Dans le domaine de la nociception, de nombreux travaux utilisent la mise en évidence de la protéine c-Fos dans les noyaux des neurones comme marqueur indirect d’activité neuronale. La technique c-Fos permet ainsi de «visualiser» au niveau de la corne dorsale de la moelle épinière, chez des animaux éveillés libres de leurs mouvements, les neurones «activés» par des stimulations périphériques nociceptives. Cet article passe en revue les principales données obtenues dans notre laboratoire en utilisant l’expression spinale de la protéine c-Fos pour les études de différents aspects physiopharmacologiques des processus nociceptifs, notamment ceux liés à l’inflammation. Cette approche nous a permis d’étudier de façon quantitative et systématique, au niveau de la moelle épinière, l’effet de différentes substances (agissant à la périphérie et/ou au niveau spinal) impliquées ou non dans les processus nociceptifs liés à l’inflammation due à l’injection intraplantaire de carragénine. De plus, nous avons étudié les effets de coadministration de diverses substances antinociceptives et anti-inflammatoires. En effet, l’intérêt majeur de ces combinaisons réside dans la diminution de doses thérapeutiques efficaces en limitant ainsi les risques d’apparition d’effets secondaires indésirables.
The effects of RB101, a complete inhibitor of enkephalin-catabolizing enzymes, alone or with a selective cholecystokinin (CCK)B receptor antagonist (CI988) or CCK(A) receptor antagonist (devazepide), on carrageenin-induced spinal c-Fos expression were investigated. Spinal c-Fos expression was observed 90 min after intraplantar carrageenin (6 mg/150 microl saline), with Fos-like-immunoreactive neurons preferentially located in the superficial laminae of the spinal dorsal horn. Intravenous RB101 (10, 20 and 40 mg/kg) dose-dependently reduced the number of superficial Fos-like-immunoreactive neurons (r2 = 0.739, P < .0001), with 63 +/- 2% (P < .0001) reduction for the highest dose. These effects were completely blocked by coadministered naloxone. Coadministration of inactive doses of i.v. RB101 (5 mg/kg) and i.p. CI988 (3 mg/kg) significantly and strongly reduced the number of carrageenin-induced, superficial, Fos-like-immunoreactive neurons (55 +/- 5% reduction of control carrageenin c-Fos expression, P < .0001). This effect was blocked by coadministered naloxone. It is important to note that coadministered RB101 and devazepide did not influence spinal c-Fos expression. None of the various drug combinations influenced the carrageenin-induced peripheral edema. These results show that RB101 dose-dependently decreases carrageenin-evoked spinal c-Fos expression. In addition, the effectiveness of RB101 can be revealed by preadministration of the CCK(B) receptor antagonist CI988. Considering the weak opioid side effects obtained with RB101 treatment and the strong increase of its effects by the CCK(B) receptor antagonist, this type of drug combination could have promising therapeutic application in the management of pain in humans.
In vitro binding of specific opioid ligands to their respective sites in membrane fractions and the contribution of individual receptor classes (mu, delta, kappa) was studied in rats after longlasting (up to 22 months) section of spinal dorsal roots at the cervical (C5-8) or thoracic (Th1-4) level. This procedure leads to autotomy or scratching of the skin on the operated side. The total number of receptors in the cervical and thoracic spinal cord was more than doubled in both operated and contralateral part of the cord in comparison with intact controls of the same age. In the cervical region, this increase mainly represented a rise in the number of free receptors, whilst in the thoracic region both free and saturated receptors were increased. On the deafferented side, receptor selectivity, especially in the delta and kappa types was decreased.
Intraplantar carrageenin (6 mg 150 μ1‐ 1 ) evoked a high level of spinal c‐Fos expression in the dorsal horn, of segments L4‐L5 of the spinal cord, and an extensive peripheral oedema; both parameters were assessed 3 h after carrageenin. Two series of experiments were performed, with the mean total number of Fos like‐immunoreactive neurones (Fos‐LI), after carrageenin, not being significantly different for the two series of experiments (266 ± 17 and 332 ± 31 Fos‐LI neurones). For both series of experiments Fos‐LI neurones were predominantly located in the superficial and deep laminae, only 10% of the total number of Fos‐LI neurones were located in the nucleus proprius and 10% were located in the ventral horn. Pre‐administration of the N‐methyl‐D‐aspartate (NMDA) receptor antagonist, (+)‐HA966 (0.5 mg kg −1 and 2.5 mg kg −1 , s.c), 30 min before carrageenin, did not significantly influence the total number of Fos‐LI neurones, as compared to control carrageenin expression. Pre‐administration of the highest dose of (+)‐HA966 (10 mg kg −1 ) significantly reduced the number of deep laminae Fos‐LI neurones (28±3% reduction of control number of Fos‐LI neurones after carrageenin, P ≤0.05), without influencing the number of superficial Fos‐LI neurones. There was a tendency towards a reduction of the number of Fos‐LI neurones in the nucleus proprius by the highest concentration of pre‐administered (+)‐HA966, (31±8% reduction), but this effect did not reach significance. Pre‐plus post‐administered (+)‐HA966 (0.5 mg kg −1 ), 30 min before and again 45 min after intraplantar carrageenin, did not significantly influence the total number of Fos‐LI neurones, as compared to control carrageenin expression. Pre‐ plus post‐administration of 2.5 mg kg −1 (+)‐HA966 significantly reduced the total number of Fos‐LI neurones, as compared to control carrageenin expression. This effect was reflected by a significant reduction in the number of Fos‐LI neurones in the nucleus proprius (36 ±7% reduction of control carrageenin c‐Fos expression respectively, P ≤ 0.05). Pre‐plus post‐administration of 10 mg kg −1 of (+)‐HA966 significantly reduced the number of Fos‐LI neurones in the superficial laminae, nucleus proprius, deep laminae and ventral horn (33±0.5%, 55±6%, 40±4% and 51±4% reduction of control carrageenin c‐Fos expression, respectively, P ≤0.05, for all areas). A single post‐administration of (+)‐HA966 (10 mg kg −1 ), 45 min after intraplantar carrageenin, did not significantly influence the number of Fos‐LI neurones in the superficial, deep laminae or ventral horn, but significantly reduced the number of Fos‐LI neurones in the nucleus proprius, as compared to control carrageenin expression (39±8% reduction of control carrageenin c‐Fos expression, P ≤0.05). None of the concentrations of (+)‐HA966 studied, irrespective of the timing of administration, influenced the peripheral carrageenin oedema. Our results illustrate a contribution of central NMDA receptor activation to carrageenin‐evoked spinal c‐Fos expression. These results extend previous studies demonstrating the contribution of the NMDA receptor to central hyperalgesia and the expression of c‐Fos.
De nombreux travaux utilisent la mise en evidence de la proteine c-Fos par immunohistochimie dans les noyaux des neurones comme marqueur d 'activite neuronale. La proteine c-Fos est codee par le gene c-fos, un gene a expression precoce immediate, qui est rapidement active a la suite de differents types de stimulations, y compris des stimulations nociceptives. Meme si l'expression de la proteine c-Fos, notamment au niveau de la moelle epiniere, est largement utilisee pour certaines etudes physiopharmacologiques, l'utilisation de cette technique dans le domaine de la douleur reste discutee. Considerant les avantages et certaines limites de cette approche, nous presentons les arguments en faveur de l'utilisation de la technique c-Fos pour aborder certains aspects physiopharmacologiques de processus nociceptifs, notamment ceux d'origine inflammatoire.
This study assesses the anti-inflammatory/analgesic effects of ketoprofen a non-steroidal anti-inflammatory drug, using the method of c-Fos immunoreactivity at the spinal cord level in two models of noxious stimulation: carrageenan-induced inflammatory pain or acute noxious heat. Ketoprofen was pre-administered intravenously or orally 25 min before an intraplantar injection of carrageenan (6 mg in 150 microliters of saline) in hindpaw of the non-anaesthetised rat or before a single noxious heat (52 degrees C, 15 sec) stimulation of hindpaw of the anaesthetised rat. Three hours after carrageenan or 2 h after noxious heat, the number of spinal c-Fos protein-like immunoreactive (c-Fos-LI) neurons in L4-L5 segments and both the ankle and paw diameter, the indicator of peripheral oedema, were assessed. Pre-administered ketoprofen (1, 3 and 10 mg/kg i.v.) dose-dependently blocks the development of the carrageenan-induced spinal c-Fos protein expression and peripheral oedema, with the highest dose influencing in parallel both parameters (75 +/- 2% diminution of total number of c-Fos-LI neurons per L4-L5 section; 64 +/- 4% and 82 +/- 6% diminution of paw and ankle oedema, respectively). The effect of ketoprofen was significantly greater on the number of c-Fos-LI neurons in deep, as compared to superficial, laminae. Furthermore, the dose-dependent effects of ketoprofen on the carrageenan-induced spinal c-Fos protein expression and both the paw and ankle oedema were correlated. Oral pre-administration of ketoprofen (20 mg/kg) produced the blockage of development of the carrageenan-induced spinal c-Fos protein expression (65 +/- 3% diminution of total number of c-Fos-LI neurons per L4-L5 section) and peripheral oedema (20 +/- 3% and 59 +/- 10% diminution of paw and ankle oedema, respectively). In contrast, the same doses of both the intravenous and oral pre-administration of ketoprofen did not influence either the spinal c-Fos protein expression nor slightly enhanced paw diameter induced by a single noxious heat stimulation. This study suggests a predominant peripheral site, without excluding a central site of action of ketoprofen in the carrageenan-induced inflammation. The method of c-Fos protein-like immunoreactivity revealed ketoprofen to be more potent in comparison to members of other families of non-steroidal anti-inflammatory drugs, previously studied in the same experimental conditions of carrageenan-induced inflammatory pain.
