Free
Meeting Abstracts  |   May 1999
Intrathecal Adenosine  : Interactions with Spinal Clonidine and Neostigmine in Rat Models of Acute Nociception and Postoperative Hypersensitivity
Author Notes
  • (Chiari) Visiting Research Scholar.
  • (Eisenach) F. M. James III Professor of Anesthesiology.
  • Received from the Department of Anesthesiology, Wake Forest University School of Medicine, Winston-Salem, North Carolina. Submitted for publication August 3, 1998. Accepted for publication December 29, 1998. Supported in part by grants GM 35523 from the National Institutes of Health (J. E.), Bethesda, Maryland, GM 48085 and the Max Kade Foundation (A. C.), Austria. Presented in part at the annual meeting of the American Society of Anesthesiologists, October 19, 1998, Orlando, Florida.
  • Address reprint requests to Dr. Eisenach: Department of Anesthesiology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157–1009. Address electronic mail to:
Article Information
Meeting Abstracts   |   May 1999
Intrathecal Adenosine  : Interactions with Spinal Clonidine and Neostigmine in Rat Models of Acute Nociception and Postoperative Hypersensitivity
Anesthesiology 5 1999, Vol.90, 1413-1421. doi:
Anesthesiology 5 1999, Vol.90, 1413-1421. doi:
INCREASED understanding of endogenous pain modulatory systems that alter nociceptive transmission in the spinal cord of animals and humans has led to the development of receptor-specific agents (opioids, [Greek small letter alpha]2-adrenergicagonists, cholinesterase inhibitors) in humans. Adenosine has also been recognized to play a role in modulation of nociceptive information at the spinal level. [1 ] Adenosine receptors are present in the substantia gelatinosa, where primary afferent neurons transmitting noxious information terminate. [2 ] Both A1 and A2 subtypes of adenosine receptors have been identified in the substantia gelatinosa on intrinsic neurons. [3 ] Intrathecal administration of adenosine receptor agonists produces antinociception in animals. [1,4 ] In addition, adenosine agonists synergistically enhance antinociception from norepinephrine and [Greek small letter alpha]2-adrenergicagonists [5,6 ] in a rat model of acute thermal nociception. [Greek small letter alpha]2-Adrenergicagonists produce antinociception in part by activation of spinal cholinergic interneurons, [7,8 ] and intrathecally applied cholinomimetic drugs and cholinesterase inhibitors produce dose-dependent antinociception in rats and cats. [9,10 ] This finding implies that spinal adenosine receptor stimulation might synergistically enhance antinociception from spinal cholinergic agents. One purpose of the current study was to test this hypothesis.
Postoperative pain has commonly been considered a form of acute pain, but pain from a surgical incision also induces mechanical hyperalgesia surrounding the wound in patients. [11 ] Recently a rat model of incisional pain has been developed that demonstrates reproducible, quantifiable mechanical allodynia lasting for several days after the incision. [12 ] It has been suggested that this model displays similarities to the human postoperative pain state. Mechanical hypersensitivity evoked by nerve injury can be powerfully reversed by [Greek small letter alpha]2-adrenergicagonists [13 ] and neostigmine. [14 ] Although intrathecal adenosine receptor agonists have been shown to reduce hypersensitivity in rat models of ligation of the 5th and 6th lumbar spinal nerve, [15 ] inflammatory hyperalgesia, [16 ] and chronic central pain, [17 ] they have not been examined in this postoperative model. A second purpose of the current study was to test the efficacy of intrathecal adenosine in this model.
All previous studies have been conducted with synthetic adenosine receptor agonists rather than with adenosine itself. Because adenosine is commercially available in a preservative-free solution in the United States and has undergone neurotoxicity testing in Sweden where it is being examined in humans, [18 ] the effects of adenosine itself in these models of pain are clinically relevant. We therefore investigated the antinociceptive properties of intrathecal adenosine itself, in the US commercially available preparation, to an acute noxious thermal stimulus and on mechanical hypersensitivity after a surgical incision. Interactions between adenosine and clonidine and neostigmine were assessed in both models.
Methods 
Animals 
Studies were approved by the Animal Care and Use Committee, Wake Forest University School of Medicine. Sixty-six male Sprague-Dawley rats (Harlan, Indianapolis, IN), weighing 250–300 g, were studied. A lumbar intrathecal catheter was inserted during general anesthesia with halothane via an incision in the atlantooccipital membrane, as previously described. [19 ] The intrathecal catheter was passed 8 cm caudally to the level of the lumbar enlargement. The end of the catheter was externalized on top of the skull and closed by melting. Before implantation, the catheter was prepared in the following way: PE 5 (polyethylene, ID 0.008 inch, OD 0.014 inch, Spectra, Colorado Springs, CO) tubing was connected with PE 10 (polyethylene, ID 0.011 inch, OD 0.024 inch, Becton-Dickenson, Sparks, MD) tubing over a metal wire using a heat gun, so that the length of the external part (PE 10) was 6 cm and the internal part (PE 5) was 8 cm. Before insertion, patency and absence of leakage of the catheter were tested with a saline flush. To confirm correct placement of the catheters, 10 [micro sign]l of 2% lidocaine were injected, followed by a 10-[micro sign]l 0.9% saline flush the day after surgery. All animals developing a bilateral motor block of the hind limbs within 30 s were included in the study. After operation and testing with lidocaine, the animals recovered for at least 5 days. Animals with a deficit in fore- or hind-limb function or other obvious neurologic damage were excluded from study. Each animal was studied twice for thermal testing experiments with a 5-day interval between studies and only once in the postoperative model, on the day of paw incision.
Drugs 
The following drugs were used in the study: adenosine (Adenocard, Fujisawa, Deerfield, IL), clonidine hydrochloride, phentolamine methanesulfonate salt, and phenylephrine (Sigma Chemical Co., St. Louis, MO), and neostigmine methylsulfate (Gensia, Irvine, CA). Adenosine and neostigmine were used in the commercially available solution with concentrations of 3 mg/ml and 1 mg/ml, respectively, and diluted with saline solution as needed. All other drugs were dissolved in normal saline solution and were injected intrathecally over 30 s in a volume of 5 [micro sign]l, followed by a 10-[micro sign]l flush.
Nociceptive Testing 
Acute thermal antinociception was measured using a commercially available paw withdrawal stimulator. [20,21 ] Rats were placed in a clear plastic container on a raised floor of clear, heat-tempered glass and allowed to habituate to the environment for 30–45 min. Then, a radiant heat source was focused on the plantar surface of one hind paw every 15 min, and the latency to paw withdrawal was measured. Both paws were tested in random order 1–2 min apart, and the average of their values was used. In absence of a response, a 30-s cutoff was used to limit possible tissue damage after exposure to the stimulus.
Postoperative mechanical hypersensitivity was tested using a recently described model, in which hypersensitivity to punctate stimuli persisted for several days. [22 ] Rats with intrathecal catheters implanted 5 days previously were anesthetized with 2% halothane in oxygen/air via a face mask, and after sterile preparation with iodine and draping, a 1-cm longitudinal incision was made with a #11 scalpel blade through skin and fascia of the plantar aspect of the right foot as previously described. [12 ] The skin was closed with two sutures of 5–0 nylon, and the wound site was covered with a mixture of polymixin B, neomycin, and bacitracin ointment. After surgery, animals were allowed to recover in their cages.
Two hours after surgery, rats were placed on an elevated plastic mesh floor in a clear plastic container and allowed to acclimate. Withdrawal responses to punctate mechanical stimulation were determined using calibrated von Frey filaments applied from underneath the cage through openings in the plastic mesh floor to an area adjacent to the wound at the heel. Starting with 41 mN, filaments were applied in a consecutive fashion, ascending or descending. In the absence of a paw withdrawal response to the initially selected filament, a stronger stimulus was presented; in the event of a paw withdrawal, the next weaker stimulus was chosen. Withdrawal threshold was calculated using the Dixon up-down paradigm. [23 ] A cutoff of 456 mN was used and recorded even if there was no response to this force. Tests were done in duplicate, with a 5-min, test-free period between withdrawal responses, and their median was used. All rats were tested only on the day of paw incision surgery. The order of drug testing was randomized, but the investigator was not blinded to drug or dosage.
Altered behavior (immobility, urination, defecation, grooming) was noted, if present, after drug administration but was not examined systematically or quantified in this study.
Study Paradigm 
After connection of intrathecal catheters with tubing and prefilled syringes, rats were allowed to habituate to the test environment. In all experiments, baseline response latencies or withdrawal thresholds were determined before any drug injection. Animals then received cumulative dosing, in 30-min intervals, of intrathecal adenosine (10, 30, 100 [micro sign]g), clonidine (5, 15, 50 [micro sign]g), or neostigmine (0.5, 1.5, 5 [micro sign]g). Timing of cumulative injections was determined by pilot experiments with each drug alone and the combinations. Thermal paw withdrawal latencies were determined every 15 min, and values obtained at 30 min after each dose were taken as the value for that dose. In separate groups of animals that had undergone paw incision on that same day, mechanical withdrawal thresholds were determined before paw incision surgery, before drug injection, and then every 30 min before injection of the next dose. Each group of rats contained 6–12 animals.
Isobolographic analyses were performed to determine the degree of interaction between adenosine and clonidine and adenosine and neostigmine in thermally evoked acute nociception as well as in postoperative mechanical hypersensitivity. Dose-response curves were constructed from % maximum possible effect (MPE), and the dose producing a 50% MPE (ED50) was calculated for each drug for antinociception as was the dose producing a 25% MPE (ED25) in reducing postoperative hypersensitivity. %MPE was defined as: 100 x (postdrug response - predrug response)/(cutoff time or threshold - predrug response). Using an ED25was necessary because maximal effects in the postoperative model in the dose range studied were <or= to 50% MPE. For drug combination dose responses, drugs were tested in a constant ratio (either of their ED50for thermal studies or ED25for postoperative studies). In the case of adenosine for acute thermal nociception, which is lacking efficacy when administered alone, the upper limit for interaction studies was arbitrarily chosen to be a cumulative dose of 30 [micro sign]g.
Two antagonist studies were performed. To test whether the postoperative effect of adenosine involved spinal [Greek small letter alpha]-adrenergic receptor stimulation, 30 [micro sign]g of intrathecal phentolamine was injected with every dose of adenosine in the postoperative model. To test adenosine-induced vasodilation as an explanation for its lack of enhancement of neostigmine in the thermal tests, 15 [micro sign]g of intrathecal phenylephrine was coadministered with every dose of adenosine and neostigmine during thermal testing.
Statistics 
Data are presented as mean +/- SD, except for paw withdrawal thresholds, which are presented as median and 25th-75th percentiles. ED50was calculated by linear regression. For the postoperative model, the isobolograms were constructed as previously described. [24 ] In isobolograms, the theoretical additive point lies on a line connecting the ED (25) or ED50values of the individual doses. 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 using the Student t test. In the case of one drug lacking efficacy (adenosine to acute thermal stimulation), any significant shift in the other agent's dose response demonstrates synergy.
Results 
Thermal Testing 
Clonidine and neostigmine, but not adenosine, produced antinociception to thermal testing (Figure 1). The ED50values were 19 +/- 6 [micro sign]g for clonidine and 3 +/- 1.2 [micro sign]g for neostigmine. Clonidine and adenosine were associated with substantial diuresis and some sedation (decreased spontaneous exploring activity, but still responsive to acoustic or tactile stimuli), and neostigmine was associated with tail grooming and licking behavior. Motor activity appeared normal in all animals, as animals moved and ambulated normally in and outside the containers.
Figure 1. Log dose-response curves for the effects of intrathecally administered adenosine, neostigmine, and clonidine on the thermal nociceptive threshold. The response is presented as % maximum possible effect (%MPE) versus log dose in micrograms. Each point on the graph represents the mean +/- SD for 6–12 rats. 
Figure 1. Log dose-response curves for the effects of intrathecally administered adenosine, neostigmine, and clonidine on the thermal nociceptive threshold. The response is presented as % maximum possible effect (%MPE) versus log dose in micrograms. Each point on the graph represents the mean +/- SD for 6–12 rats. 
Figure 1. Log dose-response curves for the effects of intrathecally administered adenosine, neostigmine, and clonidine on the thermal nociceptive threshold. The response is presented as % maximum possible effect (%MPE) versus log dose in micrograms. Each point on the graph represents the mean +/- SD for 6–12 rats. 
×
Intrathecal adenosine synergistically enhanced antinociception from intrathecal clonidine, reducing its ED50from 19 +/- 6 [micro sign]g to 4.1 +/- 3.1 [micro sign]g (Figure 2). Animals in this group exhibited intense urination and sedation. In contrast, intrathecal adenosine did not affect antinociception from intrathecal neostigmine (Figure 2; ED (50) of neostigmine alone = 3.0 +/- 1.2 and with adenosine = 2.7 +/- 1.3). Frequently observed side effects were grooming and licking behavior and urination. Addition of the [Greek small letter alpha]1-adrenergicagonist phenylephrine to every adenosine-neostigmine dose also failed to increase the antinociceptive effect (ED50of adenosine + neostigmine = 2.7 +/- 1.3 [micro sign]g and adenosine + neostigmine + phentolamine = 3.3 +/- 0.9 [micro sign]g).
Figure 2. % Maximum possible effect (%MPE) from intrathecal clonidine (upper panel) and neostigmine (lower panel) alone and without the addition of adenosine in a fixed ratio. Each value represents the mean +/- SD for 6–12 rats. 
Figure 2. % Maximum possible effect (%MPE) from intrathecal clonidine (upper panel) and neostigmine (lower panel) alone and without the addition of adenosine in a fixed ratio. Each value represents the mean +/- SD for 6–12 rats. 
Figure 2. % Maximum possible effect (%MPE) from intrathecal clonidine (upper panel) and neostigmine (lower panel) alone and without the addition of adenosine in a fixed ratio. Each value represents the mean +/- SD for 6–12 rats. 
×
Postoperative Hypersensitivity 
In all groups the mean withdrawal threshold to von Frey filaments decreased from 456 mN (cutoff) before surgery to 39 mN after incision. Intrathecal administration of clonidine, neostigmine, and also adenosine resulted in a dose-dependent increase in withdrawal thresholds (Figure 3). Phentolamine coadministered with adenosine completely abolished the antihypersensitivity effect of adenosine in all doses used (%MPE was 0 +/- 1, 1 +/- 1, and 1 +/- 1% after adenosine; 10, 30, and 100 [micro sign]g, respectively; P = NS). Because maximal effects of the given doses of adenosine, clonidine, and neostigmine were 50% MPE or less, only an ED25could be calculated with precision. According to relative potencies in single drug experiments, the dose ratios in the combination experiments were adenosine:neostigmine = 37:1 and adenosine:clonidine = 5:1. In the neostigmine-adenosine group, the effect of the combined drugs was significantly greater than the calculated additive effect (Figure 4A), yielding synergy. In contrast, the experimentally determined ED25for the antihypersensitivity response of the clonidine-adenosine mixture was not different from the theoretical additive ED25of these two drugs (Figure 4B). Side effects were similar to those observed with acute thermal testing of these compounds.
Figure 3. % Maximum possible effect (%MPE) from intrathecal adenosine, neostigmine, and clonidine on punctate mechanical hyperalgesia caused by a plantar incision. Each point on the graph represents the median with error bars showing the 25th and 75th percentile for 6–12 rats. 
Figure 3. % Maximum possible effect (%MPE) from intrathecal adenosine, neostigmine, and clonidine on punctate mechanical hyperalgesia caused by a plantar incision. Each point on the graph represents the median with error bars showing the 25th and 75th percentile for 6–12 rats. 
Figure 3. % Maximum possible effect (%MPE) from intrathecal adenosine, neostigmine, and clonidine on punctate mechanical hyperalgesia caused by a plantar incision. Each point on the graph represents the median with error bars showing the 25th and 75th percentile for 6–12 rats. 
×
Figure 4. (A) Isobologram at the ED25level for the interaction between adenosine and neostigmine in the postoperative paw incision model when coadministered in a fixed dose ratio. The straight line connecting the single drug ED25points is the theoretical additive line. The experimental point for adenosine-neostigmine mixture was found to be significantly below the theoretical additive line, indicating synergistic interaction. *P < 0.05 versus theoretical additive point. Each point on the graph represents the mean +/- SD for 6–12 rats. (B) Isobologram at the ED25level for the interaction between adenosine and clonidine in the postoperative paw incision model when coadministered in a fixed dose ratio. The straight line connecting the single drug ED25points is the theoretical additive line. The experimental point for adenosine-neostigmine mixture was found to be close to the theoretical additive line, indicating additive interaction. Each point on the graph represents the mean +/- SD for 6–12 rats. 
Figure 4. (A) Isobologram at the ED25level for the interaction between adenosine and neostigmine in the postoperative paw incision model when coadministered in a fixed dose ratio. The straight line connecting the single drug ED25points is the theoretical additive line. The experimental point for adenosine-neostigmine mixture was found to be significantly below the theoretical additive line, indicating synergistic interaction. *P < 0.05 versus theoretical additive point. Each point on the graph represents the mean +/- SD for 6–12 rats. (B) Isobologram at the ED25level for the interaction between adenosine and clonidine in the postoperative paw incision model when coadministered in a fixed dose ratio. The straight line connecting the single drug ED25points is the theoretical additive line. The experimental point for adenosine-neostigmine mixture was found to be close to the theoretical additive line, indicating additive interaction. Each point on the graph represents the mean +/- SD for 6–12 rats. 
Figure 4. (A) Isobologram at the ED25level for the interaction between adenosine and neostigmine in the postoperative paw incision model when coadministered in a fixed dose ratio. The straight line connecting the single drug ED25points is the theoretical additive line. The experimental point for adenosine-neostigmine mixture was found to be significantly below the theoretical additive line, indicating synergistic interaction. *P < 0.05 versus theoretical additive point. Each point on the graph represents the mean +/- SD for 6–12 rats. (B) Isobologram at the ED25level for the interaction between adenosine and clonidine in the postoperative paw incision model when coadministered in a fixed dose ratio. The straight line connecting the single drug ED25points is the theoretical additive line. The experimental point for adenosine-neostigmine mixture was found to be close to the theoretical additive line, indicating additive interaction. Each point on the graph represents the mean +/- SD for 6–12 rats. 
×
Discussion 
These data demonstrated that intrathecal adenosine lacks antinociceptive efficacy by itself but synergistically enhances antinociception from intrathecal clonidine in a rat model of acute thermal nociception. In addition, these data provide the first evidence of efficacy of adenosine, clonidine, and neostigmine and their interactions in a rat model of postoperative hypersensitivity. It is important to qualify these observations with the understanding that some of these agents produced sedation, which could have interfered with the behavioral end-points of these tests.
Adenosine and Its Interactions with Clonidine and Neostigmine in Acute Pain 
In contrast to previous studies with synthetic adenosine receptor agonists, which revealed dose-dependent antinociception to acute noxious stimuli in rats [1,4 ] and in spinal dorsal horn neuronal responses, [25 ] adenosine itself is inactive. Adenosine's hydrophilicity might have caused a poor penetration of the drug into the spinal cord, resulting in a too small concentration of adenosine reaching the cord to be effective alone against acute thermal stimulation. In contrast, its efficacy alone in the postoperative hypersensitivity model may reflect increased sensitivity to even these low concentrations in this model, a difference in sensory stimuli used (heat in normal animals vs. mechanical stimulation after surgery), or different intensities of stimulation in the two models. That adenosine may have poorly penetrated the spinal cord is consistent with the observation that synthetic adenosine analogs, [26 ] but not adenosine itself (current study), produces motor weakness and blockade. Adenosine's activity on both A1 and A2 receptors in the spinal cord might be another possible reason for its lack of antinociception. Although selective A2-agonists were shown to have no antinociceptive effect, [27 ] spinal adenosine A1 receptors have been clearly identified to mediate the inhibition of nociceptive input into the dorsal spinal cord. [28 ] This possibility seems less likely because intrathecally administered N-ethylcarboxamide-adenosine (NECA), which has an equal affinity for A1 and A2 receptors, showed antinociceptive effects in the tail immersion test in the rat. [4 ]
The observation that the combination of the [Greek small letter alpha]2-adrenergicagonist clonidine and adenosine yields synergy is consistent with previous reports with synthetic adenosine analogs. [5,6 ] In general, a supraadditive interaction may result from the administration of two drugs that produce their effects by different receptor mechanisms of action. Adenosine receptor activation in the spinal cord is proposed to produce antinociception by at least two different mechanisms:(1) presynaptic inhibition of excitatory neurotransmitter release with subsequent reduction of substance P concentrations in cerebrospinal fluid, [29 ] and (2) postsynaptic inhibition of the effects of excitatory neurotransmitters. [30 ] Intrathecal [Greek small letter alpha]2-adrenergicagonists mimic the effects of endogenous norepinephrine and produce antinociception by (1) decreasing the release of substance P from primary afferent nerve terminals, [31,32 ] and (2) suppressing noxiously evoked activity of wide dynamic range neurons. [33 ]
In contrast to our hypothesis, intrathecally administered adenosine did not synergistically enhance neostigmine-induced antinociception to acute thermal stimulation. Neostigmine inhibits the breakdown of endogenously released acetylcholine, which has been shown to cause antinociception by stimulation of muscarinic [10,34 ] and nicotinic [35,36 ] receptors in the spinal cord. Because rats exhibit tonic spinal cholinergic activity, [9 ] intrathecally administered cholinesterase inhibitors alone cause antinociception in this species. [34 ]
It is unexpected that adenosine's interactions with clonidine and neostigmine differ because spinal [Greek small letter alpha]2-adrenergicstimulation produces antinociception in part by causing acetylcholine release from spinal cholinergic interneurons. [7,8,37 ] One possible explanation for the different interaction between adenosine and neostigmine could be a pharmacokinetic effect, whereby vasodilation induced by adenosine resulted in more rapid clearance of neostigmine, masking a potentiating effect. We tested this hypothesis by adding phenylephrine to the neostigmine-adenosine combination to induce powerful vasoconstriction [38 ] and observed no difference in this drug interaction.
Differing interactions between adenosine and clonidine compared with neostigmine reinforce our previous hypothesis [7 ] that clonidine must be working through multiple mechanisms to produce antinociception to acute stimulation, not just stimulation of acetylcholine release.
Adenosine and Its Interactions with Clonidine and Neostigmine in a Rat Model of Postoperative Hypersensitivity 
The recently developed animal model of incisional pain [12 ] closely mimics in time course the human postoperative pain experience. The hypersensitivity to mechanical stimuli in this rat model may represent allodynia because behavior is elicited after surgery from a level of stimulation that fails to elicit behavior preoperatively. Some electrophysiologic experiments demonstrate that a surgical incision in rats induces central sensitization with subsequent mechanical hypersensitivity observed in behavioral studies. [39 ] The input from primary afferent fibers during and up to 1 h after surgery appears to be the major stimulus for the induction of postoperative hypersensitivity. [40 ] Thus, this model may offer advantages over previously established tissue or nerve injury models in predicting the ability of spinally administered agents to block central sensitization to reduce postoperative pain. [41 ] Of course, peripheral inflammation also occurs in this model, and one cannot exclude in the current experiments a peripheral component to the hypersensitivity and its response to drugs administered.
Adenosine itself is active in the postoperative hypersensitivity model. Synthetic adenosine receptor agonists decrease thermal [42 ] and inflammatory [16 ] hyperalgesia, tactile allodynia, [15 ] and chronic central pain [17 ] in animals. The clinical relevance of adenosine's potential as an antihypersensitivity agent is further supported by a case report in which intrathecal administration of a selective A1 adenosine agonist successfully abolished allodynia in a patient with chronic neuropathic pain. [43 ] When administered to human volunteers, 500–2,000 [micro sign]g of intrathecal adenosine itself has no effect on pain from ice water immersion of the foot but reduces ischemic tourniquet pain and mechanical hyperalgesia from mustard oil application, [18 ] consistent with the current study in rats.
Although the role of adenosine receptors in spinal processes involving central sensitization is not well understood, N-methyl-D-aspartate (NMDA) receptors are known to play a key role in the process of central sensitization, [44 ] and there is a close functional relationship between adenosine A1 and NMDA receptors in the spinal cord. Adenosine analogs suppress dorsal horn neuronal sensitization by subcutaneous formalin [45 ] or mustard oil application to a region of skin adjacent to their receptive fields. [25 ] In addition, adenosine reduces excitatory neurotransmitter release from nociceptive stimulation, as described previously. Because injury-induced hypersensitivity is associated in an arthritis model with chronic spinal glutamate and aspartate release, [46 ] it is conceivable that adenosine's antihypersensitivity effect reflects a reduction in ongoing excitatory neurotransmitter release.
Another possible explanation for adenosine's increased potency in the postoperative state could reflect an interaction with endogenously released norepinephrine. In rats, peripheral nerve stimulation at C-fiber intensity causes an increase in lumbar spinal cord norepinephrine release, measured by microdialysis, and this increase is reduced by thoracic spinal cord transsection. [47 ] Similarly, painful stimulation in sheep increased cerebrospinal fluid concentrations of norepinephrine. [48 ] Thus, adenosine could have enhanced endogenously released norepinephrine [5,6 ] after paw incision to produce an increased antihypersensitivity effect. Adenosine's postoperative antihypersensitivity effect was completely blocked by coadministration of phentolamine, an [Greek small letter alpha]-adrenergic antagonist, supporting this hypothesis.
[Greek small letter alpha]2-Adrenergicagonists are potent in alleviating allodynia in an animal model of nerve injury, [13 ] and epidural clonidine powerfully alleviates intractable cancer pain in patients. [49 ] Our findings in the postoperative model in rats are consistent with human clinical data [50 ] and these animal studies. Unlike acute thermal antinociception, intrathecal clonidine failed to interact synergistically with intrathecal adenosine to relieve postoperative hypersensitivity. Perhaps this finding reflects the adrenergic mechanism of adenosine's antihypersensitivity effect in the postoperative period, described previously, which would not be expected to interact synergistically with the adrenergic mechanism of clonidine.
Neostigmine [14 ] and muscarinic agonists [51 ] reduce allodynia from inflammation. [52 ] In sheep, intrathecal neostigmine has been a more effective antinociceptive agent in the acute postoperative period than at times more remote from surgery, suggesting that postoperative pain could trigger an increased cholinergic tone in that species. [53 ] Intrathecal neostigmine produces postoperative analgesia for a variety of surgical procedures in humans, [54–56 ] but side effects, such as nausea and vomiting, limit its clinical usefulness.
Unlike antinociception to acute noxious thermal stimulation, intrathecal neostigmine interacted synergistically with adenosine to reduce postoperative hypersensitivity. Because [Greek small letter alpha]2-adrenergicagonists interact synergistically with neostigmine, [34 ] this finding could again reflect an adrenergic component of adenosine's mechanism of action in the postoperative model and would provide a rationale for investigating this interaction in postoperative patients.
Clinical Implications 
In the postoperative period, pain evoked by coughing or deep breathing after upper abdominal or thoracic surgery leads to the potentially dangerous consequences: hypoventilation, atelectasis, and hypoxemia, whereas spontaneous pain at rest is generally easy to control. It has been argued that interventions that are effective in modifying punctate hypersensitivity tested by the postoperative paw incision model are likely to have a substantial effect on this aspect of postoperative pain. [41 ] Opioids are the most widely used drugs to manage postoperative pain, but bothersome and potentially dangerous side effects limit their use. [57 ] One approach to improve analgesia and reduce side effects at the same time is to combine small doses of analgesics that interact synergistically to produce analgesia without increasing side effects. The purpose of this study was not to investigate side effects of these agents alone and in combination. Because side effects were often observed to each of these drugs alone and in combination, one cannot conclude from these data that such combinations would be clinically useful. Finally, although the commercially available preparation of adenosine in the United States does not contain preservatives, it is inappropriate to administer adenosine intrathecally to humans before a toxicologic assessment has proven it to be innocuous in at least two animal species. [58 ]
In summary, intrathecal administration of adenosine alone causes no antinociception to a thermal stimulus in rats but enhances antinociception from intrathecal clonidine. A lack of improved antinociception combined with the cholinesterase inhibitor neostigmine to adenosine further suggests that the cholinergic pathway does not essentially contribute to clonidine's antinociception to acute thermal stimulation. Adenosine, clonidine, and neostigmine alone produce dose-dependent, antihypersensitivity effects in a postoperative rat model. Synergistic interaction of adenosine and neostigmine in this paradigm and additive effects of adenosine and clonidine support the hypothesis that spinal cord noradrenergic systems are activated in response to surgery. These findings may provide a rationale for combining these drugs for improved human postoperative pain treatment in the future.
REFERENCES 
REFERENCES 
Sawynok J, Sweeney MI: The role of purines in nociception. Neuroscience 1989; 32:557-69
Braas KM, Newby AC, Wilson VS, Snyder SH: Adenosine-containing neurons in the brain localized by immunocytochemistry. J Neurosci 1986; 6:1952-61
Choca JI, Green RD, Proudfit HK: Adenosine A1 and A2 receptors of the substantia gelatinosa are located predominantly on intrinsic neurons: An autoradiography study. J Pharmacol Exp Ther 1988; 247:757-64
Karlsten R, Gordh T Jr, Hartvig P, Post C: Effects of intrathecal injection of the adenosine receptor agonists R-phenylisopropyl-adenosine and N-ethylcarboxamide-adenosine on nociception and motor function in the rat. Anesth Analg 1990; 71:60-4
Aran S, Proudfit HK: Antinociceptive interactions between intrathecally administered alpha noradrenergic agonists and 5-N-ethylcarboxamide adenosine. Brain Res 1990; 519:287-93
Aran S, Proudfit HK: Antinoception produced by interactions between intrathecally administered adenosine agonists and norepinephrine. Brain Res 1990; 513:255-63
Detweiler DJ, Eisenach JC, Tong C, Jackson C: A cholinergic interaction in alpha 2 adrenoceptor-mediated antinociception in sheep. J Pharmacol Exp Ther 1993; 265:536-42
Klimscha W, Tong C, Eisenach JC: Intrathecal alpha 2-adrenergic agonists stimulate acetylcholine and norepinephrine release from the spinal cord dorsal horn in sheep. An in vivo microdialysis study. Anesthesiology 1997; 87:110-6
Zhuo M, Gebhart GF: Tonic cholinergic inhibition of spinal mechanical transmission. Pain 1991; 46:211-22
Yaksh TL, Dirksen R, Harty GJ: Antinociceptive effects of intrathecally injected cholinomimetic drugs in the rat and cat. Eur J Pharmacol 1985; 117:81-8
Tverskoy M, Oz Y, Isakson A, Finger J, Bradley EL Jr, Kissin I: Preemptive effect of fentanyl and ketamine on postoperative pain and wound hyperalgesia. Anesth Analg 1994; 78:205-9
Brennan TJ, Vandermeulen EP, Gebhart GF: Characterization of a rat model of incisional pain. Pain 1996; 64:493-501
Yaksh TL, Pogrel JW, Lee YW, Chaplan SR: Reversal of nerve ligation-induced allodynia by spinal alpha-2 adenoreceptor agonists. J Pharmacol Exp Ther 1995; 272:207-14
Lavand'homme P, Pan HL, Eisenach JC: Intrathecal neostigmine, but not sympathectomy relieves mechanical allodynia in a rat model of neuropathic pain. Anesthesiology 1998; 89:493-9
Lee YW, Yaksh TL: Pharmacology of the spinal adenosine receptor which mediates the antiallodynic action of intrathecal adenosine agonists. J Pharmacol Exp Ther 1996; 277:1642-8
Poon A, Sawynok J: Antinociception by adenosine analogs and inhibitors of adenosine metabolism in an inflammatory thermal hyperalgesia model in the rat. Pain 1998; 74:235-45
Sjolund KF, von Heijne M, Hao JX, Xu XJ, Sollevi A, Wiesenfeld-Hallin Z: Intrathecal administration of the adenosine A1 receptor agonist R-phenylisopropyl adenosine reduces presumed pain behaviour in a rat model of central pain. Neurosci Lett 1998; 243:89-92
Rane K, Segerdahl M, Goiny M, Solleri A: Intrathecal adenosine administration-A phase 1 clinical safety study in healthy volunteers, with an additional evaluation of its influence on sensory thresholds and experimental pain. Anesthesiology 1998; 89:1108-15
LoPachin RM, Rudy TA, Yaksh TL: An improved method for chronic catheterization of the rat spinal subarachnoid space. Physiol Behav 1981; 27:559-61
Hargreaves K, Dubner R, Brown F, Flores C, Joris J: A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988; 32:77-88
Dirig DM, Salami A, Rathbun ML, Ozaki GT, Yaksh TL: Characterization of variables defining hindpaw withdrawal latency evoked by radiant thermal stimuli. J Neurosci Meth 1997; 76:183-91
Zahn PK, Gysbers D, Brennan TJ: Effect of systemic and intrathecal morphine in a rat model of postoperative pain. Anesthesiology 1997; 86:1066-77
Dixon WJ: Efficient analysis of experimental observations. Ann Rev Pharmacol Toxicol 1980; 20:441-62
Tallarida RJ, Porreca F, Cowan A: Statistical analysis of drug-drug and site-site interactions with isobolograms. Life Sci 1989; 45:947-61
Sumida T, Smith MA, Haehara Y, Collins JG, Kitahata LM: Spinal R-phenyl-isopropyl adenosine spinal dorsal horn neurons responding to noxious heat stimulation in the absence and presence of sensitization. Pain 1998; 74:307-13
Sosnowski M, Stevens CW, Yaksh TL: Assessment of the role of A1/A2 adenosine receptors mediating the purine antinociception, motor and autonomic function in the rat spinal cord. J Pharmacol Pharmacol Exp Ther 1989; 250:915-22
Poon A, Sawynok J: Antinoniception by adenosine analogs and an adenosine kinase inhibitor: Dependence on formalin concentration. Eur J Pharmacol 1995; 286:177-84
Nakamura I, Ohta Y, Kemmotsu O: Characterization of adenosine receptors mediating spinal sensory transmission related to nociceptive information in the rat. Anesthesiology 1997; 87:577-84
Sjolund KF, Sollevi A, Segerdahl M, Lundeberg T: Intrathecal adenosine analog administration reduces substance P in cerebrospinal fluid along with behavioral effects that suggest antinociception in rats. Anesth Analg 1997; 85:627-32
DeLander GE, Wahl JJ: Behavior induced by putative nociceptive neurotransmitters is inhibited by adenosine or adenosine analogs coadministered intrathecally. J Pharmacol Exp Ther 1988; 246:565-70
Go VL, Yaksh TL: Release of substance P from the cat spinal cord. J Physiol 1987; 391:141-67
Kuraishi Y, Hirota N, Sato Y, Kaneko S, Satoh M, Takagi H: Nonandrenergic inhibition of the release of substance P from the primary afferents in the rabbit spinal dorsal horn neurones. Brain Res 1985; 359:177-82
Fleetwood-Walker SM, Mitchell R, Hope PJ, Molony V, Iggo A: An alpha 2 receptor mediates the selective inhibition by nonadrenaline of nociceptive responses of identified dorsal horn neurones. Brain Res 1985; 334:243-54
Naguib M, Yaksh TL: Antinociceptive effects of spinal cholinesterase inhibition and isobolographic analysis of the interaction with mu and alpha 2 receptor systems. Anesthesiology 1994; 80:1338-48
Damaj Mi, Welch Sp, Martin Br: Nicotine induced antinociception in mice: Role of G-proteins and adenylate cyclase. Pharmacol Biochem Behav 1994; 48:37-42
Bannon AW, Decker MW, Holladay MW, Curzon P, Donnelly-Roberts D, Puttfarcken PS, Bitner RS, Diaz A, Dickenson AH, Porsolt RD, Williams M, Arneric SP: Broad-spectrum, non-opioid analgesic activity by selective modulation of neuronal nicotinic acetylcholine receptors. Science 1998; 279:77-81
Hood DD, Mallak KA, Eisenach JC, Tong C: Interaction between intrathecal neostigmine and epidural clonidine in human volunteers. Anesthesiology 1996; 85:315-25
Kozody R, Palahniuk RJ, Wade JG, Cumming MO, Pucci WR: The effect of subarachnoid epinephrine and phenylephrine on spinal cord blood flow. Can Anaesth Soc J 1984; 31:503-8
Brennan TJ, Vandermeulen E, Gebhart GF: Sensitization of dorsal horn neurons by a surgical incision in the rat (abstract). Soc Neurosci 1995; 552:10
Field MJ, Holloman EF, McCleary S, Hughes J, Singh L: Evaluation of gabapentin and S-(+)-3-isobutylgaba in a rat model of postoperative pain. J Pharmacol Exp Ther 1997; 282:1242-6
Abram SE: Necessity for an animal model of postoperative pain (editorial). Anesthesiology 1997; 86:1015-7
Yamamoto T, Yaksh TL: Spinal pharmacology of thermal hyperesthesia induced by constriction injury of sciatic nerve. Excitatory amino acid antagonists. Pain 1992; 49:121-8
Karlsten R, Gordh T Jr: An A1-selective adenosine agonist abolishes allodynia elicited by vibration and touch after intrathecal injection. Anesth Analg 1995; 80:844-7
Haley JE, Sullivan AF, Dickenson AH: Evidence for spinal N-methyl-D aspartate receptor involvement in prolonged chemical nociception in the rat. Brain Res 1990; 518:218-26
Reeve AJ, Dickenson AH: The roles of spinal adenosine receptors in the control of acute and more persistent nociceptive responses of dorsal horn neurones in the anesthetized rat. Br Pharmacol 1995; 116:2221-8
Yang LC, Marsala M, Taksh TL: Characterization of time course of spinal amino acids, citrulline and PGE2 release after carrageenan/kaolin-induced knee joint inflammation: A chronic microdialysis study. Pain 1996; 67:345-54
Men DS, Matsui Y: Peripheral nerve stimulation increases serotonin and dopamine metabolites in rat spinal cord. Brain Res Bull 1994; 33:625-32
Eisenach JC, Detweiler DJ, Tong C, D'Angelo R, Hood DD: Cerebrospinal fluid norepinephrine and acetylcholine concentrations during acute pain. Anesth Analg 1996; 82:621-6
Eisenach JC, DuPen S, Duboise M, Miguel R, Allin D: Epidural clonidine analgesia for intractable cancer pain. Pain 1995; 61:391-9
Filos KS, Goduas LC, Patroni O, Polyzou V: Hemodynamic and analgesic profile after intrathecal clonidine in humans. A dose-response study. Anesthesiology 1994; 81:591-601
Kawamata M, Satoh O, Omote H, Iwasaki H, Namiki A: Intrathecal carbaschol attenuates nociceptive responses in the formalin test (abstract). Anesthesiology 1996; 85:A718
Buerkle H, Boschin M, Marcus MA, Brodner G, Wusten R, Van Aken H: Central and peripheral analgesia mediated by the acetylcholinesterase-inhibitor neostigmine in the rat inflamed knee joint model. Anesth Analg 1998; 86:1027-32
Bouaziz H, Tong C, Eisenach JC: Postoperative analgesia from intrathecal neostigmine in sheep. Anesth Analg 1995; 80:1140-4
Krukowski JA, Hood DD, Eisenach JC, Mallak KA, Parker RL: Intrathecal neostigmine for post-cesarean section analgesia: Dose response. Anesth Analg 1997; 84:1269-75
Klamt JG, Slullitel A, Garcia IV, Prado WA: Postoperative analgesic effect of intrathecal neostigmine and its influence on spinal anaesthesia. Anaesthesia 1997; 52:547-51
Lauretti GR, Reis MP, Prado WA, Klmat JG: Dose-response study of intrathecal morphine versus intrathecal neostigmine, their combination, or placebo for postoperative analgesia in patients undergoing anterior and posterior vaginoplasty. Anesth Analg 1996; 82:1182-7
Lavies N, Hart L, Rounsefell B, Runciman W: Identification of patient, medical and nursing staff attitudes to postoperative opioid analgesia: Stage 1 of a longitudinal study of postoperative analgesia. Pain 1992; 48:313-9
Yaksh TL, Collins JG: Studies in animals should precede human use of spinally administered drugs. Anesthesiology 1989; 70:4-6
Figure 1. Log dose-response curves for the effects of intrathecally administered adenosine, neostigmine, and clonidine on the thermal nociceptive threshold. The response is presented as % maximum possible effect (%MPE) versus log dose in micrograms. Each point on the graph represents the mean +/- SD for 6–12 rats. 
Figure 1. Log dose-response curves for the effects of intrathecally administered adenosine, neostigmine, and clonidine on the thermal nociceptive threshold. The response is presented as % maximum possible effect (%MPE) versus log dose in micrograms. Each point on the graph represents the mean +/- SD for 6–12 rats. 
Figure 1. Log dose-response curves for the effects of intrathecally administered adenosine, neostigmine, and clonidine on the thermal nociceptive threshold. The response is presented as % maximum possible effect (%MPE) versus log dose in micrograms. Each point on the graph represents the mean +/- SD for 6–12 rats. 
×
Figure 2. % Maximum possible effect (%MPE) from intrathecal clonidine (upper panel) and neostigmine (lower panel) alone and without the addition of adenosine in a fixed ratio. Each value represents the mean +/- SD for 6–12 rats. 
Figure 2. % Maximum possible effect (%MPE) from intrathecal clonidine (upper panel) and neostigmine (lower panel) alone and without the addition of adenosine in a fixed ratio. Each value represents the mean +/- SD for 6–12 rats. 
Figure 2. % Maximum possible effect (%MPE) from intrathecal clonidine (upper panel) and neostigmine (lower panel) alone and without the addition of adenosine in a fixed ratio. Each value represents the mean +/- SD for 6–12 rats. 
×
Figure 3. % Maximum possible effect (%MPE) from intrathecal adenosine, neostigmine, and clonidine on punctate mechanical hyperalgesia caused by a plantar incision. Each point on the graph represents the median with error bars showing the 25th and 75th percentile for 6–12 rats. 
Figure 3. % Maximum possible effect (%MPE) from intrathecal adenosine, neostigmine, and clonidine on punctate mechanical hyperalgesia caused by a plantar incision. Each point on the graph represents the median with error bars showing the 25th and 75th percentile for 6–12 rats. 
Figure 3. % Maximum possible effect (%MPE) from intrathecal adenosine, neostigmine, and clonidine on punctate mechanical hyperalgesia caused by a plantar incision. Each point on the graph represents the median with error bars showing the 25th and 75th percentile for 6–12 rats. 
×
Figure 4. (A) Isobologram at the ED25level for the interaction between adenosine and neostigmine in the postoperative paw incision model when coadministered in a fixed dose ratio. The straight line connecting the single drug ED25points is the theoretical additive line. The experimental point for adenosine-neostigmine mixture was found to be significantly below the theoretical additive line, indicating synergistic interaction. *P < 0.05 versus theoretical additive point. Each point on the graph represents the mean +/- SD for 6–12 rats. (B) Isobologram at the ED25level for the interaction between adenosine and clonidine in the postoperative paw incision model when coadministered in a fixed dose ratio. The straight line connecting the single drug ED25points is the theoretical additive line. The experimental point for adenosine-neostigmine mixture was found to be close to the theoretical additive line, indicating additive interaction. Each point on the graph represents the mean +/- SD for 6–12 rats. 
Figure 4. (A) Isobologram at the ED25level for the interaction between adenosine and neostigmine in the postoperative paw incision model when coadministered in a fixed dose ratio. The straight line connecting the single drug ED25points is the theoretical additive line. The experimental point for adenosine-neostigmine mixture was found to be significantly below the theoretical additive line, indicating synergistic interaction. *P < 0.05 versus theoretical additive point. Each point on the graph represents the mean +/- SD for 6–12 rats. (B) Isobologram at the ED25level for the interaction between adenosine and clonidine in the postoperative paw incision model when coadministered in a fixed dose ratio. The straight line connecting the single drug ED25points is the theoretical additive line. The experimental point for adenosine-neostigmine mixture was found to be close to the theoretical additive line, indicating additive interaction. Each point on the graph represents the mean +/- SD for 6–12 rats. 
Figure 4. (A) Isobologram at the ED25level for the interaction between adenosine and neostigmine in the postoperative paw incision model when coadministered in a fixed dose ratio. The straight line connecting the single drug ED25points is the theoretical additive line. The experimental point for adenosine-neostigmine mixture was found to be significantly below the theoretical additive line, indicating synergistic interaction. *P < 0.05 versus theoretical additive point. Each point on the graph represents the mean +/- SD for 6–12 rats. (B) Isobologram at the ED25level for the interaction between adenosine and clonidine in the postoperative paw incision model when coadministered in a fixed dose ratio. The straight line connecting the single drug ED25points is the theoretical additive line. The experimental point for adenosine-neostigmine mixture was found to be close to the theoretical additive line, indicating additive interaction. Each point on the graph represents the mean +/- SD for 6–12 rats. 
×