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PAIN MEDICINE

The Monoamine Reuptake Inhibitor Milnacipran Does Not Affect Nociception to Acute Visceral Distension in Rats

Sang-Wook Shin, MD^*, James C. Eisenach, MD^*, Srinias G. Rao, MD PhD, and Chuanyao Tong, MD^*

*Department of Anesthesiology, Wake Forest University School of Medicine, Winston-Salem, North Carolina; and Cypress Bioscience Inc., San Diego, California

Address correspondence to Chuanyao Tong, MD, Department of Anesthesiology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Address e-mail to ctong{at}wfubmc.edu No reprints will be available from the authors.

	Abstract

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The role of antidepressants in the treatment of visceral pain has not been extensively examined. Milnacipran, an antidepressant that inhibits monoamine reuptake, is widely used in the treatment of depression and fibromyalgia. In this study, we sought to determine the activity of milnacipran against acute visceral nociception. Female virgin rats were studied 7 days after bilateral ovariectomy. For uterine cervical distension (UCD), two metal rods were inserted into the cervical osses under general anesthesia for manual distension. Colorectal distension (CRD) was performed by insertion of a balloon catheter into the descending colon and rectum, followed by manual inflation. Two electrodes were inserted into the rectus abdominus muscle for recording UCD- or CRD-induced reflex contraction, which was quantified by electromyography (EMG). A dose response for milnacipran, administered intrathecally or IV, was obtained for UCD and CRD stimulation. Milnacipran failed to inhibit the UCD-induced EMG response, whether administered IV or intrathecally. Similarly, IV milnacipran, administered either acutely or chronically, failed to inhibit the CRD-induced EMG response. CRD and UCD are well established animal models for the study of acute visceral pain. Milnacipran, although it provides some unique advantages compared with other antidepressants, is unlikely to produce analgesia after acute administration in the setting of acute visceral pain.

IMPLICATIONS: Neither intrathecal nor IV milnacipran, a monoamine reuptake inhibitor, inhibits an acute visceral pain response induced by colorectal or uterine cervical distension.

	Introduction

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Chronic visceral pain occurs often but is often difficult to treat. Patients with irritable bowel syndrome exhibit increased sensitivity to gastrointestinal tract distension, although there is no evidence in these patients of significant tissue pathology (1–5). Chronic pelvic pain in women is common, but its etiology is multifactorial and its response to traditional treatment unpredictable. Pain in these women is often severe, and even invasive treatment such as hysterectomy often fails to eliminate the pain (6,7).

Antidepressant drugs are often administered either alone or with other traditional analgesics to treat chronic pain. The pharmacologic mechanism for their action is not clear but has been postulated to involve inhibition of norepinephrine and serotonin reuptake, especially the former. Tricyclic antidepressants (TCAs) such as amitriptyline also block N-methyl-D-aspartate receptors and sodium channels (8–11), and this could contribute to their efficacy. TCAs, especially those that inhibit norepinephrine reuptake, have been well documented to improve chronic pain relief and are well accepted by both physicians and patients.

In contrast to this experience with chronic pain, systemic and intrathecal (IT) injection of amitriptyline fails to inhibit responses to acute somatic nociception in healthy animals, although it potentiates other analgesics (12,13). There are many differences in physiology and antinociception between somatic and visceral pain; the latter has only recently received much attention, and the effect of monoamine reuptake inhibitors on acute visceral pain has not previously been examined. Milnacipran, an antidepressant drug similar to amitriptyline, has a dual action on monoamine transporters, with equivalent inhibition of norepinephrine and serotonin neuronal reuptake (14). Milnacipran exhibits a minimal affinity to postsynaptic adrenergic, muscarinic, and histamine receptors and therefore produces fewer side effects related to other drugs in the same family (11,14). In addition, in the rat formalin test (a model of persistent somatic pain), milnacipran showed a moderate antinociceptive effect (11).

We recently developed an animal model for the study of pain related to the female reproductive tract by uterine cervical distension (UCD) (15–18), which may mimic acute pain from the uterine cervix in some gynecologic and obstetric conditions. The purpose of this study was to explore the antinociceptive efficacy of systemic milnacipran in visceral pain originating from the female reproductive tract and gastrointestinal system. Because noxious input increases spinal norepinephrine release and because an inhibitory effect could be augmented by a transporter inhibitor, we also studied the IT administration of milnacipran. Finally, as a measure of assay sensitivity, we examined the effect of IT clonidine on UCD.

	Methods

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The study protocol was approved by the institutional Animal Care and Use Committee. Virgin female Sprague-Dawley rats (Harlan, Indianapolis, IN) were used for all experiments; they weighed 200–310 g at the time of experimentation. Animals were housed at 22°C with a 12-h light/dark cycle and had free access to food and water.

IT catheters were inserted as previously described (19), with minor modifications. In brief, animals were anesthetized with inhaled halothane (2%–3% in oxygen) with spontaneous ventilation, and an incision was made in the dorsal skin of the neck, followed by blunt dissection of paraspinal tissue. A small nick was made in the dura at the atlantooccipital interspace to allow the insertion of custom-made polyethylene tubing caudally, with the tip located at the T10-12 region. Catheters were flushed daily with 10 µL of normal saline, and any animal that showed signs of neurological deficit was excluded from study. A minimum recovery of 5 days was allowed before any experiment.

Animals were anesthetized with inhaled halothane with spontaneous ventilation. Bilateral flank incisions (1 cm) were made, the ovaries were identified, and the ovaries, along with surrounding fat tissue, were resected. Animals were allowed to recover at least 7 days before the experiments were started.

The experimental method for UCD has been described in detail (16). In brief, on the day of experimentation, anesthesia was induced and maintained with halothane 2%–5% in oxygen with spontaneous ventilation. Left carotid arterial and jugular venous catheters were inserted under direct vision. A tracheotomy was performed for mechanical ventilation. A lower abdominal laparotomy was performed via a midline incision, and two fine metal rods were inserted through the cervical osses, with one end fixed to a metal stand and the other end connected to a force transducer. A water-circulating pad was applied, and the body temperature was monitored and maintained at 37°C–38°C throughout the experiment.

The detailed method for colorectal distension (CRD) was previously described by Ness and Gebhart (20). Briefly, anesthesia was induced and maintained with halothane (2%–5%) in oxygen with spontaneous ventilation. Left carotid arterial and jugular venous catheters were inserted under direct vision, and a tracheotomy was performed for mechanical ventilation. A custom-made 6-cm-long latex balloon was tied onto Teflon tubing, one end of which was connected to a manual pneumatic inflation device. The balloon was inserted into the descending colon and rectum through the anus.

For electromyographic (EMG) recording, two noninsulated electrodes were inserted into the bilateral inguinal regions of the rectus abdominis muscles. The EMG activity was rectified and integrated, and the threshold was set to indicate no activity in the absence of stimulation. Total integrated EMG activity during the 10 s of stimulation for UCD and 20 s for CRD was recorded. Halothane concentration was adjusted so that the animal exhibited no spontaneous movement but showed EMG activity in response to distension. This resulted in a narrow range of halothane concentrations (0.5%–0.7%), which were maintained throughout the entire experiment.

For UCD stimulation, an 87.5 x g distending force was applied to the cervix for 10 s with an interstimulus interval of 5 min. On average, five to seven stimulations were required to reach a stable UCD-evoked EMG response. Therefore, this same distending force was applied for 10 s every 5 min until the end of the experiment. For IT injection, milnacipran or saline was administered via a microdialysis infusion pump in a cumulative dose manner, with doses separated by 15 min. The response just before the next dose was used as the effect of the previous dose, because preliminary experiments in other models showed a peak effect at 15 min. The experimenter was blinded to treatment groups. For IV injection, animals were given a single IV injection of milnacipran (10 mg/kg) or saline in a volume of 300 µL followed by UCD at 5-min intervals for up to 60 min. The injection solutions were prepared by a person not performing the experiment to maintain blinding. As a positive control, other animals received cumulative IT clonidine 0.1–3 µg in the same manner as milnacipran.

CRD stimulation was performed by manual inflation of the balloon, and the inflation pressure was monitored by a force transducer. Each stimulation was applied for 20 s, with a 5-min interval between stimuli. An initial stimulation response was obtained, and only animals with a stimulus-dependent EMG response from CRD in the 20–100 mm Hg range were included for study. For IT injection, a single dose of either saline or milnacipran was administered in a volume of 10 µL followed by a 10-µL saline flush via a microinfusion pump over 3 min. CRD stimulation was thereafter conducted every 5 min for 60 min. For IV injection, a single dose of either saline or milnacipran was injected, with CRD stimulation for 60 min as described previously. To determine the effects of chronic administration, animals were randomly assigned to receive subcutaneous (SC) saline or milnacipran (10 or 20 mg; n = 5 per group). Injections were administered twice a day for 5 days. On Day 6, animals were anesthetized with inhaled halothane and spontaneous ventilation, and a stimulus response for CRD from 20 to 100 mm Hg was performed.

Milnacipran was provided by Cypress Bioscience Inc. (San Diego, CA), dissolved in normal saline, and prepared freshly on the day of the experiment. For a single IT injection, 10 µL of either milnacipran solution or saline was injected, followed by a 10-µL flush of saline. For cumulative injections, a freshly prepared milnacipran solution was administered in a 5-µL volume through a microinfusion pump, followed by a 10-µL saline flush. For IV injection, a volume of 300 µL was administered, followed by a 100-µL saline flush. On the basis of pilot experiments, which demonstrated that IV milnacipran 30 mg/kg produced significant hypotension and bradycardia, a dose range of 3–20 mg/kg was chosen for study. Halothane was obtained from Halocarbon Laboratories (River Edge, NJ). Clonidine was obtained from Sigma Chemical Co. (St. Louis, MO).

Data are expressed as mean ± SEM. EMG data were analyzed by using a chart recorder with the rectified, integrated values but are expressed in figures for clarity as a percentage of baseline response to the submaximal stimulus. Stimulus response and cumulative drug dosing were analyzed by two-way repeated-measures analysis of variance, and P < 0.05 was considered significant.

	Results

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All animals recovered uneventfully from ovariectomy and IT catheterization before milnacipran experiments. At laparotomy for insertion of uterine cervical rods, ovariectomy had clearly resulted in a shrinkage in size of both the uterine body and cervix, with no inflammation of the cervix from the previous surgery.

Submaximal UCD stimulation produced reliable evoked EMG responses, similar to those in previous studies (16,21). Neither IT (Fig. 1) nor IV (Fig. 2) milnacipran inhibited UCD-evoked EMG activity, although there was a numeric decrease in EMG response at 15 min with IV milnacipran (P = 0.064). In contrast, IT clonidine produced a dose-dependent inhibition, which was statistically significant (Fig. 1).

View larger version (26K): [in this window] [in a new window]   Figure 1. The effects of milnacipran on uterine cervical distension stimulation. Intrathecal milnacipran (; n = 6), saline (; n = 6), and clonidine (; n = 6) were administered in a cumulative dose manner. *P < 0.05 compared with baseline; P < 0.05 compared with saline. EMG = electromyographic.

View larger version (30K): [in this window] [in a new window]   Figure 2. The effect of IV milnacipran on uterine cervical distension. Animals were randomly assigned to receive milnacipran 10 mg/kg (; n = 6) or saline (; n = 6). There was no statistical difference between groups. EMG = electromyographic.

View larger version (24K): [in this window] [in a new window]   Figure 3. The effect of intrathecal (A) or IV (B) milnacipran on colorectal distension-evoked electromyographic (EMG) activity. A, Change in EMG after colorectal distension in animals treated with intrathecal saline (;; n = 6) or intrathecal milnacipran ( 0.1 µg, n = 6; 0.3 µg, n = 6; 1.0 µg, n = 6; 10 µg, n = 6). B, Change in EMG after colorectal distension in animals treated with IV saline (, n = 6) or IV milnacipran ( 1 mg/kg, n = 6; 3 mg/kg, n = 6; 10 mg/kg, n = 6; 20 mg/kg, n = 6). There was no significant difference within or among groups. The average SEM of values from 5 to 60 min was ±34%.

View larger version (24K): [in this window] [in a new window]   Figure 4. The effect of chronic milnacipran administration on response to colorectal distension. Milnacipran was administered subcutaneously twice a day for 5 days ( 10 mg/kg; 20 mg/kg), with subcutaneous saline as the control group (). On the day of the experiment, a stimulus response to colorectal distension (20–100 mm Hg) was performed (n = 5 for each group). There were no significant differences among animals receiving milnacipran and the saline treatment.

	Discussion

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CRD is a well characterized model suitable to the study of visceral pain (20,22,23). Recently, we have developed an animal model to study pain related to the female reproductive tract by manual distension of the uterine cervix. UCD increases hypogastric nerve activity, reflex abdominal muscle activity, and expression of cFos in the thoracolumbar spinal cord (15–18). Therefore, both CRD and UCD were examined in this study to represent different types of acute visceral pain conditions that may have different mechanisms of inhibition (15,20).

The role of monoamine reuptake inhibitors in enhancing spinally-released norepinephrine and serotonin to treat acute pain is uncertain. In general, these drugs are effective in chronic inflammatory or nerve injury conditions but much less so against acute nociception. For example, milnacipran produces a moderate inhibition of formalin-induced pain behavior (11). However, we have reported that IT amitriptyline did not produce antinociception alone, but it did enhance the analgesic effects of IV morphine in rats; in humans, systemic amitriptyline produced no pain relief from capsaicin-induced allodynia (9,12). This study is in agreement that milnacipran may have a limited role in the treatment of acute pain, whether it is visceral or somatic.

IT clonidine effectively inhibited the EMG responses evoked by UCD, and IT clonidine has previously been demonstrated to inhibit the response to CRD by actions on ₂-adrenoceptors in the spinal cord (21,24,25). These results indicate that the response to acute visceral stimulation can be inhibited by stimulation of the spinal ₂-adrenoceptors on which norepinephrine acts. Previous studies have shown that acute somatic or visceral pain, including the pain of labor in humans (28), increases the spinal release of norepinephrine (26–28). However, the lack of a antinociceptive effect of milnacipran suggests that, in the current experimental conditions, both CRD and UCD are unlikely to cause an acute activation of the descending adrenergic system. Alternatively, milnacipran may not adequately penetrate the spinal cord to reach sites of action, although this is unlikely. TCAs often require several days of treatment for efficacy against chronic pain, but chronic milnacipran treatment failed to alter responses to CRD, so the lack of efficacy cannot be ascribed to too short of a treatment. This result in rats is similar to a report in healthy volunteers (29), in which a three-week treatment with amitriptyline reduced the response to experimental somatic, but not visceral, pain. Therefore, we think that milnacipran may be active in states of chronic nociception, but further study is required to explore its utility in such a chronic setting.

In summary, milnacipran produces no significant antinociception to either CRD- or UCD-induced acute visceral nociception, which suggests that milnacipran probably has a limited clinical application for the treatment of acute visceral pain.

	Acknowledgments

 
Supported in part by grants from the Foundation for Anesthesia Education and Research, from the American Society of Anesthesiologists (Rochester, MN) (CT), and from Cypress Bioscience (San Diego, CA).

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Shin, S.-W. Articles by Tong, C. Search for Related Content PubMed PubMed Citation Articles by Shin, S.-W. Articles by Tong, C. Related Collections Pain Pharmacology