Kee Won Kim

Intrathecal injection of the ς1 receptor antagonist BD1047 blocks both mechanical allodynia and increases in spinal NR1 expression during the induction phase of rodent neuropathic pain

Background: Selective blockade of spinal &sfgr;1 receptors (Sig-1R) suppresses nociceptive behaviors in the mouse formalin test. The current study was designed to verify whether intrathecal Sig-1R antagonists can also suppress chronic neuropathic pain. Methods: Neuropathic pain was produced by chronic constriction injury (CCI) of the right sciatic nerve in rats. The Sig-1R antagonist BD1047 was administered intrathecally twice daily from postoperative days 0 to 5 (induction phase of neuropathic pain) or from days 15 to 20 (maintenance phase). Western blot and immunohistochemistry were performed to determine changes in Sig-1R expression and to examine the effect of BD1047 on N-methyl-d-aspartate receptor subunit 1 expression and phosphorylation in spinal cord dorsal horn from neuropathic rats. Results: BD1047 administered on postoperative days 0–5 significantly attenuated CCI-induced mechanical allodynia, but not thermal hyperalgesia, and this suppression was blocked by intrathecal administration of the Sig-1R agonist PRE084. In contrast, BD1047 treatment during the maintenance phase of neuropathic pain had no effect on mechanical allodynia. Sig-1R expression significantly increased in the ipsilateral spinal cord dorsal horn from days 1 to 3 after CCI. Importantly, BD1047 (30 nmol) administered intrathecally during the induction, but not the maintenance phase, blocked the CCI-induced increase in N-methyl-d-aspartate receptor subunit 1 expression and phosphorylation. Conclusions: These results demonstrate that spinal Sig-1Rs play a critical role in both the induction of mechanical allodynia and the activation of spinal N-methyl-d-aspartate receptors in CCI rats and suggest a potential therapeutic role for the use of Sig-1R antagonists in the clinical management of neuropathic pain.

Intrathecal Administration of Sigma-1 Receptor Agonists Facilitates Nociception : Involvement of a Protein Kinase C-dependent Pathway

Sigma sites, originally proposed as opioid receptor subtypes, are currently thought to represent unique receptors with a specific pattern of drug selectivity, a well‐established anatomical distribution and broad range of functional roles including potential involvement in nociceptive mechanisms. We have recently demonstrated that intrathecal (i.t.) treatment with a sigma‐1 receptor antagonist reduced formalin‐induced pain behavior. In the present study, we investigated the potential role of spinal sigma‐1 receptor agonists in peripherally initiated nociception and attempted to elucidate intracellular signaling mechanisms associated with spinal cord sigma‐1 receptor activation in mice. The i.t. injection of the sigma‐1 receptor agonists PRE‐084 (PRE) or carbetapentane (CAR) significantly decreased tail‐flick latency (TFL) and increased the frequency of paw withdrawal responses to mechanical stimulation (von Frey filament, 0.6 g) as well as the amount of Fos expression in the spinal cord dorsal horn induced by noxious paw‐pinch stimulation. These PRE‐ or CAR‐induced facilitatory effects on nociception were significantly blocked by i.t. pretreatment with the sigma‐1 receptor antagonist, BD‐1047, the phospholipase C (PLC) inhibitor, U‐73,122, the Ca2+‐ATPase inhibitor, thapsigargin, and the protein kinase C (PKC) inhibitor, chelerythrine. Western blot analysis further revealed that i.t. PRE or CAR injection significantly increased pan‐PKC as well as the PKCα, ϵ, and ζ isoforms in the dorsal horn. Collectively, these findings demonstrate that calcium‐dependent second messenger cascades including PKC are involved in the facilitation of nociception associated with spinal sigma‐1 receptor activation.

Water soluble fraction (<10 kDa) from bee venom reduces visceral pain behavior through spinal α2-adrenergic activity in mice

We have previously shown that subcutaneous bee venom (BV) injection reduces visceral pain behavior in mice, but it is not clear which constituent of BV is responsible for its antinociceptive effect. In the present study, we now demonstrate that a water-soluble subfraction of BV (BVA) reproduces the antinociceptive effect of BV in acetic acid-induced visceral pain model. We further evaluated three different BVA subfractions that were separated by molecular weight, and found that only the BVAF3 subfraction (a molecular weight of <10 kDa) produced a significant antinociceptive effect on abdominal stretches and suppressed visceral pain-induced spinal cord Fos expression. Injection of melittin (MEL), a major constituent of BVAF3, also produced a visceral antinociception. However, melittins antinociception was completely blocked by boiling for 10 min at 100 degrees C, while boiling either whole BV or BVAF3 did not prevent their antinociception. The antinociceptive effect of BVAF3 was completely blocked by intrathecal pretreatment with the alpha2-adrenoceptor antagonist, yohimbine (YOH), while intrathecal pretreatment with the opioid antagonist, naloxone (NAL) or the serotonin antagonist, methysergide, had no effect. These data demonstrate that BVAF3 is responsible for the visceral antinociception of whole BV and further suggest that this effect is mediated in part by spinal alpha2-adrenergic activity.

Substantial role of locus coeruleus-noradrenergic activation and capsaicin-insensitive primary afferent fibers in bee venom's anti-inflammatory effect

Several lines of evidence indicate significant interactions between the immune and nervous systems. Our recent study reveals that bee venom (BV) induced anti-inflammatory effect (BVAI) was produced by sympathetic preganglionic neuronal activation and subsequent adrenomedullary catecholamine release in a zymosan-induced inflammation model. However, the specific peripheral input and the supraspinal neuronal systems that are involved in this BVAI remain to be defined. Here we show that subcutaneous BV injection into left hind limb significantly reduces zymosan-induced leukocyte migration and that this effect is completely inhibited by denervation of the left sciatic nerve. This BVAI was not affected by the destruction of capsaicin-sensitive primary afferent fibers using either neonatal capsaicin or resiniferatoxin (RTX) pretreatment. BV injection into the left hind limb significantly increased Fos expression in the contralateral locus coeruleus (LC) in non-inflamed mice. In zymosan-inflamed mice, BV injection produced a further increase in LC Fos expression as compared with non-inflamed mice. This BV-induced Fos increase in the LC was not affected by RTX pretreatment. Pharmacological blockage of central noradrenergic activity by either central chemical sympathectomy (i.c.v. 6-hydroxydopamine) or alpha2 adrenoceptor antagonism (i.c.v. idazoxan) completely blocked BVAI. Taken together, these results suggest that BVAI is mediated by peripheral activation of capsaicin-insensitive primary afferent fibers and subsequent central noradrenergic activation including the LC.

Intrathecal neostigmine reduces the zymosan-induced inflammatory response in a mouse air pouch model via adrenomedullary activity: Involvement of spinal muscarinic type 2 receptors

Intrathecal (IT) injection of neostigmine (a cholinesterase inhibitor) has been reported to produce a significant anti-nociceptive effect in a number of inflammatory pain models. However, a potential anti-inflammatory effect of IT neostigmine in these models has not been investigated. In the present study, we have examined the anti-inflammatory effect of IT injection of neostigmine (AI-NEO) using a standard mouse air pouch model by evaluating the effect of AI-NEO on zymosan-induced leukocyte migration and myeloperoxidase (MPO) release. IT neostigmine was found to suppress both leukocyte migration and MPO degranulation in a dose dependent manner. We then established which subtypes of cholinergic receptors were involved in this AI-NEO. IT pretreatment with atropine (a muscarinic receptor antagonist) but not hexamethonium (a nicotinic receptor antagonist) completely blocked the IT neostigmine anti-inflammatory effect. Subsequent experiments showed that IT pretreatment with methoctramine (a muscarinic type 2 (M2) receptor antagonist), but not pirenzepine (M1 receptor antagonist) or 4-DAMP (M3 receptor antagonist), suppressed the AI-NEO. We then evaluated whether adrenal glandular activity was important in the AI-NEO. Adrenalectomy significantly blocked the AI-NEO, while intraperitoneal pretreatment with the beta-adrenoceptor antagonist (propranolol), but not the corticosteroid antagonist (RU486) reversed AI-NEO. In conclusion, these results indicate that IT neostigmine facilitates the activation of spinal M2 receptors and this activation ultimately leads to release of adrenal catecholamines which contribute to the anti-inflammatory effect observed at the site of tissue inflammation.

The Antinociceptive Effect of Sigma-1 Receptor Antagonist, BD1047, in a Capsaicin Induced Headache Model in Rats

Intracranial headaches, including migraines, are mediated by nociceptive activation of the trigeminal nucleus caudalis (TNC), but the precise mechanisms are poorly understood. We previously demonstrated that selective blockage of spinal sigma-1 receptors (Sig-1R) produces a prominent antinociceptive effect in several types of pain models. This study evaluates whether the Sig-1R antagonist (BD1047) has an antinociceptive effect on capsaicin (a potent C-fiber activator) induced headache models in rats. Intracisternal infusion of capsaicin evoked pain behavior (face grooming), which was significantly attenuated by BD1047 pretreatment. BD1047 consistently reduced capsaicin-induced Fos-like immunoreactivity (Fos-LI), a neuronal activator, in the TNC in a dose-dependent manner. Moreover, capsaicin-induced phosphorylation of N-methyl-D-aspartate receptor subunit 1 was reversed by BD1047 pretreatment in the TNC. These results indicate that the Sig-1R antagonist has an inhibitory effect on nociceptive activation of the TNC in the capsaicin-induced headache animal model.

Effect of adenosine triphosphate on phosphate uptake in renal proximal tubule cells: Involvement of PKC and p38 MAPK

ATP has been known to act as an extracellular signal and to be involved in various functions of kidney. Renal proximal tubular reabsorption of phosphate (Pi) contributes to the maintenance of phosphate homeostasis, which is regulated by Na+/Pi cotransporter. However, the effects of ATP on Na+/Pi cotransporters were not elucidated in proximal tubule cells (PTCs). Thus, the effects of ATP on Na+/Pi cotransporter and its related signal pathways are examined in the primary cultured renal PTCs. In the present study, ATP inhibited Pi uptake in a time (>u20091 h) and dose (>10−6M) dependent manner. ATP‐induced inhibition of Pi uptake was correlated with the decrease of type II Na+/Pi cotransporter mRNA. ATP‐induced inhibition of Pi uptake may be mediated by P2Y receptor activation, since suramin (non‐specific P2 receptor antagonist) and RB‐2 (P2Y receptor antagonist) blocked it. ATP‐induced inhibition of Pi uptake was blocked by neomycin, U73122 (phospholipase C (PLC) inhibitors), bisindolylmaleimide I, H‐7, and staurosporine (protein kinase C (PKC) inhibitors), suggesting the role of PLC/PKC pathway. ATP also increased inositol phosphates (IPs) formation and induced PKC translocation from cytosolic fraction to membrane fraction. In addition, ATP‐induced inhibition of Pi uptake was blocked by SB 203580 [a p38 mitogen activated protein kinase (MAPK) inhibitor], but not by PD 98059 (a p44/42 MAPK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK, which was not blocked by PKC inhibitor. In conclusion, ATP inhibited Pi uptake via PLC/PKC as well as p38 MAPK in renal PTCs.

Effect of bee venom acupuncture on methamphetamine-induced hyperactivity, hyperthermia and Fos expression in mice.

Acupuncture has been used to treat drug addiction by nicotine, alcohol, cocaine and morphine. This study was designed to investigate the effect of bee venom (BV) acupuncture on hyperactivity and hyperthermia induced by acute exposure to methamphetamine (METH, 1mg/kg, s.c.) in mice. Diluted BV (20μl of 0.01, 0.1, 1 and 10mg/ml in saline, s.c.) was administered bilaterally into the Zusanli acupoint (ST36) or control points (SP9 or GB39 or tail base). BV injection into ST36 dose dependently reduced METH-induced hyperactivity and hyperthermia, while BV injection (1mg/ml) into control points did not produce these suppressive effects. METH injection significantly increased Fos expression in several brain regions including nucleus accumbens (NA), caudate putamen (CPU), ventral tegmental area (VTA), substantia nigra (SN) and locus coeruleus (LC). Interestingly, BV (1mg/ml) injection into ST36 further increased METH-induced Fos expression in NA (core and shell), SN and LC. When we performed sciatic denervation or combination treatment of BV and lidocaine (BV diluted in 5% lidocaine solution), the enhancement of Fos elevation by BV was completely blocked in the NA, SN and LC in METH-injected mice, indicating that BV-induced peripheral nerve stimulation played an important role in the BV effect. Furthermore, the effects of BV were completely blocked by the α₂-adrenoceptor antagonist, idazoxan (3mg/kg, i.p.), but not by β-adrenoceptor antagonist, propranolol (10mg/kg, i.p.). Taken together, these findings suggest that BV acupuncture into ST36 may modulate METH-induced hyperactivity, hyperthermia and Fos expression through activation of the peripheral nerve and the central α₂-adrenergic activation.

Bee venom suppresses methamphetamine-induced conditioned place preference in mice

Abstract Objectives: Although acupuncture is most commonly used for its analgesic effect, it has also been used to treat various drug addictions including cocaine and morphine in humans. This study was designed to investigate the effect of bee venom injection on methamphetamine-induced addictive behaviors including conditioned place preference and hyperlocomotion in mice. Methods: Methamphetamine (1 mg/kg) was subcutaneously treated on days 1, 3 and 5 and the acquisition of addictive behaviors was assessed on day 7. After confirming extinction of addictive behaviors on day 17, addictive behaviors reinstated by priming dose of methamphetamine (0.1 mg/kg) was evaluated on day 18. Bee venom (20 μl of 1 mg/ml in saline) was injected to the acupuncture point ST36 on days 1, 3 and 5. Results: Repeated bee venom injections completely blocked development of methamphetamine-induced acquisition and subsequent reinstatement. Single bee venom acupuncture 30 minutes before acquisition and reinstatement test completely inhibited methamphetamine-induced acquisition and reinstatement. Repeated bee venom acupunctures from day 8 to day 12 after methamphetamine-induced acquisition partially but significantly suppressed reinstatement. Discussion: These findings suggest that bee venom acupuncture has a preventive and therapeutic effect on methamphetamine-induced addiction.

239 MICROGLIA ACTIVATION IS INVOLVED IN MIRROR IMAGE ALLODYNIA AT RAT SPINAL NERVE LIGATION MODEL

possible involvement of glial cells in spinal sensitization in an acute myositis model. Methods: Myositis was induced by the administration of 400mg of carrageenan (Cg) in the right gastrocnemius (GS) muscle of rats, with saline as a control. The nociceptive threshold was evaluated using the electronic von Frey hairs. Behavioral measures were assessed before and 2 h after induction of myositis, and a decrease of the withdrawal threshold (grams) was used as a nociceptive parameter. Immunohistochemical assays to evaluate astrocyte and microglia activation (GFAP and OX-42, respectively) were performed in the lumbar dorsal horn of spinal cord. Intrathecal injection of fluorocitrate, a disruptor of glial function, was used to examine potential spinal glial mediation. Results: The administration of Cg into the GS muscle induced mechanical hyperalgesia. Immunohistochemical data showed that glial cells are activated after acute myositis, as immunostaining for GFAP and OX-42 was significantly higher in Cg-treated animals. Fluorocitrate was able to decrease Cg-induced pain sensitivity and the astrocyte and microglia activation. Conclusions: Our results suggest that glial cells are involved in the development of the nociceptive response induced by acute muscle inflammation. Understanding the role of glial cells during miositis-induced nociception may lead to new strategies for the management of muscle pain, one of the most frequent types of clinically relevant pain.