Suppressed injury-induced rise in spinal prostaglandin E2 production and reduced early thermal hyperalgesia in iNOS-deficient mice.

It is widely accepted that peripheral injury increases spinal inducible cyclooxygenase (COX-2) expression and prostaglandin E(2) (PGE(2)) formation as key mediators of nociceptive sensitization. Here, we used inducible nitric oxide synthase (iNOS) gene-deficient (iNOS-/-) mice to determine the contribution of iNOS-derived nitric oxide (NO) to this process. iNOS-/- mice exhibited reduced thermal hyperalgesia after zymosan injection. Spinal NO and PGE(2) formation both remained at baseline levels, in contrast to wild-type (wt) mice. In wt mice reduced hyperalgesia similar to that seen in iNOS-/- mice was induced by local spinal, but not by systemic treatment with the iNOS inhibitor l-NIL, suggesting that the reduced heat sensitization in iNOS-/- mice was attributable to the lack of spinal rather than peripheral iNOS. Two additional observations indicate that the antinociceptive effects of iNOS inhibition are dependent on a loss of stimulation of PG synthesis. First, intrathecal injection of the COX inhibitor indomethacin, which exerted pronounced antinociceptive effects in wt mice, was completely ineffective in iNOS-/- mice. Second, treatment with the NO donor RE-2047 not only completely restored spinal PG production and thermal sensitization in iNOS-/- mice but also its sensitivity to indomethacin. In both types of mice induction of thermal hyperalgesia was accompanied by similar increases in COX-1 and COX-2 mRNA expression. The stimulation of PG production by NO therefore involves an increase in enzymatic activity, rather than an alteration of COX gene expression. These results indicate that NO derived from spinal iNOS acts as a fast inductor of spinal thermal hyperalgesia.     

Intrathecal GM1 ganglioside and local nerve anesthesia reduce nociceptive behaviors in rats with experimental peripheral mononeuropathy.

Our previous experiments demonstrated that systemic treatment with GM1 ganglioside reduces nociceptive behaviors and spinal cord metabolic activity in a rat model of painful peripheral mononeuropathy produced by experimental sciatic nerve ligation (chronic constrictive injury, CCI). In the present study, we examined the effects of intrathecal (i.t.) GM1 treatment on thermal hyperalgesia and spontaneous pain behaviors resulting from nerve ligation in order to determine the locus of GM1 action. In addition, a local anesthetic agent, bupivacaine, given alone or combined with i.t. GM1, was applied to the injured sciatic nerve to determine if peripheral nerve anesthesia would influence post-injury nociceptive behaviors. Thermal hyperalgesia to radiant heat decreased in a dose-dependent manner when GM1 (10-80 nmol, i.t.) was administered once daily onto the lumbar segments of the spinal cord beginning 1 h after experimental nerve injury and continued for the first 9 days after nerve ligation. Moreover, this GM1 (80 nmol) treatment regimen reliably lowered spontaneous pain behavior rating scores in CCI rats suggesting the possible attenuation of spontaneous pain. The central site of i.t. GM1 action is located at the caudal (probably lumbar) spinal cord, since i.t. injection of 20 nmol GM1 onto the cervical spinal cord did not produce any protective effect. A single perinerve injection of a local anesthetic agent, bupivacaine (0.5%, 0.6 ml), on the 3rd day after nerve ligation reduced thermal hyperalgesia for at least 24 h following injection, a duration longer than that of the local anesthetic action of bupivacaine. Neither a single bupivacaine injection nor four daily i.t.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intrathecal MK-801 and local nerve anesthesia synergistically reduce nociceptive behaviors in rats with experimental peripheral mononeuropathy.

The hyperalgesia and spontaneous pain that occur following peripheral nerve injury may be related to abnormal peripheral input or altered central activity, or both. The present experiments investigated these possibilities by examining the effects of MK-801 (a non-competitive N-methyl-D-aspartate, NMDA, receptor antagonist) and bupivacaine (a local anesthetic agent) on thermal hyperalgesia and spontaneous nociceptive behaviors in rats with painful peripheral mononeuropathy. Peripheral mononeuropathy was produced by loosely ligating the rat's common sciatic nerve, a procedure which causes chronic constrictive injury (CCI) of the ligated nerve. The resulting hyperalgesia to radiant heat and spontaneous nociceptive behaviors was assessed by using a foot-withdrawal test and a spontaneous pain behavior rating method, respectively. CCI rats receiving 4 daily intraperitoneal (i.p.) MK-801 injections (0.03, 0.1, 0.3 mg/kg) beginning 15 min prior to nerve ligation exhibited less hyperalgesia (i.e., longer foot-withdrawal latencies) on days 3, 5, 7, 10, and 15 after nerve ligation as compared to those receiving saline injections. Thermal hyperalgesia also was reduced when a single MK-801 injection was given intrathecally (i.t.) onto the spinal cord lumbar segments on Day 3 after nerve ligation. This effect of postinjury MK-801 treatment was dose-dependent (2.5-20 nmol) and lasted for at least 48 h after injection. Moreover, i.t. injection of MK-801 (10 nmol) reliably lowered spontaneous pain behavior rating scores in CCI rats compared to those in the saline group. The spinal site of MK-801 action is situated within the caudal (probably lumbar) spinal cord, since i.t. injection of MK-801 (10 nmol) onto the spinal cord thoracic segments did not affect thermal hyperalgesia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Controlling pathological pain by adenovirally driven spinal production of the anti-inflammatory cytokine, interleukin-10

Gene therapy for the control of pain has, to date, targeted neurons. However, recent evidence supports that spinal cord glia are critical to the creation and maintenance of pain facilitation through the release of proinflammatory cytokines. Because of the ability of interleukin-10 (IL-10) to suppress proinflammatory cytokines, we tested whether an adenoviral vector encoding human IL-10 (AD-h-IL10) would block and reverse pain facilitation. Three pain models were examined, all of which are mediated by spinal pro-inflammatory cytokines. Acute intrathecal administration of rat IL-10 protein itself briefly reversed chronic constriction injury-induced mechanical allodynia and thermal hyperalgesia. The transient reversal caused by IL-10 protein paralleled the half-life of human IL-10 protein in the intrathecal space (t(1/2) approximately 2 h). IL-10 gene therapy both prevented and reversed thermal hyperalgesia and mechanical allodynia, without affecting basal responses to thermal or mechanical stimuli. Extra-territorial, as well as territorial, pain changes were reversed by this treatment. Intrathecal AD-h-IL10 injected over lumbosacral spinal cord led to elevated lumbosacral cerebrospinal fluid (CSF) levels of human IL-10, with far less human IL-10 observed in cervical CSF. In keeping with IL-10's known anti-inflammatory actions, AD-h-IL10 lowered CSF levels of IL-1, relative to control AD. These studies support that this gene therapy approach provides an alternative to neuronally focused drug and gene therapies for clinical pain control.     

Minocycline and fluorocitrate suppress spinal nociceptive signaling in intrathecal IL‐1β–induced thermal hyperalgesic rats

We previously demonstrated that intrathecal IL‐1β caused thermal hyperalgesia in rats. This study was conducted to examine the effects and cellular mechanisms of glial inhibitors on IL‐1β–induced nociception in rats. The effects of minocycline (20 μg), fluorocitrate (1 nmol), and SB203580 (5 μg) on IL‐1β (100 ng) treatment in rats were measured by nociceptive behaviors, western blotting of p38 mitogen‐activated protein kinase (MAPK) and inducible nitric oxide synthase (iNOS) expression, cerebrospinal fluid nitric oxide (NO) levels, and immunohistochemical analyses. The results demonstrated that intrathecal IL‐1β activated microglia and astrocytes, but not neurons, in the dorsal horn of the lumbar spinal cord, as evidenced by morphological changes and increased immunoreactivity, phosphorylated p38 (P‐p38) MAPK, and iNOS expression; the activation of microglia and astrocytes peaked at 30 min and lasted for 6 h. The immunoreactivities of microglia and astrocytes were significantly increased at 30 min (6.6‐ and 2.7‐fold, respectively) and 6 h (3.3‐ and 4.0‐fold, respectively) following IL‐1β injection, as compared with saline controls at 30 min (all P < 0.01). IL‐1β induced P‐p38 MAPK and iNOS expression predominantly in microglia and less in astrocytes. Minocycline, fluorocitrate, or SB203580 pretreatment suppressed this IL‐1β–upregulated P‐p38 MAPK mainly in microglia and iNOS mainly in astrocytes; minocycline exhibited the most potent effect. Minocycline and fluorocitrate pretreatment abrogated IL‐1β–induced NO release and thermal hyperalgesia in rats. In conclusion, minocycline, fluorocitrate, and SB203580 effectively suppressed the IL‐1β–induced central sensitization and hyperalgesia in rats. © 2012 Wiley Periodicals, Inc.     

A novel immune-to-CNS communication pathway: cells of the meninges surrounding the spinal cord CSF space produce proinflammatory cytokines in response to an inflammatory stimulus

Pain is enhanced in response to elevations of proinflammatory cytokines in spinal cerebrospinal fluid (CSF), following either intrathecal injection of these cytokines or intrathecal immune challenge with HIV-1 gp120 that induces cytokine release. Spinal cord glia have been assumed to be the source of endogenous proinflammatory cytokines that enhance pain. However, assuming that spinal cord glia are the sole source of CSF cytokines may be an underestimate, as the cellular composition of the meninges surrounding the spinal cord CSF space includes several cell types known to produce proinflammatory cytokines. The present experiments provide the first investigation of the immunocompetent nature of the spinal cord meninges. Here, we explore whether rat meninges are responsive to intrathecal gp120. These studies demonstrate that: (a) intrathecal gp120 upregulates meningeal gene expression of proinflammatory signals, including tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin 6 (IL-6), and inducible nitric oxide synthase (iNOS), and (b) intrathecal gp120 induces meningeal release of TNF-alpha, IL-1beta, and IL-6. In addition, stimulation of isolated meninges in vitro with gp120 induced the release of TNF-alpha and IL-1beta, indicating that the resident cells of the meninges are able to respond without immune cell recruitment. Taken together, these data document that the meninges are responsive to immunogenic stimuli in the CSF and that the meninges may be a source of immune products detected in CSF. The ability of the meninges to release to proinflammatory signals suggests a potential role in the modulation of pain.     

Neuronal nitric oxide synthase (nNOS) mRNA is down-regulated, and constitutive NOS enzymatic activity decreased, in thoracic dorsal root ganglia and spinal cord of the rat by a substance P N-terminal metabolite

Nitric oxide (NO) in the spinal cord plays a role in sensory and autonomic activity. Pain induced by acetic acid in the abdominal stretch (writhing) assay and hyperalgesia associated with chronic pain are highly sensitive to NO synthase (NOS) inhibitors. Because substance P (SP) is released and up-regulated in some models of chronic pain, we hypothesized that an accumulation of SP metabolites may influence NOS expression and activity. To test this hypothesis, we examined the effect of intrathecally (i.t.) injected substance P (1-7) [SP(1-7)], the major metabolite of SP in the rat, on neuronal NOS (nNOS) mRNA in the thoracic and lumbar spinal cord, dorsal root ganglia (DRG) and on the corresponding constitutive NOS (cNOS) enzyme activity. Detected using quantitative RT-PCR, nNOS mRNA content in the thoracic spinal cord was decreased 6 h after injection of 5 micromol of SP(1-7) and returned to control 2 days later. In thoracic DRG, nNOS mRNA was reduced 48 h after SP(1-7). The cNOS enzymatic activity in thoracic spinal tissue was gradually decreased to a minimum at 72 h. Down-regulation of NOS by SP(1-7) in the thoracic area appears to be highly associated with capsaicin-sensitive primary afferent neurons. No similar changes in either parameter were measured in the lumbar area after SP(1-7). These data suggest that N-terminal SP fragments, which are known to cause long-term antinociception in the writhing assay, may do so by their ability to down-regulate NO synthesis along nociceptive pathways.     

Simultaneous analysis of the time course for changes in core body temperature, activity, and nociception following systemic administration of interleukin-1beta in the rat

The aches and pains that accompany fever appear to be mediated, at least in part, by the peripheral release of cytokines such as interleukin-1beta (IL-1beta). The objective of this study was to determine, whether changes in nociceptive sensitivity produced by IL-1beta administration are temporally linked to changes in core body temperature. Experiment 1 examined nociceptive responsiveness for a period of 3 h following systemic administration of IL-1beta (1, 3, 10 and 20 microg/kg). The two highest doses of IL-1beta produced a drop in temperature beginning approximately 60 min after cytokine administration. This hypothermia lasted 90 min and was associated with hyperalgesia. Experiment 2 examined changes in temperature and nociception for 12 h following administration of IL-1beta (10 microg/kg). An early, short-lived hypothermia was followed by a significant hyperthermia from 3.25 to 6.5 h following IL-1beta administration. This late-occurring fever was accompanied by hyperalgesia. Both the hypo- and hyperthermia phases were associated with a reduction in locomotor activity. Given that repeated nociceptive testing may confound assessment of temperature and activity, Experiment 3 examined the effects of IL-1beta (10 microg/kg) administration on temperature and activity in rats that remained in their home cages. The biphasic change in temperature and the reduction in activity were nearly identical to that reported in Experiment 2, indicating that repeated nociceptive testing did not confound these data. The results of this study demonstrate that, two phases of hyperalgesia occur and coincide with the periods of altered thermoregulation produced by systemic administration of IL-1beta.     

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.     

Expression of spinal cord Fos protein in response to intrathecal adrenomedullin and CGRP in conscious rats

Adrenomedullin (AM) immunoreactivity and mRNA, in addition to a large number of specific AM-binding sites, exist in the rat spinal cord. However, no phenotype has been reported for AM in the spinal cord. Here, expression of c-fos in response to intrathecal (i.t.) administration of AM, proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP) was examined in the thoracic, lumbar and sacral regions of spinal cord in conscious rats. Two hours after i.t. administration of either CGRP (2.5 and 10 microg) or AM (10 microg), the number of c-Fos immunoreactive nuclei was increased in all the spinal regions examined in this study, with the highest increase observed in the superficial dorsal horn. Few cells with c-fos immunoreactivity were found in the spinal cord of rats 2 h after i.t. injection of either saline or PAMP. Effects of AM (10 microg) and CGRP (2.5 microg) on c-fos expression were blocked when rats were pretreated with 40 microg of intrathecal CGRP8-37 (CGRP1 receptor antagonist). Fos-like immunoreactivity induced by i.t. CGRP and/or AM were also significantly abolished by i.t. administration of the nitric oxide (NO) inhibitor, l-NAME, indicating that endogenous NO is a necessary intermediary in CGRP and AM induced c-fos expression in the rat spinal cord. In conclusion, AM induces c-fos expression in rat spinal cord when administered intrathecally, with the pattern being similar to those produced by i.t. CGRP. Effects of the two peptides are sensitive to CGRP8-37 and l-NAME.     

Is functional state of spinal microglia involved in the anti-allodynic and anti-hyperalgesic effects of electroacupuncture in rat model of monoarthritis?

Spinal microglia play a key role for creating exaggerated pain following tissues inflammation or injury. Electroacupuncture (EA) can effectively control the exaggerated pain both in humans with inflammatory disease and animals with experimental inflammatory pain. However, little is known about the relationship between spinal glial activation and EA analgesia. Using immunohistochemistry, RT-PCR analysis, and behavioral testing, the present study demonstrated that (1) Unilateral intra-articular injection of CFA produced a robust microglial activation and the up-regulation of the tumor necrosis factor (TNF)-alpha, interleukin (IL-1beta), and IL-6 mRNA levels in the spinal cord; (2) Repeated intrathecal (i.t.) injection of minocycline (100 microg), a microglial inhibitor, or EA stimulation of ipsilateral "Huantiao"(GB30) and "Yanglingquan" (GB34) acupoints significantly suppressed CFA-induced nociceptive behavioral hypersensitivity and spinal microglial activation; (3) Combination of EA with minocycline significantly enhanced the inhibitory effects of EA on allodynia and hyperalgesia. For the first time, these data provide direct evidence for the involvement of spinal microglial functional state in anti-nociception of EA. Thus, anti-neuroinflammatory effect of EA might be considered as one of the mechanisms of its anti-arthritic pain effects, and thereby a multidisciplinary integrated approach to treating symptoms related to arthritis might be raised.     

Role of Somatostatin and Somatostatin Receptor type 2 in Postincisional Nociception in Rats

Somatostatin (SST) and the somatostatin receptor type 2 (sstr2) are expressed in the superficial part (Laminae I–III) of the dorsal horn of the spinal cord. Since the neurons in these laminae also receive nociceptive sensation from the periphery, it was hypothesized that both SST and sstr2 could be involved in the modulation of nociceptive transmission. To the best of knowledge, there are no studies on the involvement of SST and sstr2 in hind paw incision model in rats, which mimics postoperative pain in humans. Sprague-Dawley rats were subjected to hind paw incision under isoflurane anaesthesia and the resulting mechanical allodynia and thermal hyperalgesia were evaluated for 5 days. In another set of animals, the spinal cord was isolated at specified time intervals after incision and examined for SST and sstr2 expression using immunohistochemistry and immunoblotting procedures. Finally, nociceptive parameters were again evaluated in incised rats, which had received SST (400 µg/kg i.p. three times per day). Blood glucose level and locomotor activity were determined after SST treatment. Both allodynia and hyperalgesia were highest immediately after incision. Spinal SST expression increased at 2 h. A further increase was noted on day 3. Expression of sstr2 increased initially but decreased at day 1. These changes could be due to exocytosis of SST and internalization of the ligand–receptor complex. SST injection significantly attenuated mechanical allodynia but not thermal hyperalgesia. Significant change in blood glucose level or locomotor activity was absent. SST appears to contribute to postincisional pain. This finding could be of clinical relevance.     

Priming enhances endotoxin-induced thermal hyperalgesia and mechanical allodynia in rats

Central inflammation is an integral component and contributor of the pathology of many debilitating diseases and has been shown to produce spontaneous pain and hyperalgesia. Recently, administration of lipopolysaccharide (LPS) into the lateral ventricle of rats was shown to elicit both thermal hyperalgesia and tactile allodynia [K. Walker, A. Dray, M. Perkins, Hyperalgesia in rats following intracerebroventricular administration of endotoxin: effect of bradykinin B₁ and B₂ receptor antagonist treatment, Pain 65 (1996) 211–219]. In this study, we have replicated the LPS model with some adaptations and correlated the nociceptive behaviors with an increased expression of activated macrophages in the central nervous system. We also examined the effects of priming on LPS-induced decreases in thermal nociceptive thresholds and mechanical response thresholds following either central or peripheral administration. Intracerebroventricular (i.c.v.) administration of LPS (0.2 μg/rat) did not alter either thermal (hot plate) or mechanical (von Frey filaments) thresholds compared to baseline values in the first few hours after injection. However, priming rats by pretreating with i.c.v. LPS (0.2 μg) 24 h prior to testing with i.c.v. LPS (0.2 μg) produced significant mechanical allodynia and thermal hyperalgesia. The mechanical allodynia had an onset of 80 min after injection and a duration of 5 h. A similar time course was observed for thermal hyperalgesia, although its expression was less pronounced. Immunohistochemical studies indicated an increased expression of activated macrophages in the brain parenchyma of primed rats but not in unprimed rats. Intraperitoneal (i.p., 2 mg/kg) administration of LPS had no significant effect on either thermal or mechanical thresholds in the first few hours after injection; however, priming rats via i.p. (0.2 mg/kg) or i.c.v. (0.2 μg) LPS produced a reduction in both thermal nociceptive thresholds and mechanical response thresholds in rats given a subsequent i.p. injection of LPS. This study demonstrates that priming is an effective protocol for the induction of central inflammation and increases the duration of these behaviors after i.c.v. administration.     

Involvement of spinal neurokinins, excitatory amino acids, proinflammatory cytokines, nitric oxide and prostanoids in pain facilitation induced by Phoneutria nigriventer spider venom

The major local symptom of Phoneutria nigriventer envenomation is an intense pain, which can be controlled by infiltration with local anesthetics or by systemic treatment with opioid analgesics. Previous work showed that intraplantar (i.pl) injection of Phoneutria nigriventer venom in rats induces hyperalgesia, mediated peripherally by tachykinin and glutamate receptors. The present study examined the spinal mechanisms involved in pain-enhancing effect of this venom. Intraplantar injection of venom into rat hind paw induced hyperalgesia. This phenomenon was inhibited by intrathecal (i.t.) injection of tachykinin NK1 (GR 82334) or NK2 (GR 94800) receptor antagonists, a calcitonin gene-related peptide (CGRP) receptor antagonist (CGRP8-37) and N-methyl-D-aspartate (NMDA; MK 801 and AP-5), non-NMDA ionotropic (CNQX), or metabotropic (AIDA and MPEP) glutamate receptor antagonists, suggesting the involvement of spinal neurokinins and excitatory amino acids. The role of proinflammatory cytokines, nitric oxide (NO), and prostanoids in spinally mediated pain facilitation was also investigated. Pharmacological blockade of tumour necrosis factor-alpha (TNFalpha) or interleukin-1beta (IL-1beta) reduced the hyperalgesic response to venom. Intrathecal injection of L-N6-(1-iminoethyl)lysine (L-NIL), but not of 7-nitroindazole (7-NI), inhibited hyperalgesia induced by the venom, indicating that NO, generated by the activity of the inducible form of nitric oxide synthase, also mediates this phenomenon. Furthermore, indomethacin, an inhibitor of cyclooxigenases (COX), or celecoxib, a selective inhibitor of COX-2, abolished venom-induced hyperalgesia, suggesting the involvement of spinal prostanoids in this effect. These data indicate that the spinal mechanisms of pain facilitation induced by Phoneutria nigriventer venom involves a plethora of mediators that may cooperate in the genesis of venom-induced central sensitization.     

Interleukin-6 mediates low-threshold mechanical allodynia induced by intrathecal HIV-1 envelope glycoprotein gp120

Spinal cord glia (microglia and astrocytes) contribute to enhanced pain states. One model that has been used to study this phenomenon is intrathecal (i.t.) administration of gp120, an envelope glycoprotein of HIV-1 known to activate spinal cord glia and thereby induce low-threshold mechanical allodynia, a pain symptom where normally innocuous (non-painful) stimuli are perceived as painful. Previous studies have shown that i.t. gp120-induced allodynia is mediated via the release of the glial pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF), and interleukin-1beta (IL-1). As we have recently reported that i.t. gp120 induces the release of interleukin-6 (IL-6), in addition to IL-1 and TNF, the present study tested whether this IL-6 release in spinal cord contributes to gp120-induced mechanical allodynia and/or to gp120-induced increases in TNF and IL-1. An i.t. anti-rat IL-6 neutralizing antibody was used to block IL-6 actions upon its release by i.t. gp120. This IL-6 blockade abolished gp120-induced mechanical allodynia. While the literature predominantly documents the cascade of pro-inflammatory cytokines as beginning with TNF, followed by the stimulation of IL-1, and finally TNF plus IL-1 stimulating the release of IL-6, the present findings indicate that a blockade of IL-6 inhibits the gp120-induced elevations of TNF, IL-1, and IL-6 mRNA in dorsal spinal cord, elevation of IL-1 protein in lumbar dorsal spinal cord, and TNF and IL-1 protein release into the surrounding lumbosacral cerebrospinal fluid. These results would suggest that IL-6 induces pain facilitation, and may do so in part by stimulating the production and release of other pro-inflammatory cytokines.     

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.     

Anti-hyperalgesic effects of intrathecally administered neuropeptide W-23, and neuropeptide B, in tests of inflammatory pain in rats

Neuropeptide W-23 (NPW23) is an endogenous ligand of both GPR7 and GPR8, and neuropeptide B (NPB) is an endogenous ligand of GPR7. GPR7 mRNA has been detected in regions of the cortex, the hippocampus, the hypothalamus, and the spinal cord in the rat, but GPR8 has not been found in rodents. GPR7 and GPR8 receptors have structural features in common with both opioid and somatostatin receptors. The effects of intrathecal (i.t.) application of NPW23 and NPB were tested in two inflammatory pain models (plantar injection of formalin or carrageenan) and two thermal nociceptive tests (52.5 degrees C and 50.5 degrees C hot plates) and one mechanical nociceptive test in the rat. I.t. injection of either NPW23 or NPB decreased the number of agitation behaviors induced by paw formalin injection and attenuated the level of mechanical allodynia, but not the level of thermal hyperalgesia, induced by paw carrageenan injection in a dose-dependent manner at a dose between 0.1 and 10 microg, significantly. The effects of either 10 microg of NPW23 or 10 microg of NPB were not antagonized by 10 microg of naloxone. I.t. injection of either NPW23 or NPB had no effect in both the 52.5 degrees C hot plate test or in the 50.5 degrees C hot plate tests at a dose between 1 and 100 microg. I.t. injection of either 10 microg of NPW23 or 10 microg of NPB had no effect in the mechanical nociceptive test. I.t. injection of either 10 microg of NPW23 or 10 microg of NPB significantly suppressed the expression of Fos-like immunoreactivity of the L4-5 spinal dorsal horn induced by paw formalin injection. These data suggest that both spinally-applied NPW23 and NPB suppressed the input of nociceptive information to the spinal dorsal horn, produced an analgesic effect in the formalin test, and attenuated the level of mechanical allodynia in the carrageenan test, and that either spinally applied NPW23 or spinally applied NPB had no effect in the physiological condition.     

Spinal somatostatin SSTR2A receptors are preferentially up-regulated and involved in thermonociception but not mechanonociception

The present study was undertaken to investigate the role of spinal somatostatin SSTR2A receptors in nociceptive processing. SSTR2A receptor-like immunoreactivity was found in a dense network in the spinal cord of normal rats. With Western blot analysis a major band of approximately 80-85 kDa was detected. Both immunohistochemistry and immunoblot analysis indicated a significant increase in SSTR2A receptor content in the spinal cord 6 h after noxious thermal stimulation that lasted for at least 24 h. However, there were no notable changes in SSTR2A receptor content 3, 6, 12, or 24 h after noxious mechanical stimulation. Effects of intrathecally administered polyclonal antiserum to SSTR2A receptor (anti-SSTR2A) on thermal and mechanical pain thresholds were determined with behavioral tests. In normal rats, pretreatment with anti-SSTR2A (1 microl, intrathecal) did not affect paw withdrawal latency or pinch threshold. Hindpaw inflammation induced by complete Freund's adjuvant led to thermal and mechanical hyperalgesia as reflected by a robust decrease in paw withdrawal latency and pinch threshold. Significant attenuation of the thermal hyperalgesia was observed 3, 5, 7, 9, and 24 h after pretreatment with anti-SSTR2A. This effect disappeared in another 24 h. In contrast, pretreatment with anti-SSTR2A failed to exert any notable effect on adjuvant-induced mechanical hyperalgesia. The present findings provide the first evidence that SSTR2A receptors are responsible for thermal, but not mechanical, nociceptive transmission in the spinal cord. The results also suggest that somatostatin has an excitatory role in spinal nociceptive processing and that there are differential receptor responses to different types of noxious stimuli.     

Role of prostaglandin receptor subtype EP1 in prostaglandin E2-induced nociceptive transmission in the rat spinal dorsal horn

It has been indicated that prostaglandin E2 (PGE2) and the receptor for PGE2 (EP receptor) are key factors contributing to the facilitated generation of nociception. This study was designed to investigate the roles of PGE2 and EP1 receptors in the spinal cord in the nociceptive transmission, using behavioral and intracellular calcium ion concentration ([Ca2+]i) assays and in situ hybridization. Experiments were conducted on Sprague-Dawley rats. In behavioral assays, withdrawal thresholds to mechanical stimuli were evaluated using von Frey filament. The effect of an intrathecally administered selective EP1 antagonist, 6-[(2S,3S)-3-(4-chloro-2-methylphenylsulfonylaminomethyl)-bicyclo[2.2.2]octan-2-yl]-5Z-hexenoic acid (ONO-8711), on the intrathecal PGE2-induced hyperalgesia was examined. In [Ca2+]i assays, we measured [Ca2+]i in the dorsal horn of spinal cord slices and examined the effects of PGE2 and ONO-8711 perfusion on the [Ca2+]i changes. In situ hybridization using EP1 digoxigenin probe was performed on the slice sections of the lumbar spinal cord and bilateral L4 and L5 dorsal root ganglions (DRGs). Mechanical hyperalgesia was observed after intrathecal PGE2 administration. Intrathecal administration of ONO-8711 attenuated the PGE2-induced mechanical hyperalgesia in a dose- and time-dependent manner. Perfusion of ONO-8711 markedly suppressed PGE2-induced [Ca2+]i increment in laminae II-VI in dorsal horn of the spinal cord slice. Moreover, in situ hybridization revealed EP1 hybridization signals in the DRG neurons, but not in the spinal cord. The results of this study suggested that spinal PGE2 activates the EP1 receptors existing on the central terminals of primary afferents, subsequently increasing in [Ca2+]i in the spinal dorsal horn, which are involved in the mechanisms of spinal PGE2-induced nociceptive transmission.