Anesthesiology 2003; 99:449–54 © 2003 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Interaction between the Spinal and Opioid Systems in a Rat Model of Neuropathic Pain Dorien H. Vrinten, M.D., Ph.D.,* Willem Hendrik Gispen, Ph.D.,† Cor J. Kalkman, M.D., Ph.D.,‡ Roger A. H. Adan, Ph.D.§

Background: The authors recently demonstrated that admin- substance P, calcitonin gene–related , cholecys- istration of the melanocortin-4 SHU9119 tokinin, and Y.6,7 We recently demon- decreased neuropathic pain symptoms in rats with a sciatic strated that such plasticity also occurs in the spinal chronic constriction injury. The authors hypothesised that there is a balance between tonic pronociceptive effects of the melanocortin system, as demonstrated by an up-regula- spinal melanocortin system and tonic antinociceptive effects of tion of melanocortin-4 (MC4) receptors in the spinal cord the spinal opioid system. Therefore, they investigated a possi- dorsal horn in a rat model for neuropathic pain, the Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/99/2/449/407606/0000542-200308000-00028.pdf by guest on 25 September 2021 ble interaction between these two systems and tested whether chronic constriction injury (CCI).8,9 Because melano- opioid effectiveness could be increased through modulation of cortins have been shown to induce hyperalgesia,10,11 the spinal melanocortin system activity. Methods: In chronic constriction injury rats, melanocortin this increase in spinal MC4 receptors might contribute to and ligands were administered through a lum- the increased sensitivity in neuropathic pain, through bar spinal catheter, and their effects on mechanical allodynia activation by the endogenous melanocortin receptor ag- were assessed by von Frey probing. onist ␣-melanocyte–stimulating hormone (␣-MSH), ␮ Results: Naloxone (10–100 g) dose-dependently increased which is also known to be present in the dorsal horn.12 ,(allodynia (percent of maximum possible effect of ؊67 ؎ 9% which is in agreement with a tonic antinociceptive effect of the An interaction between the central melanocortin and opioid system. SHU9119 decreased allodynia (percent of maxi- opioid system has been described previously. Melanocort- mum possible effect of 60 ؎ 13%), and this effect could be ins can reduce morphine-induced analgesia13,14 inhibit the blocked by a low dose of naloxone (0.1 ␮g), which by itself had development of opioid tolerance,13,15 counteract opioid ␮ no effect on withdrawal thresholds. Morphine (1–10 g) dose- addiction,16 and induce morphine withdrawal–like symp- dependently decreased allodynia (percent of maximum possi- 17 -ble effect of 73 ؎ 14% with the highest dose tested). When toms. However, it is not known whether such a func ␮g SHU9119 (percent of maximum possible effect of 47 ؎ tional antagonism between the melanocortin and opioid 0.5 14%) was given 15 min before morphine, there was an additive system also exists at the spinal level. antiallodynic effect of both compounds. It is generally accepted that in neuropathic pain, opi- Conclusions: Together, these data confirm that there is an oids are less effective. To obtain adequate pain relief, interaction between the spinal melanocortin and opioid sys- 18 tems and that combined treatment with melanocortin-4 recep- high doses of opioids are needed ; thus, their use is tor antagonists and opioids might possibly contribute to the often limited by unwanted side effects. Possible expla- treatment of neuropathic pain. nations for this right shift in the dose–response curve of opioids include a loss of opioid receptors on primary NEUROPATHIC pain is characterized by allodynia (pain afferent terminals after axotomy19,20 and an increased due to a normally nonpainful stimulus) and hyperalgesia activity of endogenous antiopioids such as dynorphin21 (increased pain in response to a normally painful stimu- or cholecystokinin22,23 in the spinal cord. Considering lus). It has become clear that numerous pathophysio- the functional antagonism between the melanocortin logic changes in response to neuronal or axonal damage and opioid system, it is possible that the aforementioned contribute to neuropathic pain.1,2 Not only do neuro- changes in the spinal melanocortin system in neuro- atomical changes occur, such as loss of axotomized pri- pathic pain9 might also contribute to the reduced anal- mary afferent fibers,3 apoptotic cell loss,4 and reorgani- gesic effect of morphine in this condition. zation of dorsal horn circuitry,5 but also the expression We therefore investigated a possible interaction be- of different and their receptors in sen- tween melanocortin and opioid systems at the spinal sory neurons is altered. Such plasticity has been de- level by administering different combinations of the opi- scribed in a number of messenger systems, including oid receptor antagonist naloxone and morphine, and the MC4 receptor antagonist SHU9119 and agonist MTII. We hypothesized that through modulation of the * Resident in Anesthesiology, Departments of Medical Pharmacology and An- esthesiology, † Professor of Neurosciences, § Professor of Molecular Pharmacol- activity of the spinal melanocortin system, it is possible ogy, Department of Medical Pharmacology, ‡ Professor of Anesthesiology, De- to increase the effectiveness of opioids in neuropathic partment of Anesthesiology. pain. Received from the Department of Medical Pharmacology and Anesthesiology, Rudolf Magnus Institute for Neurosciences, University Medical Center Utrecht, Utrecht, The Netherlands. Submitted for publication September 20, 2002. Accepted for publication March 19, 2003. Support was provided solely from institutional and/or departmental sources. Materials and Methods Address reprint requests to Dr. Vrinten: Rudolf Magnus Institute for Neuro- sciences, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Animals Utrecht, The Netherlands. Address electronic mail to: [email protected]. Individual article reprints may be purchased through the Journal Web site, Thirty male Wistar rats weighing 250–300 g at the start www.anesthesiology.org. of the study were used. Animals were housed in groups

Anesthesiology, V 99, No 2, Aug 2003 449 450 VRINTEN ET AL. of two or three in plastic cages on sawdust bedding. first drug was given 15 min before the second drug, They were kept at a 12/12-h light/dark cycle, with food because we previously demonstrated that the peak ef- and water available ad libitum. All testing procedures in fect of SHU9119 is at 15 min after injection.9 this study were performed according to the Ethical On a typical testing day, all 27 animals were randomly Guidelines of the International Association for the Study divided into three groups (n ϭ 8 or 9 each, except for of Pain24 and approved of by the Ethics Committee on simultaneous administration of MTII and morphine, Animal Experiments of Utrecht University (Utrecht, The where n ϭ 4). Each group randomly received one treat- Netherlands). ment, with the experimenter blinded to the allocation. Thereafter, animals were given at least 2 days of rest to Surgery minimize any possibility of drug interactions or develop- Animals were anesthetized with a single intramuscular ment of tolerance.9 On the next testing day, all animals Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/99/2/449/407606/0000542-200308000-00028.pdf by guest on 25 September 2021 injection of Hypnorm (Janssen Pharmaceutical Ltd., were again randomly divided into three groups, and Grove, Oxford) containing 0.315 mg/ml fentanyl citrate three other treatments were given. This design was con- and 10 mg/ml fluanisone, at a dose of 1 ml/kg body tinued until all treatments had been given to one group weight. of animals. Thus, in total, 18 groups were tested, and Four ligatures were placed around the right sciatic therefore, each animal was used multiple times on con- nerve as previously described by Bennett and Xie,8 and secutive testing days. the incision was closed with silk sutures. Two weeks after the CCI lesion, rats were anesthetized with a mix- Mechanical Stimulation Test ture of oxygen–nitrous oxide (1:2) and 2.5% halothane. Foot withdrawal threshold in response to a mechanical A lumbar spinal catheter was placed as described by stimulus was determined using a series of von Frey Storkson et al.25 and subcutaneously tunneled to the filaments (Stoelting, Wood Dale, IL), ranging from 1.08 neck region. After recovery from anesthesia and also at to 21.09 g. Animals were placed in a plastic cage with a the end of all experiments, proper placement of the metal mesh floor, allowing them to move freely. They catheters was checked by injecting 15 ␮l of lidocaine, were allowed to acclimatize to this environment before 2%, which gives an immediate and short-lasting motor the experiment. The filaments were presented to the paralysis of the hind limbs on intraspinal injection. Be- midplantar surface as described by Chaplan et al.,27 and cause of incorrect placement of the spinal catheter, 3 the smallest filament eliciting a foot withdrawal response rats were excluded from this study, thus leaving a total of was considered the threshold stimulus. 27 animals. They were allowed to recover before testing Baseline values were determined, and measurements was initiated. were repeated 60 min after drug or vehicle administra- tion (in case of a pretreatment, measurements were Drugs taken 60 min after injection of the second drug). For in vivo administration, MTII (melanotan II or cy- clo-[Nle4, Asp5, D-Phe7, Lys10]␣-MSH-(4-10)), SHU9119 Data Analysis (cyclo-[Nle4, Asp5, D-Nal(2)7, Lys10]␣-MSH-(4-10)), nalox- All data are expressed as mean Ϯ SEM for visualization one (naloxone-hydrochloride), and morphine (mor- purposes only. Effects of treatments resulting in an in- phine-hydrochloride) were used. MTII was purchased crease or decrease in mechanical allodynia are plotted from Bachem Feinchemicalien (Buberdorf, Switzerland); on a negative or positive axis, respectively. For the SHU9119 was synthesized using Fmoc solid-phase syn- mechanical stimulation test, the effect of different drug thesis as reported elsewhere.26 Naloxone and morphine treatments was quantified as the percentage of maxi- were obtained from the Utrecht University Medical Cen- mum possible effect (%MPE), using the following for- ter Pharmacy (Utrecht, The Netherlands). Drugs were mula: %MPE ϭ 100 ϫ (postdrug value Ϫ baseline value)/ dissolved in 5 ␮l saline and injected through the spinal (cutoff value Ϫ baseline value). catheter by means of a 25-␮l Hamilton syringe, followed Nonparametric tests were performed to analyze the by a saline flush (12 ␮l). data: Overall group differences were analyzed by a Kruskal-Wallis test followed by a two-tailed Mann–Whit- Treatment Paradigms ney U test to analyze differences between groups. A The following 18 different treatments were given: probability level of less than 5% was considered signifi- saline, naloxone (0.1, 10, 30, and 100 ␮g), SHU9119 cant. To assess the statistical significance of the combi- (0.5, 1, and 1.5 ␮g), morphine (1, 3, 10, and 30 ␮g), MTII nation of SHU9119 and morphine, dose–response curves (0.5 ␮g), or the following combinations: SHU9119 were generated for each compound. The composite line (1.5 ␮g) with naloxone (0.1 ␮g) pretreatment, morphine of additivity for the combination was determined and (1, 3, or 10 ␮g) with SHU9119 (0.5 ␮g) pretreatment, or compared to the experimental data using analysis of MTII (1.5 ␮g) and morphine (100 ␮g) simultaneously. In variance. These results are also represented graphically experiments in which a “pretreatment” was given, the by isobolographic analysis.

Anesthesiology, V 99, No 2, Aug 2003 SPINAL MELANOCORTIN AND OPIOID SYSTEM INTERACTION 451 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/99/2/449/407606/0000542-200308000-00028.pdf by guest on 25 September 2021 Fig. 1. Effects of naloxone on mechanical allodynia. Effects of Fig. 2. Modification of the antiallodynic effect of SHU9119 by ␮ spinal administration of naloxone on mechanical allodynia, as naloxone. Effect of spinal administration of 1.5 g SHU9119 ␮ ␮ measured by withdrawal thresholds to von Frey stimulation. (SHU 1.5), 1 g naloxone alone (Nal 1), or 1 g naloxone 15 min Doses ranged from 0 (saline) to 100 ␮g naloxone. Data are before SHU9119 (Nal 1, SHU 1.5) on mechanical allodynia. Me- presented as percent of maximum possible effect (%MPE; mean chanical withdrawal thresholds were assessed by von Frey SEM) of eight or nine rats each, 60 min after injection of probing. Data are presented as percent of maximum possible ؎ ؎ naloxone. *P < 0.01 versus saline. effect (%MPE; mean SEM) of eight or nine rats each, 60 min after the last injection. *P < 0.01 versus saline; oP < 0.01 versus SHU9119 alone.

Results Administration of Morphine with SHU9119 Baseline Mechanical Withdrawal Thresholds Pretreatment Two weeks after the CCI lesion, mean baseline with- Spinal administration of morphine (1, 3, and 10 ␮g) drawal threshold to von Frey stimulation was reduced produced a significant antiallodynic effect, as shown by from 20.3 Ϯ 0.4 g to 5.3 Ϯ 0.6 g, confirming the pres- a dose-dependent increase in withdrawal thresholds to ence of mechanical allodynia. Baseline withdrawal von Frey stimulation (fig. 4), compared to saline treat- thresholds were not altered by placement of the spinal ment. A single dose of SHU9119 (0.5 ␮g) also increased catheters and remained stable throughout the testing withdrawal thresholds, with a %MPE of 41.4 Ϯ 8.6. ␮ period. When given 15 min before morphine, 0.5 g SHU9119 resulted in a significant increase in withdrawal thresh- olds, with a maximum %MPE of 86.2 Ϯ 6.9 (0.5 ␮g Administration of Naloxone, and SHU9119 with SHU9119 and 10 ␮g morphine; fig. 4). Low-dose Naloxone Pretreatment Dose–response curves for both SHU9119 and mor- With increasing doses of naloxone (10, 30, and 100 ␮g), phine, and for their combination, were generated. From a significant decrease in withdrawal thresholds was ob- these curves, isobolograms for different levels of effect served, with a %MPE of Ϫ67.2 Ϯ 9.3 at the highest dose were plotted (fig. 5). Although these isobolograms might tested (fig. 1). suggest synergy, the comparison of the experimental SHU9119, 1.5 ␮g, increased withdrawal thresholds, data and the calculated composite additive line, as well with a %MPE of 60.0 Ϯ 13.3. When 0.1 ␮g naloxone, a dose that by itself had no effect on mechanical with- drawal thresholds, was administered 15 min before SHU9119 (1.5 ␮g), this antiallodynic effect of SHU9119 was largely decreased (%MPE was reduced to 14.8 Ϯ 10.9, which was not significantly different from saline; fig. 2).

Coadministration of High-dose Morphine and High-dose MTII Administration of 0.5 ␮g MTII significantly decreased Fig. 3. Neutralization of the proallodynic and antiallodynic ef- withdrawal thresholds, with a %MPE of Ϫ93.8 Ϯ 6.2, fects of MTII and morphine by coadministration of both drugs. whereas 30 ␮g morphine increased thresholds, with a Effect of spinal administration of 0.5 ␮g MTII (MTII 0.5) or 30 ␮g Ϯ morphine (Mor 30) alone or coadministration of 1.5 ␮g MTII %MPE of 89.6 10.4. A combination of approximately and 100 ␮g morphine (MTII 1.5, Mor 100) on mechanical allo- three times higher doses of both drugs (1.5 ␮g MTII and dynia. Mechanical withdrawal thresholds were assessed by von 100 ␮g morphine) resulted in an intermediate %MPE of Frey probing. Data are presented as percent of maximum pos- Ϯ sible effect (%MPE; mean ؎ SEM) of eight or nine rats each 12.2 9.8, which was not significantly different from (MTII and morphine alone) or four rats (combination of both saline (fig. 3). drugs). *P < 0.05 versus MTII or morphine alone.

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opposite activities on nociceptive processing (see also Adan and Gispen33). In neuropathic pain, this balance might be out of equilibrium, by increased activity of the melanocortin system34 and decreased activity of the opi- oid system.19–22 Considering this balance between the spinal melano- cortin and opioid systems, we proposed that by blocking the pronociceptive activity of the spinal melanocortin system with SHU9119,34 tonic analgesic effects of ␤-en- dorphin, through activation of opioid receptors in the dorsal horn, will predominate (see also Vrinten et al.35). Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/99/2/449/407606/0000542-200308000-00028.pdf by guest on 25 September 2021 Fig. 4. Antiallodynic effects of the combination of SHU9119 and As shown in figure 1, administration of naloxone in- morphine. Effects of spinally administered morphine (1, 3, or creased mechanical allodynia, consistent with blockade 10 ␮g; open circles) alone or 15 min after 0.5 ␮g SHU9119 (black circles) on mechanical allodynia. Mechanical withdrawal of a tonically active endogenous opioid agonist at the thresholds were assessed by von Frey probing. Black bar indi- spinal level. This confirms the results of previous work ␮ cates the antiallodynic effect of 0.5 g SHU9119. Data are pre- in which high doses of naloxone increased pain-related ؎ sented as percent of maximum possible effect (%MPE; mean SEM) of eight or nine rats each, 60 min after the last injection. behavior in mononeuropathic and spinally injured Data are presented as %MPE (mean ؎ SEM) of eight or nine rats rats.36,37 When a low dose of naloxone, which by itself had each. *P < 0.05 versus morphine alone. no effect on allodynia, was administered 15 min before SHU9119, the antiallodynic effect of SHU9119 was almost as statistical analysis of these data (as described in Mate- completely blocked, as can be seen in figure 2. rials and Methods), reveal additivity (fig. 5, inset). These data suggest that the melanocortin and opioid systems act at the same common pathway and that the Discussion level of activity of one system (i.e., the opioid system) might modulate the threshold for activation of the other In the current study, we were able to demonstrate an system (i.e., the melanocortin system), even if the nal- interaction between the spinal melanocortin and spinal oxone dose is so low that it does not affect functional opioid systems in a rat model of neuropathic pain. In the responses by itself. Other evidence for the close inter- 28,29 ␣ spinal cord, the MC4 receptor, as well as -MSH, an actions between the melanocortin and opioid systems endogenous ligand for the melanocortin receptors, and has been provided previously. For instance, chronic mor- its precursor molecule pro-opiomelanocortin12,29,30 are phine treatment is shown to induce a down-regulation of 38 shown to be expressed in the dorsal horn, an area MC4 receptors in several brain areas, whereas ablation involved in the processing of nociceptive information. of pituitary pro-opiomelanocortin neurons induces both Proteolytic cleavage from pro-opiomelanocortin yields hypothalamic pro-opiomelanocortin overexpression and not only ␣-MSH but also the endogenous opioid ␤-endor- a down-regulation of ␮-opioid receptors in several brain phin, which is also present in the dorsal horn.12 More- areas.39 Our current experiments suggest that an inter- over, both the ␮- and ␦-opioid receptors, for which action is also present at the spinal level. However, from ␤-endorphin displays a high affinity,31 are expressed in these data, it is not possible to establish the neuroana- the same area.32 Thus, both a functional melanocortin tomic origin of this interaction. The melanocortin and and an opioid system appear to be present at the same opioid systems might be organized in a linear pathway, anatomic site in the spinal cord. Possibly, there is a with the melanocortin system either upstream or down- natural balance between these two systems, each with stream of the opioid system. Another possibility is that

Fig. 5. Isobolographic analysis of the combi- nation of SHU9119 and morphine: Isobolo- grams for both 67% and 75% of maximum possible effect (%MPE), in which the doses of morphine and SHU9119 alone are plotted on the x- and y-axes, respectively. These doses are extrapolated from the respective dose– response curves. The theoretical lines of ad- ditivity (dotted lines represent 1 SEM) and the combinations of both drugs (single black dot) generating the same %MPE are also plot- ted (see Tallarida44 for details). (Inset) The effect of the combination of SHU9119 and morphine (filled circles) is not greater than the theoretical composite additive line (open circles)(P > 0.05), thus demonstrating addi- tive effects of both compounds.

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the two systems are organized in a parallel fashion. The MC4 receptor antagonists and opioids might have a place MC4 receptor and opioid receptor could be located on in the management of neuropathic pain. With concom- different neurons, with pathways converging further itant administration of MC4 receptor antagonists, the downstream, or they could be colocalized on the same total dose of opioid needed to obtain adequate analgesia neuron. The latter has been demonstrated in locus cer- might be reduced. This might diminish the incidence uleus–derived cells, in which ␣-MSH and ␤-endorphin and severity of opioid side effects, development of tol- respectively increase and decrease the level of the sec- erance, and possible tolerance-associated hyperalge- ond messenger cAMP (via adenylate cyclase) through sia.41,42 Also, by changing the interinjection interval of melanocortin receptors and ␦-opioid receptors that are SHU9119 and opioids, it could be that the combined expressed in the same cell.40 Adenylate cyclase might antiallodynic action is enhanced, as has been demon- thus function as an integrator of melanocortin- and opi- D strated for the combination of N-methyl- -aspartate re- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/99/2/449/407606/0000542-200308000-00028.pdf by guest on 25 September 2021 oid-mediated signaling, thereby regulating the output of ceptor antagonists and morphine.43 the cell in vivo. A similar mechanism has been proposed In conclusion, we were able to demonstrate an inter- to be involved in the effect of on opioid action between the spinal melanocortin and opioid sys- addiction.16 tems in a rat model of neuropathic pain. The antiallo-

To further investigate the nature of the neuroanatomic dynic actions of the MC4 receptor antagonist SHU9119 substrate underlying the interaction between the spinal were largely blocked by a low dose of naloxone, melanocortin and opioid system, we tested the effect of whereas SHU9119 and morphine had an additive antial- coadministration of a high dose of the melanocortin lodynic effect. It is conceivable that coadministration of receptor agonist MTII and morphine. As seen in figure 3, MC4 receptor antagonists and opioids might contribute each of these drugs alone already had an almost maximal to a better treatment of human neuropathic pain. effect on allodynia with a three-times-lower dose than we used for the coadministration. When the two systems are organized in a linear fashion, it would be expected that the resultant effect of the combined doses on allo- References dynia would resemble that of activation of the down- 1. 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Anesthesiology, V 99, No 2, Aug 2003