The spinal cord is the center that controls orthodromic (nociception) as well as antidromic (peripheral vasodilation) traffic in the nociceptive pathway.1 Therefore, the spinal cord could be considered a potential site for the modulation of inflammation and not just a simple transmission line from the periphery to the brain. There have been significant advances elucidating the cellular and molecular processes that mediate neuronal hyperexcitability at the periphery and in the spinal cord during an inflammatory insult.2–4
A growing body of experimental evidence5–9 suggests that the spinal cord may be an important therapeutic target for the treatment of peripheral inflammatory diseases as an alternative to systemic treatments similar to strategies already in practice for the control of some types of chronic pain.10 This evidence supports, in theory, the anti-inflammatory use of anesthetics. In line with this idea, a previous report by our laboratory7 showed that intrathecal morphine could prevent peripheral inflammatory edema by acting on opioid receptors and through the activation of the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway. Because cGMP can ultimately lead to neuron hyperpolarization by stimulating potassium channel opening, there is a reasonable possibility that the observed morphine antiedematogenic effect could be the result of the opening of potassium channels, in agreement with reported findings in nociceptive studies.11–14 The present availability of drugs that open potassium channels for clinical use offers the possibility of strengthening the concept of administration of anti-inflammatory anesthesia. Thus, the aim of the present study was to investigate the main types of K+ channels involved in the antiedematogenic effect of intrathecally administered morphine and its enhancement by a K+ channel opener in an effort to provide a clearer pharmacologic identity for this potential therapeutic strategy.
The male Wistar rats (weighing 250–350 g) used in this study were housed in a temperature-controlled (20°C ± 2°C) and light-controlled (12-hour light/dark cycle) room, with free access to water and food. All experimental procedures were conducted according to the ethical guidelines of the International Association for the Study of Pain15 and approved by the local ethics committee for animal research (CEUA-UFSC).
Inflammatory Model and Edema Measurement
We used the classical model of carrageenan (CG)-induced rat paw edema.16 Briefly, CG was diluted in physiological saline at a concentration of 3 mg/mL. This solution was boiled for 1 to 2 seconds and cooled to room temperature. The animals received 50 µL (150 µg of CG) of this solution in the hind right footpads. Inflammatory edema was measured before and at 4 timepoints after (hourly) 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 tibiotarsal joint) was accompanied by an increase in 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 to be equivalent to the paw volume.
Intrathecal drug injections were performed at the lumbar level of the spinal cord according to the method previously described by Mestre et al.17 Briefly, the animals were anesthetized with isoflurane (2% in oxygen), and a 29-gauge needle was carefully inserted between the L5 to L6 vertebrae space until a flick of the rat’s tail was observed. This reflex indicates that the spinal channel has been reached. All drugs were given intrathecally in a volume of 20 µL 30 minutes before CG administration in the hindpaw.
Drugs and Dilutions
The following substances were used: κ/λ type CG (BDH chemicals, Poole, Dorset, UK), morphine sulfate (Dimorf®, Cristália, Brazil), 4-aminopyridine (4-AP), glibenclamide (GLI), and dequalinium, dichloride (DQL) (Tocris, Bristol, UK), and nicorandil (NICO; Sigma-Aldrich, St. Louis, MO). All drugs were diluted in phosphate-buffered saline (PBS).
Evans Blue Dye Leakage
Plasma extravasation was measured after the administration of 25 mg/kg Evans blue dye into the labialis mandibularis vein located in the gingival papillae region immediately below the pair of mandibular incisive teeth of anesthetized rats (isoflurane, 2% in oxygen),18 which is a marker of protein leakage.19 The dye binds to albumins, and during inflammation, it leaks from the vessels into the extracellular space. The intensity of the blue staining of the CG-inflamed hindpaw is proportional to the intensity of inflammation. The dye was injected 30 minutes before CG injection into the paw pad. After 4 hours of CG injection, the animals were deeply anesthetized with 15% chloral hydrate (0.5 g/kg, i.p.), followed by decapitation. The inflamed paw was amputated, and the tissue was minced before being incubated with formamide/water (1:1, w/v) for 48 hours at 37°C. The optical density of the supernatants was measured at 630 nm in a spectrophotometer (model U-2001, Hitachi, Tokyo, Japan). The concentration of dye was determined from a standard curve of Evans blue in formamide, as previously described.19 Changes in vascular permeability were expressed as the difference in the amount of dye leakage between the morphine and PBS groups.
The activity of the enzyme myeloperoxidase (MPO) was evaluated using the methodology of Mullane et al.20 The animals were deeply anesthetized with 15% chloral hydrate (0.5 g/kg, i.p.), followed by decapitation 4 hours after CG injection. The right paw was amputated, and these samples were evaluated. MPO activity was expressed as the optical density at 650 nm/mg of tissue.
Four hours after CG injection, the rats were deeply anesthetized with 15% chloral hydrate (0.5 g/kg, i.p.) and decapitated. The right hindpaw was removed and postfixed in paraformaldehyde (4%; pH 7.4) for 48 hours. The skin of the paw was then removed and left again for >12 hours in paraformaldehyde 4%, embedded in paraffin, cut into 5-µm thick frontal sections, and stained with hematoxylin–eosin. The acute inflammatory reaction was graded based on the density of inflammatory infiltrates and vascular congestion. Two independent observers blind to the treatment quantified the reaction by assigning an increasing scale of 0 to 3, adapted from the study by Boettger et al.21
Data Presentation and Statistical Analysis
Statistical analysis was performed using the GraphPad Prism 5.0® software (San Diego, CA). Inflammatory edema data are presented as the relative paw volume increase compared with the normal paw volume (mean ± SEM). Multiple comparisons of time-course curves were made using 2-way analysis of variance, followed by a Bonferroni posthoc test when a P level < 0.05 was detected. Plasma leakage, MPO activity, and histologic score data are presented as mean ± SEM, and multiple comparisons were performed using 1-way analysis of variance for simple means, followed by a Tukey post hoc test when a P level < 0.05 was detected.
Effect of Intrathecal Morphine Injection
According to our previously obtained results, 37 nmol of morphine injected into the spinal channel (tested doses: 09, 18, and 37 nmol) 30 minutes before the inflammatory stimulus produced an antiedematogenic effect at all timepoints (P < 0.001, Fig. 1). Higher doses were not used in the present study, because we noted previously that they can produce reactions such as agitation, irritability, and Straub tail.7 Therefore, the dose used for the subsequent experiments was 37 nmol. At this dose, morphine also reduced plasma leakage, as detected by the Evans blue colorimetric technique (Fig. 2). However, the strong MPO reaction observed 4 hours after the inflammatory stimulus was not influenced by the opioid treatment (PBS after 4 hours = 0.1 ± 0.003, morphine after 4 hours = 0.09 ± 0.003; Fig. 3). Furthermore, histologic examination (Fig. 4) showed an intense inflammatory cell infiltrate (Figs. 4B and 5A), which was also not modified in the animals treated with morphine (Figs. 4C and 5A). On the other hand, the intense vascular congestion produced by CG (P < 0.001) was decreased in the morphine-treated group (P < 0.05; Fig. 5B).
Involvement of Potassium Channels in Morphine Effect
To identify subthreshold doses of the potassium channel blockers in the spinal cord and doses that would not interfere with the motor control of the animals, a range of concentrations was tested. These concentrations were based on those reported for the substance in neural tissue. The following figures do not show the complete series of the doses tested, but only the subthreshold doses and doses that were found to be effective on paw edema without causing motor impairment. The voltage-gated K+ channel blocker, 4-AP (10, 20 and 60 nmol), decreased paw edema only at the higher dose tested in all timepoints (P < 0.05; Fig. 6A). On the basis of this experiment, the dose that was chosen for the subsequent experiments was 10 nmol. Conversely, GLI, a KATP channel blocker (5, 20, and 40 nmol) enhanced edema at the higher dose in all timepoints (P < 0.05; Fig. 6B). The dose of 5 nmol was chosen for subsequent experiments. DQL, a low conductance Ca2+ activated K+ channel blocker (0.01 nmol, 0.1 nmol, and 1 nmol), also enhanced paw edema at 0.1nmol and 1 nmol at most timepoints (P < 0.01; Fig. 6C). The dose of 0.01 nmol was used in the subsequent experiments. Morphine (37 nmol) was coinjected with 4-AP (10 nmol), GLI (5 nmol), or DQL (0.01 nmol) 30 minutes before CG. All K+ channel blockers completely reversed morphine’s antiedematogenic effect (P < 0.001; Fig. 7).
In line with the aforementioned observations, NICO, a KATP channel opener (0.03 nmol, 0.3 nmol, and 3 nmol), decreased paw edema at the 2 higher doses in all timepoints (P < 0.05; Fig 8A). We chose the lower and subeffective dose to coinject with morphine (18 nmol) to demonstrate an enhancing effect. The K+ channel opener enhanced the morphine antiedematogenic effect at all timepoints (P < 0.01; Fig 8B).
The main finding in this study is that intrathecally injected morphine produced a peripheral antiedematogenic effect, probably due to potassium channel activation. This was supported by the antagonistic effect of 3 potassium channel blockers 4-AP, GLI, and DQL. Furthermore, the KATP activator NICO either inhibited edema by itself or enhanced the antiedematogenic effect of morphine. The present findings are an extension of our previous study,7 which indicated that this spinal effect of morphine seems to be mediated by the activation of the NO/cGMP pathway. The antiedematogenic effect of morphine therefore seems to be associated with spinal mechanisms similar to those involved in its antinociceptive effect.11 Conversely, such similarity may also support the possibility that spinal mechanisms involved in nociception are also involved in the peripheral control of inflammation in such a way that antinociceptive procedures should also diminish peripheral edema. Indeed, we have observed that some drugs used as clinical analgesics, such as indomethacin and sumatriptan, can also exert a peripheral antiedematogenic effect when administered in the spinal cord of rats.5,6 In contrast, spinal injection of prostaglandin E2 reportedly enhanced peripheral edema.5 Similarly, spinal potassium channel blockade, which antagonizes the antinociceptive effect of intrathecal morphine,22–24 enhanced peripheral edema and antagonized the morphine-induced antiedematogenic effect. However, the present study showed some differences compared with the sensitivity observed for the K+ channel blockade on the antiedematogenic and antinociceptive effects of morphine reported in the literature.
Electrophysiologic studies have demonstrated that opioid agonists open K+ channels in neurons through the activation of G proteins.25,26 Indeed, the antinociceptive effect of intrathecally injected morphine was reportedly antagonized by GLI27,22–24 and enhanced by the epidural administration of the KATP channel openers NICO and levcromakalim.28 Similarly, NICO enhanced the antiedematogenic effect of morphine, thus supporting a role for KATP channels also in this effect.
Some studies have evaluated the possible role of calcium-activated K+ channels (KCa) in morphine-induced antinociception. The slow-conductance KCa channel blocker apamin did not modify the antinociceptive effect of intrathecal morphine in the tail flick test.22 Although the number of studies available is still small, all of them suggest that the opening of KCa channels does not play an important role in morphine-induced antinociception, which is to be expected, because morphine also decreases the intracellular Ca2+ concentration. Furthermore, few studies have evaluated the role of voltage-dependent K+ channels (Kv) in the antinociceptive effect of morphine. The spinal administration of 4-AP and tetraethylammonium (TEA) did not modify the antinociception induced by morphine when given systemically or intrathecally in studies using the tail flick test.29,22 Moreover, both Kv blockers (administered in the footpad) were unable to antagonize the peripheral antinociception induced by morphine in the CG test.14 Therefore, it seems that 4-AP and TEA-sensitive K+ channels are not involved in morphine-induced antinociception. However, Fernandes et al.30 in their study showed that NO inhibition of CG-induced mouse paw edema involves Kv channels, because TEA completely blocked the NO donor’s inhibitory effect in this model. In the study by Fernandes et al., the drug was peripherally administered; however, one cannot know how much of its effect was a result of a central action, because it may have permeated the spinal cord. Comparison of these reported data with our present findings suggests that the antiedematogenic effect of intrathecally administered morphine shares some mechanisms with its antinociceptive effect. Our working hypothesis to explain the peripheral antiedematogenic effect of spinally delivered morphine7 is the inhibition of antidromic potentials generated in the primary afferents —the dorsal root reflex, or DRR—because of the nociceptive input (see the study by Willis, for a review).1 The strong DRR dependence on nociceptive input in the spinal cord may explain why the antiedematogenic effect of morphine involves K+ channels, which are also associated with the antinociceptive effect. However, the other types of K+ channels that seem to be involved in the antiedematogenic effect of morphine may be specifically involved either in the generation or propagation of DRR, probably regulating antidromic action potential propagation.31 A possible failure in the predictive value of this hypothesis was the antiedematogenic effect produced by the higher dose of 4-AP. However, in relation to the DRR, depolarization of the primary afferent may cause 2 opposing effects. It is possible that the blockade of some types of potassium channels only causes an increase in the antidromic firing rate of the fiber; however, in the case of the higher dose of 4-AP, a sustained depolarization could prevent the generation of new action potentials.1 Another concern for the present hypothesis was that GLI and DQL acted as inverse agonists in this system, exhibiting the opposite effect of morphine, when given in higher doses. Thus, there is a possibility that the antagonism produced by these inhibitors on the effect of morphine could be merely a physiologic subtraction of 2 opposing effects. We tried to circumvent this possibility by using a subthreshold dose of each inhibitor, implicating that, at least at the level of the observed effect, the inhibitors should not be having the opposite effect of morphine but hindering its mechanism of action.
Besides the effect of morphine on inflammatory edema, it is also important to examine the effect on leukocyte migration. In this study, we did not observe any change in neutrophil migration, which is the prevalent leukocyte in this acute inflammation model. Conversely, it is interesting that edema and plasma leakage were reduced, even in the presence of an unchanged neutrophil migration. The histologic analysis suggested that this antiedematogenic effect was likely due to the reduced opening of the capillary bed, which is consistent with the hypothesis that intrathecally administered morphine reduces spinal antidromic discharges with the consequent decrease of peripheral vasodilating neuropeptide release. However, even in a scenario of unchanged neutrophil migration, it is conceivable that the possible inhibition of neuropeptide release may also reduce the activity of the migrated neutrophils,32,33 thus also releasing less leukocyte edematogenic mediators. This reasoning is consistent with the observation by Boettger et al.8 that the intrathecally administered opiate can reduce the late mononuclear migration in a model of antigen-induced arthritis, considering that this late mononuclear migration can also be influenced by neutrophil activity in the early mononuclear migration.34
In conclusion, the results of the present study enhance our understanding regarding the potential peripheral anti-inflammatory effect of intrathecally administered morphine. It is conceivable that morphine stimulates the NO/guanylate cyclase/cGMP pathway7 that eventually leads to K+ channel opening. Several types of K+ channels seem to be involved in the mediation of the opiate effect, and this may help future pharmacologic clinical approaches for controlling inflammation by acting in the central nervous system. For example, there are several clinically available drugs that are K+ channel openers, such as minoxidil and NICO, which, if considered safe for intrathecal delivery, could be given with opiates in a combined spinal therapy.
Name: Vanessa R. S. Foletto, DVetMed, MSc.
Contribution: This author helped conduct the study, analyze the data, and prepare the manuscript.
Name: Maria A. Martins, DVetMed, ScD.
Contribution: This author helped conduct the study.
Name: Carlos R. Tonussi, ScD.
Contribution: This author helped advise and prepare the manuscript.
This manuscript was handled by: Quinn Hogan, MD.
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© 2013 International Anesthesia Research Society
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