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259 MICROGLIA ACTIVATION MEDIATES THE GABA(A) RECEPTOR ANTAGONIST INDUCED TACTILE ALLODYNIA
Poster Sessions / European Journal of Pain 13 (2009) S55–S285
CAPs in pulse one were used for the study of tonic inhibition of lidocaine. The difference of CAPs between pulse 10 and one was measured for phasic inhibition. Results: Lidocaine caused concentration-dependant, tonic inhibition on conduction velocity and CAPs in both A- and C-fibers. At 300 mM the average inhibition on CAPs was 55±15 and 51±8% (mean±SEM, n = 4–5) in A- and C-fibers, respectively. Electrical stimulation of A-fibers at 200 Hz exhibited concentrationdependant, phasic inhibition by lidocaine, reaching significance at 300 mM. However, no such inhibition was observed in C-fibers with the present stimulation profile. Conclusions: Lidocaine at subblocking concentrations produces phasic inhibition in A- but not C-fibers, indicating that the function of lidocaine differs between the fiber types. 256 LOCAL ADMINISTRATION OF AGMATINE DOES NOT HAVE ANTINOCICEPTION EFFECTS ON THE INDUCTION AND MAINTENANCE OF ARTHRITIC PAIN IN RATS S.S. Min *, M.J. Lee1 , P.J. Kim2 , S.H. Han3 , J.Y. Yee3 , C. Kim3 , G.H. Seol4 . 1 Department of Anesthesiology and Pain Medicine, Konkuk University School of Medicine, Chungju, South Korea; 2 Profesional Oriental Medicine Graduate School, Wonkwang University, Iksan, South Korea; 3 Department of Physiology and Biophysics, School of Medicine, Eulji University, Daejeon, South Korea; 4 Department of Basic Nursing Science, School of Nursing, Korea University, Seoul, South Korea Previous studies suggest that systemic administration of agmatine, endogenous ligand for imidazoline receptors has antihypernociceptive effects in experimental animal. However the peripheral effects of agmatine on inflammatory pain have not yet been elucidated. Here we examined the effects of intraarticular injection of agmatine in the induction and maintenance phase of arthritic pain. In addition, we sought to determine the potential contribution of imidazoline and a2 -adrenergic receptors to the antinociceptive effects using clonidine which is mixed a2 adrenoceptor and imidazoline receptor agonist. To induce arthritis in rats, 2% lambda-carrageenan (50 ml, in saline) was injected into the joint of the right hind limb under enflurane anesthesia. Either agmatine (10, 50, 100 mg/40 ml) or clonidine (10, 50, 100 mg/40 ml) was injected into the knee joint cavity immediately before or 4 hr after carrageenan injection. Weight load tests were performed to measure pain-related behavior in freely walking rats. The intraarticular injection of agmatine into the knee joint had no effects in the both phase of induction and maintenance of arthritic pain at any dose tested. However, injection of clonidine reversed arthritic pain, when injected 4 h after carrageenan injection. In rats, agmatine has no peripheral effect on inflammatory pain and imidazoline receptors in the periphery may not contribute to the anti-inflammatory pain. 257 INTRAVENOUS LIDOCAINE ASSOCIATED WITH AMITRIPTILINE ON PAIN INTENSITY AND PLASMA CONCENTRATIONS OF SEROTONINE, NORADRENALINE AND DOPAMINE IN FIBROMYALGIA PATIENTS R.K. Sakata *, R. Vlainich, A. Issy. Federal University of S˜ ao Paulo, S˜ ao Paulo, Brazil Background and Objectives: The main effect of lidocaine is central antihyperalgesic effect. The aim of the present study was to evaluate the effect of IV lidocaine associated with amitriptiline on pain intensity and plasma serotonin, norepinephrine, and dopamine in fibromyalgic patients. Methods: Thirty patients participated in this randomized doubleblinded study. All patients used 25 mg amitriptiline; G1 patients received saline, and G2, 240 mg lidocaine, once a week, for 4 weeks. Results: Pain intensity reduced after treatment in G1 (T0: 7.0 ± 1.2; T4: 4.0 ± 2.1), and G2 (T0: 7.6 ± 0.8 e T4: 4.1 ± 2.3). Serotonin
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plasma concentrations were similar in T0 (G1: 42.7 ± 32.3; G2: 65.9 ± 41.1 ng/mL) and T4 (G1: 63.7 ± 32.1; G2: 76.7 ± 55.6), as norepinephrine in T0 (G1: 189.5 ± 108.6; G2: 171.9 ± 131.0 pg/mL) and T4 (G1: 167.5 ± 85.5; G2: 191.7 ± 142.3 pg/mL); dopamine levels was higher in T4 (45.7 ± 36.9 g/mL) than in T0 (17.1 ± 9.1 pg/mL) in G1; and there was no difference in T4 (34.7 ± 33.6) and T0 (25.8 ± 15.5) in G2. Conclusion: The association of 240 mg IV lidocaine (1/week) with 25 mg amitriptiline for 4 weeks didn’t modified pain intensity and plasma concentrations of serotonine, noradrenaline, and dopamine in fibromyalgia patients. 258 DISTRIBUTION AND NEUROCHEMISTRY OF BOTULINUM TOXIN TYPE A RECEPTORS IN THE URINARY BLADDER A. Coelho1,2 *, P. Dinis2,3 , R. Pinto3 , T. Gorgal3 , C. Silva3 , A. Silva3 , J. Silva3 , C. Cruz1,2 , F. Cruz1,2,3 , A. Avelino1,2 . 1 Faculty of Medicine, University of Porto, Porto, Portugal; 2 IBMC – Instituto de Biologia Molecular e Celular, Porto, Portugal; 3 Department of Urology, Hospital S. Jo˜ ao, Porto, Portugal Pain and bladder hyperactivity are main symptoms of BPS/IC (bladder painful syndrome/interstitial cystitis). Botulinum toxin type A (BoNT/A) is used as a new treatment for these pathologies. The specificity of BONT/A is due to the presence of membrane receptors SV2 that are exposed during neurotransmitter exocytosis. Once internalized, BoNT/A cleaves SNAP25 blocking neurotransmission. In this work, we studied the distribution of SV2 and SNAP25 in three different species, including human. The neurochemistry of BONT/A sensitive structures was investigated using markers for parasympathetic, sympathetic and sensory fibers. Human bladders were obtained from cadaveric organ donors. Rodent bladders were injected with BONT/A and collected at different times. Cryostat sections were immunoreacted against SV2, SNAP25 (cleaved and uncleaved), vesicular acetylcholine transporter (VAChT), tyrosine hydroxilase (TH) and calcitonin-gene related peptide (CGRP). Immunoreactive (IR) fibers for SV2 and SNAP25 were found in the mucosa and muscular layers of all the species. No staining was found in epithelial or muscular cells. Double labeling showed extensive colocalization of both proteins. SV2 exhibited a high degree of colocalization with all types of nerve fibers. Immunostaining for cleaved SNAP25 in BoNT/A treated animals showed sparse fibers in the rat and abundant fibers in the guineapig muscular layer. Cleaved SNAP25 was abundant in VAChT-IR fibers. Our data show that: I) SV2 and uncleaved SNAP25 co-expression is high. II) BONT/A targets and BONT/A action products are abundant in cholinergic fibers. III) The guinea-pig is more sensitive than the rat to BoNT/A. Supported by an unrestricted grant from Allergan 259 MICROGLIA ACTIVATION MEDIATES THE GABA(A) RECEPTOR ANTAGONIST INDUCED TACTILE ALLODYNIA Y.W. Lee1 *, K. Ishikawa2 , H. Sasaki2 , T. Ishikawa2 . 1 Department of Anesthesiology and Pain Medicine, Anesthesia and Pain Research Institute, Yonsei University Health System, Seoul, South Korea; 2 Department of Laboratory Sciences, Division of Neurosciences, Yamaguchi University Graduate School of Medicine, Ube, Japan Background: The intrathecal (IT) GABA(A) receptor antagonists, bicuculline (BIC) results in tactile allodynia (TA) through disinhibition in the spinal cord. But a little is known about mechanisms underlying neuro-glia interaction in the developing TA. We aimed to characterize the nature of the microglia mediated modulation in relation to the analgesic effects of mynocycline (MC), SB203580 (SB) and agmatine (AG) in the BIC-induced TA model.
S82
Poster Sessions / European Journal of Pain 13 (2009) S55–S285
Methods: Male SD rats were subjected to implantations of PE-10 catheter into the lumbar subarachnoid space for drug injection. Five days after surgery, vehicles (normal saline or DMSO), or the selected drugs (MC, SB or AG) in 10 mL vehicle were injected 10 min prior to BIC (10 mg). We assessed the degree of TA (graded 0, no response; 1, mild response; 2, moderate response; 3, strong response) at every 5 min for 30 min. Results were analyzed using one-way ANOVA followed by a Tukey test for multiple comparisons (P < 0.05). Results: Rats showed no TA with IT normal saline, DMSO, MC, SB or AG alone. IT BIC-induced strong TA reached its peak and kept plateau until 20 min. Pretreatment IT MC, SB and AG significantly attenuated BIC-induced TA in dose dependent manner. Conclusions: IT BIC induces functional loss of GABA(A) receptors, which inhibit the presynaptic glutamate release and hyperpolarize the postsynaptic neuron, result in neuronal synaptic hyperexcitation and TA. This BIC-induced TA could be facilitated by the neuro-microglia interaction that was probably regulated by p38MAPK downstream signaling in the spinal microglia. 260 AMYGDALA INHIBITS BOTH THE NOXIOUS AND NON-NOXIOUS RESPONSES OF WDR NEURONS IN THE RAT MEDULLARY DORSAL HORN N. Matsumoto1 *, H. Sekiyama2 , S. Bando2 , H. Yamada2 , H. Miura2 . 1 Department of Oral Physiology, School of Dentistry, Iwate Medical University, Morioka, Japan; 2 Department of Orthodontics, School of Dentistry, Iwate Medical University, Morioka, Japan Background and Aims: Conditioning electrical stimulation of the amygdala has an inhibitory effect on responses of nociceptive neurons to electrical stimulation of the receptive field in the rat medullary dorsal horn. The purpose of the present study was to determine whether amygdala stimulation induces inhibition of responses to natural noxious and non-noxious stimulations in widedynamic range (WDR) neurons. Methods: Rats were anesthetized with N2 O-O2 (2:1) and 0.5% halothane and were immobilized with pancuronium bromide. WDR neurons were recorded in the medullary dorsal horn of the rat. Peripheral natural stimuli (brushing, pressure and pinching) were applied continuously to the facial area, while conditioning electrical stimulations (33 trains pulses at 330 Hz, 300 mA) were delivered to ipsilateral amygdala at 6-sec intervals. Results: All WDR neurons responded to the three kinds of natural stimuli, and effects of amygdala stimulation on these responses were tested. In most neurons, both noxious and non-noxious responses were inhibited and the mean inhibitory effects were nearly equivalent for those responses (40–60%). The inhibitory effect lasted for 250 msec after cessation of amygdala stimulation. However, responses of low-threshold mechanoreceptive (LTM) neurons were not affected by amygdala stimulation. Conclusions: These findings suggest that excitation of neurons in the amygdala induces postsynaptic inhibition of most WDR neurons but not of non-nociceptive neurons in the medullary dorsal horn. Acknowledgments: Supported by High-Tech Research Project (2005–2009) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 261 THE PI3Kg/AKT SIGNALING PATHWAY MEDIATES PERIPHERAL ANTINOCICEPTIVE ACTION OF DIPYRONE T. Cunha1 *, G. Souza1 , H. Duarte2 , D. Roman-Campos2 , C. Lotufo1 , W. Verri Jr.1 , J. Cruz2 , S. Ferreira2 , F. Cunha1 . 1 University of S˜ ao Paulo, Ribeirao Preto, Brazil; 2 Federal University of Minas Gerais, Belo Horizonte, Brazil Background and Aims: Dipyrone, also known as metamizol, is an over-the-counter analgesic widely used in Europe and Latin America. Although considered as NSAIDs, dipyrone directly blocks inflammatory hypernociception by acting directly on primary
nociceptive neurons which is not associated with inhibition of cyclooxygenase. There is evidence suggesting that nitric oxide pathway mediates this dipyrone peripheral effect. In the present study we tested whether the PI3Kg/AKT signaling cascade is involved the peripheral antinociceptive effect of dipyrone. Methods: Mechanical hypernociception was evaluated in PGE2 sensitized rat paws using an electronic version of the von Frey test. Phosphorylated AKT expression was analyzed by western blot of DRG culture neurons. Results: Intraplantar injection of dipyrone inhibited PGE2-induced hypernociception in a dose dependent manner (80–320 mg/paw). Non-selective (wortmannin, 3 mg/paw or LY294002, 10 mg/paw) or selective (AS605240, 10–90 mg/paw) pharmacological inhibition of PI3Kg prevented the peripheral anti-hypernociceptive action of dipyrone (160 mg/paw). Further investigating the PI3Kg downstream signaling pathway it was shown that the AKT selective inhibitor (10 mg/paw) also prevents dipyrone peripheral antinociceptive effects. Corroborating with this in vivo observation, the incubation of DRG culture neurons with dipyrone (100 mM) produce an increase in the phosphorylation of AKT. This activation was prevented by PI3Kg inhibitor (AS 605240; 1 mM) or by wortmannin (100 nM) Conclusion: The present results suggest that the peripheral blockade of hypernociception by dipyrone depends on initial activation of PI3Kg/AKT signaling pathway. 262 BASELINE PREDICTORS OF IMPROVEMENT IN PAIN USING A POOLED ANALYSIS OF 7 RANDOMIZED CONTROLLED STUDIES IN PATIENTS WITH OSTEOARTHRITIS (OA) R.A. Peloso PMoore, A. Mehta, A. Gammaitoni *. Merck Research Laboratories, Rahway, United States Background: We evaluated baseline factors predicting 15%, 30%, or 50% pain improvement in OA patients after 12 weeks of treatment. Methods: Data from 7 placebo-controlled, 12-week studies utilizing etoricoxib 30 or 60 mg, celecoxib 200 mg, naproxen 1000 mg, or ibuprofen 2400 mg were pooled. Discontinued patients were assigned 0% improvement. Potential baseline predictive factors were examined for responses of 15%, 30% or 50% improvement in WOMAC Pain Subscale (100 mm VAS) at Week 12 in a multiple logistic regression model fitted with the baseline characteristics and treatment terms. Backward selection with significance level to stay in the model at 0.10 was used. Results: Using the 30% threshold (considered clinically meaningful improvement) the following non-treatment factors (OR [95% CI]) remained in the final model: degree of flare (1.17[1.12, 1.22]; p < 0.0001); baseline WOMAC physical function (0.84[0.80, 0.88]; p < 0.0001); race (white vs. nonwhite) (0.69[0.58, 0.82]; p < 0.0001); joint (hip vs. knee) (0.80[0.67, 0.95]); p = 0.012); baseline WOMAC pain (1.17[0.97, 1.42]; p = 0.093). For treatment-related factors (vs. placebo), each treatment demonstrated significant predictive value (p < 0.0001): etoricoxib 30 mg (2.78: [2.25, 3.44]), etoricoxib 60 mg (3.13: [2.46, 4.00]), naproxen 1000 mg (2.46: [1.90, 3.18]), celecoxib 200 mg (2.30: [1.79, 2.95]), ibuprofen 2400 mg (1.88: [1.45, 2.44]). Conclusions: Etoricoxib 30 mg, 60 mg and naproxen 1000 mg provided numerically the greatest likelihood of improvement. Degree of flare before randomization, baseline WOMAC physical function score, and race were the top non-treatment predictive baseline factors. RAM has received research grants from and PP, AH, HW, & AG are employed by Merck & Co., Inc., the sponsor of the study.
CAPs in pulse one were used for the study of tonic inhibition of lidocaine. The difference of CAPs between pulse 10 and one was measured for phasic inhibition. Results: Lidocaine caused concentration-dependant, tonic inhibition on conduction velocity and CAPs in both A- and C-fibers. At 300 mM the average inhibition on CAPs was 55±15 and 51±8% (mean±SEM, n = 4–5) in A- and C-fibers, respectively. Electrical stimulation of A-fibers at 200 Hz exhibited concentrationdependant, phasic inhibition by lidocaine, reaching significance at 300 mM. However, no such inhibition was observed in C-fibers with the present stimulation profile. Conclusions: Lidocaine at subblocking concentrations produces phasic inhibition in A- but not C-fibers, indicating that the function of lidocaine differs between the fiber types. 256 LOCAL ADMINISTRATION OF AGMATINE DOES NOT HAVE ANTINOCICEPTION EFFECTS ON THE INDUCTION AND MAINTENANCE OF ARTHRITIC PAIN IN RATS S.S. Min *, M.J. Lee1 , P.J. Kim2 , S.H. Han3 , J.Y. Yee3 , C. Kim3 , G.H. Seol4 . 1 Department of Anesthesiology and Pain Medicine, Konkuk University School of Medicine, Chungju, South Korea; 2 Profesional Oriental Medicine Graduate School, Wonkwang University, Iksan, South Korea; 3 Department of Physiology and Biophysics, School of Medicine, Eulji University, Daejeon, South Korea; 4 Department of Basic Nursing Science, School of Nursing, Korea University, Seoul, South Korea Previous studies suggest that systemic administration of agmatine, endogenous ligand for imidazoline receptors has antihypernociceptive effects in experimental animal. However the peripheral effects of agmatine on inflammatory pain have not yet been elucidated. Here we examined the effects of intraarticular injection of agmatine in the induction and maintenance phase of arthritic pain. In addition, we sought to determine the potential contribution of imidazoline and a2 -adrenergic receptors to the antinociceptive effects using clonidine which is mixed a2 adrenoceptor and imidazoline receptor agonist. To induce arthritis in rats, 2% lambda-carrageenan (50 ml, in saline) was injected into the joint of the right hind limb under enflurane anesthesia. Either agmatine (10, 50, 100 mg/40 ml) or clonidine (10, 50, 100 mg/40 ml) was injected into the knee joint cavity immediately before or 4 hr after carrageenan injection. Weight load tests were performed to measure pain-related behavior in freely walking rats. The intraarticular injection of agmatine into the knee joint had no effects in the both phase of induction and maintenance of arthritic pain at any dose tested. However, injection of clonidine reversed arthritic pain, when injected 4 h after carrageenan injection. In rats, agmatine has no peripheral effect on inflammatory pain and imidazoline receptors in the periphery may not contribute to the anti-inflammatory pain. 257 INTRAVENOUS LIDOCAINE ASSOCIATED WITH AMITRIPTILINE ON PAIN INTENSITY AND PLASMA CONCENTRATIONS OF SEROTONINE, NORADRENALINE AND DOPAMINE IN FIBROMYALGIA PATIENTS R.K. Sakata *, R. Vlainich, A. Issy. Federal University of S˜ ao Paulo, S˜ ao Paulo, Brazil Background and Objectives: The main effect of lidocaine is central antihyperalgesic effect. The aim of the present study was to evaluate the effect of IV lidocaine associated with amitriptiline on pain intensity and plasma serotonin, norepinephrine, and dopamine in fibromyalgic patients. Methods: Thirty patients participated in this randomized doubleblinded study. All patients used 25 mg amitriptiline; G1 patients received saline, and G2, 240 mg lidocaine, once a week, for 4 weeks. Results: Pain intensity reduced after treatment in G1 (T0: 7.0 ± 1.2; T4: 4.0 ± 2.1), and G2 (T0: 7.6 ± 0.8 e T4: 4.1 ± 2.3). Serotonin
S81
plasma concentrations were similar in T0 (G1: 42.7 ± 32.3; G2: 65.9 ± 41.1 ng/mL) and T4 (G1: 63.7 ± 32.1; G2: 76.7 ± 55.6), as norepinephrine in T0 (G1: 189.5 ± 108.6; G2: 171.9 ± 131.0 pg/mL) and T4 (G1: 167.5 ± 85.5; G2: 191.7 ± 142.3 pg/mL); dopamine levels was higher in T4 (45.7 ± 36.9 g/mL) than in T0 (17.1 ± 9.1 pg/mL) in G1; and there was no difference in T4 (34.7 ± 33.6) and T0 (25.8 ± 15.5) in G2. Conclusion: The association of 240 mg IV lidocaine (1/week) with 25 mg amitriptiline for 4 weeks didn’t modified pain intensity and plasma concentrations of serotonine, noradrenaline, and dopamine in fibromyalgia patients. 258 DISTRIBUTION AND NEUROCHEMISTRY OF BOTULINUM TOXIN TYPE A RECEPTORS IN THE URINARY BLADDER A. Coelho1,2 *, P. Dinis2,3 , R. Pinto3 , T. Gorgal3 , C. Silva3 , A. Silva3 , J. Silva3 , C. Cruz1,2 , F. Cruz1,2,3 , A. Avelino1,2 . 1 Faculty of Medicine, University of Porto, Porto, Portugal; 2 IBMC – Instituto de Biologia Molecular e Celular, Porto, Portugal; 3 Department of Urology, Hospital S. Jo˜ ao, Porto, Portugal Pain and bladder hyperactivity are main symptoms of BPS/IC (bladder painful syndrome/interstitial cystitis). Botulinum toxin type A (BoNT/A) is used as a new treatment for these pathologies. The specificity of BONT/A is due to the presence of membrane receptors SV2 that are exposed during neurotransmitter exocytosis. Once internalized, BoNT/A cleaves SNAP25 blocking neurotransmission. In this work, we studied the distribution of SV2 and SNAP25 in three different species, including human. The neurochemistry of BONT/A sensitive structures was investigated using markers for parasympathetic, sympathetic and sensory fibers. Human bladders were obtained from cadaveric organ donors. Rodent bladders were injected with BONT/A and collected at different times. Cryostat sections were immunoreacted against SV2, SNAP25 (cleaved and uncleaved), vesicular acetylcholine transporter (VAChT), tyrosine hydroxilase (TH) and calcitonin-gene related peptide (CGRP). Immunoreactive (IR) fibers for SV2 and SNAP25 were found in the mucosa and muscular layers of all the species. No staining was found in epithelial or muscular cells. Double labeling showed extensive colocalization of both proteins. SV2 exhibited a high degree of colocalization with all types of nerve fibers. Immunostaining for cleaved SNAP25 in BoNT/A treated animals showed sparse fibers in the rat and abundant fibers in the guineapig muscular layer. Cleaved SNAP25 was abundant in VAChT-IR fibers. Our data show that: I) SV2 and uncleaved SNAP25 co-expression is high. II) BONT/A targets and BONT/A action products are abundant in cholinergic fibers. III) The guinea-pig is more sensitive than the rat to BoNT/A. Supported by an unrestricted grant from Allergan 259 MICROGLIA ACTIVATION MEDIATES THE GABA(A) RECEPTOR ANTAGONIST INDUCED TACTILE ALLODYNIA Y.W. Lee1 *, K. Ishikawa2 , H. Sasaki2 , T. Ishikawa2 . 1 Department of Anesthesiology and Pain Medicine, Anesthesia and Pain Research Institute, Yonsei University Health System, Seoul, South Korea; 2 Department of Laboratory Sciences, Division of Neurosciences, Yamaguchi University Graduate School of Medicine, Ube, Japan Background: The intrathecal (IT) GABA(A) receptor antagonists, bicuculline (BIC) results in tactile allodynia (TA) through disinhibition in the spinal cord. But a little is known about mechanisms underlying neuro-glia interaction in the developing TA. We aimed to characterize the nature of the microglia mediated modulation in relation to the analgesic effects of mynocycline (MC), SB203580 (SB) and agmatine (AG) in the BIC-induced TA model.
S82
Poster Sessions / European Journal of Pain 13 (2009) S55–S285
Methods: Male SD rats were subjected to implantations of PE-10 catheter into the lumbar subarachnoid space for drug injection. Five days after surgery, vehicles (normal saline or DMSO), or the selected drugs (MC, SB or AG) in 10 mL vehicle were injected 10 min prior to BIC (10 mg). We assessed the degree of TA (graded 0, no response; 1, mild response; 2, moderate response; 3, strong response) at every 5 min for 30 min. Results were analyzed using one-way ANOVA followed by a Tukey test for multiple comparisons (P < 0.05). Results: Rats showed no TA with IT normal saline, DMSO, MC, SB or AG alone. IT BIC-induced strong TA reached its peak and kept plateau until 20 min. Pretreatment IT MC, SB and AG significantly attenuated BIC-induced TA in dose dependent manner. Conclusions: IT BIC induces functional loss of GABA(A) receptors, which inhibit the presynaptic glutamate release and hyperpolarize the postsynaptic neuron, result in neuronal synaptic hyperexcitation and TA. This BIC-induced TA could be facilitated by the neuro-microglia interaction that was probably regulated by p38MAPK downstream signaling in the spinal microglia. 260 AMYGDALA INHIBITS BOTH THE NOXIOUS AND NON-NOXIOUS RESPONSES OF WDR NEURONS IN THE RAT MEDULLARY DORSAL HORN N. Matsumoto1 *, H. Sekiyama2 , S. Bando2 , H. Yamada2 , H. Miura2 . 1 Department of Oral Physiology, School of Dentistry, Iwate Medical University, Morioka, Japan; 2 Department of Orthodontics, School of Dentistry, Iwate Medical University, Morioka, Japan Background and Aims: Conditioning electrical stimulation of the amygdala has an inhibitory effect on responses of nociceptive neurons to electrical stimulation of the receptive field in the rat medullary dorsal horn. The purpose of the present study was to determine whether amygdala stimulation induces inhibition of responses to natural noxious and non-noxious stimulations in widedynamic range (WDR) neurons. Methods: Rats were anesthetized with N2 O-O2 (2:1) and 0.5% halothane and were immobilized with pancuronium bromide. WDR neurons were recorded in the medullary dorsal horn of the rat. Peripheral natural stimuli (brushing, pressure and pinching) were applied continuously to the facial area, while conditioning electrical stimulations (33 trains pulses at 330 Hz, 300 mA) were delivered to ipsilateral amygdala at 6-sec intervals. Results: All WDR neurons responded to the three kinds of natural stimuli, and effects of amygdala stimulation on these responses were tested. In most neurons, both noxious and non-noxious responses were inhibited and the mean inhibitory effects were nearly equivalent for those responses (40–60%). The inhibitory effect lasted for 250 msec after cessation of amygdala stimulation. However, responses of low-threshold mechanoreceptive (LTM) neurons were not affected by amygdala stimulation. Conclusions: These findings suggest that excitation of neurons in the amygdala induces postsynaptic inhibition of most WDR neurons but not of non-nociceptive neurons in the medullary dorsal horn. Acknowledgments: Supported by High-Tech Research Project (2005–2009) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 261 THE PI3Kg/AKT SIGNALING PATHWAY MEDIATES PERIPHERAL ANTINOCICEPTIVE ACTION OF DIPYRONE T. Cunha1 *, G. Souza1 , H. Duarte2 , D. Roman-Campos2 , C. Lotufo1 , W. Verri Jr.1 , J. Cruz2 , S. Ferreira2 , F. Cunha1 . 1 University of S˜ ao Paulo, Ribeirao Preto, Brazil; 2 Federal University of Minas Gerais, Belo Horizonte, Brazil Background and Aims: Dipyrone, also known as metamizol, is an over-the-counter analgesic widely used in Europe and Latin America. Although considered as NSAIDs, dipyrone directly blocks inflammatory hypernociception by acting directly on primary
nociceptive neurons which is not associated with inhibition of cyclooxygenase. There is evidence suggesting that nitric oxide pathway mediates this dipyrone peripheral effect. In the present study we tested whether the PI3Kg/AKT signaling cascade is involved the peripheral antinociceptive effect of dipyrone. Methods: Mechanical hypernociception was evaluated in PGE2 sensitized rat paws using an electronic version of the von Frey test. Phosphorylated AKT expression was analyzed by western blot of DRG culture neurons. Results: Intraplantar injection of dipyrone inhibited PGE2-induced hypernociception in a dose dependent manner (80–320 mg/paw). Non-selective (wortmannin, 3 mg/paw or LY294002, 10 mg/paw) or selective (AS605240, 10–90 mg/paw) pharmacological inhibition of PI3Kg prevented the peripheral anti-hypernociceptive action of dipyrone (160 mg/paw). Further investigating the PI3Kg downstream signaling pathway it was shown that the AKT selective inhibitor (10 mg/paw) also prevents dipyrone peripheral antinociceptive effects. Corroborating with this in vivo observation, the incubation of DRG culture neurons with dipyrone (100 mM) produce an increase in the phosphorylation of AKT. This activation was prevented by PI3Kg inhibitor (AS 605240; 1 mM) or by wortmannin (100 nM) Conclusion: The present results suggest that the peripheral blockade of hypernociception by dipyrone depends on initial activation of PI3Kg/AKT signaling pathway. 262 BASELINE PREDICTORS OF IMPROVEMENT IN PAIN USING A POOLED ANALYSIS OF 7 RANDOMIZED CONTROLLED STUDIES IN PATIENTS WITH OSTEOARTHRITIS (OA) R.A. Peloso PMoore, A. Mehta, A. Gammaitoni *. Merck Research Laboratories, Rahway, United States Background: We evaluated baseline factors predicting 15%, 30%, or 50% pain improvement in OA patients after 12 weeks of treatment. Methods: Data from 7 placebo-controlled, 12-week studies utilizing etoricoxib 30 or 60 mg, celecoxib 200 mg, naproxen 1000 mg, or ibuprofen 2400 mg were pooled. Discontinued patients were assigned 0% improvement. Potential baseline predictive factors were examined for responses of 15%, 30% or 50% improvement in WOMAC Pain Subscale (100 mm VAS) at Week 12 in a multiple logistic regression model fitted with the baseline characteristics and treatment terms. Backward selection with significance level to stay in the model at 0.10 was used. Results: Using the 30% threshold (considered clinically meaningful improvement) the following non-treatment factors (OR [95% CI]) remained in the final model: degree of flare (1.17[1.12, 1.22]; p < 0.0001); baseline WOMAC physical function (0.84[0.80, 0.88]; p < 0.0001); race (white vs. nonwhite) (0.69[0.58, 0.82]; p < 0.0001); joint (hip vs. knee) (0.80[0.67, 0.95]); p = 0.012); baseline WOMAC pain (1.17[0.97, 1.42]; p = 0.093). For treatment-related factors (vs. placebo), each treatment demonstrated significant predictive value (p < 0.0001): etoricoxib 30 mg (2.78: [2.25, 3.44]), etoricoxib 60 mg (3.13: [2.46, 4.00]), naproxen 1000 mg (2.46: [1.90, 3.18]), celecoxib 200 mg (2.30: [1.79, 2.95]), ibuprofen 2400 mg (1.88: [1.45, 2.44]). Conclusions: Etoricoxib 30 mg, 60 mg and naproxen 1000 mg provided numerically the greatest likelihood of improvement. Degree of flare before randomization, baseline WOMAC physical function score, and race were the top non-treatment predictive baseline factors. RAM has received research grants from and PP, AH, HW, & AG are employed by Merck & Co., Inc., the sponsor of the study.