Department of Pharmacology, College of Medicine, University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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Ozaki, Noriyuki,
J. N. Sengupta, and
G. F. Gebhart.
Mechanosensitive Properties of Gastric Vagal Afferent Fibers
in the Rat.
J. Neurophysiol. 82: 2210-2220, 1999.
Single, teased fiber recordings were made from the
decentralized right cervical vagus nerve (hyponodal) of the rat. A
total of 67 afferent fibers that responded to gastric distension (GD) were studied: 9 fibers were stimulated by phasic balloon GD, 58 by more
natural fluid GD. All balloon GD-responsive fibers had resting
activity (3.1 imp/s), and 57/58 fluid GD responsive fibers had resting
activity (1.3 imp/s). All balloon GD-responsive fibers exhibited a
dynamic response to phasic distension followed by slow adaptation,
whereas fluid GD-responsive fibers exhibited increasing responses as
intragastric pressure increased, followed typically by slow adaptation.
Responses to graded GD were studied in all fibers, and all gave
increasing responses to increasing pressures (5-60 mmHg). Thresholds
for response varied between 0 and 18 mmHg. Mean response thresholds for
two durations of fluid GD (30 and 60 s) were 5.6 and 3.9 mmHg; the
mean response threshold to phasic balloon GD (30 s duration) was 5.3 mmHg. The potential sensitizing effect of platelet activating factor
(PAF, 50 or 100 ng · kg1 · min
1 for 20 min) infused into the gastric artery was studied in 20 fibers. Fifteen
fibers exhibited an increase in spontaneous activity; intragastric
pressure also slightly increased during PAF infusion. The increase in
activity produced by PAF was attenuated in the presence of the PAF
receptor antagonist WEB 2086. After PAF-induced acute inflammation of
the stomach, three of five fibers studied did not exhibit any change in
response to graded GD. The present study characterized
distension-sensitive afferent fibers in the right cervical vagus
innervating the stomach of the rat by balloon GD and fluid GD. The
results document that all distension-sensitive gastric vagal afferent
fibers encoded the intensity of GD, but none had response thresholds in
what might be considered the noxious range. PAF infusion activated
mechanosensitive gastric vagal afferent fibers, but acute inflammation
produced by PAF did not sensitize responses to GD.
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INTRODUCTION |
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The upper abdominal gastrointestinal (GI) tract receives dual innervation by the vagus and spinal visceral nerves (primarily the splanchnic nerve). These afferent nerve fibers play an integral part in conveying information from the gut to the CNS relative to regulatory functions (e.g., absorption, secretion, storage, propulsion, and emptying) and conscious sensations (e.g., satiety, nausea, discomfort, and pain). The most compelling sensations that arise from the GI tract are discomfort and pain. Although considerable information has accumulated about the morphology and neuropeptide contents of afferent fibers innervating the GI tract, our understanding of the functional role of these fibers remains limited.
Most nonhuman animal studies have focused on the roles of visceral
afferent fibers in GI motility, secretion, and other regulatory functions (for reviews see Cervero 1994; Sengupta
and Gebhart 1994
; 1995
). It is generally held
that vagal afferent fibers do not convey nociceptive information, but
vagal afferent fibers innervating the inferior-posterior surface of the
heart (Meller and Gebhart 1992
) and the upper
respiratory tract (Barnes 1991
; Coleridge and
Coleridge 1984
) may contribute to visceral pain sensation.
Previous studies of vagal afferent fibers have addressed their
involvement in regulatory mechanisms (e.g., motility) and chemosensitivity under normal physiological conditions. Their mechanosensitivity, particularly associated with discomfort and pain,
has not been studied.
Functional bowel disorders are characterized by altered sensory
perceptions (e.g., bloating, discomfort, and pain), which can arise
from changes in peripheral or/and central neuron excitability (see
Mayer and Gebhart 1994). The aim of this study was to
gain information about the response characteristics of vagal afferent fibers innervating the stomach. Accordingly, we quantitatively characterized the mechanosensitive properties of vagal afferent fibers
innervating the stomach, investigating the reproducibility and
intensity-coding properties of these afferent fibers to graded fluid
and balloon gastric distension (GD). To determine whether acute
inflammation of the stomach would provide insight into afferent mechanisms that could contribute to altered sensations, we also examined the effect of platelet activating factor (PAF) on gastric afferent fibers and their response to GD. We chose to use PAF because
it is an endogenous mediator of inflammation (Zimmerman et al.
1992
), and its content in both gastric juice and mucosa is
increased in patients with ulcers (Ackerman et al. 1990
;
Sobhani et al. 1992
). Some of these data have been
presented previously in abstract form (Ozaki et al.
1998
; Sengupta et al. 1997
).
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METHODS |
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General procedures
Experiments were performed on 52 male Sprague-Dawley rats
(Harlan, Indianapolis, IN; 400-500 g). Food, but not water, was withheld for 24 h before surgery. The rats were anesthetized
initially with an intraperitoneal injection of pentobarbital sodium
(Nembutal, Abbott Laboratories, Abbott Park, IL; 45-50 mg/kg) and
subsequently maintained with a constant intravenous infusion of
pentobarbital (5-10 mg · kg1 · h
1). The right femoral vein was cannulated for
infusion of fluid and anesthetic. The right femoral artery was
cannulated and connected to a pressure transducer for monitoring blood
pressure and heart rate. The mean arterial pressure was maintained at
>80 mmHg with supplemental intravenous injection of 5% dextrose in
saline administered in a bolus of 1-1.5 ml as required. The trachea
was intubated to permit artificial ventilation with room air. The rat
was paralyzed with pancuronium bromide (0.2-0.3 mg/kg iv) and
mechanically ventilated with room air (~70 strokes/min, 2-2.5 ml
stroke volume). Supplemental doses of pancuronium bromide (0.2-0.3 mg
· kg
1 · h
1) were
given to maintain paralysis during the experiment. Core body
temperature was maintained at 36°C by a hot water circulating heating
pad placed under the rat and an overhead feedback-controlled heat lamp
(thermoprobe inserted into the rectum; Yellow Springs Instrument,
Yellow Springs, OH). At the end of experiments, rats were killed with
an overdose of pentobarbital. The experimental protocol was approved by
the Institutional Animal Care and Use Committee, The University of Iowa.
The abdomen was opened by a transverse epigastric incision 4-5 cm in length. To measure afferent fiber conduction velocity, a bipolar wire electrode was placed on the vagal trunk at the esophageal-gastric junction in some experiments. The right vagus nerve was isolated from the esophagus, and a pair of Teflon-coated, 40-gauge stainless steel wires stripped at the tips were placed around the nerve and sealed with nonreactive Wacker gel (Wacker Silicone, Adrian, MI).
For phasic balloon GD (n = 8 rats), a 2.0-2.5 cm long,
2-3 cm diam flaccid, flexible latex balloon was placed surgically in the stomach through the fundus. The balloon occupied approximately two-thirds of the proximal stomach. The pylorus was not obstructed, and
there was no blockage of gastric emptying. The outside diameter of the
balloon when inflated was greater than the intraluminal diameter of the
stomach of the rat. Therefore the pressure measured during GD reflected
actual intragastric pressure. The balloon catheter was connected to a
distension control device via a low-volume pressure transducer (see
Gebhart and Sengupta 1996 for details).
For fluid GD (n = 44 rats), the stomach was intubated with flexible plastic tubing (Tygon, Fisher Scientific Co., Pittsburgh, PA; 2.3 mm OD, 1.3 mm ID) via the mouth, esophagus, and cardia. The catheter was secured by a ligature around the esophageal-gastric junction. Another Tygon tube (3.9 mm OD, 2.4 mm ID) was introduced distally through the pylorus and was secured by a ligature placed caudal to the pyloric sphincter; the duodenum was ligated close to the pyloric ring. For GD, the oral catheter was connected to a reservoir containing saline at room temperature. Constant pressure distension was controlled by the distension control device with the distal catheter clamped. Intragastric pressure was monitored by connecting the distal catheter via a three-way stopcock to a low-volume pressure transducer. The left gastric artery was freed from connective tissue under a stereomicroscope and cannulated for infusion of PAF in 22 rats. The abdomen was closed with silk sutures.
Recording of afferent nerve activity
The right vagus nerve was exposed by a ventral midline incision in the neck. The sternocleidomastoid, sternohyoid, and omohyoid muscles were removed. The skin was reflected laterally and tied to the frame to make a pool for warm mineral oil (37°C). The nerve was dissected away from the carotid tissue sheath, decentralized close to its entry to the nodose ganglion, and placed over a black micro-base plate. The perineural sheath was removed in the pool of warm mineral oil (37°C), the nerve was split into thin bundles, and fine filaments were teased from the bundle to obtain a single unit. Electrical activity of the single unit was recorded by placing the fiber over one arm of a bipolar silver-silver chloride electrode. A fine strand of connective tissue was placed over the other pole of the electrode for differential recording.
Action potentials were monitored continuously by analogue delay and displayed on a storage oscilloscope after low noise AC differential amplification. Action potentials were processed through a window discriminator and counted (1-s binwidth) on-line using the SPIKE2/CED 1401 data acquisition program. Peristimulus time histograms, intragastric pressure, and blood pressure were displayed on-line continuously. Data were also recorded on tape for later analysis.
Experimental protocol
Mechanosensitive gastric muscle afferents in the vagus nerve
were identified by response to a test stimulus of GD (40 mmHg, <5 s).
This intensity of GD was chosen because it is in excess of the mean
response threshold for high-threshold mechanosensitive fibers
innervating other viscera (see Sengupta and Gebhart
1995). If a fiber responded to 40 mmHg GD, a stimulus-response
function (SRF) to distending pressures of 5, 10, 20, 30, 40, and 60 mmHg, 30 or 60 s at 4-min intervals was determined.
To measure conduction velocity, the vagus nerve was stimulated with a
single 0.5-ms square-wave pulse at 3-8 mA, and the conduction delay
(time between stimulus artifact and evoked response) was recorded. The
conduction distance was measured postmortem. Fibers were classified on
the basis of their conduction velocities; those with conduction
velocities <2.5 m/s were considered unmyelinated C-fibers, and those
with conduction velocities >2.5 m/s were considered thinly myelinated
A-fibers.
The effect of PAF on mechanosensitive gastric afferent fibers was
tested as were responses to GD following the acute inflammation produced by PAF. PAF in 0.25% bovine serum albumin (BSA in saline) was
infused via the left gastric artery (50 or 100 ng · kg1 · min
1 for 20 min). The volume of infusion was <0.1 ml/min. Because PAF produces
smooth muscle contraction (Stimler and O'Flaherty 1983
), changes in activity of these mechanosensitive fibers to PAF may reflect an indirect effect due to contraction. To test whether
responses to PAF were direct or secondary to muscle contraction, 12 rats were pretreated with loperamide (50 mg/kg iv) 5 or 25 min before
the experiment in an attempt to eliminate gastric smooth muscle
contractions; one rat was treated with loperamide during PAF infusion.
The effect of intragastric artery infusion of vehicle (0.25% BSA in
saline) was tested in nine rats. To determine whether drug effects were
produced at the PAF receptor, the PAF receptor antagonist WEB 2086 (Hines and Fisher 1992
) was administered intravenously 5 min before starting the intragastric artery infusion of PAF. WEB 2086 was given in a dose of 250 mg/kg, and PAF was infused at 50 ng · kg
1 · min
1 for 20 min. In addition, WEB 2086 was tested in other experiments where it was
injected 5 min after starting the infusion of PAF. The effect of acute
inflammation produced by PAF was tested on seven mechanosensitive
afferent fibers. Responses of the fibers to graded GD were determined
30 and 90 min after the 20-min infusion of PAF.
At the end of the protocol for each fiber, the abdomen was opened, and the mechanosensitive receptive field was located by probing the stomach with a fine, blunt glass rod. At the end of an experiment, stomachs from treated and untreated rats were removed, opened along the greater curvature, pinned flat (mucosal side up), and examined under a dissecting microscope to qualitatively assess inflammation.
Drugs
Platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was obtained from Sigma Chemical (St. Louis, MO). An aliquot of a stock 2 mg/ml solution of PAF in chloroform was evaporated and reconstituted in 0.25% (wt/vol) BSA in 0.9% (wt/vol) saline when required. WEB 2086 was kindly provided by Dr. Rory A. Fisher (University of Iowa) and was dissolved in 0.9% saline immediately before use. Loperamide hydrochloride was obtained from Sigma and was dissolved in 0.9% saline.
Data analysis
The resting activity of a fiber was counted for 60 s before GD, and the response to GD was determined as the increase in discharge during GD above its resting activity (imp/s). Stimulus-response functions (SRF) to graded GD were plotted for each individual fiber, and a least-squares regression line was obtained from the linear part of the SRF. The regression line then was extrapolated to the ordinate (representing distension pressure) to estimate response threshold. The response to PAF infusion was determined as the maximum discharge during infusion (imp/s).
All data are expressed as means ± SE. Results were analyzed using Student's t-test or ANOVA. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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Fiber sample
A total of 67 vagal afferent fibers (in 52 rats) were studied, all of which responded to GD. In the presence of the balloon in the stomach (n = 9 fibers in 8 rats), fibers exhibited ongoing activity (3.1 ± 1.5 imp/s, mean ± SE; range: 0.02-14.2 imp/s). Of the 58 fibers (in 44 rats) that responded to fluid GD, 1 had no spontaneous activity, and 57 were spontaneously active (1.3 ± 0.2 imp/s; range: 0.01-5.5 imp/s; P < 0.05 vs. balloon GD). This group of 57 spontaneously active fibers consisted of 29 in which the gastric artery had been cannulated (1.4 ± 0.3 imp/s) and 28 in which the artery was not cannulated (1.3 ± 0.3 imp/s).
Conduction velocities of 27 fibers that responded to fluid GD were measured by electrical stimulation of the vagus nerve. All tested fibers were unmyelinated C-fibers (mean conduction velocity: 0.70 ± 0.06 m/s, range 0.4-2.2 m/s).
Responses to GD
The nine fibers tested with balloon GD typically gave an initial dynamic response followed by a slowly adapting response during maintained GD (60 mmHg; e.g., Fig. 1A). Slow adaptation to a tonic discharge was generally observed at all intensities of GD.
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Fibers tested with fluid GD (60 mmHg) typically gave slowly incrementing responses as intragastric pressure increased, and either began to slowly adapt during maintained pressure or sustained responses until termination of distension. Examples of responses to fluid GD are shown in Figs. 1 and 2. Because distension pressure typically became maximal at the end of 30-s fluid GD, there was no opportunity to observe adaptation during distension (e.g., see Fig. 1B). Fibers tested with longer duration, 60-s fluid GD (n = 46) did permit observation of adaptation during stimulation: 38 fibers were slowly adapting (e.g., Fig. 2C). The remaining eight fibers were either nonadapting (e.g., Fig. 1C) or appeared to quickly adapt near or at peak distending pressure as if the stomach had relaxed (although intragastric pressure did not decrease).
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Following termination of either phasic balloon or fluid GD, many fibers gave evidence of a period of poststimulus inhibition (Fig. 1). The frequency of discharge fell below the resting level of activity after termination of GD in 5/9 fibers tested with 30-s balloon GD, 3/12 fibers tested with 30-s fluid GD, and 17/46 fibers tested with 60-s fluid GD. Most fibers tested also exhibited prolonged afterdischarge at rates greater than resting. Although we did not follow the duration of all afterdischarges, afterdischarges in six of nine fibers tested with 30-s balloon GD continued for 23 s to >145 s after termination of 60 mmHg GD. Eight of 12 fibers tested with 30-s fluid GD gave afterdischarges that ranged from 43 to >220 s (Fig. 2B). Fourteen of 16 fibers tested with 60-s fluid GD and 26/30 fibers tested with 60-s fluid GD in stomachs with the gastric artery catheterized gave afterdischarges that ranged from 12 to >190 s (e.g., Fig. 2C). There was no relationship between afterdischarge and response thresholds of the fibers (see Table 1). The characteristics of gastric vagal afferent fibers to 60 mmHg GD are summarized in Table 1.
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Eighteen of 67 fibers tested with GD exhibited intermittent phasic changes in resting activity that were maintained during and after termination of distension (Fig. 3). These changes were not noted to be associated with spontaneous changes in intragastric pressure.
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Stimulus-response functions (SRFs)
Responses to graded balloon or fluid GD were studied in all 67 fibers. SRFs of fibers in the different experimental groups are given in Fig. 4. Extrapolation of the linear portion of individual SRFs revealed that gastric vagal afferent fibers exhibited a range of thresholds for response to GD. The mean response threshold to 30-s balloon GD was 5.3 ± 1.7 mmHg (range 0-13.8 mmHg), to 30-s fluid GD 5.6 ± 1.8 mmHg (range 0-15.5 mmHg), to 60-s fluid GD 3.9 ± 1.1 mmHg (range 0-13.2 mmHg), and to 60-s fluid GD in gastric artery-catheterized stomach 5.7 ± 1.0 mmHg (range 0-18.3 mmHg; Fig. 5). Mean response thresholds did not differ between groups.
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The mean SRFs of vagal afferent fibers of the SRF to GD are presented in Fig. 6. When responses to the more natural fluid filling of the stomach were compared with responses to phasic balloon GD, response magnitude generally was greater to fluid GD, particularly during the second half of the period of distension. Although GD was delivered at a constant pressure whether by balloon or fluid, the rate at which the final pressure was achieved and the duration of the target pressure differed for the two types of GD. We would have expected responses to phasic balloon GD to be of greater magnitude than fluid GD, but this was apparent for only the first 10 s of GD. The slope and mean response magnitudes of afferent fibers to 60-s fluid GD in gastric artery catheterized stomach were significantly lower than responses to the same stimulus in naïve stomach (F = 32.8; P < 0.01).
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Gastric receptive fields (RFs)
Receptive fields in the stomach were found for 2/12 fibers tested with 30-s fluid GD, 2/16 fibers tested with 60-s fluid GD, and 9/30 fibers tested with 60-s fluid GD (gastric artery catheterized). No RFs were located in the cardia or fundus; all 13 RFs were associated with the corpus. Characteristics of these fibers are presented in Table 2.
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Effect of PAF on gastric mechanosensitive vagal afferent fibers
The effect of gastric artery infusion of PAF (20 min) was tested
in 20 fibers, in the absence or presence of loperamide, 13 during 50 ng · kg1 · min
1 PAF and
7 during 100 ng · kg
1 · min
1 PAF.
Spontaneous activity increased in 9/13 and 6/7 fibers
during 50 and 100 ng · kg
1 · min
1
infusion of PAF. Mean spontaneous activity increased from 1.3 ± 0.3 imp/s to a mean maximum response 16.9 ± 2.9 imp/s
(n = 9) during 50 ng · kg
1
· min
1 PAF infusion and from 0.5 ± 0.3 imp/s to
15.2 ± 2.7 imp/s (n = 6) during 100 ng · kg
1 · min
1 PAF. Figure
7 shows examples of the effect of
infusion of 100 ng · kg
1 · min
1 PAF.
One fiber was tested to a second infusion of PAF started 15 min after
conclusion of the first infusion. The fiber responded to the first PAF
infusion but did not respond to the second PAF infusion (Fig.
7B).
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The effect of gastric artery infusion of 0.25% BSA was tested in nine fibers. No fibers exhibited increases or decreases in spontaneous activity during BSA infusion. In six fibers, BSA was tested before PAF infusion, five of which gave increases in spontaneous activity to PAF (Fig. 8). In two fibers, BSA was tested 15 min after PAF infusion. Both fibers responded to PAF; neither of them responded to BSA.
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Intragastric pressure increased during PAF infusion (see Figs. 7 and
8). The mean maximum increase in intragastric pressure was 4.1 ± 0.4 mmHg (n = 7). To test whether the response of
afferent fibers to PAF was due to direct activation of the nerve ending or secondary to changes in tension of the smooth muscle during PAF-induced contractions, the effect of PAF was tested in 13 fibers 5 min after loperamide (50 mg/kg iv) administration. Seven of 10 fibers
tested with 50 ng · kg1 · min
1 PAF
and 2/3 fibers tested with 100 ng · kg
1 · min
1 PAF exhibited a response to PAF after loperamide
treatment, suggesting a direct action of PAF. The response to PAF
(doses combined) in the absence of loperamide was 15.9 ± 2.2 imp/s (n = 6) and in the presence of loperamide was
14.8 ± 3.0 imp/s (n = 9;
P > 0.05). However, the intragastric pressure
still increased slightly (mean maximum increase: 3.7 ± 0.4 mmHg,
n = 13) during infusion of PAF.
To test whether the PAF receptor was involved in the increases in spontaneous activity during PAF infusion, the effect of the specific PAF receptor antagonist WEB 2086 (125 mg/kg iv) was tested in five fibers. The antagonist was injected into the femoral vein during PAF infusion into the gastric artery in five fibers and clearly attenuated the response to PAF in three fibers without affecting the PAF-induced increase in intragastric pressure (Fig. 9). The effect of WEB 2086 administered during PAF infusion was to attenuate the PAF-induced increase in activity to 60.3% of the activity just before WEB 2086 administration. The PAF-induced increase in intragastric pressure was maintained after WEB 2086 administration (102.3% of intragastric pressure before WEB 2086).
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The mechanosensitive properties of five fibers were tested after
infusion of 50 or 100 ng · kg1 · min
1
PAF (20 min) into the gastric artery. The spontaneous activity of three
fibers increased during PAF infusion. Responses to GD did not change at
any of the pressures of GD tested (5-60 mmHg) either 30 or 90 min
after PAF infusion. Figure 10 shows an
example of responses of an afferent fiber to graded GD before, 30 and 90 min after infusion of PAF, and Fig.
11 illustrates the mean SRFs of fibers
before and 30 and 90 min after PAF infusion.
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Macroscopic examination of PAF-treated stomachs at the end of experiments revealed acute damage relative to stomachs taken from untreated rats. Damage consisted of both hemorrhagic and nonhemorrhagic lesions. A nonhemorrhagic lesion was defined as one that had a shallow erosion without any trace of blood; hemorrhagic lesions had deeper damage with blood at the erosion site.
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DISCUSSION |
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The principal findings of the present study are that high-threshold mechanosensitive receptors were not present in the sample of vagal afferent C-fibers studied and that PAF did not sensitize these receptors to GD. These results suggest that gastric vagal afferent fibers likely play no role in acute gastric pain. The fibers studied, however, did encode increasing intensities of GD and thus likely contribute to altered gastric sensations. Phasic balloon GD-responsive fibers typically exhibited a dynamic response followed by slow adaptation during GD, whereas fluid GD-responsive fibers exhibited an increasing response as intragastric pressure increased, followed by either slow adaptation or no adaptation during GD. SRFs to graded GD revealed that gastric vagal C-fibers have a range of thresholds for response, but no response thresholds exceeded what might be considered an intensity in the noxious range. Gastric distension-responsive fibers also responded to PAF, an effect mediated by the PAF receptor, but were not sensitized by PAF-produced gastric inflammation.
Vagal innervation of the stomach
Electron microscopic studies of the abdominal vagus nerve in the
rat estimate that there are 10,000 unmyelinated fibers (Gabella and Pease 1973). Quantitative studies of subdiaphragmatic
dorsal and ventral vagal branches innervating abdominal visceral organs revealed that dorsal and ventral gastric branches, celiac branches, and
hepatic branches of the vagus consist primarily of unmyelinated afferent fibers; the total number of unmyelinated fibers in the rat has
been estimated to exceed 99% of all vagal fibers (Prechtl and
Powley 1990
).
The conduction velocity of vagal afferent fibers in the stomach has
been reported to range from 6.5 to 13 m/s (Paintal
1954b) or <1.3 m/s (Iggo 1957
) in the cat,
2-12 m/s in the goat (Iggo 1955
), 1.1 ± 0.1 m/s
in the dog (Takeshima 1971
, 1974
),
1.0-1.7 m/s in the sheep (Falempin et al. 1978
),
0.91 ± 0.21 m/s in the ferret (Andrews et al.
1980
), and 0.4-1.6 m/s in the rat (Davison and Clarke
1978
, 1988
). The majority of gastric muscle
afferent fibers in the rat are unmyelinated C-fibers. The present
results are in accord with these morphological and electrophysiological studies. Conduction velocities of 27 fibers tested were unmyelinated C-fibers, 26 having a conduction velocity
1.0 m/s. Because it was not
possible in all experiments to place stimulating electrodes at the
gastroesophageal junction, some of the fibers studied may have been
more rapidly conducting A
-fibers.
Gastrointestinal distension-responsive afferent fibers are generally
considered to be muscle afferent fibers. The location of
distension-sensitive afferent fibers in muscle layers has been demonstrated by removal of the underlying mucosa and submucosa (Cottrell 1984; Iggo 1957
;
Takeshima 1971
) without impairing responses to
distension. Recent morphological studies have also confirmed the
locations and structures of the sensory endings in the stomach. Berthoud and Powley (1992)
injected DiI in the nodose
ganglion of the rat and reported that labeled vagal afferent fiber
endings terminated in longitudinal and circular muscle layers of the
stomach and duodenum, sending collaterals to the myenteric plexus,
mucosa, and submucosa.
Vagal afferent fibers supplying the stomach have ongoing activity, and
some exhibit periodic bursts with the respiratory cycle (Andrews
et al. 1980; Blackshaw et al. 1987
;
Davison and Clarke 1988
; Grundy et al.
1995
; Iggo 1955
; Paintal 1954b
;
Takeshima 1971
). The spontaneous activity of gastric
afferent fibers in these reports ranged from 1.5 to 38 imp/s,
consistent with resting activity between <1 and 14 imp/s in the
present study. We also observed phasic, regular changes in activity
that were maintained during GD (e.g., Fig. 3). These bursts, however,
did not cycle with respiration (~70/min).
Response to GD
Responses of GD-sensitive vagal afferent fibers to phasic gastric
distension (constant volume) were reported to consist of three major
components during and immediately after distension: 1) a
dynamic response (initial burst), 2) a static or sustained response, with or without random fluctuations during maintained GD, and
3) a pause or silent period after the termination of GD (Blackshaw et al. 1987; Davison and Clarke
1988
; Falempin et al. 1978
; Iggo
1955
, 1957
; Leek 1969
;
Paintal 1954b
; Takeshima 1971
, 1974
). The dynamic response consists of a burst of
discharges at the onset of phasic distension. This high-frequency of
discharge is likely due to an initial, high tension that develops
during active resistance offered by smooth muscle and local excitatory reflexes produced by intrinsic nerves. The afferent discharge then
declines during the period of distension, believed to be due to
reflexive relaxation of the stomach. With termination of the distending
stimulus, the discharge of fibers typically falls below predistension
spontaneous activity; occasionally there is transient silence. The
duration of the silent period has been suggested to be related to the
magnitude of distension and to the rate of deflation (Davison
and Clarke 1988
). It has also been reported that during phasic,
step inflation of the stomach of the rat, the initial dynamic response
was greatest at low volumes of distension; as the distension volume was
increased, the dynamic response decreased. However, the static response
of the fibers remained unaltered and followed linearly the step
increases in gastric volume (Davison and Clarke 1978
).
In the present study, constant pressure, phasic balloon distension (30 s duration) led typically to 1) a dynamic, initial burst,
2) a slowly adapting response, and 3) a pause or
silent period at the termination of GD. However, the initial dynamic
response was not greatest at low pressures of GD as was reported for
low volumes of distension. This likely reflects differences in the
methods of GD as well as the 4-min intertrial interval in the present
study (rather than step increases) between distensions. Davison
and Clarke (1978)
incremented intragastric volume stepwise with
a 10-s interval or no interval between steps. Gastric vagal afferent
fiber responses to fluid distension exhibited slowly increasing
responses clearly associated with increasing intragastric pressure.
When gastric pressure plateaued (during 60-s fluid distension), fibers
typically exhibited slow adaptation. In contrast with balloon GD, and
consistent with the rate of pressure increase, fibers did not give
dynamic responses at the onset of fluid GD because fluid distension
slowly increases initial tension that may not develop active resistance in smooth muscle. Some investigators have reported that a small number
of afferent fibers in the vagus nerve exhibit rapid adaptation during
phasic distension (Falempin et al. 1978
; Niijima
1962
; Takeshima 1971
, 1974
),
which was observed in a small number of fibers studied here.
Some fibers exhibited spontaneous fluctuations in discharge at
rest as well as during and after distension. Such fluctuations suggest
gastric motor activity (peristalsis), although we did not observe
intragastric pressure waves when studying these fibers. The
fluctuations may alternatively reflect muscle contraction of localized
peristalsis in the stomach; localized peristalsis may not affect or be
reflected in intragastric pressure of the whole stomach. Two types of
distension-sensitive afferent fibers were identified in the vagus nerve
of dog (Takeshima 1971, 1974
) and ferret
(Andrews et al. 1980
). Stretch receptors (sensitive to
distension) primarily innervate the fundus and corpus, and tension
receptors (sensitive to distension and contraction) primarily innervate
the antrum and pylorus in these animals. In the present study, those
fibers exhibiting spontaneous fluctuations may have been associated
with tension receptors, although we did not discern a pattern of
response properties associated with location of receptive field.
Response patterns of vagal afferent fibers to balloon and fluid GD
differ (Andrews et al. 1980; Blackshaw et al.
1987
; Davison and Clarke 1978
,
1988
; Falempin et al. 1978
; Grundy
et al. 1995
; Iggo 1955
, 1957
;
Leek 1969
; Niijima 1962
; Paintal
1953
, 1954a
,b
; Yoshida-Yoneda et al.
1996
). Certainly, fluid GD more closely approximates a natural
stimulus, although the stomach is not normally filled as quickly as in
these (and other) experiments. Fluid GD also permits measurement of
changes in intragastric pressure more accurately, likely because the
stomach is more uniformly filled by fluid than by the balloon, which is
surgically inserted into the fundus and accordingly distends the fundus
more than corpus, antrum, and pyloric regions.
The right gastric artery was catheterized in some experiments. We noted that the slope and magnitude of the SRF to 60-s fluid GD in catheterized stomach were significantly lower than responses to the same stimulus in naive stomach. One might have expected responses to GD to be greater (i.e., sensitized) if not the same when the additional insult of catheterization was added to the experimental protocol. Interestingly, further insult by inflammation with PAF also did not sensitize fiber responses to GD (see Effect of PAF infusion).
Thresholds for response to distending stimuli have been reported
for many hollow viscera (for review see Sengupta and Gebhart 1995). Afferent fibers having low and high thresholds for
response have been described, and function has been assigned on the
basis of response threshold. Low thresholds for response are
interpreted as indicating a role for the fibers in regulatory functions
in addition to conscious sensations associated with nonpainful
distention (e.g., fullness, bloating, nausea, etc.) (Cervero and
Jänig 1992
). High thresholds for response have been taken
as evidence for the presence of specific nociceptors that give rise to
discomfort and pain (Cervero 1994
). In the present
study, gastric vagal afferent fibers that responded, whether to balloon
or fluid GD, exhibited a range of thresholds, none of which exceed 20 mmHg. These findings are consistent with previous reports
(Andrews et al. 1980
; Davison and Clarke
1978
, 1988
; Iggo 1957
) and
suggest the absence of high-threshold, potentially nociceptive, gastric
vagal afferent fibers. The absence of high-threshold, mechanosensitive
afferent fibers may be characteristic of vagal afferents in general. No
high-threshold esophageal vagal afferents were found in an earlier
study of the opossum esophagus (Sengupta et al. 1989
).
Effect of PAF infusion
PAF is an endogenous phospholipid that is an important
mediator of inflammation (Zimmerman et al. 1992). PAF is
also a potent vasodilator (Blank et al. 1979
); PAF
increases vascular permeability (McManus et al. 1981
)
and stimulates polymorphonuclear leukocytes to aggregate
(O'Flaherty et al. 1981
). An elevated content of PAF is
present in the gastric juice of patients with erosive gastritis and
oesophagitis (Sobhani et al. 1992
). PAF content has also
been reported to be increased in fundic, antral, and duodenal mucosa from patients with duodenal ulceration (Ackerman et al.
1990
). We thus examined the effect of PAF on gastric vagal
afferent fibers.
Local intra-arterial infusion of PAF causes an initial,
dose-related contraction of the rat stomach followed by a second
contraction after termination of infusion of PAF (Espulgues and
Whittle 1989). Peripheral infusion of tetrodotoxin reduces the
initial contraction and abolishes the following contraction produced by
PAF. It has been suggested that stimulation of neuronal activity by PAF
underlies the gastric motility response. In the present study,
GD-sensitive fibers responded to PAF infusion, an effect mediated by
the PAF receptor. Intragastric pressure slightly increased during PAF infusion, perhaps reflecting smooth muscle contraction of the stomach.
We attempted to address this issue through the use of loperamide to
prevent smooth muscle contraction. Loperamide did not affect fiber
responses to PAF infusion or eliminate the increase in intragastric
pressure produced by PAF. Because WEB 2086 attenuated the effects of
PAF on fiber activity, but not intragastric pressure, the PAF-induced
increase in fiber activity is unrelated to smooth muscle contraction.
Acute inflammation of the stomach was also produced by PAF.
Administration of exogenous PAF to laboratory animals causes gastric ulcerations in the rat (Esplugues and Whittle 1988;
Rosam et al. 1986
) and small bowel inflammation
(Gonzalez-Crussi and Hsueh 1983
; Hsueh et al.
1986
; Wallace and Whittle 1986
). Chronic
idiopathic dyspepsia (CID), a functional bowel disorder, is a clinical
syndrome of the upper abdominal GI tract without any identifiable cause by diagnostic evaluation (Talley and Phillips 1988
). It
has been speculated that the etiology of CID involves either
hypersensitivity of primary afferent fibers and/or CNS neurons (for
reviews see Mayer and Gebhart 1994
; Mayer and
Raybould 1990
). Although not a model of CID, we used PAF to
determine whether inflammation induces sensitization of fiber responses
to GD. None of the vagal afferent fibers studied exhibited altered
responses to the range of GD pressures tested (5-60 mmHg), unlike the
sensitization of visceral primary afferent fibers documented for other
nerves (Häbler et al. 1993
; Sengupta et al.
1996
; Su et al. 1997a
,b
). Decreasing blood
drainage from the mucosa by aggregation of neutrophils (Valone and Goetzl 1983
), neutrophil-derived free radicals
(Droy-Lefaix et al. 1991
), or increased vascular
permeability (Hatakeyama et al. 1991
) could contribute
to PAF-induced gastrointestinal damage. Mustard oil, which has been
documented to sensitize afferent fibers innervating the urinary bladder
(Häbler et al. 1993
; Su et al. 1997a
) and colon (Su et al. 1997b
), is
considered to be a neurogenic inflammatory agent that directly
activates unmyelinated afferents and releases various peptides
(Koltzenburg and McMahon 1986
; Patacchini et al.
1990
). Different mechanisms for producing inflammation may
affect the induction of sensitization of visceral primary afferent
fibers. Alternatively, GD-responsive vagal afferent fibers in the rat
may sensitize to stimuli not tested here.
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ACKNOWLEDGMENTS |
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We thank M. Burcham for preparation of the figures and S. Birely for secretarial assistance.
This research was supported by National Institute of Neurological Disorders and Stroke Grants NS-19912 and NS-35790.
Present address of J. N. Sengupta: Gastrointestinal Pharmacology, Preclinical R&D, Astra Hassle Ab., S-431 83 Molndal, Sweden.
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FOOTNOTES |
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Address for reprint requests: G. F. Gebhart, Dept. of Pharmacology, The University of Iowa College of Medicine, 2-471 Bowen Science Bldg., Iowa City, IA 52242.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 March 1999; accepted in final form 4 June 1999.
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REFERENCES |
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