermal hyperalgesia evoked by CR. In addition, there was a positive correlation between the percent change in paw volume and the expression of Fos positive neurons in the spinal cord. These results indicate that BV pretreatment has both antinociceptive and anti-inflammatory effects in CR-induced inflammatory pain. These data also suggest that BV administration may be useful in the treatment of the pain and edema associated with chronic inflammatory diseases.

Effects of dizocilpine pretreatment on parvalbumin immunoreactivity and Fos expression after cerebral ischemia in the hippocampus of the Mongolian gerbil.

The mechanisms of ischemic neuronal death have been focused on glutamate receptor activation and subsequent elevation of intracellular Ca2+ concentration. The purpose of this study was to evaluate the effects of dizocilpine, an NMDA receptor antagonist, pretreatment on Fos expression and parvalbumin (PV, calcium binding protein) immunoreactivity in the hippocampus of the mongolian gerbil after global ischemic insults. The number of PV-immunoreactive (PV-ir) neurons in CA1 were significantly decreased from 1 day after cerebral ischemia, while dizocilpine pretreatment completely suppressed the loss of PV-ir neurons in CA1. Dizocilpine pretreatment also protected the structural loss of microtubule-associated protein 2 immunoreactivity in CA1 after ischemic insults. In addition, dizocilpine pretreatment increased Fos expression in both hippocampal CA3 and CA4 after 3 hr ischemic reperfusion as compared to that of the saline pretreated group. Subsequently, the Fos-defined cellular activity of PV-ir neurons was slightly increased by dizocilpine pretreatment in the hippocampal area. This study demonstrated that NMDA receptor mediated calcium influx was associated with the loss of PV-ir neurons in CA1 hippocampal region, and that dizocilpine pretreatment increased Fos expression and the neuronal activity of PV-ir neurons in the non-vulnerable region of hippocampus after cerebral ischemia. Based on this data, we conclude that the protective effect of dizocilpine may be induced by the regulation of calcium overload, or by the upregulation of a neuroregenerative initiator such as Fos protein.

General pharmacology studies on beta-domain deleted recombinant factor VIII.

beta-Domain deleted recombinant factor VIII (GC-rAHF), newly developed by Korea Green Cross Co., is a novel therapeutic for hemophiliacs and is currently under clinical evaluation. The general pharmacological properties of this drug were evaluated using mice, rats, guinea pigs and rabbits. Intravenous doses of 5 to 500 IU/kg were assayed in several tests to analyze their effects in vivo on various systems. The effect of the substance under study was also tested in vitro on isolated guinea pig ileum preparations at final concentrations of 5 to 50 IU/kg. The result of this study showed that GC-rAHF did not affect general behavior in the Irwin test. Similarly the drug was not found to affect neither normal body temperature nor the spontaneous activity in mice. In addition, it was not found to induce pharmacologically significant alterations of the cardiovascular and respiratory parameters in rats. No effects were observed either in the pentobarbital sodium-induced sleep-induction time and duration, in writhing test or in the test of pentetrazole-induced convulsion. Finally, the tested drug did not modify the gastrointestinal motility, acetylcholine or histamine-induced contraction of the isolated guinea pig ileum, nor gastric secretion. The results demonstrated that GC-rAHF has no effects on the central nervous, cardiovascular, respiratory and digestive systems in the doses of 5, 50 and 500 IU/kg in vivo and 5, 10, 50 and 100 IU/kg in vitro.

Different frequencies of electroacupuncture modified the cellular activity of serotonergic neurons in brainstem.

In this study, we evaluated whether different frequencies of electroacupuncture (EA) modified the activities of serotonergic neurons in the dorsal raphe (DR) and raphe magnus (RMg) using double labeling immunohistochemistry for Fos and serotonin. The results demonstrated that both high and low frequency EA increased the colocalization between Fos and serotonin in the DR, not in RMg as compared with anesthesia control. In addition, high frequency EA more potently increased the serotonergic activity in the DR rather than low frequency EA, suggesting that serotonergic pathway from the DR plays an important role in the high frequency EA analgesia.

Effect of 7-nitroindazole, a selective neuronal nitric oxide synthase inhibitor, on parvalbumin immunoreactivity after cerebral ischaemia in the hippocampus of the Mongolian gerbil.

Previous studies have demonstrated that a loss of parvalbumin-immunoreactive (PV-ir) neurones is observed in the hippocampus after transient cerebral ischaemia. However, whether the loss of parvalbumin (PV) immunoreactivity is related to the over-production of nitric oxide (NO) during cerebral ischaemia has not been evaluated. This study was designed to test the effect of 7-nitroindazole pre-treatment (7-NI, 50 mg/kg), a selective neuronal NO synthase inhibitor, on PV immunoreactivity and its cellular activity following forebrain ischaemia. PV-ir neurones in the hippocampus of the control group were widely distributed in the pyramidal cell layer and stratum oriens of CA1 and CA3, and the granular cell layer of dentate gyrus. 7-NI pre-treatment completely suppressed the reduction of PV immunoreactivity in CA1 that was observed in the ischaemia-induced group. Subsequently, 7-NI pre-treatment also protected against the structural loss of microtubule-associated protein 2 (MAP2) immunoreactivity in CA1 after ischaemic insult. In addition, the Fos-defined neuronal activity of PV-ir neurones was slightly increased by the 7-NI pre-treatment 3 h after ischaemia. Based on these data, we conclude that the neuronal toxicity of NO may be involved in the loss of PV-ir neurones after cerebral ischaemia.