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Keywords:

  • baclofen;
  • Fos;
  • functional dyspepsia;
  • nerve afferent;
  • visceral pain;
  • visceromotor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Background  The amino acid γ-aminobutyric acid (GABA) is an important modulator of pain but its role in visceral pain syndromes is just beginning to be studied. Our aims were to investigate the effect and mechanism of action of the GABAB receptor agonist, baclofen, on gastric hypersensitivity in a validated rat model of functional dyspepsia (FD).

Methods  10-day-old male rats received 0.2 mL of 0.1% iodoacetamide in 2% sucrose daily by oral gavages for 6 days. Control group received 2% sucrose. At 8–10 weeks rats treated with baclofen (0.3, 1, and 3 mg kg−1 bw) or saline were tested for behavioral and electromyographic (EMG) visceromotor responses; gastric spinal afferent nerve activity to graded gastric distention and Fos protein expression in dorsal horn of spinal cord segments T8–T10 to noxious gastric distention.

Key Results  Baclofen administration was associated with a significant attenuation of the behavioral and EMG responses (at 1 and 3 mg kg−1) and expression of Fos in T8 and T9 segments in neonatal iodoacetamide sensitized rats. However, baclofen administration did not significantly affect splanchnic nerve activity to gastric distention. Baclofen (3mg kg−1) also significantly reduced the expression of spinal Fos in response to gastric distention in control rats to a lesser extent than sensitized rats.

Conclusions & Inferences  Baclofen is effective in attenuating pain associated responses in an experimental model of FD and appears to act by central mechanisms. These results provide a basis for clinical trials of this drug in FD patients.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The amino acid γ-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system (CNS). Its near ubiquitous expression in this system corresponds to the multiple functional activities ascribed to it including sedation and anxiolysis, muscle relaxation, analgesia and anticonvulsant activities.1–3 The effects of GABA are mediated through one of two receptor complexes – either the ionotropic GABAA or the metabotropic GABAB.4,5 In general, activation of the latter results in significant analgesia, possibly by presynaptic inhibition of neurotransmitter release from the central endings of primary nociceptors in the spinal cord. Baclofen is a prototypical GABAB receptor agonist that is also the most well studied because of its widespread clinical use as a muscle relaxant and adjuvant in pain relief regimens. Experimental evidence for baclofen as an effective analgesic is almost exclusively derived from the somatic literature and includes clinical and experimental conditions associated with neuropathy, chronic constriction injury, and inflammation.1–11

In gastroenterology, baclofen has been best studied as a possible treatment for gastroesophageal reflux disease, by inhibiting vagally-mediated transient lower esophageal relaxations.4,12–16 By contrast, the therapeutic potential of baclofen in visceral pain has received little, if any, attention and both the efficacy and mechanism of action are far from settled. A recent paper suggests that baclofen attenuated visceromotor and cardiovascular reflexes in response to colorectal distention in rats.17 However, the rats were otherwise healthy (i.e. not sensitized) and no direct evidence was provided that the observed changes in reflexes specifically represented an analgesic effect. Further, by contrast to what is believed to be a predominantly central effect in modulating somatic pain, baclofen has been shown to inhibit the activity of peripheral mechanosensitive vagal afferents raising the possibility of a similar effect on nociceptive afferents.4 The aim of this study was therefore to establish the effects of baclofen in a previously established rodent model of functional dyspepsia (FD) with associated visceral hyperalgesia18 and to localize their primary site of action.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Animals

Male Sprague–Dawley (Harlan, Indianapolis, IN, USA) rats were used in all the experiments. The animal protocol was approved by the Institutional Animal Care and Use Committee of the Stanford University Medical Center. We followed our previously described protocol for inducing long-lasting gastric hypersensitivity.18 Briefly, 10-day-old rat pups received 0.2 mL of 0.1% iodoacetamide in 2% sucrose daily for 6 days by oral gavages (FD rats). Control rat pups received 0.2 mL of 2% sucrose (Fig. 1).

image

Figure 1.  Experimental procedures and time line: 10-day-old neonates were treated with 0.1% iodoacetamide for 6 days. Rats were then tested at 8–10 weeks.

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Implantation of balloon and electrodes for behavior and electromyographic (EMG) testing

A total of 24 FD rats were used for behavioral and EMG testing. Rats were fasted overnight prior to surgery having free access to water. Rats were anesthetized with a cocktail of intra-peritoneal ketamine (80 mg kg−1) and xylazine (7 mg kg−1) and all surgical procedures were performed under sterile conditions. Balloons for gastric distention: 2.5 cm long (made from latex condoms) balloon was attached to a catheter (PE-240). A 3–4 cm long left lateral epigastric incision was made, and the balloon was placed in the stomach through a small hole made at the tip of the fundus. When placed properly and not inflated, the pylorus is not obstructed, and there is no blockage of gastric emptying. The hole is then securely tied to avoid leakage of gastric fluid into the peritoneum. The polyethylene tubing for air inflation of the gastric balloon was exteriorized at the back of the neck along with the EMG electrode leads. After balloon implantation, the abdominal cavity and skin incisions were closed.

Electrode implantation: A sterile multistranded, Teflon-insulated, 40-gauge stainless steel wires (Cooner Wire, Chatsworth, CA, USA) were implanted into the acromiotrapezius (a superficial neck) muscle and the incision was closed with 4-0 silk suture.

Behavior and EMG testing

One week post surgery all the rats were tested for behavioral and visceromotor responses to graded gastric balloon distention (20–80 mmHg) after 0.3, 1, and 3 mg kg−1 baclofen (from Sigma, St. Louis, MO, USA) or saline administered subcutaneously (n = 6 per each dose). Behavioral responses (the abdominal withdrawal reflex, AWR) were graded as previously described18: 0, no behavioral response to gastric distention; 1, brief head movement followed by immobility; 2, contraction of abdominal muscles; 3, lifting of abdomen; 4, body arching, lifting of pelvic structures and stretching of body. Visceromotor responses to gastric distention were recorded simultaneously before and 0.5, 2 and 4 h after treatment and EMG activity was quantified.

Greater splanchnic nerve (GSN) dissection and recording

The effect of baclofen on primary nerve afferent to gastric distention was measured by recording GSN activity to gastric distention. Five separate FD rats were used in this experiment. The surgical procedure of nerve dissection and afferent nerve recording was described in detail.18 Briefly, 8-week-old rats were anesthetized with sodium pentobarbital (50 mg kg−1, intraperitoneally) and maintained with a constant infusion of 50 mg sodium pentobarbital in 9mL, 0.9% NaCl at 1.0 mL h−1 through the jugular vein. Tracheotomy was performed for artificial ventilation and rats were paralyzed with gallamine triethiodide. Body temperature was maintained at 37 °C by a rectal probe feedback-controlled electric heating blanket (Harvard Apparatus, Holliston, MA, USA). A flank incision was made to expose the left GSN. The skin and abdominal muscles were retracted using silk sutures to create a pool. The GSN was cut just below the diaphragm and the distal segment was placed in a pool of warm mineral oil on a black base plate. The nerve was then teased into fine bundles and recorded by draping over one arm of the bipolar silver electrode while an equally fine bundle of connective tissue was placed on the other arm of the electrode as reference.

Action potentials after amplification were processed and recorded through a window discriminator using the CED 1401/SPIKE2 program. The GSN fibers responsive to gastric distention were identified and used in baclofen treatment when the fiber activity was increased ≥30% from baseline in response to a short test stimulus (60 mmHg). Single unit recordings were differentiated and compiled into rate histograms (1 s bin width) using wave-mark template in SPIKE 2. Graded intragastric pressure [low-threshold 20 mmHg, and high-threshold 60 mmHg] was produced by inflating the balloon for 20 s with 5 min intervals via a sphygmomanometer. The GSN response was recorded before and 0.5 h after baclofen treatment.

Fos immunohistochemistry

Separate groups of FD and control rats were used to examine spinal Fos expression (n = 6 for each group). Eight week old FD and control rats were implanted with balloons for gastric distention. One week post surgery, rats were treated with baclofen (3mg kg−1, n = 3 per group) or saline (n = 3 per group). Thirty minutes after baclofen or saline treatment these rats received a noxious gastric distention (80 mmHg for 20 s). Twenty minutes later spinal cords from these rats were harvested for Fos immunohistochemistry as previously described.19 Briefly, anesthetized rats were transcardially perfused with saline (0.9% w/v NaCl) followed by 4% paraformaldehyde in 0.1 mol L−1 phosphate buffer (PB) in pH 7.4. Spinal cord segment corresponding to DRG T10, T9 and T8 identified by counting proximally from the last rib that corresponds to DRG T13 was cut and postfixed in the fixative solution over night and incubated in 30% sucrose in 0.1 mol L−1 PBS (24 h at 4 °C) followed by freezing on dry ice with optimal cutting temperature (OCT). Frozen serial coronal sections (35μm) from segments T8, T9, and T10 were cut on a cryostat, collected in separate wells of a 24 well plate and stored floating in antifreeze at −20 °C. For Fos immunohistochemistry, a total of six sections per segment were processed. The sections were stained with a rabbit polyclonal primary antiserum directed against Fos (ab7963 from Abcam, Cambridge, MA, USA) diluted (1 : 2000) in PBS containing 3% normal goat serum and 0.1% Triton X-100, for 24 h at 4 °C. Antibody staining was visualized with biotinylated goat anti-rabbit IgG (1 : 1000) and avidin–biotin peroxidase complex (Vector Labs Inc., Burlingame, CA, USA) with hydrogen peroxide and diaminobenzidine as substrate. Sections were mounted on Superfrost Plus slides and dehydrated. Regions of laminae I and II were defined by the border along the substantia gelatinosa under darkfield optics. Mean numbers of Fos-positive cells were counted under ×40 optics in lamina I/II from six slices per individual spinal segment (T8–T10) per animal(by a blinded investigator).

Data analysis

All values were presented as means ± SE. Student’s t-test or two-way repeated measures anova were used for comparison. Post hoc comparisons were made using the Student–Newman–Keuls test. Statistical analysis was performed using StatView (Abacus Concepts Inc, Berkeley, CA, USA); P < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Baclofen administration is followed by inhibition of behavioral and visceromotor responses to graded intragastric distension pressure in FD rats

In preliminary experiments, we found that the effect of baclofen on behavioral and visceromotor reflexes peaked at 30 min after injection and all further results were therefore presented at this time point. Baclofen inhibited the behavioral and EMG response of FD rats to gastric distention in a dose-dependent manner (Figs 2A and B). These results were significant for both baclofen treatment and distention by two-way anova. Bonferroni post hoc testing revealed that the lowest dose (0.3 mg kg−1) had no significant effect on either behavior or EMG; 1 mg kg−1 resulted in a significant effect only at 60 and 80 mmHg of pressure for both behavioral and EMG responses and the highest dose (3 mg kg−1) resulted in a significant effect on behavior at all pressures and on EMG responses only at 60 and 80 mmHg pressure.

image

Figure 2.  Behavioral and electromyographic (EMG) responses to gastric distention of functional dyspepsia (FD) rats treated with increasing doses of baclofen. (A) Behavior (a) and EMG (b) responses 0.5 h after baclofen treatment were significantly reduced (P < 0.05) in a dose-dependent manner (see text for details) (B) Representative EMG response at 80 mmHg before and 0.5, 2 and 4 h after treatment with 1 and 3 mg kg−1 baclofen or saline.

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Baclofen administration does not affect GSN afferent activity in response to noxious gastric distention

A total of five gastric distension-sensitive GSN afferent single unit responses from five FD rats treated with baclofen were studied. Based on the above results, 3 mg kg−1 baclofen was used to test the effects of baclofen on GSN afferent activity to physiological (20 mmHg) and noxious (60 mmHg) gastric distention pressures. All the isolated nerve units displayed spontaneous frequency of discharge without gastric distention and a clear increase in response above baseline during gastric distention (Fig. 3A). No significant change in either baseline spontaneous frequency or responses (mean spikes per second) to 20 and 60 mmHg stimulation (2.6 ± 0.4 vs 2.5 ± 0.4, 5.0 ± 0.7 vs 5.0 ± 1.0 and 9.0 ± 1.8 vs 8.5 ± 1.7, P > 0.05, Fig. 3B) was observed following baclofen treatment.

image

Figure 3.  Greater splanchnic nerve (GSN) response to 20 and 60 mmHg gastric distention pre and postbaclofen (3 mg kg−1) treatment: (A) Representative GSN single unit response to pre and post-treatment with baclofen. (B) GSN baseline and response to gastric distention was not significantly different between pre and post-treatment (at 30 min) with baclofen.

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Baclofen administration is followed by reduction of spinal Fos expression in response to gastric distention

Baclofen (3mg kg−1) significantly reduced dorsal horn spinal cord Fos expression to 80 mmHg gastric distention for 20 min in both control and FD rats (Fig. 4A) The number of Fos-positive neurons in lamina I-II of T8, T9, and T10 spinal dorsal horn was significantly reduced in baclofen-treated control rats compared with saline-treated control rats (29.94 ± 2.22 vs 36.44 ± 1.77 (T8), 28.89 ± 1.16 vs 35.08 ± 1.45 (T9), and 30.92 ± 0.99 vs 39.08 ± 1.54 (T10) respectively P < 0.05 and 0.001, Fig. 4B). In baclofen-treated FD rats, the number of Fos-positive neurons in lamina I-II of T8, T9, and T10 spinal dorsal horn was also significantly reduced compared with saline-treated FD rats (26.28 ± 1.98 vs 40.31 ± 3.56 (T8), 32.22 ± 1.29 vs 42.97 ± 3.10(T9), and 29.81 ± 2.63 vs 49.25 ± 3.05(T10) respectively P < 0.001, Fig. 4B). However, the percent change in reduction of the Fos-positive neurons in T8 and T10 in baclofen-treated FD rats was much greater compared with baclofen-treated control rats (64.93 ± 4.88 vs 82.20 ± 6.1 and 60.52 ± 5.34 vs 79.1 ± 2.53, respectively; P < 0.05 and 0.001). In T9, this difference did not reach statistical significance (74.98 ± 3.0 vs 82.3 ± 3.29, P = 0.11). Finally, Fos expression was significantly increased in saline-treated FD rats as compared with saline-treated controls in T9 and T10 (Fig. 4B).

image

Figure 4.  Spinal Fos expression in response to noxious (80 mmHg) gastric distention was reduced after baclofen. (A) Representative photographs of spinal cord sections (T8, T9 and T10) for each (30 min postbaclofen or saline) treatment group stained for Fos, 20 and 40× magnification. Drawing of a hemi-section of the spinal cord under 20× showing specific regions that was analyzed for Fos immunoreactivity. The arrow refers to representative Fos immunoreactivity. (B) Average number of Fos-positive cells in the dorsal horn of T8, T9 and T10 in response to gastric distention were significantly reduced after 3 mg kg−1 of baclofen treatment. (*: FD rats + saline (n = 3) compared with control rats + saline (n = 3); Δ: FD rats + baclofen (n = 3) compared with FD rats + saline; &: Control rats + baclofen (n = 3) compared with control rats + saline. The single symbol represents a P value of <0.05; the double symbol represents a P value of <0.01).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Our previous studies have shown that treatment with 0.1% iodoacetamide orally in neonatal rats can induce mild but transient damage to gastric surface epithelium and chronically sensitize gastric sensory afferents during adulthood in the absence of overt structural abnormalities.18 This rat model displays important characteristics of the human condition including gastric hypersensitivity to mechanical distention and impaired accommodation. It is therefore a suitable model to study the effects of drugs that may be used for chronic visceral hyperalgesia. The present study demonstrated that baclofen, presumably by activation of GABAB receptors, significantly attenuates behavior and visceromotor responses to gastric mechanical distention. This is associated with reduced spinal Fos expression in the dorsal horn but no significant change in peripheral mechanosensitive afferent activity to noxious gastric distention. Although the reduction in response to baclofen was proportionately greater in the FD group, even control rats responded to the drug suggesting that the analgesic effects were not specific to the sensitized state.

We used doses of baclofen that have been previously tested in somatic models of pain,6 and observed a dose-dependent effect of baclofen on the response to mechanical gastric distention, with the lowest dose (0.3mg kg−1) producing little or no effect and the middle dose (1mg kg−1) having an intermediate effect. The most robust effect was seen at the highest dose (3mg kg−1). Previous studies have suggested 40 mmHg to be the noxious threshold in mechanically evoked gastric distention paradigms.18 Our results showed that high dose baclofen (3mg kg−1) inhibited behavior and visceromotor responses caused at both physiological (20 mmHg) and noxious (40–80 mmHg) distention pressures, similar to that shown in previous studies.6,17,20

Animal studies have shown that baclofen has antinociceptive effects in acute pain models and inhibits allodynia and hyperalgesia in a chronic neuropathic pain model.6 A recent study has shown that baclofen inhibits visceral pain-related response to colorectal distension in conscious rats.17 However, those experiments were performed on otherwise healthy rats and therefore the results cannot be extrapolated to conditions where chronic sensitization may exist such as in functional dyspepsia or irritable bowel syndrome. Further, the assays used were unable to reliably distinguish a specific analgesic effect from the confounding factors of generalized sedation or muscle relaxant which may also interfere with visceromotor responses or other reflexes. Our study addressed the latter issue by directly examining the expression of Fos in the dorsal horn, an immediate early gene that is a validated measure of the spinal response to nociceptive stimulation of the corresponding somatic or visceral region and has been widely used in various nociceptive studies.10,21–24 Retrograde tracing studies have shown splanchnic nerve afferent fibers from stomach enter the spinal cord through T3-L3 dorsal roots and most labeled cells in ganglia T8–T10.25–27 Our results from the study of Fos expression are consistent with the observed changes in behavior and EMG reflexes and that these reflect in part at least, a true analgesic effect.

Our results also showed that there were no significant changes in gastric afferent response to either low or high gastric distention pressures after baclofen treatment suggesting that the predominant mechanism of the analgesic effect is central and not peripheral. This is consistent with the somatic literature and the current paradigm of GABA being an important inhibitory neurotransmitter in descending pathways that converge on and dampen incoming signal to the spinal cord via presynaptic receptors.5,28 Both GABAA and GABAB receptors are distributed in the lamina I-III of spinal dorsal horn, with a higher expression of the latter than the former.5,21,29 It should be noted that baclofen has been noted to have a peripheral effect on vagal primary afferent responses to gastric distention.4,15,16 This pathway appears to be mainly involved in conveying sensation of nausea, fullness or bloating, while spinal afferents are predominantly responsible for conveying nociceptive signals.30,31 Thus, splanchnectomy but not vagotomy can prevent the relay of acute noxious information from the stomach to CNS.25 We did not study vagal afferent signaling and it is possible that baclofen may have effects on this pathway as well in our model. Such effects may modulate not only non-noxious gastric sensation but also physiological reflexes involving gastric accommodation and contractility.

Finally, it should be noted that at the doses we used, considerable sedation was observed in rats, raising the question of whether baclofen is a practical drug for this indication. In this regard positive allosteric modulators such as CGP7930 that are being developed for reflux, may have an advantage as they are not agonists in their own right but augment the effects of endogenous GABA.17 Further studies in this and other models of chronic visceral hyperalgesia will need to be done to test their efficacy. In the meantime, our study validates the GABAergic pathway as a potential target for the treatment of functional dyspepsia and related conditions.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Supported in part by a grant from Procter and Gamble.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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