Low Dose Alpha-2 Antagonist Paradoxically Enhances Rat Norepinephrine and Clonidine Analgesia

Ultralow-dose opioid antagonists prolong opioid antinociception and block tolerance. In this study we determined whether low doses of the &agr;-2 adrenergic receptor (A2-R) antagonist, atipamezole, similarly influenced A2-R-induced antinociception and tolerance. In rats, intrathecal norepinephrine (NE) or clonidine in combination with atipamezole was tested using tail-flick and paw pressure tests. Acute tolerance to NE was induced by serial injections. Low-dose atipamezole significantly prolonged NE and clonidine-induced antinociception. Coadministration of atipamezole with A2-R agonists also prevented loss of agonist potency in the acute tolerance model. This study demonstrates paradoxical effects of low-dose A2-R antagonists augmenting A2-R agonist-induced analgesia.

gic receptor agonists, like opioids, produce potent analgesia, and their repeated administration induces tolerance. 1,2 Numerous reports, including clinical pilot studies, have demonstrated that ultralow doses of opioid receptor antagonists paradoxically augment spinal opioid-induced analgesia and block acute and chronic tolerance. [3][4][5][6] Considering the interaction between opioid and adrenergic receptors, 7,8 we examined whether ultralow doses of the selective ␣-2 antagonist, atipamezole, can similarly influence NE-induced spinal antinociception and tolerance. The specific objectives of the current investigation were to determine whether ultralow-dose atipamezole influenced spinal NE and clonidine-induced antinociception and the development of acute tolerance to repeated administration of spinal NE.

METHODS
Experiments were performed on male Sprague-Dawley rats (275 to 300 g) in accordance with the Guidelines of the Canadian Council on Animal Care after approval from the Queen's University Animal Care Committee. Animals were maintained on a normal light-dark cycle and allowed free access to food and water. Indwelling intrathecal catheter (7.5 cm, PE-10) implantation was performed under isoflurane anesthesia, as has been previously described. 9 Drugs under investigation were administered through the exteriorized portion of the catheter in a 10-L volume and flushed with 10 L of 0.9% saline. Control animals received equivalent injections of saline. All behavioral testing was performed without knowledge of the treatment between 0800 to 1400 hours.
The tail-flick test 10 evaluated the response to a brief thermal stimulus applied to the tail (5 cm from the end) with an analgesia meter. 11 Baseline latencies were set between 2 to 3 seconds and cutoff time at 10 seconds to prevent tissue damage. The paw pressure test evaluated responses to a brief mechanical nociceptive stimulus applied to the dorsal hindpaw by using an inverted air-filled syringe linked to a pressure gauge (baseline 80 to 100 mm Hg, cutoff time at 300 mm Hg). 12

Acute Tolerance
Tolerance to NE (30 g) or in combination with atipamezole (0.8 or 0.008 ng) was induced by 3 serial injections (intrathecal) delivered at 90-minute intervals with animals tested over a 240-minute time period. Twenty-four hours after these serial injections, cumulative dose-response curves to the acute effects of NE were derived to obtain quantitative estimates of agonist potency (ED 50 values). Tolerance was indicated by a loss of agonist potency (i.e., an increase in ED 50 value).
All tail-flick and paw pressure values were converted to percentage of maximum possible effect (M.P.E.) (% MPE ϭ 100 ϫ (postdrug response Ϫ baseline response)/(cutoff response Ϫ baseline response)). Data are expressed as mean Ϯ sem for n ϭ 4 to 11 per group. ED 50 values were determined using nonlinear regression analysis. A 2-way repeated-measures analysis of variance (ANOVA) with time as a within-subject factor and treatment as a betweensubjects factor was used to account for the repeatedmeasures design. A Time ϫ Treatment interaction was included to test for differences in the longitudinal response patterns. Tukey post hoc tests were conducted where appropriate.
Three successive intrathecal injections of NE (30 g) 90 minutes apart resulted in a progressive loss of NE-induced antinociception illustrating acute tolerance. NE-induced antinociceptive effects at 240 minutes were reduced to nearly 35% of the initial response value at 30 minutes in the tail-flick test (Fig. 3A) and 22% of the initial response at 30 minutes in the paw pressure test (Fig. 3B). In addition, repeated intrathecal NE administration was associated with an approximately 2.5-fold increase in the ED 50 value (Fig.  3C). In contrast, the combination of NE with a low dose of atipamezole prevented both the decline of the antinociceptive response (0.8 ng, Fig. 3) and the increase in ED 50 values (0.8, 0.008 ng, Fig. 3C).

DISCUSSION
The results demonstrate that ultralow doses of an ␣-2 adrenergic receptor antagonist have the ability to augment agonist-mediated acute antinociception and to block the development of tolerance at doses far below those previously reported to have antagonistic effects at the ␣-2 adrenergic receptor. 13 The antitolerance effect of atipamezole is indicated by both the prevention of the progressive loss of the pharmacological response and decline in the agonist potency. The ultralow doses of atipamezole were selected by extrapolation from doses of the drug shown previously to influence morphine analgesia and tolerance. 13 Our goal was to investigate the lowest dose that produced consistent effects on analgesia without visible adverse effects on the mobility of animals. Scouting experiments were performed to establish doses that met this criteria and hence the difference between the doses used in the clonidine and NE experiments. We have previously demonstrated that low-dose ␣-2 receptor antagonists augment spinal morphine analgesia and inhibit the development of tolerance. 13 Although the mechanisms underlying this unusual effect of atipamezole remain unclear, it may be that it acts in this regard on the opioid-␣-2 receptor complex 13,14 to prevent excitatory signaling by opiates, block the latent stimulatory response to the agonist, prevent hyperalgesia, and thus augment analgesia. Similar to the excitatory effects of opiates, first reported in electrophysiologic studies on the dorsal root ganglion neurons, 15,16 a low dose of clonidine can increase dorsal horn neuronal activity stimulated by nociceptive input, 17 and prolonged spinal administration of clonidine to rats causes thermal hypersensitivity, which reflects hyperalgesia. 18 A low dose of the antagonist may minimize or prevent the acute desensitization of receptor population-mediating analgesia or prevent the adaptive pronociceptive responses (such as N-methyl-d-aspartate receptor activation) that serve to limit adrenergic analgesia and contribute to the induction of analgesic tolerance. 18   In panels A and B, NE (30 g, intrathecal) was injected at 0, 90, and 180 minutes to induce acute tolerance, as is indicated by the arrows. Atipamezole was coadministered with NE as a single injection. Behavioral testing was performed at 30-minute intervals. A 2-way repeated-measures analysis of variance (ANOVA) of the tail-flick data revealed a significant effect of Treatment (F (2,72) ϭ 46.00, ***P Ͻ 0.001), Time (F (8,72) ϭ 97.20,***P Ͻ 0.001), and a Time ϫ Treatment interaction (F (16,72) ϭ 30.26, ***P Ͻ 0.001). Likewise, a 2-way repeated-measures ANOVA of the paw pressure data revealed a significant effect of Treatment (F (2,72) ϭ 19.70, **P Ͻ 0.01), Time (F (8,72) ϭ 19.70,***P Ͻ 0.001), and a Time ϫTreatment interaction (F (16,72) ϭ 17.20, ***P Ͻ 001). Tukey post hoc comparisons revealed which corresponding data points between the NE-only and NE ϩ ATPZ treatment groups were significantly different (*P Ͻ 0.05, **P Ͻ 0.01, ***P Ͻ 0.001). In panel C, the NE ED 50 values were obtained from cumulative dose-response curves to the antinociceptive action of NE 24 hours after the initial intrathecal drug treatment (panels A and B). One-way ANOVAs revealed significant differences between treatments both within the tail-flick paradigm (F (3,14) ϭ 524.1, ***P Ͻ 0.001) and within the paw pressure paradigm (F (3,14) ϭ 1008, ***P Ͻ 0.001). Tukey post hoc analysis were used to compare each treatment group with the saline controls (*P Ͻ 0.05, ***P Ͻ 0.001) and with the NE-only-induced effects (ϩϩϩP Ͻ 0.001). M.P.E. ϭ maximum possible effect.