Spinal Adrenergic and Cholinergic Receptor Interactions Activated by Clonidine in Postincisional Pain

The study was approved by the Animal Care and Use Committee of the Wake Forest University School of Medicine (Winston Salem, NC). Male Sprague-Dawley rats (250–300 g) obtained from Harlan (Indianapolis, IN) were used in all experiments. Animals were housed under a 12-h light–dark cycle, with food and water ad libitum . For intrathecal drug administration, sterilized 32-gauge tubing (RecathCo, Allison Park, PA) connected to 8.5-cm Tygon® external tubing (Saint-Gobain Performance Plastics, Akron, OH) was inserted during halothane anesthesia, as previously described. 10The catheter was passed caudally from the cisterna magnum to the level of lumbar enlargement (7.5–8.0 cm). Only animals without evidence of neurologic dysfunction after catheter insertion were studied. Correct position of the lumbar catheter was verified in a subset (n = 68) of animals after surgery. All studies were performed at least 7 days after insertion of the intrathecal catheter. Paw incision was performed as described by Brennan et al.  11Animals were anesthetized with halothane; the plantar surface of the left hind paw was prepared with 70% ethanol; and a 1-cm longitudinal incision was made through the skin and fascia, starting 0.5 cm from the edge of the heel and extending toward the toes. The plantaris muscle was elevated and incised longitudinally. The wound was closed with two silk 5.0 sutures.

For determining withdrawal threshold, rats were placed individually in plastic cages with a plastic mesh floor. Animals were tested after acclimation to the environment, typically 30 min after being placed in the cage. Withdrawal threshold to punctuate mechanical testing was determined using calibrated von Frey filaments (Stoelting, Wood Dale, IL), beginning with the 2.0-gauge filament. Filaments were applied vertically to an area adjacent to the wound at the heel for 4 s while the hair was bent. Brisk withdrawal or paw flinching was considered a positive response. In the absence of a response, the filament of next greater force was applied. In the presence of a response, the filament of next lower force was applied. The tactile stimulus producing a 50% likelihood of withdrawal was determined using the up–down method, as described by Chaplan et al.  12Tests were performed in duplicate, with an approximate 3-min test-free period between withdrawal responses, and their average was used. Studies were performed on the first day after paw incision surgery.

All drugs experimental treatment was carried out 24 h after plantar incision.

The α²-adrenoceptor agonists used in this study were clonidine hydrochloride (non α²-adrenoceptor subtype selective, molecular weight of 267 g/mol, nonsubtype selective; Sigma Chemical, St. Louis, MO) and ST91 hydrochloride (α²non-A preferring, molecular weight of 254 g/mol; Boehringer Ingelheim, Ridgefield, CT). Animals received cumulative dosing at 40-min intervals of intrathecal clonidine (19, 56, 190 nmol) or at 60-min intervals of intrathecal ST91 (20, 59, 197 nmol). Timing of cumulative injections and dose range were determined in pilot experiments with each drug. Dose–response curves were constructed after conversion of withdrawal thresholds to percent maximum possible effect. Percent maximum possible effect was defined as: 100 × (postdrug response − baseline)/(prepaw incision threshold − baseline). Agonists were administered intrathecally in volumes of 10 μl, and thresholds for withdrawal were determined 40 min after clonidine administration and 60 min after ST91 injection.

The α²-adrenoceptor subtype antagonists were BRL44408, selective for the α²-A-adrenoceptor subtype, and ARC239, selective for α²-non-A-adrenoceptor subtypes (both from Tocris Cockson, Ballwin, MO). Antagonists or vehicles were injected intrathecally in a volume of 10 μl 15 min prior to agonist administration. Based on pilot experiments, we used probe doses of 94 nmol clonidine (25 μg) and 59 nmol ST91 (15 μg), and doses of 9.4 or 94 nmol BRL (n = 7–10 animals) and of 9.4 or 94 nmol ARC (n = 5–9 animals). All studies were conducted with the investigator blinded to antagonist or vehicle administered. Drugs were dissolved in normal saline or, when necessary, in 2-hydroxypropyl-β-cyclodextrin (Sigma Chemical).

Muscarinic and nicotinic cholinergic receptor antagonism was performed with intrathecal injection of atropine sulfate (44 nmol) and mecamylamine hydrochloride (491 nmol). We have previously shown that the intrathecal doses of selected antagonists inhibit corresponding cholinergic and α²-adrenoceptor ligands in rats subjected to mechanical stimuli. 8,13 

Data are represented as mean ± SD. Dose–dependence of clonidine and ST91 was evaluated by linear regression analysis. Paw withdrawal thresholds in response to mechanical stimulation before and after paw incision were compared using a paired Student t  test. Effects of individual drugs on withdrawal thresholds were determined using a two-way analysis of variance for repeated measures followed by the Bonferroni correction for appropriate multiple comparisons. P < 0.05 was considered significant.

One hundred eighty-nine rats were included in the study. The average withdrawal threshold to punctuate mechanical stimulus before paw incision surgery was 40 ± 14 g and decreased to 4 ± 3 g within 24 h after plantar incision (P < 0.05).

ST91 produced antihypersensitivity to von Frey filaments with similar potency but significantly greater efficacy than clonidine (fig. 1). ST91 produced transient serpentine tail movements, which were no longer present when withdrawal thresholds were evaluated. Larger doses of clonidine could not be studied due to intense behavioral sedation.

Intrathecal injection of 94 nmol of clonidine increased the withdrawal threshold significantly 40 min after injection (figs. 2A and 2B). Intrathecal injection of BRL44408 and ARC239 significantly inhibited the antihypersensitivity effect of intrathecal clonidine in a dose-dependent manner (figs. 2A and 2B).

Intrathecal injection of 59 nmol ST91 significantly increased the withdrawal threshold 60 min after injection (fig. 3A and B). Intrathecal injection of ARC239 inhibited the antihypersensitivity effect of intrathecal ST91 in a dose-dependent manner (fig. 3A). In contrast, intrathecal injection of BRL44408 failed to influence the antihypersensitivity effect of ST91 (fig. 3B). Intrathecal injection of vehicle or either antagonist alone did not alter the withdrawal threshold (control time points:figs. 2 and 3).

Intrathecal injection of 94 nmol of clonidine increased the withdrawal threshold significantly 40 min after injection (fig. 4A). Intrathecal injection of atropine sulfate and mecamylamine significantly inhibited the antihypersensitivity effect of intrathecal clonidine (fig. 4A).

Intrathecal injection of 59 nmol ST91 increased the withdrawal threshold significantly 60 min after injection (fig. 4B). Neither intrathecal injection of atropine nor of mecamylamine altered the antihypersensitivity effect of intrathecal ST91 (fig. 4B). Intrathecal injection of vehicle or either antagonist alone failed to alter the withdrawal threshold (control time points:fig. 4).

Anesthesiologists deal most commonly with acute pain associated with surgery. Despite the continuing evidence that most patients after surgery experience episodes of severe pain, 14which can lead to development of chronic pain, 15most pain research focuses on chronic inflammatory, metabolic, or neuropathic pain conditions. Seminal work by Brennan et al.  describing the behavioral, 11neurophysiologic, 16and pharmacologic 17consequences and properties of surgery clearly indicate that postoperative pain differs in many fundamental ways from these other conditions, and we and others have argued that the relative lack of basic research in this area hampers better treatment for our postoperative patients. 18The current study uses the method developed by Brennan et al.  11,16,17and carries important implications for drug development and drug mechanisms.

Like opioids, cyclooxygenase inhibitors, and glutamate receptor antagonists, the analgesic potency and maximum efficacy of intrathecal or epidural clonidine depends heavily on the type of clinical pain it is used to treat. This was first noted when the bolus or continuous infusion doses of epidural or intrathecal clonidine to effectively treat neuropathic pain 19were found to be less than 25% those needed to treat postoperative 20–22or obstetricpain. 23Similarly, potency of clonidine to reduce experimentally induced allodynia was increased compared to treatment of acute noxious stimuli in normal volunteers, 24indicating a shift in drug action or mechanism in the presence of central sensitization. These clinical observations are paralleled in studies with rodents, where generation of hypersensitivity from nerve injury increases potency and efficacy of intrathecal clonidine. 25,26The current study in rats suggests that hypersensitivity to mechanical stimuli surrounding a skin incision lies closer in this regard to the normal condition than that following nerve injury. Intrathecal clonidine's potency in the current study of postoperative hypersensitivity is similar to that observed in rats to acute noxious stimuli. 27 

All three α²-adrenoceptor subtypes (A, B, and C) are present in rat spinal cord, 28with a preponderance of α²A and α²C subtypes. 29Anatomic and pharmacologic evidence suggests a shift in analgesic mechanisms of intrathecal clonidine from α²A predominant in the normal rat to α²C predominant after nerve injury. In the normal rat, antinociception of clonidine is reduced by acute knockdown of α²A adrenoceptors using antisense oligodeoxynucleotides 30and reversed by the α²A adrenoceptor preferring antagonist, BRL44408. 8Following nerve injury there is a loss of α²A adrenoceptor immunoreactivity in the spinal cord, but no change or an increase in α²C adrenoceptor immunoreactivity, 31and antinociception of clonidine in this case is unresponsive to antagonism by BRL44408, but reversed by the α²non-A-adrenoceptor antagonist, ARC239. 8 

The current study suggests that the mix of α²-adrenoceptor subtypes responsible for clonidine antinociception following surgery is intermediate between that in the normal animal and that in the neuropathic animal. Thus, BRL44408 reversed clonidine's effect in the current study, indicating a role for α²A adrenoceptors, unlike the results after nerve injury. ARC239 was similarly potent to BRL44408 to reverse clonidine's effect in normal animals, 8but was nearly 10-fold more potent to reverse clonidine's effect after surgery in the current study, indicating a partial, but not complete shift to an α²non-A-adrenoceptor mechanism.

Stimulation of spinal cholinergic systems by intrathecal injection of α²-adrenoceptor agonists is widely documented in humans and animals. Thus, intrathecal clonidine increases acetylcholine concentrations in cerebrospinal fluid 32and analgesic effect of clonidine is potentiated by intrathecal neostigmine in humans. 33The reliance of antinociception from clonidine on this spinal cholinergic interaction varies between normal and nerve-injured animals. Thus, intrathecal clonidine antinociception to acute noxious thermal stimuli in normal rats is unaffected by intrathecal atropine or mecamylamine (unpublished observations, Xavier Paqueron, M.D., Research Fellow, Department of Anesthesiology, Wake Forest University School of Medicine, June 2000), but the reversal of hypersensitivity by clonidine following spinal nerve ligation is completely blocked by intrathecal atropine and partially blocked by intrathecal mecamyla-mine. 13The current study suggests that postoperative hypersensitivity most resembles nerve injury–induced hypersensitivity in this regard since the effect of clonidine was completely antagonized by atropine and partially by mecamylamine. Should these data apply to humans, they provide a rationale for the study of epidural clonidine–neostigmine combinations for postoperative analgesia.

ST91, the diethyl derivative of clonidine, was described more than 25 yr ago to explain the lack of hypotensive and sedative properties of clonidine when administered systemically, originally ascribed to its hydrophilic nature. 34Shortly thereafter, it was demonstrated to produce antinociception after intrathecal injection in rats, 35but with a different structure activity relation for blockade by α²-adrenoceptor antagonists than clonidine or dexmedetomidine, 6,36and with differential cross-tolerance with these agents, 37suggesting it acted on different α²-adrenoceptor subtypes. More recent observations of synergistic interactions between dexmedetomidine and ST91 reinforce these early studies. 7As in normal animals and those with spinal nerve ligation-induced hypersensitivity, 8the effect of ST91 after surgery in the current study was reversed by the α²non-A-adrenoceptor antagonist ARC239 but was unaffected by the α²A-adrenoceptor–preferring antagonist BRL44408 or the cholinergic antagonists.

ST91 could represent an improvement on clonidine in the treatment of postoperative pain. Although ST91 exhibits less intrinsic activity than clonidine or dexmedetomidine in normal rats, 38it is more effective than dexmedetomidine for antinociception in some strains of rats and in some thermal assays. 7ST91 was more efficacious than clonidine in the current study, although whether this was due to a partial agonist effect of clonidine could not be determined, due to dose-limiting sedation by this agent. Sedation and hypotension limit the use of clonidine in the treatment of postoperative pain. 39ST91 does not cause sedation after systemic or intrathecal administration, nor does it reduce blood pressure after intrathecal administration in several species. 5,34,35,40Of course, human trials must await proper preclinical chemistry, formulation, stability, toxicity, and neurotoxicity studies.

In summary, intrathecal clonidine partially reverses the reduced withdrawal threshold to mechanical stimulation near a paw incision in rats through the actions of α²A- and α²non-A-adrenoceptor subtypes. This is also a mechanism that involves stimulation of spinal cholinergic receptors. These data suggest that the proportion of α²-adrenoceptor subtypes activated by clonidine to produce antinociception in the postoperative state differs from the normal animal with acute noxious stimuli and the nerve-injured animal with hypersensitivity. ST91 was more efficacious than clonidine, perhaps because larger relative doses could be administered without sedation, and it relied entirely on α²non-A-adrenoceptor subtypes. These data add to the growing literature indicating that postoperative pain exhibits a unique pharmacology of analgesia compared with acute experimental or chronic nerve injury pain. The data also provide the rationale for the clinical study of epidural clonidine–neostigmine combinations and for development of ST91 for clinical trials.