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Keywords:

  • antral peristalsis;
  • electrical vagal stimulation;
  • frequency–dependency;
  • gastric pressure;
  • in vitro stomach;
  • receptive relaxation;
  • vagus

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. In vitro stomach preparation
  6. Experimental protocol
  7. Image acquisition
  8. Image processing and generation of diameter maps (D-map)
  9. Quantitative analysis of motion from D-maps and statistical analysis
  10. Statistical procedures
  11. Results
  12. Discussion
  13. Vagal tone
  14. Vagal modulation of antral peristaltic contractions
  15. Vagal effect on fundus
  16. Conclusions and Perspectives
  17. Acknowledgments
  18. References

AbstractOur aim was to evaluate topographically specific gastric motility changes induced by graded vagal activation. A recently developed method of constructing spatio-temporal maps of motility from video movies was adapted to the in vitro perfused guinea-pig stomach with an intact vagal nerve supply. In the unstimulated preparation, spontaneous activity was low or absent. Bilateral vagal stimulation with frequencies as low as 0.2 Hz triggered weak anally, and in some cases orally, propagating antral contractions at rates of about 5–6 min−1. Upon stimulation with higher frequencies, antral contractions increased significantly in length (starting more proximally) and amplitude, and produced large pressure peaks of up to 25 hPa, with maximal effects at 2–4 Hz. In contrast, the speed of propagation and the interval between peristaltic waves did not change with vagal stimulation at any frequency. Vagal stimulation also produced a significant and frequency-dependent enlargement of the fundus with a maximal effect at 4 Hz. It is concluded that a very low tonic vagal activity is apparently necessary and sufficient to express basic antral motility, while more sustained vagal activity is necessary for high-amplitude gastric contractions and significant sustained fundic relaxation. The constant interval between propagating contractions supports the concept that vagal input impinges on intrinsic enteric neural circuits that have a modulatory role in the myogenic mechanism underlying slow-wave peristalsis, rather than directly on gastric musculature.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. In vitro stomach preparation
  6. Experimental protocol
  7. Image acquisition
  8. Image processing and generation of diameter maps (D-map)
  9. Quantitative analysis of motion from D-maps and statistical analysis
  10. Statistical procedures
  11. Results
  12. Discussion
  13. Vagal tone
  14. Vagal modulation of antral peristaltic contractions
  15. Vagal effect on fundus
  16. Conclusions and Perspectives
  17. Acknowledgments
  18. References

The major functions of the mammalian stomach are storage, acidification, mixing, and controlled delivery of ingested food to the small intestine. Both receptive and adaptive relaxation reflexes of primarily the proximal or fundic stomach are responsible for the stomach's reservoir function. Mixing of foods and delivery of chyme to the duodenum are accomplished by coordinated actions of the muscularis externa and the pyloric sphincter muscle.

Activity of gastric smooth muscle is directly controlled through two interacting systems, the network of interstitial pacemaker cells1 and the enteric nervous system.2 Evidence for both excitatory and inhibitory reflexes with regard to smooth muscle contraction has been found in vivo and in vitro. Cannon3,4 described propagating contractions and receptive relaxation in the in vivo dog stomach at the turn of the century. Experiments in vagotomized rats,5 ferrets,6 as well as in isolated guinea-pig stomach7,8 have shown that these basic reflex motions can occur in the absence of central nervous system (CNS) control.

However, because gastric functions are crucial for the overall process of digestion and the flux of nutrients, sensors in other parts of the alimentary canal, and external sensors such as visual and olfactory stimuli, can exert powerful control over gastric motility via a number of hormones and both vagal and sympathetic innervation. Vagal control over gastric functions is particularly strong, as shown by both the large number of reflexes with an efferent vagal limb, and the density of vagal preganglionic innervation.9 Several of the gastro–entero–pancreatic hormones such as secretin,10 glucagon-like peptide 1 (GLP-1),11 cholecystokinin (CCK),12 and pancreatic polypeptide,13 originally thought to affect gastrointestinal and pancreatic function directly, have recently been shown to engage vago-vagal reflexes involving brainstem circuitry. Furthermore, gastric motility effects induced by duodenal nutrient infusion and distension,14 by glucose signals from the portal vein and brainstem,15 and by nociceptive signals16–18 have also been shown to be at least partially mediated by vagal efferents. Finally, a number of peptides such as atrial natriuretic factor (ANF),19 tumour necrosis factor α (TNF-α),20 peptide YY (PYY), neuropeptide Y (NPY), Thyrotropin-releasing hormone (TRH) pituitary adenylate cyclare activating polypeptide (PACAP) PYY and NPY,21 TRH,22 substance P,23 PACAP and vasoactive intestinal peptide (VIP)24 injected into the cisterna magna or dorsal vagal complex have been shown to powerfully influence gastric motility. Elements of this strong vagal control are also increasingly recognized to play a role in the abnormal motility patterns linked to diabetes.25

At least two types of vagal efferent fibres to the stomach have been characterized in classical stimulation experiments. Larger diameter fibres with a low threshold to electrical stimulation are mainly responsible for cholinergic excitatory input to the gastric smooth muscle, producing strong contractions, while high-threshold smaller fibres mainly produce nonadrenergic, noncholinergic (NANC) muscle relaxation as well as gastric acid secretion.26,27

In most animal studies, gastric motility was measured indirectly with manometry or localized strain gauge transducers,13,28,29 which are not able to provide a high spatial resolution of specific motility changes. Roentgenographic methods using barium sulphate3,30,31 or radio-opaque pellets,31 ultrasound,25,32 radioscintigraphy using radionuclides such as 99Tc,28,33 magnetic field goniometry using small ingested magnets,34 or magnetic field decay using magnetized particles,35,36 and most recently magnetic resonance imaging37 can provide somewhat better spatial resolution, but for practical reasons are mainly used in clinical studies.

A new videographic method using high-resolution spatio-temporal maps has recently been introduced to study intestinal peristalsis in vitro.38,39 Intestinal diameter is expressed as intensity value of pixels, or third dimension, in three-dimensional maps with the other two dimensions being location and time. These maps are then used to quantify changes in the length and diameter of pieces of intestine as well as the speed of propagation and amplitude of peristaltic waves. This simple but powerful method has now been adapted to circular segments of large intestine,40 and was used in the present experiment to study vagal stimulation-induced gastric motility changes in guinea-pig stomach. This method has allowed us, for the first time, to show the complex motility changes induced by vagal stimulation with varying frequencies, occurring throughout the entire stomach with a high temporal and spatial resolution.

In vitro stomach preparation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. In vitro stomach preparation
  6. Experimental protocol
  7. Image acquisition
  8. Image processing and generation of diameter maps (D-map)
  9. Quantitative analysis of motion from D-maps and statistical analysis
  10. Statistical procedures
  11. Results
  12. Discussion
  13. Vagal tone
  14. Vagal modulation of antral peristaltic contractions
  15. Vagal effect on fundus
  16. Conclusions and Perspectives
  17. Acknowledgments
  18. References

Nonfasted guinea-pigs (IMVS Institute of Medical and Veterinary Science coloured) of either sex, weighing 230–400 g were killed by cervical dislocation, followed by bleeding from the carotid arteries (protocol approved by the Animal Welfare Committee of Flinders University). After a midline laparotomy, a block of tissue containing stomach, oesophagus, proximal duodenum, pancreas and liver was quickly excised and placed in ice-cold carboxygenated Krebs solution with the following composition (mmol L−1) NaCl, 118; KCl, 4.7; NaH2PO4, 1.0; NaHCO3, 25; MgSO4, 1.2; d-glucose, 11.0; CaCl2, 2.5; pH 7.4. The stomach was then rinsed with cold Krebs by gentle, repeated push-and-pull from a 10-ml syringe via a short piece of soft silastic tubing (4 mm O.D.) introduced into the lumen through the pyloric sphincter. Both vagal trunks were fitted with black silk ligatures at the level of the heart, and carefully dissected from the oesophagus under steromicroscopic guidance. The common hepatic vagal branch was preserved by carefully dissecting along its path near the liver hilus, hepatic and gastroduodenal arteries. The coeliac branches were cut, and all non-gastric tissue removed except for a 5-mm length of distal oesophagus and 5 mm of proximal duodenum.

The stomach was then suspended (greater curvature down) in a special chamber by fixing an oesophageal fistula (2 mm OD) and a pyloric fistula (3 mm OD) in vertical orientation to a bar above the chamber (Fig. 1). The bar was then lowered so that the hanging stomach was completely submerged into the chamber. The chamber was filled with non-circulating, warm (36–37 °C) carboxygenated Krebs solution. Constant bath temperature was guaranteed by thermostatically controlled water flow in a compartment surrounding the bath chamber. The pyloric fistula was connected to a syringe pump containing Krebs solution, allowing filling of the stomach with various volumes at a given rate (see below). In preliminary experiments we found that the pH within the gastric lumen remained constant, so that continuous flow was not necessary. The oesophageal fistula was hooked up with a pressure transducer (Statham P23X), connected to a MacLab data acquisition system (AD Instruments, Sydney, Australia). Finally, both vagal trunks were laid on a bipolar platinum hook electrode positioned near the surface of the bath solution, and connected to a stimulator (Grass model S44) via a stimulus isolation unit.

image

Figure 1. Schematic diagram showing experimental setup. The stomach is hung in a bath chamber filled with warm and carboxygenated saline, with only the oesophageal and pyloric cannulas held in place. Bath temperature is held constant by a thermostatically controlled chamber surrounding the bath (not shown). The stomach is illuminated by a fibre-optic light source to produce high contrast view against black back panel. Both vagal trunks are separated from the oesophagus and laid over a bipolar hook electrode just above the bath level.

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Experimental protocol

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. In vitro stomach preparation
  6. Experimental protocol
  7. Image acquisition
  8. Image processing and generation of diameter maps (D-map)
  9. Quantitative analysis of motion from D-maps and statistical analysis
  10. Statistical procedures
  11. Results
  12. Discussion
  13. Vagal tone
  14. Vagal modulation of antral peristaltic contractions
  15. Vagal effect on fundus
  16. Conclusions and Perspectives
  17. Acknowledgments
  18. References

The stomach was initially filled with a mean of 10.8 ml (9–12, n = 5) of Krebs, depending on the bodyweight of the animal, and after a resting period of 15 min the first electrical stimulation series began. The volume was then increased to a mean of 16.6 ml (14–18, n = 5), and a second series of stimulations was carried out. Because the stomachs were not completely empty at the beginning of the experiment (emptying by suction was not considered because of possible injury), the exact volumes were back-calculated on the basis of the final contents and the volumes infused. Each stimulation series consisted of two parts, one with no stimulation (0 Hz) background, and one with a low-frequency (0.2–1.0 Hz) stimulation background. The latter was chosen to reinstate basal vagal tone in the vagotomized preparation, and the criterion for the chosen baseline frequency was to produce visible, low-level antral contractions. Both series consisted of 1-min stimulation bouts with increasing frequency of 1, 2, 4, 8 and 16 Hz, delivered every 5 min. Stimulation consisted of rectangular pulses of 50–100 V and 0.2 ms pulse duration. These supramaximal stimulation parameters were determined in pilot experiments.

Image acquisition

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. In vitro stomach preparation
  6. Experimental protocol
  7. Image acquisition
  8. Image processing and generation of diameter maps (D-map)
  9. Quantitative analysis of motion from D-maps and statistical analysis
  10. Statistical procedures
  11. Results
  12. Discussion
  13. Vagal tone
  14. Vagal modulation of antral peristaltic contractions
  15. Vagal effect on fundus
  16. Conclusions and Perspectives
  17. Acknowledgments
  18. References

A video camera (Panasonic WV-CL504) with a 50-mm lens was positioned in front of the bath (Fig. 1), to capture the brightly lit dorsal surface of the stomach on the dark background provided by the black acrylic back panel of the bath. The camera was connected to a high-resolution S-VHS video recorder (Panasonic AG-7355) in PAL format at 25 frames s−1. Frames of select sequences of the video were digitized on an Apple Power MacIntosh (7600/132) at 5 per second, using a modified version of the image analysis software developed by the National Institute of Health (NIH Image 1.62).

The maximum resolution achievable using S-VHS video tape is approximately 410 columns by 625 rows. Depending on the final magnification (approximately × 2.5), this corresponded to a vertical resolution of 0.15–0.25 mm and a horizontal resolution of 0.2–0.3 mm. To achieve a reasonable balance between the capture rate and the size of the digitized image, a half-sized capture screen (384 × 256 pixels) was used, enabling the video stream to be captured at a rate of 5 s−1. The temporal accuracy of capture was very stable, with time discrepancies of <1 s h−1. The correlation between the video and the pressure recordings was established by using visual markers synchronized to time points in the pressure recording.

Image processing and generation of diameter maps (D-map)

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. In vitro stomach preparation
  6. Experimental protocol
  7. Image acquisition
  8. Image processing and generation of diameter maps (D-map)
  9. Quantitative analysis of motion from D-maps and statistical analysis
  10. Statistical procedures
  11. Results
  12. Discussion
  13. Vagal tone
  14. Vagal modulation of antral peristaltic contractions
  15. Vagal effect on fundus
  16. Conclusions and Perspectives
  17. Acknowledgments
  18. References

The digitized frames of selected sequences were first subjected to a thresholding procedure to define the edges of the stomach. Digitized images were converted to binary (1 bit/black or white) with white representing the stomach surface and black the background. Next, the digitized sequences were cleaned by removing (black fill) the fixed parts of oesophagus and pylorus, and using paintbrush to cover artefacts produced by, e.g. air bubbles, and/or median filters to smoothen the gastric contours if necessary.

In order to construct diameter maps, the stomach was arbitrarily divided into three zones: fundus, corpus and antrum, with the corpus defined by vertical lines drawn through the outer edge of the oesophageal fistula and the inner edge of the pyloric fistula (Fig. 2). The diameter of the corpus region at every pixel column was determined by the number of white pixels in each column, and then represented as single pixels, with the smallest diameter coded as white and the largest diameter as black. Intermediate diameters were assigned a proportional level of grey. The grey-scale coded pixels from each frame of the movie sequence were used to construct a single row, and sequential rows were placed underneath each other in a separate image, to produce a spatio-temporal map of diameters. Because of the curved nature of the fundic and antral regions of the stomach, instead of vertical pixel columns, a line rotating around a pivot point was used to determine the diameter (Fig. 2). The pivot point for the fundus was chosen near the gastric cardia, and the pivot point for the antrum near the pyloric sphincter. Thus, three separate diameter maps were first generated, and then joined together.

image

Figure 2. Generation of spatio-temporal diameter maps of in vitro guinea-pig stomach. The three top panels show dorsal views of suspended stomach. The arrows in panel A point to the two pivot points near the oesophagus (E) and pylorus (P) around which a line is rotated to measure the diameter of the curved fundus (F) and antral (A) regions. Three-dimensional maps with one axis representing the location along the circumference, the other axis representing time, and the grey level of each pixel representing the relative state of contraction at a given location and time were constructed by aligning each pixel column obtained from consecutive video frames (see Methods section for details). Aborally-propagating contraction (arrow heads) is shown in two frames 2.5 s apart in B and C, and on diameter map in D. Fundic enlargement is indicated by short arrows in B and D. White lines placed over antral contractions in D were used to determine amplitude (maximal change in diameter), length, speed (slope of line) and frequency (1 per distance between lines) of contractions. Intragastric pressure is shown in panel E.

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Quantitative analysis of motion from D-maps and statistical analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. In vitro stomach preparation
  6. Experimental protocol
  7. Image acquisition
  8. Image processing and generation of diameter maps (D-map)
  9. Quantitative analysis of motion from D-maps and statistical analysis
  10. Statistical procedures
  11. Results
  12. Discussion
  13. Vagal tone
  14. Vagal modulation of antral peristaltic contractions
  15. Vagal effect on fundus
  16. Conclusions and Perspectives
  17. Acknowledgments
  18. References

Frequency, speed of propagation and length of antral peristaltic waves was measured by placing lines along the white ridges of contraction on the map (Fig. 2). A macro developed for the purpose identified the maximal contraction at every point and calculated the slope of the least square regression line of minimum diameter points of each contraction. The slope of this line was taken as the average speed of propagation of the contraction. The length of the contraction was determined by the observer on visual inspection of the white streak. A test of validity was performed by reconstructing the silhouette of the stomach from the map and establishing that the area of contraction had a diameter consistently smaller than the surrounding area. In addition, the interval between the lines and the length of the lines (contractions) was also calculated and transformed into frequency by the macro. The amplitude of contractions was measured by placing a horizontal line in the distal antrum (20% of the total circumference from tip of fundus to pylorus in basal condition), and using a plotting tool of image the maximal and minimal diameters were calculated. The difference between maximal and minimal diameters was calculated and taken as amplitude. The degree of stimulation-induced fundic relaxation was estimated by measuring the increase of perimeter length from the spatio-temporal maps and expressed as percentage changes of perimeter length before stimulation.

Statistical procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. In vitro stomach preparation
  6. Experimental protocol
  7. Image acquisition
  8. Image processing and generation of diameter maps (D-map)
  9. Quantitative analysis of motion from D-maps and statistical analysis
  10. Statistical procedures
  11. Results
  12. Discussion
  13. Vagal tone
  14. Vagal modulation of antral peristaltic contractions
  15. Vagal effect on fundus
  16. Conclusions and Perspectives
  17. Acknowledgments
  18. References

Statistical comparisons were made by applying three sets of anovas to each independent measure. In one set, separate mixed-procedure, two-way doubly repeated measures anovas were carried out for low- and high-volume experiments on baseline (prestimulus), during stimulation and poststimulation scores. In another set, separate mixed-procedure two-way repeated measures anovas were carried out across both volumes on baseline, stimulus and post-stimulus scores. Finally in the third set, separate two-way repeated measures anovas were carried out on difference scores between prestimulation (baseline) and stimulated values, with prestimulus as covariant for amplitude, length, speed and intervals. (PROC MIXED in SAS 6.12).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. In vitro stomach preparation
  6. Experimental protocol
  7. Image acquisition
  8. Image processing and generation of diameter maps (D-map)
  9. Quantitative analysis of motion from D-maps and statistical analysis
  10. Statistical procedures
  11. Results
  12. Discussion
  13. Vagal tone
  14. Vagal modulation of antral peristaltic contractions
  15. Vagal effect on fundus
  16. Conclusions and Perspectives
  17. Acknowledgments
  18. References

Although a moderate degree of motility in the form of antral peristaltic waves was observed in response to gastric filling, spontaneous motility was either very low or absent after the 15-min resting period before the vagal stimulation series. Vagal stimulation with as low as 0.2 Hz induced weak antral peristaltic waves, accompanied by small variations in intragastric pressure (Fig. 3).

image

Figure 3. Spatio-temporal diameter map demonstrating effect of bilateral electrical vagal stimulation at 0.2 Hz (vertical bar) on motility of in vitro guinea-pig stomach. There was no detectable motility before vagal stimulation. Vagal stimulation induced shallow contractions beginning at mid corpus and propagating towards the pylorus.

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Stimulation with higher frequencies (0 to 1, 2, 4 and 8 Hz) generated much stronger antral peristaltic waves with synchronized large pressure increases and other motility changes (not shown). Overall, there was considerable variability in the amplitude and directionality of motility elicited by vagal stimulation, including some cases of reversed peristalsis (Fig. 4B). Inspection of the videos and a limited number of D-maps with a stimulation step from 0 Hz to 1–16 Hz showed no fundamental differences in response compared with the series with a step from low (0.2–0.7 Hz) to 1–16 Hz. Because low vagal stimulation appeared to rescue basal gastric motility and to more accurately reflect the normal physiological state with low vagal tone, we only analysed in depth the latter series with low vagal background stimulation in this report. Increasing vagal stimulation from this low level (range 0.2–0.8 Hz, mean 0.5 Hz) to 1, 2 or 4 Hz under low-volume conditions (Fig. 4), and (range 0.3–1.4 Hz, mean 0.8 Hz) to 2, 4, 8 and 16 Hz under high-volume conditions (Fig. 5) increased antral peristalsis and intragastric pressure waves.

image

Figure 4. Vagal stimulation-induced motility and pressure changes of guinea-pig stomachs under low-volume (9–11 mL) conditions. Examples with electrical stimulation at frequencies of 0.5 Hz (A), 1 Hz (B), 2 Hz (C) and 4 Hz (D) are shown. Note that stimulation with 4 Hz (D) induces only three strong aborally propagating contractions, with two much weaker or ‘skipped’ contractions (arrows), and corresponding peaks in intragastric pressure.

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image

Figure 5. Vagal stimulation-induced motility and pressure changes of guinea-pig stomachs under high-volume (14–18 mL) conditions. Examples with electrical stimulation at frequencies of 2 Hz (A), 4 Hz (B), 8 Hz (C) and 16 Hz (D) are shown. Stimulation with 8 Hz for 40 s (C) produced three strong antral contractions and considerable enlargement of the fundus, resulting in relatively modest pressure increases. Stimulation with 16 Hz for 30 s (D) produced three relatively modest antral contractions and almost no fundic enlargement, resulting in a large pressure increase with three superimposed peaks.

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In addition, the fundic portion of the stomach increased in size by expanding its rostral pole towards the oesophagus, an effect that was clearly linked to the antral peristaltic activity and its associated peak pressure waves, and was more or less frequency-dependent for the low-volume condition (Figs 4 and 10).

image

Figure 10. Effect of electrical stimulation with different frequencies on increase of perimeter compared with low background stimulation of guinea-pig stomach under low and high volume conditions. Under low- but not high-volume conditions the perimeter increased in a frequency-dependent manner (P < 0.05). Bars not sharing the same letter are significantly different from each other (P < 0.05).

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Further analysis of D-maps showed that the amplitude (Fig. 6) and length (Fig. 7), but not the frequency (Fig. 8) and propagation speed (Fig. 9) of the peristaltic waves were significantly changed by increasing vagal stimulation. All changes in motility and pressure returned to baseline levels upon cessation of stimulation, although in a few cases, particularly with higher stimulation frequencies, after-contractions were seen.

image

Figure 6. Effect of electrical stimulation with different frequencies on amplitude of contractions of guinea-pig stomach under low- and high-volume conditions. Amplitude of contraction is shown as mean ± SEM (n = 5) during stimulation (shaded bars) and before and after stimulation (open bars). Stimulation with all frequencies and under both low- and high-volume conditions significantly increased amplitude of contraction compared with before and after stimulation (*P < 0.01). There was no significant effect of stimulation frequency.

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image

Figure 7. Effect of electrical stimulation with different frequencies on length of contractions of guinea-pig stomach under low- and high-volume conditions. Length of contraction is shown as mean ± SEM (n = 5) during stimulation (shaded bars) as well as before and after stimulation (open bars). Stimulation with all frequencies, except for 1 Hz per low volume significantly increased length of contraction compared with before and after stimulation (*P < 0.01).

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image

Figure 8. Effect of electrical stimulation with different frequencies on interval between contractions of guinea-pig stomach under low- and high-volume conditions. The interval between contractions is shown as mean ± SEM (n = 5) during stimulation (shaded bars) as well as before and after stimulation (open bars). There was no significant effect of stimulation or frequency.

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image

Figure 9. Effect of electrical stimulation with different frequencies on speed of contractions of guinea-pig stomach under low- and high-volume conditions. Speed of contraction is shown as mean ± SEM (n = 5) during stimulation (shaded bars) as well as before and after stimulation (open bars). There was no significant effect of stimulation or frequency.

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The amplitude of antral peristaltic waves was significantly increased during stimulation compared with the prestimulation baseline and poststimulation period under both low-volume (F[2,8] = 25.3, P < 0.0003) and high-volume (F[2,8] = 23.7, P < 0.0004) conditions. Although there was a trend for a frequency–response relationship at the lower end of stimulation frequencies (1–2 Hz with low volume, and 2–4 Hz with high volume; Fig. 6), there was no overall significant effect of stimulation frequency whether anova was applied to both volumes (F[5,18] = 0.44, n.s.) or to low- and high-volume trials separately. The relative size of the amplitude (with the maximum diameter taken as 100%) at optimal stimulation frequency was 25% (2 Hz) for low-volume and 32% (4 Hz) for high-volume conditions. Following all stimulations, amplitudes reverted back to prestimulation levels.

Stimulation-induced increases in amplitude were almost exclusively due to decreases in minimum diameter, without much change in maximum diameter. Under high volume conditions, minimum diameters were significantly lower compared with their respective baseline and poststimulation periods for all stimulation frequencies (F[2,20] = 6.26 16.52, P < 0.01). Under low-volume conditions the same trend was observed, but the decreases did not reach statistical significance.

Similar to the amplitude, the length of contractions was also significantly increased by vagal stimulation (F[2,8] = 16.42, P = 0.0015 for low volume; F[2,8] = 25.65, P = 0.0003, high volume; Fig. 7). The contractions started further back in the corpus (Figs 4 and 5). The effect was maximal with 2 Hz both under low-volume (F[2,16] = 22.08, P < 0.0001) and high-volume (F[2,20] = 15.36, P < 0.0001) conditions. Under high-volume conditions there was an inverse relationship between frequency of stimulation and length of contraction, resulting in a significant main effect of frequency (F[5,18] = 3.24, P = 0.029).

In contrast to the above two measures, the interval between contractions was not significantly changed by any stimulation and volume condition (Fig. 8). There was a tendency for slightly longer intervals under high-volume conditions, but this trend was not statistically significant. The interval was remarkably constant between 10.3 and 11.9 s (5.0–5.8 contractions min−1) for the low-volume, and between 10.5 and 13 s (4.6–5.7 contractions min−1) for the high-volume condition. Although some contractions were skipped, particularly at the beginning of stimulation, the following contractions remained phase-locked to the basal rhythm of around 12 s (Figs 4 and 5). When the stimulation step started, the effects were immediate, so that the basal peristaltic wave was not finished before the stronger contractions started. At the end of the step, however, resumption of basal activity was not immediate, in that already started large-amplitude peristaltic contractions and the associated pressure peaks typically finished their course (Figs 4 and 5).

The speed of contraction propagation was also not significantly changed by vagal stimulation (Fig. 9). Note that only the average speed of propagation was estimated by the imaginary lines, when in fact the speed often changed as the contraction moved closer to the pylorus (Fig. 9).

Fundus size and volume were dramatically increased during vagal stimulation. Not only did the perimeter (as viewed from ventral by the camera) increase, but also the diameter in a ventral–dorsal direction (not detected by the camera). With higher stimulation frequencies the tip of the fundus often expanded into two horns, hugging the cannulated oesophagus, and making it difficult to assess the entire perimeter. Thus, our percentage increase in total perimeter is only a conservative estimate of fundic enlargement (Fig. 10). For the low-volume series the perimeter increased in a frequency-dependent manner (F[2,8] = 5.30, P = 0.034), reaching 24% at 4 Hz (Fig. 10). For the high-volume series it was maximal at 21% at 4 Hz, with no further increase at 8 and 16 Hz.

Intragastric pressure changes were often but not always synchronized with peristaltic contractions, with peaks occurring when the contraction was located about midway between its origin and the pyloric sphincter. Pressure peaks were typically larger at the beginning of the stimulation step, probably owing to the fact that the fundus had not yet maximally enlarged. However, during stimulation, intragastric pressure typically did not quite return to basal levels between antral contractions, despite a significantly enlarged fundus. This suggests that besides the peristaltic contractions, the more distal stomach was in a general contractile state during stimulation.

In addition to the antral peristaltic contractions and fundic enlargement, other vagal stimulation-induced motility changes were observed. In some preparations, peristaltic contractions were observed on the lateral surfaces of the fundus. Because they did not impinge on the greater curvature, they were not picked up by the frontal video, and were thus not quantified. Another motility effect, as mentioned above, may have been a general contraction of the more distal stomach, particularly with higher stimulation frequencies. This general contraction may not have been detected because the change occurred mainly in the dorso-ventral dimension not picked up by our camera position.

Vagal tone

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. In vitro stomach preparation
  6. Experimental protocol
  7. Image acquisition
  8. Image processing and generation of diameter maps (D-map)
  9. Quantitative analysis of motion from D-maps and statistical analysis
  10. Statistical procedures
  11. Results
  12. Discussion
  13. Vagal tone
  14. Vagal modulation of antral peristaltic contractions
  15. Vagal effect on fundus
  16. Conclusions and Perspectives
  17. Acknowledgments
  18. References

Without vagal stimulation there was little basic motility in our in vitro guinea-pig stomach. The question arises as to whether this absence of motility is normal, or because of the procedures during dissection, such as severing the vagal trunks and connective tissues, aspirating the gastric contents, and exposing the preparation to ice-cold saline.

There are contrasting views on the role of the vagus in the expression of normal gastric motility. On the one hand, it is clear that the stomach can express the major motions of propagating contractions and adaptive relaxation in the absence of vagal input.6–8 On the other hand, there are clearly acute effects41 and chronic deficits in gastric emptying of solids and liquids42,43 after vagal blockade, and research into the anatomical and neurochemical aspects of vagal efferent innervation points to a very strong representation of gastric functions in the brain. The importance of vagal input in maintaining gastric tone has been demonstrated in dogs using transient vagal cooling,41 which led to a decrease in gastric tone, and atropine alone or in combination with adrenergic antagonists produced gastric relaxation that was not further increased by vagal cooling. This suggests that physiological vagal input to cholinergic enteric neurones is necessary to maintain a basal gastric tone. This interpretation is also consistent with observations on firing rate of vagal efferent fibres to the stomach. Vagal efferent discharge contains a strong low-frequency component around 0.1 Hz in the basal state.44 Because both vagal trunks are severed in this preparation, it is a possibility that the absence of detectable motility was due to the lack of vagal efferent input. Bilateral vagal stimulation with as little as one single pulse every 5 s (0.2 Hz) was able to ‘rescue’ basal gastric motility.

Because the criterion used to set the baseline frequency was to obtain low-level antral waves, baseline stimulation frequency varied among individual stomachs, and tended to be higher for the high-volume conditions. We do not believe, however, that these differences in baseline stimulation frequency obscured the major effects of the stepwise-increased vagal stimulation on the various parameters. Partial analysis of the other series of stimulation with a consistent 0 Hz baseline showed that the fundamental responses to full vagal stimulation with 1–16 Hz were similar.

Vagal modulation of antral peristaltic contractions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. In vitro stomach preparation
  6. Experimental protocol
  7. Image acquisition
  8. Image processing and generation of diameter maps (D-map)
  9. Quantitative analysis of motion from D-maps and statistical analysis
  10. Statistical procedures
  11. Results
  12. Discussion
  13. Vagal tone
  14. Vagal modulation of antral peristaltic contractions
  15. Vagal effect on fundus
  16. Conclusions and Perspectives
  17. Acknowledgments
  18. References

The powerful effect of vagal input was demonstrated by the large-amplitude contractions starting often near the proximal corpus, and moving all the way to the pyloric sphincter, obtained with frequencies well in the physiological range. In spite of these large increases of amplitude and length, the period and speed of peristaltic waves did not change much. The period of about 10–12 s (5–6 contractions min−1) was close to the period measured when phasic pressure waves were evoked by rapid gastric filling.8 The speed of propagation also was not significantly changed by increasing vagal stimulation.

These results corroborate earlier findings in anaesthetized ferrets45 and vagotomized dogs,46 indicating a modulatory rather than controlling effect of vagal input, with the basic rhythm and speed of propagation of contractions paced by an intrinsic mechanism and not dependent on vagal input. Interstitial cells of Cajal may play the role of autonomous pacemakers that generate slow wave activity.1,47 However, the fact that under some vagal stimulation conditions, individual contractions were skipped shows that vagal input can affect the initiation of muscle contractions that propagate towards the pylorus. This could be accomplished by inhibitory vagal input to more distal parts of the stomach. It was already reported by Langley48 that in some experiments the distal part of the dog stomach relaxed, and in the ferret, vagal stimulation under ‘NANC conditions’ inhibited spontaneous antral contractions.45 Alternatively this effect could be an artefact due to the simultaneous stimulation of all efferent vagal fibres as is the case with electrical stimulation. Under physiological conditions vagal efferent fibres might be activated in a particular temporal and spatial pattern, resulting in different overall motility effects.

The maximal effect of vagal stimulation on amplitude and length of contraction was already reached at 2–4 Hz, with no further increase at 8 Hz. This is in line with reported spike frequencies recorded from vagal efferent fibres in anaesthetized dogs,49 although peak instantaneous frequencies can be much higher, as recorded in rats.44 Vagal preganglionic fibres responsible for excitatory motor effects such as antral contractions are thought to be low-threshold fibres because they can be activated with relatively low-strength electrical stimulation, compared with high-threshold fibres responsible for inhibition (relaxation) and gastric acid secretion.27 We have chosen suprathreshold parameters (as determined in preliminary experiments) in order to activate both putative types of fibres. Because low-threshold fibres have larger diameters and are faster conducting than high-threshold thin-calibre fibres,50 it could have been expected that they can follow stimulation frequencies higher than 4 Hz. However, stimulation with continuous impulses may be inherently less effective than stimulation with bursts of impulses. Stimulation with a natural burst pattern obtained from recording of vagal afferent fibre discharge gave rise to gastric contractions of larger amplitude than either artificial burst or continuous stimulation in anaesthetized ferrets.51 The natural burst pattern consists of very low to relatively high instantaneous frequencies,44 somehow resulting in higher transmitter release or a different temporal interaction with other modulatory influences on the postganglionic neurones.

Vagal effect on fundus

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. In vitro stomach preparation
  6. Experimental protocol
  7. Image acquisition
  8. Image processing and generation of diameter maps (D-map)
  9. Quantitative analysis of motion from D-maps and statistical analysis
  10. Statistical procedures
  11. Results
  12. Discussion
  13. Vagal tone
  14. Vagal modulation of antral peristaltic contractions
  15. Vagal effect on fundus
  16. Conclusions and Perspectives
  17. Acknowledgments
  18. References

Vagal stimulation produced dramatic enlargements of the fundus. Unable to measure fundic volume changes directly, we estimated this enlargement by measuring the percentage increase in total gastric perimeter. Vagal stimulation-induced fundic enlargement may be the result of active relaxation by the inhibitory vagal pathways or passive dilatation concomitant with increased intragastric pressure as induced by increased antral tone and phasic contractions.

It has been convincingly demonstrated that vagal stimulation-induced fundic relaxation is mediated by nitric oxide (NO) acting as a neurotransmitter at the enteric neuronal-smooth muscle junction.52 Vagal preganglionic neurones have been shown to terminate on NO-producing enteric neurones in the rat fundus.53 There is also some evidence for the additional involvement of adenosine triphosphate transmission.54,55 As the maximum effect was achieved with 4-Hz stimulation under both volume conditions, this response exhibited about the same frequency–response relationship as antral excitation. If these NANC inhibitory effects were because of the recruitment of thin-calibre high-threshold vagal preganglionic fibres,27 they were expected to show a frequency–response relationship reaching its maximum at low frequencies, perhaps lower than for the excitatory effects. The maximum effective frequency of about 4 Hz is low and comparable with the optimal frequency to stimulate gastric acid secretion in the rat,56 but not lower than for antral contractions. Measuring relaxation by means of force transducers sewn on the fundic serosa, the optimal frequency for vagal trunk stimulation was 10 Hz in the dog57 and 2.5 Hz (rapid transient relaxation) or 10 Hz (prolonged relaxation) in the rat.12 In the latter study, the optimum frequency to produce phasic contractions was 5 Hz. Collectively these studies demonstrate that it is impossible to clearly distinguish the two putatively different vagal fibre populations on the basis of a different frequency–response relationship.

Fundic expansion during vagal stimulation could also be passively induced by increased intragastric pressure resulting from either increased antral tone and/or the phasic antral contractions. With the increased temporal resolution used in this study, it is possible to detect both components. The peaks of phasic distensions of the fundus were often associated with the initiation of antral peristaltic waves in the distal corpus/orad antrum. Contractions in this region were broad and are likely to cause significantly greater fluid displacement than the narrower contractions in the distal antrum. This phasic displacement of volume and higher intraluminal pressures are likely to expand the thin distensible wall of the fundus passively, as it is unlikely that intrinsic inhibitory reflexes activated by intraluminal distension are activated so quickly. However, the ‘background’ level of fundic distensibility is likely to be set by ongoing release of NO from inhibitory neurones, activated by vagal inhibitory pathways, or intrinsic reflex pathways.

The results of the present study do not allow distinguishing between active and passive fundic enlargement. Additional experiments using pharmacological intervention will be necessary. Preliminary observations in a few preparations showed that in the presence of the Nitro orginine NO inhibitor NOARG, fundic enlargement was substantially reduced, while in the presence of the cholinergic blocker scopolamine, fundic enlargement was reduced but not abolished.

Conclusions and Perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. In vitro stomach preparation
  6. Experimental protocol
  7. Image acquisition
  8. Image processing and generation of diameter maps (D-map)
  9. Quantitative analysis of motion from D-maps and statistical analysis
  10. Statistical procedures
  11. Results
  12. Discussion
  13. Vagal tone
  14. Vagal modulation of antral peristaltic contractions
  15. Vagal effect on fundus
  16. Conclusions and Perspectives
  17. Acknowledgments
  18. References

Clear advantages of this new video method are its continuous high temporal and spatial resolution and the possibility for controlled pharmacological intervention. If a second camera were used to capture gastric motility in an orthogonal plane, regional volume changes over time could additionally be estimated. Addition of pharmacological blockers to a few preparations in this study and earlier in vitro preparations8 also demonstrate the great potential of this method to study the multiple transmission mechanisms and their interactions on the final integrated motor output.

It is less clear whether this preparation is suitable to study the selective activation of ‘function-specific’ vagal efferent pathways. Although autonomic outflow has been traditionally thought of as monolithic effector systems that are globally active at a certain level (tone), there is now considerable evidence for function-specific sympathetic outflow.58 Function-specific vagal outflow is less understood, but the variable effects on gastrointestinal and pancreatic functions of numerous peptides applied to the vagal motor nucleus point to such an organization. If the video method could be adapted to an ex vivo preparation, with an intact brainstem and vagal connections to the stomach, it should be possible to selectively activate specific populations of vagal efferent pathways and observe the resulting motility effects with the same high temporal and spatial fidelity.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. In vitro stomach preparation
  6. Experimental protocol
  7. Image acquisition
  8. Image processing and generation of diameter maps (D-map)
  9. Quantitative analysis of motion from D-maps and statistical analysis
  10. Statistical procedures
  11. Results
  12. Discussion
  13. Vagal tone
  14. Vagal modulation of antral peristaltic contractions
  15. Vagal effect on fundus
  16. Conclusions and Perspectives
  17. Acknowledgments
  18. References

This study was part of a sabbatical leave project of H-R. B. at the Department of Human Physiology, Flinders University. The research was partially supported by the National Health and Medical Research Council of Australia (Grant no. 27593) and the National Institute of Diabetes and Digestive and Kidney Diseases (Grant DK 57242 to H-R. B.). We thank Nan Bao for help with the data collection and analysis.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. In vitro stomach preparation
  6. Experimental protocol
  7. Image acquisition
  8. Image processing and generation of diameter maps (D-map)
  9. Quantitative analysis of motion from D-maps and statistical analysis
  10. Statistical procedures
  11. Results
  12. Discussion
  13. Vagal tone
  14. Vagal modulation of antral peristaltic contractions
  15. Vagal effect on fundus
  16. Conclusions and Perspectives
  17. Acknowledgments
  18. References
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