Altered effects of an A1 adenosine receptor agonist on the evoked responses of spinal dorsal horn neurones in a rat model of mononeuropathy

Altered Effects of an A, Adenosine Receptor Agonist on the Evoked Responses of Spinal Dorsal Horn Neurones in a Rat Model of Mononeuropathy Rie Suzuki, Anabelle Gale, and Anthony H. Dickenson Abstract:

There is clinical evidence that adenosine might be a useful treatment for neuropathic pain states, although little is known regarding its mechanisms. In this study, we use the selective (L5/L6) spinal nerve ligation model to investigate the effects of an adenosine A, receptor agonist, N6-cyclopentyladenosine (CPA), on the evoked responses of dorsal horn neurones after nerve injury in vivo. Two weeks after surgery, the responses of dorsal horn neurones to controlled electrical and natural (mechanical and thermal) stimuli were recorded and the effects of intrathecal CPA were compared between nerve-ligated and sham-operated rats. CPA produced significant inhibitions of the C-fiber-evoked response, postdischarge, wind-up, mechanical, and thermal-evoked responses in both groups, but only minor inhibitions of the Aj3-fiber response. Overall, the drug effects in spinal nerve-ligated rats were greater than those of sham-operated rats. Spinal theophylline reversed these inhibitions. In contrast, CPA produced marked facilitations of the A&fiber-evoked neuronal responses in sham-operated animals, yet this effect was completely absent after nerve injury. These results suggest that nerve injury induces plasticity in the spinal A, receptor system, which might form the basis for the therapeutic use of adenosine. Key words: Adenosine, CPA, nerve injury, antinociception, spinal cord.

s

ubstantial evidence exists for a role for adenosine in modulating primary afferent transmission through spinal neurones of the dorsal horn.‘,* Adenosine 5’-triphosphate (ATP) has been reported to be released from small diameter primary afferents and subsequently can be converted to adenosine through a series of metabolic pathways.3 Receptors for this purine have been identified in the substantia gelatinosa of the spinal cord, where they are localized on intrinsic neurones, and 2 subtypes of adenosine receptors (A,/A,) have been described so far.4-6 Although evidence exists for the involvement of A2 receptors in spinallymediated antinociception,7 it appears to be pre-

Received October 12, 1999; Revised December 6, 1999; Accepted December 8, 1999 From the Department of Pharmacology, University College London, Gower Street, London, United Kingdom. Supported in part by the European Community Biomed II program BMH4-CT95-0172, the Wellcome Trust, an Overseas Research Scholarship, and a Nuffield Vacation Studentship. Address reprint request to Rie Suzuki, PhD, Department of Pharmacology, University College London,Gower Street, London. WCIE 68T, United Kingdom. E-mail: [email protected] 0 2000 by the American Pain Society 1526.5900/00/0102-0006$8.00/O doi:10.1054/xb.2000.6055

dominantly the A, receptor subtype that plays the major role in inhibiting the nociceptive input in the dorsal spinal cord.8,g There have been numerous studies showing the antinociceptive effects of both adenosine and receptorselective analogs in animal behavioral models, with relatively few electrophysiological investigations.*,1° Adenosine has been shown to produce hyperpolarizations of the postsynaptic membrane, and depress excitatory postsynaptic currents (EPSC) in the substantia gelatinosa of the spinal cord. l1 Similarly, iontophoretic application of adenosine 5’-monophosphate (AMP), which is readily converted to adenosine, inhibits the ongoing activity and responses of dorsal horn neurones to noxious and innocuous peripheral stimuli.3 In addition, in vivo studies have shown that intrathecal adenosine agonists attenuate the acute and persistent nociceptive responses of dorsal horn neurones,g and are effective in reducing noxious evoked activity in the presence and absence of sensitization.‘* These results, observed across several models of pain, strongly support the potential clinical use of these agents in various pain states. There is now a growing interest in the development of therapeutic agents that interact with adenosine

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systems for the treatment of neuropathic pain. One characteristic of neuropathic pain states is their relative refractoriness to opioids, making pain management difficult13,14; yet, clinical and animal studies have reported adenosine analogs to be effective against symptoms of neuropathic pain. 15-18 Although very little is known regarding the mechanisms behind these effects, we have proposed an association between adenosine and the N-methyl-D-aspartate (NMDA) receptor in normal animals9 This might relate not only to the well-established role of adenosine receptors in antinociception, but also in the mechanisms underlying longterm potentiation (LTP),lgr20 as this class of receptors appears to have an important role in regulating the expression of LTP through their ability to indirectly modulate synaptic transmission. A recent study reported that LTP can be induced in the superficial dorsal horn by acute nerve injury.21 The direct and indirect manipulation of the adenosine system might prove to be a useful approach in the modulation of longterm changes in synaptic events, such as those contributing to spinal nociception. Most previous studies on adenosine systems have been behavioral and, generally, have studied only drug effects on a single sensory modality. Thus, previous studies have rarely compared the effectiveness of adenosine A, agonists on a wide range of stimuli under identical conditions or suggested possible underlying neuronal mechanisms for the observed actions of the purine. Here, we examine the effect of intrathecal CPA, an adenosine A, receptor agonist, on the evoked neuronal responses to controlled natural (mechanical/thermal) and electrical stimuli in an animal model of neuropathic pain. 22 We have compared the effectiveness of CPA between spinal nerve-ligated (SNL) and sham-operated rats to investigate whether plasticity in the spinal purinergic system takes place after peripheral nerve injury and to provide a possible basis for the treatment of the multiple symptoms of neuropathic pain.

Materials and Methods Animals Twelve adult male Sprague-Dawley rats (Central Biological services, University College London, London,United Kingdom) weighing 140 to 150 g were used in this study. The animals were housed in groups of 4 to 5 in plastic cages under a 12 hour day/l2 hour night cycle for a week before to surgery. Animals were divided into a control group (sham operation, n = 6) and an experimental group (L5/L6 spinal nerve ligation, n = 6). All experimental procedures were

approved by the Home Office and follow the guidelines under the International Association for the Study of Pain. 23 The procedure for the L5/L6 spinal nerve ligation was performed as previously described by Kim and Chung (1992).

Surgical Procedure for Spinal Nerve Ligation Under halothane anesthesia (1.2% to 2.5% in 50% N,O and 50% O,), the rat was placed in a prone position and the left paraspinal muscles were separated from the spinous processes at the L4-S2 levels. A part of the L6 transverse process was carefully removed with rongeurs and the L4-L6 spinal nerves were exposed and identified. The left L5 and L6 spinal nerves were isolated and tightly ligated with 6-O silk thread. A complete hemostasis was confirmed and the wound was sutured. The surgical procedure for the sham-operated group was identical to that of the experimental group, except that the spinal nerves were not ligated.

Behavioral Tests After surgery, rats were maintained under the same conditions as during the preoperative period. The weight gain and general behavior of the operated rats were carefully monitored throughout the postoperative period. Careful observations were made on the position and posture of the foot. Behavioral tests were carried out in the morning, after surgery at 2, 3, 5, 7, 9, 12, and 14 days. Rats were placed in transparent plastic cubicles on a mesh floor and a period of acclimatization was allowed before to testing. The sensitivity of the rats’ hindpaws to mechanical and cooling stimuli were assessed through 2 forms of behavioral testing. Mechanical sensitivity was assessed through the measurement of foot withdrawal frequencies to a sequential series of calibrated von Frey filaments of weights 1, 5, and 9 g (9.9, 49.5, and 89.1 mN, respectively) applied to the plantar surface of the foot. A single trial of stimuli consisted of applications of a von Frey filament repeated 10 times, each application not lasting for more than 3 seconds. Von Frey filaments were applied in an ascending order and the tests were separated by periods of 3 minutes. The occurrence of foot withdrawal for each trial was quantified and expressed as the difference score: difference score = (number of foot withdrawals on contralateral paw) (number of foot withdrawals on ipsilateral paw) The sensitivity of the hindpaw to cooling was assessed through the application of a drop of

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acetone onto the plantar region of the foot using a syringe connected to a small polyethylene tubing. Care was taken not to apply the acetone in an abrupt squirt to avoid evoking a mechanical response rather than a response to the cooling effect of acetone itself. Each trial consisted of 5 applications of acetone per hindpaw and a period of 5 minutes was allowed between each application. The number of foot withdrawals was expressed as the difference score as described above.

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Electrophysiological Studies After the above behavioral testing period, the operated animals (250 to 300 g) were subsequently used for electrophysiological studies when allodynia reached its maximal level (14 to 17 days after surgery). Experimental procedures for the electrophysiological studies have been described previously. 24 Briefly, anesthesia was induced in rats with 2.0% to 2.5% halothane in a gaseous mixture of 66% N20 and 33% 02, after which tracheal cannulation was performed. The rats were subsequently placed in a stereotaxic frame to ensure stability during electrophysiological recordings. A laminectomy was performed at the Ll-L3 level and segments L4-L5 of the spinal cord were exposed. The cord was held rigid by clamps caudal and rostra1 to the exposed section, and 2 rods were inserted into the lateral processes of the vertebrae to improve stability at the recording site. The level of anesthesia was reduced to 1.2% to 1.8% halothane, which produced a state of complete areflexia. The core body temperature of the rat was monitored and maintained (36.5”C to 37°C) by means of a heating blanket connected to a rectal thermal probe by an automatic feedback control unit. Extracellular recordings of convergent dorsal horn neurones, ipsilateral to spinal nerve ligation or sham procedure were made with parylenecoated tungsten electrodes that were descended through the cord by a SCAT microdrive. The depth of the neurone from the surface of the dorsal horn of the spinal cord was recorded for each neurone. Transcutaneous electrical stimuli were applied by 2 needles inserted to the receptive field of the recorded neurone. A train of 16 stimuli was applied at 3 times the threshold current for C-fibers (0.5 Hz), and a poststimulus histogram was constructed. AD-, A&, and C-fiberevoked neuronal responses were separated and quantified on the basis of latency measurements (AD-fiber, 0 to 20 milliseconds; AS-fiber, 20 to 90 milliseconds; C-fiber, 90 to 300 milliseconds) based on the known conduction velocities for the various fiber types (Fig IB). Any neuronal responses occurring after the C-fiber latency

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1. (A) A single-sweep oscilloscope trace of the response of a single dorsal horn neurone to an electrical stimulus applied to the peripheral receptive field. (B) A typical example of a poststimulus histogram following a train of 16 electrical stimuli. The responses evoked by the different fibers were separated and quantified on the basis of latency measurements. (C) Typical responses of a single convergent dorsal horn neurone to various natural stimuli: brush, a blunt prod stimulus, von Frey hairs, and heat. All stimuli were applied to the center of the peripheral receptive field. The duration of the applied stimulus (10 seconds) is indicated as a horizontal bar below each evoked neuronal response. B, brush; P, prod.

band resulting from the hyperexcitability of the neurone (300 to 800 milliseconds) were taken to be the postdischarge of the neurone. Windup was calculated as the difference between the number of action potentials evoked at 3 times the C-fiber threshold after 16 stimuli and the baseline. The baseline response (the nonpotentiated response that would have occurred in the absence of windup) was calculated as the number of action potentials in the response produced by the first stimulation, multiplied by the total num-

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2. The difference score of foot withdrawal frequencies to the application of (A) von Frey (9 g) and (B) a drop of acetone in SNL (filled circles) and sham operated rats (open squares). Difference scores were calculated as: difference score = number of foot withdrawals on contralateral paw-number of foot withdrawals on the ipsilateral paw. Negative values indicate a greater frequency of foot withdrawal on the ipsilateral hindpaw, which was interpreted to be a manifestation of mechanical allodynia. The data are presented as the mean foot withdrawal frequency f SEM.

I. A Corn arisonof the MeanCellDepth,C-fiber Thresholdand Bredru EvokedResponses of S inal Neuronesto ElectricaPand NaturalStimuli in 5RL and Sham-Operated Rats14to 17 DaysAfter Surgery

Table

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SHAM

Cell depth (pm) C-fiber threshold (mA) A/3-fiber evoked response A&fiber evoked response C-fiber evoked response Wind-up (AP) Postdischarge (AP) von Frey 9g (AP) von Frey 509 (AP) Heat (AP)

(AP) (AP) (AP)

710 + 1.8 f 93 + 115+28 400 2 273 i 297 f 176+77 649+ 871 f

38 0.2 23 95 62 119 112 221

638 212 1.4 k 0.2 842 13 85+ 10 330 f 55 245 r?: 70 280 i 59 187-+40 603+ 112 684 i 142

NOTE: The magnitudes of the electrical and natural evoked responses before drug administration were comparable between both animal groups for all measures. Abbreviations: SNL, spinal nerve ligated; AR (the mean number of) action potentials.

ber of stimuli in the stimulus train (16). Data was captured and analyzed by a CED 1401 interface coupled to a Pentium computer with Spike 2 software (rate and poststimulus histogram functions) (Cambridge Electronic Design, Cambridge, United Kingdom). The peripheral neuronal receptive field also was stimulated using various natural stimuli (brush, prod, mechanical punctate, and heat). Typical examples of the neuronal responses to electrical and natural stimuli are shown in Fig 1.

Pharmacological Studies on Evoked Afeuronal Responses Before drug administration, stable responses to peripheral electrical and

control natural

stimuli were established. Tests to natural stimuli involved the application of low- and high-intensity mechanical punctate (von Frey weights 9 g and 50 g) and thermal stimuli (45°C) to the receptive field of the recorded neurone. Not all natural stimuli (brush, prod, mechanical punctate, heat; see Fig IC) were employed in the pharmacological study to avoid potential peripheral changes in the hindpaw. Thermal stimulation involved the application of a constant jet of water (45°C) onto the center of the receptive field. 25 All stimuli were applied for a period of 10 seconds, and the frequency of the neuronal response to the applied stimulus was quantified. CPA (Research Biochemicals International [RBI]) was administered to the rat intrathecally, directly onto the surface of the spinal cord (cumulative doses of 0.5 pg, 5 pg, and 50 pg). The effect of the drug was observed over a period of 40 minutes per dose, and tests were carried out at lominute intervals to determine the effects of the drug on electrically and naturally evoked responses. The effects of the highest dose of CPA (50 pg) were reversed with intrathecal theophylline, a nonspecific A, / A2 receptor antagonist (1000 pg) to investigate whether the effects of CPA could be reversed with an antagonist applied to the spinal cord.

5 ta tistical Analysis Data are presented mean (SEM) unless effects are expressed age of the predrug otherwise. Statistical performed using the

as mean f: standard error of stated otherwise. Drug as mean maximal percentcontrol value unless stated analysis of drug effects was Wilcoxon signed rank test.

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Figure 3. The effect of intrathecal administration of CPA on the (A) C-fiber-evoked response, ( E3) postdischarge, (C) A& fiber-evoked response, and (D) A@fiber-evoked response of spinal neurones in SNL and sham-operated rats. CPA produced a marked facilitation of the Ad-fiber evoked response in sham-operated rats; however, this was completely absent in SNL rats (5 mg, P= .05; 50 mg, P = .02). * Indicates a significant difference (P < .05) between animal groups. Data are presented as percentage inhibitions of the predrug control values 2 SEM.

The Mann-Whitney test was employed for the comparison of drug effects between different animal groups. All statistical tests were performed on raw data. Level of significance was taken to be a P-value of .05.

Results Behavioral Studies After surgery, the rats maintained good health and exhibited normal weight gain. The rats displayed no signs of distress, abnormal aggressive behavior, or autotomy. SNL rats exhibited a guarding behavior of the ipsilateral hindpaw. Such behavior was not seen in sham-operated rats. SNL rats displayed behavioral signs of mechanical and cold allodynia of the ipsilateral hind paw. Rats exhibited an abrupt and brisk foot withdrawal in response to normally innocuous mechanical stimuli (von Freys 1 g, 5 g, and 9g) and to the application of acetone. Foot withdrawal was often accompanied by aversive behaviors such as shaking and licking of the ipsilateral paw. These exaggerated responses were interpreted to be a manifestation of mechanical and cold allodynia and were observed as early as two days after surgery. There was an increase in the level of allodynia over the postoperative period, and this was maintained throughout the whole of the testing period (2 to 14 days after

surgery). The contralateral paw of SNL rats did not develop such modified mechanical/cold sensitivity. Examples of these behavioral responses are shown in Fig 2. Sham-operated rats did not exhibit any signs of mechanical or cold allodynia on either hindpaw (ipsilateral or contralateral) and rarely responded to even the strongest von Frey filament (9 g) employed in the behavioral study.

ElectrophysiologicaI Studies A total of 12 dorsal horn neurones (SNL, n = 6; sham-operated, n = 6) were employed in the study. The mean depths of the neurones were similar between SNL and sham-operated rats and corresponded to lamina V of the dorsal horn. Both the C-fiber threshold and the predrug electrically and naturally evoked responses of spinal neurones were comparable between the 2 animal groups (Table 1).

Effect of CPAAdministration on Electrical Evoked Neuronal Responses The administration of CPA produced a significant and dose-dependent inhibition of the Cfiber-evoked response of SNL rats (0.5 ug, P = .03; 5 ug and 50 pg, P = .02). Similarly, the Cfiber-evoked response of sham-operated rats was reduced after CPA (0.5 ug, 5 ug, and 50 ug,

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4. (A) The effect of intrathecal administration of CPA Data are presented as percentage inhibitions of the predrug between animal groups. Typical examples of the effect of CPA rats. Trains of 16 electrical stimuli were given at 3 times the evoked per stimulus was plotted against the stimulus number drug CPA (50 pg).

on the windup of spinal neurones in SNL and sham-operated rats. a significant difference (P < .05) control values * SEM. * Indicates on the windup of spinal neurones in (B) SNL and (C) sham-operated threshold current for C-fibers and the number of action potentials before (open squares) and after (filled circles) administration of the

P = .03). The magnitude of the inhibition was greater in SNL rats (maximal inhibition, 72 * 7% of predrug control value) compared with that of sham-operated rats (55 + 16% of predrug control); however, this difference was nonsignificant (Fig 3A). CPA produced a significant inhibition of the postdischarge of spinal neurones in SNL (0.5 ug, P = .03; 5 pg and 50 ug, P = .02) and shamoperated rats (50 ug, P = .03). The effect of the drug was greater in SNL rats (maximal inhibition, 94 f 3%) as compared with that of sham-operated rats (70 + 18%); however, again, this difference was nonsignificant between the 2 groups. As with the C-fiber-evoked response, the increased effect of CPA after nerve injury was manifested as a parallel leftward shift in the dose-response curve (Fig 3B). The windup of spinal neurones was significantly inhibited by CPA in SNL rats (5 pg and 50 pg, P = .03). The inhibition of the windup was greater in SNL rats (maximal inhibition, 85 f 9%) compared with that of sham-operated rats (56 + 19%) (5 pg, P = .04). This shift was not seen with the lowest dose of CPA (Fig 4). Interestingly, the effect of CPA on the A&fiber-evoked response was quite different after nerve injury. In complete contrast to the dose-related inhibitory effects seen with CPA on the C-fiber-evoked response and measures of hyperexcitability (windup and postdischarge), CPA produced a marked facilitation of the Adfiber-evoked response of sham-operated rats

(Fig 3C). After the administration of 50 pg of the drug, CPA produced a clear enhancement of the A&fiber-evoked response in sham-operated animals (an increase of 194 f 55% compared with predrug control values). Remarkably, in complete contrast, CPA produced little effect on the Ah-fiber-evoked response of SNL rats and the facilitation was entirely absent in this group of animals (Fig 3C). CPA produced only a minor inhibition of the AP-fiber-evoked response in both SNL (maximal inhibition, 20 it 4%) and shamoperated rats (23 -c 5%). The maximal inhibition did not exceed 25% even with the highest dose of CPA employed in this study, and the effects were comparable between the animal groups (Fig 3D).

Effect of CPAAdministration on Naturally Evoked Meuronal Responses CPA produced dose-dependent inhibitions of the mechanically evoked responses of SNL and sham-operated rats (Figs 5A and 58). Both the 9 g von Frey (0.5 ug, P = .03; 5 and 50 ug, P = .02) and 50 g von Frey-evoked responses (0.5 ug, P = .03; 5 pg and 50 ug, P = .02) of SNL rats were reduced by the administration of CPA. Similarly, inhibitions also were observed in sham-operated rats (von Frey 9 g: 5 ug and 50 ug, P = .03; von Frey 50 g: 5 pg and 50 ug, P = .03). CPA produced a significantly greater inhibition of the 9 g von

ORIGINAL REPORT/Suzuki et al

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Figure 6. Typical time course of the effect of fiber-evoked response of a single dorsal Cumulative doses of CPA were administered and their effects were observed over a period per dose. CPA produced an inhibition of the response, and this was reversed with spinal after the final dose of CPA (5Opg).

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Figure 5. The effect of intrathecal administration of CPA on the (A) mechanical 9 g von Frey evoked response, (B) 50 g von Frey-evoked response and (C) thermal stimuli evoked response (45Y) of 5NL and sham-operated rats. Data are presented as percentage inhibitions of the predrug control values * SEM. * Indicates a significant difference (P < .05) between animal groups. Frey-evoked response in SNL rats (5 ug, P = .Ol) (Fig 5A). The inhibition of the 50 g von Frey-evoked response also tended to be greater in SNL rats (maximal inhibition 88 & 6%) compared with sham-operated rats (77 * 5%) (Fig 5B). The administration of CPA produced a significant reduction of the thermally evoked response of SNL (0.5 pg, 5pg, and 50 pg, P = .03) and sham-operated rats (0.5 pg, 5 ug, and 50 pg, P = .04) (Fig 5C). The inhibition was greater in SNL rats (maximal inhibition 88 f 7%) compared with that of sham-operated rats (74 f 10%) (0.5 ug, P = .03; 5 ug, P = .04). Maximal effects of all doses of CPA were observed between 20 to 30

CPA on the Chorn neurone. intrathecally, of 40 minutes C-fiber-evoked theophylline

minutes after administration of the drug (Fig 6). Thus, our use of 40-minute intervals between cumulative dosing is justified, because the doseeffect had reached a stable maximum at this point (eg, mean maximal effect for C-fiber inhibition: 0.5 ug: 28 + 3 minutes after CPA administration; 5 ug: 26 f 3 minutes; 50 ug: 31 f 2 min). Consistent with previous findings, the effects of spinal CPA were reversed to near predrug control values with spinal theophylline (1000 ug) in both groups of animals. For example, the inhibitions of the C-fiber-evoked response and the 50 g von Frey-evoked response produced by 50 ug CPA were reversed to 101 + 2% and 97 * 4% of predrug control values with the administration of theophylline, respectively. These findings confirmed that the effects of intrathecal CPA were produced through actions at adenosine receptors in the spinal cord.

Discussion In the present study, we have replicated an animal model of neuropathic painZ2 and conducted an electrophysiological study to quantify the effects of an A, adenosine agonist, CPA, on the responses of spinal dorsal horn neurones. Selective ligation of the L5/L6 spinal nerves produced reproducible behavior in rats that was characterized by changes in foot posture and modifications of the ipsilateral hindpaw sensitivity to mechanical and cold stimuli. The model of neuropathy employed in the pre-

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sent study involves the selective ligation of 2 of the 3 spinal nerves that make up the sciatic nerve.** The tight ligation of the spinal nerves produces a complete block of sensory transmission through these nerves; hence, results in a significant decrease in the sensory input into these segments of the spinal cord. Using presynaptic opioid receptors as markers of presynaptic terminals, a previous autoradiographic study quantified the contribution of afferent fibers in a single dorsal root to adjacent spinal segments in the cervical spinal cord. 26 If the same pattern holds for the lumbar spinal cord, it could be expected that the overall input to the L4 segment (which receives information from the intact L4 spinal nerve and also from the L5 and L6 spinal nerves) is considerably reduced by about 37% as a result of the partial deafferentation. This might only relate to unmyelinated small diameter fibers, because opioid receptors are predominantly located on these fibers so that the exact contribution of large myelinated fibers from adjacent spinal segments is unknown. Evidence for the loss of primary afferents following spinal nerve ligation is lacking; however, a considerable proportion of spinal input is expected to be lost in the L4-L6 segments of SNL rats, as a result of the L5/L6 nerve ligation. However, our present electrophysiological study has shown that despite this possible loss of spinal input, there was no dramatic shift in the predrug control responses of spinal neurones 2 weeks after nerve injury (Table 1). This would suggest that there is a marked increase in spinal cord excitability that compensates for this loss of input. A previous study has reported a high level of spontaneous activity in spinal neurones of SNL rats25 and this could in part contribute to the overall increased spinal hyperexcitability. Additionally, although the evoked neuronal responses were comparable after nerve injury, thresholds for responses to natural stimuli can be lower, as we have previously reported.27 Possible correlations between alterations in dorsal horn neuronal responses and the behavioral and clinical symptoms in this model of nerve injury have been discussed.25 In this study, we show that the intrathecal administration of CPA, an adenosine A, receptor agonist, produces inhibitions of the responses of dorsal horn neurones to electrical and natural stimulation of the peripheral receptive field. CPA produced significant inhibitions of the Cfiber-evoked response, postdischarge and windup of spinal neurones, and reduced the neuronally evoked responses to low- and highintensity natural stimuli. In contrast, the lowthreshold AD-fiber-evoked response remained relatively unaffected, despite the fact that CPA reduced the low-intensity 9 g von Frey-evoked

response. It is likely that this natural mechanical stimulus, which might include a contribution from both AP- and As-fibers, would produce a pattern of activity more amenable to inhibition than the suprathreshold synchronized responses elicited by the electrical activation of AP-fibers. Therefore, our results indicate a broad spectrum of action of CPA that might result from a combination of presynaptic and postsynaptic actions. Adenosine-like immunoreactivity is found within the substantia gelatinosa of the spinal cord dorsal horn,6,28 where a high density of adenosine receptors (AI/A,) are localized, primarily on neurones postsynaptic to primary afferents.3-5,2g In general, adenosine exerts its actions through the inhibition of excitatory postsynaptic sensory pathways, by activating K+ channels and consequently producing hyperpolarization.3,11,30*31 Furthermore, a presynaptic action of adenosine has been proposed on the basis of an inhibition of primary afferent Ca*+ currents and the release of neuropeptides in various experimental preparations.32-36 Adenosine also has been shown to decrease transmitter release from retinotectal terminals,37 as well as from rat hippocampal slices38-3g and brainstem/ spinal cord preparations40 Whereas presynaptic receptors associated with small-diameter afferents could explain the effects of CPA on the C-fiber and noxious-evoked activity, the broad inhibitory effects, especially on the postdischarge and N-methyl-D-aspartate (NMDA) receptor-mediated windup, imply postsynaptic receptors, as does the inhibition of the low-intensity mechanical stimuli. Although A, receptors are predominantly located in the superficial dorsal horn, their location on interneuronal systems or dendritic trees of the deeper neurones from which we recorded could be responsible for the observed effects. Post synaptic actions are supported by our finding that the dose-response curves for the inhibitory effects of CPA were similar for all modalities of stimuli. interestingly, the inhibitory effects of CPA were more marked after nerve injury and a leftward-shift in the dose-response curves was observed for the SNL group at 2 weeks after surgery. In direct contrast to these powerful inhibitory effects, CPA produced a dose-dependent facilitation of the A&fiber-evoked response in shamoperated rats, an observation also previously reported in naive ratsgr41 Because the A, receptor is inhibitory, this observed facilitation is likely to result from a postsynaptic disinhibitory action. The A&fiber terminals in the spinal cord, more so than other fiber terminals, are known to be contacted by GABAergic neurones.42r43 Activation of the A, receptor could hyperpolar-

ORIGINAL REPORT/Suzuki et al

ize the inhibitory gamma-aminobutyric acid (GABA) neurones, and this reduction in inhibitory tone could then result in a net facilitation of the As-fiber-evoked response (ie, disinhibition), as observed in the present study. However, during neuropathy, there appears to be a progressive loss of inhibitory interneurones, such as the GABA system.44,45 The loss of inhibitory controls might explain why such facilitations of the AZ-fiber-evoked responses were absent in SNL rats. Interestingly, in a recent study in which intrathecal adenosine was administered to patients with chronic neuropathic pain, transient pain was reported in some patients on spinal injection, which was followed by a prolonged pain relief.46 In addition to the mechanisms that have been proposed, it is possible that the pain experienced by these patients is caused by the disinhibitory action of adenosine on AS-fibers, causing a net facilitation in areas of the spinal cord outside the neuropathic zone, evoking a pain sensation.46 Previous studies have proposed possible interactions between adenosine and glutamate in the spinal cord,47 and between NMDA receptor activation and adenosine release elsewhere in the brain.48 From these studies, the A, receptor has been implicated in indirectly controlling the spinal NMDA polysynaptic nociceptive pathway, and this is suggested to be one mechanism underlying adenosine-mediated antinociception at the spinal level. g,41 Adenosine receptors have been shown to play an important inhibitory role in the development and maintenance of central sensitization of spinal dorsal horn neurones.12 After neuropathy, there appears to be an enhanced role of the NMDA receptor system in both low- and high-threshold signaling in the spinal cord.4g,50 Spinal hyperexcitability, which occurs partly as a result of a sustained afferent drive from the injury site, is likely to produce a greater degree of NMDA receptor activation following peripheral nerve injury. This, in turn, would lead to an increased release of adenosine, resulting in an eventual depletion of the purine. A recent study revealed that patients with neuropathic pain have deficits in circulating and cerebrospinal fluid (CSF) adenosine levels.51 It could then be envisaged that any disruption of the endogenous purinergic inhibitory tone as a result of nerve injury could augment spinal nociceptive transmission, contributing to facilitated sensory transmission (hyperalgesia) and miscoding of innocuous information (allodynia). Furthermore, any reduction in the level or release of adenosine would be expected to induce receptor upregulation. To date, changes in adenosine receptor expression after nerve injury have not been reported; however, the

107

enhanced effectiveness of CPA after nerve injury would support this idea.

The Role of Adenosine and its Analogs in Neuropathic Pain Substantial evidence exists for the effectiveness of adenosine analogs in animal models of neuropathic pain. The administration of R-PIA (Rphenylisopropyl-adenosine), an AI receptor agonist, was shown to attenuate the spontaneous scratching behaviors2 and allodynia of chronic constriction injury (Ccl) rats.15 Administration of intrathecal R-PIA also relieved allodynia in SNL rats18,53 and in rats with spinal cord injury.54 The spinal administration of NECA (5’-N-ethylcarboxamide-adenosine), a nonselective A,/A, receptor agonist, also was shown to be effective against thermal hyperalgesia in CCI rats.55 Furthermore, a recent study investigated the effects of adenosine itself and showed that spinal administration relieves allodynia in SNL rats.53 In contrast to this study, intrathecal adenosine did not produce antiallodynic effects in rats with spinal cord injury, 56 whereas R-PIA was found to be effective against this measure. Because none of these animal behavioral studies have used a broad range of stimuli, the complete role of the AI receptor has been difficult to assess in terms of its effects on different sensory modalities. The results of our electrophysiological study support these behavioral studies in which adenosine analogs were found to be effective against some of the symptoms of nerve injury states, and possibly provide a neuronal basis for these findings. Consistent with previous studies, we have shown that CPA reduces the acute nociceptive responses of dorsal horn neurones and further extend these observations by showing that the controls exerted by the A, receptor include a large range of modalities (low- and high-intensity mechanical and thermal stimuli). Clinically, there has been a considerable interest in the potential therapeutic use of adenosine in neuropathic pain states. In patients with peripheral neuropathic pain, systemic infusion of adenosine was shown to provide significant relief of spontaneous pain and tactile allodynia.17,57 lntrathecal administration of adenosine46 and its analogue also was shown to be effective against allodynia in patients with chronic neuropathic pain.58 Therefore, our present finding, that CPA produces inhibitions of the electrical and natural stimuli-evoked responses of spinal neurones, supports a role for adenosine analogs as potential therapeutics for the treatment of neuropathic pain. Further trials of the effectiveness of adenosine and its analogs in neuropathic pain conditions are required. One limitation in the use of

Adenostrw

108

adenosine analogs is the occurrence effects (eg, motor dysfunction and effects) that are associated with higher the drug.7,‘8,56,5g The motor dysfunction primarily to be an A,-mediated effect

of sidecardiac doses of appears in rats,5g

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