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Inhibition of Neuropathic Pain by Selective Ablation of Brainstem Medullary Cells Expressing the ␮-Opioid Receptor

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Inhibition of Neuropathic Pain by Selective Ablation of Brainstem Medullary Cells Expressing the ␮-Opioid Receptor The Journal of Neuroscience, July 15, 2001, 21(14):5281–5288 Inhibition of Neuropathic Pain by Selective Ablation of Brainstem Medullary Cells Expressing the ␮-Opioid Receptor Frank Porreca,1 Shannon E. Burgess,1 Luis R. Gardell,1 Todd W. Vanderah,1 T. Philip Malan Jr,1 Michael H. Ossipov,1 Douglas A. Lappi,2 and Josephine Lai1 1Departments of Pharmacology and Anesthesiology, University of Arizona, Tucson, Arizona 85724, and 2Advanced Targeting Systems, San Diego, California 92121 Neurons in the rostroventromedial medulla (RVM) project to SNL-induced experimental pain, and no pretreatment affected spinal loci where the neurons inhibit or facilitate pain transmis- the responses of sham-operated groups. This protective effect sion. Abnormal activity of facilitatory processes may thus rep- of dermorphin–saporin against SNL-induced pain was blocked resent a mechanism of chronic pain. This possibility and the by ␤-funaltrexamine, a selective ␮-opioid receptor antagonist, phenotype of RVM cells that might underlie experimental neu- indicating specific interaction of dermorphin–saporin with the ropathic pain were investigated. Cells expressing ␮-opioid re- ␮-opioid receptor. RVM microinjection of dermorphin–saporin, ceptors were targeted with a single microinjection of saporin but not of dermorphin or saporin, in animals previously under- conjugated to the ␮-opioid agonist dermorphin; unconjugated going SNL showed a time-related reversal of the SNL-induced saporin and dermorphin were used as controls. RVM dermor- experimental pain to preinjury baseline levels. Thus, loss of phin–saporin, but not dermorphin or saporin, significantly de- RVM ␮ receptor-expressing cells both prevents and reverses creased cells expressing ␮-opioid receptor transcript. RVM experimental neuropathic pain. The data support the hypothe- dermorphin, saporin, or dermorphin–saporin did not change sis that inappropriate tonic-descending facilitation may underlie baseline hindpaw sensitivity to non-noxious or noxious stimuli. some chronic pain states and offer new possibilities for the Spinal nerve ligation (SNL) injury in rats pretreated with RVM design of therapeutic strategies. dermorphin–saporin failed to elicit the expected increase in sensitivity to non-noxious mechanical or noxious thermal stim- Key words: neuropathic pain; descending facilitation; RVM; uli applied to the paw. RVM dermorphin or saporin did not alter ␮-opioid receptors; saporin; ON cells The presence of endogenous pain-modulating mechanisms is well tralateral hemisections, and selective lesions of the ipsilateral or established. Although pain inhibitory systems projecting from the contralateral (relative to the side of peripheral nerve injury) brainstem to the spinal cord offer obvious survival advantages, the dorsal columns block nerve injury-induced pain (Bian et al., 1998; role of descending projections that facilitate pain transmission is Sung et al., 1998; Sun et al., 2001). Lidocaine injection into the less clear (Watkins and Mayer, 1982; Wiertelak et al., 1992; ipsilateral (relative to the side of peripheral nerve injury), but not Mason et al., 1999). Nevertheless, the presence of pain facilitation contralateral, nucleus gracilis also blocks nerve injury-induced mechanisms raises the possibility that their abnormal sustained pain (Sun et al., 2001). These studies support a role for central activity may underlie chronic pain. processes in the mediation of experimental neuropathic pain. Injury to nerves may elicit neuropathic pain characterized, in Blockade of established nerve injury-induced pain is also pro- part, by increased sensitivity to normally non-noxious and nox- duced by lidocaine microinjection into the rostral ventromedial ious stimuli (allodynia and hyperalgesia, respectively) (Payne, medulla (RVM), indicating additionally the importance of de- 1986; Chaplan and Sorkin, 1997). Aspects of this human condi- scending modulatory pathways (Pertovaara et al., 1996; tion have been modeled by injury to spinal nerves in animals Kovelowski et al., 2000) and the possibility of tonic discharge of (Bennett and Xie, 1988; Seltzer et al., 1990; Kim and Chung, cells mediating descending facilitation (Fields et al., 1991; Fields, 1992; Kim et al., 1997). Increased spontaneous and persistent 1992). The importance of descending facilitation is also sup- afferent discharge may be critical in eliciting hypersensitivity of ported by observations that lesions of the dorsolateral funiculus spinal neurons (i.e., central sensitization) (Kirk, 1974; Wall and (DLF) block nerve injury-induced pain (Ossipov et al., 2000). Gutnick, 1974; Devor, 1991, 1994; Kajander et al., 1992). Both One characteristic of RVM cells that may mediate descending injured as well as adjacent uninjured fibers become spontaneously facilitation is their sensitivity to opioid ␮ receptor agonists active after injury (Li et al., 2000; Wu et al., 2001). Manipulations (Fields et al., 1983; Fields and Heinricher, 1989; Pan et al., 1990; designed to interfere with ascending, large-fiber projections to Heinricher et al., 1994). These findings suggest that RVM cells brainstem nuclei, including spinal transection, ipsilateral and con- that may mediate descending facilitation in chronic pain states Received March 15, 2001; revised April 26, 2001; accepted May 1, 2001. might be identified by their expression of this opioid receptor. We thank Dr. Naomi Rance for access to the image-assisted mapping equipment The present experiments tested this hypothesis by targeting and software as well as for her critical evaluation of the in situ hybridization data. RV M ␮ receptor-expressing cells with the cytotoxin saporin, by Correspondence should be addressed to Dr. Frank Porreca, Department of conjugating the toxin to a potent opioid ␮ receptor agonist, Pharmacology, College of Medicine, University of Arizona Health Sciences Center, Tucson, AZ 85724. E-mail: [email protected]. dermorphin (Broccardo et al., 1981; Braga et al., 1984). The goal Copyright © 2001 Society for Neuroscience 0270-6474/01/215281-08$15.00/0 was to determine the role of RVM ␮-opioid receptor-expressing 5282 J. Neurosci., July 15, 2001, 21(14):5281–5288 Porreca et al. • Medullary Opioid ␮ Receptor Cells Mediate Neuropathic Pain cells in preventing or reversing nerve injury-induced pain. A The incision was closed, and the animals were allowed to recover for 5 d. similar approach has been successfully used to lesion spinal cord Rats that exhibited motor deficiency or failure to exhibit subsequent increased sensitivity to innocuous mechanical stimulation were excluded lamina I projection cells with a substance P–saporin conjugate from further testing (Ͻ5% of the animals were not used). Sham control (Mantyh et al., 1997; Nichols et al., 1999). rats underwent the same operation and handling as the experimental animals, but without SNL. Evaluation of response thresholds was per- MATERIALS AND METHODS formed on the hindpaw ipsilateral to SNL or sham SNL using the procedures described above. Radioligand binding. Radioligand binding was performed using crude In situ hybridization of opioid ␮ receptor mRNA. For tissue preparation, membrane preparations from NG 108-15 (expresses mouse opioid ␦ ␮ male Sprague Dawley rats were deeply anesthetized with ketamine and receptors) and from transfected cells that express the rat opioid perfused transcardially with PBS treated with 0.1% diethylpyrocarbonate receptors (MORs). All radioligand binding assays were performed in (DEPC), followed by 4% paraformaldehyde. Whole rat brains were duplicate in 50 mM Tris, pH 7.4, in the presence of 0.5 mg/ml bovine ␮ ␮ removed and post-fixed in fixative overnight, cryoprotected in 30% su- serum albumin (BSA) and protease inhibitors (30 M bestatin, 10 M crose in PBS treated with 0.1% DEPC, and stored at 4°C. Frozen frontal captopril, 0.37 U/ml bacitracin, and 0.1 mM phenylmethylsulfonyl fluo- sections (20–40 ␮m) were prepared from the brainstem caudal to the site ride). All reactions were performed at 25°C for 3 hr in a total reaction Ϫ Ϫ of incision of the cannulas and mounted on positively charged slides. A volume of 1 ml. At least 10 concentrations of dermorphin (10 14 to 10 5 Ϫ 14 Ϫ 7.5 single-stranded, fluorescein-labeled partial cDNA probe corresponding M) or dermorphin–saporin (10 to 10 M) were used. The concen- ␮ 3 2 4 5 to nucleotides 628–965 of the coding region of the rat opioid receptor tration of H-[D-Ala , NMPhe , Gly-01 ]enkephalin (2.2 nM) was based cDNA was synthesized by PCR by use of a 100:1 ratio of antisense on the Kd value of the radioligand determined from saturation analysis. (3Ј)-to-sense (5Ј) primers and a mixture of fluorescein-labeled dUTP and The reaction was terminated by rapid filtration through Whatman GF/B unlabeled dNTP. The PCR was performed for 30 cycles (45 sec at 95°C, filters presoaked in polyethyleneimine and washed three times with 4 ml 60 sec at 60°C, and 2 min at 72°C); the product was purified by ethanol of ice-cold PBS. Nonspecific binding was defined as that in the presence ϫ ␮ precipitation. The probe was reconstituted in RNase-free 1 Tris– of 10 M naloxone. Radioactivity in the samples was determined by liquid EDTA and analyzed by agarose gel electrophoresis and Southern trans- scintillation counting. Data were analyzed by nonlinear regression anal- fer to determine the yield. A corresponding sense probe was synthesized ysis using GraphPad Prism (Graph Pad, San Diego, CA). The Ki value(s) under the same conditions except using a 1:100 ratio of antisense-to- for each ligand
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