Stage-dependent changes in the modulation of spinal nociceptive neuronal activity during the course of inflammation.

Spinal and supraspinal controls can tonically or phasically modulate the output of spinal nociceptive neurons. Alterations of these modulatory systems have been described during the acute stage of inflammation. In the present study in the rat, tonic descending controls were assessed during acute (24--48 h) and chronic (3--4 weeks) stages of monoarthritis of the ankle. The electrophysiological properties of spinal convergent neurons with ankle input were compared before and after spinalization. In a parallel series of experiments, spinal convergent neurons were recorded from the normal side in order to assess the propriospinal and supraspinal inhibitory controls triggered by nociceptive stimulation of the inflamed ankle. Tonic descending inhibition of convergent neurons with input from the inflamed ankle was enhanced during the acute stage and then decreased during the chronic stage of monoarthritis. Contralateral-induced inhibitions exhibited a similar temporal evolution. Time-dependent changes in the spinal transmission of nociceptive signals were shown by removing descending modulation in animals with monoarthritis; sensitization of spinal neurons with input from the inflamed ankle was demonstrated during the acute stage of monoarthritis, whereas a crossed transmission between inflamed and normal sides was observed during the chronic stage of the disease. These results show that dynamic and stage-dependent modifications of descending controls tend to dampen the central changes associated with inflammation.

Alteration of descending modulation of nociception during the course of monoarthritis in the rat.

Diffuse noxious inhibitory controls (DNIC), which involve supraspinal structures and modulate the transmission of nociceptive signals, were investigated at different stages during the development of adjuvant-induced monoarthritis in the rat. After behavioral evaluation, recordings of trigeminal convergent neurons were performed in anesthetized animals with acute (24-48 hr) or chronic (3-4 weeks) monoarthritis of the ankle. Inhibitions of C-fiber-evoked neuronal responses during and after the application of noxious conditioning stimuli to the ankle were measured to evaluate DNIC. The conditioning stimuli consisted of mechanical (maximal flexion and graded pressures) and graded thermal stimuli and were applied alternately to normal and arthritic ankles. Behaviorally, the two groups of animals exhibited a similar increased sensitivity to mechanical stimuli applied to the arthritic joint (i.e., an increased ankle-bend score and a decreased vocalization threshold to pressure stimuli). However, they showed different electrophysiological profiles. In the animals with acute monoarthritis, the DNIC-induced inhibitions produced by mechanical or thermal stimulation of the arthritic joint were significantly increased at all intensities compared with the normal joint. In contrast, in the chronic stage of monoarthritis, the DNIC-induced inhibitions triggered by thermal or pressure stimuli were similar for both ankles, except with the most intense mechanical stimuli. This discrepancy between the behavioral and electrophysiological findings suggests that inputs activated during chronic monoarthritis may fail to recruit DNIC and may thus be functionally different from those activated in the acute stage of inflammation.

Unilateral joint inflammation induces bilateral and time-dependent changes in neuropeptide FF binding in the superficial dorsal horn of the rat spinal cord: implication of supraspinal descending systems.

Using quantitative autoradiography, the effects of acute and chronic inflammation on specific 125I-1DMethyl-FLFQPQRFamide binding were investigated in the rat spinal cord dorsal horn superficial layers, at 6 and 24 h and 2, 4, 6 and 12 weeks after induction of monoarthritis produced by injection of killed Mycobacterium butyricum suspended in Freund adjuvant in one tibio-tarsal joint. Six hours after monoarthritis induction, no modification in specific 125I-1DMethyl-FLFQPQRFamide binding was observed, whereas a significant bilateral increase occurred after 24 h and 2 weeks in L4/L5 dorsal horns, with a return to control values at 4, 6 and 12 weeks. Specific 125I-1DMethyl-FLFQPQRFamide binding was also investigated 24 h after monoarthritis induction in rats submitted 4 days before the induction to spinal cord lesions at the thoracic level (T9-T10). Hemisection of the spinal cord contralateral to the affected ankle prevented the transient bilateral increase in specific 125I-1DMethyl-FLFQPQRFamide binding, whereas total spinal cord section induced a significant bilateral decrease. All of these modifications were restricted to the spinal segments receiving afferent input from the arthritic ankle (L4/L5); no modifications were found at the levels L1 or C6-C8. These data suggest that FLFQPQRFamide is involved in spinal nociceptive processing during sustained peripheral nociceptor activation. The effects of spinal cord lesions in monoarthritic rats indicate that the modifications seen in the FLFQPQRFamide system activity, during sustained peripheral inflammation, depend on afferent fiber activation as well as on supraspinal controls.

Sympathectomy does not modify the levels of dopa or dopamine in the rat dorsal root ganglion.

In the present study we report on the levels of dihydroxyphenylalanine (DOPA), dopamine and their metabolites, 3-O-methyl-DOPA, homovanillic acid and dihydroxyphenylacetic acid, in the dorsal root ganglion (DRG) where dopaminergic sensory neurons have recently been identified. HPLC with electrochemical detection was used. Chemical sympathectomy, induced by daily injection of 30 mg/kg/sc of guanethidine over 4 days, did not modify these levels. DOPA, dopamine and their metabolites were also detected in the dorsal root in both intact and sympathectomized rats. The present results show that in the DRG, DOPA and dopamine do not derive from sympathetic nerves, and agree with previous reports suggesting that there is a peripheral dopaminergic sensory innervation. In addition the identification of 3-O-methylDOPA, the extraneuronal metabolite of DOPA is not only the precursor of dopamine in the DRG but may be released from DRG cells. The high 3-O-methyl-DOPA/DOPA ratio found in the DRG and the dorsal root suggests that DOPA could play a role as neurotransmitter or modulator in the peripheral sensory innervation, as it has been proposed for the central nervous system and the sympathetic system.

[Role of the spinal serotoninergic, noradrenergic and dopaminergic systems in the mechanism of analgesia]. / Rôle des systèmes sérotonergique, noradrénergique et dopaminergique spinaux dans les mécanismes d'analgésie.

The aminergic systems arising from the supraspinal structures which innervate the spinal cord are involved in the control of the activity of 1) the sensory neurons 2) the motor neurons 3) at the thoracic level the neurons of vegetative system. During the last two decades numerous publications have been devoted to the study of the role of the serotonergic and the noradrenergic systems. Several groups have also identified a spinal dopaminergic innervation arising from supraspinal structures and suggested that this innervation is, like the two other systems, involved in those controls. All these studies have more particularly suggested that the serotonergic, noradrenergic and dopaminergic fibers which innervate the dorsal horn of the spinal cord play an important role in the control of the transmission of the nociceptive messages and are implicated in the mechanisms of analgesia. Data supporting this hypothesis are reported in the present paper. The criticisms which have been expressed about the hypothesis related to the role of the serotonergic system will be also presented.

Hindbrain structures involved in pain processing as revealed by the expression of c-Fos and other immediate early gene proteins.

We have used the evoked expression of the immediate early gene-encoded proteins (c-Fos, Fos B, Jun B, Jun D, c-Jun and Krox-24) to monitor sensory processing in the hindbrain structures of rats undergoing somatic inflammation. Experiments were performed on freely moving animals that did not experience constraints other than those imposed by the disease itself. Local injections of chemicals were used to cause subcutaneous inflammation of the plantar foot or monoarthritis by intracapsular injection. Labelling was studied at survival times that corresponded either to the time points of maximum labelling in the spinal cord (4 h for the subcutaneous model, 24 h and two weeks for the monoarthritis model) or at survival times that corresponded to the chronic phase of monoarthritis evolution (six, nine and 15 weeks). Controls consisted of freely moving, unstimulated animals. Basal expression was observed for all immediate early genes and in a variety of structures, but always remained moderate. All immediate early gene-encoded protein expressions except c-Jun were evoked, but except for c-Fos, and to a lesser extent Jun D, intensities of staining always remained faint. The following results will be mainly based on c-Fos expression, as this protein proved to be the most effective marker for all the survival times studied. Somatic pain evoked c-Fos expression in a subset of discrete subregions of both the caudal medulla oblongata and transitional areas of the pontomesencephalic junction. In the caudal medulla oblongata, structures involved were the caudal intermediate reticular nucleus, the subnucleus reticularis dorsalis, the ventrolateral reticular formation and the lateral paragigantocellular nucleus. Structures involved at the pontomesencephalic junction level mostly included the superior and dorsal lateral subnuclei of the parabrachial area, the nucleus cuneiformis and the most caudal portions of the lateral central gray, also including the laterodorsal tegmental nucleus; labelling in other lateral subnuclei of the parabrachial area always remained moderate. Staining in the caudal reticular areas was evident only at short survival times (4 and 24 h survival times in subcutaneous and monoarthritis models, respectively). Staining in nuclei of the pontomesencephalic junction was evident in all cases except for the very long survival periods (six to 15 weeks) of monoarthritis. In all cases staining was bilateral with contralateral predominance with regard to the stimulated limb. The present work demonstrates that hindbrain structures involved in somatic pain processing can be effectively identified in behaving animals and that c-Fos is the most reliable activity marker in this case.(ABSTRACT TRUNCATED AT 400 WORDS)

Dopamine as trace amine in the dorsal root ganglia.

It has been shown that in the chick dorsal root ganglion (DRG) about 8% of neurons, belonging to both the A and B classes of sensory neurons exhibit a clear dopamine immunoreactivity. In the present study are reported the results of measurements, by mean of HPLC-electrochemical detection (HPLC-ED), of DA and of the DA metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the rat DRG and their central nerves. Very low levels of DA, about 10 folds lower than the levels found in the dorsal horn of the spinal cord, were found in the DRG. However the levels of DOPAC and HVA were approximately equivalent to the levels found in the cord. The immunocytochemical study performed in parallel has shown that some dopaminergic-immunoreactive fibers in the DRG are located around the blood vessels. Few dopamine-immunoreactive sensory neurons were identified in the DRG and immunoreactive fibers, not linked to blood vessels, were identified in the dorsal root nerves. The present work indicates that there is a dopaminergic innervation of the blood vessels in the rat DRG but that dopamine may also be, as in the chick, a transmitter of primary afferent fibers.

Differential time course and spatial expression of Fos, Jun, and Krox-24 proteins in spinal cord of rats undergoing subacute or chronic somatic inflammation.

We have used the evoked expression of both immediate early gene (IEG)-encoded proteins (Krox-24, c-Fos, Fos B, Jun D, Jun B, c-Jun), and dynorphin to monitor sensory processing in the spinal cords of rats undergoing subacute or chronic somatic inflammation (i.e., subcutaneous inflammation of the plantar foot and monoarthritis, respectively). Behavioral and immunocytochemical approaches were conducted in parallel up to 15 weeks postinjection in order to detect possible relationships between clinical evolution and spatiotemporal pattern of IEG-encoded protein expression. Each disease had specific characteristics both in terms of their clinical evolution and pattern of evoked protein expression. All IEG proteins were expressed in both cases. Most of the staining was observed in both the superficial layers of the dorsal horn and deep dorsal horn (laminae V-VII and X). Monoarthritis was distinguished by a high level of total protein expression. Staining was especially dense in the deep dorsal horn. More labelled cells were observed at 1-2 days and at 2 weeks postinjection, corresponding to the initiation and progressive phases of the disease, respectively. Subcutaneous inflammation was characterized by a moderate level of total IEG expression. More labelled cells were observed in the first day following injection. It is the relative degree of expression of each IEG-encoded protein with regard to the others that characterized the progression of the diseases. Early stages of the diseases coincided with the expression of all Fos and Jun proteins, while late stages showed an increase in Jun D and Fos B involvement; Krox-24 was induced mostly during the early phases and/or periods of paroxysm of the diseases. Persistent stimulation was characterized by a predominant expression in deep versus superficial layers of the dorsal horn. Evoked expression of c-Jun in motoneurons was only observed in monoarthritis. The peak of dynorphin expression was late in regard to both the induction of inflammation and period of maximal IEG-encoded protein expression. The present work indicates that the neural processing that takes place during progression of these diseases can be monitored well at the spinal cord level by using the expression of an array of IEG-encoded proteins. Study of long term evolutive diseases and especially those that evolve into chronicity can largely benefit from such an approach.

Dorsal and ventral dopaminergic innervation of the spinal cord: functional implications.

Several studies have demonstrated that a descending dopaminergic pathway innervates the dorsal and the intermediate gray matter of the spinal cord and have suggested that this pathway is involved in pain modulation and in the control of autonomic functions. Other studies have also demonstrated the presence of dopamine (DA) and DA metabolites as well as of DA receptors in the ventral cord. There is also evidence for the implication of DA in the control of motor functions at the spinal level. The occurrence of a dopaminergic innervation in the ventral horn has been, however, disputed until recently. But recent work has demonstrated that the motoneural cell groups in the ventral horn (lamina IX) are a target for descending dopaminergic fibers. In addition, the possibility that DA is a mediator of primary afferent fibers has also been postulated. Finally, the occurrence of dopaminergic cell bodies has been suggested in the spinal cord. This indicates that DA is probably implicated in a complex manner in spinal functions. In the present paper the possible involvement of DA in sensory and in motor functions at spinal level will be discussed in view of neurochemical observations made in polyarthritic rats, in which pain-related behavior and reduction of locomotor activity associated with a marked decrease in mobility, are observed.

Monoarthritis induces complex changes in mu-, delta- and kappa-opioid binding sites in the superficial dorsal horn of the rat spinal cord.

Recently, an experimental model of monoarthritis was described in the rat induced by injection with Freund's adjuvant of the tibio-tarsal joint of one hindlimb. After injection, the clinical and behavioural signs of arthritis are stable from weeks 2 to 6 post-injection. Our purpose was to study the regulation of mu-, delta- and kappa-opioid binding sites in the superficial layers (laminae I-II) of the lumbar and cervical enlargements of the spinal cord 2, 4 and 6 weeks post-injection. Using quantitative receptor autoradiography and highly selective opioid ligands, we found complex changes consisting of a bilateral increase in specific [3H]DAMGO (Tyr*-D-Ala-Gly-NMe-Phe-Gly-ol) and [3H]pCl-DPDPE (Tyr*-D-Pen-Gly-Cl-Phe-D-Pen) binding at 2 weeks post-injection and a bilateral decrease in [3H]U-69593 ((5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1- oxaspiro(4,5)dec-8-yl]) specific binding at 4 weeks post-injection. These changes were restricted to the lumbar level. At 6 weeks post-injection, there was a bilateral increase in [3H]pCl-DPDPE specific binding at both lumbar and cervical levels. Altogether, these results suggest that, after probable local changes in endogenous opioid peptides, the three types of opioid binding sites are differentially involved in the development of the pathological process. These results contrast with the lack of significant modification in mu-, delta- and kappa-opioid binding classically reported at various levels of the spinal cord in polyarthritic rats at 3 weeks post-injection and verified for 2, 4 and 6 weeks post-injection in the present study.

Decreased cerebrospinal fluid neuropeptide-converting enzyme activity in monoarthritic rats.

The activity in rat cerebrospinal fluid (CSF) of dynorphin-converting enzyme (DCE) and substance P endopeptidase (SPE) was determined in control animals and in rats with monoarthritis. Enzymatic activities were measured with specific radioimmunoassays toward the N-terminal products Leu-enkephalin-Arg6 and substance P1-7, respectively. A monoarthritis stable during weeks 2-6 post-injection was induced by injection (0.05 ml) into one joint with Freund's adjuvant. Both SPE and DCE were significantly decreased 15 days after the intraarticular injection. Despite the degree of arthritis that was sustained equally at four weeks after inoculation, both DCE and SPE were back to control levels at that time. It can therefore be concluded that arthritis from a single joint is sufficient to elicit changes in CSF convertase activities, and that these effects disappear between 2 and 4 weeks after injection, although the arthritis persists.

Effect of aging on dopamine metabolism in the rat cerebral cortex: a regional analysis.

Age-related changes in the levels of dopamine (DA) and its metabolites were measured in seven cerebral cortical areas and in the striatum of 3, 10 and 27 month-old Sprague-Dawley rats. An age-related increase in DA levels was observed in the somatomotor (SM) cortex. In contrast, a decrease was observed in the temporal (T) cortex. Decreases in homovanillic acid (HVA) levels were observed in prelimbic (PL), pyriform (PY) and T cortex of aged rats, whereas significant increases in the levels of 3-methoxytyramine (3-MT) were observed in PL, prefrontal (PF), cingulate (C) as well as in T cortex. In the striatum, DA and HVA were decreased but the level of 3-MT remained unchanged. Norepinephrine (NE) levels increased in rats from 3 to 27 months in all the cortical areas. The increase in the levels of the DA extraneuronal metabolite, 3-MT, confirms our previous results showing that the release of DA might be increased with age in some cortical areas. The present results show that there is no general age-related decrease in the level of monoamines and of their metabolites in the rat cerebral cortex and that the changes display a complex, area-specific pattern.

Increase in "pain sensitivity" induced by exercise applied during the onset of arthritis in a model of monoarthritis in the rat.

We have recently developed, in the rat, a model with a limited arthritic process for chronic pain studies. Intra-articular injection (0.05 ml) of complete adjuvant containing 300 micrograms Mycobacterium butyricum in the tibio-tarsal joint produces a predictable monoarthritis stable clinically and behaviourly from weeks 2 through 6 post-injection. This model appears to be a suitable alternative for the polyarthritic rat for chronic studies based on both its ethical and scientific advantages. In the present work we report results of experiments on the effects of exercise on the pain behaviour and development of arthritis in this model. A group of rats prepared with the above protocol was submitted at 2 weeks post-inoculation to mild exercise (swimming [water 37 degrees C] three times per week) increasing from 5 to 15 min during 4 weeks. As revealed by analyses of the arthritis score, the stiffness score and the mobility score, no aggravation of arthritis occurred in these rats. However the threshold for struggle in response to paw pressure was further decreased (as compared to control arthritic rats) in these animals. These results are discussed in view of observations made in human studies.

Further evidence for the involvement of the diencephalo-dopaminergic system in pain modulation: a neurochemical study on the effect of morphine in the arthritic rat.

Numerous studies from the past two decades suggest that the bulbospinal serotonergic and noradrenergic systems are involved in pain modulation at the spinal level. More recently the occurrence of a diencephalo-dopaminergic system has been demonstrated and there is evidence that this pathway may also participate in pain modulation. Several works have been devoted to the effect of morphine on the activity of the dopaminergic system in supraspinal areas but it is not at present known if opiates modify the activity of this system in the spinal cord sensory areas. In the present study the effect of morphine on the metabolism of dopamine (DA), 5-hydroxytryptamine (5-HT) and norepinephrine (NE) in different laminae of the lumbar spinal cord of arthritic rats was investigated. A relatively small dose of morphine (2.5 mg/kg s.c.), which produces marked analgesia in these animals, induces a naloxone-reversible increase of DA metabolism in the different laminae of the spinal cord that contain neurons which respond to the activation of articular nociceptors. In contrast, the same dose of morphine does not significantly affect 5-HT and NE metabolism in the same areas. These observations suggest that the diencephalo-dopaminergic system is probably of equal importance to (or more important than) the two other monoaminergic systems in pain modulation at spinal level.

Simultaneous measurements of tryptophan and its metabolites, kynurenine and serotonin, in the superficial layers of the spinal dorsal horn. A study in normal and arthritic rats.

It has been demonstrated that 5-hydroxytryptamine (5-HT) is not the only neuroactive metabolite of tryptophan (TRP) in the CNS. The presence of kynurenine (KYN) and its metabolites has been reported in the brain of several mammalian species and the neuroactive properties of these compounds are now well established. In the present study, we report the identification of KYN in the superficial layers of the rat spinal dorsal horn. KYN was measured simultaneously with TRP. 5-hydroxytryptophan, 5-HT, 5-hydroxyindoleacetic acid and 5-HT-O-sulfate by means of liquid chromatography with coulometric electrode array detection. The results observed in the normal rat and in an animal model of persistent pain, the arthritic rat, are discussed in view of the hypothesis relating to the involvement of the bulbospinal serotonergic system in pain mechanisms and of the possible participation of KYN and its metabolites in these mechanisms.

Regional distribution of 3-O-methyl-DOPA in the cortex and subcortical structures in the rat: effect of aging.

Dihydroxyphenylalanine (DOPA) and its metabolite 3-O-methyl-DOPA (3-OMDOPA) have been identified as normal constituents in blood, CSF and brain in human and several animal species. In the present study, we report results of 3-OMDOPA measurements in seven cortical areas, hippocampus and striatum of young (3-month-old), middle aged (10-month-old) and aged (27-month-old) rats. 3-OMDOPA was identified in all the areas considered. Regional differences were observed, with the highest levels being found in the hippocampus and the somatomotor and pyriform cortex. A marked increase in the level of 3-OMDOPA was observed in all the cortical areas and in the hippocampus of aged rats. In contrast, this level remained unchanged in the striatum. These modifications were compared to those observed in the levels of dopamine (DA) in the same areas.

Effect of deafferentation on the levels of uric acid in the spinal cord of the rat.

In previous studies we reported that the rat spinal cord contains relatively high levels of uric acid and that the levels in a rat model of bilateral chronic pain, experimental adjuvant arthritis. In this report we evaluate the changes in UA in the unilaterally deafferented rat, a preparation which has also been used to study chronic pain. Uric acid was measured by high-pressure liquid chromatography with electrochemical detection in the spinal cord of rats that underwent unilateral, multiple cervical dorsal rhizotomy. Compared to control and sham-operated rats, there was a significant increase in the level of uric acid in the dorsal quadrant of the spinal cord ipsilateral to the dorsal rhizotomy. This increase was present at 1 and 4 weeks after surgery. At 1 week, we also observed a small but statistically insignificant increase in uric acid levels in the dorsal quadrant contralateral to the deafferentation and in sham-operated rats. Four weeks after surgery the levels of UA in all regions except for the deafferented dorsal quadrant returned to normal. The possibility was raised that the changes in uric acid reflect an increase in purinergic metabolism in the spinal cord secondary to the increased activity of the dorsal horn neurons that occurs with deafferentation.

Age-related changes in dopaminergic and serotonergic indices in the rat forebrain.

Age-related changes in the content of dopamine (DA), homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in anterior cerebral cortex, hippocampus and striatum of the rat have been investigated using HPLC with electrochemical detection. A significant decrease in HVA was observed in the striatum and hippocampus of the aged (27 months) animals, as compared to the controls (2.4 to 2.6 months). A significant decrease in DA levels was also observed in the hippocampus but not in the striatum. In contrast, the level of DA in the cerebral cortex was markedly increased in the aged animals. A concomitant increase in 3-MT level was observed. Finally the level of 5-HIAA was significantly increased in striatum and hippocampus.