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Involvement of spinal CCL3 in the mechanism of tactile hypersensitization after peripheral nerve injury
Abstracts / Neuroscience Research 68S (2010) e109–e222
perfused intracardially with paraformaldehyde at 2 hr after thermal stimulation and lumbar spinal cords were removed. Frozen transverse serial sections from the spinal cord were processed for immunohistochemical staining for c-Fos by using the ABC method. The numbers of c-Fos immunopositive neurons in the spinal cord were manually counted under optical microscope. Milnacipran significantly attenuated FLI in the superficial layer of the spinal dorsal horn of both neuropathic pain model rats. On the other hand, the both paroxetine and fluvoxamine significantly attenuated FLI in the diabetic rat, but these inhibitory effects were less than that of milnacipran. The inhibitory effect of ADs on the induction of FLI following innocuous thermal stimulation seems likely to be in rats with neuropathy but not in normal animals. doi:10.1016/j.neures.2010.07.2293
P1-j05 Elimination of IB4-positive neurons in the trigeminal caudal nucleus enhanced nociceptive responses of the formalin-induced pain-related behavior at second phase in the upper lip in rats Aiko Oyamaguchi 1 , Shinichi Sugiyo 2 , Tetsuya Masawaki 1 , Hitoshi Niwa 1 , Motohide Takemura 2
e163
conditions. Under cell-attached conditions, LC neuron tested fired spontaneously. Cutaneous noxious stimuli transiently increased the frequency of the spontaneous action potentials. Under current-clamp conditions, the resting membrane potential was ranged from-35 to −65 mV, and spontaneous firing was also detected at the similar frequency compared with that obtained from cell-attached recordings. Under voltage-clamp conditions at a holding potential of −70 mV, LC neurons exhibited spontaneous excitatory postsynaptic currents (EPSCs). Spontaneous inhibitory postsynaptic currents (IPSCs) were evoked in the same neurons at a holding potential of 0 mV. The EPSCs and IPSCs, respectively, were inhibited by application of CNQX and bicuculline to the dorsal surface of the brain stem. The frequency of spontaneous EPSCs was much lower than that of IPSCs, suggesting that LC neurons in vivo receive abundant inhibitory synaptic inputs. This newly-developed in vivo recording technique enabled us to analyze excitatory and inhibitory synaptic responses evoked in LC neurons and is therefore useful to study actions of analgesic drugs on descending noradrenergic system at the synaptic level. doi:10.1016/j.neures.2010.07.2295
Abe 3 , Aya
1
Dent. Anesthesiol., Osaka Univ. Grad Sch. of Dent.,Osaka 2 Oral Anat. and Neurobiology. Osaka Univ. Grad Sch. of Dent., osaka 3 Dept. Dent Oral Surg Hyogo Col Med, Nishinomiya Small-sized sensory fibers, most of them are nociceptors, but not exclusive, can be classified into two groups, peptidergic and non-peptidergic. The non-peptidergic fibers have an affinity to a lectin Griffonia simplicifolia IB4(IB4) and are sensitive to GDNF, while peptidergic fibers are sensitive to NGF. The functional significance of these two categories of nociceptors is largely unknown. This study examined effects of selective ablation of neurons-having binding affinity to IB4 in the medullary dorsal horn(Vc) by intra-cisterna magnal injection of IB4 conjugated to saporin(IB4-Sap) on the formalin-induced nociceptive responses and c-Fos expression in the Vc. In rats 2–4 weeks after pretreated with IB4-Sap, the IB4 binding immunoreactivity in lamina II of the Vc decreased compared with rats pretreated with saline or blank-saporin. Eye wipes after a drop of ammonium hydroxide was placed into one eye process was used to verify of the first pain. Number of Eye wipes in 10 s significantly increased in rats pretreated with IB4-Sap compared with pretreated with Sal or Bl-Sap. Injection of formalin into the upper lip of saline-pretreated control rats evoked time-dependent increases in number of pain-related behavior(PRB). From 0–15 min after formalin injection, abrupt increase in the PRB was observed(phase 1), which was followed by more active behavior at 15-30 min(phase 2), and a gradual decrease of behavior with some fluctuation from 30-45 min(phase 3). In rats pretreated with IB4Sap, the formalin injection induced significant increased number of PRB at second phase compared with pretreated Sal or Bl-Sap. The PRB showed the increasing tendency in the rats that had pretreated IB4-Sap. Two hrs after formalin injection, c-Fos immunoreactivity in the superficial layers of the Vc showed a tendency of increase in the IB4-Sap-pretreated rats. These results indicate that IB4-binding neurons are involved in the inhibition of the 2nd phase of the inflammatory pain. doi:10.1016/j.neures.2010.07.2294
P1-j07 Changes in skin temperature in humans elicited by scratching on the itchy skin Natsu Koyama Department of Physiology, Shiga University of Medical Science Itch as well as pain is the sensation mediated by the unmyelinated C-fibers. We have performed two kind of analysis to evaluate the skin temperature changes induced by the C-fiber mediated-painful sensation using computerassisted thermography. The first one is the analysis of the neurogenic inflammation. Intradermal injection of the algogenic substance such as the bee venom melittin increased the local skin temperature around the injection site, with a latency of a few minutes after on set of pain. The second one is to analyze the remote temperature in the index finger which is richly innervated by sympathetic vasoconstrictor nerves. Melittin injection into the forearm produced skin temperature decrease in finger, which was interpreted as a cutaneous vasoconstrictor reflex response occurred simultaneously with pain sensation. Aim of the present study is to verify that the skin temperature changes are produced by itch stimulation similarly to painful stimulation. Although skin scratching with a fingernail dose not usually evoke itch sensation for healthy subject, it evoked itch sensation for the healthy subjects but with dermographia. Skin-scratching evoked itch sensation produced the visible flare, vasodilatation and the skin temperature increase, corresponding to these produced by the painful stimulation. However, it did not affect the finger temperature decrease. These results suggested that the stimulation evoked itch sensation produced the axon reflex-neurogenic inflammation, but did not evoke a cutaneous vasoconstrictor reflex responses. doi:10.1016/j.neures.2010.07.2296
P1-j08 Involvement of spinal CCL3 in the mechanism of tactile hypersensitization after peripheral nerve injury Hidetoshi Saitoh , Makoto Tsuda, Kazuaki Ueda, Kazuhide Inoue Dept. Mol. Sys. Pharmacol., Graduate Sch. Pharmaceut. Sci., Kyushu Univ
P1-j06 In vivo patch-clamp analysis of synaptic responses evoked in the rat locus coeruleus neurons
Daisuke Sugiyama 1,2 , Keiji Imoto 2,3 , Mikito Kawamata 1 , Hidemasa Furue 2,3 1 Dept of Anesthesiol & Resuscitol, Shinshu Univ School of Medicine, Matsumoto 2 Dept of Information Physiol, National Institute for Physiological Sciences, Okazaki 3 School of life Science, The Graduate University for Advanced studies
The nucleus locus coeruleus (LC) is a major source of noradrenergic projections to the spinal dorsal horn. There is considerable evidence that the bulbospinal noradrenergic system plays a significant role in pain modulation. In this study, we developed an in vivo patch-clamp recording technique from LC neurons to study the modulatory role at the synaptic level. Male rats (4-6weeks old) were anesthetized with urethane and the animal was fixed in a stereotaxic frame. After a craniotomy was performed, the cerebellum was removed to expose the dorsal surface of the brain stem. The patch pipette was advanced into the LC by using a micromanipulator. After established a gigaohm seal formation, cell-attached and whole-cell patch-clamp recordings were made from LC neurons under current- and voltage-clamp
Neuropathic pain is an expression of pathological operation of the nervous system, which occurs after peripheral nerve injury and one hallmark of which is tactile allodynia (pain hypersensitivity to innocuous stimuli). The mechanisms by which nerve injury develops tactile allodynia have remained largely unknown. Recent studies have revealed that several molecules of chemokine family is involved in the pathogenesis of neuropathic pain. However, chemokine family and also chemokine receptor family consist of large number of molecules, and there are sophisticated signal crosstalk between each ligand and receptor set. Hence their remains a possibility for the other chemokines to be involved in the pathogenesis of neuropathic pain. Here we found that the level of CC chemokine ligand 3(CCL3) mRNA was significantly increased in ipsilateral spinal cord after nerve injury. This increase was observed from day1 to day14 after nerve injury. A single intrathecal administration of recombinant CCL3 produce tactile allodynia. The paw withdrawal threshold (PWT) to mechanical stimulation decreased over first 15 min and the decrease persisted for 5 h. However the PWT decreased again from 24 h to 10 days after administration. These results suggest that the release of CCL3 was induced by nerve injury, and CCL3 signaling may be involved in neuropathic pain. Thus, CCL3 might be a new therapeutic target for pain induced after nerve injury.
e164
Abstracts / Neuroscience Research 68S (2010) e109–e222
doi:10.1016/j.neures.2010.07.2297
P1-j09 ATP-induced suppression of mechanical response and sensitization to acid of muscle C-fiber afferents in vitro Teru
Matsuda 1,2
, Toru
Taguchi 1 ,
Kazue
1
Mizumura 1
Dept. Neurosci. II, Res. Inst. Environ. Med., Nagoya Univ., Nagoya Phys. Ther., Coll. Life Health Sci., Chubu Univ., Kasugai
2
Dept.
Subcutaneous injection of ATP is known to induce pain and hyperalgesia to heat and mechanical stimulation in the skin. In addition, decrease of interstitial pH by inflammation or exercise is thought to be one of the causes for pain and hyperalgesia. In this experiment we examined whether ATP and pH have any effect on muscle thin fiber afferent activities. Single fiber activities were recorded in vitro from EDL muscle-common peroneal nerve preparations excised from anesthetized rats. The ramp mechanical stimulation (0–196 mN in 10 s) was applied every 10 min. ATP (1 M, 100 M, 1 mM) (ATP group) or Krebs solution (control group) was superfused for 5 min before 2nd mechanical stimulation. The P2 receptor antagonist PPADS (100 M) or suramin (300 M) was superfused 1 min before until the end of ATP (100 M) administration. Thereafter different pH (7.4, 7.0, 6.6 and 6.2) solutions were superfused for 30 s. 1 mM ATP induced excitation in about half of muscle thin-fiber afferents tested. It significantly increased the mechanical threshold and decreased the response magnitude to the following mechanical stimulation. 100 M ATP significantly increased the mechanical threshold but did not change the response magnitude. 1 M ATP had similar tendencies. PPADS and suramin reversed the increased mechanical threshold induced by 100 M ATP. None of pH solutions (∼ pH 6.2) induced significant increase in discharges in the control group, whereas pH 6.2 solution significantly increased discharges in the ATP group. PPADS and suramin suppressed the delayed increase of pH6.2-induced discharges. Presently observed suppressive effect of ATP on mechanical response of muscle C-fibers is different from previous reports, but both this suppressive effect on mechanical response and delayed facilitation of pH response was reversed by PPADS and suramin, demonstrating that these effects are mediated by P2 receptors. To validate these observations, behavioral pain test is now being studied. doi:10.1016/j.neures.2010.07.2298
P1-j10 The Effects of Endogenous Dopamine on Mechanical Nociceptive Responses recorded in the Prefrontal Cortex Shoichi Sogabe , Yoriko Kawakami Department of Physiology, Tokyo Women’s Medical University, Tokyo We examined effects of dopamine (DA) on nociceptive responses recorded in the cingulate area of the prefrontal cortex (PFC). Experiments using extracellular single unit recording were performed to determine whether endogenous dopamine levels affect nociceptive neuronal activity in the PFC. [Mehtods & Results] Nociceptive responses, which were evoked by mechanical pressure stimulation (for 2 s at 500 g constant force) applied to the tails of urethane-anesthetized rats, were recorded in PFC. High-frequency stimulation (HFS) (50 Hz, 250 A for 30 s) delivered to VTA, which has been reported to increase PFC dopamine concentrations (Gurden, 1999), significantly decreased nociceptive responses at 10 and 30 min after HFS. We studied effects of microinjections of dopamine D1 and D2 receptor antagonists into PFC on the depressive effects of HFS delivered to VTA. The results suggested that the depression of nociceptive responses induced by HFS of VTA was mediated by DA higher concentration. On the contrary, the microinjection of -opioid into the VTA inhibited dopaminergic neurons projecting to PFC (Margolis, 2006). We investigated changes in nociceptive neuronal activity after a microinjection of -opioid receptor agonist, U-50488, into the VTA.[Discussion] DA has been considered to regulate attention in PFC (Seamans, 2004). Basic DA concentration in PFC is tonically maintained and VTA lesion markedly decreased of DA (Devoto P, 2008). Dopamine level of brain is also suggested to modify pain sensation in Parkinson’s disease. In clinical reports of Parkinson’s disease, patients frequently complain pain before motor disorder (Chaudhuri, 2006). Neuropathic pain highly appears in Parkinson’s disease patients compared to normal adults (Defazio, 2008). DA system may be involved in processes of conscious pain. The mechanisms of pain symptoms in Parkinson’s disease may associate with disorders of the endogenous dopamine system as well as motor symptoms. doi:10.1016/j.neures.2010.07.2299
P1-j11 Nociceptive stimulation induces the c-fos-mRFP fusion gene expression in the spinal cord and the hypothalamus of a transgenic rat Toru Ishikura 1 , Hitoshi Suzuki 1,2 , Hiroaki Fujihara 1 , Hideo Ohnishi 2 , Yoichi Ueta 1 1 Department of Physiology, School of Medicine, University of Occupational and Environmental Health 2 Department of Orthopaedics, School of Medicine, University of Occupational and Environmental Health
The expression of the c-fos gene has been widely used as a marker of neuronal activity in the central nervous system (CNS). Recently, we generated transgenic rats expressing the c-fos and monomeric red fluorescent protein (mRFP) fusion gene in the CNS after adequate stimulation. In the present study, we observed the induction of the c-fos-mRFP fusion gene in the spinal cord and the hypothalamus after nociceptive stimulation in c-fosmRFP transgenic rats. Ninety minutes after a subcutaneous injection of saline or 5% formalin into the dorsal surface of hindpaw, animals were perfused transcardially by 4% paraformaldehyde solution after anesthesia. Abundant nuclear mRFP fluorescences were observed in the dorsal horn of the spinal cord and the several central regions including the hypothalamic paraventricular nucleus in formalin-injected rats but a few in the saline-injected rats under fluorescent microscopy. This c-fos-mRFP transgenic rat is a new useful animal to study the central response to nociceptive stimulation. doi:10.1016/j.neures.2010.07.2300
P1-j12 Pain modulation following air-puff startle response in the rat Junichiro Tamaki , Masayoshi Tsuruoka, Masako Maeda, Bunsho Hayashi, Tomio Inoue Department of Physiology, Showa University School of Dentistry An air puff elicits a startle response in mammals. Following the air–puff startle reaction, rats assume a defensive–like, immobile posture (DIP) of approximately 2–5 s in length. We previously reported that bilateral lesions of the nucleus coeruleus/subcoeruleus (LC/SC) reduce the DIP period. The LC/SC is one of the structures that play an important role in endogenous pain control. Our particular interest is whether the nociceptive inputs are inhibited by activation of the LC/SC during the DIP period. Rats were tested for behavioral nociception with heat stimuli. The heat source was attached to the tail; rats bit the heat source following heating. The bite latency (BL) was measured as an indicator of pain threshold. The BL and the DIP period were measured with a digital stop watch. For air–puff stimulation, compressed house air (14.4 psi in strength, 0.1 s in duration) was presented as a transient through a vinyl tube suspended 2.5 cm above the rat’s head. Two weeks before the experiment, the rats received bilateral injections of 6 g of the neurotoxin 6–hydroxydopamine to specifically lesion noradrenaline–containing neurons of the LC/SC. When heat stimuli and air–puff stimuli were simultaneously applied, BLs were significantly prolonged in comparison with those induced by only heat stimuli. In the LC/SC–lesioned rats (n = 10), air–puff–induced prolongation of BLs was not observed in post–lesions, whereas air–puff stimulation significantly prolonged BLs in pre–lesions. In contrast, in the sham-lesioned rats (n = 10), air–puff stimulation significantly prolonged BLs in both pre- and postlesions. When the percentage change of BLs between pre–and post–lesions was plotted against that of DIP periods between pre–and post–lesions, regression analysis showed a strong correlation (R2 = 0.67). These results suggest that (1) air–puff stimulation produces pain reduction, (2) the LC/SC is involved in pain reduction, and (3) pain reduction is related to the DIP period. doi:10.1016/j.neures.2010.07.2301
P1-j13 Macrophage inflammatory protein-1␣ mediates the development of neuropathic pain following peripheral nerve injury Norikazu Kiguchi , Takehiko Fukazawa, Shiroh Kishioka
Maeda, Yuka
Kobayashi, Yohji
Dept Pharmacol, Wakayama Med Univ, Wakayama Although accumulating evidence indicates that several inflammatory mediators derived from glial cells and immune cells participate in the pathogenesis of neuropathic pain, the roles of chemokines are poorly understood. In this study, we investigated the role of the macrophage inflammatory protein-1␣ (MIP-1␣; CCL3) in the development of neuropathic pain following partial
perfused intracardially with paraformaldehyde at 2 hr after thermal stimulation and lumbar spinal cords were removed. Frozen transverse serial sections from the spinal cord were processed for immunohistochemical staining for c-Fos by using the ABC method. The numbers of c-Fos immunopositive neurons in the spinal cord were manually counted under optical microscope. Milnacipran significantly attenuated FLI in the superficial layer of the spinal dorsal horn of both neuropathic pain model rats. On the other hand, the both paroxetine and fluvoxamine significantly attenuated FLI in the diabetic rat, but these inhibitory effects were less than that of milnacipran. The inhibitory effect of ADs on the induction of FLI following innocuous thermal stimulation seems likely to be in rats with neuropathy but not in normal animals. doi:10.1016/j.neures.2010.07.2293
P1-j05 Elimination of IB4-positive neurons in the trigeminal caudal nucleus enhanced nociceptive responses of the formalin-induced pain-related behavior at second phase in the upper lip in rats Aiko Oyamaguchi 1 , Shinichi Sugiyo 2 , Tetsuya Masawaki 1 , Hitoshi Niwa 1 , Motohide Takemura 2
e163
conditions. Under cell-attached conditions, LC neuron tested fired spontaneously. Cutaneous noxious stimuli transiently increased the frequency of the spontaneous action potentials. Under current-clamp conditions, the resting membrane potential was ranged from-35 to −65 mV, and spontaneous firing was also detected at the similar frequency compared with that obtained from cell-attached recordings. Under voltage-clamp conditions at a holding potential of −70 mV, LC neurons exhibited spontaneous excitatory postsynaptic currents (EPSCs). Spontaneous inhibitory postsynaptic currents (IPSCs) were evoked in the same neurons at a holding potential of 0 mV. The EPSCs and IPSCs, respectively, were inhibited by application of CNQX and bicuculline to the dorsal surface of the brain stem. The frequency of spontaneous EPSCs was much lower than that of IPSCs, suggesting that LC neurons in vivo receive abundant inhibitory synaptic inputs. This newly-developed in vivo recording technique enabled us to analyze excitatory and inhibitory synaptic responses evoked in LC neurons and is therefore useful to study actions of analgesic drugs on descending noradrenergic system at the synaptic level. doi:10.1016/j.neures.2010.07.2295
Abe 3 , Aya
1
Dent. Anesthesiol., Osaka Univ. Grad Sch. of Dent.,Osaka 2 Oral Anat. and Neurobiology. Osaka Univ. Grad Sch. of Dent., osaka 3 Dept. Dent Oral Surg Hyogo Col Med, Nishinomiya Small-sized sensory fibers, most of them are nociceptors, but not exclusive, can be classified into two groups, peptidergic and non-peptidergic. The non-peptidergic fibers have an affinity to a lectin Griffonia simplicifolia IB4(IB4) and are sensitive to GDNF, while peptidergic fibers are sensitive to NGF. The functional significance of these two categories of nociceptors is largely unknown. This study examined effects of selective ablation of neurons-having binding affinity to IB4 in the medullary dorsal horn(Vc) by intra-cisterna magnal injection of IB4 conjugated to saporin(IB4-Sap) on the formalin-induced nociceptive responses and c-Fos expression in the Vc. In rats 2–4 weeks after pretreated with IB4-Sap, the IB4 binding immunoreactivity in lamina II of the Vc decreased compared with rats pretreated with saline or blank-saporin. Eye wipes after a drop of ammonium hydroxide was placed into one eye process was used to verify of the first pain. Number of Eye wipes in 10 s significantly increased in rats pretreated with IB4-Sap compared with pretreated with Sal or Bl-Sap. Injection of formalin into the upper lip of saline-pretreated control rats evoked time-dependent increases in number of pain-related behavior(PRB). From 0–15 min after formalin injection, abrupt increase in the PRB was observed(phase 1), which was followed by more active behavior at 15-30 min(phase 2), and a gradual decrease of behavior with some fluctuation from 30-45 min(phase 3). In rats pretreated with IB4Sap, the formalin injection induced significant increased number of PRB at second phase compared with pretreated Sal or Bl-Sap. The PRB showed the increasing tendency in the rats that had pretreated IB4-Sap. Two hrs after formalin injection, c-Fos immunoreactivity in the superficial layers of the Vc showed a tendency of increase in the IB4-Sap-pretreated rats. These results indicate that IB4-binding neurons are involved in the inhibition of the 2nd phase of the inflammatory pain. doi:10.1016/j.neures.2010.07.2294
P1-j07 Changes in skin temperature in humans elicited by scratching on the itchy skin Natsu Koyama Department of Physiology, Shiga University of Medical Science Itch as well as pain is the sensation mediated by the unmyelinated C-fibers. We have performed two kind of analysis to evaluate the skin temperature changes induced by the C-fiber mediated-painful sensation using computerassisted thermography. The first one is the analysis of the neurogenic inflammation. Intradermal injection of the algogenic substance such as the bee venom melittin increased the local skin temperature around the injection site, with a latency of a few minutes after on set of pain. The second one is to analyze the remote temperature in the index finger which is richly innervated by sympathetic vasoconstrictor nerves. Melittin injection into the forearm produced skin temperature decrease in finger, which was interpreted as a cutaneous vasoconstrictor reflex response occurred simultaneously with pain sensation. Aim of the present study is to verify that the skin temperature changes are produced by itch stimulation similarly to painful stimulation. Although skin scratching with a fingernail dose not usually evoke itch sensation for healthy subject, it evoked itch sensation for the healthy subjects but with dermographia. Skin-scratching evoked itch sensation produced the visible flare, vasodilatation and the skin temperature increase, corresponding to these produced by the painful stimulation. However, it did not affect the finger temperature decrease. These results suggested that the stimulation evoked itch sensation produced the axon reflex-neurogenic inflammation, but did not evoke a cutaneous vasoconstrictor reflex responses. doi:10.1016/j.neures.2010.07.2296
P1-j08 Involvement of spinal CCL3 in the mechanism of tactile hypersensitization after peripheral nerve injury Hidetoshi Saitoh , Makoto Tsuda, Kazuaki Ueda, Kazuhide Inoue Dept. Mol. Sys. Pharmacol., Graduate Sch. Pharmaceut. Sci., Kyushu Univ
P1-j06 In vivo patch-clamp analysis of synaptic responses evoked in the rat locus coeruleus neurons
Daisuke Sugiyama 1,2 , Keiji Imoto 2,3 , Mikito Kawamata 1 , Hidemasa Furue 2,3 1 Dept of Anesthesiol & Resuscitol, Shinshu Univ School of Medicine, Matsumoto 2 Dept of Information Physiol, National Institute for Physiological Sciences, Okazaki 3 School of life Science, The Graduate University for Advanced studies
The nucleus locus coeruleus (LC) is a major source of noradrenergic projections to the spinal dorsal horn. There is considerable evidence that the bulbospinal noradrenergic system plays a significant role in pain modulation. In this study, we developed an in vivo patch-clamp recording technique from LC neurons to study the modulatory role at the synaptic level. Male rats (4-6weeks old) were anesthetized with urethane and the animal was fixed in a stereotaxic frame. After a craniotomy was performed, the cerebellum was removed to expose the dorsal surface of the brain stem. The patch pipette was advanced into the LC by using a micromanipulator. After established a gigaohm seal formation, cell-attached and whole-cell patch-clamp recordings were made from LC neurons under current- and voltage-clamp
Neuropathic pain is an expression of pathological operation of the nervous system, which occurs after peripheral nerve injury and one hallmark of which is tactile allodynia (pain hypersensitivity to innocuous stimuli). The mechanisms by which nerve injury develops tactile allodynia have remained largely unknown. Recent studies have revealed that several molecules of chemokine family is involved in the pathogenesis of neuropathic pain. However, chemokine family and also chemokine receptor family consist of large number of molecules, and there are sophisticated signal crosstalk between each ligand and receptor set. Hence their remains a possibility for the other chemokines to be involved in the pathogenesis of neuropathic pain. Here we found that the level of CC chemokine ligand 3(CCL3) mRNA was significantly increased in ipsilateral spinal cord after nerve injury. This increase was observed from day1 to day14 after nerve injury. A single intrathecal administration of recombinant CCL3 produce tactile allodynia. The paw withdrawal threshold (PWT) to mechanical stimulation decreased over first 15 min and the decrease persisted for 5 h. However the PWT decreased again from 24 h to 10 days after administration. These results suggest that the release of CCL3 was induced by nerve injury, and CCL3 signaling may be involved in neuropathic pain. Thus, CCL3 might be a new therapeutic target for pain induced after nerve injury.
e164
Abstracts / Neuroscience Research 68S (2010) e109–e222
doi:10.1016/j.neures.2010.07.2297
P1-j09 ATP-induced suppression of mechanical response and sensitization to acid of muscle C-fiber afferents in vitro Teru
Matsuda 1,2
, Toru
Taguchi 1 ,
Kazue
1
Mizumura 1
Dept. Neurosci. II, Res. Inst. Environ. Med., Nagoya Univ., Nagoya Phys. Ther., Coll. Life Health Sci., Chubu Univ., Kasugai
2
Dept.
Subcutaneous injection of ATP is known to induce pain and hyperalgesia to heat and mechanical stimulation in the skin. In addition, decrease of interstitial pH by inflammation or exercise is thought to be one of the causes for pain and hyperalgesia. In this experiment we examined whether ATP and pH have any effect on muscle thin fiber afferent activities. Single fiber activities were recorded in vitro from EDL muscle-common peroneal nerve preparations excised from anesthetized rats. The ramp mechanical stimulation (0–196 mN in 10 s) was applied every 10 min. ATP (1 M, 100 M, 1 mM) (ATP group) or Krebs solution (control group) was superfused for 5 min before 2nd mechanical stimulation. The P2 receptor antagonist PPADS (100 M) or suramin (300 M) was superfused 1 min before until the end of ATP (100 M) administration. Thereafter different pH (7.4, 7.0, 6.6 and 6.2) solutions were superfused for 30 s. 1 mM ATP induced excitation in about half of muscle thin-fiber afferents tested. It significantly increased the mechanical threshold and decreased the response magnitude to the following mechanical stimulation. 100 M ATP significantly increased the mechanical threshold but did not change the response magnitude. 1 M ATP had similar tendencies. PPADS and suramin reversed the increased mechanical threshold induced by 100 M ATP. None of pH solutions (∼ pH 6.2) induced significant increase in discharges in the control group, whereas pH 6.2 solution significantly increased discharges in the ATP group. PPADS and suramin suppressed the delayed increase of pH6.2-induced discharges. Presently observed suppressive effect of ATP on mechanical response of muscle C-fibers is different from previous reports, but both this suppressive effect on mechanical response and delayed facilitation of pH response was reversed by PPADS and suramin, demonstrating that these effects are mediated by P2 receptors. To validate these observations, behavioral pain test is now being studied. doi:10.1016/j.neures.2010.07.2298
P1-j10 The Effects of Endogenous Dopamine on Mechanical Nociceptive Responses recorded in the Prefrontal Cortex Shoichi Sogabe , Yoriko Kawakami Department of Physiology, Tokyo Women’s Medical University, Tokyo We examined effects of dopamine (DA) on nociceptive responses recorded in the cingulate area of the prefrontal cortex (PFC). Experiments using extracellular single unit recording were performed to determine whether endogenous dopamine levels affect nociceptive neuronal activity in the PFC. [Mehtods & Results] Nociceptive responses, which were evoked by mechanical pressure stimulation (for 2 s at 500 g constant force) applied to the tails of urethane-anesthetized rats, were recorded in PFC. High-frequency stimulation (HFS) (50 Hz, 250 A for 30 s) delivered to VTA, which has been reported to increase PFC dopamine concentrations (Gurden, 1999), significantly decreased nociceptive responses at 10 and 30 min after HFS. We studied effects of microinjections of dopamine D1 and D2 receptor antagonists into PFC on the depressive effects of HFS delivered to VTA. The results suggested that the depression of nociceptive responses induced by HFS of VTA was mediated by DA higher concentration. On the contrary, the microinjection of -opioid into the VTA inhibited dopaminergic neurons projecting to PFC (Margolis, 2006). We investigated changes in nociceptive neuronal activity after a microinjection of -opioid receptor agonist, U-50488, into the VTA.[Discussion] DA has been considered to regulate attention in PFC (Seamans, 2004). Basic DA concentration in PFC is tonically maintained and VTA lesion markedly decreased of DA (Devoto P, 2008). Dopamine level of brain is also suggested to modify pain sensation in Parkinson’s disease. In clinical reports of Parkinson’s disease, patients frequently complain pain before motor disorder (Chaudhuri, 2006). Neuropathic pain highly appears in Parkinson’s disease patients compared to normal adults (Defazio, 2008). DA system may be involved in processes of conscious pain. The mechanisms of pain symptoms in Parkinson’s disease may associate with disorders of the endogenous dopamine system as well as motor symptoms. doi:10.1016/j.neures.2010.07.2299
P1-j11 Nociceptive stimulation induces the c-fos-mRFP fusion gene expression in the spinal cord and the hypothalamus of a transgenic rat Toru Ishikura 1 , Hitoshi Suzuki 1,2 , Hiroaki Fujihara 1 , Hideo Ohnishi 2 , Yoichi Ueta 1 1 Department of Physiology, School of Medicine, University of Occupational and Environmental Health 2 Department of Orthopaedics, School of Medicine, University of Occupational and Environmental Health
The expression of the c-fos gene has been widely used as a marker of neuronal activity in the central nervous system (CNS). Recently, we generated transgenic rats expressing the c-fos and monomeric red fluorescent protein (mRFP) fusion gene in the CNS after adequate stimulation. In the present study, we observed the induction of the c-fos-mRFP fusion gene in the spinal cord and the hypothalamus after nociceptive stimulation in c-fosmRFP transgenic rats. Ninety minutes after a subcutaneous injection of saline or 5% formalin into the dorsal surface of hindpaw, animals were perfused transcardially by 4% paraformaldehyde solution after anesthesia. Abundant nuclear mRFP fluorescences were observed in the dorsal horn of the spinal cord and the several central regions including the hypothalamic paraventricular nucleus in formalin-injected rats but a few in the saline-injected rats under fluorescent microscopy. This c-fos-mRFP transgenic rat is a new useful animal to study the central response to nociceptive stimulation. doi:10.1016/j.neures.2010.07.2300
P1-j12 Pain modulation following air-puff startle response in the rat Junichiro Tamaki , Masayoshi Tsuruoka, Masako Maeda, Bunsho Hayashi, Tomio Inoue Department of Physiology, Showa University School of Dentistry An air puff elicits a startle response in mammals. Following the air–puff startle reaction, rats assume a defensive–like, immobile posture (DIP) of approximately 2–5 s in length. We previously reported that bilateral lesions of the nucleus coeruleus/subcoeruleus (LC/SC) reduce the DIP period. The LC/SC is one of the structures that play an important role in endogenous pain control. Our particular interest is whether the nociceptive inputs are inhibited by activation of the LC/SC during the DIP period. Rats were tested for behavioral nociception with heat stimuli. The heat source was attached to the tail; rats bit the heat source following heating. The bite latency (BL) was measured as an indicator of pain threshold. The BL and the DIP period were measured with a digital stop watch. For air–puff stimulation, compressed house air (14.4 psi in strength, 0.1 s in duration) was presented as a transient through a vinyl tube suspended 2.5 cm above the rat’s head. Two weeks before the experiment, the rats received bilateral injections of 6 g of the neurotoxin 6–hydroxydopamine to specifically lesion noradrenaline–containing neurons of the LC/SC. When heat stimuli and air–puff stimuli were simultaneously applied, BLs were significantly prolonged in comparison with those induced by only heat stimuli. In the LC/SC–lesioned rats (n = 10), air–puff–induced prolongation of BLs was not observed in post–lesions, whereas air–puff stimulation significantly prolonged BLs in pre–lesions. In contrast, in the sham-lesioned rats (n = 10), air–puff stimulation significantly prolonged BLs in both pre- and postlesions. When the percentage change of BLs between pre–and post–lesions was plotted against that of DIP periods between pre–and post–lesions, regression analysis showed a strong correlation (R2 = 0.67). These results suggest that (1) air–puff stimulation produces pain reduction, (2) the LC/SC is involved in pain reduction, and (3) pain reduction is related to the DIP period. doi:10.1016/j.neures.2010.07.2301
P1-j13 Macrophage inflammatory protein-1␣ mediates the development of neuropathic pain following peripheral nerve injury Norikazu Kiguchi , Takehiko Fukazawa, Shiroh Kishioka
Maeda, Yuka
Kobayashi, Yohji
Dept Pharmacol, Wakayama Med Univ, Wakayama Although accumulating evidence indicates that several inflammatory mediators derived from glial cells and immune cells participate in the pathogenesis of neuropathic pain, the roles of chemokines are poorly understood. In this study, we investigated the role of the macrophage inflammatory protein-1␣ (MIP-1␣; CCL3) in the development of neuropathic pain following partial