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Dual role of 5-HT3 receptors in a rat model of delayed stress-induced visceral hyperalgesia
Pain 130 (2007) 56–65 www.elsevier.com/locate/pain
Dual role of 5-HT3 receptors in a rat model of delayed stress-induced visceral hyperalgesia q Sylvie Bradesi a,e,f, Lijun Lao a,e,f, Peter G. McLean g, Wendy J. Winchester g, Kevin Lee g, Gareth A. Hicks h, Emeran A. Mayer a,b,c,d,e,* a
b
Center for Neurovisceral Sciences and Women’s Health, Department of Medicine, VAGLAHS CURE Building 115, Room 223, 11301 Wilshire Boulevard, Los Angeles, CA 90073, USA Department of Physiology, Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA c Department of Neurobiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA d Brain Research Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA e CURE: Digestive Diseases Research Center, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA f VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA g GlaxoSmithKline, Neurology and GI Centre of Excellence for Drug Discovery, Harlow, UK h Novartis Pharmaceuticals Corporation, East Hanover, NJ 07936, USA Received 3 August 2006; received in revised form 22 September 2006; accepted 26 October 2006
Abstract Despite its beneficial effect in IBS patients, the mechanism of action of the 5-HT3 receptor (5-HT3R) antagonist alosetron is still incompletely understood. We aimed to characterize the effect and site(s) of action in a model of stress-induced sensitization of visceral nociception in rats. Adult male Wistar rats were equipped for recording of visceromotor response (VMR) to phasic colorectal distension (CRD; 10–60 mm Hg). VMR to CRD was recorded 24 h after an acute session of water avoidance (WA) stress (postWA). Baseline and post-WA responses were measured in rats exposed to WA or sham-WA, treated with alosetron at 0.3 mg/kg subcutaneously (s.c.) 25 nmol intrathecally (i.t.) or vehicle before post-WA CRD. Some rats were treated with capsaicin/vehicle on the cervical vagus nerve and received alosetron (0.3 mg/kg, s.c.) 15 min before post-WA CRD. WA stress led to visceral hyperalgesia 24 h later. Alosetron (0.3 mg/kg, s.c.) failed to inhibit WA-induced exacerbation of VMR to CRD. Stress-induced visceral hyperalgesia was abolished when alosetron was injected intrathecally (P < 0.05) in intact rats or subcutaneously (0.3 mg/kg) in capsaicin-pretreated animals (P < 0.05). Capsaicin-pretreatment did not affect the exacerbating effect of stress on visceral sensitivity. Alosetron had no inhibitory effect on normal visceral pain responses when administered subcutaneously or intrathecally. We demonstrated that 5-HT3Rs on central terminals of spinal afferents are engaged in the facilitatory effect of stress on visceral sensory information processing. In addition, we showed that stress-induced sensitization of visceral nociception is independent of 5-HT3R activation on vagal afferents. 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: 5-HT3 receptors; Visceral nociception; Hyperalgesia; Stress
q
Grant support: This work was supported by GlaxoSmithKline (Neurology and GI Centre of Excellence for Drug Discovery), Harlow, UK, and NIH Grants P50 DK64539 (E.A.M.), R24 AT00281 (E.A.M.). * Corresponding author. Tel.: +1 310 312 9276; fax: +1 310 794 2864. E-mail address: [email protected] (E.A. Mayer).
1. Introduction Irritable bowel syndrome (IBS), a common functional gastrointestinal disorder, is characterized by recurrent abdominal pain and discomfort associated with altered bowel habits (Drossman, 2006). Two selective and
0304-3959/$32.00 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2006.10.028
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potent 5-HT3 receptor (5-HT3R) antagonists have been demonstrated to be effective in providing global symptom relief, as well as a reduction in abdominal pain, in IBS patients with diarrhea (Camilleri et al., 2000; Lembo et al., 2001; Chey and Cash, 2005). However, the mechanism of action of 5-HT3R antagonists in relieving IBS symptoms is incompletely understood. Both pre-clinical and clinical evidence support the concept that the effect of alosetron on motility and secretion is mediated by an action on receptors located within the GI tract, whereas its effect on abdominal pain may involve a centrally (spinal and/or supraspinal) mediated mechanism of action. 5-HT3Rs are expressed by myenteric and submucosal neurons in rats (Glatzle et al., 2002), and on peripheral nerve terminals of both vagal and spinal primary afferent neurons innervating the rat gut (Kidd et al., 1993). 5-HT3Rs have also been described in the spinal cord and throughout the brain (Hamon et al., 1989; Morales et al., 1998; Zeitz et al., 2002; Conte et al., 2005). Recent preclinical evidence is consistent with the involvement of 5-HT3R participation in a spino-bulbospinal reflex loop engaged in the amplification of somatic pain (Suzuki et al., 2004b). It has been proposed that the activation of neurokinin 1 receptors (NK1Rs) containing ascending spinal neurons in the dorsal horn results in an increased engagement of descending serotoninergic systems. 5-HT released from these descending systems activates presynaptic 5-HT3Rs on the central terminals of spinal afferents, thereby increasing the spinal transmission in the entire dorsal horn, resulting in increased pain and reflex responses (Suzuki et al., 2002). Inhibition of these 5-HT3Rs by intrathecal administration of ondansetron prevents the development of chronic pain in this model (Suzuki et al., 2002, 2004a). This mechanism could explain the reported effectiveness of this class of drugs for other conditions characterized by chronic pain and discomfort, such as migraine, fibromyalgia and functional dyspepsia (Riering et al., 2004). The current study aimed to characterize the effect of alosetron on a previously reported stress-induced visceral hyperalgesia model in rats (Schwetz et al., 2004). Similar to the neuropathic pain model referenced above, persistent hyperalgesia in this model is dependent on the increased expression of NK1Rs on spinal neurons (Schwetz et al., 2004; Bradesi et al., 2006). By evaluating the possible involvement of spinal 5-HT3Rs in the effect of alosetron on visceral response to colorectal distension (CRD) in basal and stress conditions, we wanted to test the general hypothesis that 5-HT3Rs are part of a spinal pain amplification system, which is driven by NK1Rs in the spinal dorsal horn. Since 5-HT3Rs are also located on vagal afferents and we have previously demonstrated that vagotomy affects visceromotor responses (VMR) to CRD (Gschossmann et al., 2002), we also wanted to test
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the hypothesis that peripheral 5-HT3Rs on vagal afferents are involved in the modulation of stress-induced visceral hyperalgesia. 2. Methods The study includes three series of experiments as detailed in Section 2.5. Briefly, in a first series of experiments, we tested the effect of peripheral injection of alosetron, or vehicle, on the VMR to CRD following acute WA stress or sham WA. In a second series of experiments, we assessed the effect of spinal injection of alosetron, or vehicle, on VMR to CRD after acute WA stress or sham WA. Finally, the third series of experiments consisted in testing the effect of peripheral injection of alosetron or vehicle, on acute WA stress-induced delayed visceral hyperalgesia in rats previously exposed to vagotomy by perineural capsaicin exposure or controls. 2.1. Animals Adult male Wistar rats (275–300 g) were purchased from Harlan (Indianapolis, IN). Animals were maintained on a normal light–dark cycle, housed in pairs or singly when equipped with a chronic intrathecal catheter. They were provided with food and water ad libitum. All protocols were approved by the Institutional Animal Care and Use Committee at the VA Greater Los Angeles Healthcare System, Los Angeles. 2.2. Surgery 2.2.1. Implantation of electromyographic (EMG) electrodes Adult male rats were deeply anesthetized with sodium pentobarbital (45 mg/kg, Nembutal, Abbott Labs, North Chicago, IL) administered intraperitoneally (i.p.). Electrodes (Teflon-coated stainless steel wire, AstraZeneca, Mo¨lndal, Sweden) were stitched into the external oblique musculature, just superior to the inguinal ligament, for EMG recordings as previously described (Ness and Gebhart, 1988). Electrode leads were then tunneled subcutaneously (s.c.) and externalized laterally for future access. Wounds were closed in layers with 4–0 silk. Following surgery, rats were allowed to recover for 5–7 days. Wounds were tested for tenderness to ensure complete recovery from surgery before testing. 2.2.2. Implantation of chronic intrathecal catheter Rats were deeply anesthetized with sodium pentobarbital (45 mg/kg, Nembutal, Abbott Labs, North Chicago, IL) administered intraperitoneally. Animals were placed in a stereotaxic frame and a small incision was made at the back of the neck. A small puncture was made in the atlanto-occipital membrane of the cisterna magna and a 32 Gauge polyurethane catheter of 8.5 cm (ReCathCo, LLC, PA) was inserted such that the caudal tip reached the lumbar enlargement of the spinal cord. The dead volume of the catheter was 10 lL. The rostral end of the catheter was exteriorized at the top of the head and sutures were used to secure the placement of the catheter and close the wound. The rats were allowed to recover from the surgery for 5 to 7 days. Rats exhibiting any sign of neurological or motor impairment, as evidenced by paralysis, abnormal gait, weight loss, or negligent grooming, were
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excluded from the study. Rats were housed separately to ensure catheter patency. After completion of drug testing, the catheter position was verified in each animal by post-mortem examination of the spinal cord. 2.2.3. Vagotomy: Perineural capsaicin treatment This procedure is described in detail elsewhere (Raybould and Tache´, 1988). Briefly, rats were anesthetized with sodium pentobarbital (60 mg/kg i.p.). The cervical vagi were exposed and the vagal trunks freed from the surrounding tissue. Cotton wool soaked in capsaicin (1 mg/ml) was placed around the nerve trunk for 30 min after which the area was thoroughly rinsed with physiological saline. Animals in which the vagi were treated with vehicle (10% Tween 80 in olive oil) served as vehicle controls. Rats were used in experiments 10–14 days after treatment. 2.3. Assessment of visceromotor response to colorectal distension The visceral stimulus employed was distension of the descending colon and rectum using a well-established and validated method for the evaluation and quantification of visceral nociceptive responses (Ness and Gebhart, 1988). Briefly, under light Halothane anesthesia, a flexible latex balloon (6 cm) was inserted intra-anally (after the distal part of the rectum was gently cleared by massage) such that its end was 1 cm proximal to the anus. Once recovered from anesthesia, animals equipped with the balloon were placed in a Plexiglas cylinder for 30 min before the CRD procedure was initiated. The CRD procedure consisted of two series of phasic distensions to constant pressures of 10, 20, 40, and 60 mm Hg (20-s duration; 4min inter-stimulus interval). The VMR to CRD was quantified by measuring EMG activity in the external oblique musculature. EMG activity was recorded 20 s before (baseline), 20 s during, and 20 s after termination of CRD. The EMG activity was rectified, and the increase in the area under the curve (AUC) of EMG amplitude during CRD over the baseline period before CRD was recorded as the response. In the following, we will use the term EMG referring to the VMR to CRD. Animals showing an EMG signal-to-noise ratio <0.05 were excluded from the study. 2.4. Water avoidance (WA) stress protocol The test apparatus consisted of a Plexiglas tank (25 · 25 · 45 cm) with a block (8 · 8 · 10 cm) affixed to the center of the floor. The tank was filled with fresh room temperature water (25 C) to within 1 cm of the top of the block. The animals were placed on the block for an acute period of 1 h (WA stress, WA). The sham WA stress (sham WA) consisted in placing the rats on the same platform in a waterless container. This well-characterized test represents a potent psychological stressor with large elevations of ACTH and corticosterone within 30 min (Million et al., 1999). 2.5. Experimental design Adult male Wistar rats were used in this study. All the animals were equipped with electrodes in the abdominal muscles for EMG recording. After placement of the electrodes, rats were allowed to recover for 5–7 days. Acclimation to the
experimental conditions was performed for 3 days preceding the start of the experiment. Each day, animals were transported to the testing room, and placed for 30 min in the Plexiglas cylinders used for partial restraint during the CRD experiments. 2.5.1. Effect of peripheral injection of alosetron on acute WA stress-induced delayed visceral hyperalgesia Four groups of rats were used in this series of experiments. On Day 1, a baseline VMR response to CRD was evaluated (CRD#1). Then, rats were exposed to either WA stress or sham WA for 1 h. The VMR response to CRD was recorded again 24 h after the WA or sham WA session on day 2 (CRD#2). Alosetron (0.3 mg/kg, provided by GlaxoSmithKline, Harlow, UK) or vehicle (saline, 1 ml/kg) was injected subcutaneously 15 min before the start of CRD#2 on day 2. The effect of alosetron or vehicle, injected subcutaneously on the VMR response to CRD at day 2, was tested according to the following group distribution: • Two groups of rats were subjected to WA stress and were
injected with either alosetron (0.3 mg/kg, s.c., n = 14) or vehicle (1 ml/kg, s.c., n = 7) before CRD#2. • Two groups of rats were exposed to sham WA stress and injected with either alosetron (0.3 mg/kg, s.c., n = 8) or vehicle (1 ml/kg, s.c., n = 6) before CRD#2. 2.5.2. Effect of spinal injection of alosetron on acute WA stressinduced delayed visceral hyperalgesia Four different groups of rats were used to test the effect of spinal injection of alosetron on VMR to CRD in WA and sham WA rats. At the time of surgical placement of EMG electrodes, animals were equipped with intrathecal catheters implanted chronically. After recovery and acclimation to experimental conditions, a baseline recording of VMR to CRD was performed on day 1. Rats were then exposed to WA or sham WA for 1 h. Twenty-four hours later, rats were intrathecally (i.t.) administered a dose of alosetron (25 nmol in 30 ll) or vehicle (30 ll) via the spinal catheter in place. The catheter was then flushed with vehicle to ensure full distribution of the solution volume. Thirty minutes later, a post- injection measurement of VMR to CRD#2 was performed. Alosetron and vehicle injections were performed according to the following group distribution: • Two groups of rats were subjected to WA stress and were
injected with either alosetron (25 nmol, i.t., n = 10) or vehicle (30 ll, i.t., n = 12) 30 min before CRD#2. • Two groups of rats were exposed to sham WA stress and injected with either alosetron (25 nmol, i.t., n = 8) or vehicle (30 ll, i.t., n = 8) 30 min before CRD#2. 2.5.3. Effect of peripheral injection of alosetron on acute WA stress-induced delayed visceral hyperalgesia in rats previously exposed to vagotomy by perineural capsaicin exposure Four different groups of rats were used in this series of experiments. Two groups were subjected to application of capsaicin on vagal trunks in the cervical region. The two remaining groups were control groups treated with vehicle only (10%
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Tween 80 in olive oil). At the time of treatment with capsaicin or vehicle, rats were equipped with EMG electrodes for further recording of VMR to CRD. After 10 days of recovery and acclimation to the experimental conditions, a baseline VMR to CRD was recorded. Rats were then exposed to WA stress only. Twenty-four hours later, rats were injected with either alosetron (0.3 mg/kg, s.c.) or vehicle (1 ml/kg, s.c.). Measurement of VMR to CRD#2 was performed 15 min after injection. Groups were distributed according to the following: • Two groups were treated with capsaicin and exposed to WA
stress. One group was treated with alosetron (0.3 mg/kg, s.c.), whereas the second group received vehicle (1 ml/kg, s.c.). • Two groups were treated with the control vehicle for capsaicin and exposed to WA stress. One group was treated with alosetron (0.3 mg/kg, s.c.), whereas the second group received vehicle (1 ml/kg, s.c.).
2.6. Data presentation and statistical analysis To examine the pressure-response relationship, EMG amplitudes were normalized as percent of the baseline response for the highest pressure (60 mm Hg) for each rat, and averaged for each group of rats. This type of normalization has generally been used to account for individual variations of the EMG signal (Schwetz et al., 2004). The effect of pharmacological treatments on the EMG response to CRD within one group of animals was analyzed by comparing the post-treatment measurements to the baseline values at each distension pressure using a repeated measures two-way ANOVA followed by Bonferroni post-test comparisons. The analysis of the effect of pharmacological treatment compared with vehicle control (in WA or sham WA groups) was performed by assessing and comparing the mean difference ± standard error of the mean EMG response from baseline in each group using a Sudent’s t test. This method of analysis has been previously validated in a similar model of EMG measurement in response to CRD (Schwetz et al., 2004).
3. Results 3.1. Effect of peripheral injection of alosetron on acute WA stress-induced delayed visceral hyperalgesia Rats exposed to acute WA stress and injected s.c. with vehicle developed a delayed increase of VMR to CRD observed 24 h later (response to CRD#2) compared with baseline (CRD#1). The increase was significant for the 40- and 60-mm Hg pressures (P < 0.05, repeated measures ANOVA followed by Bonferroni post-test; Fig. 1A). Similarly, we observed an increased VMR to CRD 24 h after WA stress in rats treated with alosetron (0.3 mg/kg, s.c.) 15 min before CRD#2. The response was significantly increased from baseline for the 40- (P < 0.05) and 60-mm Hg pressures (P < 0.01; Fig. 1B). The comparison of the mean change from
Fig. 1. Effect of peripheral injection of alosetron on stress-induced visceral hyperalgesia. (A) Rats exposed to acute water avoidance (WA) stress and injected with vehicle exhibit enhanced visceromotor response (VMR) to colorectal distension (CRD) 24 h after WA stress, compared with baseline, for the pressures of 40 and 60 mm Hg (twoway ANOVA followed by Bonferroni post-test, n = 7). (B) Alosetron injected subcutaneously (s.c.) (0.3 mg/kg) fails to inhibit the exacerbated response 24 h post-WA compared with baseline (two-way ANOVA followed by Bonferroni post-test, n = 14). (C) Comparison of the mean change from baseline in both groups revealed no significant effect of alosetron treatment compared with vehicle on stress-induced visceral hyperalgesia (student t-test). Data are means ± SEM.
baseline after vehicle and alosetron treatment 24 h after WA stress demonstrated that subcutaneous alosetron had no effect on the delayed stress-induced increase in the VMR to CRD compared with vehicle (Fig. 1C). In the control sham WA stress condition, the VMR to
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( 5.24 ± 17.78 versus 53.68 ± 36.79; Student’s t test (Fig. 2C).
P = 0.0346),
3.2. Effect of spinal administration of alosetron on acute WA stress-induced delayed visceral hyperalgesia
Fig. 2. Effect of peripheral injection of alosetron on basal visceral response in control sham water avoidance (sham WA) rats. (A) Sham WA rats treated with vehicle exhibit no change in visceromotor response (VMR) to colorectal distension (CRD) compared with baseline (n = 6). (B) Whereas not reaching statistical significance, alosetron injected subcutaneously (s.c.) (0.3 mg/kg) 24 h post-WA tends to exacerbate the visceral response compared with baseline (n = 8). (C) Compared with vehicle, alosetron significantly exacerbates VMR to CRD in the basal sham WA condition for the highest pressure of distension, 60 mm Hg (student t-test). Data are means ± SEM.
CRD observed at 24 h in vehicle-treated rats was not different from the baseline response (Fig. 2A). However, when rats previously exposed to sham WA stress were injected with alosetron (0.3 mg/kg, s.c.) 15 min before the CRD at 24 h, they exhibited a trend for an increased VMR for the 40- and 60-mm Hg pressures (Fig. 2B). When analyzing this effect by comparison of the mean change from baseline after vehicle and alosetron treatments, we identified an augmenting effect of alosetron on the response to the 40- ( 9.26 ± 11.82 versus 38.27 ± 21.63; P = 0.0866) and 60-mm Hg pressures
In order to evaluate a possible role of spinal 5-HT3Rs, presumably located presynaptically on central terminals of DRG neurons (Hamon et al., 1989; Zeitz et al., 2002; Conte et al., 2005), we evaluated the effect of intrathecal application of alosetron on the VMR to CRD. Exposure to acute WA stress resulted in an increased VMR to CRD 24 h later in the group of rats injected intrathecally with vehicle 30 min before CRD#2. The increase in VMR to CRD at 24 h was statistically significant compared with baseline for the 40- (P < 0.05) and 60-mm Hg pressures (P < 0.01) as assessed by repeated measures ANOVA followed by Bonferroni post-test (Fig. 3A). In contrast, intrathecal treatment with alosetron (25 nmol, i.t.) at 24 h postWA stress abolished the stress-induced increase of the VMR to CRD as shown by the lack of difference of the response after WA stress compared with baseline in rats treated with intrathecal alosetron (Fig. 3B). The inhibitory effect of intrathecal alosetron on stressinduced exacerbation of the response at 24 h post-WA stress is further illustrated by the significant reduction of the mean difference from baseline in rats treated with alosetron compared with vehicle. The effect was significant for the 60-mm Hg pressure (P < 0.01) and reached P = 0.0980 for the 40-mm Hg pressure (Student’s t test; Fig. 3C). In the sham WA condition, neither vehicle nor alosetron treatments affected the VMR to CRD at 24 h. There was no change of the VMR to CRD after injection at 24 h compared with baseline in either case (Fig. 4A and B). The lack of effect of alosetron compared with vehicle on the VMR to CRD 24 h after sham WA is illustrated by comparison of the mean change from baseline in Fig. 4C showing no difference between treatments. 3.3. Effect of peripheral injection of alosetron on acute WA stress-induced delayed visceral hyperalgesia in rats previously exposed to vagotomy by perineural capsaicin exposure In order to test the possible involvement of 5-HT3Rs on vagal afferent pathways, we tested the response to peripheral injection of alosetron in rats with afferent vagotomy. Rats were subjected to perivagal capsaicin treatment to obliterate vagal afferent pathways (Raybould and Tache´, 1988) (see Section 2 for details). Afferent vagotomy had no effect on weight or rat behavior. Rats were exposed to acute WA stress 10–14 days following the capsaicin treatment and the effect of
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Fig. 3. Effect of intrathecal (i.t.) injection of alosetron on stressinduced visceral hyperalgesia. (A) Rats exposed to acute water avoidance stress (WA) and injected with vehicle exhibit enhanced visceromotor response (VMR) to colorectal distension (CRD), compared with baseline 24 h later, for the pressures of 40 and 60 mm Hg (two-way ANOVA followed by Bonferroni post-test, n = 12). (B) Intrathecal injection of alosetron in rats exposed to WA completely abolishes visceral hyperalgesia. The VMR post-alosetron injection was similar to baseline (n = 10). (C) Compared with vehicle, alosetron inhibits stress-induced exacerbation of VMR to CRD. The effect of alosetron was significant for the 60 mm Hg pressure (two-way ANOVA followed by Bonferroni post-test). Data are means ± SEM.
vehicle and alosetron (0.3 mg/kg, s.c.) treatments was assessed on the VMR to CRD at 24 h post-WA. In the group treated with vehicle, animals exhibited an enhanced VMR to CRD at the 40- and 60-mm Hg pressures (Fig. 5A). The increase was significant compared to baseline for the 60-mm Hg pressure (P < 0.05). In contrast, stressed rats previously subjected to perivagal
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Fig. 4. Effect of intrathecal (i.t.) injection of alosetron on basal visceral response to CRD in control sham water avoidance (sham WA) rats. (A) Vehicle-treated rats previously exposed to sham WA show unchanged visceromotor response (VMR) to colorectal distension (CRD) post-injection compared with baseline. (B) Intrathecal injection of alosetron did not affect the VMR to CRD compared with baseline. (C) Compared with vehicle, alosetron had no effect on the VMR to CRD in sham WA rats. Data are means ± SEM.
capsaicin application and treated with alosetron (0.3 mg/kg, s.c.) failed to show an increased response to WA stress at 24 h compared with baseline. In this group, rats exhibited a decreased VMR to CRD for the 60-mm Hg pressure only (P < 0.05; Fig. 5B). The effect of alosetron compared to vehicle on the stress response in capsaicin-treated rats was assessed by comparing the mean change from baseline in both groups. Statistical analysis revealed a significant inhibition of the post-WA stress VMR after alosetron treatment compared with vehicle. The difference was significant for the 40- ( 3.83 ± 12.44 versus 35.16 ± 12.87) and 60-mm Hg pressures ( 28.14 ± 10.58 versus 47.45 ± 24.56; Fig. 5C).
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Fig. 5. Effect of subcutaneous (s.c.) injection of alosetron on acute stress-induced visceral hyperalgesia in rats previously exposed to perivagal capsaicin. (A) Water avoidance (WA) rats treated with vehicle and previously exposed to perivagal capsaicin treatment exhibit enhanced visceromotor response (VMR) to colorectal distension (CRD). Increased VMR compared with baseline is significant for the highest pressure of distension, 60 mm Hg (two-way ANOVA followed by Bonferroni post-test, n = 6). (B) Alosetron injection in sham WA rats previously exposed to perivagal capsaicin significantly abolishes stress-induced visceral hyperalgesia for the highest pressure of distension, 60 mm Hg, compared with baseline (two-way ANOVA followed by Bonferroni post-test, n = 7). (C) Compared with vehicle, alosetron inhibits stress-induced exacerbation of VMR to CRD. Comparison of the mean change from baseline revealed a significant inhibitory effect of alosetron, compared with vehicle, for the pressures 40 and 60 mm Hg (two-way ANOVA followed by Bonferroni post-test). Data are means ± SEM.
In the control capsaicin vehicle-treated group, stressed rats injected with vehicle exhibited enhanced VMR to CRD, with a significant increase for the
Fig. 6. Effect of subcutaneous (s.c.) injection of alosetron on acute stress-induced visceral hyperalgesia in rats previously exposed to perivagal vehicle for capsaicin (sham ‘‘vagotomy’’). (A) WA rats treated with vehicle and previously exposed to perivagal vehicle control for capsaicin (10% Tween 80 in olive oil) treatment exhibit enhanced visceromotor response (VMR) to colorectal distension (CRD). Increased VMR compared with baseline is significant for the highest pressure of distension, 60 mm Hg (two-way ANOVA followed by Bonferroni post-test, n = 9). (B) Alosetron injection in WA rats previously exposed to perivagal vehicle for capsaicin failed to inhibit stress-induced visceral hyperalgesia. Increased VMR to CRD was observed for the 40 mm Hg pressure (two-way ANOVA followed by Bonferroni post-test, n = 9). (C) Compared with vehicle, alosetron had no significant effect on VMR to CRD (two-way ANOVA followed by Bonferroni post-test). Data are means ± SEM.
60-mm Hg pressure (P < 0.01; Fig. 6A). Similarly, capsaicin vehicle-treated rats exposed to WA stress and administered alosetron showed increased VMR to CRD compared with baseline. The increase was significant for the 40-mm Hg pressure (P < 0.05; Fig. 6B).
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These data illustrated in Fig. 6 are consistent with the results observed in naı¨ve stressed rats and confirm the lack of effect of the capsaicin vehicle treatment on VMR to CRD. 4. Discussion Using a model of stress sensitization, we demonstrated that alosetron abolishes visceral hyperalgesia without effect on baseline responses. However, this antihyperalgesic effect was only seen when the compound was given intrathecally, or peripherally in the absence of an intact vagal afferent innervation. Viewed together with previous reports (Miura et al., 1999), these findings suggest that the inhibitory effect of alosetron is only seen under conditions of visceral hyperalgesia, and is primarily mediated by 5-HT3Rs on central terminals of spinal afferents. In addition, the findings suggest that the activation of 5-HT3Rs on vagal afferents by 5-HT released from intestinal enterochromaffin cells may play a role in the inhibitory modulation of basal visceral nociception. 4.1. Peripherally administered alosetron does not produce a viscero analgesic or antihyperalgesic effect We tested the effect of peripheral administration of alosetron on both normal response and visceral hyperalgesia in a previously validated model of acute WA stress (Schwetz et al., 2004). Surprisingly, alosetron injected subcutaneously not only failed to reduce visceral hyperalgesia observed 24 h after acute WA stress, but enhanced the response in both groups of animals with and without stress sensitization. This proalgesic effect reached only statistical significance in the control animals. Reported effects of peripherally administered 5-HT3R antagonists on visceral pain responses in rats are inconsistent, but primarily support an antihyperalgesic effect, while no effect on basal pain responses: granisetron and zacopride was found to reduce colonic hypersensitivity in a model of intracolonic acetic acid, whereas ondansetron and tropisetron were found ineffective in the same model (Langlois et al., 1996). Alosetron was shown to inhibit Fos like immunoreactivity in the spinal cord induced by prolonged noxious phasic (80 mm Hg) CRD (Kozlowski et al., 2000). Even though the authors suggested that the effect was mediated by a viscero analgesic effect of alosetron on peripheral nerve terminals of extrinsic primary afferent neurons, the findings were most consistent with an antihyperalgesic effect of peripherally administered alosetron on central sensitization (induced by prolonged, repeated noxious CRD), possibly mediated by 5-HT3R antagonism in the dorsal horn.
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4.2. Role of spinal 5-HT3Rs in the effect of acute WA stress on visceral sensitivity To test the hypothesis that spinal 5-HT3Rs play a role in mediating the stress-induced visceral hyperalgesia in our model, we evaluated the effect of intrathecal alosetron on basal and sensitized visceral pain responses. Intrathecal alosetron inhibited visceral hyperalgesia without affecting basal visceral nociception in control sham WA rats. In the spinal cord, 5-HT3Rs are predominantly localized in the superficial dorsal horn lamina where they are expressed on presynaptic nerve terminals of small-diameter afferents and on a small population of dorsal horn neurons (Morales et al., 1998; Raybould et al., 2003). A series of electrophysiological and behavioral evidence using animal models of peripheral inflammation or injury suggests a pronociceptive function of these presynaptic 5-HT3Rs (Green et al., 2000; Suzuki et al., 2004a) presumably through enhancement of transmitter release from nerve terminals. Recently, Suzuki et al. (2004b) proposed a model of descending serotoninergic facilitatory pathways which increase dorsal horn excitability via 5-HT3Rs. According to this model, the descending pathway is part of a spino-bulbo-spinal pain amplification loop activated in models of peripheral tissue irritation or nerve injury, and driven by lamina I neurons expressing the NK1R. We have previously shown that delayed visceral hyperalgesia observed after a single WA stress session was abolished by spinal application of a NK1R antagonist, while it had no effect on baseline pain responses (Schwetz et al., 2004). In Suzuki’s model it was suggested that while the primary stimulus for upregulation of the spinal pain amplification loop comes from tissue injury or irritation in the periphery, higher corticolimbic inputs to the bulbar side of this loop could modulate the overall degree of pain amplification (Suzuki et al., 2004b). Our results are most consistent with a model in which corticolimbic influences, induced by a brief, mild stressor, can upregulate the NK1R/5-HT3R pain amplification system, without a peripheral tissue irritation. We have recently demonstrated that the exacerbated visceral sensitivity observed in a chronic WA stress model is related to the upregulation of NK1Rs on dorsal horn neurons (Bradesi et al., 2006). In this model, visceral hyperalgesia was abolished by spinal administration of alosetron, whereas such treatment did not affect basal nociception (Bradesi et al., 2005). No significant change in the A and B subunit expression of the 5-HT3R was observed in this model (unpublished observations). Even though we did not investigate gene expression of the NK1 and 5-HT3 receptors in the current study, our findings in the chronic stress model suggest that increased spinal NK1R expression may result in increased 5-HT3R-mediated pain amplification, without any stress-induced changes in 5-HT3R expression itself.
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Future studies will need to confirm this hypothesis in the delayed WA stress model. 4.3. Role of 5-HT3Rs on vagal afferents in stress-induced delayed visceral hyperalgesia The discrepancy between the observed effects with peripheral and spinal application of alosetron may be related to a pronociceptive peripheral effect of alosetron on a subset of pain modulating vagal afferents (Randich and Gebhart, 1992). Sensory information from the gut is conveyed in part by vagal afferents projecting to the brain stem where they make synaptic connections with second order neurons connected to central nervous system structures involved in the descending control of spinal nociceptive transmission. A biphasic response of spinal nociceptive neurons (inhibition and facilitation) involved in the tail flick reflex, depending on the intensity of stimulation, has been described after electrical stimulation of cervical vagal afferents (Ren et al., 1993). Furthermore, 5HT3Rs on vagal afferents have been implicated in the mediation of chemotherapy-induced nausea (Simpson et al., 2000) in the pathophysiology of eating disorders (Washburn et al., 1994). In the current study, we demonstrate that the development of delayed stress-induced visceral hyperalgesia is unaffected by afferent vagotomy arguing against a necessary role of vagal afferents in the modulatory effect of psychological stress on visceral sensitivity. However, the pronociceptive effect of peripherally administered alosetron on VMR to CRD in control animals was no longer observed after vagotomy. In contrast, alosetron under these conditions demonstrated a clear antihyperalgesic effect, similar to the effect seen with intrathecal administration. 4.4. Possible model involving peripheral and central 5HT3Rs in visceral pain modulation Our findings are consistent with the following model: 5-HT3Rs on a subset of vagal afferents and on a subset of central terminals of spinal afferents play opposite roles in the modulation of visceral pain responses in a rat model of stress-induced visceral hyperalgesia. 5-HT3Rs located on vagal afferents are involved in the descending inhibitory modulation of visceral pain responses, which is reduced both in stressed and control rats after peripheral administration of alosetron. The reduction of vagal afferent activity results in an increased nociceptive response to CRD. This hypothesis is supported by a previous report by Gschossmann et al. (2002), demonstrating a significantly enhanced VMR to CRD in rats with afferent vagotomy produced by perineural trunk vagal capsaicin. In that study, the role of opioids in the
antinociceptive pathways engaged by vagal stimulation during visceral stimulation was suggested by the sensitivity of the effect to naloxone treatment. The current data suggest the possible involvement of 5-HT3Rs on vagal afferents in this vagally driven, opioid-dependent antinociceptive system. The fact that alosetron had a significant antihyperalgesic effect when applied intrathecally, or peripherally in animals with afferent vagotomy, suggests that peripherally administered alosetron may act on both vagal afferents and spinal dorsal horn neurons, and that the combined effects cancel each other out. Central nervous system penetration of alosetron, to a certain degree, has been previously documented (unpublished source) and an increased central nervous system penetration of alosetron due to a stress condition cannot be excluded. Stress may increase the blood–brain barrier permeability and may enhance the ability of certain agents to act on central targets when systemically administered (Sharma et al., 1991). Based on the recent evidence for a role of spinal 5-HT3Rs in the spinalbulbo-spinal loop engaged in persistent pain (Suzuki et al., 2004b), we suggest that the net antihyperalgesic effect of alosetron observed in vagotomized rats exposed to stress is mediated by 5-HT3Rs at the spinal cord level. 5. Conclusion The present findings confirm the participation of 5HT3Rs in the modulation of visceral nociceptive responses, contributing to both inhibitory and facilitatory influences on nociceptive response, depending on the anatomical site of activation. We demonstrate that 5HT3Rs on vagal afferents are involved in a tonic inhibitory control of visceral nociception, independent of stress sensitization, whereas 5-HT3Rs on central terminals of spinal afferents are engaged in the facilitatory effect of stress on visceral sensory information processing. These observations may explain the inconsistent results obtained from the testing of alosetron in several animal (Moss and Sanger, 1990; Miura et al., 1999; Kozlowski et al., 2000) and clinical experimental studies (Zerbib et al., 1994; Delvaux et al., 1998). One may speculate that depending on the dose, central nervous system penetration and state of activation of the spino-bulbospinal pain amplification system, the antihyperalgesic effect of 5-HT3Rs may vary considerably in different patient populations.
Acknowledgements The authors thank Miss Teresa Olivas for her invaluable editorial assistance in preparing this manuscript.
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References Bradesi S, Kokkotou E, Simeonidis S, Patierno S, Ennes HS, Mittal Y, et al. The role of neurokinin 1 receptors in the maintenance of visceral hyperalgesia induced by repeated stress in rats. Gastroenterology 2006;130:1729–42. Bradesi S, Ohning G, McLean PG, Winchester WJ, Mayer EA. Role of spinal 5-HT3 receptors in chronic stress-induced visceral hyperalgesia. Gastroenterology 2005;128:A-221. Camilleri M, Northcutt AR, Kong S, Dukes GE, McSorley D, Mangel AW. Efficacy and safety of alosetron in women with irritable bowel syndrome: a randomised placebo-controlled trial. Lancet 2000;355:1035–40. Chey WD, Cash BD. Cilansetron: a new serotonergic agent for the irritable bowel syndrome with diarrhoea. Expert Opin Investig Drugs 2005;14:185–93. Conte D, Legg ED, McCourt AC, Silajdzic E, Nagy GG, Maxwell DJ. Transmitter content, origins and connections of axons in the spinal cord that possess the serotonin (5-hydroxytryptamine) 3 receptor. Neuroscience 2005;134:165–73. Delvaux M, Louvel D, Mamet JP, Campos-Oriola R, Frexinos J. Effect of alosetron on responses to colonic distension in patients with irritable bowel syndrome. Aliment Pharmacol Ther 1998;12:849–55. Drossman DA. The functional gastrointestinal disorders and the Rome III process. Gastroenterology 2006;130:1377–90. Glatzle J, Sternini C, Robin C, Zittel TT, Wong H, Reeve Jr JR, Raybould HE. Expression of 5-HT3 receptors in the rat gastrointestinal tract. Gastroenterology 2002;123:217–26. Green GM, Scarth J, Dickenson A. An excitatory role for 5-HT in spinal inflammatory nociceptive transmission: state-dependent actions via dorsal horn 5-HT(3) receptors in the anaesthetized rat. Pain 2000;89:81–8. Gschossmann JM, Mayer EA, Miller JC, Raybould HE. Subdiaphragmatic vagal afferent innervation in activation of an opioidergic antinociceptive system in response to colorectal distension in rats. Neurogastroenterol Motil 2002;14:403–8. Hamon M, Gallissot MC, Menard F, Gozlan H, Bourgoin S, Verge D. 5-HT3 receptor binding sites are on capsaicin-sensitive fibres in the rat spinal cord. Eur J Pharmacol 1989;164:315–22. Kidd EJ, Laporte AM, Langlois X, Fattaccini CM, Doyen C, Lombard MC, et al. 5-HT3 receptors in the rat central nervous system are mainly located on nerve fibres and terminals. Brain Res 1993;612:289–98. Kozlowski CM, Green A, Grundy D, Boissonade FM, Bountra C. The 5-HT3 receptor antagonist alosetron inhibits the colorectal distention induced depressor response and spinal c-fos expression in the anaesthetised rat. Gut 2000;46:474–80. Langlois A, Pascaud X, Junien JL, Dahl SG, Riviere PJ. Response heterogeneity of 5-HT3 receptor antagonists in a rat visceral hypersensitivity model. Eur J Pharmacol 1996;318:141–4. Lembo T, Wright RA, Bagby B, Decker C, Gordon S, Jhingran P, Carter E. Team Lotronex Investigator. Alosetron controls bowel urgency and provides global symptom improvement in women with diarrhea-predominant irritable bowel syndrome. Am J Gastroenterol 2001;96:2662–70. Million M, Tache´ Y, Anton P. Susceptibility of Lewis and Fisher rats to stress-induced worsening of TNB-colitis: protective role of brain CRF. Am J Physiol Gastrointest Liver Physiol 1999;276:G1027–36.
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Miura M, Lawson DC, Clary EM, Mangel AW, Pappas TN. Central modulation of rectal distension-induced blood pressure changes by alosetron, a 5-HT3 receptor antagonist. Dig Dis Sci 1999;44:20–4. Morales M, Battenberg E, Bloom FE. Distribution of neurons expressing immunoreactivity for the 5-HT3 receptor subtype in the rat brain and spinal cord. J Comp Neurol 1998;402:385–401. Moss HE, Sanger GJ. The effects of granisetron, ICS 205-930 and ondansetron on the visceral pain reflex induced by duodenal distension. Br J Pharmacol 1990;100:497–501. Ness TJ, Gebhart GF. Colorectal distension as a noxious visceral stimulus: physiological and pharmacological characterization of pseudoaffective reflexes in the rat. Brain Res 1988;450:153–69. Randich A, Gebhart GF. Vagal afferent modulation of nociception. Brain Res Brain Res Rev 1992;17:77–99. Raybould HE, Glatzle J, Robin C, Meyer JH, Phan T, Wong H, et al. Expression of 5-HT3 receptors by extrinsic duodenal afferents contribute to intestinal inhibition of gastric emptying. Am J Physiol Gastrointest Liver Physiol 2003;284:G367–72. Raybould HE, Tache´ Y. Cholecystokinin inhibits gastric motility and emptying via a capsaicin-sensitive vagal pathway in rats. Am J Physiol 1988;255:G242–6. Ren K, Zhuo M, Randich A, Gebhart GF. Vagal afferent stimulationproduced effects on nociception in capsaicin-treated rats. J Neurophysiol 1993;69:1530–40. Riering K, Rewerts C, Zieglgansberger W. Analgesic effects of 5-HT3 receptor antagonists. Scand J Rheumatol Suppl 2004:19–23. Schwetz I, Bradesi S, McRoberts JA, Sablad M, Miller JC, Zhou H, et al. Delayed stress-induced colonic hypersensitivity in male Wistar rats: role of neurokinin-1 and corticotropin-releasing factor-1 receptors. Am J Physiol Gastrointest Liver Physiol 2004;286:G683–91. Sharma HS, Cervos-Navarro J, Dey PK. Increased blood–brain barrier permeability following acute short-term swimming exercise in conscious normotensive young rats. Neurosci Res 1991;10:211–21. Simpson K, Spencer CM, McClellan KJ. Tropisetron: an update of its use in the prevention of chemotherapy-induced nausea and vomiting. Drugs 2000;59:1297–315. Suzuki R, Morcuende S, Webber M, Hunt SP, Dickenson AH. Superficial NK1-expressing neurons control spinal excitability through activation of descending pathways. Nat Neurosci 2002;5:1319–26. Suzuki R, Rahman W, Hunt SP, Dickenson AH. Descending facilitatory control of mechanically evoked responses is enhanced in deep dorsal horn neurones following peripheral nerve injury. Brain Res 2004a;1019:68–76. Suzuki R, Rygh LJ, Dickenson AH. Bad news from the brain: descending 5-HT pathways that control spinal pain processing. Trends Pharmacol Sci 2004b;25:613–7. Washburn BS, Jiang JC, Cummings SL, Dixon K, Gietzen DW. Anorectic responses to dietary amino acid imbalance: effects of vagotomy and tropisetron. Am J Physiol 1994;266:R1922–7. Zeitz KP, Guy N, Malmberg AB, Dirajlal S, Martin WJ, Sun L, et al. The 5-HT3 subtype of serotonin receptor contributes to nociceptive processing via a novel subset of myelinated and unmyelinated nociceptors. J Neurosci 2002;22:1010–9. Zerbib F, Bruley des Varannes S, Oriola RC, McDonald J, Isal JP, Galmiche JP. Alosetron does not affect the visceral perception of gastric distension in healthy subjects. Aliment Pharmacol Ther 1994;8:403–7.
Dual role of 5-HT3 receptors in a rat model of delayed stress-induced visceral hyperalgesia q Sylvie Bradesi a,e,f, Lijun Lao a,e,f, Peter G. McLean g, Wendy J. Winchester g, Kevin Lee g, Gareth A. Hicks h, Emeran A. Mayer a,b,c,d,e,* a
b
Center for Neurovisceral Sciences and Women’s Health, Department of Medicine, VAGLAHS CURE Building 115, Room 223, 11301 Wilshire Boulevard, Los Angeles, CA 90073, USA Department of Physiology, Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA c Department of Neurobiology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA d Brain Research Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA e CURE: Digestive Diseases Research Center, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA f VA Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA g GlaxoSmithKline, Neurology and GI Centre of Excellence for Drug Discovery, Harlow, UK h Novartis Pharmaceuticals Corporation, East Hanover, NJ 07936, USA Received 3 August 2006; received in revised form 22 September 2006; accepted 26 October 2006
Abstract Despite its beneficial effect in IBS patients, the mechanism of action of the 5-HT3 receptor (5-HT3R) antagonist alosetron is still incompletely understood. We aimed to characterize the effect and site(s) of action in a model of stress-induced sensitization of visceral nociception in rats. Adult male Wistar rats were equipped for recording of visceromotor response (VMR) to phasic colorectal distension (CRD; 10–60 mm Hg). VMR to CRD was recorded 24 h after an acute session of water avoidance (WA) stress (postWA). Baseline and post-WA responses were measured in rats exposed to WA or sham-WA, treated with alosetron at 0.3 mg/kg subcutaneously (s.c.) 25 nmol intrathecally (i.t.) or vehicle before post-WA CRD. Some rats were treated with capsaicin/vehicle on the cervical vagus nerve and received alosetron (0.3 mg/kg, s.c.) 15 min before post-WA CRD. WA stress led to visceral hyperalgesia 24 h later. Alosetron (0.3 mg/kg, s.c.) failed to inhibit WA-induced exacerbation of VMR to CRD. Stress-induced visceral hyperalgesia was abolished when alosetron was injected intrathecally (P < 0.05) in intact rats or subcutaneously (0.3 mg/kg) in capsaicin-pretreated animals (P < 0.05). Capsaicin-pretreatment did not affect the exacerbating effect of stress on visceral sensitivity. Alosetron had no inhibitory effect on normal visceral pain responses when administered subcutaneously or intrathecally. We demonstrated that 5-HT3Rs on central terminals of spinal afferents are engaged in the facilitatory effect of stress on visceral sensory information processing. In addition, we showed that stress-induced sensitization of visceral nociception is independent of 5-HT3R activation on vagal afferents. 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: 5-HT3 receptors; Visceral nociception; Hyperalgesia; Stress
q
Grant support: This work was supported by GlaxoSmithKline (Neurology and GI Centre of Excellence for Drug Discovery), Harlow, UK, and NIH Grants P50 DK64539 (E.A.M.), R24 AT00281 (E.A.M.). * Corresponding author. Tel.: +1 310 312 9276; fax: +1 310 794 2864. E-mail address: [email protected] (E.A. Mayer).
1. Introduction Irritable bowel syndrome (IBS), a common functional gastrointestinal disorder, is characterized by recurrent abdominal pain and discomfort associated with altered bowel habits (Drossman, 2006). Two selective and
0304-3959/$32.00 2006 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2006.10.028
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potent 5-HT3 receptor (5-HT3R) antagonists have been demonstrated to be effective in providing global symptom relief, as well as a reduction in abdominal pain, in IBS patients with diarrhea (Camilleri et al., 2000; Lembo et al., 2001; Chey and Cash, 2005). However, the mechanism of action of 5-HT3R antagonists in relieving IBS symptoms is incompletely understood. Both pre-clinical and clinical evidence support the concept that the effect of alosetron on motility and secretion is mediated by an action on receptors located within the GI tract, whereas its effect on abdominal pain may involve a centrally (spinal and/or supraspinal) mediated mechanism of action. 5-HT3Rs are expressed by myenteric and submucosal neurons in rats (Glatzle et al., 2002), and on peripheral nerve terminals of both vagal and spinal primary afferent neurons innervating the rat gut (Kidd et al., 1993). 5-HT3Rs have also been described in the spinal cord and throughout the brain (Hamon et al., 1989; Morales et al., 1998; Zeitz et al., 2002; Conte et al., 2005). Recent preclinical evidence is consistent with the involvement of 5-HT3R participation in a spino-bulbospinal reflex loop engaged in the amplification of somatic pain (Suzuki et al., 2004b). It has been proposed that the activation of neurokinin 1 receptors (NK1Rs) containing ascending spinal neurons in the dorsal horn results in an increased engagement of descending serotoninergic systems. 5-HT released from these descending systems activates presynaptic 5-HT3Rs on the central terminals of spinal afferents, thereby increasing the spinal transmission in the entire dorsal horn, resulting in increased pain and reflex responses (Suzuki et al., 2002). Inhibition of these 5-HT3Rs by intrathecal administration of ondansetron prevents the development of chronic pain in this model (Suzuki et al., 2002, 2004a). This mechanism could explain the reported effectiveness of this class of drugs for other conditions characterized by chronic pain and discomfort, such as migraine, fibromyalgia and functional dyspepsia (Riering et al., 2004). The current study aimed to characterize the effect of alosetron on a previously reported stress-induced visceral hyperalgesia model in rats (Schwetz et al., 2004). Similar to the neuropathic pain model referenced above, persistent hyperalgesia in this model is dependent on the increased expression of NK1Rs on spinal neurons (Schwetz et al., 2004; Bradesi et al., 2006). By evaluating the possible involvement of spinal 5-HT3Rs in the effect of alosetron on visceral response to colorectal distension (CRD) in basal and stress conditions, we wanted to test the general hypothesis that 5-HT3Rs are part of a spinal pain amplification system, which is driven by NK1Rs in the spinal dorsal horn. Since 5-HT3Rs are also located on vagal afferents and we have previously demonstrated that vagotomy affects visceromotor responses (VMR) to CRD (Gschossmann et al., 2002), we also wanted to test
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the hypothesis that peripheral 5-HT3Rs on vagal afferents are involved in the modulation of stress-induced visceral hyperalgesia. 2. Methods The study includes three series of experiments as detailed in Section 2.5. Briefly, in a first series of experiments, we tested the effect of peripheral injection of alosetron, or vehicle, on the VMR to CRD following acute WA stress or sham WA. In a second series of experiments, we assessed the effect of spinal injection of alosetron, or vehicle, on VMR to CRD after acute WA stress or sham WA. Finally, the third series of experiments consisted in testing the effect of peripheral injection of alosetron or vehicle, on acute WA stress-induced delayed visceral hyperalgesia in rats previously exposed to vagotomy by perineural capsaicin exposure or controls. 2.1. Animals Adult male Wistar rats (275–300 g) were purchased from Harlan (Indianapolis, IN). Animals were maintained on a normal light–dark cycle, housed in pairs or singly when equipped with a chronic intrathecal catheter. They were provided with food and water ad libitum. All protocols were approved by the Institutional Animal Care and Use Committee at the VA Greater Los Angeles Healthcare System, Los Angeles. 2.2. Surgery 2.2.1. Implantation of electromyographic (EMG) electrodes Adult male rats were deeply anesthetized with sodium pentobarbital (45 mg/kg, Nembutal, Abbott Labs, North Chicago, IL) administered intraperitoneally (i.p.). Electrodes (Teflon-coated stainless steel wire, AstraZeneca, Mo¨lndal, Sweden) were stitched into the external oblique musculature, just superior to the inguinal ligament, for EMG recordings as previously described (Ness and Gebhart, 1988). Electrode leads were then tunneled subcutaneously (s.c.) and externalized laterally for future access. Wounds were closed in layers with 4–0 silk. Following surgery, rats were allowed to recover for 5–7 days. Wounds were tested for tenderness to ensure complete recovery from surgery before testing. 2.2.2. Implantation of chronic intrathecal catheter Rats were deeply anesthetized with sodium pentobarbital (45 mg/kg, Nembutal, Abbott Labs, North Chicago, IL) administered intraperitoneally. Animals were placed in a stereotaxic frame and a small incision was made at the back of the neck. A small puncture was made in the atlanto-occipital membrane of the cisterna magna and a 32 Gauge polyurethane catheter of 8.5 cm (ReCathCo, LLC, PA) was inserted such that the caudal tip reached the lumbar enlargement of the spinal cord. The dead volume of the catheter was 10 lL. The rostral end of the catheter was exteriorized at the top of the head and sutures were used to secure the placement of the catheter and close the wound. The rats were allowed to recover from the surgery for 5 to 7 days. Rats exhibiting any sign of neurological or motor impairment, as evidenced by paralysis, abnormal gait, weight loss, or negligent grooming, were
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excluded from the study. Rats were housed separately to ensure catheter patency. After completion of drug testing, the catheter position was verified in each animal by post-mortem examination of the spinal cord. 2.2.3. Vagotomy: Perineural capsaicin treatment This procedure is described in detail elsewhere (Raybould and Tache´, 1988). Briefly, rats were anesthetized with sodium pentobarbital (60 mg/kg i.p.). The cervical vagi were exposed and the vagal trunks freed from the surrounding tissue. Cotton wool soaked in capsaicin (1 mg/ml) was placed around the nerve trunk for 30 min after which the area was thoroughly rinsed with physiological saline. Animals in which the vagi were treated with vehicle (10% Tween 80 in olive oil) served as vehicle controls. Rats were used in experiments 10–14 days after treatment. 2.3. Assessment of visceromotor response to colorectal distension The visceral stimulus employed was distension of the descending colon and rectum using a well-established and validated method for the evaluation and quantification of visceral nociceptive responses (Ness and Gebhart, 1988). Briefly, under light Halothane anesthesia, a flexible latex balloon (6 cm) was inserted intra-anally (after the distal part of the rectum was gently cleared by massage) such that its end was 1 cm proximal to the anus. Once recovered from anesthesia, animals equipped with the balloon were placed in a Plexiglas cylinder for 30 min before the CRD procedure was initiated. The CRD procedure consisted of two series of phasic distensions to constant pressures of 10, 20, 40, and 60 mm Hg (20-s duration; 4min inter-stimulus interval). The VMR to CRD was quantified by measuring EMG activity in the external oblique musculature. EMG activity was recorded 20 s before (baseline), 20 s during, and 20 s after termination of CRD. The EMG activity was rectified, and the increase in the area under the curve (AUC) of EMG amplitude during CRD over the baseline period before CRD was recorded as the response. In the following, we will use the term EMG referring to the VMR to CRD. Animals showing an EMG signal-to-noise ratio <0.05 were excluded from the study. 2.4. Water avoidance (WA) stress protocol The test apparatus consisted of a Plexiglas tank (25 · 25 · 45 cm) with a block (8 · 8 · 10 cm) affixed to the center of the floor. The tank was filled with fresh room temperature water (25 C) to within 1 cm of the top of the block. The animals were placed on the block for an acute period of 1 h (WA stress, WA). The sham WA stress (sham WA) consisted in placing the rats on the same platform in a waterless container. This well-characterized test represents a potent psychological stressor with large elevations of ACTH and corticosterone within 30 min (Million et al., 1999). 2.5. Experimental design Adult male Wistar rats were used in this study. All the animals were equipped with electrodes in the abdominal muscles for EMG recording. After placement of the electrodes, rats were allowed to recover for 5–7 days. Acclimation to the
experimental conditions was performed for 3 days preceding the start of the experiment. Each day, animals were transported to the testing room, and placed for 30 min in the Plexiglas cylinders used for partial restraint during the CRD experiments. 2.5.1. Effect of peripheral injection of alosetron on acute WA stress-induced delayed visceral hyperalgesia Four groups of rats were used in this series of experiments. On Day 1, a baseline VMR response to CRD was evaluated (CRD#1). Then, rats were exposed to either WA stress or sham WA for 1 h. The VMR response to CRD was recorded again 24 h after the WA or sham WA session on day 2 (CRD#2). Alosetron (0.3 mg/kg, provided by GlaxoSmithKline, Harlow, UK) or vehicle (saline, 1 ml/kg) was injected subcutaneously 15 min before the start of CRD#2 on day 2. The effect of alosetron or vehicle, injected subcutaneously on the VMR response to CRD at day 2, was tested according to the following group distribution: • Two groups of rats were subjected to WA stress and were
injected with either alosetron (0.3 mg/kg, s.c., n = 14) or vehicle (1 ml/kg, s.c., n = 7) before CRD#2. • Two groups of rats were exposed to sham WA stress and injected with either alosetron (0.3 mg/kg, s.c., n = 8) or vehicle (1 ml/kg, s.c., n = 6) before CRD#2. 2.5.2. Effect of spinal injection of alosetron on acute WA stressinduced delayed visceral hyperalgesia Four different groups of rats were used to test the effect of spinal injection of alosetron on VMR to CRD in WA and sham WA rats. At the time of surgical placement of EMG electrodes, animals were equipped with intrathecal catheters implanted chronically. After recovery and acclimation to experimental conditions, a baseline recording of VMR to CRD was performed on day 1. Rats were then exposed to WA or sham WA for 1 h. Twenty-four hours later, rats were intrathecally (i.t.) administered a dose of alosetron (25 nmol in 30 ll) or vehicle (30 ll) via the spinal catheter in place. The catheter was then flushed with vehicle to ensure full distribution of the solution volume. Thirty minutes later, a post- injection measurement of VMR to CRD#2 was performed. Alosetron and vehicle injections were performed according to the following group distribution: • Two groups of rats were subjected to WA stress and were
injected with either alosetron (25 nmol, i.t., n = 10) or vehicle (30 ll, i.t., n = 12) 30 min before CRD#2. • Two groups of rats were exposed to sham WA stress and injected with either alosetron (25 nmol, i.t., n = 8) or vehicle (30 ll, i.t., n = 8) 30 min before CRD#2. 2.5.3. Effect of peripheral injection of alosetron on acute WA stress-induced delayed visceral hyperalgesia in rats previously exposed to vagotomy by perineural capsaicin exposure Four different groups of rats were used in this series of experiments. Two groups were subjected to application of capsaicin on vagal trunks in the cervical region. The two remaining groups were control groups treated with vehicle only (10%
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Tween 80 in olive oil). At the time of treatment with capsaicin or vehicle, rats were equipped with EMG electrodes for further recording of VMR to CRD. After 10 days of recovery and acclimation to the experimental conditions, a baseline VMR to CRD was recorded. Rats were then exposed to WA stress only. Twenty-four hours later, rats were injected with either alosetron (0.3 mg/kg, s.c.) or vehicle (1 ml/kg, s.c.). Measurement of VMR to CRD#2 was performed 15 min after injection. Groups were distributed according to the following: • Two groups were treated with capsaicin and exposed to WA
stress. One group was treated with alosetron (0.3 mg/kg, s.c.), whereas the second group received vehicle (1 ml/kg, s.c.). • Two groups were treated with the control vehicle for capsaicin and exposed to WA stress. One group was treated with alosetron (0.3 mg/kg, s.c.), whereas the second group received vehicle (1 ml/kg, s.c.).
2.6. Data presentation and statistical analysis To examine the pressure-response relationship, EMG amplitudes were normalized as percent of the baseline response for the highest pressure (60 mm Hg) for each rat, and averaged for each group of rats. This type of normalization has generally been used to account for individual variations of the EMG signal (Schwetz et al., 2004). The effect of pharmacological treatments on the EMG response to CRD within one group of animals was analyzed by comparing the post-treatment measurements to the baseline values at each distension pressure using a repeated measures two-way ANOVA followed by Bonferroni post-test comparisons. The analysis of the effect of pharmacological treatment compared with vehicle control (in WA or sham WA groups) was performed by assessing and comparing the mean difference ± standard error of the mean EMG response from baseline in each group using a Sudent’s t test. This method of analysis has been previously validated in a similar model of EMG measurement in response to CRD (Schwetz et al., 2004).
3. Results 3.1. Effect of peripheral injection of alosetron on acute WA stress-induced delayed visceral hyperalgesia Rats exposed to acute WA stress and injected s.c. with vehicle developed a delayed increase of VMR to CRD observed 24 h later (response to CRD#2) compared with baseline (CRD#1). The increase was significant for the 40- and 60-mm Hg pressures (P < 0.05, repeated measures ANOVA followed by Bonferroni post-test; Fig. 1A). Similarly, we observed an increased VMR to CRD 24 h after WA stress in rats treated with alosetron (0.3 mg/kg, s.c.) 15 min before CRD#2. The response was significantly increased from baseline for the 40- (P < 0.05) and 60-mm Hg pressures (P < 0.01; Fig. 1B). The comparison of the mean change from
Fig. 1. Effect of peripheral injection of alosetron on stress-induced visceral hyperalgesia. (A) Rats exposed to acute water avoidance (WA) stress and injected with vehicle exhibit enhanced visceromotor response (VMR) to colorectal distension (CRD) 24 h after WA stress, compared with baseline, for the pressures of 40 and 60 mm Hg (twoway ANOVA followed by Bonferroni post-test, n = 7). (B) Alosetron injected subcutaneously (s.c.) (0.3 mg/kg) fails to inhibit the exacerbated response 24 h post-WA compared with baseline (two-way ANOVA followed by Bonferroni post-test, n = 14). (C) Comparison of the mean change from baseline in both groups revealed no significant effect of alosetron treatment compared with vehicle on stress-induced visceral hyperalgesia (student t-test). Data are means ± SEM.
baseline after vehicle and alosetron treatment 24 h after WA stress demonstrated that subcutaneous alosetron had no effect on the delayed stress-induced increase in the VMR to CRD compared with vehicle (Fig. 1C). In the control sham WA stress condition, the VMR to
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( 5.24 ± 17.78 versus 53.68 ± 36.79; Student’s t test (Fig. 2C).
P = 0.0346),
3.2. Effect of spinal administration of alosetron on acute WA stress-induced delayed visceral hyperalgesia
Fig. 2. Effect of peripheral injection of alosetron on basal visceral response in control sham water avoidance (sham WA) rats. (A) Sham WA rats treated with vehicle exhibit no change in visceromotor response (VMR) to colorectal distension (CRD) compared with baseline (n = 6). (B) Whereas not reaching statistical significance, alosetron injected subcutaneously (s.c.) (0.3 mg/kg) 24 h post-WA tends to exacerbate the visceral response compared with baseline (n = 8). (C) Compared with vehicle, alosetron significantly exacerbates VMR to CRD in the basal sham WA condition for the highest pressure of distension, 60 mm Hg (student t-test). Data are means ± SEM.
CRD observed at 24 h in vehicle-treated rats was not different from the baseline response (Fig. 2A). However, when rats previously exposed to sham WA stress were injected with alosetron (0.3 mg/kg, s.c.) 15 min before the CRD at 24 h, they exhibited a trend for an increased VMR for the 40- and 60-mm Hg pressures (Fig. 2B). When analyzing this effect by comparison of the mean change from baseline after vehicle and alosetron treatments, we identified an augmenting effect of alosetron on the response to the 40- ( 9.26 ± 11.82 versus 38.27 ± 21.63; P = 0.0866) and 60-mm Hg pressures
In order to evaluate a possible role of spinal 5-HT3Rs, presumably located presynaptically on central terminals of DRG neurons (Hamon et al., 1989; Zeitz et al., 2002; Conte et al., 2005), we evaluated the effect of intrathecal application of alosetron on the VMR to CRD. Exposure to acute WA stress resulted in an increased VMR to CRD 24 h later in the group of rats injected intrathecally with vehicle 30 min before CRD#2. The increase in VMR to CRD at 24 h was statistically significant compared with baseline for the 40- (P < 0.05) and 60-mm Hg pressures (P < 0.01) as assessed by repeated measures ANOVA followed by Bonferroni post-test (Fig. 3A). In contrast, intrathecal treatment with alosetron (25 nmol, i.t.) at 24 h postWA stress abolished the stress-induced increase of the VMR to CRD as shown by the lack of difference of the response after WA stress compared with baseline in rats treated with intrathecal alosetron (Fig. 3B). The inhibitory effect of intrathecal alosetron on stressinduced exacerbation of the response at 24 h post-WA stress is further illustrated by the significant reduction of the mean difference from baseline in rats treated with alosetron compared with vehicle. The effect was significant for the 60-mm Hg pressure (P < 0.01) and reached P = 0.0980 for the 40-mm Hg pressure (Student’s t test; Fig. 3C). In the sham WA condition, neither vehicle nor alosetron treatments affected the VMR to CRD at 24 h. There was no change of the VMR to CRD after injection at 24 h compared with baseline in either case (Fig. 4A and B). The lack of effect of alosetron compared with vehicle on the VMR to CRD 24 h after sham WA is illustrated by comparison of the mean change from baseline in Fig. 4C showing no difference between treatments. 3.3. Effect of peripheral injection of alosetron on acute WA stress-induced delayed visceral hyperalgesia in rats previously exposed to vagotomy by perineural capsaicin exposure In order to test the possible involvement of 5-HT3Rs on vagal afferent pathways, we tested the response to peripheral injection of alosetron in rats with afferent vagotomy. Rats were subjected to perivagal capsaicin treatment to obliterate vagal afferent pathways (Raybould and Tache´, 1988) (see Section 2 for details). Afferent vagotomy had no effect on weight or rat behavior. Rats were exposed to acute WA stress 10–14 days following the capsaicin treatment and the effect of
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Fig. 3. Effect of intrathecal (i.t.) injection of alosetron on stressinduced visceral hyperalgesia. (A) Rats exposed to acute water avoidance stress (WA) and injected with vehicle exhibit enhanced visceromotor response (VMR) to colorectal distension (CRD), compared with baseline 24 h later, for the pressures of 40 and 60 mm Hg (two-way ANOVA followed by Bonferroni post-test, n = 12). (B) Intrathecal injection of alosetron in rats exposed to WA completely abolishes visceral hyperalgesia. The VMR post-alosetron injection was similar to baseline (n = 10). (C) Compared with vehicle, alosetron inhibits stress-induced exacerbation of VMR to CRD. The effect of alosetron was significant for the 60 mm Hg pressure (two-way ANOVA followed by Bonferroni post-test). Data are means ± SEM.
vehicle and alosetron (0.3 mg/kg, s.c.) treatments was assessed on the VMR to CRD at 24 h post-WA. In the group treated with vehicle, animals exhibited an enhanced VMR to CRD at the 40- and 60-mm Hg pressures (Fig. 5A). The increase was significant compared to baseline for the 60-mm Hg pressure (P < 0.05). In contrast, stressed rats previously subjected to perivagal
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Fig. 4. Effect of intrathecal (i.t.) injection of alosetron on basal visceral response to CRD in control sham water avoidance (sham WA) rats. (A) Vehicle-treated rats previously exposed to sham WA show unchanged visceromotor response (VMR) to colorectal distension (CRD) post-injection compared with baseline. (B) Intrathecal injection of alosetron did not affect the VMR to CRD compared with baseline. (C) Compared with vehicle, alosetron had no effect on the VMR to CRD in sham WA rats. Data are means ± SEM.
capsaicin application and treated with alosetron (0.3 mg/kg, s.c.) failed to show an increased response to WA stress at 24 h compared with baseline. In this group, rats exhibited a decreased VMR to CRD for the 60-mm Hg pressure only (P < 0.05; Fig. 5B). The effect of alosetron compared to vehicle on the stress response in capsaicin-treated rats was assessed by comparing the mean change from baseline in both groups. Statistical analysis revealed a significant inhibition of the post-WA stress VMR after alosetron treatment compared with vehicle. The difference was significant for the 40- ( 3.83 ± 12.44 versus 35.16 ± 12.87) and 60-mm Hg pressures ( 28.14 ± 10.58 versus 47.45 ± 24.56; Fig. 5C).
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Fig. 5. Effect of subcutaneous (s.c.) injection of alosetron on acute stress-induced visceral hyperalgesia in rats previously exposed to perivagal capsaicin. (A) Water avoidance (WA) rats treated with vehicle and previously exposed to perivagal capsaicin treatment exhibit enhanced visceromotor response (VMR) to colorectal distension (CRD). Increased VMR compared with baseline is significant for the highest pressure of distension, 60 mm Hg (two-way ANOVA followed by Bonferroni post-test, n = 6). (B) Alosetron injection in sham WA rats previously exposed to perivagal capsaicin significantly abolishes stress-induced visceral hyperalgesia for the highest pressure of distension, 60 mm Hg, compared with baseline (two-way ANOVA followed by Bonferroni post-test, n = 7). (C) Compared with vehicle, alosetron inhibits stress-induced exacerbation of VMR to CRD. Comparison of the mean change from baseline revealed a significant inhibitory effect of alosetron, compared with vehicle, for the pressures 40 and 60 mm Hg (two-way ANOVA followed by Bonferroni post-test). Data are means ± SEM.
In the control capsaicin vehicle-treated group, stressed rats injected with vehicle exhibited enhanced VMR to CRD, with a significant increase for the
Fig. 6. Effect of subcutaneous (s.c.) injection of alosetron on acute stress-induced visceral hyperalgesia in rats previously exposed to perivagal vehicle for capsaicin (sham ‘‘vagotomy’’). (A) WA rats treated with vehicle and previously exposed to perivagal vehicle control for capsaicin (10% Tween 80 in olive oil) treatment exhibit enhanced visceromotor response (VMR) to colorectal distension (CRD). Increased VMR compared with baseline is significant for the highest pressure of distension, 60 mm Hg (two-way ANOVA followed by Bonferroni post-test, n = 9). (B) Alosetron injection in WA rats previously exposed to perivagal vehicle for capsaicin failed to inhibit stress-induced visceral hyperalgesia. Increased VMR to CRD was observed for the 40 mm Hg pressure (two-way ANOVA followed by Bonferroni post-test, n = 9). (C) Compared with vehicle, alosetron had no significant effect on VMR to CRD (two-way ANOVA followed by Bonferroni post-test). Data are means ± SEM.
60-mm Hg pressure (P < 0.01; Fig. 6A). Similarly, capsaicin vehicle-treated rats exposed to WA stress and administered alosetron showed increased VMR to CRD compared with baseline. The increase was significant for the 40-mm Hg pressure (P < 0.05; Fig. 6B).
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These data illustrated in Fig. 6 are consistent with the results observed in naı¨ve stressed rats and confirm the lack of effect of the capsaicin vehicle treatment on VMR to CRD. 4. Discussion Using a model of stress sensitization, we demonstrated that alosetron abolishes visceral hyperalgesia without effect on baseline responses. However, this antihyperalgesic effect was only seen when the compound was given intrathecally, or peripherally in the absence of an intact vagal afferent innervation. Viewed together with previous reports (Miura et al., 1999), these findings suggest that the inhibitory effect of alosetron is only seen under conditions of visceral hyperalgesia, and is primarily mediated by 5-HT3Rs on central terminals of spinal afferents. In addition, the findings suggest that the activation of 5-HT3Rs on vagal afferents by 5-HT released from intestinal enterochromaffin cells may play a role in the inhibitory modulation of basal visceral nociception. 4.1. Peripherally administered alosetron does not produce a viscero analgesic or antihyperalgesic effect We tested the effect of peripheral administration of alosetron on both normal response and visceral hyperalgesia in a previously validated model of acute WA stress (Schwetz et al., 2004). Surprisingly, alosetron injected subcutaneously not only failed to reduce visceral hyperalgesia observed 24 h after acute WA stress, but enhanced the response in both groups of animals with and without stress sensitization. This proalgesic effect reached only statistical significance in the control animals. Reported effects of peripherally administered 5-HT3R antagonists on visceral pain responses in rats are inconsistent, but primarily support an antihyperalgesic effect, while no effect on basal pain responses: granisetron and zacopride was found to reduce colonic hypersensitivity in a model of intracolonic acetic acid, whereas ondansetron and tropisetron were found ineffective in the same model (Langlois et al., 1996). Alosetron was shown to inhibit Fos like immunoreactivity in the spinal cord induced by prolonged noxious phasic (80 mm Hg) CRD (Kozlowski et al., 2000). Even though the authors suggested that the effect was mediated by a viscero analgesic effect of alosetron on peripheral nerve terminals of extrinsic primary afferent neurons, the findings were most consistent with an antihyperalgesic effect of peripherally administered alosetron on central sensitization (induced by prolonged, repeated noxious CRD), possibly mediated by 5-HT3R antagonism in the dorsal horn.
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4.2. Role of spinal 5-HT3Rs in the effect of acute WA stress on visceral sensitivity To test the hypothesis that spinal 5-HT3Rs play a role in mediating the stress-induced visceral hyperalgesia in our model, we evaluated the effect of intrathecal alosetron on basal and sensitized visceral pain responses. Intrathecal alosetron inhibited visceral hyperalgesia without affecting basal visceral nociception in control sham WA rats. In the spinal cord, 5-HT3Rs are predominantly localized in the superficial dorsal horn lamina where they are expressed on presynaptic nerve terminals of small-diameter afferents and on a small population of dorsal horn neurons (Morales et al., 1998; Raybould et al., 2003). A series of electrophysiological and behavioral evidence using animal models of peripheral inflammation or injury suggests a pronociceptive function of these presynaptic 5-HT3Rs (Green et al., 2000; Suzuki et al., 2004a) presumably through enhancement of transmitter release from nerve terminals. Recently, Suzuki et al. (2004b) proposed a model of descending serotoninergic facilitatory pathways which increase dorsal horn excitability via 5-HT3Rs. According to this model, the descending pathway is part of a spino-bulbo-spinal pain amplification loop activated in models of peripheral tissue irritation or nerve injury, and driven by lamina I neurons expressing the NK1R. We have previously shown that delayed visceral hyperalgesia observed after a single WA stress session was abolished by spinal application of a NK1R antagonist, while it had no effect on baseline pain responses (Schwetz et al., 2004). In Suzuki’s model it was suggested that while the primary stimulus for upregulation of the spinal pain amplification loop comes from tissue injury or irritation in the periphery, higher corticolimbic inputs to the bulbar side of this loop could modulate the overall degree of pain amplification (Suzuki et al., 2004b). Our results are most consistent with a model in which corticolimbic influences, induced by a brief, mild stressor, can upregulate the NK1R/5-HT3R pain amplification system, without a peripheral tissue irritation. We have recently demonstrated that the exacerbated visceral sensitivity observed in a chronic WA stress model is related to the upregulation of NK1Rs on dorsal horn neurons (Bradesi et al., 2006). In this model, visceral hyperalgesia was abolished by spinal administration of alosetron, whereas such treatment did not affect basal nociception (Bradesi et al., 2005). No significant change in the A and B subunit expression of the 5-HT3R was observed in this model (unpublished observations). Even though we did not investigate gene expression of the NK1 and 5-HT3 receptors in the current study, our findings in the chronic stress model suggest that increased spinal NK1R expression may result in increased 5-HT3R-mediated pain amplification, without any stress-induced changes in 5-HT3R expression itself.
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Future studies will need to confirm this hypothesis in the delayed WA stress model. 4.3. Role of 5-HT3Rs on vagal afferents in stress-induced delayed visceral hyperalgesia The discrepancy between the observed effects with peripheral and spinal application of alosetron may be related to a pronociceptive peripheral effect of alosetron on a subset of pain modulating vagal afferents (Randich and Gebhart, 1992). Sensory information from the gut is conveyed in part by vagal afferents projecting to the brain stem where they make synaptic connections with second order neurons connected to central nervous system structures involved in the descending control of spinal nociceptive transmission. A biphasic response of spinal nociceptive neurons (inhibition and facilitation) involved in the tail flick reflex, depending on the intensity of stimulation, has been described after electrical stimulation of cervical vagal afferents (Ren et al., 1993). Furthermore, 5HT3Rs on vagal afferents have been implicated in the mediation of chemotherapy-induced nausea (Simpson et al., 2000) in the pathophysiology of eating disorders (Washburn et al., 1994). In the current study, we demonstrate that the development of delayed stress-induced visceral hyperalgesia is unaffected by afferent vagotomy arguing against a necessary role of vagal afferents in the modulatory effect of psychological stress on visceral sensitivity. However, the pronociceptive effect of peripherally administered alosetron on VMR to CRD in control animals was no longer observed after vagotomy. In contrast, alosetron under these conditions demonstrated a clear antihyperalgesic effect, similar to the effect seen with intrathecal administration. 4.4. Possible model involving peripheral and central 5HT3Rs in visceral pain modulation Our findings are consistent with the following model: 5-HT3Rs on a subset of vagal afferents and on a subset of central terminals of spinal afferents play opposite roles in the modulation of visceral pain responses in a rat model of stress-induced visceral hyperalgesia. 5-HT3Rs located on vagal afferents are involved in the descending inhibitory modulation of visceral pain responses, which is reduced both in stressed and control rats after peripheral administration of alosetron. The reduction of vagal afferent activity results in an increased nociceptive response to CRD. This hypothesis is supported by a previous report by Gschossmann et al. (2002), demonstrating a significantly enhanced VMR to CRD in rats with afferent vagotomy produced by perineural trunk vagal capsaicin. In that study, the role of opioids in the
antinociceptive pathways engaged by vagal stimulation during visceral stimulation was suggested by the sensitivity of the effect to naloxone treatment. The current data suggest the possible involvement of 5-HT3Rs on vagal afferents in this vagally driven, opioid-dependent antinociceptive system. The fact that alosetron had a significant antihyperalgesic effect when applied intrathecally, or peripherally in animals with afferent vagotomy, suggests that peripherally administered alosetron may act on both vagal afferents and spinal dorsal horn neurons, and that the combined effects cancel each other out. Central nervous system penetration of alosetron, to a certain degree, has been previously documented (unpublished source) and an increased central nervous system penetration of alosetron due to a stress condition cannot be excluded. Stress may increase the blood–brain barrier permeability and may enhance the ability of certain agents to act on central targets when systemically administered (Sharma et al., 1991). Based on the recent evidence for a role of spinal 5-HT3Rs in the spinalbulbo-spinal loop engaged in persistent pain (Suzuki et al., 2004b), we suggest that the net antihyperalgesic effect of alosetron observed in vagotomized rats exposed to stress is mediated by 5-HT3Rs at the spinal cord level. 5. Conclusion The present findings confirm the participation of 5HT3Rs in the modulation of visceral nociceptive responses, contributing to both inhibitory and facilitatory influences on nociceptive response, depending on the anatomical site of activation. We demonstrate that 5HT3Rs on vagal afferents are involved in a tonic inhibitory control of visceral nociception, independent of stress sensitization, whereas 5-HT3Rs on central terminals of spinal afferents are engaged in the facilitatory effect of stress on visceral sensory information processing. These observations may explain the inconsistent results obtained from the testing of alosetron in several animal (Moss and Sanger, 1990; Miura et al., 1999; Kozlowski et al., 2000) and clinical experimental studies (Zerbib et al., 1994; Delvaux et al., 1998). One may speculate that depending on the dose, central nervous system penetration and state of activation of the spino-bulbospinal pain amplification system, the antihyperalgesic effect of 5-HT3Rs may vary considerably in different patient populations.
Acknowledgements The authors thank Miss Teresa Olivas for her invaluable editorial assistance in preparing this manuscript.
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