Effect of antisense knock-down of α 2a- and α 2c-adrenoceptors on the antinociceptive action of clonidine on trigeminal nociception in the rat

Abstract

Although activation of α 2-adrenoceptors is known to play an important role in mediating antinociception, the contribution of various α 2-adrenoceptor subtypes in modulating trigeminal nociception remains unknown since subtype specific agonists and antagonists are not available. The present study investigated the functional role of α 2-adrenoceptor subtypes in modulating the N-methyl- d-aspartate-induced nociceptive behavior in the medullary dorsal horn by using antisense oligodeoxynucleotides to selectively knock-down the receptor subtypes. Microinjection of N-methyl- d-aspartate (2 nmol in 10 μl) through a cannula implanted dorsal to the medullary dorsal horn produced a total of 164.9±8.8 scratches in the facial region ( n=14), and the scratching behavior lasted for 77.8±5.2 s ( n=14). Microinjection of clonidine, an α 2-agonist (7 μg in 5 μl), 15 min prior to administration of N-methyl- d-aspartate, produced a reduction of 71.6% ( n=12) in the number of scratches and a reduction of 57.5% ( n=12) in the duration. The inhibitory effect of clonidine was blocked by idazoxan ( n=4) and yohimbine ( n=4), α 2 antagonists. In rats pretreated with the antisense probe to the α 2A adrenoceptor, clonidine only produced a reduction of 7.3% in the number of scratches ( n=12) and a reduction of 9% in the duration ( n=12). The antinociceptive effect of clonidine recovered completely 4 days after termination of the α 2A antisense oligodeoxynucleotide treatment. In contrast to the α 2A antisense-treated animals, clonidine reduced the number of scratches and the duration by 85.5% ( n=9) and 82.1% ( n=9), respectively, in rats pretreated with the sense probe to the α 2A adrenoceptor. The effect of clonidine was not altered in rats pretreated with the antisense or the sense probes to the α 2C adrenoceptor. In the α 2C antisense pretreated rats, clonidine reduced the number of scratches and the duration by 60.8% ( n=11) and 44.5 % ( n=11), respectively. In the sense-pretreated rats, clonidine produced a reduction of 69.1% in the number of scratches ( n=9) and a reduction of 55.1% in the duration ( n=9). In order to assess the effectiveness of the antisense treatment, the receptor expression was examined by immunohistochemistry. Antisense treatment reduced α 2A and α 2C receptor immunoreactivity in the medullary dorsal horn compared to the sense and the vehicle-treated animals. Quantitative image analysis revealed a significant decrease in pixel intensity following the antisense treatment. These results indicate that activation of α 2A adrenoceptor plays an important role in mediating the antinociceptive effect of clonidine in the medullary dorsal horn in the rat.