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Perisurgical amitriptyline produces a preventive effect on afferent hypersensitivity following spared nerve injury
PAINÒ 146 (2009) 308–314
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Perisurgical amitriptyline produces a preventive effect on afferent hypersensitivity following spared nerve injury Andre Arsenault, Jana Sawynok * Department of Pharmacology, Dalhousie University, Halifax, NS, Canada B3H 1X5
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Article history: Received 6 May 2009 Received in revised form 13 July 2009 Accepted 10 August 2009
Keywords: Amitriptyline Nerve injury Preventive analgesia Noradrenaline
a b s t r a c t Following surgery, nerve injury can lead to persistent neuropathic pain. Pre-emptive and preventive analgesic treatments in the perioperative period aim to minimize nerve injury-induced pain. Here we demonstrate that a perioperative regimen of amitriptyline (10 mg/kg i.p. 30 min before and immediately after surgery, followed by oral amitriptyline 15–18 mg/kg/day in the drinking water for 7 days post-surgery) prevents hypersensitivity to a chemogenic stimulus (ab-MeATP, a ligand for P2X3 receptors, together with noradrenaline or NA) in the spared nerve injury (SNI) model in rats. It also prevents hyposensitivity to capsaicin and NA. However, amitriptyline treatment had no effect on the development of mechanical allodynia. We investigated the role of NA mechanisms in the action of amitriptyline by using the neurotoxin 6-hydroxydopamine (6-OHDA) and by examining desipramine. Intrathecal treatment with 6-OHDA on the day of surgery reversed the preventive effect of amitriptyline on hypersensitivity to ab-MeATP/NA, and desipramine exhibited a similar effect to amitriptyline. We also examined the effect of antibodies to the nerve growth factors glial-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), given intrathecally three times (days 0, 3 and 7) on the action of amitriptyline and observed that the interruption of GDNF and BDNF signaling impaired the prevention of hypersensitivity to ab-MeATP/ NA. This study indicates that tricyclic antidepressants given in the perioperative period may be useful in preventing nerve injury-induced sensory changes that contribute to the development of chronic post-surgical neuropathic pain. Ó 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
1. Introduction Surgical interventions can lead to persistent pain long after recovery from the surgical event. The prevalence of such pain varies depending on the procedure, but can occur in up to 50% of patients [21]. Damage to peripheral nerves causes maladaptive changes at every level of the nervous system, and can include altered function at the initial injury site, at neuronal cell bodies located in the dorsal root ganglion and at synapses of the central nervous system [36]. Injured nerves also affect cellular processes in adjacent uninjured nerves, as well as at remote sites along the neural axis [7]. This altered neuronal function, or plasticity, contributes to the development of neuropathic pain. Studies in animals, and in the clinical setting, have attempted to prevent pain that results from surgery by using pre-emptive or preventive analgesia strategies [21]. The rationale for this approach is to use analgesics in the perioperative period to prevent or minimize nervous system changes that contribute to the development of neuropathic pain. Reviews of randomized controlled trials * Corresponding author. Tel.: +1 902 494 2596; fax: +1 902 494 1388. E-mail address: [email protected] (J. Sawynok).
employing preventive analgesia strategies report limited success using such strategies [20,26,28]. There are, however, some preventive analgesia reports that appear promising using antidepressants [4,32]. Tricyclic antidepressants have long been the primary pharmacological treatment for neuropathic pain [14]; these act on several aspects of pain signaling relevant to neuropathic pain – they increase the availability of noradrenaline (NA) and 5-hydroxytryptamine (5-HT) at central synapses, block Na+ channels, activate K+ channels, inhibit Ca2+ reuptake, and activate opioid and adenosine systems [25]. More recently, the nerve growth factors glial-cellline-derived neurotrophic factor (GDNF) [18,19] and brain-derived neurotrophic factor (BDNF) [11,17] have been implicated in the actions of antidepressants. The role of these factors in the development of neuropathic pain and in the preventive analgesic actions of antidepressants will be discussed later. In the present study, we investigated whether amitriptyline, a tricyclic antidepressant, could alter the development of chemogenic and mechanical hypersensitivity following surgical nerve injury in rats. We used the spared nerve injury (SNI) model and a lateral hindpaw testing site which allows for the assessment of the effects of stimuli in a sensory field influenced by injured fibres in the upper reaches of the nerve, in ganglia and in the spinal cord [9].
0304-3959/$36.00 Ó 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2009.08.003
A. Arsenault, J. Sawynok / PAINÒ 146 (2009) 308–314
The chemogenic stimulus used was a combination of ab-methylene adenosine triphosphate (ab-MeATP, which activates P2X3 receptors) and NA; this previously was shown to produce an augmented response at the lateral site following SNI and its actions are relevant to sympathetic nerve influences on sensory transmission [23]. Mechanical responses were determined using von Frey hairs and previously were shown to be hypersensitive following the nerve injury [9]. When a preventive effect was shown to occur, we subsequently examined the involvement of spinal cord NA, GDNF and BDNF in the preventive action of amitriptyline. Finally, a chemogenic stimulus which exhibits hyposensitivity following nerve injury (capsaicin/NA) [23] was examined, as clinical neuropathic pain can involve sensory hyposensitivity (sensory loss) as well as hypersensitivity (spontaneous pain, allodynia, and hyperalgesia) [1].
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regimen [12]. Fluid intake, and therefore drug dosing, was monitored by weighing the drinking bottles. In one experiment desipramine was given instead of amitriptyline, using the same regimen. 2.5. 6-Hydroxydopamine treatment Immediately following nerve injury, and while rats were still anaesthetized, the neurotoxin 6-hydroxydopamine (6-OHDA) was delivered to the spinal cord via an acutely inserted intrathecal cannula [22]. The cannula (polyethylene – 10 tubing) was inserted into the sub-arachnoid space 7.5 cm caudally through a small opening in the cisterna magna. 6-OHDA (100 lg) was dissolved in 0.1% ascorbic acid and delivered in a final volume of 8 ll. After drug delivery, the cannula was left in place for 2–3 min before slow removal; the wound was then closed with sutures. This dose of 6OHDA has previously been shown to deplete spinal cord NA levels by >80% [35,37].
2. Methods 2.6. Antibody studies 2.1. Animals Male Sprague–Dawley rats (Charles River Laboratory, Que., Canada) weighing 175–225 g at the time of surgery were used in most experiments. The mouse study used male C57/BL6 mice (Charles River Laboratory, Que., Canada) weighing 20–25 g. Animals were housed under a 12–12 h light–dark cycle and allowed to acclimate to the facility for a period of 1 week prior to surgery. All animals were given free access to food and water. All studies were approved by the University Committee on Laboratory Animals (Dalhousie University) and complied with the Canadian Council of Animal Care guidelines for the ethical use of animals.
BDNF (Millipore) and GDNF (AbCam) antibodies were dissolved in normal saline. Antibody (15 lg/injection in a final volume of 10 ll) was delivered by acute intrathecal lumbar puncture (the needle was inserted between the L5 and L6 vertebrae) immediately after surgery while the rat was still anaesthetized, and then 3 and 7 days following surgery (again under anaesthesia). An equivalent concentration of bovine serum albumen, dissolved in saline, was used as a vehicle control. 2.7. Behavioural analysis
All drugs and reagents were obtained from Sigma–Aldrich (Canada) with the exception of the BDNF (Millipore, Canada) and GDNF (AbCam, USA) antibodies.
All behavioural analysis was conducted between 09:00 and 14:00 h. Sensitivity of the paw ipsilateral to the nerve injury was assessed using chemical and mechanical modalities. Animals were placed in a plexiglass box (30 30 30 cm) and permitted to habituate for 30 min. ab-MeATP (150 nmol), or capsaicin (3 lg dissolved in 10% DMSO), and NA (25 nmol) were injected in a volume of 30 ll s.c. into the lateral side of the injured paw. Vigorous flinches of the paw were recorded in 1 min intervals for 15 min. Testing was performed between 14 and 28 days post-injury, although some determinations were done at 42 days. Mechanical threshold to the application of a von Frey probe was assessed using the Dixon up-down method [10]. Animals were placed in a plastic container with an elevated wire mesh floor, allowing access to the plantar surface of the hindpaw. Using an established testing procedure [33], the lateral surface of the plantar hindpaw was probed for 2–3 s. A positive response was identified as a sharp withdrawal, or biting and licking, immediately following the application of the von Frey hair. In the case of a positive response the next lightest hair was used, and in the case of a negative response the next heaviest hair was applied. Four more stimuli were applied after an initial change was observed, and the pattern of responses was translated into a 50% von Frey threshold using the following equation; 10(xf+jd)/10,000 where xf = value (in log units) of the final von Frey hair used, j = tabular value for the pattern of the last 6 positive/negative responses and d = mean (in log units) between stimuli.
2.4. Drug treatments
2.8. Statistics
Animals received an i.p. injection of amitriptyline (10 mg/kg) or an equivalent amount of vehicle (normal saline) before and after surgery. Following surgery, amitriptyline (or no drug) was then administered in the drinking water (16 mg/kg/day) for a period of 7 days. This dosage level was based on a previous study which demonstrated antihyperalgesic effects of amitriptyline using this
All statistical analyses were performed using Sigmastat v1.0. Data were analysed for each particular time following surgery. In some cases, this involved use of the Student’s t-test (Figs. 1 and 2); in others, a one-way analysis of variance (ANOVA) with the Student–Newman–Keuls (SNK) post hoc analysis was performed (Figs. 3–5 and 7). An ANOVA on ranks and the Dunn’s post hoc test was used for the
2.2. Surgery The spared nerve injury procedure was performed as previously described with minor surgical modifications [9]. Briefly, animals were placed under anaesthesia (2% isoflurane) and an incision was made along the back of the thigh. The biceps femoris muscle was separated to expose the three terminal branches of the sciatic nerve; the common peroneal, tibial and sural nerves. The tibial and common peroneal nerves were tightly ligated using 6.0 silk and transected distal to the ligation removing 2–4 mm of nerve stump. The sural nerve was left intact and the wound was closed with cutaneous sutures. Rats were housed individually for 48 h before being paired with their original cage mate. Animals displaying autotomy (1 animal per group) were euthanized. In mice, the spared nerve injury was performed in the same manner as in rats except the tibial and common peroneal nerves were not ligated before being transected. Sham surgery controls were performed by exposing the nerve bundle, but not manipulating the nerves, and then closing the wound. 2.3. Drugs and reagents
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Fig. 1. Spared nerve injury (SNI) produces sustained hypersensitivity to coadministration of ab-MeATP/NA. Lateral hindpaw injection of ab-MeATP/NA (150/ 25 nmol) produced increased flinching behaviours over 15 min, 14–42 days after SNI compared to sham animals. Values depict mean + SEM; ***P < 0.001, Student’s ttest, n = 7–9 per group.
mechanical allodynia experiments (Fig. 6). Data are presented as means, and error bars represent the standard error of the mean. 3. Results 3.1. Chemogenic hypersensitivity following nerve injury and the effect of amitriptyline A long-lasting enhanced response to ab-MeATP/NA (150/ 25 nmol) was observed on days 14, 28 and 42 following SNI, when compared to sham surgery (Fig. 1). Amitriptyline, administered pre- and post-surgically (10 mg/kg i.p.) and then orally in the drinking water for one week following surgery (16 mg/kg/day) produced a significant reduction in the hypersensitivity response observed 14–42 days following surgery (Fig. 2). In this experiment, the SNI group that received no drug declined over time, and flinches in response to ab-MeATP/NA were reduced significantly
Fig. 2. Amitriptyline given pre- and post-operatively and in the drinking water for 7 days following SNI diminishes hypersensitivity to ab-MeATP/NA. Animals received amitriptyline (ami) perioperatively (10 mg/kg i.p. 30 min pre- and then again post-operatively) followed by oral amitriptyline (16 mg/kg/day in the drinking water for 7 days). Values depict mean + SEM; *P < 0.05, **P < 0.01, ***P < 0.001 compared to the corresponding vehicle-treated group, Student’s t-test; n = 7–9 per group.
Fig. 3. Intrathecal administration of 6-OHDA inhibits the preventive analgesic effects of amitriptyline following SNI. Intrathecal injection of 6-OHDA (100 lg) on the day of surgery had no effect on flinches over 15 min in response to lateral hindpaw injection of ab-MeATP/NA (150/25 nmol) compared to the intrathecal vehicle (veh; 0.1% ascorbic acid) group; however, it reversed the preventive effect of the amitriptyline (ami) regimen. Values depict mean + SEM; fP < 0.05 compared to veh group; *P < 0.05 compared to veh + ami group; ANOVA and SNK post hoc analysis; n = 7–8 per group.
from day 14 values by day 42 (P < 0.05, ANOVA and SNK post hoc test). Rats were not tested on day 7, as any effect observed at that time would reflect an acute drug action. Both ab-MeATP and NA given alone into the lateral hindpaw following SNI also exhibit a hypersensitivity response [23]; however, these individual agents were not tested in this set of experiments, as we wished to work with the more robust chemogenic response shown by their combination [39]. 3.2. Involvement of noradrenaline in the preventive effect of amitriptyline Animals given an acute injection of 100 lg 6-OHDA into the spinal sub-arachnoid space immediately following nerve injury
Fig. 4. Desipramine given pre- and post-operatively and in the drinking water for 7 days following SNI diminishes hypersensitivity to ab-MeATP/NA. Animals received desipramine (des) perioperatively (10 mg/kg i.p. 30 min pre- and then again post-operatively) followed by oral desipramine (16 mg/kg/day in the drinking water for 7 days). Values depict mean + SEM; ***P < 0.001 compared to corresponding vehicle-treated group, Student’s t-test; n = 7–9 per group.
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Fig. 5. Intrathecal injection of GDNF antibodies inhibits the preventive analgesic actions of amitriptyline following SNI. Intrathecal injection of antibody (ab) to GDNF, day 0, 3 and 7 following surgery, had no effect on flinches over 15 min in response to lateral hindpaw injection of ab-MeATP/NA (150/25 nmol) compared to the vehicle (veh; bovine serum albumin) group; however, it reversed the preventive effect of the amitriptyline (ami) regimen. Values depict mean + SEM; fP < 0.05 compared to veh group; *P < 0.05 compared to veh + ami group; ANOVA and SNK post hoc analysis; n = 6–8 per group.
exhibited no altered behaviours compared to vehicle controls in SNI rats when tested 17 days post-injury (left column pair, Fig. 3). Animals that received 6-OHDA, in conjunction with amitriptyline, following surgery exhibited a reversal of the reduction in hypersensitivity response to ab-MeATP/NA exhibited by the amitriptyline regimen group (right column pair, Fig. 3). To further explore the role of NA in the preventive action of amitriptyline, a group of animals was given desipramine (10 mg/kg preand post-operatively, 16 mg/kg/day for 7 days) in the same manner as amitriptyline. As with amitriptyline, there was a reversal of the hypersensitivity to ab-MeATP/NA with desipramine 14 and 28 days following surgery (Fig. 4). Again, control responses to abMeATP/NA were reduced on day 42 compared to day 14 (P < 0.05, ANOVA and SNK post hoc test). [This decline may represent a lessening of hypersensitivity with time, or a desensitization to the stimulus following repeated injections to the same site. It is not clear why it
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Fig. 6. Intrathecal injection of BDNF antibodies inhibits the preventive analgesic actions of amitriptyline. Intrathecal injection of antibody (ab) to BDNF, days 0, 3 and 7 following surgery, had no effect on flinches over 15 min in response to lateral hindpaw injection of ab-MeATP/NA (150/25 nmol) compared to the vehicle (veh; bovine serum albumin) group; however, it reversed the preventive effect of the amitriptyline (ami) regimen. Values depict mean + SEM; fP < 0.05 compared to veh group; *P < 0.05 compared to veh + ami group; ANOVA and SNK post hoc analysis; n = 7–8 per group.
was somewhat variable in presentation, being clearly present in experiments depicted in Figs. 2 and 4, but not in Fig. 1.] 3.3. Effect of GDNF and BDNF antibodies on the preventive effect of amitriptyline Intrathecal injection of GDNF antibodies by acute lumbar puncture on the day of surgery and on days 3 and 7 following surgery had no effect on nerve injury-induced hypersensitivity to abMeATP/NA versus vehicle controls 14 days following surgery (Fig. 5; left column pair). In animals that received amitriptyline, the antibody reversed the suppression of hypersensitivity produced by amitriptyline (Fig. 5; right column pair). Intrathecal delivery of BDNF antibodies using the same injection schedule also produced a significant reversal of the amitriptyline effect on day 14 but had no effect on controls (Fig. 6).
Fig. 7. A preventive perioperative regimen of amitriptyline has no significant impact on mechanical allodynia following SNI. Nerve injured rats (A) and mice (B) displayed a significant reduction in withdrawal thresholds as compared to sham animals. Rats (A) and mice (B) that received amitriptyline perioperatively (10 mg/kg i.p. pre-and postoperatively) followed by amitriptyline (16 mg/kg/day) in the drinking water for 7 days, displayed no significant difference in mechanical threshold of the injured hindpaw (measured by the Dixon’s Up-Down method) compared to the vehicle-treated nerve injury group. Values depict mean + SEM; *P < 0.05 compared to sham animals, ANOVA on ranks and Dunn’s post hoc analysis, n = 6–8 per group.
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Fig. 8. Treatment with amitriptyline pre-and post-operatively and for 7 days in the drinking water inhibits SNI-induced hyposensitivity to capsaicin/NA. Nerve injury led to a reduction in flinching behaviours over 15 min in response to lateral hindpaw injection of capsaicin/NA (3 lg/25 nmol in 10% DMSO). In animals that received the perioperative amitriptyline regimen, this hyposensitivity was reversed. Values depict mean + SEM; fP < 0.05 compared to sham vehicle group; *P < 0.05 compared to SNI vehicle group; ANOVA and SNK post hoc analysis; n = 6–7 per group.
3.4. Amitriptyline and mechanical allodynia Administration of amitriptyline, perisurgically and for one week following surgery, did not alter the development of mechanical allodynia 14 days following surgery in rats (Fig. 7A). Given that chronic administration of amitriptyline did not influence mechanical allodynia following nerve injury in rats [12], but did alleviate mechanical allodynia in another nerve injury model in mice [3], we also tested the effect of the amitriptyline regimen in mice with SNI. However, amitriptyline did not alter the development of mechanical allodynia 14 days following SNI in mice (Fig. 7B). 3.5. Amitriptyline and chemogenic hyposensitivity In contrast to ab-MeATP/NA, lateral hindpaw injection of capsaicin/NA following SNI leads to a hyposensitivity response (Fig. 8) (see also [23]). The same peri- and post-surgical regimen of amitriptyline that reversed hypersensitivity to ab-MeATP/NA also resulted in a reversal of the hyposensitivity response to capsaicin/ NA 14 and 21 days following surgery (Fig. 8). Responses to capsaicin alone were not altered by SNI [23] and were not evaluated in this series of experiments. 3.6. Further characterization of amitriptyline actions Several additional aspects of the action of amitriptyline were examined in paired groups in which only the treatment and the addi-
tional intervention were examined; in each case, when no effect was observed, further control experiments were not conducted. (a) We determined whether only pre- and post-operative exposure to amitriptyline was sufficient to produce a preventive effect, but noted that it was not – thus, the group that received only this treatment still exhibited hypersensitivity to ab-MeATP/NA (flinches remained at the hypersensitive level of 80–100/15 min, see Figs. 1, 2 and 4) (Table 1A). (b) We determined whether the co-presence of morphine for 3 days following surgery had any effect on the preventive amitriptyline regimen – it did not appear to, as both the saline- and morphine-treated groups were statistically indistinguishable and exhibited apparent reversal of the hyperalgesia (flinches of 40–50/ 15 min were noted, compared to the 80–100/15 min control levels seen in Figs. 1, 2 and 4) (Table 1B). (c) We determined whether caffeine could alter the preventive effect of the amitriptyline regimen – however, oral coadministration of caffeine in the drinking water along with the amitriptyline for one week had no effect on the action of amitriptyline 14 days following surgery (Table 1C). 4. Discussion 4.1. Preventive effect of amitriptyline following nerve injury The present study demonstrates that treatment with amitriptyline, perisurgically and for 1 week following surgery, prevents long-term chemogenic hypersensitivity responses to ab-MeATP/ NA and hyposensitivity responses to capsaicin/NA. These changes were primarily apparent 14–28 days following surgery with some effects also observed at 42 days following surgery. Given that the first determination occurs one week following drug discontinuation, it is unlikely that acute pharmacological effects contribute to drug action at these times. This effect is, therefore, a preventive action on aberrant sensory responses that result from nerve injury. Clinical neuropathic pain can involve both hypersensitivity (spontaneous pain, allodynia, and hyperalgesia) and hyposensitivity (sensory deficits) [1], and reversing bidirectional abnormal signaling in sensory afferents may be important in preventing manifestations of neuropathic pain. Both P2X3 receptors and TRPV1 receptors are localized on C-fibres [6,38], and augmentation of their actions by NA may reflect interactions between a1-adrenergic and these receptors on afferent neurons [34,39]. The differing direction of the effect of nerve injury on these responses may reflect differential mechanisms of coupling between adrenergic and respective ion channel receptors on sensory neurons [23]. In contrast to chemogenic responses, amitriptyline did not prevent development of mechanical allodynia in either rats or mice. Mechanical allodynia following nerve injury in rats exhibits a differential sensitivity to capsaicin compared to thermal hyperalgesia, and appears to reflect activity in A-fibres rather than in C-fibres [13,29]. The differential effect of amitriptyline on the two end points may reflect actions of the drug on different fibre types. A recent study in mice demonstrated that chronic administration of amitriptyline leads to alleviation of mechanical allodynia in a cuff
Table 1 Further characterization of the preventive effect of the pre- and post-operative drinking water regimen of amitriptyline following SNI in rats. Treatment
Perioperative amitriptyline
Presence of treatment
Observations on day 14 (# of flinches over 15 min)
A. Only pre- and post-operative amitriptyline (10 mg/kg i.p.)
+ + + +
+ + +
81 ± 6 90 ± 11 (NS) 38 ± 6 53 ± 11 (NS) 69 ± 7 62 ± 8 (NS)
B. Post-operative morphine (10 mg/kg i.p.) days 0, 1, 2 and 3 after SNI surgery C. Oral caffeine (6–8 mg/kg/day) in drinking water along with amitriptyline for 7 days following SNI surgery NS P > 0.05.
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model of neuropathic pain [3], but we did not observe a preventive effect on allodynia with our amitriptyline regimen in mice using the SNI model. While the lack of preventive effect on allodynia could reflect the need for a different dosing regimen (Benbouzid et al. [3] administered amitriptyline for 10 days before an antiallodynic effect was observed), it is important to note that the 7-day regimen was clearly sufficient to reverse chemogenic responses in C-fibres in rats in this study, and that a 21-day regimen still did not reverse allodynia in rats [23]. While it appears that there may be a species difference in antidepressant responses, testing a wider range of sensory modalities in the two species would be helpful for further understanding these differences. 4.2. Role of NA in the preventive effect of amitriptyline Amitriptyline is well known as a dual NA and 5-HT reuptake inhibitor, and its ability to inhibit the uptake of both amines is implicated in acute antinociceptive actions [2,25]. Here we demonstrate that the destruction of spinal cord NA systems, using intrathecal delivery of the neurotoxin 6-OHDA, reverses the preventive action of amitriptyline against chemogenic hypersensitivity. While we did not measure amine levels in this study, previous studies in this laboratory have demonstrated a marked depletion (>80%) in spinal cord NA levels following such spinal delivery of 6-OHDA [35,37]. Further implicating NA in the current preventive action is the observation that desipramine, a more selective NA reuptake inhibitor than amitriptyline, also prevented nerve injury-induced hypersensitivity to ab-MeATP/NA. The involvement of NA in spinal pain signaling has been extensively reviewed [30]. Administration of NA to the spinal cord produces dose-dependent antinociceptive effects due to actions on a-2-adrenoceptors, and a-2 adrenoceptor agonists have been proposed as part of multimodal regimens to prevent development of central sensitization [31]. While we cannot exclude an involvement of spinal dopamine systems contributing to the 6OHDA effect in our experiments, it is interesting to note that descending dopaminergic regulation of neuropathic hypersensitivity responses also involves a-2 adrenoceptors [41]. 4.3. Role of GDNF in the preventive effect of amitriptyline Recent work has implicated GDNF in the mechanism of action of amitriptyline, as well as in the development of neuropathic pain. Thus, in rats subjected to spinal nerve ligation or chronic constriction injury to the sciatic nerve, GDNF expression was shown to decrease following injury [27]. Furthermore, intrathecal infusion of GDNF following spinal nerve ligation prevented mechanical allodynia and thermal hyperalgesia [40]. In vitro studies demonstrate that amitriptyline induces synthesis and release of GDNF from glial cells [19]. Here we demonstrate that antiserum to GDNF reverses the preventive effect of amitriptyline on chemogenic hypersensitivity following nerve injury. Based on these results, it is reasonable to infer that the effect of amitriptyline on the synthesis and release of GDNF provides a form of neuroprotection that prevents long-term changes that result in sensory hypersensitivity. Intrathecal injection of GDNF has been reported to reverse the down-regulation of P2X3 receptors in L4/5 dorsal root ganglia following complete sciatic nerve axotomy [5], and changes in receptor expression could contribute to observed behavioural changes. While the above line of reasoning implicates GDNF, there are, however, issues that need to be considered. In the spared nerve injury model used in our study, overall expression of P2X3 receptors remained unchanged, and increased behavioural responses to P2X3 receptor ligands were attributed to increased cell surface expression of the receptor [8]. Also, in the works previously mentioned, delivery of GDNF alleviated mechanical allodynia, whereas our amitriptyline treatment did not. The discrepancies in expres-
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sion patterns of P2X3 receptors and manifestations of behavioural responses may be due to the different natures of the nerve injury models used in these studies. While it is clear that GDNF plays a role in the development of persistent pain, the exact mechanism by which this occurs requires further elaboration. 4.4. Role of BDNF in the preventive effect of amitriptyline There is a body of research that has focused on the role of BDNF in the development of neuropathic pain, and the possible involvement of BDNF in the mechanism of action of tricyclic antidepressants. Depressed human patients receiving amitriptyline showed higher serum concentrations of BDNF versus control subjects [17]. Following axotomy, BDNF mRNA expression decreases in damaged small neurons but increases in medium to large DRG neurons and their central terminals in the dorsal horn [24]. In the spinal nerve ligation model, BDNF was shown to induce spinal noradrenergic fibre sprouting in the dorsal spinal cord following nerve injury [16]. This mechanism may contribute to the results observed in our experiments. Thus, following nerve injury enhanced BDNF levels may contribute to amitriptyline preventive analgesia by inducing spinal noradrenergic fibre sprouting. This mechanism needs to be elaborated using additional approaches. 4.5. Further characterization of the preventive actions of amitriptyline We further characterized the preventive amitriptyline regimen by examining the potential influences of morphine and caffeine on this action, and by reducing the drug regimen to acute perisurgical exposure (Table 1). Initially, we varied the amitriptyline regimen to determine which component of administration was essential for action. We found that chronic administration of amitriptyline was required for a preventive analgesic effect because amitriptyline given only at the time of surgery did not have any effect on the development of chemogenic hypersensitivity (Table 1A). Chronic, but not acute, exposure to amitriptyline is required to produce a preventive effect on sensory changes. Secondly, as opioids are used routinely to control pain following surgery, examining the effect of morphine on the preventive action of amitriptyline is potentially clinically relevant. However, a 3-day regimen of morphine given post-operatively did not modify the preventive action of amitriptyline (Table 1B). Finally, American adults consume, on average, 193 mg of caffeine per day [15], and, as previous work has shown that chronic caffeine consumption blocks the analgesic effect of amitriptyline in a nerve injury model [12], we determined whether caffeine could also modify the preventive effect of amitriptyline. However, caffeine had no impact on this action (Table 1C). This observation also dissociates the mechanisms by which amitriptyline produces acute and preventive analgesia, as the former involves adenosine (caffeine blocks adenosine A1 and A2 receptors) but the latter does not. Importantly, these experiments demonstrate that two commonly used drugs, morphine and caffeine, do not influence the preventive analgesic actions of amitriptyline, and both observations are of clinical interest given the widespread use of morphine in a post-operative setting, and of caffeine as a part of dietary intake. 4.6. Concluding comments The present results indicate a preventive effect of a regimen of peri- and post-surgical amitriptyline on hypersensitivity and hyposensitivity responses in rats following nerve injury. Tricyclic antidepressants have been used to treat neuropathic pain for decades, and our results indicate that they also may be useful for preventing longterm effects of damage to nerves that can inadvertently result from surgery. There are two clinical reports that indicate a preventive
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[19] Hisaoka K, Takebayashi M, Tsuchioka M, Maeda N, Nakata Y, Yamawaki S. Antidepressants increase glial cell line-derived neurotrophic factor production through monoamine-independent activation of protein tyrosine kinase and extracellular signal-regulated kinase in glial cells. J Pharmacol Exp Ther 2007;321:148–57. [20] Katz J, Cohen L. Preventive analgesia is associated with reduced pain disability 3 weeks but not 6 months after major gynecologic surgery by laparotomy. Anesthesiology 2004;101:169–74. [21] Kehlet H, Jensen TS, Woolf CJ. Persistent postsurgical pain: risk factors and prevention. Lancet 2006;367:1618–25. [22] Malkmus SA, Yaksh TL. Intrathecal catheterization and drug delivery in the rat. Methods Mol Med 2004;99:109–21. [23] Meisner JG, Reid AR, Sawynok J. Adrenergic regulation of P2X3 and TRPV1 receptors: differential effects of spared nerve injury. Neurosci Lett 2008;444:172–5. [24] Michael GJ, Averill S, Shortland PJ, Yan Q, Priestley JV. Axotomy results in major changes in BDNF expression by dorsal root ganglion cells: BDNF expression in large trkB and trkC cells, in pericellular baskets, and in projections to deep dorsal horn and dorsal column nuclei. Eur J Neurosci 1999;11:3539–51. [25] Mico JA, Ardid D, Berrocoso E, Eschalier A. Antidepressants and pain. Trends Pharmacol Sci 2006;27:348–54. [26] Moiniche S, Kehlet H, Dahl JB. A qualitative and quantitative systematic review of preemptive analgesia for postoperative pain relief: the role of timing of analgesia. Anesthesiology 2002;96:725–41. [27] Nagano M, Sakai A, Takahashi N, Umino M, Yoshioka K, Suzuki H. Decreased expression of glial cell line-derived neurotrophic factor signaling in rat models of neuropathic pain. Br J Pharmacol 2003;140:1252–60. [28] Ochroch EA, Gottschalk A, Augostides J, Carson KA, Kent L, Malayaman N, Kaiser LR, Aukburg SJ. Long-term pain and activity during recovery from major thoracotomy using thoracic epidural analgesia. Anesthesiology 2002;97:1234–44. [29] Ossipov MH, Bian D, Malan Jr TP, Lai J, Porreca F. Lack of involvement of capsaicin-sensitive primary afferents in nerve-ligation injury induced tactile allodynia in rats. Pain 1999;79:127–33. [30] Pertovaara A. Noradrenergic pain modulation. Prog Neurobiol 2006;80:53–83. [31] Reuben SS, Buvanendran A. Preventing the development of chronic pain after orthopaedic surgery with preventive multimodal analgesic techniques. J Bone Joint Surg Am 2007;89:1343–58. [32] Reuben SS, Makari-Judson G, Lurie SD. Evaluation of efficacy of the perioperative administration of venlafaxine XR in the prevention of postmastectomy pain syndrome. J Pain Symptom Manage 2004;27:133–9. [33] Ringkamp M, Eschenfelder S, Grethel EJ, Habler HJ, Meyer RA, Janig W, Raja SN. Lumbar sympathectomy failed to reverse mechanical allodynia- and hyperalgesia-like behavior in rats with L5 spinal nerve injury. Pain 1999;79:143–53. [34] Sawynok J, Reid A, Meisner J. Pain behaviors produced by capsaicin: influence of inflammatory mediators and nerve injury. J Pain 2006;7:134–41. [35] Sawynok J, Reid A, Nance D. Spinal antinociception by adenosine analogs and morphine after intrathecal administration of the neurotoxins capsaicin, 6hydroxydopamine and 5,7-dihydroxytryptamine. J Pharmacol Exp Ther 1991;258:370–80. [36] Scholz J, Woolf CJ. Can we conquer pain? Nat Neurosci 2002;5:1062–7. [37] Sweeney MI, White TD, Sawynok J. Intracerebroventricular morphine releases adenosine and adenosine 30 ,50 -cyclic monophosphate from the spinal cord via a serotonergic mechanism. J Pharmacol Exp Ther 1991;259:1013–8. [38] Szallasi A, Cortright DN, Blum CA, Eid SR. The vanilloid receptor TRPV1: 10 years from channel cloning to antagonist proof-of-concept. Nat Rev Drug Discov 2007;6:357–72. [39] Waldron JB, Sawynok J. Peripheral P2X receptors and nociception: interactions with biogenic amine systems. Pain 2004;110:79–89. [40] Wang R, Guo W, Ossipov MH, Vanderah TW, Porreca F, Lai J. Glial cell linederived neurotrophic factor normalizes neurochemical changes in injured dorsal root ganglion neurons and prevents the expression of experimental neuropathic pain. Neuroscience 2003;121:815–24. [41] Wei H, Viisanen H, Pertovaara A. Descending modulation of neuropathic hypersensitivity by dopamine D2 receptors in or adjacent to the hypothalamic A11 cell group. Pharmacol Res 2009;59:355–63.
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Perisurgical amitriptyline produces a preventive effect on afferent hypersensitivity following spared nerve injury Andre Arsenault, Jana Sawynok * Department of Pharmacology, Dalhousie University, Halifax, NS, Canada B3H 1X5
a r t i c l e
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Article history: Received 6 May 2009 Received in revised form 13 July 2009 Accepted 10 August 2009
Keywords: Amitriptyline Nerve injury Preventive analgesia Noradrenaline
a b s t r a c t Following surgery, nerve injury can lead to persistent neuropathic pain. Pre-emptive and preventive analgesic treatments in the perioperative period aim to minimize nerve injury-induced pain. Here we demonstrate that a perioperative regimen of amitriptyline (10 mg/kg i.p. 30 min before and immediately after surgery, followed by oral amitriptyline 15–18 mg/kg/day in the drinking water for 7 days post-surgery) prevents hypersensitivity to a chemogenic stimulus (ab-MeATP, a ligand for P2X3 receptors, together with noradrenaline or NA) in the spared nerve injury (SNI) model in rats. It also prevents hyposensitivity to capsaicin and NA. However, amitriptyline treatment had no effect on the development of mechanical allodynia. We investigated the role of NA mechanisms in the action of amitriptyline by using the neurotoxin 6-hydroxydopamine (6-OHDA) and by examining desipramine. Intrathecal treatment with 6-OHDA on the day of surgery reversed the preventive effect of amitriptyline on hypersensitivity to ab-MeATP/NA, and desipramine exhibited a similar effect to amitriptyline. We also examined the effect of antibodies to the nerve growth factors glial-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), given intrathecally three times (days 0, 3 and 7) on the action of amitriptyline and observed that the interruption of GDNF and BDNF signaling impaired the prevention of hypersensitivity to ab-MeATP/ NA. This study indicates that tricyclic antidepressants given in the perioperative period may be useful in preventing nerve injury-induced sensory changes that contribute to the development of chronic post-surgical neuropathic pain. Ó 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
1. Introduction Surgical interventions can lead to persistent pain long after recovery from the surgical event. The prevalence of such pain varies depending on the procedure, but can occur in up to 50% of patients [21]. Damage to peripheral nerves causes maladaptive changes at every level of the nervous system, and can include altered function at the initial injury site, at neuronal cell bodies located in the dorsal root ganglion and at synapses of the central nervous system [36]. Injured nerves also affect cellular processes in adjacent uninjured nerves, as well as at remote sites along the neural axis [7]. This altered neuronal function, or plasticity, contributes to the development of neuropathic pain. Studies in animals, and in the clinical setting, have attempted to prevent pain that results from surgery by using pre-emptive or preventive analgesia strategies [21]. The rationale for this approach is to use analgesics in the perioperative period to prevent or minimize nervous system changes that contribute to the development of neuropathic pain. Reviews of randomized controlled trials * Corresponding author. Tel.: +1 902 494 2596; fax: +1 902 494 1388. E-mail address: [email protected] (J. Sawynok).
employing preventive analgesia strategies report limited success using such strategies [20,26,28]. There are, however, some preventive analgesia reports that appear promising using antidepressants [4,32]. Tricyclic antidepressants have long been the primary pharmacological treatment for neuropathic pain [14]; these act on several aspects of pain signaling relevant to neuropathic pain – they increase the availability of noradrenaline (NA) and 5-hydroxytryptamine (5-HT) at central synapses, block Na+ channels, activate K+ channels, inhibit Ca2+ reuptake, and activate opioid and adenosine systems [25]. More recently, the nerve growth factors glial-cellline-derived neurotrophic factor (GDNF) [18,19] and brain-derived neurotrophic factor (BDNF) [11,17] have been implicated in the actions of antidepressants. The role of these factors in the development of neuropathic pain and in the preventive analgesic actions of antidepressants will be discussed later. In the present study, we investigated whether amitriptyline, a tricyclic antidepressant, could alter the development of chemogenic and mechanical hypersensitivity following surgical nerve injury in rats. We used the spared nerve injury (SNI) model and a lateral hindpaw testing site which allows for the assessment of the effects of stimuli in a sensory field influenced by injured fibres in the upper reaches of the nerve, in ganglia and in the spinal cord [9].
0304-3959/$36.00 Ó 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2009.08.003
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The chemogenic stimulus used was a combination of ab-methylene adenosine triphosphate (ab-MeATP, which activates P2X3 receptors) and NA; this previously was shown to produce an augmented response at the lateral site following SNI and its actions are relevant to sympathetic nerve influences on sensory transmission [23]. Mechanical responses were determined using von Frey hairs and previously were shown to be hypersensitive following the nerve injury [9]. When a preventive effect was shown to occur, we subsequently examined the involvement of spinal cord NA, GDNF and BDNF in the preventive action of amitriptyline. Finally, a chemogenic stimulus which exhibits hyposensitivity following nerve injury (capsaicin/NA) [23] was examined, as clinical neuropathic pain can involve sensory hyposensitivity (sensory loss) as well as hypersensitivity (spontaneous pain, allodynia, and hyperalgesia) [1].
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regimen [12]. Fluid intake, and therefore drug dosing, was monitored by weighing the drinking bottles. In one experiment desipramine was given instead of amitriptyline, using the same regimen. 2.5. 6-Hydroxydopamine treatment Immediately following nerve injury, and while rats were still anaesthetized, the neurotoxin 6-hydroxydopamine (6-OHDA) was delivered to the spinal cord via an acutely inserted intrathecal cannula [22]. The cannula (polyethylene – 10 tubing) was inserted into the sub-arachnoid space 7.5 cm caudally through a small opening in the cisterna magna. 6-OHDA (100 lg) was dissolved in 0.1% ascorbic acid and delivered in a final volume of 8 ll. After drug delivery, the cannula was left in place for 2–3 min before slow removal; the wound was then closed with sutures. This dose of 6OHDA has previously been shown to deplete spinal cord NA levels by >80% [35,37].
2. Methods 2.6. Antibody studies 2.1. Animals Male Sprague–Dawley rats (Charles River Laboratory, Que., Canada) weighing 175–225 g at the time of surgery were used in most experiments. The mouse study used male C57/BL6 mice (Charles River Laboratory, Que., Canada) weighing 20–25 g. Animals were housed under a 12–12 h light–dark cycle and allowed to acclimate to the facility for a period of 1 week prior to surgery. All animals were given free access to food and water. All studies were approved by the University Committee on Laboratory Animals (Dalhousie University) and complied with the Canadian Council of Animal Care guidelines for the ethical use of animals.
BDNF (Millipore) and GDNF (AbCam) antibodies were dissolved in normal saline. Antibody (15 lg/injection in a final volume of 10 ll) was delivered by acute intrathecal lumbar puncture (the needle was inserted between the L5 and L6 vertebrae) immediately after surgery while the rat was still anaesthetized, and then 3 and 7 days following surgery (again under anaesthesia). An equivalent concentration of bovine serum albumen, dissolved in saline, was used as a vehicle control. 2.7. Behavioural analysis
All drugs and reagents were obtained from Sigma–Aldrich (Canada) with the exception of the BDNF (Millipore, Canada) and GDNF (AbCam, USA) antibodies.
All behavioural analysis was conducted between 09:00 and 14:00 h. Sensitivity of the paw ipsilateral to the nerve injury was assessed using chemical and mechanical modalities. Animals were placed in a plexiglass box (30 30 30 cm) and permitted to habituate for 30 min. ab-MeATP (150 nmol), or capsaicin (3 lg dissolved in 10% DMSO), and NA (25 nmol) were injected in a volume of 30 ll s.c. into the lateral side of the injured paw. Vigorous flinches of the paw were recorded in 1 min intervals for 15 min. Testing was performed between 14 and 28 days post-injury, although some determinations were done at 42 days. Mechanical threshold to the application of a von Frey probe was assessed using the Dixon up-down method [10]. Animals were placed in a plastic container with an elevated wire mesh floor, allowing access to the plantar surface of the hindpaw. Using an established testing procedure [33], the lateral surface of the plantar hindpaw was probed for 2–3 s. A positive response was identified as a sharp withdrawal, or biting and licking, immediately following the application of the von Frey hair. In the case of a positive response the next lightest hair was used, and in the case of a negative response the next heaviest hair was applied. Four more stimuli were applied after an initial change was observed, and the pattern of responses was translated into a 50% von Frey threshold using the following equation; 10(xf+jd)/10,000 where xf = value (in log units) of the final von Frey hair used, j = tabular value for the pattern of the last 6 positive/negative responses and d = mean (in log units) between stimuli.
2.4. Drug treatments
2.8. Statistics
Animals received an i.p. injection of amitriptyline (10 mg/kg) or an equivalent amount of vehicle (normal saline) before and after surgery. Following surgery, amitriptyline (or no drug) was then administered in the drinking water (16 mg/kg/day) for a period of 7 days. This dosage level was based on a previous study which demonstrated antihyperalgesic effects of amitriptyline using this
All statistical analyses were performed using Sigmastat v1.0. Data were analysed for each particular time following surgery. In some cases, this involved use of the Student’s t-test (Figs. 1 and 2); in others, a one-way analysis of variance (ANOVA) with the Student–Newman–Keuls (SNK) post hoc analysis was performed (Figs. 3–5 and 7). An ANOVA on ranks and the Dunn’s post hoc test was used for the
2.2. Surgery The spared nerve injury procedure was performed as previously described with minor surgical modifications [9]. Briefly, animals were placed under anaesthesia (2% isoflurane) and an incision was made along the back of the thigh. The biceps femoris muscle was separated to expose the three terminal branches of the sciatic nerve; the common peroneal, tibial and sural nerves. The tibial and common peroneal nerves were tightly ligated using 6.0 silk and transected distal to the ligation removing 2–4 mm of nerve stump. The sural nerve was left intact and the wound was closed with cutaneous sutures. Rats were housed individually for 48 h before being paired with their original cage mate. Animals displaying autotomy (1 animal per group) were euthanized. In mice, the spared nerve injury was performed in the same manner as in rats except the tibial and common peroneal nerves were not ligated before being transected. Sham surgery controls were performed by exposing the nerve bundle, but not manipulating the nerves, and then closing the wound. 2.3. Drugs and reagents
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Fig. 1. Spared nerve injury (SNI) produces sustained hypersensitivity to coadministration of ab-MeATP/NA. Lateral hindpaw injection of ab-MeATP/NA (150/ 25 nmol) produced increased flinching behaviours over 15 min, 14–42 days after SNI compared to sham animals. Values depict mean + SEM; ***P < 0.001, Student’s ttest, n = 7–9 per group.
mechanical allodynia experiments (Fig. 6). Data are presented as means, and error bars represent the standard error of the mean. 3. Results 3.1. Chemogenic hypersensitivity following nerve injury and the effect of amitriptyline A long-lasting enhanced response to ab-MeATP/NA (150/ 25 nmol) was observed on days 14, 28 and 42 following SNI, when compared to sham surgery (Fig. 1). Amitriptyline, administered pre- and post-surgically (10 mg/kg i.p.) and then orally in the drinking water for one week following surgery (16 mg/kg/day) produced a significant reduction in the hypersensitivity response observed 14–42 days following surgery (Fig. 2). In this experiment, the SNI group that received no drug declined over time, and flinches in response to ab-MeATP/NA were reduced significantly
Fig. 2. Amitriptyline given pre- and post-operatively and in the drinking water for 7 days following SNI diminishes hypersensitivity to ab-MeATP/NA. Animals received amitriptyline (ami) perioperatively (10 mg/kg i.p. 30 min pre- and then again post-operatively) followed by oral amitriptyline (16 mg/kg/day in the drinking water for 7 days). Values depict mean + SEM; *P < 0.05, **P < 0.01, ***P < 0.001 compared to the corresponding vehicle-treated group, Student’s t-test; n = 7–9 per group.
Fig. 3. Intrathecal administration of 6-OHDA inhibits the preventive analgesic effects of amitriptyline following SNI. Intrathecal injection of 6-OHDA (100 lg) on the day of surgery had no effect on flinches over 15 min in response to lateral hindpaw injection of ab-MeATP/NA (150/25 nmol) compared to the intrathecal vehicle (veh; 0.1% ascorbic acid) group; however, it reversed the preventive effect of the amitriptyline (ami) regimen. Values depict mean + SEM; fP < 0.05 compared to veh group; *P < 0.05 compared to veh + ami group; ANOVA and SNK post hoc analysis; n = 7–8 per group.
from day 14 values by day 42 (P < 0.05, ANOVA and SNK post hoc test). Rats were not tested on day 7, as any effect observed at that time would reflect an acute drug action. Both ab-MeATP and NA given alone into the lateral hindpaw following SNI also exhibit a hypersensitivity response [23]; however, these individual agents were not tested in this set of experiments, as we wished to work with the more robust chemogenic response shown by their combination [39]. 3.2. Involvement of noradrenaline in the preventive effect of amitriptyline Animals given an acute injection of 100 lg 6-OHDA into the spinal sub-arachnoid space immediately following nerve injury
Fig. 4. Desipramine given pre- and post-operatively and in the drinking water for 7 days following SNI diminishes hypersensitivity to ab-MeATP/NA. Animals received desipramine (des) perioperatively (10 mg/kg i.p. 30 min pre- and then again post-operatively) followed by oral desipramine (16 mg/kg/day in the drinking water for 7 days). Values depict mean + SEM; ***P < 0.001 compared to corresponding vehicle-treated group, Student’s t-test; n = 7–9 per group.
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Fig. 5. Intrathecal injection of GDNF antibodies inhibits the preventive analgesic actions of amitriptyline following SNI. Intrathecal injection of antibody (ab) to GDNF, day 0, 3 and 7 following surgery, had no effect on flinches over 15 min in response to lateral hindpaw injection of ab-MeATP/NA (150/25 nmol) compared to the vehicle (veh; bovine serum albumin) group; however, it reversed the preventive effect of the amitriptyline (ami) regimen. Values depict mean + SEM; fP < 0.05 compared to veh group; *P < 0.05 compared to veh + ami group; ANOVA and SNK post hoc analysis; n = 6–8 per group.
exhibited no altered behaviours compared to vehicle controls in SNI rats when tested 17 days post-injury (left column pair, Fig. 3). Animals that received 6-OHDA, in conjunction with amitriptyline, following surgery exhibited a reversal of the reduction in hypersensitivity response to ab-MeATP/NA exhibited by the amitriptyline regimen group (right column pair, Fig. 3). To further explore the role of NA in the preventive action of amitriptyline, a group of animals was given desipramine (10 mg/kg preand post-operatively, 16 mg/kg/day for 7 days) in the same manner as amitriptyline. As with amitriptyline, there was a reversal of the hypersensitivity to ab-MeATP/NA with desipramine 14 and 28 days following surgery (Fig. 4). Again, control responses to abMeATP/NA were reduced on day 42 compared to day 14 (P < 0.05, ANOVA and SNK post hoc test). [This decline may represent a lessening of hypersensitivity with time, or a desensitization to the stimulus following repeated injections to the same site. It is not clear why it
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Fig. 6. Intrathecal injection of BDNF antibodies inhibits the preventive analgesic actions of amitriptyline. Intrathecal injection of antibody (ab) to BDNF, days 0, 3 and 7 following surgery, had no effect on flinches over 15 min in response to lateral hindpaw injection of ab-MeATP/NA (150/25 nmol) compared to the vehicle (veh; bovine serum albumin) group; however, it reversed the preventive effect of the amitriptyline (ami) regimen. Values depict mean + SEM; fP < 0.05 compared to veh group; *P < 0.05 compared to veh + ami group; ANOVA and SNK post hoc analysis; n = 7–8 per group.
was somewhat variable in presentation, being clearly present in experiments depicted in Figs. 2 and 4, but not in Fig. 1.] 3.3. Effect of GDNF and BDNF antibodies on the preventive effect of amitriptyline Intrathecal injection of GDNF antibodies by acute lumbar puncture on the day of surgery and on days 3 and 7 following surgery had no effect on nerve injury-induced hypersensitivity to abMeATP/NA versus vehicle controls 14 days following surgery (Fig. 5; left column pair). In animals that received amitriptyline, the antibody reversed the suppression of hypersensitivity produced by amitriptyline (Fig. 5; right column pair). Intrathecal delivery of BDNF antibodies using the same injection schedule also produced a significant reversal of the amitriptyline effect on day 14 but had no effect on controls (Fig. 6).
Fig. 7. A preventive perioperative regimen of amitriptyline has no significant impact on mechanical allodynia following SNI. Nerve injured rats (A) and mice (B) displayed a significant reduction in withdrawal thresholds as compared to sham animals. Rats (A) and mice (B) that received amitriptyline perioperatively (10 mg/kg i.p. pre-and postoperatively) followed by amitriptyline (16 mg/kg/day) in the drinking water for 7 days, displayed no significant difference in mechanical threshold of the injured hindpaw (measured by the Dixon’s Up-Down method) compared to the vehicle-treated nerve injury group. Values depict mean + SEM; *P < 0.05 compared to sham animals, ANOVA on ranks and Dunn’s post hoc analysis, n = 6–8 per group.
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Fig. 8. Treatment with amitriptyline pre-and post-operatively and for 7 days in the drinking water inhibits SNI-induced hyposensitivity to capsaicin/NA. Nerve injury led to a reduction in flinching behaviours over 15 min in response to lateral hindpaw injection of capsaicin/NA (3 lg/25 nmol in 10% DMSO). In animals that received the perioperative amitriptyline regimen, this hyposensitivity was reversed. Values depict mean + SEM; fP < 0.05 compared to sham vehicle group; *P < 0.05 compared to SNI vehicle group; ANOVA and SNK post hoc analysis; n = 6–7 per group.
3.4. Amitriptyline and mechanical allodynia Administration of amitriptyline, perisurgically and for one week following surgery, did not alter the development of mechanical allodynia 14 days following surgery in rats (Fig. 7A). Given that chronic administration of amitriptyline did not influence mechanical allodynia following nerve injury in rats [12], but did alleviate mechanical allodynia in another nerve injury model in mice [3], we also tested the effect of the amitriptyline regimen in mice with SNI. However, amitriptyline did not alter the development of mechanical allodynia 14 days following SNI in mice (Fig. 7B). 3.5. Amitriptyline and chemogenic hyposensitivity In contrast to ab-MeATP/NA, lateral hindpaw injection of capsaicin/NA following SNI leads to a hyposensitivity response (Fig. 8) (see also [23]). The same peri- and post-surgical regimen of amitriptyline that reversed hypersensitivity to ab-MeATP/NA also resulted in a reversal of the hyposensitivity response to capsaicin/ NA 14 and 21 days following surgery (Fig. 8). Responses to capsaicin alone were not altered by SNI [23] and were not evaluated in this series of experiments. 3.6. Further characterization of amitriptyline actions Several additional aspects of the action of amitriptyline were examined in paired groups in which only the treatment and the addi-
tional intervention were examined; in each case, when no effect was observed, further control experiments were not conducted. (a) We determined whether only pre- and post-operative exposure to amitriptyline was sufficient to produce a preventive effect, but noted that it was not – thus, the group that received only this treatment still exhibited hypersensitivity to ab-MeATP/NA (flinches remained at the hypersensitive level of 80–100/15 min, see Figs. 1, 2 and 4) (Table 1A). (b) We determined whether the co-presence of morphine for 3 days following surgery had any effect on the preventive amitriptyline regimen – it did not appear to, as both the saline- and morphine-treated groups were statistically indistinguishable and exhibited apparent reversal of the hyperalgesia (flinches of 40–50/ 15 min were noted, compared to the 80–100/15 min control levels seen in Figs. 1, 2 and 4) (Table 1B). (c) We determined whether caffeine could alter the preventive effect of the amitriptyline regimen – however, oral coadministration of caffeine in the drinking water along with the amitriptyline for one week had no effect on the action of amitriptyline 14 days following surgery (Table 1C). 4. Discussion 4.1. Preventive effect of amitriptyline following nerve injury The present study demonstrates that treatment with amitriptyline, perisurgically and for 1 week following surgery, prevents long-term chemogenic hypersensitivity responses to ab-MeATP/ NA and hyposensitivity responses to capsaicin/NA. These changes were primarily apparent 14–28 days following surgery with some effects also observed at 42 days following surgery. Given that the first determination occurs one week following drug discontinuation, it is unlikely that acute pharmacological effects contribute to drug action at these times. This effect is, therefore, a preventive action on aberrant sensory responses that result from nerve injury. Clinical neuropathic pain can involve both hypersensitivity (spontaneous pain, allodynia, and hyperalgesia) and hyposensitivity (sensory deficits) [1], and reversing bidirectional abnormal signaling in sensory afferents may be important in preventing manifestations of neuropathic pain. Both P2X3 receptors and TRPV1 receptors are localized on C-fibres [6,38], and augmentation of their actions by NA may reflect interactions between a1-adrenergic and these receptors on afferent neurons [34,39]. The differing direction of the effect of nerve injury on these responses may reflect differential mechanisms of coupling between adrenergic and respective ion channel receptors on sensory neurons [23]. In contrast to chemogenic responses, amitriptyline did not prevent development of mechanical allodynia in either rats or mice. Mechanical allodynia following nerve injury in rats exhibits a differential sensitivity to capsaicin compared to thermal hyperalgesia, and appears to reflect activity in A-fibres rather than in C-fibres [13,29]. The differential effect of amitriptyline on the two end points may reflect actions of the drug on different fibre types. A recent study in mice demonstrated that chronic administration of amitriptyline leads to alleviation of mechanical allodynia in a cuff
Table 1 Further characterization of the preventive effect of the pre- and post-operative drinking water regimen of amitriptyline following SNI in rats. Treatment
Perioperative amitriptyline
Presence of treatment
Observations on day 14 (# of flinches over 15 min)
A. Only pre- and post-operative amitriptyline (10 mg/kg i.p.)
+ + + +
+ + +
81 ± 6 90 ± 11 (NS) 38 ± 6 53 ± 11 (NS) 69 ± 7 62 ± 8 (NS)
B. Post-operative morphine (10 mg/kg i.p.) days 0, 1, 2 and 3 after SNI surgery C. Oral caffeine (6–8 mg/kg/day) in drinking water along with amitriptyline for 7 days following SNI surgery NS P > 0.05.
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model of neuropathic pain [3], but we did not observe a preventive effect on allodynia with our amitriptyline regimen in mice using the SNI model. While the lack of preventive effect on allodynia could reflect the need for a different dosing regimen (Benbouzid et al. [3] administered amitriptyline for 10 days before an antiallodynic effect was observed), it is important to note that the 7-day regimen was clearly sufficient to reverse chemogenic responses in C-fibres in rats in this study, and that a 21-day regimen still did not reverse allodynia in rats [23]. While it appears that there may be a species difference in antidepressant responses, testing a wider range of sensory modalities in the two species would be helpful for further understanding these differences. 4.2. Role of NA in the preventive effect of amitriptyline Amitriptyline is well known as a dual NA and 5-HT reuptake inhibitor, and its ability to inhibit the uptake of both amines is implicated in acute antinociceptive actions [2,25]. Here we demonstrate that the destruction of spinal cord NA systems, using intrathecal delivery of the neurotoxin 6-OHDA, reverses the preventive action of amitriptyline against chemogenic hypersensitivity. While we did not measure amine levels in this study, previous studies in this laboratory have demonstrated a marked depletion (>80%) in spinal cord NA levels following such spinal delivery of 6-OHDA [35,37]. Further implicating NA in the current preventive action is the observation that desipramine, a more selective NA reuptake inhibitor than amitriptyline, also prevented nerve injury-induced hypersensitivity to ab-MeATP/NA. The involvement of NA in spinal pain signaling has been extensively reviewed [30]. Administration of NA to the spinal cord produces dose-dependent antinociceptive effects due to actions on a-2-adrenoceptors, and a-2 adrenoceptor agonists have been proposed as part of multimodal regimens to prevent development of central sensitization [31]. While we cannot exclude an involvement of spinal dopamine systems contributing to the 6OHDA effect in our experiments, it is interesting to note that descending dopaminergic regulation of neuropathic hypersensitivity responses also involves a-2 adrenoceptors [41]. 4.3. Role of GDNF in the preventive effect of amitriptyline Recent work has implicated GDNF in the mechanism of action of amitriptyline, as well as in the development of neuropathic pain. Thus, in rats subjected to spinal nerve ligation or chronic constriction injury to the sciatic nerve, GDNF expression was shown to decrease following injury [27]. Furthermore, intrathecal infusion of GDNF following spinal nerve ligation prevented mechanical allodynia and thermal hyperalgesia [40]. In vitro studies demonstrate that amitriptyline induces synthesis and release of GDNF from glial cells [19]. Here we demonstrate that antiserum to GDNF reverses the preventive effect of amitriptyline on chemogenic hypersensitivity following nerve injury. Based on these results, it is reasonable to infer that the effect of amitriptyline on the synthesis and release of GDNF provides a form of neuroprotection that prevents long-term changes that result in sensory hypersensitivity. Intrathecal injection of GDNF has been reported to reverse the down-regulation of P2X3 receptors in L4/5 dorsal root ganglia following complete sciatic nerve axotomy [5], and changes in receptor expression could contribute to observed behavioural changes. While the above line of reasoning implicates GDNF, there are, however, issues that need to be considered. In the spared nerve injury model used in our study, overall expression of P2X3 receptors remained unchanged, and increased behavioural responses to P2X3 receptor ligands were attributed to increased cell surface expression of the receptor [8]. Also, in the works previously mentioned, delivery of GDNF alleviated mechanical allodynia, whereas our amitriptyline treatment did not. The discrepancies in expres-
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sion patterns of P2X3 receptors and manifestations of behavioural responses may be due to the different natures of the nerve injury models used in these studies. While it is clear that GDNF plays a role in the development of persistent pain, the exact mechanism by which this occurs requires further elaboration. 4.4. Role of BDNF in the preventive effect of amitriptyline There is a body of research that has focused on the role of BDNF in the development of neuropathic pain, and the possible involvement of BDNF in the mechanism of action of tricyclic antidepressants. Depressed human patients receiving amitriptyline showed higher serum concentrations of BDNF versus control subjects [17]. Following axotomy, BDNF mRNA expression decreases in damaged small neurons but increases in medium to large DRG neurons and their central terminals in the dorsal horn [24]. In the spinal nerve ligation model, BDNF was shown to induce spinal noradrenergic fibre sprouting in the dorsal spinal cord following nerve injury [16]. This mechanism may contribute to the results observed in our experiments. Thus, following nerve injury enhanced BDNF levels may contribute to amitriptyline preventive analgesia by inducing spinal noradrenergic fibre sprouting. This mechanism needs to be elaborated using additional approaches. 4.5. Further characterization of the preventive actions of amitriptyline We further characterized the preventive amitriptyline regimen by examining the potential influences of morphine and caffeine on this action, and by reducing the drug regimen to acute perisurgical exposure (Table 1). Initially, we varied the amitriptyline regimen to determine which component of administration was essential for action. We found that chronic administration of amitriptyline was required for a preventive analgesic effect because amitriptyline given only at the time of surgery did not have any effect on the development of chemogenic hypersensitivity (Table 1A). Chronic, but not acute, exposure to amitriptyline is required to produce a preventive effect on sensory changes. Secondly, as opioids are used routinely to control pain following surgery, examining the effect of morphine on the preventive action of amitriptyline is potentially clinically relevant. However, a 3-day regimen of morphine given post-operatively did not modify the preventive action of amitriptyline (Table 1B). Finally, American adults consume, on average, 193 mg of caffeine per day [15], and, as previous work has shown that chronic caffeine consumption blocks the analgesic effect of amitriptyline in a nerve injury model [12], we determined whether caffeine could also modify the preventive effect of amitriptyline. However, caffeine had no impact on this action (Table 1C). This observation also dissociates the mechanisms by which amitriptyline produces acute and preventive analgesia, as the former involves adenosine (caffeine blocks adenosine A1 and A2 receptors) but the latter does not. Importantly, these experiments demonstrate that two commonly used drugs, morphine and caffeine, do not influence the preventive analgesic actions of amitriptyline, and both observations are of clinical interest given the widespread use of morphine in a post-operative setting, and of caffeine as a part of dietary intake. 4.6. Concluding comments The present results indicate a preventive effect of a regimen of peri- and post-surgical amitriptyline on hypersensitivity and hyposensitivity responses in rats following nerve injury. Tricyclic antidepressants have been used to treat neuropathic pain for decades, and our results indicate that they also may be useful for preventing longterm effects of damage to nerves that can inadvertently result from surgery. There are two clinical reports that indicate a preventive
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