福尔马林模型熟悉

RESEARCH PAPER Dexmedetomidine and ST-91 analgesia in the formalin model is mediated by a2A-adrenoceptors: a mechanism of action distinct from morphine A Nazarian1,2, CA Christianson1, X-Y Hua1 and TL Yaksh1 1Department of Anesthesiology, University of California-San Diego, La Jolla, CA, USA and 2Department of Pharmaceutical Sciences, Western University of Health Sciences, Pomona, CA, USA Background and purpose: Intrathecal administration of a2-adrenoceptor agonists produces potent analgesia. This study addressed the subtype of spinal a2-adrenoceptor responsible for the analgesic effects of i.t. dexmedetomidine and ST-91 in the formalin behavioural model and their effects on primary afferent substance P (SP) release and spinal Fos activation. Experimental approach: The analgesic effects of i.t. dexmedetomidine and ST-91 (a2 agonists) were tested on the formalin behavioural model. To determine the subtype of a2-adrenoceptor involved in the analgesia, i.t. BRL44408 (a2A antagonist) or ARC239 (a2B/C antagonist) were given before dexmedetomidine or ST-91. Moreover, the ability of dexmedetomidine and ST-91 to inhibit formalin-induced release of SP from primary afferent terminals was measured by the internalization of neurokinin1 (NK1) receptors. Finally, the effects of dexmedetomidine on formalin-induced Fos expression were assessed in the dorsal horn. Key results: Intrathecal administration of dexmedetomidine or ST-91 dose-dependently reduced the formalin-induced paw- flinching behaviour in rats. BRL44408 dose-dependently blocked, whereas ARC239 had no effect on the analgesic actions of dexmedetomidine and ST-91. Dexmedetomidine and ST-91 had no effect on the formalin-induced NK1 receptor internalization, while morphine significantly reduced the NK1 receptor internalization. On the other hand, both dexmedetomidine and morphine diminished the formalin-induced Fos activation. The effect of dexmedetomidine on formalin-induced Fos activation was reversed by BRL44408, but not ARC239. Conclusion and implications: These findings suggest that a2A-adrenoceptors mediate dexmedetomidine and ST-91 analgesia. This effect could be through a mechanism postsynaptic to primary afferent terminals, distinct from that of morphine. British Journal of Pharmacology (2008) 155, 1117–1126; doi:10.1038/bjp.2008.341; published online 1 September 2008 Keywords: Dexmedetomidine; ST-91; a2-adrenoceptors; substance P; neurokinin 1 receptor; primary afferent; nociception; morphine; BRL44408; ARC239 Abbreviations: PBS, sodium phosphate buffer; SP, substance P Introduction Activation of spinal a2-adrenoceptors by noradrenaline or synthetic agonists produces potent analgesia in animals and humans (Eisenach et al., 1996; Pertovaara, 2006). There are three different subtypes of a2-adrenoceptors (a2A, a2B and a2C), which have similar homology and signal transduction mechanisms (Bylund et al., 1994; Kable et al., 2000). Histo- logical evidence indicates that the predominant subtypes of a2-adrenoceptors in the spinal cord are the a2A- and a2C-adrenoceptors. a2A-Adrenoceptors are largely located on substance P (SP)-containing C-fibre primary afferent terminals; although a smaller population also exists on other sites within the superficial dorsal horn (Stone et al., 1998; Chen et al., 2007). On the other hand, a2C-adrenoceptors are found on inhibitory interneurons and axons of projections neurons (Olave and Maxwell, 2002). The spinal cord contains negligible levels of a2B-adrenoceptors (Zeng and Lynch, 1991; Nicholas et al., 1993). On the basis of the distribution of binding and findings from transgenic animals, spinally administered a2-adrenoceptor agonists are considered to produce their analgesic actions through activation of a2A-adrenoceptors. That is, disabling a2A-adrenoceptor func- tion by a point mutation in the a2A gene resulted in the inability of clonidine and dexmedetomidine to be analgesic in acute thermal nociception and SP-elicited pain behaviour in mice (Lakhlani et al., 1997; Stone et al., 1997). The distribution of a2A-adrenoceptors raises the possibility that spinal a2-adrenoceptor agonists produce their analgesic effects by a presynaptic mechanism. Indeed, previous Received 22 February 2008; revised 11 July 2008; accepted 22 July 2008; published online 1 September 2008 Correspondence: Dr A Nazarian, Department of Pharmaceutical Sciences, Western University of Health Sciences. 309 East Second Street, Pomona, CA 91766-1854, USA. E-mail: anazarian@westernu.edu British Journal of Pharmacology (2008) 155, 1117–1126 & 2008 Macmillan Publishers Limited All rights reserved 0007–1188/08 $32.00 www.brjpharmacol.org findings have shown that dexmedetomidine, clonidine and ST-91 reduced the evoked release of SP from spinal cord preparations (Pang and Vasko, 1986; Ono et al., 1991; Takano et al., 1993). Work from our laboratory and others (Honore et al., 1999; Nazarian et al., 2008) demonstrated that intraplantar formalin evokes the release of SP, as measured by the internalization of neurokinin1 (NK1) receptors (NK1r). Since a2A-adrenoceptors are involved in the analgesia produced by a2-adrenoceptor agonists, it would be reason- able to predict that a2A-adrenoceptors are also involved in the presynaptic inhibition of SP release. Thus, in this study, it was determined whether a2A-adrenoceptors mediate the analgesic effects of dexmedetomidine and ST-91 in the formalin-induced pain behaviour model, as well as regu- lating formalin-induced primary afferent SP release, as measured by NK1r internalization. Finally, the effects of dexmedetomidine on formalin-evoked Fos expression in the dorsal horn were also measured. The present findings indicate that dexmedetomidine and ST-91 produce their analgesic actions through a2A-adrenoceptors in the formalin behaviour test; however, this effect appears to be mediated postsynaptically in the spinal dorsal horn. Materials and methods Animals Male Holtzman Sprague–Dawley rats (250–350 g; Harlan, Indianapolis, IN, USA) were individually housed in standard cages and maintained on a 12-h light/dark cycle (lights on at 07 h). Testing occurred during the light cycle. Animal care was in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publica- tion 85–23, Bethesda, MD, USA) and approved by the Institutional Animal Care and Use Committee of the University of California, San Diego. Food and water were available ad libitum. Intrathecal catheter implantation Rats were implanted with a single i.t. catheter for drug delivery, as described previously (Malkmus and Yaksh, 2004). In brief, rats were anaesthetized by induction with 4% isoflurane in a room air/oxygen mixture (1:1), and the anaesthesia was maintained with 2% isoflurane delivery by mask. The animal was placed in a stereotaxic headholder with the head flexed forward. A midline incision was made on the back of the occipital bone and the neck to expose the cisternal membrane. The membrane was carefully opened with a stab blade, and a single lumen polyethylene-5 (outer diameter 0.36 mm) catheter (8.5 cm) was inserted and passed into the i.t. space surrounding L3–L4 spinal segments. The other end of the catheter was jointed to a polyethylene-10, which was tunnelled s.c. to exit through the top of the head. Catheters were flushed with 10 ml of saline and then plugged. The rats were given 5 mL of lactated ringer’s solution s.c. and allowed to recover under a heat lamp; those showing motor weakness or signs of paresis on recovery from anaesthesia were killed immediately. The rats were allowed to recover for 5–7 days before the experiment. a2-Adrenoceptor agonists and antagonists on formalin-induced flinching Formalin-induced flinching was measured using an auto- mated detection system (Yaksh et al., 2001). A soft metal band (10 mm wide and 27 mm long, shaped into a C, and weighingB0.5 g) was placed around the left hindpaw of the animal being tested. Animals were allowed to acclimate in individual Plexiglas chambers for 30 min before experimen- tal manipulations. For dexmedetomidine and ST-91 dose– response studies, rats were administered with dexmedetomi- dine (0.01, 0.1, 0.3, 1 or 2 mg), ST-91 (0.3, 0.9, 3, 10 or 30 mg) or saline 10 min before a s.c. injection of formalin (5%, 50 mL) into the dorsal side of the banded paw. Immediately after the formalin injection, rats were placed into the test chamber and nociceptive behaviour was quantified by automatic counting of spontaneous flinching and shaking of the injected paw. Flinches were counted in 1-min intervals for 60 min. The data are expressed as total number of flinches observed during phase 1 (0–9 min) and phase 2 (10–60 min). In studies with the antagonist drugs, the a2A-adrenoceptor antagonist, BRL44408 (Young et al., 1989) or the a2B/C- adrenoceptor antagonist, ARC239 (Bylund et al., 1992) were administered 10 min before the administration of dexmede- tomidine or ST-91. All drugs were injected i.t. in a volume of 10 mL followed by a 10 mL saline flush. a2-Adrenoceptor agonists on formalin- and paw compression- induced NK1 receptor internalization After recovery from i.t. catheter implantation, rats were administered i.t. with dexmedetomidine (0.3–10 mg), ST-91 (30 mg), morphine (20 mg) or saline. Five minutes after i.t. drug administration, rats were anaesthetized with sodium pentobarbital (50 mg kg�1, i.p.). For formalin-induced NK1r internalization, intraplantar formalin injection (5%, 50 mL) to the left hindpaw occurred 5 min after the anaesthesia. Rats were killed and transcardially perfused with fixative 8 min after the formalin injection. For paw compression-induced NK1r internalization, dexmedetomidine (1 or 10 mg), ST-91 (30 mg), morphine (20 mg) or saline was administered i.t. Five minutes after i.t. drug administration, rats were anaesthe- tized with sodium pentobarbital (50 mg kg�1, i.p.) and 5 min after anaesthesia, the hindpaw was positioned perpendicu- larly across the jaws of a 6-inch mosquito forceps with non- serrated jaws. The jaws were closed to the third click of the hemostat ratchet. Compression was applied for 60 s. Applica- tion of the forceps produces an evident compression of the soft tissue resulting in approximately a 30% reduction in paw thickness during the compression. This stimulus has been previously used to evoke NK1r internalization (Ghilardi et al., 2004; Kondo et al., 2005; Nazarian et al., 2008). Rats were transcardially perfused 5 min after the paw compression (see Immunocytochemistry). Dexmedetomidine and a2-adrenoceptor antagonists on formalin- induced Fos expression After the rats had recovered from i.t. catheter implantation, they were administered i.t. with BRL44408 (100mg), ARC239 (100mg) or saline. Ten minutes after antagonist administration, a2-Adrenoceptor analgesia mechanism of action A Nazarian et al1118 British Journal of Pharmacology (2008) 155 1117–1126 rats were administered with dexmedetomidine (1 mg), mor- phine (20 mg) or saline. Intraplantar formalin (5%, 50 mL) was injected 10 min after agonist administration. Rats were anaesthetized with sodium pentobarbital (50 mg kg�1, i.p.), 1 h 55 min after formalin administration. Five minutes after induction of anaesthesia rats were transcardially perfused. Tissue preparation and immunocytochemistry Anaesthetized rats were transcardially perfused with 0.9% NaCl followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PBS), pH 7.4. The lumbar spinal cord was removed and postfixed overnight. After cryoprotection in 20% sucrose, coronal sections were made using a sliding microtome (30 mm for NK1r staining and 50 mm for Fos staining). Immunofluorescent staining was performed to examine NK1r expression in the spinal dorsal horn. In brief, sections were incubated in a rabbit anti-NK1r polyclonal antibody overnight at room temperature. The antibody was diluted to a concentration of 1:3000 in 0.01 M PBS contain- ing 10% normal goat serum and 0.3% Triton X-100. After rinses in PBS, sections were then incubated for 90 min at room temperature in a goat anti-rabbit secondary antibody conjugated with Alexa 488 diluted at 1:1000 in 0.01 M PBS containing 10% normal goat serum and 0.3% Triton X-100. All sections were finally rinsed and mounted on glass slides and coverslipped with ProLong mounting medium. Immunocytochemical staining for Fos consisted of blocking endogenous peroxidate activity by incubating the spinal cord sections in 0.3% H2O2 for 30 min. Non-specific binding sites were blocked in 0.5% Triton X-100 and 5% normal goat serum in PBS. For primary antibody incubation, tissues were incubated in rabbit anti-Fos polyclonal antibody (1:10 000) for 72 h at 4 1C. The binding sites were visualized by using an ABC kit. The signal was developed in diaminobenzidine–tetrahydrochloride solution containing H2O2 in PBS. Upon the presence of a light background, the reaction was stopped by two washes in distilled water. Sections were mounted on slides, air-dried, dehydrated through graded ethanol solution followed by citrisolve and then coverslipped with Permount mounting medium. Quantification of NK1 receptor internalization and Fos expression Neurokinin1 receptor internalization was counted using an Olympus fluorescence microscope at �60 magnification and followed the standard of previous reports (Mantyh et al., 1995; Abbadie et al., 1997). The total number of NK1r- immunoreactive neurons in lamina I, with or without NK1r internalization, was counted and taken as a ratio of cells showing NK1r internalization vs all NK1r-positive cells and then converted into a percentage of NK1r-immunoreactive cells. Neuronal profiles that had 10 or more endosomes in their soma and the contiguous proximal dendrites were considered to have internalized NK1rs. NK1r-positive neurons in both sides of the dorsal horn were counted. The person counting the neurons was blinded to the experi- mental treatments. Mean counts from three to five sections per segment of the lumbar spinal cord were used as representative counts for a given animal. Three to five animals per drug treatment group were used for statistical analysis (n¼3–5). Fos immunoreactivity was quantified by counting Fos- positive cells in lamina I of the lumbar L3–4 segments of the spinal cord ipsilateral and contralateral to the formalin-treated paw. The mean counts from five sections were used as representative counts for a given animal. Four animals per drug treatment group were used for the statistical analysis (n¼4). Confocal microscopy and image processing Confocal images of representative NK1r cells were acquired by a Leica TCS SP2 confocal system equipped with AOBS with a �63 objective (1.4 numerical aperture) and an argon 488 nm laser line with a pinhole of 1 airy unit. Images were acquired at a digital size of 1024�1024 pixels. Five to ten adjacent optical sections (B0.5-mm separation) along the z-axis were projected together for demonstration. Images were processed with Adobe Photoshop software (version 8.0) by using the ‘curves’ option to adjust image contrast. Drugs, antibodies and materials Dexmedetomidine (Precedex, Hospira Inc., Lake Forest, IL, USA) was purchased from the pharmacy of the University of California, San Diego Medical Center. Dexmedetomidine in powder form was kindly provided by Dr Donna L Hammond from the University of Iowa. ST-91 (N-(2,6-diethyl- phenyl)-4,5-dihydro-1H-imidazol-2-amine hydrochloride) was provided by Boehringer Ingelheim (Ridgefield, CT, USA), Formalin solution was purchased from Sigma Chemicals (St Louise, MO, USA). BRL44408 (2-[(4,5-dihydro-1H-imida- zol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole maleate) and ARC239 (2-[2-(4-(2-methoxyphenyl)piperazin-1-yl)ethyl]- 4,4-dimethyl-1,3-(2 H,4 H)-isoquinolindione dihydrochloride) were purchased from Tocris Biosciences (Ellisville, MO, USA). Dexmedetomidine, ST-91 and BRL44408 were dissolved in saline, whereas ARC239 was dissolved in 10% 2-hydroxy- propyl-b-cyclodextrin. All drugs were prepared on the day of testing. The rabbit anti-NK1r polyclonal antibody was purchased from the Advanced Targeting Systems (San Diego, CA, USA) and the rabbit anti-Fos polyclonal antibody was purchased from Calbiochem-EMD Biosciences (La Jolla, CA, USA). Secondary Alexa 488-conjugated antibody was purchased from Invitrogen (Eugene, OR, USA). ProLong mounting medium was obtained from Invitrogen, ABC kit from Vector Laboratories (Burlingame, CA, USA) and Permount mounting medium was from Fisher Scientific (Pittsburgh, PA, USA). Nomenclature for drugs and receptors conform with the guide to receptors and channels of the British Journal of Pharmacology (Alexander et al., 2008). Statistical analysis Changes in formalin-induced paw-flinching behaviour were analysed using separate one-way ANOVA for phases 1 and 2. Upon detection of a significant ANOVA, Tukey’s post hoc tests were performed for pair-wise comparisons of drug-treated a2-Adrenoceptor analgesia mechanism of action A Nazarian et al 1119 British Journal of Pharmacology (2008) 155 1117–1126 groups with their relative control within phase 1 or 2. The analyses for NK1r internalization data consisted of one-way or two-way ANOVAs. To detect the differences in the presence of a significant one-way ANOVA, Tukey’s post hoc analysis was conducted. In the presence of a significant two- way ANOVA, Bonferroni post hoc tests were applied. The analysis of the Fos data consisted of a paired t-test and a one-way ANOVA. Tukey’s post hoc test was used for pair-wise comparisons. In all analysis, probability to detect the differences was set at the 5% level (Po0.05). Results Effects of dexmedetomidine and ST-91 on formalin-induced paw-flinching behaviour The effects of dexmedetomidine and ST-91 on formalin- induced paw-flinching behaviour are shown in Figures 1a and b, respectively. Dexmedetomidine dose-dependently reduced the formalin-induced paw flinches in phase 1: F(5,21)¼6.42, Po0.001; with doses of 1 and 2 mg producing a significant reduction in flinching. In phase 2, dexmede- tomidine reduced the formalin flinches at doses of 0.3, 1 and 2 mg: F(5,21)¼8.05, Po0.001. In this experiment, the 2mg dose of dexmedetomidine produced mild sedation, but the 1 mg dose of dexmedetomidine did not appear to have sedative effects; similarly, i.t. dexmedetomidine (1 mg) was not found to alter the rotarod performance in rats (Ida¨npa¨a¨n-Heikkila¨ et al., 1994). Accordingly, the 1mg dose of dexmedetomidine was typically employed in subsequent experiments, unless otherwise noted. ST-91 dose-depen- dently reduced the formalin-induced paw flinches in phase 1: F(5,31)¼5.74, Po0.001, with 10 and 30 mg doses produ- cing a significant reduction in flinching. In phase 2, ST-91 at doses of 3, 10 and 30 mg reduced the formalin flinching: F(5,31)¼11.36, Po0.001, with no effect upon arousal noted at the highest dose. a2-Adrenoceptor antagonists on dexmedetomidine and ST-91 analgesia The effects of the a2A-adrenoceptor antagonist, BRL44408 on dexmedetomidine and ST-91 analgesia are presented in Figures 2a and b. In phase 1, dexmedetomidine reduced the formalin-flinching behaviour: F(4,22)¼13.14, Po0.001, Whereas the i.t. pretreatment with BRL44408 (10 and 100 mg) blocked the dexmedetomidine-induced reduction in forma- lin flinching. Equally, in phase 2, dexmedetomidine reduced the formalin-flinching behaviour; whereas pretreatment with BRL44408 (10 and 100 mg) blocked the dexmedetomi- dine-induced reduction in formalin-flinching behaviour: F(4,22)¼17.72, Po0.001. ST-91 did not significantly alter phase 1 formalin-flinching behaviour and BRL44408 pre- treatment had no effect on ST-91-treated rat