Endothelin ET B Receptor Antagonist Reduces Mechanical Allodynia in Rats with Trigeminal Neuropathic Pain

Abstract

Trigeminal neuropathic pain, which is associated with marked orofacial mechanical allodynia, is frequently refractory to currently available drugs. Because endothelins (ETs) can contribute to nociceptive changes in animal models of inflammatory, cancer, and diabetic neuropathic pain, the present study evaluated the influence of ET_A and ET_B receptor antagonists on orofacial mechanical allodynia in a rat model of trigeminal neuropathic pain. Unilateral constriction (C) of the infraorbital nerve (ION) caused pronounced and sustained bilateral mechanical allodynia, evaluated by application of von Frey hairs to the vibrissal pad. Mechanical allodynia on postoperative days 12–15 after nerve injury was abolished for up to 90 mins by subcutaneous administration of 2.5 mg/kg morphine, but was fully refractory to intravenous (iv) administration of 10 mg/kg of the dual ET_A plus ET_B or selective ET_A receptor antagonists, bosentan and atrasentan, respectively. In sharp contrast, iv administration of 20 mg/kg of the selective ET_B receptor antagonist, A-192621, caused a net 61 ± 15% reduction of mechanical threshold, lasting 2 hrs. Co-injection of atrasentan plus A-192621 did not modify ION injury-induced mechanical allodynia. Injection of 10 pmol ET-1 into the upper lip of naive rats caused ipsilateral mechanical allodynia lasting up to 5 hrs. Thus, ET_B receptor–mediated mechanisms contribute to orofacial mechanical allodynia induced by CION injury, but, some-how, functional ET_A receptors are required for expression of the antiallodynic effect of ET_B receptor blockade.

Introduction

Trigeminal neuralgia (TN) is a form of neuropathic pain characterized by severe lancinating pain in orofacial regions innervated by the trigeminal nerve. Because currently available medical or surgical procedures for TN treatment fail to provide consistent and permanent pain relief in all patients, further studies characterizing its underlying mechanisms and exploring new effective treatment strategies are clearly warranted.

Chronic constriction (C) of the infraorbital nerve (ION), a branch of the trigeminal nerve, has been developed as an experimental model that reproduces important aspects of TN, including signs of abnormal spontaneous pain-related behavior, mechanical allodynia (1, 2), heat hyperalgesia (3), and inflammatory hypersensitivity (4). Enhanced responsiveness to mechanical stimulation is frequently observed in patients with TN and other chronic neuropathic pain forms (5). On the other hand, mechanical allodynia in the hind paw of rats with diabetic neuropathy is temporarily attenuated by atrasentan, a highly selective antagonist of endothelin (ET)_A receptors (6). ET_A and ET_B receptors can be expressed in central and peripheral neurons (7, 8), and are specifically targeted by ETs, a family of peptides that includes ET-1, ET-2, and ET-3 (9, 10) and can be produced by many cell types in the central nervous system (11, 12), as well as in peripheral sensory ganglia, including cells of the trigeminal ganglion (13). ET-1 can trigger pain or overt nociception in humans and animals (14–17), as well as hyperalgesia to noxious chemical, mechanical, and thermal stimuli (17–20). Moreover, endogenous ETs contribute significantly to pain and/or hyperalgesia of inflammatory, immune, neuropathic, and neoplasic origins (6, 17, 21–23).

In light of these considerations, the present study aimed to evaluate the influence of CION on nocifensive responses of rats to mechanical stimuli (using Von Frey hairs) applied to the snout, as well as the susceptibility of the resultant changes to reversal by treatment with morphine and ET_A and/or ET_B receptor antagonists.

Materials and Methods

Animals.

Experiments were conducted on male Wistar rats weighing 180–200 g, housed five to a cage at 22 ± 1°C on a 12:12-hr light:dark cycle (lights on at 0700 hrs) with free access to laboratory chow and tap water. All experimental procedures were previously approved by Universidade Federal de Santa Catarina’s Committee on the Ethical Use of Animals, where the study was conducted.

Surgical Procedure.

The method for producing CION differed slightly from that originally proposed by Vos et al. (2). Briefly, rats were anesthetized with a mixture of 50 mg/kg ketamine and 10 mg/kg xylazine, and a small incision was made on the snout, under the right eye, approximately 3 mm caudal to the mystacial pads. The rostral end of the ION was exposed, at the point at which it emerged from the infraorbital fissure, and two silk 4–0 ligatures were tied loosely around it. The wound was closed with additional 4–0 silk sutures. Sham-operated rats were treated identically, but no ligatures were applied to the ION. After surgery, all rats were treated with intramuscular administration of 60 mg/kg oxytetracycline and maintained in a warm room until they recovered from anesthesia.

Mechanical Stimulation.

Before each testing session, animals were placed in individual plastic cages and left to adapt to the environment for at least 1 hr. The mechanical threshold was measured using a graded series of 10 von Frey filaments ranging from 0.02 to 10 g (Semmes-Weinstein monofilaments, Stoelting, Wood Dale, IL) as described previously (24). Each filament was applied near the center of the vibrissal pad, to the point of bending, three times, at 30-sec intervals on the contralateral side and then on the nerve-injured side, for a total of six applications per rat. Each stimulation series began with the filament producing the lowest force, and proceded up to the filament that evoked one of the following nocifensive behaviors twice: brisk head withdrawal, escape or attack reactions, or short-lasting facial grooming. Only rats that did not react to application of the 10-g filament in the preoperative tests were included in this study, to avoid unspecific responses.

Drug Treatments.

The influence of various drug treatments on CION-induced changes in nocifensive responsiveness was assessed on Days 12 or 15 after surgery, by determining the mechanical threshold for evoking responses before drug administration and then at 30-min intervals up to 8 hrs, thereafter. Drug treatments included: subcutaneous (sc) administration of 2.5 mg/kg morphine hydrochloride, intravenous (iv) administration of the nonpeptidic ET receptor antagonists, bosentan (dual ET_A plus ET_B; 10 or 20 mg/kg), atrasentan (selective ET_A; 10 mg/kg), and A-192621 (selective ET_B; 20 mg/kg), prepared daily in heated (50°C) water alone (bosentan) or warm water containing 3% ethanol and 100 μl of 0.1 N NaOH. In some rats, 10 pmol ET-1 or its vehicle (phosphate-buffered saline [PBS]) was injected into the upper lip, and its effect on responses to mechanical stimulation was evaluated for up to 6 hrs. Control rats were always treated identically with the corresponding vehicle.

Drugs.

The drugs used were: bosentan (kindly provided by Actelion A.G., Allschwil, Switzerland), atrasentan and A-192621.1 ([2R-{2a,3b,4a}]-4-[1,3-benzodioxol-5-yl]-1-[2-{2,6-diethylphenyl}amino]-2-oxoethyl-2-[4-propoxyphenyl]-3 pyrrolidinecarboxylic acid; both kindly provided by Abbott Laboratories, Abbott Park, IL), morphine hydrochloride (Merck AG, Darmstadt, Germany), ET-1 (Bachem, Natick, MA), and oxytetracycline (Terramicina; Pfizer, Guarulhos, Brazil).

Statistical Analysis.

Results are presented as mean ± SEM at different time points after surgery for each group and were analyzed using the nonparametric Kruskal-Wallis ANOVA, followed by the Mann-Whitney U test for individual comparisons. Differences with P values less than 0.05 were considered significant.

Results

CION induced bilateral mechanical allodynia starting 9 days after surgery, which was maximal on both sides by Day 12 and remained at this level at least up to Day 120. Treatment of rats with 2.5 mg/kg morphine hydrochloride suppressed mechanical allodynia in the period between 30 and 90 mins after injection (Fig. 1). On the other hand, bilateral mechanical allodynia was unaffected by iv administration of either the dual ET_A plus ET_B receptor antagonist, bosentan (10 or 20 mg/kg; data not shown), or the selective ET_A receptor antagonist, atrasentan (10 mg/kg; Fig. 2 ). In sharp contrast, significant short-lasting and bilateral inhibitory effects were observed between 1 and 2 hrs after iv injection of the selective ET_B receptor antagonist A-192621 (20 mg/kg; Figs. 1 and 2 show only results regarding stimulation of the ipsilateral side). Combined iv treatment of rats with atrasentan plus A-192621, at the same doses used previously, failed to affect mechanical allodynia significantly (Fig. 2 ). Local sc injection of 50 μl of 10 pmol ET-1 caused mechanical allodynia that was restricted to the injected side and lasted up to 5 hrs (Fig. 3 ).

Discussion

The current study demonstrates that CION induces a state of persistent orofacial bilateral mechanical allodynia in rats that is amenable to temporary relief by opiates (morphine) or selective antagonists of ET_B receptors, yet is fully resistant to alleviation by selective ET_A receptor antagonists or to simultaneous blockade of ET_A and ET_B receptors, with either bosentan or a combination of atrasentan plus A-192621.

Previous studies have disclosed differential roles for ET receptors in controlling nocifensive responsiveness in experimental pain models. In some models, ET-1–induced nociception occurs exclusively through ET_A receptor activation (17, 19, 21), which agrees well with their expression in small-sized peptidergic and nonpeptidergic sensory neurons in dorsal root ganglia (8). Likewise, ET_A receptor antagonists abolish hyperalgesic responses to thermal and chemical stimuli evoked by ET-1 in mice and attenuate the tactile allodynia in rats displaying diabetes-induced neuropathy (6, 17, 19, 20). However, the nociceptive effects of exogenous ETs in the writhing test and mechanical inflammatory hyperalgesia caused by carrageenan or complete Freund’s adjuvant in the hind paw of mice involve both ET_A and ET_B receptors (15, 20), whereas phenylbenzoquinone-induced abdominal writhes in mice (22), articular nociception induced by lipopolysaccharide in a previously inflamed knee joint (21), and mechanical hyperalgesia induced by ET-1, interleukin (IL)-18, and IL-12 in rats seem to be mediated by ET_B (but not ET_A) receptors (25, 26).

Jarvis et al. (6) showed that mechanical allodynia detected in the hind paw of rats with diabetic neuropathy can be reduced temporarily by blockade of ET_A receptors with atrasentan, but not by blockade of ET_B receptors with A-192621. Corroborating such findings, the current study demonstrates that ETs can mediate mechanical allodynia in a distinct neuropathic model, that is, orofacial pain induced by CION. Nevertheless, it is important to emphasize that the receptors mediating the allodynic effects of the ET system in the diabetes and CION models are clearly opposite (ET_A and ET_B receptors, respectively). Despite this significant difference, the analgesic/antiallodynic effect produced by selective antagonism of one receptor type in both the CION (present study) and diabetes neuropathy (6) models was attenuated by simultaneous blockade of the other receptor type. Specifically, suppression of CION-induced orofacial mechanical allodynia by ET_B receptor blockade the CION model was fully abrogated by concomitant antagonism of ET_A receptors, either by coadministration of A-192621 with atrasentan, or by treatment with bosentan. One could speculate that ET_A receptor blockade with atrasentan may have reduced the intensity of ET_B receptor blockade afforded by A-192621, by augmenting the concentration of ET-1 and, hence, its ability to compete with the latter antagonist. If this happens to be the case, the failure of ET_A receptor blockade alone to enhance mechanical allodynia might be caused by a floor effect of CION on noxious mechanical threshold (i.e., a maximal increase in sensitivity to mechanostimulation). A somewhat similar scenario could be envisaged to account for the lack of an antiallodynic effect of bosentan. However, the true mechanisms underlying this apparent interaction between ET_A and ET_B receptors on CION-induced orofacial mechanical allodynia clearly remain to be clarified.

On the other hand, because atrasentan does not cross the blood-brain barrier (27) and A-192621 displays a closely related chemical structure to atrasentan, it seems likely that peripheral, rather than central, ET_B receptor–dependent mechanisms are responsible for the mechanical allodynia induced by CION. This view is strengthened by a report that the trigeminal ganglion expresses a high density of ET_B receptors (28).

In summary, our results show that ET-dependent mechanisms mediated via ET_B receptors contribute to orofacial mechanical allodynia induced by CION in the rat. The results obtained with A-192621 suggest that ET_B receptor antagonists may represent promising candidates for the treatment of TN in humans.

Figure 1.

Effects of morphine hydrochloride or the selective ET_B receptor antagonist, A-192621, on CION-induced orofacial mechanical allodynia in rats. On Day 12 after surgery, rats received sc administration of 2.5 mg/kg morphine, iv administration of 20 mg/kg A-192621, or the respective vehicles. The mechanical threshold was assessed with von Frey filaments at different time intervals. Values represent the mean ± SEM of the minimal force required to elicit a nocifensive response when applied to the snout on the side ipsilateral to the nerve injury (n = 6–8). *P < 0.05 when compared with corresponding values of sham-operated rats; # and ⊗ indicate P < 0.05 when compared with corresponding value of CION rats treated with vehicle.

Figure 2.

Effects of ET_A and/or ET_B receptor antagonists on CION- induced orofacial mechanical allodynia in rats. On Day 15 after surgery, rats received iv administration of 10 mg/kg atrasentan (ET_A receptor antagonist) or 20 mg/kg A-192621 (ET_B receptor antagonist) alone or in combination, or the respective vehicle. The mechanical threshold was assessed with von Frey filaments. Values represent the mean ± SEM of the minimal force required to elicit nocifensive response when applied to the snout on the side ipsilateral to the nerve injury before (basal) and 1 or 2 hrs after drug treatment (n =6–8). Asterisks and fences denote P < 0.05 when compared with corresponding value of sham-operated or CION rats treated with vehicle, respectively.

Figure 3.

Effect of unilateral injection of 10 pmol ET-1 into the upper lip on the mechanical threshold of naive rats. Values represent the mean ± SEM of the minimal force required to elicit a nocifensive response from either ipsilateral (ips) or contralateral (cl) sides of the snout, relative to injection (n = 6). *P < 0.05 when compared with corresponding values of the control group.

Footnotes

The study was supported by the Brazilian National Research Council (CNPq), Coordenação de Aperfeicoamento de Pessoal de Nível Superior, Fundação de Amparo a Ciência e Tecnologia do Estado de Santa Catarina, and the Pronex program of the Brazilian Ministry of Science and Technology. J.G.C. is the recipient of a CNPq doctoral scholarship.

Acknowledgements

The kind donations of bosentan by Dr. Martine Clozel (Actelion Pharmaceuticals, Allschwil, Switzerland), and atrasentan and A-192621 by Dr. Terry Opgenorth (Abbott Laboratories, Abbott Park, IL) are gratefully acknowledged.

References

Vos BP, Maciewicz RJ. Behavioral changes following ligation of the infraorbital nerve in rat: an animal model of trigeminal neuropathic pain. In: Besson JM, Guilbaud G, Eds. Lesions of Primary Afferent Fibers as a Tool for the Study of Clinical Pain. Amsterdam: Elsevier, pp147–158, 1991.

Vos BP, Strassman AM, Maciewicz RJ. Behavioral evidence of trigeminal neuropathic pain following chronic constriction injury to the rat’s infraorbital nerve. J Neurosci 14:2708–2723, 1994.

Imamura Y, Kawamoto H, Nakanishi O. Characterization of heat-hyperalgesia in an experimental trigeminal neuropathy in rats. Exp Brain Res 116:97–103, 1997.

Anderson LC, Vakoula A, Veinote R. Inflammatory hypersensitivity in a rat model of trigeminal neuropathic pain. Arch Oral Biol 48:161–169, 2003.

Eide PK, Rabben T. Trigeminal neuropathic pain: pathophysiological mechanisms examined by quantitative assessment of abnormal pain and sensory perception. Neurosurgery 43:1103–1109, 1998.

Jarvis MF, Wessale JL, Zhu CZ, Lynch JJ, Dayton BD, Calzadilla SV, Padley RJ, Opgenorth TJ, Kowaluk EA. ABT-627, an endothelin ET(A) receptor-selective antagonist, attenuates tactile allodynia in a diabetic rat model of neuropathic pain. Eur J Pharmacol 388:29–35, 2000.

Kurokawa K, Yamada H, Ochi J. Topographical distribution of neurons containing endothelin type A receptor in the rat brain. J Comp Neurol 389:348–360, 1997.

Pomonis JD, Rogers SD, Peters CM, Ghilardi JR, Mantyh PW. Expression and localization of endothelin receptors: implications for the involvement of peripheral glia in nociception. J Neurosci 21:999–1006, 2001.

Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto KE, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:411–415, 1988.

10.

Inoue A, Yanagisawa M, Kimura S, Kasuya Y, Miyauchi T, Goto K, Masaki, T. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci U S A 86:2863–2867, 1989.

11.

Giaid A, Gibson SJ, Herrero MT, Gentleman S, Legon S, Yanagisawa M, Masaki T, Ibrahim NB, Roberts GW, Rossi ML. Topographical localization of endothelin mRNA and peptide immunoreactivity in neurons of the human brain. Histochemistry 95:303–314, 1991.

12.

Yamada H, Kurokawa K. Histochemical studies on endothelin and the endothelin-A receptor in the hypothalamus. J Cardiovasc Pharmacol 31(Suppl 1):S215–S218, 1998.

13.

Milner P, Loesch A, Burnstock G. Endothelin immunoreactivity and mRNA expression in sensory and sympathetic neurons following selective denervation. Int J Dev Neurosci 18:727–734, 2000.

14.

Ferreira SH, Romitelli M, de Nucci G. Endothelin-1 participation in overt and inflammatory pain. J Cardiovasc Pharmacol 13(Suppl 1):S220–S222, 1989.

15.

Raffa RB, Schupsky JJ, Jacoby HI. Endothelin-induced nociception in mice: mediation by ETA and ETB receptors. J Pharmacol Exp Ther 276:647–651, 1996.

16.

Davar G, Hans G, Fareed MU, Sinnott C, Strichartz G. Behavioral signs of acute pain produced by application of endothelin-1 to rat sciatic nerve. Neuroreport 9:2279–2283, 1998.

17.

Piovezan AP, D’Orleans-Juste P, Souza GE, Rae GA. Endothelin-1-induced ET(A) receptor-mediated nociception, hyperalgesia and oedema in the mouse hind-paw: modulation by simultaneous ET(B) receptor activation. Br J Pharmacol 129:961–968, 2000.

18.

Piovezan AP, D’Orleans-Juste P, Tonussi CR, Rae GA. Effects of endothelin-1 on capsaicin-induced nociception in mice. Eur J Pharmacol 351:15–22, 1998.

19.

Menendez L, Lastra A, Hidalgo A, Baamonde A. Nociceptive reaction and thermal hyperalgesia induced by local ET-1 in mice: a behavioral and Fos study. Naunyn-Schmiedeberg’s Arch Pharmacol 367:28–34, 2003.

20.

Baamonde A, Lastra A, Villazon M, Bordallo J, Hidalgo A, Menendez L. Involvement of endogenous endothelins in thermal and mechanical inflammatory hyperalgesia in mice. Naunyn-Schmiedeberg’s Arch Pharmacol 369:245–251, 2004.

21.

De-Melo JD, Tonussi CR, D’Orleans-Juste P, Rae GA. Articular nociception induced by endothelin-1, carrageenan and LPS in naive and previously inflamed knee-joints in the rat: inhibition by endothelin receptor antagonists. Pain 77:261–269, 1998.

22.

Griswold DE, Douglas SA, Martin LD, Davis TG, Davis L, Ao Z, Luttmann MA, Pullen M, Nambi P, Hay DW, Ohlstein EH. Endothelin B receptor modulates inflammatory pain and cutaneous inflammation. Mol Pharmacol 56:807–812, 1999.

23.

Wacnik PW, Eikmeier LJ, Ruggles TR, Ramnaraine ML, Walcheck BK, Beitz AJ, Wilcox GL. Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain. J Neurosci 21:9355–9366, 2001.

24.

Christensen D, Gautron M, Guilbaud G, Kayser V. Combined systemic administration of the glycine/NMDA receptor antagonist, (+)-HA966 and morphine attenuates pain-related behaviour in a rat model of trigeminal neuropathic pain. Pain 83:433–440, 1999.

25.

Verri WA Jr, Schivo IR, Cunha TM, Liew FY, Ferreira SH, Cunha FQ. Interleukin-18 induces mechanical hypernociception in rats via endothelin acting on ETB receptors in a morphine-sensitive manner. J Pharmacol Exp Ther 310:710–717, 2004.

26.

Verri WA Jr, Molina RO, Schivo IR, Cunha TM, Parada CA, Poole S, Ferreira SH, Cunha FQ. Nociceptive effect of subcutaneously injected interleukin-12 is mediated by endothelin acting on ETB receptors in rats. J Pharmacol Exp Ther 315:609–615, 2005.

27.

Peters CM, Lindsay TH, Pomonis JD, Luger NM, Ghilardi JR, Sevcik MA, Mantyh PW. Endothelin and the tumorigenic component of bone cancer pain. Neuroscience 126:1043–1052, 2004.

28.

Brändli P, Loffler BM, Breu V, Osterwalder R, Maire JP, Clozel M. Role of endothelin in mediating neurogenic plasma extravasation in rat dura mater. Pain 64:315–322, 1996.