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Department of Clinical Neuroscience, Section of Neurosurgery, Karolinska Institutet, Stockholm, Sweden
Address correspondence and reprint requests to Bengt Linderoth, MD, PhD, Section of Neurosurgery, Karolinska Hospital, SE-171 76 Stockholm. Address e-mail to bengt.linderoth{at}kus.se
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Abstract |
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2-adrenoceptor agonist, clonidine, is extensively used as an adjunct to spinal morphine and is suggested to be particularly effective for neuropathic pain, but its clinical use is limited by side effects such as sedation and hypotension. In this study, we investigated the dose-response characteristics of IT clonidine, and whether a subeffective dose of clonidine could enhance the effect of SCS in nerve-injured rats with tactile hypersensitivity (allodynia). Results showed that clonidine, in doses of 1–20 µg, reduced the hypersensitivity in a dose-dependent manner. In rats in which SCS per se failed to suppress tactile hypersensitivity, the combination of SCS and a subeffective dose of clonidine appeared to be highly synergistic and markedly attenuated the hypersensitivity. These results suggest that small doses of IT clonidine may be combined with SCS in neuropathic pain patients who do not obtain satisfactory relief with SCS alone. IMPLICATIONS: Pain after nerve injury is often difficult to manage, but some patients may respond to electrical stimulation of the spinal cord (SCS) with satisfactory pain relief. This study in the rat suggests that spinal delivery of a drug (clonidine) in combination with SCS for pain relief warrants clinical investigation.
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Introduction |
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Spinal cord stimulation (SCS) has proven to be an effective treatment of nerve injury-induced pain, and up to 60%–70% of well selected patients experience satisfactory pain relief (1). Although the mechanism of action of SCS is only partially understood, experimental data have suggested that a combination of SCS and drugs may be useful to improve pain relief. Previous studies in our laboratory have demonstrated that the effect of SCS on neuropathy-related behavior in nerve-lesioned rats can be enhanced by simultaneous spinal administration of small doses of the gamma-aminobutyric acid (GABA)B receptor agonist baclofen (2), the adenosine A1 receptor agonist R-PIA (3), and the two anticonvulsants gabapentin and pregabalin (4). Furthermore, subsequent clinical trials have indicated that both intrathecal (IT) baclofen and adenosine may be effective adjuvants to SCS in patients experiencing pain from nerve injury and in whom stimulation treatment alone is insufficient (5).
Clonidine, an 2-adrenoceptor agonist, was originally introduced as an antihypertensive drug. Within pain therapy, it has been used to control nociceptive pain (e.g., postoperative pain) and as an adjunct to both systemic and spinal opioids (6,7). Moreover, clonidine has been demonstrated to produce analgesia in both human neuropathic pain (6) and in various animal models of neuropathy (8–10). Some observers even assert that clonidine is more effective in reducing neuropathic than acute pain (11). Clonidine is one of the most common drugs administered epidurally or IT in chronic-pain patients, but its use is limited by cardiovascular and sedative side effects (although less so with the IT route of administration) (7).
The aim of this study was to analyze the capability of a subeffective dose of IT clonidine to potentiate the effect of SCS on tactile hypersensitivity in neuropathic rats when stimulation per se was ineffective. In addition, both dose-response and time-response characteristics of IT clonidine were studied. Interestingly, it was recently reported that systemic administration of clonidine enhances the analgesic effect of transcutaneous electrical nerve stimulation, a stimulation technique related to SCS, in an animal model of inflammatory pain (12).
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Methods |
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Male Sprague-Dawley rats (B&K Universal AB, Sweden) weighing 175–400 g were used. The relatively wide weight range was due to the fact that many rats were used for all parts of the study, which extended over months. The rats were housed at the local animal department, exposed to a 12:12-h light/dark cycle, and provided with ready access to food and water.
All surgery was performed with general halothane anesthesia delivered through an open mask system. Anesthesia was induced by 3% Fluothane® (AstraZeneca, Sweden) and was maintained with 1%–2% in a 1:1 mixture of air and oxygen at a flow rate of 2 L/min. During surgery, the body temperature was maintained at 37°C ± 0.5°C by an automatic heating pad (CMA/150; CMA Microdialysis AB, Stockholm, Sweden).
Mononeuropathy was created according to the method of Seltzer et al. (13). In brief, the left sciatic nerve was exposed at the proximal thigh level under microscope magnification. An 8-0 silk suture with a 3/8 curved and reverse cutting minineedle was inserted into the dorsal part of the nerve and strictly tied, severing approximately one third to one half of the nerve. The skin was then closed, and the rats were allowed to recover in separate cages.
The behavioral studies were performed by one examiner in a quiet room. The rat was placed in a circular observation cage with a metal mesh floor and was allowed to adapt for at least 15 min before the beginning of the test.
To quantify the degree of tactile hypersensitivity, withdrawal thresholds to static tactile stimulation were evaluated with von Frey nylon monofilaments. Regularly calibrated filaments with stiffnesses corresponding to a range from 0.5 to 30 g were applied to the midplantar surface of the hindpaw until the filament bent. As a control, the withdrawal thresholds were compared for the same parts of the hindpaw of the intact and the nerve-injured paw. The test was always started with the softest filament and continued in ascending order of stiffness alternately applied to the intact and the injured paws. A brisk withdrawal of the hindlimb was considered a positive response.
Only rats that had developed tactile hypersensitivity, defined as withdrawal to at least 5 of 10 applications of a filament corresponding to 
8 g, were included in the experiments. This value was designated as the 50% withdrawal threshold (14). The filament corresponding to 30 g was selected as a cutoff.
Nerve-injured rats that developed a consistent decrease of the paw withdrawal threshold to innocuous tactile stimuli were subjected to implantation of an IT catheter and an electrode system for SCS. A polyethylene-10 catheter was inserted via a 21-gauge cannula between the L5 and L6 lamina and was advanced in a caudorostral direction up to the lumbar enlargement. The catheter was then fixed to the fascia, tunneled subcutaneously, and fixed to the neck skin. The catheter location was verified physiologically by the injection of 300 µg of lidocaine (Xylocaine®; AstraZeneca, Sweden), which induces a transient flaccid paralysis of the hindlimbs.
Implantation of a monopolar electrode system for SCS in the rat has previously been described by Cui et al. (2). After exposure of the spine, a laminectomy was performed at T12, and the cathode (a solid rectangular silver plate, 3 x 1.5 mm; thickness, 0.25 mm) was placed in the dorsal epidural space. The anode (a solid silver disk, diameter = 6 mm; thickness, 0.25 mm) was implanted in the left paravertebral subcutaneous tissue. Insulated steel wires connecting the two poles were tunneled subcutaneously to a microcontact, which was rigidly fixed to the neck skin. To avoid damage of the microcontact and the IT catheter (see below), the rat was placed in a separate cage after surgery and allowed to recover for 48 h before further experiments. Rats with signs of neurological sequelae after the surgery were excluded from the subsequent experiments and killed.
The rat was placed in a circular observation cage, and the microcontact of the SCS electrode was connected, via an electric swivel mounted on a balancing arm (GH Medic, Sweden), to a Grass S44 stimulator with a constant current unit (CCU1; Grass Instruments). This arrangement was designed to allow the rat to move freely in the cage during the experiment.
Monopolar electrical stimulation was applied with a frequency of 50 Hz, a pulse width of 0.2 ms, and a stimulation intensity individually set to two thirds of the intensity required to produce a short-lasting motor response in the lower trunk muscles. The variables used in this study were chosen to correspond to those used in clinical practice and were the same as those used in our previous studies (2). The motor threshold was assessed 15–20 min before each SCS session.
SCS was applied for 30 min, and the withdrawal responses to tactile stimuli were assessed every 10 min starting just before the initiation of SCS or IT clonidine. This continued until the pretreatment threshold values were reinstituted.
Only rats that did not respond to SCS with a significant suppression of tactile hypersensitivity were included in the experiments (approximately 85% of all implanted rats with significant hypersensitivity). The current intensity used varied between 0.35 and 0.85 mA (i.e., two thirds of the intensity needed to induce a motor response).
The subeffective doses of clonidine used were individually determined for each rat in the preceding experiments to avoid side effects and to evaluate the antiallodynic effect when clonidine was combined with SCS. Clonidine hydrochloride (Sigma, Sweden) was dissolved in saline and prewarmed to 38°C before administration. IT injections of clonidine were given in volumes of 10 µL followed by 10 µL of prewarmed saline to rinse the catheter. After drug administration, side effects were assessed by observing the presence of sedation, general posture, ambulation, and frequency of urination. Sedation was defined by a significant decrease in spontaneous activity and a loss of the orienting response to light touch stimulation. Motor weakness was evaluated by observing ambulation and weight bearing.
In the first part of the study, dose-response relations between IT clonidine in doses of 1, 5, 10, and 20 µg and tactile hypersensitivity were determined. In the second part of the study, individually screened subeffective doses of clonidine were administered in combination with SCS, and tactile hypersensitivity was evaluated. The subeffective dose for each animal was defined as the largest dose tested that did not suppress tactile hypersensitivity (withdrawal threshold <8 g) and that produced no side effects.
In 3 rats, 10 µL of prewarmed saline was IT injected as control, and no response was observed in these rats. Because we have applied the same method in several previous studies (15), it was not considered necessary to use saline injections as a routine control in the subsequent experiments.
The Friedman nonparametric analysis of variance followed by Dunn’s post hoc test was used to analyze changes in withdrawal responses within the same group over time. For comparison of withdrawal responses after the different treatments (SCS, clonidine, or SCS and clonidine combined) at the same time points after intervention, the one-tailed Wilcoxon’s signed rank test for paired observations was used.
All data are presented as mean ± SEM, and P < 0.05 was considered significant in all statistical tests. Graphics and calculations were performed with GraphPad Prism Version 3.03 (GraphPad) and Microsoft Excel Version 5.0 (Microsoft Corp.).
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Results |
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The administration of IT clonidine produced a dose-dependent and transient increase of the withdrawal threshold in the nerve-injured but not in the intact paw (Fig. 1). A dose of 1 µg did not produce any significant effect, whereas with 5 µg there was a tendency toward a minor, although significant, threshold increase. However, 10 and 20 µg of clonidine produced a marked increase of the withdrawal threshold with an early onset and a maximum effect occurring at 40 min after injection. The antihypersensitive effect remained significantly different from baseline until 60 min after injection. In most rats, the withdrawal threshold was restored to baseline levels after 120 min.
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Discussion |
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The aims of the dose-response study were to determine the range of doses at which clonidine has no effect and to evaluate the side effects produced with these doses. A problem with the interpretation of dose-response curves for clonidine is the occurrence of side effects (motor weakness and sedation) associated with IT doses 
10 µg (17). It is our impression that the dose-specific response relationship is highly unreliable when side effects are present, although it has been argued that motor weakness and sedation are not likely to significantly influence the pain-relieving effect of clonidine (10).
Although only approximately 15% of all rats that displayed behavioral signs of neuropathy responded to SCS with a significant suppression of tactile hypersensitivity, most tended to respond with a partial increase of the withdrawal threshold (Fig. 2). This small number of rats with a robust response to SCS is in line with our previous results (3,4).
Experimental evidence suggests that the pain-alleviating effects of SCS are due to changes in neurotransmitter release (5), which is associated with a suppression of neuronal hyperexcitability (19,20) that is primarily mediated via antidromic activation of the dorsal columns in the spinal cord. For example, a decrease in glutamate and an increase in GABA release have been demonstrated to occur simultaneously in the spinal dorsal horn after SCS in neuropathic rats (2,21). However, it is conceivable that other transmitter systems are involved as well, and our understanding of the mechanisms behind the pain-relieving effects of SCS remains incomplete.
Even though the analgesic mechanisms of action of clonidine are not yet fully known, a number of studies indicate that pain relief is produced through the activation of 2-adrenoceptors (11). It has been proposed that the effect of clonidine in neuropathy may be attributed to an inhibitory effect on sympathetic outflow by a direct effect on spinal sympathetic preganglionic neurons (10), although experimental data do not support the notion of sympaticolysis as a major analgesic mechanism in neuropathy (22). Instead, there is strong evidence that the actions of IT clonidine involve cholinergic mechanisms that lead to a release of acetylcholine and a synthesis of nitric oxide in the spinal dorsal horn (23). Furthermore, it has been demonstrated that both muscarinic receptor agonists and acetylcholinesterase inhibitors produce analgesia by activating GABAergic interneurons in the dorsal horn (24). Considering that SCS increases the spinal release of GABA, as demonstrated in our previous experimental studies (2,21), a combined effect on the GABAergic system may explain the synergy between SCS and IT clonidine, as observed in this study.
Recent studies have demonstrated that the analgesic effect of clonidine in neuropathy may involve the activation of cholinergic spinal interneurons, and it has been suggested that this could lead to an attenuation of the enhanced neuronal activity induced by nerve injury (25). The fact that SCS produces a similar inhibitory effect (20) may therefore indicate a possible neurophysiological mechanism behind the synergy between SCS and clonidine. This notion warrants further study of a possible direct effect of SCS on the spinal cholinergic system.
Pain conditions diagnosed as lumbosacral rhizopathy are often associated with lumbar pain due to spinal degenerative disease or surgery, and they may thus comprise both nociceptive and neuropathic components. Although it is generally assumed that SCS affects mainly the neuropathic component when applied in these conditions (5), some authors (26) have reported good effects on nociceptive low back pain as well. Because clonidine also influences nociceptive components of pain (27), a combination of clonidine and SCS might, therefore, be a possible approach for controlling such complex pain states.
In conclusion, these results suggest that the combination of SCS and a subeffective dose of clonidine suppresses tactile hypersensitivity in a rat model of mononeuropathy in which SCS itself proved ineffective. Future clinical studies in this area could contribute to the development of a new combined therapeutic strategy with fewer side effects for the treatment of neuropathic pain.
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Acknowledgments |
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The authors thank Göte Hammarström for excellent technical assistance.
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