Differential roles of spinal neurokinin 1/2 receptors in development of persistent spontaneous nociception and hyperalgesia induced by subcutaneous bee venom injection in the conscious rat

To evaluate the roles of spinal neurokinin receptors in the development of persistent nociception and hyperalgesia to thermal and mechanical stimuli induced by subcutaneous (s.c.) bee venom injection, effects of intrathecal (i.t.) pre- or post-treatment with a non-selective antagonist of (NK1/2) receptors, [D-Arg1,D-Trp7,9,Leu11] substance P (spantide), and a selective NK3 receptor antagonist, (S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl)propyl)-4-phenylpiperidin-4-yl)-N-methyl acetamide (SR142801) were assessed in conscious rat. Injection of bee venom s.c. into the plantar surface of one hind paw resulted in a pathological pain phenomenon characterized by a 1-2 h single phase of persistent spontaneous nociceptive behaviors (continuously flinching the injected paw) and a 72-96 h profound primary thermal and mechanical hyperalgesia in the injection site and a secondary thermal hyperalgesia in the non-injected hindpaw. Pre-treatment with spantide i.t. at 0.05 microg, 0.5 microg and 5 microg produced a dose-related suppression of the bee venom-induced flinching reflex during the whole time course and the inhibitory rate was 24 +/- 12.60% (35.38 +/- 4.12 flinches/5 min, n=5), 48 +/- 6.75% (24.53 +/- 2.90 flinches/5 min, n=5) and 60 +/- 7.69% (18.88 +/- 3.58 flinches/5 min, n=5) respectively when compared with the saline control group (46.80 +/- 2.60 flinches/5 min, n=5). Post-treatment of spantide i.t. at the highest dose (5 microg) used in the present study 5 min after bee venom injection also produced a 49% suppression of the flinching reflex in the control group [post-spantide vs saline: 19.42 +/- 3.15 (n=5) vs 38.42 +/- 3.25 flinches/5 min (n=5)]. Moreover, i.t. pre-treatment with 5 microg spantide partially prevented the primary and secondary thermal hyperalgesia from occurring, while it did not show any influence on the development of primary mechanical hyperalgesia. Neither the established thermal nor mechanical hyperalgesia identified in the above sites was affected by i.t. post-treatment with the same dose of spantide 3 h after bee venom injection. Pre and post-treatment of SR142801 did not produce any significant effect on the bee venom-induced spontaneous pain and thermal and mechanical hyperalgesia. Our present result suggests that activation of spinal NK1/2 receptors is involved in both induction and maintenance of the persistent spontaneous nociception, while it is only involved in induction of the primary and secondary thermal, but not primary mechanical hyperalgesia induced by s.c. bee venom injection. The spinal NK3 receptor seems not likely to be involved in the bee venom-induced behavioral response characterized by spontaneous pain and thermal and mechanical hyperalgesia.     

Differential effect of peripheral glutamate (NMDA,non-NMDA) receptor antagonists on bee venom-induced spontaneous nociception and sensitization

This study aimed to investigate the role of peripheral N-methyl-d-aspartate (NMDA) and non-NMDA receptor on (1). spontaneous nociception and (2). on sensitization induced by subcutaneous (s.c.) injection of bee venom (0.2mg/50 micro l) in rats. Peripheral s.c. administration of the competitive NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (AP5), the non-competitive NMDA receptor channel blocker MK-801, and the competitive non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were performed before (pre-treatment) and after (post-treatment) bee venom-induced inflammation. Pre-treatment with AP5 (10mM, 50 micro l) and both pre-treatment and post-treatment with MK-801 (2mM, 50 micro l) into the same area of the bee venom injection site markedly reduced the bee venom-increased spontaneous responses of wide-dynamic range (WDR) neuron of the spinal cord. Post-treatment with the same dose of AP5 as well as pre-treatment and post-treatment with CNQX (5mM, 50 micro l) did not produce any inhibitory effects. Additionally, the role of peripheral NMDA and non-NMDA receptors on bee venom-induced mechanical allodynia and hyperalgesia were investigated and assessed by the paw withdrawal reflex to the innocuous and noxious mechanical stimulation. Peripheral administration of AP5, but not CNQX, reduced mechanical allodynia and hyperalgesia. The data suggest that the peripheral NMDA receptor, but not non-NMDA receptor, plays a pivotal role in the bee venom-induced persistent nociception and hyperexcitability.     

Recombinant peptide derived from the venom the Phoneutria nigriventer spider relieves nociception by nerve deafferentation

The avulsion of nerve roots of the brachial plexus that is commonly seen in motorcycle accidents is a type of neuropathy due to deafferentation. This type of pain is clinically challenging since therapeutical protocols fail or have severe side effects. Thus, it is proposed to evaluate the antinociceptive activity of the recombinant CTK 01512-2 peptide that is derived from the venom of the Phoneutria nigriventer spider, as a future new therapeutical option. The neuropathic pain was surgically induced by avulsion of the upper brachial plexus trunk in groups of male Wistar rats and after 17 days, they were treated intrathecally with morphine, ziconotide, and CTK 01512–2. Behavioral tests were performed to evaluate mechanical and thermal hyperalgesia, cold allodynia, the functional activity of the front paw, and exploratory locomotion after the treatments. The peripheral blood samples were collected 6 h after the treatments and a comet assay was performed. The spinal cord was removed for the lipoperoxidation dosing of the membranes. The cerebrospinal fluid was analyzed for the dosage of glutamate. The recombinant peptide showed an antinociceptive effect when compared to the other drugs, without affecting the locomotor activity of the animals. Mechanical and thermal hyperalgesia, as well as cold allodynia, were reduced in the first hours of treatment. The levels of glutamate and the damage by membrane lipoperoxidation were shown to be improved, and genotoxicity was not demonstrated. In a scenario of therapeutical failures in the treatment of this type of pain, CTK 01512–2 was shown as a new effective alternative protocol. However, further testing is required to determine pharmacokinetics.     

PhTx4, a new class of toxins from Phoneutria nigriventer spider venom, inhibits the glutamate uptake in rat brain synaptosomes.

We report the characterization of a new class of glutamate uptake inhibitors isolated from Phoneutria nigriventer venom. Glutamate transport activity was assayed in rat cerebrocortical synaptosomes by using [(3)H]-L-glutamate. PhTx4 inhibited glutamate uptake in a dose dependent manner. The IC(50) value obtained was 2.35+/-0.9 microg/ml which is in the observed range reported for glutamate uptake blockers. Tx4-7, one of PhTx4 toxins, showed the strongest inhibitory activity (50.3+/-0.69%, n=3).     

Involvement of tachykinin NK1 receptors in nociceptin-induced hyperalgesia in mice.

Intrathecal (i.t.) injection of nociceptin at small doses (3.0 and 30.0 fmol) produced a significant hyperalgesic response as assayed by the tail-flick test. This hyperalgesic effect peaked at 15 min following i.t. administration of nociceptin (3.0 fmol) and returned to control level within 30 min. Hyperalgesia elicited by nociceptin was inhibited dose-dependently by i.t. co-administration of tachykinin NK1 receptor antagonists, CP-99,994 and sendide. A significant antagonistic effect of [D-Phe7, D-His9] substance P (6-11), a selective antagonist for substance P, was observed against the nociceptin-induced hyperalgesia. Pretreatment with i.t. substance P antiserum and i.t. capsaicin resulted in a complete block of the reduced threshold produced by nociceptin. The NK2 receptor antagonist, MEN-10,376 and pretreatment with neurokinin A antiserum did not alter the behavioural effect of nociceptin. The N-methyl-D-aspartate (NMDA) receptor antagonists, dizocilpine (MK-801) and D(-)-2-amino-5-phosphonovaleric acid (D-APV), and L-NG-nitro arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, failed to inhibit nociceptin-induced hyperalgesia. The results obtained suggest that the hyperalgesic effect of nociceptin may be mediated through tachykinin NK1 receptors in the spinal cord.     

Intrathecally administered big dynorphin,a prodynorphin-derived peptide,produces nociceptive behavior through an N-methyl-D-aspartate receptor mechanism

Intrathecal (i.t.) administration of big dynorphin (1-10 fmol), a prodynorphin-derived peptide consisting of dynorphin A and dynorphin B, to mice produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank, which peaked at 5-15 min after an injection. Dynorphin A produced a similar response, though the doses required were higher (0.1-30 pmol) whereas dynorphin B was practically inactive even at 1000 pmol. The behavior induced by big dynorphin (3 fmol) was dose-dependently inhibited by intraperitoneal injection of morphine (0.125-2 mg/kg) and also dose-dependently, by i.t. co-administration of D(-)-2-amino-5-phosphonovaleric acid (D-APV) (1-4 nmol), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (0.25-4 nmol), an NMDA ion-channel blocker, and ifenprodil (2-8 pmol), an inhibitor of the NMDA receptor ion-channel complex interacting with the NR2B subunit and the polyamine recognition site. On the other hand, naloxone, an opioid receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA glutamate receptor antagonist, 7-chlorokynurenic acid, a competitive antagonist of the glycine recognition site on the NMDA receptor ion-channel complex, [D-Phe(7),D-His(9)]-substance P(6-11), a specific antagonist for substance P (NK1) receptors, and MEN-10376, a tachykinin NK2 receptor antagonist, had no effect. These results suggest that big dynorphin-induced nociceptive behavior is mediated through the activation of the NMDA receptor ion-channel complex by acting on the NR2B subunit and/or the polyamine recognition site but not on the glycine recognition site, and does not involve opioid, non-NMDA glutamate receptor mechanisms or tachykinin receptors in the mouse spinal cord.     

Contractile effect of tachykinins on Suncus murinus (house musk shrew) isolated ileum

Recent studies used Suncus murinus to investigate the anti-emetic potential of NK(1) tachykinin receptor antagonists. However, the pharmacology of tachykinin receptors in this species has not been fully characterized. In the present studies, therefore, we examined a range of tachykinin receptor agonists for a capacity to induce contractions of the isolated ileum. The tachykinin NK1 receptor preferring agonists substance P, septide and [Sar9Met(O2)11] substance P, and the tachykinin NK2 preferring agonists neurokinin A and GR 64349 (Lys-Asp-Ser-Phe-Val-Gly-R-gamma-lactam-Leu-Met-NH2) caused concentration dependent contractions with EC50 values in the nanomolar range. However, the tachykinin NK3 preferring agonists neurokinin B and senktide (1nM-1microM) induced only weak contractions. The action of senktide, but not [Sar9Met(O2)11] substance P, septide, or GR 64349, was antagonized significantly by atropine (P<0.05); tetrodotoxin and hexamethonium were inactive. The tachykinin NK1 receptor antagonist CP-99,994 ((+)-[(2S,3S)-3-(2-methoxy-benzyl-amino)-2-phenylpiperidine]) (10-100nM) inhibited substance P- and septide-induced contractions non-competitively. The pA2 value estimated for CP-99,994 against septide was 7.3+/-0.1. It also non-competitively antagonized the contractile responses induced by [Sar9Met(O2)11] substance P with a pA2 of 7.4+/-0.1. CP-99,994 also had a slight inhibitory action on neurokinin A-induced contractions, but did not modify the action of GR 64349. Conversely, the tachykinin NK2 receptor antagonist, saredutant, competitively antagonized GR 64349-induced contractions with a pA2 of 7.34+/-0.02. On the other hand, the presence of both CP-99,994 and saredutant competitively antagonized substance P-induced contraction. The present studies indicate that tachykininNK1 and NK2 receptors exist in the ileum of S. murinus and are involved in mediating contractions directly on smooth muscle, whereas tachykinin NK3 receptors may play a minor role involving a release of acetylcholine.     

Local and descending circuits regulate long-term potentiation and zif268 expression in spinal neurons

Long-term potentiation (LTP), a use dependent long-lasting modification of synaptic strength, was first discovered in the hippocampus and later shown to occur in sensory areas of the spinal cord. Here we demonstrate that spinal LTP requires the activation of a subset of superficial spinal dorsal horn neurons expressing the neurokinin-1 receptor (NK1-R) that have previously been shown to mediate certain forms of hyperalgesia. These neurons participate in local spinal sensory processing, but are also the origin of a spino-bulbo-spinal loop driving a 5-hydroxytryptamine 3 receptor (5HT3-R)- mediated descending facilitation of spinal pain processing. Using a saporin-substance P conjugate to produce site-specific neuronal ablation, we demonstrate that NK1-R expressing cells in the superficial dorsal horn are crucial for the generation of LTP-like changes in neuronal excitability in deep dorsal horn neurons and this is modulated by descending 5HT3-R-mediated facilitatory controls. Hippocampal LTP is associated with early expression of the immediate-early gene zif268 and knockout of the gene leads to deficits in long-term LTP and learning and memory. We found that spinal LTP is also correlated with increased neuronal expression of zif268 in the superficial dorsal horn and that zif268 antisense treatment resulted in deficits in the long-term maintenance of inflammatory hyperalgesia. Our results support the suggestion that the generation of LTP in dorsal horn neurons following peripheral injury may be one mechanism whereby acute pain can be transformed into a long-term pain state.     

The role of neuropeptides and capsaicin-sensitive fibres in glutamate-induced nociception and paw oedema in mice

This study sought to establish whether sensory neuropeptides and the capsaicin-sensitive fibres are involved in the nociception and oedema formation caused by intraplantar (i.pl.) injection of glutamate into the mouse paw. The i.pl. co-injection of the selective neurokinin (NK) NK(2) (SR 48968, 0.05-0.5 nmol/paw), and to a lesser extent the selective NK(1) (FK 888, 0.25-1.0 nmol/paw) receptor antagonists, resulted in a significant inhibition of glutamate-induced nociception. The percentages of inhibition were 82 and 37%, respectively. In contrast, the selective NK(3) receptor antagonist (SR 142801, 0.25-1.0 nmol/paw) failed to significantly affect glutamate-induced nociception. SR 48968, but not FK 888 or SR 142801, significantly inhibited (36%) glutamate-induced paw oedema formation. The i.pl. injection of kinin B(1) receptor antagonist des-Arg(9)-[Leu(8)]-BK (0.2-0.8 nmol/paw), but not the B(2) receptor antagonist HOE 140 (1.0-4.0 nmol/paw), together with glutamate, also inhibited glutamate-induced nociception (53%) in a graded manner, without affecting glutamate-induced paw oedema. The i.pl. co-injection of the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37) (1 nmol/paw) failed to significantly inhibit glutamate-induced nociception or oedema. Finally, neonatal-capsaicin (50 mg/kg, s.c.) treatment inhibited glutamate-induced nociception by 69% and to a lesser extent glutamate-mediated oedema formation (30%). Collectively, the current results indicate that the nociception caused by i.pl. injection of glutamate in mice is clearly mediated by capsaicin-sensitive fibres and by release of neurokinins from sensory neurones that activate NK(2) receptors and to a lesser extent NK(1) receptors. Furthermore, kinins acting at B(1) (but not at B(2)) receptors also largely account for glutamate-mediated nociceptive behaviour response. In contrast, glutamate-induced paw oedema seems to be primarily mediated via activation of NK(2) receptors and stimulation of capsaicin-sensitive C-fibres. CGRP receptors do not seem to be involved in either of the glutamate responses.     

NK(3) receptors in the feline nucleus tractus solitarius are not involved with the muscle pressor response

Isometric muscle contractions cause an increase in mean arterial pressure and heart rate. Previously, we showed that substance P (SP) is released from sites in the feline medial nucleus tractus solitarius (mNTS) in response to isometric muscle contractions, and that it most likely interacted with NK(1) tachykinin receptors at these sites. This study was undertaken to determine whether other tachykinin receptors in this area of the brainstem are involved with the muscle pressor response. Receptor autoradiography, using [(125)I]Bolton-Hunter SP and [(125)I] [MePhe(7)] neurokinin B to label NK(1) and NK(3) receptors, respectively, indicated that NK(3) tachykinin receptors are as abundant as NK(1) and NK(3) receptors, respectively, indicated that NK(3) tachykinin receptors are as abundant as NK(1) receptors in this region of the feline brainstem Injections of the specific NK(3) receptor antagonist, SR 142801 (0.1 to 10 microM) into the mNTS did not modify the pressor response or the heart rate response to isometric muscle contractions. Injection of SR142801 into the NTS prior to the injection of the NK(1) antagonist, GR82334 did not affect the action of GR82334 to attenuate the muscle pressor reflex. We conclude that NK(3) receptors in the NTS are not involved with the regulation of cardiovascular function during activation of the muscle pressor response.     

Spinal serotonergic receptors mediate facilitation of a nociceptive reflex by subcutaneous formalin injection into the hindpaw in rats

It is documented that spinal nociceptive transmission receives descending facilitatory and inhibitory modulation from supraspinal structures. The rostral ventral medulla (RVM), including the nucleus raphe magnus (NRM), nuclei reticularis gigantocellularis (NGC) and gigantocellularis pars alpha (NGCα), is the major bulbar relay of descending modulatory influences. Pharmacological studies show that facilitation of a spinal nociceptive tail-flick (TF) reflex induced by stimulation in the NGC and NGCα is mediated by spinal serotonergic receptors. The present series of experiments provide evidence that activation of spinal serotonergic systems are critical for both induction and maintenance of secondary hyperalgesia induced by subcutaneous injection of formalin into one hindpaw. Subcutaneous injection of formalin produced facilitation of tail withdrawal (mechanical) and the TF reflex (thermal). Facilitatory effects persisted for at least 30 min. Peripheral blockade of the activity by local injection of a hydrophilic lidocaine derivative (QX-314, 5%) into the injected hindpaw abolished both mechanical and thermal facilitation, indicating that peripheral input is important to maintain long-lasting facilitation. Intrathecal application of a serotonergic receptor antagonist methysergide at a dose (64 nmol) which completely blocked descending facilitation produced by electrical- or chemical-stimulation in the NGC and NGCα also significantly attenuated or completely abolished facilitation of tail withdrawal and the TF reflex induced by formalin. Methysergide was effective whether the injection was performed before or after the formalin injection. These results suggest that activation of descending facilitatory serotonergic influences by a prolonged noxious stimulation could contribute to secondary hyperalgesia observed at the tail.     

Intrathecal PKA-selective siRNA treatment blocks sustained morphine-mediated pain sensitization and antinociceptive tolerance in rats

Sustained morphine treatment has been shown to produce paradoxical pain sensitization (opioid-induced hyperalgesia) and also causes increase in spinal pain neurotransmitter, such as calcitonin gene related peptide (CGRP), concentration in experimental animals. Studies have also shown that cyclic adenosine-monophosphate (cAMP)-dependent protein kinase (PKA) plays a major role in the regulation of presynaptic neurotransmitter (such as CGRP and substance P) synthesis and release. We have previously shown that in cultured primary sensory dorsal root ganglion (DRG) neurons sustained in vitro opioid agonist treatment upregulates cAMP levels (adenylyl cyclase (AC) superactivation) and augments basal and capsaicin evoked CGRP release in a PKA dependent manner. In the present study, we investigated the in vivo role of PKA in sustained morphine-mediated pain sensitization. Our data indicate that selective knock-down of spinal PKA activity by intrathecal (i.th.) pretreatment of rats with a PKA-selective small interference RNA (siRNA) mixture significantly attenuates sustained morphine-mediated augmentation of spinal CGRP immunoreactivity, thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance. The present findings indicate that sustained morphine-mediated activation of spinal cAMP/PKA-dependent signaling may play an important role in opioid induced hyperalgesia.     

Nociceptive transmitter release in the dorsal spinal cord by capsaicin-sensitive fibers after noxious gastric stimulation

Little is known about transmitters that encode noxious gastric stimuli in the spinal cord. The release of glutamate, substance P, and CGRP from the spinal cord was therefore investigated in response to acid injury of the gastric mucosa. Dorsal halves of the caudal thoracic spinal cord (T7-T13) were removed 6 h after oral application of 0.5 M HCl or saline, transferred to a superfusion chamber, and the basal and capsaicin-stimulated (3.3 microM) transmitter release was determined. After acid injury, basal glutamate release increased 134% as compared to saline-treated animals. Capsaicin-stimulated release of CGRP and SP was 48% and 58% lower in acid- than in saline-treated animals, indicating that capsaicin-sensitive fibers in the dorsal spinal cord were already partially depleted by acid treatment. Capsaicin denervation reduced basal glutamate release by 33% after acid injury as compared to non-denervated acid-treated animals. Gastric origin and capsaicin sensitivity of glutamatergic, CGRP- and SP-containing primary afferents in thoracic dorsal root ganglia were then determined by retrograde tracing with True Blue and immunohistochemical labeling with the vanilloid receptor TRPV1. About 65% of True Blue-labeled cells were glutamatergic and more than 73% of this population expressed the TRPV1 receptor. Nearly all True Blue/CGRP (85%)- and True Blue/SP-positive cells (97%) coexpressed TRPV1. We conclude that noxious gastric stimulation with acid induces release of glutamate, SP, and CGRP from capsaicin-sensitive sensory afferents in the dorsal horn of the spinal cord where they may play an important role in gastric nociception and hyperalgesia.     

Effects of pre-emptively administered nociceptin on the development of thermal hyperalgesia induced by two models of experimental mononeuropathy in the rat

Pre-emptive analgesia is thought to be produced by the prevention of spinal facilitation evoked by nociceptive input to the spinal cord. Opioid receptor-like 1 (ORL1) receptor agonist has been reported to inhibit the development of spinal facilitation. We investigated the effect of nociceptin, an ORL1 receptor agonist, on the development of thermal hyperalgesia and the expression of Fos-like immunoreactivity (Fos-LI) in the spinal dorsal horn induced by two neuropathic pain models, the chronic constriction injury model and the partial sciatic nerve injury model. Chronic constriction injury is created by placing four loosely tied ligatures around the right sciatic nerve. Partial sciatic nerve injury was created by tight ligation of one third to one half of the right sciatic nerve. All drugs were injected intrathecally 10 min before the nerve injury. The anti-hyperalgesic effect of drugs was evaluated by the measurement of the paw withdrawal latency (PWL) against thermal nociceptive stimulation. The PWLs of the injured paws were measured 7, 14 and 21 days after the nerve injury. Expression of Fos-LI was examined 2 h after the nerve injury. Intrathecal injection of nociceptin significantly delayed the development of thermal hyperalgesia and decreased the expression of Fos-LI induced by chronic constriction injury, but not that induced by partial sciatic nerve injury. These data indicate that pre-emptive administration of nociceptin might be one strategy for the prevention of the development of neuropathic pain.     

An initial investigation of spinal mechanisms underlying pain enhancement induced by fractalkine, a neuronally released chemokine

Fractalkine is a chemokine that is tethered to the extracellular surface of neurons. Fractalkine can be released, forming a diffusible signal. Spinal fractalkine (CX3CL1) is expressed by sensory afferents and intrinsic neurons, whereas its receptor (CX3CR1) is predominantly expressed by microglia. Pain enhancement occurs in response both to intrathecally administered fractalkine and to spinal fractalkine endogenously released by peripheral neuropathy. The present experiments examine whether fractalkine-induced pain enhancement is altered by a microglial inhibitor (minocycline) and/or by antagonists/inhibitors of three putative glial products implicated in pain enhancement: interleukin-1 (IL1), interleukin-6 (IL6) and nitric oxide (NO). In addition, it extends a prior study that demonstrated that intrathecal fractalkine-induced mechanical allodynia is blocked by a neutralizing antibody to the rat fractalkine receptor, CX3CR1. Here, intrathecal anti-CX3CR1 also blocked fractalkine-induced thermal hyperalgesia. Furthermore, blockade of microglial activation with minocycline prevented both fractalkine-induced mechanical allodynia (von Frey test) and thermal hyperalgesia (Hargreaves test). Microglial activation appears to lead to the release of IL1, given that pretreatment with IL1 receptor antagonist blocked both fractalkine-induced mechanical allodynia and thermal hyperalgesia. IL1 is not the only proinflammatory cytokine implicated, as a neutralizing antibody to rat IL6 also blocked fractalkine-induced pain facilitation. Lastly, NO appears to be importantly involved, as l-NAME, a broad-spectrum NO synthase inhibitor, also blocked fractalkine-induced effects. Taken together, these data support that neuronally released fractalkine enhances pain via activation of spinal cord glia. Thus, fractalkine may be a neuron-to-glia signal triggering pain facilitation.     

Interactions of 5-HT2 receptor agonists with acetylcholine in spinal analgesic mechanisms in rats with neuropathic pain

Serotonin type 2 (5-HT(2)) receptors reportedly inhibit neuropathic pain in the spinal cord, but little is known about how spinal 5-HT(2) receptors might act against such abnormal sensitivity. We examined whether the cholinergic and tachykinin systems were involved in the antiallodynic effect of intrathecally administered 5-HT(2) receptor agonists in rats with nerve injury. Allodynia was produced by tight ligation of the left L5 and L6 spinal nerves, and determined by applying von Frey hairs to the left hindpaw. Effects of intrathecal pretreatment with 5-HT(2) receptor antagonists (ketanserin and RS-102221), muscarinic receptor antagonists (atropine and scopolamine), a choline uptake blocker (hemicholium-3), and an NK(1) receptor antagonist (L-706336) were assessed in rats subsequently given a 100- micro g intrathecal dose of a 5-HT(2) receptor agonist either alpha-methyl-5-HT or iododimethoxy aminopropane (DOI). Antiallodynic effects of 5-HT(2) receptor agonists were attenuated by the 5-HT(2A) receptor antagonist ketanserin (30 micro g), but not by the 5-HT(2C) receptor antagonist RS-102221 (40 micro g). Muscarinic receptor antagonists (30 micro g each), the choline uptake blocker (10 micro g), and the NK(1) receptor antagonist (30 micro g) also inhibited the antiallodynic effects of 5-HT(2) receptor agonists. Antiallodynic effects of intrathecally administered 5-HT(2) receptor agonists may be mediated by spinal release of acetylcholine induced via 5-HT(2A) and NK(1) receptors.     

Evidence for the interaction of glutamate and NK1 receptors in the periphery

It is known that Substance P (SP) enhances glutamate- and N-methyl- -aspartate (NMDA)-induced activity in spinal cord dorsal horn neurons and that this enhancement is important in the generation of wind-up and central sensitization. It is now known that SP and glutamate receptors are present on sensory axons in rat glabrous skin. This raises the issue as to whether SP and glutamate interact in the periphery. Using the tail skin in rats, the present study demonstrates 1) that unmyelinated axons at the dermal–epidermal junction immunostain for antibodies directed against NMDA, non-NMDA or SP (NK1) receptors; 2) that glutamate injected into the tail skin results in dose-dependent nociceptive behaviors interpreted as mechanical hyperalgesia, mechanical allodynia and thermal hyperalgesia, which are blocked following co-injection with glutamate antagonists; 3) that peripheral injection of SP potentiates glutamate-induced nociceptive behaviors in that the co-injection of SP+glutamate results in a significantly longer duration of behavioral responses compared to the responses seen following injection of either substance alone. These data provide support for the hypothesis that primary afferent neurons might well be subject to similar mechanisms that result in wind-up or central sensitization of spinal cord neurons.     

Altered Calcitonin Gene-related Peptide, Substance P and Enkephalin Immunoreactivities and Receptor Binding Sites in the Dorsal Spinal Cord of the Polyarthritic Rat

The dorsal horn of the spinal cord, which forms the locus of first synapses in pain pathways, is an important site of interaction between calcitonin gene-related peptide (CGRP), substance P and enkephalin—the neuropeptides considered to be especially involved in the regulation of pain perception. Since adjuvant-induced arthritic rats provide a suitable model for peripheral inflammation and hyperalgesia, the possible alterations of immunoreactive CGRP, substance P and enkephalin as well as the binding sites for [¹²⁵I]hCGRPα, [¹²⁵I]substance Plneurokinin-1, (NK1) and [¹²⁵I]FK-33-824/μ-opioid receptors were studied in the dorsal horn of the spinal cord receiving projections from the inflamed limbs. In arthritic rats compared to control animals, a bilateral increase in CGRP- and substance P-immunoreactive fibres and the presence of enkephalin-immunoreactive cell bodies were noted in the dorsal horn of the spinal cord. As for receptors, while a significant decrease in [¹²⁵I]hCGRPα and [¹²⁵I]substance P/NK1 binding sites was observed in selective layers, no measurable alteration in [¹²⁵I]FK-33-824/μ-opioid binding sites was noted in any regions of the arthritic rat dorsal horn compared to the unaffected control rats. Following unilateral section of the peripheral nerve prior to induction of arthritis, CGRP- and substance P-immunoreactive fibres were markedly depleted and no enkephalin-positive neurons were observed in the ipsilateral dorsal horn. Analysis of receptor binding sites in denervated arthritic rats, however, exhibited differential responses, i.e. a significant increase in [¹²⁵I]hCGRPα, a marked decrease in [¹²⁵I]FK-33-824/μ-opioid and apparently no alteration in [¹²⁵I]substance P/NK1 receptor binding sites were observed in the ipsilateral dorsal horn compared to the intact contralateral side. These results taken together provide anatomical evidence for a concerted role of these peptides in the regulation of adjuvant-induced hyperalgesia accompanying peripheral inflammation.     

The p75 receptor is associated with inflammatory thermal hypersensitivity

Inflammatory pain, characterized by a decrease in the nociceptive threshold, arises through the actions of inflammatory mediators, and one of the key molecules is nerve growth factor (NGF). Here we report that the administration of neutralizing antibody to the neurotrophin receptor p75 (p75(NTR)) blocks hyperalgesia, which develops with complete Freund's adjuvant (CFA)-induced inflammation or with an intraplantar injection of NGF. Although CFA injection results in the up-regulation of calcitonin gene-related peptide (CGRP) levels in the primary sensory neurons, blocking p75(NTR) abolishes this effect. We further demonstrate that pro-NGF is the predominant ligand of p75(NTR) in vivo. Plasmin treatment, which is intended to decompose pro-NGF, ameliorates CFA-induced hyperalgesia. In addition, an intraplantar injection of pro-NGF induces hyperalgesia. These data together suggest that pro-NGF, as well as mature NGF, binding to p75(NTR) plays an important role in inflammation-induced hyperalgesia. Interference in the binding may provide a therapeutic approach for the treatment of inflammatory pain.     

Descending control of persistent pain: inhibitory or facilitatory?

The periaqueductal gray matter (PAG) and the nucleus raphe magnus and adjacent structures of the rostral ventromedial medulla (RVM), with their projections to the spinal dorsal horn, constitute the “efferent channel” of a pain-control system that “descends” from the brain onto the spinal cord. Considerable evidence has recently emerged regarding participation of this system in persistent pain conditions such as inflammation and neuropathy. Herein, this evidence is reviewed and organized to support the idea that persistent nociception simultaneously triggers descending facilitation and inhibition. In models of inflammation, descending inhibition predominates over facilitation in pain circuits with input from the inflamed tissue, and thus attenuates primary hyperalgesia, while descending facilitation predominates over inhibition in pain circuits with input from neighboring tissues, and thus facilitates secondary hyperalgesia. Both descending facilitation and inhibition mainly stem from RVM. The formalin-induced primary hyperalgesia, although considered a model for inflammation, is mainly facilitated from RVM. Also, formalin-induced secondary hyperalgesia is facilitated by RVM. Again, formalin triggers a concomitant but concealed descending inhibition. The (primary) hyperalgesia and allodynia of the neuropathic syndrome are also facilitated from RVM. Simultaneously, there is an inhibition of secondary neuronal pools that is partly supported from the PAG. Because in all these models of peripheral damage descending facilitation and inhibition are triggered simultaneously, it will be important to elucidate why inhibition predominates in some neuronal pools and facilitation in others. Therapies that enhance descending inhibition and/or attenuate descending facilitation are furthermore an important target for research in the future.