Spinal Adenosine Receptor Activation Reduces Hypersensitivity after Surgery by a Different Mechanism Than after Nerve Injury

The study was approved by the Animal Care and Use Committee of the Wake Forest University School of Medicine (Winston-Salem, North Carolina). Male Sprague-Dawley rats (weight, 250 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 administration, a sterilized 32-gauge polyethylene catheter (RecathCo, Allison Park, PA) connected to 8.5 cm Tygon external tubing (Saint-Gobain Performance Plastics, Akron, OH) was inserted under halothane anesthesia, as previously described. 16The catheter was passed caudally 7.5 cm from the cisterna magnum to the lumbar enlargement. Only animals without evidence of neurologic dysfunction after catheter insertion were used for studies. Paw incision as described by Brennan et al.  13was performed 5 days after intrathecal catheter implantation. For this, rats were anesthetized with halothane, and after sterile preparation with 70% ethanol, a 1-cm long incision was made in the plantar aspect of the left hind paw, starting 0.5 cm from edge of the heel toward the toe. The plantaris muscles was elevated and incised longitudinally. The wound was closed with two mattress sutures of 5.0 silk.

Rats were placed individually in a plastic cage with a plastic mesh floor and were allowed to acclimate to the environment for 30 min. Withdrawal threshold 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 for 6 s while the filament was gently bent. 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.  17General behavior, including ambulation and activity level, was assessed throughout the time of testing. The investigator was blinded to drug treatment for all studies.

Drug testing was performed 24 h after paw incision. Rats received intrathecal T62 (0.3, 0.5, 1, and 3 μg) or clonidine (1, 3, and 10 μg). The withdrawal threshold was determined before (prepaw incision threshold) and 24 h after incision (baseline) and then every 30-min for 4 h after intrathecal injection, using an up–down method with von Frey filaments. Dose–response curves were conducted from a peak effect at each dose after conversion of withdrawal thresholds to percentages of maximum possible effect (%MPE), where %= 100 × (Postdrug Response − Baseline)/(Prepaw Incision Threshold − Baseline).

After determining the efficacy of each drug, isobolographic analysis was performed to determine the type of interaction between T62 and clonidine. The dose producing a 40% MPE (ED⁴⁰) was calculated from dose– response curves for each drug in reducing postoperative hypersensitivity. Using an ED⁴⁰was necessary because maximal effects of T62 in the dose range studied were not greater than 50% MPE. For drug combination dose responses, a fixed-ratio combination of T62 and clonidine in a ratio of 0.16:1 wt/wt was administered. Because the time of peak effect for T62 was 30 min later than clonidine, T62 was administered first, followed by clonidine injection 30 min later.

An antagonist study was performed to test whether the effect of T62 in the postoperative pain model involves α²-adrenergic receptor stimulation. The selective α²-adrenoceptor antagonist idazoxan, 30 μg, or saline was administered intrathecally 15 min before T62 injection. The effect of idazoxan alone was tested in another group of animals.

Drugs were administered intrathecally in a volume of 5 μl and followed by a 10-μl saline injection to flush the catheter. T62 was dissolved in 20% dimethyl sulfoxide, except for the highest dose, which was dissolved in 30% dimethyl sulfoxide because of its low solubility. Other drugs were dissolved in normal saline. T62 was obtained from King Pharmaceuticals (Cary, NC). Clonidine and idazoxan were purchased from Sigma Chemical Co. (St. Louis, MO).

Data were normally distributed and are shown as mean ± SEM. Time course and dose–response effects of T62 or clonidine were analyzed using a two-way analysis of variance, followed by a Student-Newman-Keuls post hoc  test. ED⁴⁰was calculated by linear regression analysis. The isobolograms were constructed as previously described. 18In isobolographic analysis, the theoretical additive points lies on a line connecting the ED⁴⁰values of the each drug. Experimental values that lie on or near that line are considered to have additive interactions. Values that lie below and to the left of this additive line are considered to be synergistic, whereas values that lie above and to the right of that line demonstrate a less-than-additive interaction. The difference between the theoretical additive point and the experimentally determined value was compared by the Student t  test. A P  value of less then 0.05 was considered to indicate statistical significance.

Overall, paw incision resulted in a reduced paw withdrawal threshold 24 h after surgery compared with pre-surgical values (4.7 ± 0.19 g after, 31 ± 1.2 g before; P <0.05; n = 102), with no differences across treatment groups. Intrathecal administration of T62, 0.3–1 μg, produced a dose-dependent antihypersensitivity effect, with a peak 60 min after injection (fig. 1). T62 did not affect ambulation or the level of general activity. The maximum dose studied, 3 μg, was less effective than the effect after administration of 1 μg (fig. 2). Clonidine also produced a dose-dependent antihypersensitivity effect, with a peak effect 30 min after injection (fig. 1). Dose responses at the time of peak antihypersensitivity for T62 and clonidine are shown in figure 2. The ED40 (95% confidence interval) for T62, based on the 0.3- to 1-μg doses, was 0.77 (0.63–0.91) μg, and that for clonidine was 1.23 (0.56 –1.9) μg. The peak antihypersensitivity effects of intrathecal injection of the fixed-ratio combination of two drugs were observed 30 min after injection of clonidine (fig. 3).ED⁴⁰(total dose) was only 0.36(0.35–0.37) μg, just 31% of the theoretical additive total dose (1.16 μg; P < 0.05). Isobolographic analysis indicated a synergistic interaction between T62 and clonidine (fig. 4). Intrathecal pretreatment with 30 μg idazoxan only partially inhibited (by approximately 55%) the antihypersensitivity effect of 1 μg T62 (fig. 5). Intrathecal administration of 30 μg idazoxan alone did not alter the withdrawal threshold (the thresholds before and 15, 45, and 75 min after idazoxan injection were 5.0 ± 0.46, 4.6 ± 0.49, 4.2 ± 0.30, and 4.4 ± 0.27 g, respectively; n = 6).

Intrathecal administration of T62 inhibits postoperative hypersensitivity in this animal model of postoperative pain without motor or sedative effects, as determined by gross ambulatory and spontaneous activity. T62 also exhibits activity in other settings of hypersensitivity, such as mechanical allodynia in rats with spinal nerve ligation 10,11and thermal hypersensitivity in carrageenin-inflamed rats, 12but is inactive for acute thermal and mechanical pain tests in normal rats. 12These observations are consistent with the results from previous studies with intrathecal injection of adenosine itself, which suppresses mechanical allodynia after nerve injury, 7,14but shows minimal effect in acute thermal pain. 6In the current study, intrathecal administration of T62 produced only modest effects in this model of postoperative pain, exhibiting a ceiling effect at 1 μg and an inverted U–shaped dose response. This dose–response characteristic is not unexpected because it is similar to the bell-shaped concentration response for allosteric adenosine receptor modulators on agonist binding to the to A1 receptor in vitro, thought to reflect receptor antagonism at high drug concentrations. 8 

Several previous observations have suggested that reduction in hypersensitivity elicited by intrathecal adenosine is mediated by a spinal circuit that involves the release of norepinephrine and actions on α²adrenoceptors. Therefore, the antihypersensitivity effect of intrathecally administered adenosine in rats with spinal nerve ligation is completely abolished by intrathecal pretreatment with idazoxan, 30 3g, or by destruction of spinal noradrenergic nerve terminals using neurotoxins.11,14 Spinal perfusion of adenosine releases norepinephrine in rats with spinal nerve ligation but not in normal rats,15 suggesting that this interaction does not occur in the normal animal, in which adenosine is inactive. The mechanisms of norepinephrine release by activation of spinal adenosine A1 receptors after nerve injury are not understood. Such mechanisms must involve disinhibition because A1 adenosine receptors are themselves inhibitory, and the circuitry involved in such disinhibition is under active investigation in our laboratory. A recent report shows that spinal noradrenergic innervation density to the lumbar dorsal horn is increased after nerve injury. 19Adenosine receptors are present on intrinsic interneurons in the dorsal horn. 2It is conceivable that these adenosine receptor–containing neurons interact with novel noradrenergic sprouts after injury to produce antihypersensitivity.

In the current study, the combination of T62 and clonidine produced a synergistic interaction in rats with incisional pain. Synergy usually indicates that the two drugs have different final pathways to produce their effect, although other levels of interaction, such as altered drug disposition, can also be responsible. We did not measure tissue concentrations of drugs, so we cannot exclude a pharmacokinetic mechanism of synergy in the current study. Nonetheless, the observation of synergy is somewhat surprising if, as indicated by studies with spinal nerve ligation, the effect of T62 relies entirely on stimulating spinal norepinephrine release, which acts on α²adrenoceptors. One would in that case expect an additive interaction, and intrathecal adenosine and clonidine do interact additively in spinal-ligated rats. 14In contrast, a synthetic adenosine agonist interacts synergistically with clonidine in acute thermal nociception tests in normal rats. 20In addition, idazoxan only partially reversed the antihypersensitivity effect of T62 in the current study. Taken together, these results indicate that, in the postoperative model, T62 acts via  a mechanism different from that following the spinal nerve ligation model: Both spinal activation and other mechanisms are involved in the antihypersensitivity effect of intrathecally administered T62 after surgery. Adenosine receptor activation in the spinal cord is proposed to produce antinociception via  presynaptic inhibition of excitatory neurotransmitter release 21and postsynaptic inhibition of excitatory transmission. 22These mechanisms, at least in part, may be involved in the antinociceptive effects of T62 in the postoperative pain model. In the current study, we tested T62 24 h after paw incision, and it is likely that the potency and efficacy of T62 changes in a complex fashion over time after surgery.

These results add to an emerging literature to suggest that postoperative pain exhibits a unique pharmacology of analgesia compared with other sustained pain models. For example, although spinal N -methyl-d-aspartate receptor antagonists attenuate hypersensitivity in most models of persistent pain, these are not effective in this postoperative pain model. 23Surprisingly, intrathecal administration of non–N -methyl-D-aspartate receptor antagonists 24and neurokinin-1 receptor antagonists 25are effective in this model. Further, hypersensitivity after incisional surgery is suppressed by intrathecal cyclooxygenase 1 but not by cyclooxygenase 2 inhibitors. 26These observations suggest that mechanisms of hypersensitivity after incision differ those after inflammatory or peripheral nerve injury.

Interactions between T62 and clonidine may also be important for practical reasons. Clonidine is effective in nerve injury–induced hypersensitivity 27and is approved for treatment of chronic neuropathic pain. Because clonidine therapy is limited by hypotension in some patients with chronic pain, 28the current observation of synergistic interactions between clonidine and T62 suggests that the clonidine dose, and potentially its adverse effects, could be reduced by the addition of T62.

In summary, intrathecal administration of T62 produces antinociception in the rat postoperative pain model. By isobolographic analysis, T62 interacts synergistically with intrathecal clonidine and is only partially inhibited by idazoxan. These results indicate that the mechanisms of antinociception by T62 in the postoperative pain model differ from those in the neuropathic pain model.