Kontinen, Vesa K. MD; Paananen, Sami MB; Kalso, Eija MD, PhD
Injury of a peripheral nerve may lead to neuropathic pain. Currently, the most widely used drugs in neuropathic pain are tricyclic antidepressants, which inhibit the reuptake of norepinephrine. alpha2-Adrenergic agonists acting in the descending inhibitory tracts of the spinal cord are effective in acute nociceptive  and inflammatory  pain. The role of the alpha2-adrenergic system in neuropathic pain has been studied using various pharmacological interventions and animal models [3-5]. Neuropathic pain can be sympathetically maintained or at least partly controlled by sympathetic activity. Several mechanisms in the periphery, along the nerve or at the level of the dorsal root ganglion, could explain coupling between sympathetic and sensory afferent fibers in an injured nerve . Surgical sympathectomy and sympathetic blocks alleviate mechanical [7-9] and thermal  allodynia (painful response to a stimulus that is normally not painful) in some partial nerve injury models. However, in other experimental settings, sympathectomy does not reduce hyperalgesia (increased response to a painful stimulus) [10,11] and may even increase the symptoms [9,11], depending on the timing of the intervention and the nerve injury model used. Based on these results, some of the experimental models of neuropathic pain have been described as models of sympathetically maintained pain [7-9,11] or sympathetically independent pain . The spinal nerve root ligation neuropathy model  is considered to represent sympathetically maintained pain [7,8], but this has been recently questioned .
Trauma-induced afferent barrage is one of the factors that may lead to plastic changes in the central nervous system sustaining neuropathic pain. In a sciatic axotomy model, pretreatment with alpha2-adrenergic agonists mitigates this activity  but does not prevent autotomy (self-mutilation of toes), which is considered a sign of spontaneous pain in an axotomized limb [14,15]. In the chronic constriction injury model, subcutaneous (SC) injections of clonidine given 30 minutes before and 6 hours after the ligation of the sciatic nerve prevent mechanical hyperalgesia . Intrathecal (IT) administration of clonidine before the ligation delays the development of thermal hyperesthesia, but it is without effect when given after the injury .
It is usually not possible to preempt neuropathic pain in the clinical setting. Therefore, drugs that attenuate neuropathic pain, when started after the nerve injury, are needed. alpha2-Adrenergic, but not alpha1-adrenergic, agonists reduce tactile allodynia after tight ligation of the L5-6 spinal nerves . This effect of IT clonidine has been enhanced by the N-methyl-D-aspartate-antagonist MK-801 . After sciatic axotomy, the IT administration of the alpha2-adrenergic agonists clonidine and dexmedetomidine suppresses autotomy [15,20], and termination of the treatment results in increased autotomy. However, similar treatment with the alpha2-adrenergic agonists guanfacine or ST-91 has no effect on autotomy .
Experimental data on the chronic administration of alpha2-adrenergic agonists in the treatment of neuropathic pain are limited. IT infusion of the alpha (2-adrenergic) agonist tizanidine started one week after nerve injury does not reduce mechanical hyperalgesia, although acute IT administration of tizanidine restores normal ambulation and increased paw pinch withdrawal latencies . Mechanical allodynia after sciatic cryoneurolysis is attenuated by perineural infusion of dexmedetomidine, the alpha1-adrenergic antagonist prazosin, or a small dose (2.0 micro g) of clonidine .
The aim of the present study was to assess the possibility of treating neuropathic pain with the SC administration of the selective alpha2-adrenergic agonist, dexmedetomidine. Both prevention of neuropathy with a pre-injury injection of dexmedetomidine and treatment of the symptoms with long-term infusions of dexmedetomidine were studied in rats with unilateral peripheral mononeuropathy caused by tight ligation of the L5 and L6 spinal nerves.
Male Sprague-Dawley rats weighing 150-175 g at the beginning of the experiment were used. Laboratory chow and water were available to the rats ad libitum. Rats were housed in groups of six in plastic cages with artificial lighting and a fixed 12-h light/dark cycle. We adhered to the guidelines for animal research set forth by local authorities and the International Association for the Study of Pain . The study protocol was approved by our institutional animal investigation committee.
The model of neuropathic pain previously described by Kim and Chung  was used. In brief, the animals were anesthetized with halothane (0.5%-1% Trothane[registered sign]; ISC Chemicals, Bristol, UK) in N2 O/O2 (70:30). The left L5 and L6 spinal nerves were exposed by removing a small piece of the paravertebral muscle and a small piece of the left spinous process of the L5 lumbar vertebra. The L5 and L6 spinal nerves were then carefully isolated and ligated tightly with 6-0 silk. After checking hemostasis, the muscle and the adjacent fascia were closed with sutures, and the skin was closed with metal clips.
For the assessment of mechanical and cold allodynia, the rats were placed on a metal mesh covered with a plastic dome, and they were allowed to habituate until exploratory behavior diminished. The threshold for mechanical allodynia was measured by using a series of von Frey hairs (Semmes-Weinstein, Stoelting, IL) [24,25]. The ventral surface of the paw was touched with different von Frey hairs with a bending force from 0.217 to 12.5 g until the threshold force that induced paw withdrawal in more than half of the stimuli was found. The testing was begun by finding the allodynic areas of the ventral surface of the paw with the 12.5-g von Frey hair. If the rat responded to the stimulation by withdrawing the paw, the next weaker hair was used until the threshold was found. To avoid excessive stimulation, the testing was started in the following sessions with the weakest hair that had elicited withdrawal responses in the previous session. If the strongest hair did not give a response, 12.5 g was recorded as the threshold. Cold allodynia was measured as the number of foot withdrawal responses after application of cold stimuli to the plantar surface of the paw . A drop of acetone was gently applied to the heel of the rat with a syringe connected to a thin polyethylene tube. A brisk foot withdrawal response after the spread of acetone over the plantar surface of the paw was considered a sign of cold allodynia. The testing was started with the paw contralateral to the nerve injury and repeated five times for both paws with an interval of approximately 2 min between each test. Thermal (heat) nociception was measured using the paw flick test . The intensity of the light beam was set to 40 (units of the scale of the apparatus 0-90), and a cut-off time of 16 s was used to avoid tissue damage. The stimulus was begun only when the tested paw was set on the glass floor of the device [27,28]. The values of five repeated paw flick measurements were averaged for both sides.
The effect of dexmedetomidine administered SC before the nerve injury was examined in a group of rats (Pretreatment Group) that received a single dose of dexmedetomidine (120 micro g/kg) or saline (1.0 mL/kg) injected SC 30 min before the ligation of the spinal nerves . To study the effects of chronic administration of an alpha2-adrenergic agonist, an osmotic minipump (Alzet 2001; Alza Corporation, Palo Alto, CA; infusion rate 1.0 micro L/d) delivering either dexmedetomidine 60 micro g in 24 h for 7 days or saline was placed under the rats' skin on the side contralateral to the ligation after the spinal nerves were ligated and the paravertebral muscle was closed (Early Infusion Group). In the third study group, the development of neuropathy was followed for 14 days after the ligation. The rats that developed signs of significant mechanical hyperalgesia (withdrawal response to stimulation with a von Frey hair corresponding to 4.2 g or less) were anesthetized with halothane for the implantation of an osmotic minipump (Late Infusion Group) infusing either 30 micro g of dexmedetomidine in 24 h or saline for 7 days. The rats in each infusion group were tested every other day for 28 days after the start of the drug treatment. An acute challenge dose of the alpha2-adrenergic antagonist atipamezole (1 mg/kg SC) was given to rats in the Late Infusion Group that had not developed neuropathic signs. The presence of mechanical and cold allodynia was tested 15, 30, 45, and 60 min after the injection of atipamezole. Atipamezole and saline control injections were given to these rats in a cross-over manner so that half of the rats received saline first. The interval between the challenges was 3 days. At the end of the study, all rats were killed with an overdose of halothane.
Dexmedetomidine hydrochloride and atipamezole hydrochloride (Orion Corporation, Turku, Finland) were dissolved in sterile physiological saline and stored at 4[degree sign]C before use.
Analysis of variance for repeated measurements, followed by a t-test with Bonferroni's correction when appropriate, was used for the statistical analysis of the behavioral symptoms over time and between the treatment groups and the control groups.
Significant mechanical and cold allodynia developed in all study groups after the ligation of the spinal nerves. Mechanical allodynia developed within 10 days after the operation and remained relatively stable during the rest of the observation period (Figure 1, Figure 2 and Figure 3). Cold allodynia was strongest between 2 and 14 days after the operation and seemed to slightly decrease thereafter. However, this change was not statistically significant. The animals did not show any autotomy, and there were no apparent signs of distress or altered social behavior.
In the thermal (heat) nociceptive test, the mean (+/- SEM) paw flick latency was 5.4 +/- 0.2 s in the tests performed before the neuropathy operation. There were no significant differences between the latencies in the operated and the contralateral paws in either of the treatment groups. The paw flick latencies did not change significantly over time in any of the study groups. The dexmedetomidine pretreatment or the infusions did not change the paw flick latencies significantly compared with the control groups (data not shown).
After the start of the dexmedetomidine infusion, the rats were considered sedated in observation and lost weight for a period of 4 and 2 days, respectively, in both the Early (mean change +/- SEM 21 +/- 5.3 g, percent change 12.5%) and the Late (13 +/- 2.5 g, 4.6%) Infusion Groups. However, later during the infusion, the weight gain was comparable to the control groups.
There were no differences between the Pretreatment Group and the corresponding control group in either mechanical or cold allodynia in the operated paw (Figure 1). Also, the Early Infusion Group was not different from the respective control group in either mechanical or cold allodynia (Figure 2). Of the 27 rats that were to be used in the Late Infusion Group, 13 fulfilled the criteria for neuropathy 14 days after the operation. There were no differences between the treatment group and the corresponding control group in mechanical allodynia in the operated paw. However, in the contralateral paw, there was a statistically significant increase in mechanical allodynia (Figure 3) during treatment in the Late Infusion Group (F[15,150] = 6.9, P = 0.002 for time; F[15,150] = 5.8, P = 0.01 for the interaction of treatment and time).
The remaining 14 rats not included in the Late Infusion Group were used in the acute atipamezole challenge, as described in Methods. The atipamezole challenge increased both mechanical and cold allodynia, but only the change in cold allodynia was statistically significant (F[1,268] = 4.4, P = 0.04) when compared with the saline challenge (Figure 4). This change was reversible, and the maximal effect was seen 30 min after the SC injection of atipamezole.
The observed pattern of the development of mechanical and cold allodynia after the ligation of the spinal nerves in the control groups is comparable to results reported previously [5,26]. In the rat model used in this study, the neuropathic pain-like behavior is fully developed within two weeks after the ligation of the spinal nerves and remains relatively stable for at least a few weeks . The methods used to measure mechanical and cold allodynia seem to be sensitive, and a clinically relevant effect of a given treatment could have been measured in this study.
Three clinically different situations were modeled in this study. We designed the pretreatment experiment to give information relevant to a situation in which we were attempting to prevent the consequences of an unavoidable nerve injury. If a treatment that prevents neuropathic pain could be found, it should be used as part of premedication or induction of anesthesia, at least in operations that have a high risk of postoperative neuropathic pain. However, most nerve injuries, and even more importantly, the development of neuropathic pain, cannot be anticipated. The information from the early infusion experiment could be useful when an injury of a peripheral nerve is diagnosed either during surgery or acutely after trauma. The late infusion experiment simulated treatment of fully developed neuropathic pain. Systemic administration of dexmedetomidine was used at doses close to the largest tolerated by the rat without significant side effects. The late infusion experiment was performed with a smaller dose than that used in the early infusion experiment because the rats in the early infusion group lost weight during the first days of the infusion, possibly because of the sedative effect of the alpha2-adrenergic agonists. However, strong sedation could also interfere with the behavior of the animals in the nociceptive tests, increasing the measured nociceptive thresholds. This was not seen in this experiment.
There were no differences between the treatment groups and the corresponding control groups in mechanical allodynia in the operated paw. The development of cold allodynia in the operated paw was not different in any of the treatment groups compared with the respective control groups. Previous reports vary in five basic characteristics: the nerve injury model chosen, and the route, timing, duration, and drug used in the treatment. Using the same model as in the present study, Yaksh et al.  reported that single IT injections of an alpha2-adrenergic agonist reduce tactile allodynia. Administration of an alpha2-adrenergic agonist before, but not after, the injury is effective , but the opposite was seen in another study  using different nerve injury models. In some earlier studies [15,17-20,22] alpha (2-adrenergic) agonists were administrated IT. However, if systemic administration were effective, the treatment would be available to more patients with lesser risks and cost. Systemically administered clonidine prevents the development of hyperalgesia to mechanical stimuli . There is limited information about chronic treatments, but in one study, the IT infusion of tizanidine did not reduce mechanical hyperalgesia .
Surprisingly, an increase in mechanical allodynia was seen during the dexmedetomidine infusion in the Late Infusion Group in the contralateral, but not in the operated, paw. Contralateral changes have been reported in previous studies (for review, see ), but in the spinal nerve ligation model, there are normally limited or no changes in the contralateral paw , which was true also in the present study. Sciatic nerve transection also triggers sprouting of sympathetic fibers to the dorsal root ganglion in the side contralateral to the injury (for review, see ). The function of these new fibers is largely unknown and could be changed by systemic, chronic alpha2-adrenergic agonist treatment.
We did not observe any differences between the operated paw and the contralateral paw during the thermal nociceptive test. The withdrawal latencies remained stable over time after surgery and were comparable to the presurgery levels. This could indicate that thermal nociception in the paw is not altered after ligation of the L5-6 spinal nerves, or that thermal nociception in the neuropathic paw cannot be reliably studied by using the paw flick test. In a previous report, spinal nerve ligation induced thermal hyperalgesia  that was abolished by sympathectomy . However, this nerve injury-induced shift in the thermal nociceptive threshold was relatively small (18%-30% of the baseline) compared with the very large changes seen in mechanical and cold allodynia . Changes in the position of the paw after nerve injury due to changes in the muscle balance or mechanical hyperalgesia and guarding behavior may lead to loose contact between the glass surface and the paw, altering the transmission of the heat stimulus. Loose contact between the paw and the glass surface has been shown to lead to shortened withdrawal latencies, probably because of loss of the heat sink effect of the glass . This could theoretically counteract the effect of putative thermal hyperalgesia, leading to normal latencies.
In rats that did not develop significant mechanical hyperalgesia within the two weeks after nerve injury, SC administration of atipamezole, an alpha2-adrenergic antagonist, increased both mechanical and cold allodynia in a reversible manner. Although the change was small, it could be interpreted as a sign of increased endogenous adrenergic activity in rats that did not develop neuropathic symptoms after a standardized surgical injury, compared with the rats that showed allodynia. There are previous reports of increased neuropathic pain-like behavior after blockade of endogenous opioid pathways . However, more evidence is needed to reach this conclusion. A direct effect of atipamezole on the vasculature of the paw could change the skin temperature and, thereafter, the cooling sensation caused by an acetone drop.
Dexmedetomidine has affinity to all three alpha2-adrenoceptor subtypes . The role of the different subtypes of alpha2-adrenoceptors in neuropathic pain is unclear. alpha2A- and alpha2C-receptors may have a role in the inhibition of nociceptive pain [21,33]. However, it has been proposed that the number and the relative proportions of the alpha2-adrenergic receptor subtypes and also the functional coupling of the receptors might change in an injured nerve . Subtype-selective pharmacological tools are needed to further elucidate the possibility of prevention and treatment of neuropathic pain by modulating the alpha2-adrenergic system.
In conclusion, the rats with a ligation injury in the L5-6 spinal nerves developed both mechanical and cold allodynia, but no thermal hyperalgesia. The alpha (2-adrenergic) mechanisms seem important in the development of these symptoms, but systemic treatment with dexmedetomidine neither prevents nor attenuates mechanical and cold allodynia with the doses used in this study. Larger systemic doses are not tolerated because of adverse effects. Subtype-selective alpha2-adrenergic agonists are needed for further studies.
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