Concomitant Administration of Morphine and an N-Methyl-D-Aspartate Receptor Antagonist Profoundly Reduces Inflammatory Evoked Spinal c-Fos Expression

Experiments were performed on 66 adult male albino Sprague-Dawley rats (Charles River, France), weighing 225-250 g. Guidelines on ethical standards for investigations of experimental pain in conscious animals were followed. [45]Rats were kept in a room at a constant temperature of 22 degrees C, with a 12-h alternating light-dark cycle.

In this study, neither nonstimulated control animals nor control animals receiving an intraplantar injection of saline were included because we have previously shown that in these two groups, there was almost no labeling (less than 5 Fos-LI neurons per section). [33,40]By comparison, in rats receiving intraplantar carrageenin, we previously observed, 3 h after intraplantar carrageenin, more than 100 labeled neurons per 40-micro meter section. [33].

In the first series of experiments, the effects of morphine (morphine hydrochloride, injectable solution, 10 mg/ml, Meram, diluted in saline), (+)-HA966 ((+)-(3R)-3-Amino-1-hydroxy-pyrrolidin-2-one, Tocris Cookson (Bristol, England), dissolved in bidistilled water) and coadministration of morphine and (+)-HA966 on c-Fos expression and peripheral edema induced at 1.5 h after carrageenin were studied. Morphine (0.3 mg/kg, n = 5) was injected intravenously in the tail 10 min before carrageenin (6 mg/150 micro liter, in saline). (+)-HA966 (2.5 mg/kg, n = 5) was injected subcutaneously 30 min before carrageenin. The coadministration group received 0.3 mg/kg intravenous morphine 10 min before carrageenin and 2.5 mg/kg subcutaneous (+)-HA966 30 min before carrageenin (n = 5). A control group of carrageenin-stimulated rats received an equal volume of saline and water, before carrageenin, under the same conditions as the coadministration group (n = 5).

In the second series of experiments, the effects of 0.3 mg/kg intravenous morphine administered 10 min before carrageenin (n = 5), 2.5 mg/kg subcutaneous (+)-HA966 administered 30 min before carrageenin (n = 4), and coadministration of 0.3 mg/kg intravenous morphine and 2.5 mg/kg subcutaneous (+)-HA966 (n = 5) on c-Fos expression and peripheral edema induced 3 h after administration of carrageenin were studied. A control group of carrageenin-stimulated rats received an equal volume of saline and water, before carrageenin, under the same conditions as the coadministration group (n = 4).

In the third series of experiments, the effects of 3 mg/kg intravenous morphine administered 10 min before carrageenin (n = 5), 2.5 mg/kg subcutaneous (+)-HA966 administered 30 min before carrageenin (n = 4), and coadministration of 3 mg/kg intravenous morphine and 2.5 mg/kg subcutaneous (+)-HA966 (n = 5) on c-Fos expression and peripheral edema induced at 3 h after carrageenin were studied. In addition, the effect of naloxone on the effects of coadministered morphine and (+)-HA966 on c-Fos expression and peripheral edema was investigated. One group of rats received 1 mg/kg intravenous naloxone (naloxone hydrochloride, injectable solution, 0.4 mg/ml, Du Pont de Nemours, Nemours, France) 10 min before carrageenin and because of its short duration of action a second injection of naloxone was administered 30 min after carrageenin (n = 4) and another group of rats received a coadministration of 3 mg/kg intravenous morphine 10 min before carrageenin and 2.5 mg/kg subcutaneous (+)-HA966 30 min before carrageenin plus 1 mg/kg intravenous naloxone 10 min before and 30 min after carrageenin (n = 5). A control group of carrageenin-stimulated rats received an equal volume of saline and water, before carrageenin, under the same conditions as the coadministration group of morphine, (+)-HA966 plus naloxone (n = 5).

In the fourth series of experiments, the effects of 0.3 or 3 mg/kg intravenous morphine 10 min before carrageenin (n = 5), 2.5 mg/kg subcutaneous (+)-HA966 30 min before carrageenin (n = 5), and coadministration of 0.3 or 3 mg/kg intravenous morphine and 2.5 mg/kg subcutaneous (+)-HA966 (n = 5 in each group) on body temperature were studied. A control group of carrageenin-stimulated rats received an equal volume of saline and water, before carrageenin, under the same conditions as the coadministration group of morphine, (+)-HA966 (n = 5). Rectal temperature was measured 30 min and 10 min before intraplantar carrageenin injection, just before intraplantar carrageenin injection and 10, 20, 30, 40, 90, and 180 min after intraplantar carrageenin injection.

At 1.5 h or 3 h, after the carrageenin injection, the animals were deeply anesthetized with 55 mg/kg intraperitoneal pentobarbital (Sanofi, Libourne, France) and underwent intracardiac perfusion with 200 ml phosphate-buffered saline 0.1 M followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The spinal cord was then removed and postfixed for 4 h in the same fixative, and cryoprotected overnight in 30% sucrose in PB. Frontal frozen sections, 40-micro meter thick, were cut and collected in PB to be processed immunohistochemically as free floating sections.

The serial sections from the lumbar segment were immunostained for c-Fos-like protein according to the avidin-biotin-peroxidase method. [46]The tissue sections were incubated for 30 min at room temperature in a blocking solution of 3% normal goat serum in phosphate buffer 0.1 M + saline 0.9% (phosphate-buffered saline) with 0.3% Triton-X (NGST) and were then incubated overnight at 4 degrees C in the primary antiserum directed against the c-Fos protein. The c-Fos antibody (Oncogene Science; Ab-2 solution, 0.1 mg/ml; diluted at 1:4,000) is a rabbit polyclonal antibody directed against residues 4-17 of the N-terminal region of the peptide. The incubated sections were washed three times in 1% NGST and incubated in biotinylated goat anti-rabbit immune globulin G for 1 h at room temperature, then washed twice in 1% NGST and incubated for 1 h in avidin-biotin-peroxidase complex (Vectastain, Vector Laboratories). Finally, the sections were washed three times in phosphate-buffered saline and developed in 1-naphtol ammonium carbonate solution (89.5 ml 0.1 M PB, 10 ml ammonium carbonate (1% in distilled water), 0.5 ml 1-naphtol (N-199-2 Aldrich, 10% in absolute alcohol) and 0.1 ml hydrogen peroxide 30% (w/w) solution) for 5 min, and were washed three times in PB to stop the staining reaction. The sections were mounted on gelatine-subbed slides and air dried for the stain to be intensified and made alcohol resistant through basic dye enhancement in 0.025% crystal violet (42555 Aldrich, St. Quentin Fallavier, France) in PB for 3 min. After 2 short PB rinses to take off the excess stain, sections were differentiated in 70% alcohol and the differentiation time was evaluated under the microscope. After being air dried, the slides were coverslipped. To test the specificity of the primary antibody, controls were performed; preabsorption with the corresponding synthetic peptide or omission of any stage in the protocol abolished the staining.

Tissue sections were first examined using dark field microscopy to determine the segmental level according to Molander et al., [47]as well as the gray matter landmarks. The sections were then examined under light field microscopy at x10 to localize c-Fos positive cells. Labeled nuclei were counted using a camera lucida attachment. To study the laminar distribution, four regions were defined: superficial dorsal horn (laminae I-II; superficial), nucleus proprius (laminae III-IV; nucleus proprius), neck of the dorsal horn (laminae V-VI; neck) and the ventral gray (laminae VII-X; ventral).

After intraplantar carrageenin, Fos-LI nuclei, which were stained to a variable degree, were located in the ipsilateral dorsal horn of the spinal cord. All Fos-LI nuclei were analyzed without considering the intensity of the staining. We have previously shown that the most numerous c-Fos positive neurons were localized in the L4-L5 segments, so for all the pharmacologic studies, for each rat, two sets of analyses were made: (1) the total number of Fos-LI neurons in the gray matter for ten sections through L4-L5 segments, and (2) the number of Fos-LI neurons per specific defined region of the spinal gray matter in these ten sections.

To assess the level of the peripheral inflammation, we considered two inflammatory parameters at the time the animals were killed, the diameters of both the ankle and paw, ipsilateral and contralateral to carrageenin injection, measured with a caliper square.

Statistical analysis was performed to compare the total number of Fos-LI neurons, using one-way analysis of variance for the different groups of animals, and two-way analysis of variance for the different groups of animals and the laminar region. To compare the ankle or the paw diameters we used one-way analysis of variance for the different groups of animals. For multiple comparisons, the Fisher's protected least significant difference test was used. The investigator responsible for plotting and counting the Fos-LI neurons was blind to the experimental situation of each animal.

The total number of Fos-LI neurons observed 1.5 h after carrageenin was 66+/-8 Fos-LI neurons per section, in segments L4-L5 of the spinal cord. The Fos-LI neurons were preferentially located in the superficial laminae (I-II) of the dorsal horn (67+/-6%), whereas fewer neurons were observed in the nucleus proprius (2+/-1%), the deep laminae (V-VI; 24+/-6%), and the ventral horn (7+/- 2%). The total number of Fos-LI neurons observed 3 h after administration of carrageenin was 219+/-14 Fos-LI neurons per section, in segments L4-L5 of the spinal cord. At this later time, Fos-LI neurons were more equally distributed in the superficial laminae (I-II) and deep laminae (V-VI) of the dorsal horn (48+/-3% and 37+/-3%, respectively). The number of Fos-LI neurons in the ventral horn was moderate (11+/-1%), whereas very few neurons in the nucleus proprius expressed c-Fos (4+/-1%).

Prior administration of morphine (0.3 mg/kg) or (+)-HA966 (2.5 mg/kg) was ineffective at reducing the number of spinal Fos-LI neurons induced at 1.5 h or 3 h after intraplantar carrageenin (Figure 1and Figure 2and Table 1). However, prior coadministration of morphine (0.3 mg/kg) and (+)-HA966 (2.5 mg/kg) significantly, and strongly, reduced the total number of spinal Fos-LI neurons induced at 1.5 h after intraplantar carrageenin (60+/-7% reduction of control carrageenin c-Fos expression, P < 0.01). These effects were essentially observed at the level of the superficial laminae (58+/-7% reduction of control carrageenin c-Fos expression, P < 0.01). In addition, these effects were significantly different from the lack of effect of morphine alone (P < 0.01) and (+)-HA966 alone (P < 0.05; Figure 1(A-B), Figure 3, and Table 1).

In contrast, prior coadministration of the same doses of morphine (0.3 mg/kg) and (+)-HA966 (2.5 mg/kg) did not significantly influence the total number of spinal Fos-LI neurons induced 3 h after intraplantar carrageenin (16+/-6% reduction of control carrageenin c-Fos expression; Figure 1(C) and Table 1).

In this series of experiments, the total number of Fos-LI neurons observed 3 h after carrageenin administration was 120+/-9 Fos-LI neurons per section, in segments L4-L5 of the spinal cord. Again, Fos-LI neurons were predominantly distributed in the superficial laminae (I-II) and deep laminae (V-VI) of the dorsal horn (59+/-3% and 32 +/-4%, respectively). The number of Fos-LI neurons in the ventral horn was moderate (8+/-1%), whereas very few neurons in the nucleus proprius expressed c-Fos (1+/-1%).

Prior administration of a larger dose of morphine (3 mg/kg) significantly reduced the total number of spinal Fos-LI neurons induced 3 h after intraplantar carrageenin (40+/-7% reduction of control carrageenin c-Fos expression, P < 0.001; Figure 3and Figure 4and Table 2). Coadministration of morphine (3 mg/kg) with the already mentioned ineffective dose of (+)-HA966 (2.5 mg/kg) further reduced the total number of spinal Fos-LI neurons induced 3 h after intraplantar carrageenin (80+/-7% reduction of control carrageenin c-Fos expression, P < 0.0001). The effects of coadministered morphine and (+)-HA966 on c-Fos expression in the superficial and deep laminae (80 +/-8% and 79+/-9% reduction of control carrageenin c-Fos expression, P < 0.0001 for both, respectively) were virtually equivalent. The effect of coadministered morphine and (+)-HA966 were significantly different from the effect of morphine alone (P < 0.001) and the lack of effect of (+)-HA966 alone (P < 0.001). The significant reduction of c-Fos expression by coadministered morphine and (+)-HA966 was substantially blocked by a coadministration of naloxone (74+/-8% of control carrageenin c-Fos expression, P < 0.05), which injected alone had no effect (Figure 3and Figure 4and Table 2).

The peripheral edema associated with carrageenin was extensive, both the paw and ankle diameters of the injected hind paw were increased (first series: 200+/-5% and 113+/-6%; second series: 282 +/-10% and 197+/-10%; third series: 257+/-12% and 181+/-11%, paw and ankle diameters of nonstimulated rats, respectively). Neither morphine (0.3 or 3 mg/kg intravenous), (+)-HA966 (2.5 mg/kg subcutaneous) or coadministration of morphine (0.3 or 3 mg/kg intravenous) and (+)-HA966 (2.5 mg/kg subcutaneous) influenced the carrageenin induced peripheral edema. In addition, neither naloxone, nor coadministration of naloxone, morphine, and (+)-HA966 modified the peripheral edema.

As can been seen in Table 3, before any drug administration, the group of rats that were to receive morphine (3 mg/kg intravenous) had a body temperature significantly greater than that of the control group. In all the tested groups, the body temperature slightly increased throughout the experiments, and in the 3 mg/kg intravenous morphine group, body temperature was significantly different from the control group (as before any drug injection), with a maximal increase of 1.8+/-1% at 1.5 h after carrageenin administration. Three hours after carrageenin injection, there was no significant difference in body temperature with the various drug combinations.

In this article, we present evidence that coadministrations of ineffective doses, doses that do not influence carrageenin-induced c-Fos expression, of morphine and an antagonist at the glycine site of the NMDA receptor, (+)-HA966, strongly decrease spinal c-Fos expression induced at 1.5 h after intraplantar carrageenin. The same coadministration of morphine and (+)-HA966 was ineffective at reducing spinal c-Fos expression when assessed 3 h after carrageenin injection. However, increasing the dose of morphine, coadministered with (+)-HA966, resulted in a strong reduction of spinal c-Fos expression induced at 3 h after carrageenin, these effects were partially blocked by a coadministration of naloxone. Furthermore, none of the drug combinations had effect on the peripheral edema induced 1.5 h or 3 h after intraplantar carrageenin or on body temperature monitored throughout the experiments.

In agreement with previous study of carrageenin-evoked c-Fos expression, [33]Fos-LI neurons were observed in lumbar segments L2-L6, with maximal labeling in segments L4 and L5, in all the experiments.

At 1.5 h after intraplantar carrageenin, spinal c-Fos expression was essentially located in the superficial laminae of the dorsal horn of the spinal cord, whereas at 3 h after intraplantar carrageenin, the number of Fos-LI neurons was increased, both in the superficial and deep laminae of the spinal dorsal horn, thus agreeing with the results of our previous study. [33]The number of spinal Fos-LI neurons observed 3 h after intraplantar carrageenin in the second and third experiments were different (219+/-14 and 120+/-9 spinal Fos-LI neurons per section), reflecting variations between experiments. However, as in the same experiments, all the experimental groups were performed simultaneously and immunohistochemically stained simultaneously, we can compare the various experimental groups in the same experiment. The number of Fos-LI neurons in the contralateral dorsal horn was not significantly different from the well-established low number of spinal Fos-LI neurons in nonstimulated rats, < 5 Fos-LI neurons per section. [40].

Prior systemic administration of the smaller dose of morphine (0.3 mg/kg) did not influence the spinal c-Fos expression observed at 1.5 h or 3 h after intraplantar carrageenin. In contrast, prior systemic administration of the larger dose of morphine (3 mg/kg) reduced the number of spinal Fos-LI neurons observed 3 h after intraplantar carrageenin. Our results are in good agreement with previous studies that have shown that systemic morphine reduces noxiously evoked spinal c-Fos expression in a naloxone-reversible manner. [34,39,41,42].

It is noteworthy that intravenous injection of morphine alone has been shown not to increase spinal c-Fos expression. [41,42]In our study, none of the doses of morphine had an effect on the peripheral edema, these results agreed with our previous study, [43]but contrast with the study of Hargreaves et al. [29]in which a considerably smaller dose of intraplantar carrageenin was used. Furthermore, none of the drug combinations used in this study influenced rat body temperature, rejecting possible nonspecific effects of the drugs on inflammation and as consequences on spinal c-Fos expression.

Prior administration of (+)-HA966 did not influence the spinal c-Fos expression or the peripheral edema observed 1.5 h or 3 h after intraplantar carrageenin. These findings agree with our previous study in which we have shown that this dose of (+)-HA966 had no effect on the two parameters [15]and with other studies illustrating that NMDA receptor antagonism does not influence peripheral edema. [10].

The major finding of our study is the demonstration of the presence of a supraadditive effect of coadministered morphine and (+)-HA966 on carrageenin-induced spinal c-Fos expression. In addition, the peripheral edema was not influenced by coadministered morphine and (+)-HA966, suggesting that the location of the interaction between morphine and (+)-HA966 and the effects on c-Fos expression is at the spinal or supraspinal level. Our results are in good agreement with previous studies showing a potentiation between the effects of intrathecal morphine and NMDA antagonists on windup of C-fiber-evoked responses of spinal dorsal horn neurons [24]and additive effects on thermal hyperalgesia in a model of neuropathic pain. [25]However, our results contrast, in part, with a previous study showing that only the effects of low doses of morphine were enhanced by coadministration of NMDA antagonist. [26]Indeed, in our study, coadministration of a subthreshold dose of morphine plus (+)-HA966 decreased the number of Fos-LI neurons induced 1.5 h but not 3 h after intraplantar carrageenin, whereas coadministration of a larger dose of morphine, plus (+)-HA966 resulted in a strong reduction of spinal c-Fos expression induced 3 h after carrageenin administration. Our results suggest that the interactions between these two drugs are not based on a modification of the pharmacokinetic parameters of the action of morphine (i.e., increasing the duration of action of morphine, which is normally about 70 min with variations depending of the experimental conditions) but a pharmacodynamic interaction because 0.3 mg/kg intravenous morphine had no effect given alone on either spinal c-Fos expression induced 1.5 h or 3h after intraplantar carrageenin, but becoming active when (+)-HA966 is added, only on carrageenin-induced spinal c-Fos expression observed at 1.5 h, and one could suggest that if there was a pharmacokinetic interaction, the combination of inactive dose of morphine plus (+)-HA966 would have also decreased carrageenin-induced spinal c-Fos expression observed at 3 h. Furthermore, Kest et al. have previously shown that the increase of the effects of morphine by MK-801 was related to intensity of action and not duration of action. [26].

The behavioral hyperalgesia associated with the carrageenin model of inflammation has been shown to be mediated, at least in part, by spinal NMDA receptor activation. [11,12]The prototype experimental model of NMDA receptor-mediated events, electrically evoked windup has, as discussed earlier, a relatively low sensitivity to morphine. We have previously shown that a component of carrageenin-evoked c-Fos expression is NMDA receptor-mediated, [15]and that owing to the short time course of systemic morphine relative to the duration of carrageenin-evoked c-Fos stimulation that comparatively high concentrations of opioids are required to reduce the c-Fos expression. [43,48].

At low doses, morphine is thought to act preferentially at presynaptically located micro-opioid receptors, with higher concentrations acting postsynaptically. [49]Spinal opioids are thought to exert their antinociceptive effects by diminishing the release of neurotransmitters from small primary afferent fibers, in particular decreasing the amount of spinal glutamate release induced by various types of peripheral stimulations [50-52]and by acting postsynaptically to hyperpolarize dorsal horn neurons, which are driven by C-fiber inputs. [53-55]Morphine has been shown to readily reduce the steady-state C-fiber-evoked responses, whereas higher concentrations were required to reduce windup, [34]however, moderate doses of morphine delay the onset of windup without inhibiting the process itself. [56]In contrast, NMDA receptor antagonists selectively reduce windup. [3,4]With consideration of these previous studies, it seems highly probable that the ability of coadministration of a low dose of morphine plus (+)-HA966 to dramatically reduce c-Fos expression reflects a reinforcing dual site of action of morphine, on presynaptic sites, reducing but not completely blocking the input onto the dorsal horn neurons, and NMDA antagonism at postsynaptic sites, reducing the degree of the windup response, culminating in a pronounced antinociceptive effect at the spinal level. It is noteworthy that this effect of the low dose of morphine plus (+)-HA966 had a limited time course, as would be expected from the duration of action of systemic morphine, and a predominant presynaptic site of action of low doses of morphine. [49]However, increasing the concentration of morphine coadministered with (+)-HA966 resulted in a dramatic reduction of spinal c-Fos expression at 3 h after carrageenin administration, this may reflect higher concentrations of morphine acting at both presynaptic and postsynaptic sites, [49]thus further reinforcing the interaction with postsynaptic NMDA receptor antagonism. Finally, it is worth bearing in mind a possible presynaptic effect of NMDA antagonism because 30% of the spinal NMDA receptors has been shown to be located on the primary afferent fibers and are thus potential autoreceptors. [57].

From our study it appears that concurrent micro-opioid receptor activation and NMDA receptor antagonism results in a strong reduction of nociceptive transmission at the level of the spinal cord, as shown by the strong reduction of carrageenin-evoked c-Fos expression. Such enhanced antinociceptive effects of coadministered morphine and NMDA antagonists have important implications for the reduction, or prevention of the long-term manifestations associated with sustained nociceptive processing, with a reduced side effect liability. [58].