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PAIN MEDICINE

Antinociception of Intrathecal Adenosine Receptor Subtype Agonists in Rat Formalin Test

Department of Anesthesiology and Pain Medicine, Chonnam National University, Medical School, Gwangju, Korea

Address correspondence and reprint requests to Myung Ha Yoon, MD, Professor in Department of Anesthesiology and Pain Medicine, Chonnam National University, Medical School, 8 Hakdong, Dongku, Gwangju 501–757, Korea. Address e-mail to mhyoon{at}chonnam.ac.kr.

	Abstract

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Adenosine has shown antinociceptive action via spinal adenosine receptors. There are four types of adenosine receptors: A₁, A_2A, A_2B, and A₃. We characterized the nature of types of adenosine receptors for the control of nociception at the spinal level. For nociception, formalin solution (5%, 50 µL) was injected into the hindpaw of male Sprague-Dawley rats. The effects of intrathecal adenosine A₁ (CPA), A_2A (DPMA), and A₃ (IB-MECA) receptor agonists were examined. CPA and IB-MECA produced limited or no effect on the early phase response of the formalin test, respectively, but the two drugs depressed the late phase response. DPMA suppressed both phase responses. CPA was the most potent drug among the three in the late phase. These results suggest that spinal adenosine A₁ and A_2A receptors may be involved in the modulation of the early and the late phase responses of the formalin test, whereas adenosine A₃ receptor may be involved in the regulation of the late phase response.

	Introduction

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An endogenous purine compound adenosine functions as an extracellular signaling molecule within the central and peripheral nervous systems (1,2). Adenosine is released locally at tissue sites in response to adverse events such as trauma and ischemia and interacts with specific receptors. Four types of adenosine receptors have been identified and cloned as A₁, A_2A, A_2B, and A₃ (1,3). Experimental data have elucidated the role of adenosine in the modulation of nociceptive transmission at the spinal level (4–7). Although many pharmacologic studies (8–13) have investigated the effect of adenosine receptor agonists for the nociceptive state, the role of types of adenosine receptors at the spinal level have not been definitely established.

The purpose of the present study was to understand the physiological relevance of subtypes of adenosine receptors in the control of nociception at the spinal level. Thus, we examined the effects of several selective adenosine receptor agonists given intrathecally in the formalin test, which shows an early phase of acute nociceptive response followed by a late phase response being related to more complex inflammatory reactions. Further, we sought to determine the antinociceptive potency of these agonists under the same nociceptive conditions.

	Methods

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All animal protocols were reviewed and approved by The Institutional Animal Care Committee of the Research Institute of Medical Science at Chonnam National University. Adult Male Sprague-Dawley rats (250–300 g) were housed in groups of 4 in standard clear plastic cages and maintained in a temperature-controlled room (20°C ± 1°C) on a 12-h night/day cycle. Food and water were provided at ad libitum. Rats were implanted with chronic intrathecal cathe-ters under enflurane anesthesia according to a method described elsewhere (14). A midline incision was made over the atlantooccipital junction. Each polyethylene-10 catheter extended from the cisterna to the rostral edge of the lumbar enlargement and was externalized through the anterior part of the scalp. The outer end of the catheter was plugged with a steel wire and the skin was closed with 3–0 silk sutures. Only rats with normal motor function were used; the others were killed by volatile anesthetic overdose. After recovery from anesthesia, animals were individually housed in cages and monitored for at least 4–5 days before experiments.

Drugs used in this study were as follows: 2-chloro-N⁶-cyclopentyladenosine (CPA, Research Biochemical Internationals [RBI], USA), DPMA (RBI) and IB-MECA (Tocris Cookson Ltd., UK). All the drugs were dissolved in dimethylsulfoxide and intrathecally administered using a hand-driven, gear-operated syringe in a volume of 10 µL solution followed by an additional 10 µL of saline to flush the catheter.

The formalin test was used to measure pain state. Formalin 50 µL 5% solution was injected subcutaneously into the plantar aspect of the hindpaw using a 30-gauge needle. Formalin injection produces the specific behavior of flinching/shaking of the affected paw. This formalin-induced behavior was regarded as a pain response and observed for 60 min. The number of flinching/shaking response was counted for 1-min periods at 1 to 2 min and 5 to 6 min and at intervals of 5 min from 10 to 60 min. The flinching response is typically observed in two phases after formalin injection. Thus, the early and late phases of the formalin test were defined as the period of time immediately after injection of formalin until 10 min or 10 to 60 min after formalin injection, respectively. Rats were killed by volatile anesthetic overdose at the end of the formalin test.

Rats were placed in a restraining cylinder 4–5 days after surgery for the study. After a habituation period of 20 min, rats were assigned to one of the drug treatment groups. Dimethylsulfoxide was used as a control (n = 6). Ninety-eight rats were used and each group comprised 6–8 rats. Rats received only one dose of drug. The formalin test was performed only once in each rat.

After intrathecal administration of adenosine agonists, motor function was assessed by placing-stepping and righting reflexes (n = 15). The former was assessed by placing the rat horizontally with its back on the table, which normally causes an immediate coordinated twisting of the body to an upright position. The latter was evaluated by drawing the dorsum of either hindpaw of the rat across the edge of the table, which normally causes the rat to try to put the paw ahead into a position to walk. Motor function was measured at 5, 10, 20, 30, 40, 50, and 60 min after intrathecal administration of adenosine agonists at maximum doses used in this study.

For evaluation of the time course and dose-response of the effect of adenosine receptors agonists, A₁ agonist (CPA, 0.3, 0.8, 2.7 nmol), A_2A agonist (DPMA, 5.8, 19.2, 57.6, 191.8 nmol), and A₃ agonist (IB-MECA, 19.6, 58.8, 196, 587.9 nmol) were intrathecally administered 10 min before the formalin injection. On the other hand, intrathecal CPA resulted in motor disturbance at 8.1 nmol; therefore we used 2.7 nmol of CPA as a maximal dose. Each ED₅₀ value (effective dose producing a 50% reduction of control formalin response) of the three drugs was separately determined.

Data are expressed as mean ± sem. The time response data are presented as the number of flinches. The dose-response data are presented as the sum of the number of flinches in each phase. To calculate the ED₅₀ values of each drug, the number of flinches was converted to "percentage of control" as follows: % of control = (Sum of flinching number with drug in phase 1 [2])/(Sum of flinching number in control phase 1 [2]) x 100.

To compare the potency, each ED₅₀ and 95% confidence intervals (CI) in 2 phases were estimated according to Tallarida and Murray (15). Dose response data were analyzed by one-way analysis of variance with Scheffé testing for post hoc. The level of statistical significance was set at P < 0.05.

	Results

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Subcutaneous injection of formalin into the plantar region of the hindpaw resulted in a biphasic flinching response in the injected paw. The time course effects of intrathecal CPA, DPMA, and IB-MECA are shown in Figure 1. Intrathecal CPA produced a limited (approximately 54% of control) suppression of the early phase response of the formalin test, while it produced a dose-dependent suppression of the late phase response (Fig. 2). Intrathecal DPMA dose-dependently blocked the flinching response during the early and the late phases of the formalin test (Fig. 2). Intrathecal IB-MECA reduced the late phase response without affecting the early phase response (Fig. 2).

View larger version (35K): [in this window] [in a new window]   Figure 1. Time course curves of intrathecal CPA (A), DPMA (B), and IB-MECA (C) for the flinching response in the formalin test. Drugs were administered 10 min before formalin (F) injection. Data are presented as the number of flinches. Each point on the graph represents the mean ± sem of 6–8 rats. *P < 0.05 versus control.

View larger version (18K): [in this window] [in a new window]   Figure 2. Dose response curves of intrathecal CPA, DPMA, and IB-MECA for the flinching response during phase 1 (A) and phase 2 (B) in the formalin test. Data are presented as the percentage of control. CPA produced a limited reduction of the flinching response during phase 1 in the formalin test, but it dose-dependently decreased the flinching response during phase 2. DPMA produced a dose-dependent inhibition of flinching response in both phases in the formalin test. IB-MECA did not reduce the flinching response during phase 1 in the formalin test, but it dose-dependently decreased the flinching response during phase 2. Each point on the graph represents the mean ± sem of 6–8 rats. C = control. *P < 0.05, P < 0.01, P < 0.001 versus control.

The calculated ED₅₀ values with 95% CI of CPA, DPMA, and IB-MECA in the early or late phase are shown in Table 1. The rank order of potency (defined by ED₅₀ in nmol) of the late phase in the formalin test was as follows: CPA > DPMA >> IB-MECA.

Placing-stepping and righting reflexes were normal after intrathecal delivery of CPA, DPMA, and IB-MECA at maximum doses and no other motor dysfunction was observed.

	Discussion

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The behavioral pain response to formalin injection is characterized by two phases corresponding to basically different processes, with the early short-lasting phase (phase 1) of acute pain and the late phase of prolonged pain (phase 2). The phase 1 response results essentially from the direct stimulation of nociceptors of the primary afferent. The phase 1 response is therefore considered to correspond to the high level of activity in the primary afferent. On the other hand, the phase 2 response seems to originate from the continuous low level of small afferent input. The afferent input generated by formalin is believed to be from release of glutamate and substance P, which initiate a cascade through N-methyl-d-aspartate and neurokinin1 receptors. The resulting cascade leads to a state of facilitation that appears to be more than anticipated, considering the diminished level of afferent input (16).

In the present study, intrathecal CPA (adenosine A₁ receptor agonist) had a limited effect on the early phase response of the formalin test but decreased the late phase response. Intrathecal DPMA (adenosine A_2A receptor agonist) attenuated formalin-induced pain behavior during both phases. Intrathecal IB-MECA (adenosine A₃ receptor agonist) suppressed the flinching response during the late phase but not during the early phase. These observations suggest that adenosine A₁ and A_2A receptors may be minimally or actively involved in the modulation of acute nociception in the spinal cord, respectively. In contrast, the spinal adenosine A₃ receptor may not contribute to the control of acute nociception. On the other hand, spinal adenosine A₁, A_2A and A₃ receptors may play a critical role in the modulation of the inflammatory process occurring during formalin pain.

Adenosine may play an important role in the modulation of nociceptive inputs through adenosine A₁ and A₂ receptors identified in the dorsal horn of the spinal cord (7,17). It has been reported that intrathecal adenosine A₁ receptor agonists attenuated not only the inflammatory hyperalgesia but also acute nociception (8,9,10,12). Moreover, adenosine A₁ receptor agonist inhibited excitatory transmission in the spinal cord (18). Thus, the role of spinal adenosine A₁ receptor in this study is in accordance with results from previous studies. Meanwhile, intrathecal adenosine A_2A receptor agonist failed to modify the frequency of flinching/lifting during the entire formalin test, but it induced a limited amount of suppression of the late phase licking/biting responses without affecting early phase responses (8). Moreover, spinal adenosine A_2A receptor agonist produced a modest antinociception in the inflammatory thermal hyperalgesia model (10). However, intraperitoneal adenosine A_2A receptor agonist counteracted the flinching response induced by the formalin test only during the early phase but not the late phase (11). Although the activation of adenosine A_2A receptor exerts different actions, it is not clear if the different effects observed could be, at least in part, ascribed to the difference in drugs, the activation of the receptor, the route of drugs, or the different types of tested stimuli. Adenosine receptor activation in the spinal cord is proposed to produce an antinociception by at least two distinct mechanisms: presynaptic inhibition of excitatory neurotransmitter release with subsequent reduction of substance P concentration in cerebrospinal fluid (9) and postsynaptic inhibition of the effects of excitatory neurotransmitters (19). The above findings jointly suggest the adenosine A_2A receptor contributing to antinociceptive action in the spinal cord.

Unfortunately, we did not evaluate the role of adenosine A_2B receptor for the modulation of nociception because there are no available adenosine A_2B-selective agonists.

Subcutaneous administration of adenosine A₃ receptor agonist produced nociceptive behavior (13). However, stimulation of spinal adenosine A₃ receptor possibly exerted an inhibitory influence on the release of pain-related neuropeptide in the spinal cord (20), which may have contributed to the antinociceptive role for spinal adenosine A₃ receptor in the control of nociception. Moreover, spinal adenosine A₃ receptor agonist reduced the late phase response of the formalin test in the present study. This is the first report of the antinociceptive role of spinal adenosine A₃ receptor in formalin-induced inflammatory hyperalgesia.

In the current study, intrathecal CPA showed a minimal effect (54% of control) in phase 1 and IB-MECA also had no effect in the same condition. Furthermore, intrathecal CPA caused motor dysfunction at 8.1 nmol. Therefore, we could not compare the potency of the above three drugs during phase 1 in the formalin test. However, maximum of % of control of intrathecal CPA, DPMA, and IB-MECA was similar in phase 2, which made it possible to compare their potency. Thus, the rank order of potency in phase 2 was CPA > DPMA >> IB-MECA at the spinal level. These observations suggest that spinal adenosine A₁ receptor may be more involved than adenosine A_2A and A₃ receptors in the modulation of the inflammatory hyperalgesia. Therefore, agonists for adenosine A₁ receptor other than adenosine A_2A and A₃ receptors may be effective in the management of the inflammatory hyperalgesia in the spinal cord. One interesting finding was the relative effectiveness of DPMA on phase 1 and 2 responses of the formalin rest. A three-fold larger ED₅₀ for phase 2 was observed compared with that for phase 1 with DPMA. These data suggest that adenosine A_2A receptor seems to be much more effective on early pain than on inflammatory hyperalgesia, which in turn supports the theory that agonists for adenosine A₁ receptor may be useful in the treatment of acute pain in the spinal cord.

In conclusion, adenosine A₁ and A_2A receptors but not adenosine A₃ receptor exhibit an antinociceptive profile in acute nociception in the spinal cord. However, all adenosine A_1, A_2A and A₃ receptors are involved in the control of a formalin-induced inflammatory hyperalgesia.

	Footnotes

 
Supported, in part, by Research Institute of Medical Science, Chonnam National University.

Accepted for publication May 25, 2005.

	References

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Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999