mRNA In Dorsal Root Ganglia Research Articles

5-HT2A receptor subtype in the peripheral branch of sensory fibers is involved in the potentiation of inflammatory pain in rats

One of the major serotonin (5-HT) receptor subtypes expressed in the rat dorsal root ganglion (DRG) neurons is the 5-HT2A receptor. We have previously shown that 5-HT2A receptors in the peripheral sensory terminals are responsible for 5-HT-induced pain and hyperalgesia. In the present study, we characterized neurons expressing 5-HT2A receptors in the rat DRG neurons by means of in situ hybridization, immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and behavioral tests. In situ hybridization on consecutive sections revealed that 5-HT2A receptor mRNA is colocalized with calcitonin-gene related peptide (CGRP) mRNA (100/104; 96.2%) but not with c-Ret mRNA (1/115; 0.9%). Signals for 5-HT2A receptor mRNA were found in 9.4±2.2% of normal DRG (L5) neurons, most of which were small to medium in size. Four days of complete Freund's adjuvant-induced inflammation of the hindpaw doubled the incidence of 5-HT2A receptor mRNA-expressing neurons to 19.3±2.8%. The level of 5-HT2A receptor mRNA in DRGs of normal and various pathological conditions was then determined by RT-PCR. The level was up-regulated by peripheral inflammation, but not by axotomy or chronic constriction of the peripheral nerve. Systemic administration of 5-HT2A receptor antagonist (Sarpogrelate HCI) produced analgesic effects on thermal hyperalgesia caused by peripheral inflammation, but failed to attenuate thermal hyperalgesia in chronic constriction injury model. These findings suggest that 5-HT2A receptors are mainly expressed in CGRP-synthesizing small DRG neurons and may be involved in the potentiation of inflammatory pain in the periphery.

Lack of Effects of Transforming Growth Factor-α Gene Knockout on Peripheral Nerve Regeneration May Result from Compensatory Mechanisms

Transforming growth factor-α (TGF-α), previously identified as a major member of the epidermal growth factor (EGF) family of growth factors, plays a role in proliferation, differentiation, and survival of neuronal and glial precursors and is implicated in development of the nervous system. However, its roles in nerve injury-induced responses remain obscure. The current study examined roles of endogenous TGF-α in peripheral nerve regeneration using sciatic nerve injury models with TGF-α knockout mice. Three weeks after a sciatic nerve crush, no significant differences were found between TGF-α wild-type and mutant mice in the number of retrogradely labeled L5 dorsal root ganglion (DRG) sensory neurons and L5 spinal cord motor neurons and in the morphology of myelinated regenerating nerve fibers, indicating that TGF-α is not essential for sensory and motor nerve regeneration. To assess a possible functional redundancy among TGF-α-related ligands in response to a nerve injury, mRNA expression of the EGF family was analyzed by RT-PCR in L4/L5 DRG pools and distal degenerating sciatic nerve segments after sciatic nerve ligation. Prior to and 1 day after ligation, there was a higher level of EGF-R mRNA in DRGs and in nerve in TGF-α null mice compared to wild types, and there was an induction of ligand amphiregulin mRNA in DRGs in mutant mice in place of the TGF-α upregulation present in wild types. These results indicate that TGF-α gene knockout does not affect peripheral nerve regeneration, probably due to a functional redundancy within the EGF family through a compensatory expression mechanism at both the receptor and ligand levels in TGF-α knockout mice.

Induction of alpha-calcitonin gene-related peptide mRNA expression in rat dorsal root ganglia by heat stress involves the heme oxygenase-1/carbon monoxide pathway

SD rats were pretreated with whole body hyperthermia (rectal 42°C) for 15min. The level of calcitonin gene-related peptide (CGRP) in plasma, and α- and β-CGRP mRNA as well as heme oxygenease-1 and heme oxygenase-2 mRNA in dorsal root ganglia were determined by radioimmunoassay and semi-quantitative reverse-transcription polymerase chain reaction, respectively. Heat stress induced only the expression of α-CGRP or heme oxygenease-1 but not β-CGRP or heme oxygenase-2 mRNA, and the release of CGRP and induction of α-CGRP mRNA expression were abolished by pretreatment with Zinc protoporphyrin IX, the heme oxygenase inhibitor, or methylene blue, the inhibitor of soluble guanylate cyclase. These results indicate that induction of α-CGRP mRNA expression in rat DRG by heat stress involves the heme oxygenase-1/carbon monoxide pathway.

Vanilloid (Capsaicin) Receptors Influence Inflammatory Sensitivity in Response to Particulate Matter

The signs of airway inflammation and hyperresponsiveness that occur in animals exposed to air pollutants are often strain- and species-specific. To investigate the underlying causes of this phenomenon, BALB/c and C57bl/6 mice were exposed intratracheally to residual oil fly ash (ROFA, 3 mg/kg) and examined after 24 h for signs of airway inflammation. BALB/c showed significantly higher numbers of neutrophils and increased airway hyperresponsiveness in response to methacholine challenge, whereas B6 mice showed no significant change in either inflammatory endpoint. To determine the underlying cause of this strain specificity, cultures of dorsal root ganglion (DRG) sensory neurons, which innervate the upper airways in situ, were explanted from both BALB/c and B6 fetal mice. After 5–7 days in culture, they were exposed to ROFA, other urban and industrial particulate matter (PM; e.g., oil fly ash, woodstove, Mt. St. Helen, St. Louis, Ottawa, coal fly ash) or to prototype irritants (e.g., capsaicin 3–10 μM, pH 5.0 and 6.5). In all instances (except for woodstove), DRG neurons from BALB/c mice released significantly higher levels of the pro-inflammatory cytokine IL-6 into their nutrient media relative to neurons from B6 mice. This cytokine release could be significantly reduced for all PM treated cultures (except woodstove) by pretreatment of cultures with capsazepine (CPZ), a competitive antagonist of vanilloid receptors.DRG neurons, cultured from BALB/c and B6 neonates, were loaded with Fluo-3 AM and exposed to the prototype irritants, acid pH (5.0, 6.5), or capsaicin (3, 10 μM). Analysis of their increases in intracellular calcium showed that significantly higher numbers of BALB/c neurons responded to these prototype irritants, relative to B6 neurons. Morphometric analysis of BALB/c neurons, histochemically stained with cobalt to label neurons bearing capsaicin-sensitive receptors, showed a significantly higher level of stained neurons relative to B6 neurons. Finally, semiquantitative RT-PCR showed a higher expression of VR1 receptor mRNA in DRG and spinal cord taken from neonatal BALB/c mice relative to B6 mice. Taken together, these data suggest that capsaicin and acid-sensitive irritant receptors, located on somatosensory cell bodies and their nerve fiber terminals, subserve PM-induced airway inflammation and are quantitatively different in responsive and nonresponsive mouse strains.

α2-Adrenergic Receptors in Human Dorsal Root Ganglia

Nonselective alpha2-adrenergic receptor (alpha2AR) agonists (e.g., clonidine) mediate antinociception in part through alpha2ARs in spinal cord dorsal horn; however, use of these agents for analgesia in humans is limited by unwanted sedation and hypotension. The authors previously demonstrated alpha2a approximately alpha2b > > > alpha2c mRNA in human spinal cord dorsal horn cell bodies. However, because 20% of dorsal horn alpha2ARs derive from cell bodies that reside in the associated dorsal root ganglion (DRG), it is important to evaluate alpha2AR expression in this tissue as well. Therefore, the authors evaluated the hypothesis that alpha2b mRNA, alpha2c mRNA, or both are present in human DRG. Molecular approaches were used to determine alpha2AR expression in 28 human DRGs because of low overall receptor mRNA expression and small sample size. After creation of synthetic competitor cDNA and establishment of amplification conditions with parallel efficiencies, competitive reverse transcription polymerase chain reaction was performed using RNA isolated from human DRG. Overall expression of alpha2AR mRNA in DRG is low but reproducible at all spinal levels. alpha2b and alpha2cAR subtype mRNAs predominate (alpha2b approximately alpha2c), accounting for more than 95% of the total alpha2AR mRNA in DRG at all human spinal nerve root levels. Predominance of alpha2b and alpha2cAR mRNA in human DRG is distinct from alpha2AR mRNA expression in cell bodies originating in human spinal cord dorsal horn, where alpha2a and alpha2b predominate with little or absent alpha2c expression. These findings also highlight species heterogeneity in alpha2AR expression in DRG. If confirmed at a protein level, these findings provide an additional step in unraveling mechanisms involved in complex neural pathways such as those for pain.

Decreased Depressor Response Mediated by Calcitonin Gene-Related Peptide(CGRP)-Containing Vasodilator Nerves to Spinal Cord Stimulation and Levels of CGRP mRNA of the Dorsal Root Ganglia in Spontaneously Hypertensive Rats.

The depressor response to electrical stimulation of the spinal cord and the level of calcitonin gene-related peptide (CGRP) mRNA in the dorsal root ganglion (DRG) in the spontaneously hypertensive rat (SHR) was compared with the normotensive Wistar Kyoto rat (WKY) and Wistar rat (WR). The animals were pithed by inserting a stainless-steel rod into the spinal cord. Pithed rats were treated with hexamethonium (2 mg/kg/min i.v.) to block autonomic outflow, and mean arterial blood pressure (MBP) was maintained at approximately 100 mmHg with continuous infusion of methoxamine (10 to 15 microg/kg/min i.v.). Electrical stimulation (2 and 4 Hz for 30 s) of the lower thoracic spinal cord (T9-12) via the pithing rod caused a frequency-dependent depressor response without a change in heart rate. The depressor response to spinal cord stimulation was significantly smaller in SHR than in WKY and WR. Long-term treatment of 8 week-old SHR with captopril (0.1% in drinking water) for 7 weeks restored the reduced depressor response to spinal cord stimulation. The level of CGRP mRNA in DRG of SHR was significantly lower than that in WKY. These results suggest that the function of CGRP-containing nerves from the spinal cord decreases in SHR and captopril treatment prevents its reduction.

Increased spinal cholecystokinin activity after systemic resiniferatoxin: electrophysiological and in situ hybridization studies

The present study assessed the effect of a single subcutaneous injection of resiniferatoxin (RTX), an ultrapotent capsaicin analogue, on the activity of spinal cholecystokinin (CCK) systems, by using electrophysiological and in situ hybridization techniques. Subcutaneous RTX at 0.3 mg/kg, but not vehicle, produced marked thermal hypoalgesia in rats on the hot plate and tail flick tests. Partial recovery from hypoalgesia occurred in some (<50%), but not all, RTX-treated rats after 2 weeks. The flexor reflex in response to activation of high threshold afferents was recorded 15–35 days after RTX- or vehicle-treatment. There was no obvious difference between RTX- and vehicle-treated rats in the baseline flexor reflex. Intravenous morphine at 1 mg/kg caused a depression of the flexor reflex in vehicle- and in RTX-treated rats. The reflex depressive effect of morphine was significantly briefer in RTX-treated, non-recovered rats than vehicle-treated rats. Furthermore, CI-988, a high affinity antagonist of CCKB receptors, caused a minor depression of the reflex in vehicle- and RTX-treated rats that had partially recovered, whereas the reflex depressive effect of CI-988 was significantly enhanced in RTX-treated, non-recovered rats. In situ hybridization showed that RTX treatment caused a marked and significant increase in the number of dorsal root ganglion (DRG) neurone profiles expressing CCKB receptor mRNA, whereas only a small increase was observed for CCKA receptor mRNA expressing neurone profiles. Significantly more DRG neurone profiles expressed CCKB receptor mRNA in RTX-treated, non-recovered rats compared to partially recovered rats. RTX-treatment did not influence the expression of CCK mRNA in DRGs. Since CCK functions as a physiological antagonist of morphine, it is suggested that RTX treatment enhances the activity of spinal CCK systems, leading to the reduced effect of morphine and increased effect of the CCKB receptor antagonist CI-988. This may mainly be due to upregulation of CCKB receptors in DRG neurones.

Expression of muscarinic m2 receptor mRNA in dorsal root ganglia of neonatal rat

The expression of mRNA coding for m2 subtype of muscarinic cholinergic receptors was assessed in dorsal root ganglia (DRG) of 15-day post-natal rats. Northern blot analysis on total RNA using a mixture of two different oligonucleotide probes, indicated the presence of a single prominent band of approximately 6.5 kb in rat DRG; a band of the same size was observed both in brainstem and cortex taken as positive controls. Analysis by RT-PCR of the mRNA coding for a region of the third cytoplasmic loop of m2 receptor showed a single signal both in rat DRG and hippocampus. In situ hybridization was then used to identify the neuronal subpopulations expressing the mRNA for M 2. The transcripts were preferentially localized in medium–small neurons of the ganglion as well as in satellite cells surrounding the neuron cell body. Large neurons were usually negative. Finally, competition binding experiments, performed in the presence of [ 3 H ]-quinuclidinyl benzilate (QNB) and methoctramine (a selective competitor for M 2 receptors), demonstrated the presence of M 2 receptor protein ( K i=100 nM), as previously observed in chick DRG. The preferential localization of M 2 in medium–small neurons of the ganglion suggests the involvement of this receptor subtype in the transduction of nociceptive stimuli.

Differential Expression of the mRNA for the Vanilloid Receptor Subtype 1 in Cells of the Adult Rat Dorsal Root and Nodose Ganglia and Its Downregulation by Axotomy

Sensitivity to the pungent vanilloid, capsaicin, defines a subpopulation of primary sensory neurons that are mainly polymodal nociceptors. The recently cloned vanilloid receptor subtype 1 (VR1) is activated by capsaicin and noxious heat. Using combined in situ hybridization and histochemical methods, we have characterized in sensory ganglia the expression of VR1 mRNA. We show that this receptor is almost exclusively expressed by neurofilament-negative small- and medium-sized dorsal root ganglion cells. Within this population, VR1 mRNA is detected at widely varying levels in both the NGF receptor (trkA)-positive, peptide-producing cells that elicit neurogenic inflammation and the functionally less characterized glial cell line-derived neurotrophic factor-responsive cells that bind lectin Griffonia simplicifolia isolectin B4 (IB4). Cells without detectable levels of VR1 mRNA are found in both classes. A subpopulation of the IB4-binding cells that produce somatostatin has relatively low levels of VR1 mRNA. A previously uncharacterized population of very small cells that express the receptor tyrosine kinase (RET) and that do not label for trkA or IB4-binding has the highest relative levels of VR1 mRNA. The majority of small visceral sensory neurons of the nodose ganglion also express VR1 mRNA, in conjunction with the BDNF receptor trkB but not trkA. Axotomy results in the downregulation of VR1 mRNA in dorsal root ganglion cells. Our data emphasize the heterogeneity of VR1 mRNA expression by subclasses of small sensory neurons, and this may result in their differential sensitivity to chemical and noxious heat stimuli. Our results also indicate that peripherally derived trophic factors may regulate levels of VR1 mRNA.

Sciatic nerve transection evokes lasting up-regulation of angiotensin AT 2 and AT 1 receptor mRNA in adult rat dorsal root ganglia and sciatic nerves

The angiotensin AT 2 receptor is involved in tissue repair and cellular stress responses in non-neuronal cells. We have previously observed that the AT 2 receptor-induced neurite formation in PC12W cells is paralleled by a reduced neurofilament M expression as it occurs in nerve fiber regeneration. Here we show that transection and crush of sciatic nerve fibers of adult rats results in dramatic changes of AT 2, AT 1a and AT 1b receptor mRNA in dorsal root ganglion neurons (DRGs) and in sciatic nerves 3, 14 and 28 days after axotomy and crush. The expression patterns were determined by reverse transcription polymerase chain reaction (RT-PCR) assay, and the specificity of amplification products was verified by Southern blot hybridization. Whereas axotomy evoked a transient increase of AT 2 receptor mRNA by more than 1000% after 3 days in proximal and after 14 days in distal sciatic nerve stumps (510%), the maximum expression in DRGs was observed after 14 days (1100%). Sciatic nerve crush resulted in a time-dependent up-regulation of AT 2 receptor mRNA in sciatic nerve segments coinciding with the successful regeneration of nerve fibers. In sciatic nerves, AT 1a and AT 1b receptor mRNA levels were increased within different time-courses and to different extents with a maximum expression of 570%. In contrast to AT 1a receptor mRNAs, AT 1b receptor mRNA levels were increased in DRGs by maximally 800%. These results suggest that AT 2 and AT 1 receptor-mediated pathways are involved in Schwann cell-mediated myelination and in neuroregenerative responses of DRGs.

Peripheral morphine analgesia in dental surgery

The recent identification of opioid receptors on peripheral nerve endings of primary afferent neurons and the expression of their mRNA in dorsal root ganglia support earlier experimental data about peripheral analgesic effects of locally applied opioids. These effects are most prominent under localized inflammatory conditions. The clinical use of such peripheral analgesic effects of opioids was soon investigated in numerous controlled clinical trials. The majority of these have tested the local, intraarticular administration of morphine in knee surgery and have demonstrated potent and long-lasting postoperative analgesia. As the direct application of morphine into the pain-generating site of injury and inflammation appears most promising, we examined direct morphine infiltration of the surgical site in a unique clinical model of inflammatory tooth pain. Forty-four patients undergoing dental surgery entered into this prospective, randomized, double-blind study. Before surgery they received, together with a standard local anesthetic solution (articaine plus epinephrine) a submucous injection of either 1 mg of morphine (group A) or saline (group B). Postoperative pain intensity was assessed using the visual analog scale (VAS) and numeric rating scale (NRS) at 2, 4, 6, 8, 10, 12, 16, 20 and 24 h after surgery. In addition, patients recorded the occurrence of side effects and the supplemental consumption of diclofenac tablets. Results of 27 patients were analyzed (group A: n=14, group B: n=13). Pain scores which were moderate to severe preoperatively were reduced to a similar extent in both groups up to 8 h postoperatively. Thereafter, pain scores in group A were significantly lower than those in group B for up to 24 h, demonstrating the analgesic efficacy of additional morphine. The time to first analgesic intake and the total amount of supplemental diclofenac were less in group A than in group B. No serious side effects were reported. Our results show that 1 mg of morphine added to a local anesthetic for dental surgery results in significant improvement of postoperative analgesia. Since the majority of dental surgeries is accompanied with an inflammatory reaction, supplemental morphine may be of benefit for the relief of postoperative dental pain.

Peripheral axotomy influences the in vivo release of cholecystokinin in the spinal cord dorsal horn—possible involvement of cholecystokinin-B receptors

An increased expression of cholecystokinin (CCK) messenger RNA (mRNA) as well as CCK-B receptor mRNA in dorsal root ganglion (DRG) cells following peripheral axotomy has previously been demonstrated. In the present in vivo microdialysis study, the effect of unilateral sciatic nerve section on basal and potassium-induced release of CCK-like (CCK-LI) immunoreactivity in the rat dorsal horn was investigated. We also compared the effects of the CCK-B receptor antagonist CI988 on basal and potassium-stimulated CCK-LI release in intact animals and in chronically axotomized rats. Perfusion of the microdialysis probe with KCl (100 mM) induced a more than 6-fold increase of the extracellular level of CCK-LI in control animals. In contrast, following unilateral sciatic nerve section the same KCl stimulation failed to evoke a release of CCK-LI ipsilaterally. However, after systemic administration of CI988 (1 mg kg −1, i.v.), 100 mM KCl induced a significant increase of the extracellular CCK-LI level in axotomized rats, similar to that observed in control animals. In control animals no effect of CI988 on KCl-stimulated CCK-LI release could be detected. CI988 by itself had no influence on the extracellular CCK-LI level in either nerve injured or control animals. The present data suggest that axotomy reduces the release of CCK-like immunoreactivity in the spinal cord by a mechanism involving the CCK-B receptor binding site.

The immunosuppressant FK506 increases GAP-43 mRNA levels in axotomized sensory neurons

FK506, an immunosuppressant drug used to prevent allograft rejection in organ transplantations, accelerates functional recovery and nerve regeneration in the rat sciatic nerve crush model. While the mechanism by which FK506 increases regeneration is unknown, in contrast to immunosuppression, it does not involve calcineurin inhibition. Using the reverse-transcriptase polymerase chain reaction (RT-PCR) technique and a digoxigenin-labeled probe, we show that subcutaneous injections of FK506 (10 mg/kg/day) markedly increases the level of axotomy-induced growth-associated protein (GAP-43) mRNA in dorsal root ganglion (DRG) neurons. Quantitation of DRG neurons revealed that FK506 produced a 33% increase in the numbers of neurons exhibiting intense staining. Increased synthesis of GAP-43 may play a role in FK506's ability to speed nerve regeneration.

Multiple isoforms of neuregulin are expressed in developing rat dorsal root ganglia.

Accumulating evidence suggests that neuregulin (NRG) plays special roles in the development of the mammalian nervous system. We have already identified NRG as a survival factor for Schwann cells during development. In this report, we have studied all possible NRG isoforms and expression of NRG in the developing rat dorsal root ganglia (DRG) and compared them with those of brain and spinal cord. Neural NRG isoforms comprise common immunoglobulin and epidermal growth factor domains. Various different transcripts were characterized, which arose by alternative splicing in several regions: N-terminal (exon 1 or 2), spacer (exon 5), juxtamembrane (exon 9 or 10), and cytoplasmic (exon 12, 13, or 14) domains. At least 13 novel isoforms among 16 splice variants were identified. The transmembrane isoforms of NRG are dominant forms in developing rat DRG. The mRNA expression of NRG isoforms in DRG is similar to that in spinal cord, while in brain the expression is much less. The mRNA in DRG was found at similar levels from birth to postnatal day 7 of the premyelinating stage, and it decreased afterward. Our results suggest that several NRGs, including isoforms not reported before, play a role as survival factors for Schwann cells in the premyelinating stage.

Impaired molecular regenerative responses in sensory neurones of diabetic rats: gene expression changes in dorsal root ganglia after sciatic nerve crush.

This study investigated changes in gene expression in lumbar dorsal root ganglia (DRG), contralateral and ipsilateral to a sciatic nerve crush in control and streptozotocin (STZ)-induced diabetic rats. After 10 weeks of diabetes, the left sciatic nerves of all rats were crushed at mid-thigh level, and the rats were maintained for a further 2 weeks. Northern blots, with internal standards, were made from L4 and L5 (pooled) DRG on each side to compare RNA hybrids from ganglia attached to crushed nerves with those attached to intact nerves. The expression of growth-associated proteins, GAP-43 and Talpha1 alpha-tubulin mRNA in DRG, was stimulated (all P < 0.05) by crush injury in control and diabetic rats. Steady-state expression of transcripts for neurofilament (NF) proteins (NF-L, NF-H) and the high-affinity NGF receptor, trkA was decreased by diabetes in the contralateral ganglia to the crush (all P < 0.05). Crush injury further decreased expression of these transcripts in both control and diabetic rats (all P < 0.05). This reduced expression of mRNA coding for both growth-associated proteins, and neurofilament proteins in ganglia of diabetic rats could participate in the reduced competence of the regenerative response to nerve crush.

Increase of calcitonin gene-related peptide (CGRP) release and mRNA levels in endotoxic rats.

Previously we showed that calcitonin gene-related peptide (CGRP) is released into the circulation during pathogenesis of septic shock in humans and endotoxicosis in rats and pigs. The present study examines the changes of both CGRP release and synthesis during endotoxicosis in rats. Endotoxin was administered as a bolus (5 mg/kg,i.v.) to rats lightly anesthetized with ether. Blood samples and lumbar dorsal root ganglia (DRG), neuronal cell bodies synthesizing CGRP, were taken from rats at 0, .5, 3, or 8 h after endotoxin administration. CGRP levels in plasma and the steady-state levels of mRNA for CGRP in DRG were determined by methods of radioimmunoassay and semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR), respectively. Endotoxin elevated plasma CGRP by 62%, but not mRNA at .5 h. Endotoxin increased plasma CGRP by 142% and CGRP mRNA in DRG by 36% at 3 h, and further increased plasma CGRP by 216% and CGRP mRNA in DRG by 88% at 8 h. The data suggest that both CGRP expression and release in sensory nerves are increased during development of endotoxicosis in rats. CGRP synaptic transmission may be maintained during the course of endotoxicosis in rats. DRG undergo an elevation of CGRP expression in response to endotoxicosis, presumably as an adaptation of CGRP release during the inflammation. CGRP, because of its extremely high potency in hypotensive, tachycardiac and immunosuppressive effects, may play an important role in the pathogenesis of septic shock.

Effects of the capsaicin analogue resiniferatoxin on spinal nociceptive mechanisms in the rat: behavioral, electrophysiological and in situ hybridization studies

The effect of a single subcutaneous (s.c.) injection of the ultrapotent capsaicin analogue resiniferatoxin (RTX) on responses of adult rats to noxious thermal and mechanical stimulation was examined. The effects of RTX treatment on the nociceptive flexor reflex and activity-dependent increase in spinal excitability after conditioning C-fiber stimulation (CS) were also assessed. Finally, the expression of galanin message associated peptide (GMAP) mRNA in dorsal root ganglion (DRG) cells and the effects of the high affinity galanin receptor antagonist M35 on the flexor reflex in RTX-treated rats were evaluated. RTX, but not vehicle, produced marked thermal hypoalgesia on the hot plate test with partial recovery in about 50% of animals after about 2 weeks and no recovery in the remaining rats after 4 weeks. In all animals there was only a transient and moderate increase in paw withdrawal threshold to mechanical pressure. The flexor reflex in response to a C-fiber CS train was recorded 15–35 days after RTX or vehicle treatment. There was no difference between RTX and vehicle treated rats on baseline response, but RTX treatment lead to less wind-up during the CS and reduced hyperexcitability. This was particularly the case for rats which did not recover from RTX-induced hypoalgesia. The C-fiber mediated hyperexcitability was potentiated by the galanin receptor antagonist M35, more so in the non-recovered rats than in the partially recovered rats. The number of DRG cells expressing GMAP mRNA was significantly higher in non-recovered than in partially recovered rats. Thus, RTX produced marked and prolonged impairment of capsaicin-sensitive afferents and upregulation of the inhibitory neuropeptides GMAP and galanin in DRG neurons, which may underlie the prolonged effect of RTX.

Enkephalinergic and dynorphinergic neurons in the spinal cord and dorsal root ganglia of the polyarthritic rat — in vivo release and cDNA hybridization studies

Complex and contradictory data have been reported regarding the changes in spinal opioidergic systems associated with chronic inflammatory pain in the rat. In an attempt to solve these discrepancies, the in vivo release of met-enkephalin and dynorphin and the expression of the corresponding propeptide genes were investigated at the spinal level in arthritic rats and paired controls. A dramatic increase in the concentration of prodynorphin mRNA (+300–550%) and a less pronounced elevation of that of dynorphin-like material (+40–50%) were found in the dorsal part of cervical and lumbar segments of the spinal cord in rats rendered arthritic by an intradermal injection of Freund's adjuvant four weeks prior to these measurements. In addition, the spinal release of dynorphin-like material (assessed through an intrathecal perfusion procedure in halothane-anaesthetized animals) was approximately twice as high in arthritic rats as in controls. In spite of significant elevations in the levels of both met-enkephalin (+30–70%) and proenkephalin A mRNA (+40−50%) in the dorsal part of cervical and lumbar segments, the spinal release of met-enkephalin-like material was decreased (−50%) in arthritic rats as compared to paired controls. Proenkephalin A mRNA (but not prodynorphin mRNA) could be measured in dorsal root ganglia, and its levels were dramatically reduced in ganglia at the lumbar segments in arthritic rats. Such parallel reductions in the spinal release of met-enkephalin-like material and the levels of proenkephalin A mRNA in dorsal root ganglia of arthritic rats support the idea that the activity of primary afferent enkephalinergic fibres decreases markedly during chronic inflammatory pain.

508 Studies on the centrally administered nitric oxide synthesis inhibitor on the cardiovascular regulation system

An increased expression of cholecystokinin (CCK) messenger RNA (mRNA) as well as CCK-B receptor mRNA in dorsal root ganglion (DRG) cells following peripheral axotomy has previously been demonstrated. In the present in vivo microdialysis study, the effect of unilateral sciatic nerve section on basal and potassium-induced release of CCK-like (CCK-LI) immunoreactivity in the rat dorsal horn was investigated. We also compared the effects of the CCK-B receptor antagonist CI988 on basal and potassium-stimulated CCK-LI release in intact animals and in chronically axotomized rats. Perfusion of the microdialysis probe with KCl (100 mM) induced a more than 6-fold increase of the extracellular level of CCK-LI in control animals. In contrast, following unilateral sciatic nerve section the same KCl stimulation failed to evoke a release of CCK-LI ipsilaterally. However, after systemic administration of CI988 (1 mg kg−1, i.v.), 100 mM KCl induced a significant increase of the extracellular CCK-LI level in axotomized rats, similar to that observed in control animals. In control animals no effect of CI988 on KCl-stimulated CCK-LI release could be detected. CI988 by itself had no influence on the extracellular CCK-LI level in either nerve injured or control animals. The present data suggest that axotomy reduces the release of CCK-like immunoreactivity in the spinal cord by a mechanism involving the CCK-B receptor binding site.

Studies on the centrally administered nitric oxide synthesis inhibitor on the cardiovascular regulation system

An increased expression of cholecystokinin (CCK) messenger RNA (mRNA) as well as CCK-B receptor mRNA in dorsal root ganglion (DRG) cells following peripheral axotomy has previously been demonstrated. In the present in vivo microdialysis study, the effect of unilateral sciatic nerve section on basal and potassium-induced release of CCK-like (CCK-LI) immunoreactivity in the rat dorsal horn was investigated. We also compared the effects of the CCK-B receptor antagonist CI988 on basal and potassium-stimulated CCK-LI release in intact animals and in chronically axotomized rats. Perfusion of the microdialysis probe with KCl (100 mM) induced a more than 6-fold increase of the extracellular level of CCK-LI in control animals. In contrast, following unilateral sciatic nerve section the same KCl stimulation failed to evoke a release of CCK-LI ipsilaterally. However, after systemic administration of CI988 (1 mg kg−1, i.v.), 100 mM KCl induced a significant increase of the extracellular CCK-LI level in axotomized rats, similar to that observed in control animals. In control animals no effect of CI988 on KCl-stimulated CCK-LI release could be detected. CI988 by itself had no influence on the extracellular CCK-LI level in either nerve injured or control animals. The present data suggest that axotomy reduces the release of CCK-like immunoreactivity in the spinal cord by a mechanism involving the CCK-B receptor binding site.