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From the Departamento de Farmacologia, CCB, Universidade Federal de Santa Catarina, Brasil.
Address correspondence and reprint requests to Carlos Rogério Tonussi, Departamento de Farmacologia, CCB, Universidade Federal de Santa Catarina, Florianópolis, SC, 88040-900, Brasil. Address e-mail to tonussi{at}farmaco.ufsc.br.
Abstract
BACKGROUND: Morphine can inhibit inflammatory edema in experimental animals. The mechanisms and sites by which opioids exert this effect are still under debate. Since the spinal level is a site for modulation of the neurogenic component of inflammation, we investigated the effect of intrathecal (IT) administration of morphine, and the involvement of spinal nitric oxide (NO)/cyclic-guanosine monophosphate-GMP pathway in carrageenan (CG)-induced paw edema.
METHODS: Male Wistar rats received IT injections of drugs (20 µL) 30 min before paw stimulation with CG (150 µg). Edema was measured as paw volume increase (mL), and neutrophil migration was evaluated indirectly by myeloperoxidase (MPO) assay.
RESULTS: Morphine (37, 75, and 150 nmol) inhibited inflammatory edema, but had no effect on MPO activity. Coinjection with naloxone (64 nmol) reversed the effect of morphine. The corticosteroid synthesis inhibitor, aminoglutethimide (50 mg/kg, v.o.), administered 90 min before morphine injection did not modify its antiedematogenic effect. Low doses of the NO synthase inhibitor, N
-nitro-l-arginine (L-NNA; 10 and 30 pmol) increased, while higher doses (3 and 30 nmol) inhibited edema. The guanylate cyclase inhibitor 1H-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 21 and 42 nmol) increased, while the phosphodiesterase type 5 inhibitor sildenafil (0.15 and 1.5 nmol) inhibited paw edema. Coadministration of a subeffective dose of L-NNA (3 pmol) or ODQ (10 nmol) with morphine prevented its antiedematogenic effect, but sildenafil (0.15 nmol) rendered a subeffective dose of morphine effective (18 nmol). ODQ also prevented the antiedematogenic effect of the NO donor S-nitroso-N-acethyl-penicilamine.
CONCLUSION: These results support the idea that morphine can act on opioid receptors at the spinal level to produce antiedematogenic, and that the NO/cGMP pathway seems to be an important mediator in this effect.
In addition to the powerful analgesic activity of systemic morphine, this substance can inhibit inflammatory edema induced by several stimuli such as carrageenan (CG), yeast, and capsaicin, Freund's complete adjuvant, and even snake venom.1–7 Besides this antiedematogenic effect produced by opioid agonists, a proinflammatory effect produced by some opioid antagonists, such as naloxone and naltrexone, suggested that the inflammatory process may be under endogenous opioid inhibitory control.4,8,9 The mechanisms underlying this effect may be essentially peripheral, including the inhibition of vasoactive peptide released from nerve endings,2,10–12 inhibition of synthesis and release of cytokines from leukocytes,13 inhibition of local release of nerve growth factor,7 and inhibition of plasma extravazation.14 This idea is corroborated by the finding that a peripheral opioid receptor antagonist reversed the antiedematogenic effect of morphine administered systemically;15 however, experiments using local treatment with opioid agonists generally did not inhibit inflammatory edema.4,16,17 Whether the peripheral action of systemically-given opioids is sufficient to inhibit inflammatory edema, a central mechanism for morphine's antiedematogenic effect has also been suggested. This effect seemed to be indirectly mediated by endogenous corticosteroids.18 Nonetheless, Whiteside et al.19 argued that it may be the most important mechanism in the antiedematogenic effect of opioids.19
The spinal cord has been implicated in the modulation of peripheral edema in different inflammatory models.20–23 Several lines of evidence identify the spinal segmental modulation of dorsal root reflex (DRR) generation as mainly responsible for peripheral control of the neurogenic component of inflammation.21
DRR are antidromic action potentials generated in the central branches of nociceptors, mainly due to depolarization induced by 
-aminobutyric acid (GABA)ergic dorsal horn interneurons. These interneurons seems to be activated by glutamate release due to enhanced nociceptive input as observed in inflammatory lesions.21 Indeed, dorsal horn GABA was found to be upregulated by the increase of noxious inflow conveyed by C-fiber nociceptors from inflamed tissues.24 DRR, likely, significantly contribute to inflammatory edema by enhancing peripheral vasoactive neuropeptide21,25 and prostaglandin (PG) release.26
Opioid receptors in the spinal cord can inhibit neurotransmitter release from nociceptor central terminals,27,28 which may also affect DRR generation. In this context, the spinal cord could well be a potential site for the antiedematogenic action of opioids, and our aim in this work was to test this hypothesis.
METHODS
Animals
Experiments were performed on male Wistar rats (250–350 g) which were housed in temperature-controlled rooms (20–22°C) under a 12/12 h light/dark cycle with free access to water and food. All experiments were conducted according to the ethical guidelines of the International Association for the Study of Pain,29 and approved by the local ethical committee for animal research (CEUA-UFSC).
Inflammatory Model and Edema Measurement
We used the classical model of CG-induced rat paw edema.30 Briefly, CG was diluted in physiological saline at a concentration of 3 mg/mL. This solution was boiled for 1 to 2 s, and cooled to room temperature. The animals received 50 µL (150 µg of CG) of this solution in the hind left footpads. Inflammatory edema was measured before and hourly after CG injection by immersing the injected paw into a cuvette (10 mL) filled with a 2.5% lauryl sulfate water solution (v/v). The cuvette was fixed on the plate of an electronic balance so that the immersion of the paw (at the level of the tibio-tarsal joint) was accompanied by an increase of the weight displayed (Archimedes’ principle). Because the weight in grams is directly correlated to the volume of the immersed paw (cuvette solution density = 1 g/mL), the value displayed by the balance was assumed as equivalent to the paw volume. In some groups, CG injection and edema measurements were made in the forepaw.
Intrathecal Injections
Drug injections at the lumbar level of the spinal cord were performed according to the method previously described by Mestre et al.31 Briefly, the animals were anesthetized with halotane (2% in oxygen), and a 29-gauge needle was carefully inserted between the L5–6 vertebrae space until a flick of the rat's tail was observed. This reflex indicates the spinal channel has been reached. Intrathecal (IT) injections did not exceed 20 µL, and were done 30 min before CG administration in the hindpaw.
Drugs and Dilutions
The following drugs were used: multiple type CG (BDH chemicals, UK), morphine sulfate (Dimorf®, Cristália, Brazil), naloxone (Sigma, USA), aminoglutethimide (Orimeten®, Novartis, Brazil), S-nitroso-N-acetylpenicillamine (SNAP) (Sigma, USA), N-nitro-l-arginine (L-NNA) (Sigma, USA), ODQ (1H-oxadiazolo[4,3-a]quinoxalin-1-one) (Tocris, USA), and sildenafil citrate (kindly donated by Pfizer Global Research, UK). All drugs were diluted in saline. Aminoglutethimide was administered orally, but otherwise all drugs were given IT. CG dilution was described above.
Myeloperoxidase Assay
Animals were killed just after the experimental session (4 h after CG injection) and samples of inflamed tissue (or control noninflamed) were removed and frozen. These samples were homogenized for 10 s in a sodium phosphate buffer solution (50 mM; pH 5.4) of hexadecyltrimethylammonium bromide (HTAB; 0.5% w/v), and sonicated for 10 s at 4°C. The resulting homogenate was centrifuged (15 min, 10,000 rpm, 4°C), and a 25 µL aliquot of the supernatant was incubated for 5 min with tetramethylbenzidine (1.6 mM) and hydrogen peroxide (H2O2; 0.5 mM) in 80 mM sodium phosphate buffer (pH 5.4) at 37°C. The reaction was stopped by adding 100 µL of sulfuric acid (H2SO4; 1 M). Light absorbance readings were made at 450 nm. Another 200 µL aliquot of the previous supernatant was diluted in 800 µL of buffered HTAB solution for absorbance readings at 280 nm. The total protein content was estimated assuming that each unit of absorbance at 280 nm was equivalent to 1 mg of protein. Myeloperoxidase (MPO) activity was expressed as the optical density at 450 nm per mg of protein.
Data Presentation and Statistical Analysis
Data were presented as the relative paw volume increase from normal paw volumes (mean ± se mean). In some figures, the results were expressed as the percentage of the control values for each hour. Statistical analyses were made with Graphpad Prism 3.0 (graphpad.com). Multiple comparisons were made using one-way ANOVA for repeated measures, and when a significance level of at least P < 0.05 was detected, analysis was followed by the Tukey's post hoc test. Unless indicated otherwise, the significance levels are always related to the respective control group.
RESULTS
Inhibition of Paw Edema by IT Morphine
Morphine injected IT (18, 37, 75, and 150 nmol) produced a dose-related antiedematogenic effect on CG-induced inflammatory paw edema (Fig. 1). The lowest dose produced a significant inhibitory effect in the first hour after CG injection, but not when the whole curve was compared with the saline-treated control (ANOVA for repeated measures). Thus, this dose was considered subeffective for further experiments. The maximal antiedematogenic effect was obtained with the 150 nmol dose (P < 0.01), but it also produced significant behavioral reactions such as agitation, irritability, and Straub's tail, indicating that supraspinally driven stereotyped behavior could be reached by this dose. Therefore, the dose used for the subsequent experiments was 37 nmol. This IT dose was approximately 14-fold less than an ineffective s.c. dose (1 mg/Kg) evaluated in a systemic dose-response experiment (Fig. 1, inset here). The specificity of the IT opioid effect was confirmed by coinjecting naloxone (64 nmol) with morphine (37 nmol), which did not produce the antiedematogenic effect as observed for morphine alone (Fig. 2). As morphine is thought to modulate the hypotalamus–pituitary–adrenal axis, potentially interfering with the inflammatory response,32,33 the aromatase-inhibitor aminoglutethimide (Fig. 2) was given orally 2 h before CG injection (50 mg/kg). This treatment was intended to circumvent the possible increase of plasma corticosteroid levels induced by morphine, thus avoiding endocrine interference in inflammatory edema.34 In this situation, the antiedematogenic effect of morphine (37 nmol) was not different from that produced with aminoglutethimide pretreatment. Neutrophil migration to the inflamed paw after spinal treatment with morphine was evaluated indirectly by MPO assay 4 h after CG injection. The opioid agonist did not modify this activity, even at the highest dose (saline = 0.51 ± 0.1 OD/mg; morphine 18 nmol = 0.51 ± 0.1 OD/mg; 37 nmol = 0.62 ± 0.1 OD/mg; 150 nmol = 0.77 ± 0.09 OD/mg). The IT injection of the effective dose of morphine (37 nmol) also did not affect forepaw inflammation induced by CG (saline 4th h = 0.28 ± 0.03 mL SEM, morphine 4th h = 0.29 ± 0.02 mL SEM).
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Involvement of Nitric Oxide–Cyclic Guanosine Monophosphate Pathway
Pretreatment with L-NNA (3, 10, and 30 pmol, 3 and 30 nmol), a nonselective inhibitor of nitric oxide (NO) synthase, produced dose-related opposing effects. Figure 3A shows the significant increase in edema that was observed with doses in pmol range (10 pmol, P < 0.05; 30 pmol, P < 0.001). In contrast, Figure 3B shows the higher doses (3 and 30 nmol), which caused edema inhibition (P < 0.001). The soluble GC is an usual target for NO in most cellular systems, and its potential role in the spinal modulation of paw edema was verified by injecting the GC inhibitor ODQ (10, 21, and 42 nmol), which increased paw edema with the two higher doses (P < 0.001) (Fig. 4). Further experiments showed that the IT injection of the cyclic guanosine monophosphate (cGMP) hydrolysis inhibitor sildenafil (0.15 and 1.5 nmol) significantly inhibited edema (P < 0.01) (Fig. 5A).
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Next, we evaluated the possible involvement of the NO/cGMP pathway in morphine's antiedematogenic effect. The subeffective dose of sildenafil (0.15 nmol) coinjected with the subeffective dose of morphine (18 nmol) enhanced the antiedematogenic effect of morphine alone (P < 0.001) (Fig. 5B), while coadministration of the subeffective dose of L-NNA (3 pmol) with morphine (37 nmol) prevented the antiedematogenic effect (Fig. 6A). This antiedematogenic effect produced by morphine was also inhibited by coinjection with the subeffective dose of ODQ (10 nmol). In addition, this dose of ODQ also inhibited the antiedematogenic effect produced by the NO donor SNAP (Fig. 6B).
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DISCUSSION
In the foregoing sections, we have reported the antiedematogenic effect of the opioid agonist morphine injected at the lumbar level of the spinal channel in a model of CG-induced hindpaw edema. This effect was naloxone-reversible and can be attributed to a central action of the drug, since a 14-fold higher dose given subcutaneously was not able to inhibit paw edema, which means that the drug could not be diffusing out from the spinal channel to act in the peripheral tissue. However, it also could not be attributed to an action at higher sites in the central nervous system, since the effective IT dose did not affect edema induced by CG injection in the forepaws. Furthermore, IT injection of morphine produced an antiedematogenic effect in animals pretreated with aminoglutethimide; therefore, it is unlikely that an increase in plasma corticosteroid level is involved in this effect. Thus, the effect of spinally injected morphine on peripheral edema presupposes a segmental action at the lumbar level, probably on afferent fibers that directly communicate with the peripherally affected tissue within the spinal cord. Our working hypothesis was that morphine inhibits the generation of DRR, thus inhibiting paw edema. This idea is well supported by the presence of opioid receptors in the nociceptor central terminals, which can affect nociceptive terminal excitation to the point of inhibiting nociceptor neurotransmitter release,27,28 and consequent GABA interneuron activation. Indeed, morphine was also found to inhibit GABA release from dorsal horn slices.35 These effects could have a direct inhibitory role on DRR generation, as well.
Notwithstanding, it remains to be determined whether this spinal effect explains the antiedematogenic effect observed after subcutaneous morphine injection. The systemic dose range that was antiedematogenic in the present study was also shown to effectively inhibit tonic nociception, and the simultaneous spinal glutamate release induced by formalin.36 In addition, the maximal cerebrospinal fluid concentration achieved after the same systemic doses37 was similar to that found to inhibit glutamate and GABA release in spinal cord slices after dorsal root electrical stimulation.35 However, a 10-fold higher dose was needed to inhibit neuropeptide release in the spinal cord due to tonic mechanical paw pressure.24 These findings, although suggesting that morphine asymmetrically inhibits amino acid and neuropeptide release from nociceptors, also support the idea that spinal glutamate and GABA release inhibition may explain morphine's antiedematogenic effect after systemic injection.
The antiedematogenic effect induced by IT injection of morphine was not observed on the parallel inhibition of neutrophil migration. This observation reinforces the importance of nociceptor activity for edema formation, and suggests that neutrophil products must interact, at least in part, with nociceptor products to produce edema. Actually, although vasoactive peptides are thought to be released most abundantly by nociceptors at the peripheral level, PG E2, which unequivocally contributes to edema formation, can also be released from peripheral terminals of polymodal nociceptors.26 The importance of PG over neutrophil migration to edema formation may be determined by the remarkable effect of the cyclooxygenase inhibitor indomethacin. This nonsteroidal antiinflammatory drug (NSAID) produces a potent antiedematogenic effect despite its enhancing effect on leukocyte infiltration.38 We speculate that the reduction of nociceptor antidromic activity during an inflammatory process, such as the CG model, could also reduce the overall release of PGE2 in addition to the reduction of neuropeptide release, thus resulting in the antiedematogenic effect we observed.
The involvement of the NO/cGMP pathway in this spinal action of morphine was suggested by the blockade of morphine's antiedematogenic effect when the opioid was coinjected with the NO synthase inhibitor L-NNA, or with the GC inhibitor ODQ. Furthermore, the cGMP phosphodiesterase inhibitor sildenafil enhanced a subeffective dose of morphine. This molecular pathway has been implicated in the inhibitory action of opiates on nociceptors, as evidenced by different nociceptive approaches.39–42 The coincident mechanism of morphine on nociception and spinal modulation of peripheral edema reinforces the notion that morphine is acting on nociceptors to inhibit increasing inflammation.
Besides the opioid effect, L-NNA, ODQ, and sildenafil given alone also affected paw edema, suggesting that there is a continuing formation of NO and cGMP in the spinal cord during peripheral inflammation. Low doses of L-NNA produced an increase, while higher doses produced a decrease of peripheral edema. This dual effect may be correlated with similar findings in nociceptive studies. Indeed, there are reports showing that NO may exert opposing effects on neuronal activity,43 and IT administration of low doses of NO donor-induced antinociception while high doses increased the nociceptive response.44 The idea that low doses of L-NNA, and ODQ, increased edema by enhancing the DRR is supported by the findings of Hoheisel et al.45 who have shown that, at the spinal level and under normal conditions, a lack of cGMP leads to an increase in background activity of nociceptive dorsal horn neurones which was comparable with that of a lack of NO.45 Furthermore, the spinal administration of sildenafil decreased dorsal horn activity under peripheral inflammatory conditions,46 which is compatible with our hypothesis that spinally administered sildenafil reduced peripheral edema by decreasing DRR generation.
Spinal injection of opiates is a common technique to obtain segmental analgesia during some surgical procedures. The present results, in addition to our previous reports with cyclooxygenase inhibition and serotonergic modulation,22,23 support the notion that this technique may also be improved to obtain a better antiinflammatory effect with potentially fewer undesirable reactions than those commonly associated with NSAIDs. We also suggest that sildenafil may be an useful adjuvant for the opioid effect.
Footnotes
Accepted for publication November 13, 2007.
Supported by the Brazillian funding agencies CAPES and FAPESC-CNPq/PRONEX.
REFERENCES
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