INTRODUCTION

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THE INNERVATION of the rat stomach is organized along two distinct pathways: the sympathetic nerves and the two branches of the vagus. Traditionally, it has been held that visceral pain and sensation originating from the stomach are conducted along sympathetic afferent fibers (16). The vagus nerve is primarily a sensory nerve, with afferent fibers constituting as much as 70-90% of its total fiber content (17). The various functions of afferent vagal fibers have yet to be fully described, but their importance in activities such as ingestive behavior, satiety, nausea, and vomiting has been established. Vagal afferents are not believed to play a major role in the perception of visceral sensations or to be involved in nociception (13).

Woodsworth and Sherrington (23) defined a pseudoaffective response as the autonomic and somatic reflexes produced in response to a nociceptive stimulus. In healthy volunteers, we recently showed that mechanical distension of the distal esophagus is associated with a slight decrease in heart rate and a significant shift in sympathovagal balance toward increased vagal modulation, as measured by power spectral analysis of heart rate variability (20). Preliminary studies in the rat have suggested that gastric distension may elicit a similar response (4), but the neural pathways involved in the response are poorly characterized (8).

In the present study, we examined the cardiovascular response to gastric distension over a wide range of distension volumes and pressures in ketamine- and xylazine-anesthetized rats and determined the respective involvement of vagal and sympathetic afferent pathways as well as of capsaicin-sensitive unmyelinated C afferents. The effects of cholinergic and/or adrenergic blockade on the cardiac response to gastric distension were also examined.

	MATERIAL AND METHODS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

Prior approval for this study was obtained from the Animal Care Committee of McMaster University, and all procedures were conducted in accordance with the Guidelines of the Canadian Council on Animal Care.

Animals

Experimental Protocol

A midline laparotomy was performed, and a distension device consisting of a 2 × 2-cm latex balloon (Forabco Dealers, Milwaukee, WI) affixed to polyethylene (PE-160) tubing (Becton-Dickinson, Parsippany, NJ) was inserted into the stomach through a small incision in the proximal duodenum (1.5 cm from the pylorus) and advanced, in a retrograde manner, through the pylorus and into the stomach.

A midline cervical incision was performed, through which a cannula was then inserted in the left internal jugular vein for intravenous drug administration, and a tracheostomy was performed to allow passage of a tracheal cannula for maintenance of the airway.

In some animals, after measuring the cardiac response to gastric distension (see below), a bilateral subdiaphragmatic vagotomy was performed by identifying the anterior and the posterior vagal branches immediately below the diaphragm, suturing the two branches, and then cutting the distal end. In other animals, the celiac plexus was identified and carefully dissected out until completely removed. In some of these animals, a bilateral vagotomy was done subsequently to the removal of the celiac plexus.

Control animals underwent cervical incision and laparotomy (for insertion of gastric balloon), but neither vagotomy nor celiac plexus removal.

Gastric balloon distension. We studied the gastric distension volume-cardiac response relationship by randomly applying a wide range of gastric distension volumes, using air, for a period of 60 s (n = 18). To let the animal recover from the previous distension, 30 min separated each gastric distension. The results of three trials of distension were used at each volume. The volumes used for gastric distension ranged from 0 to 18 ml (3, 6, 9, 12, 15, and 18 ml). In another group of animals, we subsequently examined responses over varying duration of stimulation (15, 30, 60, and 90 s, n = 6 in each group, volume 9 ml). In another group of animals, we measured the intragastric pressure associated with distension volumes ranging from 3 to 18 ml (n = 6).

Electrocardiogram Acquisition and Analysis

Pharmacological Agents and Drug Administration

To confirm the effects of capsaicin in adult rats, another group of rats (n = 14) were given capsaicin (50 mg/kg) within 48 h of birth (7), using the same anesthetic and analgesic regimen in the following 72 h. These animals were studied at the same age and in the same manner as the other animals.

In addition, we compared the effects of perivagal capsaicin treatment with those of systemic capsaicin, using the approach previously described by Raybould and Taché (15). In these animals (n = 6), after ketamine and xylazine anesthesia, atropine (0.5 mg/kg ip) was first given to reduce the acute effects of capsaicin on the heart and respiratory system. A midline laparotomy was performed, and both the anterior and posterior subdiaphragmatic branches of the vagus were isolated. Parafilm and 2 × 2 gauze were placed around the vagus to protect the surrounding tissues from the damaging effects of capsaicin. Cotton tips were soaked in capsaicin solution and applied to the nerve trunks for 30 s. The area was then thoroughly rinsed with olive oil first and then with physiological saline. It was subsequently dried using sterile cotton swabs, and the incision was closed. Any subsequent experiments were conducted 4 days later.

In all experiments, capsaicin was dissolved in a solution of 10% ethanol, 10% Tween 80, and 80% normal saline in a concentration of 12.5 mg/ml. Control animals received the vehicle without capsaicin.

Atropine sulfate (7.5 mg/kg) was dissolved in normal saline and given subcutaneously in a volume of 1 ml 5-15 min before gastric distension (n = 8). Bretylium tosylate (10 mg/kg iv, n = 7) was given as a bolus in a concentration of 50 mg/ml in normal saline (Burroughs Wellcome, Kirkland, QC, Canada). This dose has previously been shown to be an effective adrenergic ganglionic blocker (3, 21). Propranolol, 1.5 mg/kg in 1 ml normal saline, was given intravenously (n = 5; Ref. 3). Control animals received equivalent volumes of normal saline.

Statistical Analysis

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

Heart Rate Response to Gastric Distension: Gastric Distension Volume-Heart Rate Response Relationship

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Volume-dependent decrease in heart rate associated with increasing gastric distension volumes. Data presented as means ± SE of %change in heart rate after gastric distension compared with resting heart rate. For actual heart rates, see RESULTS. * P < 0.05 vs. resting heart rate.

All volumes associated with a decreased heart rate resulted in increased intragastric pressures (Fig. 2; n = 6).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Intragastric pressures produced by increasing intragastric distension volumes. Data presented as mean intragastric pressures ± SE at each distension volume.

The cardiac response of bradycardia occurred almost simultaneously with the onset of gastric distension and was maximal ~20 s after onset of distension. The bradycardia was sustained at larger volumes but returned quickly to baseline values within 60 s. The greater the gastric distension volume, the longer it took for the heart rate to return to its original baseline. Varying the duration of gastric distension had no effect on the magnitude of the bradycardic response to gastric distension (see Table 1).

Effect of Bilateral Subdiaphragmatic Vagotomy or Celiac Plexus Ganglionectomy on the Cardiac Response to Gastric Distension

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effects of bilateral subdiaphragmatic vagotomy and celiac plexus removal on cardiac response to 9 ml (filled bar), 12 ml (open bar), and 15 ml (hatched bar) gastric distension. Control was response obtained before either vagotomy or celiac plexus. Data presented as means ± SE of %change (decrease) in heart rate during gastric distension compared with resting heart rate in control rats. * P < 0.05 vs. resting heart rate before distension; # P < 0.05 vs. heart rate response to gastric distension in control animals using a similar volume of distension; + P < 0.05 vs. heart rate response to gastric distension after celiac plexus removal.

Heart Rate Response to Gastric Distension in Capsaicin-Treated Animals

In control animals treated with vehicle only, mean resting heart rate was 214.9 ± 37.42 beats/min. With gastric distension of 6, 9, 12, and 15 ml, mean decreases in heart rate were, respectively, 5.34, 9.53, 18.08, and 29.92% (P < 0.05 from resting heart rate). Mean resting heart rates in rats treated with neonatal capsaicin or in adult rats given systemic capsaicin 2 wk before were, respectively, 226.28 ± 37.35 and 199.46 ± 22.50 beats/min and not significantly different from resting heart rate noted in vehicle-treated animals.

In animals given neonatal capsaicin, gastric distension with 6, 9, 12, and 15 ml produced a markedly smaller decrease in heart rate during distension than in vehicle-treated animals (decreases of 0.02, 1.10, 0.36, and 2.58%, respectively, P < 0.007 vs. vehicle-treated animals). In adult rats treated with capsaicin 2 wk before, resting mean heart rate actually increased by 0.63% with 6 ml and 1.35% with 9 ml and decreased by only 0.01% with volumes of 12 and 15 ml (P < 0.002 vs. vehicle-treated animals).

Perivagal capsaicin treatment decreased the cardiac response to gastric distension, but the overall effect was smaller than seen with systemic capsaicin administration in the neonatal period or in adult animals (P < 0.05). In animals treated with perivagal capsaicin, gastric distension with 6, 9, 12, and 15 ml reduced mean heart rate by 1.69, 6.81, 8.93, and 18.72%, respectively, from baseline heart rate (180.35 ± 25.82 beats/min; Fig. 4, n = 6 for all groups).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effect of pretreatment with vehicle (filled bars), systemic capsaicin administration in adult animals (125 mg/kg, 2 wk before; hatched bars), neonatal capsaicin (50 mg/kg, within 48 h of birth; open bars), and perivagal capsaicin (1 mg directly to vagus; crosshatched bars) on cardiac response to 6, 9, 12, and 15 ml of gastric distension. Data presented as mean %decrease in heart rate during gastric distension (±SE). * P < 0.05 vs. resting heart rate; # P < 0.05 vs. response in vehicle-treated animals; + P < 0.05 vs. perivagal capsaicin.

Pharmacological Modulation of the Heart Rate Response to Gastric Distension

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effect of normal saline (control, filled bars), atropine (7.5 mg/kg, hatched bars), propranolol (1.5 mg/kg, open bars), and bretylium (10 mg/kg, crosshatched bars) administration on heart rate response to 9, 12, and 15 ml gastric distension. Data presented as means ± SE of %change in heart rate during gastric distension. * P < 0.05 vs. resting heart rate; # P < 0.05 vs. response in saline-treated animals (control group); + P < 0.05 vs. heart rate response in animals given either saline, propranolol, or bretylium.

The postganglionic -receptor antagonist propranolol (1.5 mg/kg iv) decreased resting heart rate by 30% (from 266 ± 19 to 185 ± 6 beats/min) over saline-treated controls. Propranolol decreased the bradycardia observed during gastric distension at all distension volumes, but the effect was only significant with 15-ml gastric distension volumes (Fig. 5). The residual heart rate decrease still observed after propranolol administration was nearly completely abolished by atropine (7.5 mg).

Bretylium (10 mg/kg iv), a quaternary ammonium adrenergic blocker, decreased resting heart rate by 18% (from 266 ± 18 to 218 ± 11 beats/min). At higher gastric distension volumes (12 or 15 ml), it also decreased the heart rate response to gastric distension (Fig. 5, n = 7). The magnitude of the decrease was similar to that observed with propranolol.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION REFERENCES

Involvement of Vagal Afferents in the Cardiac Response to Gastric Distension

Capsaicin Prevents the Cardiac Response to Gastric Distension

Although capsaicin spares as much as 30-60% of unmyelinated fibers (6), it is nevertheless clear from the present study that the afferent fibers involved in the cardiac response to gastric distension are capsaicin sensitive, which is not to imply that all unmyelinated fibers are capsaicin sensitive. There are few data on the specific effects of capsaicin on vagal afferent fibers, but it has been shown that there is relative sparing of enteric neurons after neonatal administration, whereas in adult animals, the administration of capsaicin has relatively little effect on sympathetic and enteric neurons (6). These observations further support, although only indirectly, that the effect of capsaicin on the cardiac response to gastric distension is primarily on vagal afferents pathways, because enteric and sympathetic neurons are unaffected by capsaicin.

Perivagal administration of capsaicin provides similar results to those observed with systemic capsaicin, but the magnitude of the capsaicin effect on the cardiac response to gastric distension is smaller with perivagal administration than with systemic capsaicin. There are few data on the effects of perivagal capsaicin on unmyelinated vagal afferents. One could hypothesize that the local application of capsaicin spares a proportion of these unmyelinated vagal afferents, as it does with sympathetic and enteric nerves. As the response is largely abolished by vagotomy, it could also mean that other types of vagal afferents (e.g., myelinated afferents) are also involved in addition to unmyelinated afferents. However, the complete abolition of the response with either systemic or neonatal capsaicin administration suggests that the dose, the interval between administration and gastric distension (4 days), or the method of application is not as effective and results in functional sparing of some vagal afferents.

Role of Cholinergic and Adrenergic Efferent Pathways

Pseudoaffective Autonomic Response to Noxious Visceral Stimuli

A recent study that examined the cardiac response to esophageal distension also found that vagal afferent pathways were the primary pathways involved (11), although Kidd et al. (8) described the involvement of afferent fibers closely associated with sympathetic nerves in the increased blood pressure associated with gastric distension. This was unaffected by bilateral vagotomy. This study of Kidd et al. failed to obtain any effect of gastric distension on heart rate, possibly because urethan, which was the anesthetic used, depresses parasympathetic activity. In the cat, under -chloralose anesthesia, gastric distension failed to produce any effects on heart rate (10), whereas in pigs, gastric distension actually increased heart rate, a response prevented by a prior subdiaphragmatic vagotomy, indicating again involvement of vagal afferent pathways in the response (21, 22).

In a recent study, Loomis et al. (11) carefully examined the cardiac response to esophageal distension in urethan-anesthetized rats and found a pressure-dependent increase in heart rate in response to esophageal distension, a response that was also primarily mediated via vagal afferent pathways. The intraesophageal pressures used by Loomis et al. (25-150 mmHg) are very similar to the intragastric pressures we obtained in our study (which ranged from 30 mmHg with 3-ml distension to 150 mmHg with 15-18 ml), and which, at least in the higher ranges, would likely be perceived as increasingly painful without the presence of a general anesthetic. Therefore the heart rate response we describe in response to gastric distension likely represents a pseudoaffective autonomic response to a noxious stimulus, as proposed by Sherrington (18), which, in our model, is mediated though vagal afferent pathways.

In humans, electrical or mechanical esophageal stimulation reproducibly increases vagal cardiac outflow while decreasing sympathetic efferent cardiac outflow (2, 20). Because the cardiac response to gastric distension observed in the present study is almost completely abolished after atropine administration, the response must be largely mediated through atropine-sensitive cholinergic vagal efferent pathways. However, given the attenuation of the cardiac response to gastric distension after administration of bretylium and propranolol, it is likely that adrenergic sympathetic pathways are partly involved. The magnitude of the effect observed with atropine is much greater than with adrenergic blockers, suggesting that cholinergic pathways are primarily involved.

These observations are in agreement with those of Grundy and Davison (4), who also found the cardiac response to gastric distension to be atropine sensitive in rats. However, Pittam et al. (14) did not find that atropine had any effect on the cardiac response in rats, which was instead found to be particularly sensitive to -blockade, suggesting a sympathetic modulation. The different responses noted in the present study are likely due in part to the different anesthetics used in our respective studies.

In pigs, gastric distension also has cardiac effects, which are also abolished by subdiaphragmatic vagotomy. However, in pigs the cardiac response was also clearly dependent on sympathetic efferent pathways as it was completely blocked by bretylium tosylate but unaffected by atropine administration, albeit with a much smaller dose of atropine than in the present study (22).

Clinical Perspectives

Studies, such as those of Loomis et al. (11) and the present one, provide models suitable for the examination of the possible mechanisms underlying the altered autonomic responsiveness seen in conditions such as dyspepsia and noncardiac chest pain. In addition, by providing a quantifiable and dose-dependent physiological response to a noxious visceral stimulus, it also represents a pseudoaffective model for the study of these noxious visceral stimuli in animals that are much better suited to the examination of the mechanisms and pharmacological modulators of nociceptive visceral perception.