Characterization of mechanosensitive splanchnic nerve afferent fibers innervating the rat stomach

Noriyuki Ozaki and G. F. Gebhart

Department of Pharmacology, College of Medicine, University of Iowa, Iowa City, Iowa 52242


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Splanchnic nerve fibers innervating the stomach were studied in anesthetized rats; 997 fibers in the T9 or T10 dorsal roots were identified by electrical stimulation of the splanchnic nerve. Thirty-one fibers responded to gastric distension. Extrapolated response thresholds ranged between 0 and 53 mmHg; seven fibers had thresholds for response >= 30 mmHg. Thermo- and/or chemosensitivity was tested in 18 of the 31 fibers. Four of twelve fibers responded to intragastric perfusion of heated saline; none of eight fibers tested responded to perfusion of cold saline. Infusion of glucose, L-arginine, or potassium oleate produced no change in resting activity. Intragastric instillation of 12% glycerol or an inflammatory soup (bradykinin 10-5 M, PGE2 10-5 M, serotonin 10-5 M, histamine 10-5 M, and KCl 10-3 M) and prior heat stimulation sensitized responses to distension. The results reveal the presence of low- and high-threshold mechanosensitive fibers in the splanchnic innervation of the stomach. These fibers have the ability to sensitize, and they likely contribute to pain and altered sensations that can arise from the stomach.

visceral pain; sensitization; thermosensitivity; chemosensitivity; functional gastrointestinal disorders


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AFFERENT (sensory) nerve fibers in the vagus or splanchnic nerves that innervate the upper gastrointestinal tract provide information to the central nervous system that leads to a variety of consciously perceived sensations. These sensations include satiety, nausea, bloating, discomfort, and pain, the latter associated with mechanoreceptor endings in muscle that respond to stretch or distension of the organ (see Refs. 11 and 64 for review).

The presence of populations of low- and high-threshold mechanosensitive afferent fibers that innervate hollow viscera has been well documented (7, 10, 13, 27-29, 34, 60-63, 68). Low thresholds for response are interpreted as indicating a role in regulatory functions (e.g., storage, propulsion, emptying) in addition to conscious sensations associated with nonpainful mechanical stimulation (e.g., fullness, bloating, nausea) (12). High thresholds for response have been taken as evidence for the presence of nociceptors that give rise to discomfort and acute pain (11, 64, 65). Previous studies have not identified high-threshold mechanosensitive afferent fibers in the vagus nerve (3, 16, 17, 36, 50, 51), thus indirectly supporting clinical evidence (5, 75) and the generally held notion that mechanosensitive gastric vagal afferent fibers are not important to acute stomach pain.

Visceral receptors have been classified anatomically on the basis of their location (mucosa, muscle, serosa) or physiologically on the basis of the stimuli to which they respond (mechanical, thermal, chemical), although many respond to more than one stimulus (for review, see Ref. 64). Much of the recent literature has focused on the mechanosensitivity of gastrointestinal receptors (7, 32, 39, 50, 51, 62, 63) and their potential role in visceral nociception. To our knowledge, no such study of gastric splanchnic afferent fibers has been reported. The principal aim of the present study was thus to characterize the mechanosensitivity of gastric splanchnic afferent fibers. Because mechanical distension of the stomach was achieved by fluid distension, we were also able to examine responses of mechanosensitive fibers to some chemical stimuli and to thermal stimuli. Splanchnic afferent fibers innervating the stomach have their cell bodies located in lower thoracic and upper lumbar spinal ganglia (T4-L2) in the rat (46, 67). However, the distributions of cell bodies from the three principal divisions of the stomach (fundus, corpus, and pyloric antrum) are not known, and we undertook a preliminary investigation of this question before starting the electrophysiological phase of the study. Some of these data have been presented previously in abstract form (49).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All experiments were approved by the University of Iowa Institutional Animal Care and Use Committee.

Retrograde Labeling

The origin of the primary afferent innervation of the stomach was examined by retrograde tracing using the fluorescent dye fluorogold (FG; Fluorochrome, Denver, CO). Experiments were performed on six male Sprague-Dawley rats (400-500 g; Harlan, Indianapolis, IN). Food, but not water, was withheld for 24 h before surgery. The animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (Nembutal, 45-50 mg/kg; Abbott Laboratories, Abbott Park, IL). The stomach was exposed by midline abdominal incision, and 2.5-µl injections of a 4% (wt/vol) suspension of FG in saline were made in the ventral (8 sites) and dorsal (8 sites) walls of the fundus, corpus, or pyloric antrum of the stomach with a Hamilton microsyringe. The needle was advanced for a distance of 0.5-1 cm from the point of insertion and was left in place for up to 1 min after injection to prevent leakage of the dye along the needle track. Immediately after withdrawal of the needle, the insertion hole was sealed with cyanoacrylate (Borden, Columbus, OH). The stomach was then thoroughly washed and swabbed with saline before the abdomen was closed.

Ten to sixteen days after FG injection, rats were deeply anesthetized with an overdose of pentobarbital sodium and perfused via the aorta with saline followed by ice-cold 4% paraformaldehyde in 0.1 M PBS. Dorsal root ganglia (DRG) bilaterally from spinal levels T4-L2 and stomachs were removed from rats and postfixed for a further hour in 4% paraformaldehyde at 4°C. Tissue was then washed extensively in 20% sucrose phosphate buffer for at least 24 h before use.

DRGs were sectioned on a cryostat at 10 µm, -20°C; sections were thaw-mounted onto slides and air dried for 1-3 h. Sections were mounted in Vectashield (Vector Laboratories, Burlingame, CA), coverslipped, and examined with a Leitz Diaplan microscope with epi-illumination using filter block A (ultraviolet excitation at 340- to 380-nm wavelength). Only cells containing FG and a visible nucleolus were counted as labeled. Counts were made at all serial sections cut across the main axis of the ganglion. Data are presented as the number of FG-labeled cells for each ganglion. Data on the cell diameters of FG-labeled cells are based on the means of the minimum and maximum axes for cells with a visible nucleolus.

Electrophysiology

General procedures. Experiments were performed on 71 male Sprague-Dawley rats (400-500 g). Food, but not water, was withheld for 24 h before surgery. The animals were anesthetized initially with an intraperitoneal injection of pentobarbital sodium (45-50 mg/kg) and subsequently maintained with a constant intravenous infusion of pentobarbital (5-10 mg · kg-1 · h-1). The right femoral vein was cannulated for infusion of fluid and pentobarbital. 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- to 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 37°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.

Surgical procedures. The abdomen was opened by a transverse epigastric incision 4-5 cm in length. The left greater splanchnic nerve was isolated from the surrounding fatty tissues, and a pair of Teflon-coated, 40-gauge stainless steel wires stripped at the tips were placed around the nerve and sealed with hydrophilic vinyl polysiloxane (Reprosil, Dentsply International, Milford, DE).

The stomach was intubated with flexible Tygon tubing (2.3-mm OD, 1.3-mm ID) and Intramedic tubing (PE50; 0.965-mm OD, 0.58-mm ID) via the mouth, esophagus, and cardia. The catheter was secured by a ligature around the esophageal-gastric junction, with care being taken not to damage the vagus nerve. The blood supply and nerves innervating the stomach remained intact. Another Tygon tube (4.0-mm OD, 2.4-mm ID) was introduced distally through the pylorus and secured by a ligature placed caudal to the pyloric sphincter; the duodenum was ligated close to the pyloric ring. For gastric distension (GD), the distal catheter was connected to a pressurized reservoir containing saline. The abdomen was closed with silk sutures.

The thoracolumbar spinal cord was exposed by laminectomy (T7-T11), and the rat was suspended from thoracic and lumbar spinal clamps. The anatomic study showed that afferent fibers innervating the stomach had their cell bodies predominantly in the T9-T10 DRG (see RESULTS). The left T9-T10 dorsal roots were identified and decentralized close to their entry to the spinal cord. The intercostal nerves were isolated and transected. The dorsal skin was reflected laterally and tied to make a pool for mineral oil. The dura was carefully removed, and the spinal cord was covered with warm (37°C) mineral oil.

Recording of afferent nerve action potentials. Recordings were made from the distal cut end of the central processes of primary afferent fibers. A length of nerve fiber was draped over a black microbase plate immersed in warm (37°C) mineral oil. The dorsal rootlet 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 analog 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 bin width) on-line using the spike2/CED 1401 data acquisition program (Cambridge Electronic Design, Cambridge, UK). Peristimulus time histograms, intragastric pressure, intragastric temperature, and blood pressure were displayed on-line continuously. Data were also recorded on tape for later analysis.

Experimental Protocol

Splanchnic nerve input to the T9-T10 dorsal root was identified first by electrical stimulation of the left greater splanchnic nerve (a single 0.5-ms square-wave pulse at 1-15 V). Single fibers were classified on the basis of conduction velocity (CV) determined by estimating with a piece of thread the distance between stimulation and recording sites postmortem and dividing the conduction distance by conduction time (time between stimulus artifact and evoked response). Fibers with a CV < 2.5 m/s were considered unmyelinated C-fibers, and those with a CV > 2.5-25.0 m/s were considered thinly myelinated Adelta -fibers. The isolated stomach was connected to a pressurized fluid reservoir through the pyloric catheter. The fluid reservoir was connected to a distension control device (22, 69); intragastric pressure was monitored via an in-line low-volume pressure transducer. At rest, saline (0 mmHg) remained in the stomach. For phasic, constant-pressure distension (5-80 mmHg, 30 s), saline was introduced via the pyloric (distal) catheter while the oral catheter was clamped. Mechanosensitive gastric afferents in the splanchnic nerve were identified by response to a test stimulus of GD (60 mmHg, <10 s). If a fiber responded to GD, a stimulus-response function (SRF) to distending pressures of 5, 10, 20, 30, 40, and 60 or 80 mmHg (30 s at 4-min intervals) was determined.

Thermal and chemical stimulation of the stomach was produced by changing the temperature or composition of the saline solution with which the stomach was perfused. To monitor the temperature of the perfusate, a thermoprobe (Physitemp type IT-1E) was introduced into the stomach with the distal catheter. After responses to graded intensities of GD were characterized, responses to thermal (heat and/or cold) stimulation generally were tested before examining responses to chemical stimulation (see below). Thermal stimulation of the stomach was produced by ramp increases or decreases in temperature (37-55°C or 37-12°C, ~480 s) without changing intragastric pressure while outflow was open. Ten minutes after thermal stimulation, responses to graded GD (5-80 mmHg, 30 s) with 37°C saline were tested by clamping the oral catheter. The responses of some fibers to repeated heat or cold stimuli were also tested.

Chemical stimulation of the stomach was produced by adding chemicals to the perfusate or by perfusion with an inflammatory soup (IS; bradykinin 10-5 M, PGE2 10-5 M, serotonin 10-5 M, histamine 10-5 M, and KCl 10-3 M, pH 5.5; Refs. 30, 69). The pH of the perfusate was adjusted by adding HCl or NaOH to the saline solution. The effects of these chemical stimuli on spontaneous activity and responses of fibers to graded intensities of GD were determined with the chemical-containing fluid in the stomach; the stomach was flushed with 37°C saline solution after chemical stimulation. Testing of other stimuli followed a recovery interval of 40-60 min (at which time responses to GD returned to control).

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 [impulses(imp)/s] during GD above resting activity. SRFs 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. To estimate the response threshold to thermal stimulation, the mean ± SD of the resting activity was determined. Threshold was defined as the temperature at which unit activity increased >2 SD above resting activity. For fibers with no or low background activity, the response threshold was considered as that temperature at which the fiber began and continued to discharge. Unit activity during thermal stimulation was counted in 10-s bins, and the maximum response during thermal stimulation was defined as that bin with the greatest number of counts.

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.

Chemicals and Drugs

D-Glucose, glycerol, hydrochloric acid, and sodium hydroxide were purchased from Fisher Scientific (Fair Lawn, NJ); L-arginine and potassium oleate were purchased from Sigma (St. Louis, MO) and dissolved in saline. Histamine hydrochloride (mol wt 184.1), serotonin hydrochloride (mol wt 212.7), PGE2 (mol wt 352.5), and bradykinin (mol wt 1,060.2) were purchased from Sigma and dissolved in saline.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Retrograde Labeling

FG injected into the stomach labeled cells bilaterally in DRG. There were similar numbers of cells on each side. Labeled cells were distributed in DRGs T4-L2 with peak distribution at T9 or T10 (Fig. 1B). The distributions in DRG of labeled neurons after FG injections into the fundus, corpus, and pyloric antrum were similar. FG-labeled cells ranged in size from 17 to 47 µm in diameter, most being in the 30- to 40-µm range (mean diameter 35.3 ± 0.2 µm; Fig. 1C). The distribution of cell diameters was unimodal. Relatively few cells were smaller than 30 µm or larger than 40 µm. Labeling in the DRG was distributed throughout the ganglion; no localized distribution was observed.


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Fig. 1.   A: photomicrograph of a retrogradely labeled cell containing fluorogold (FG) and a visible nucleolus in the T9 dorsal root ganglia. B: segmental distribution (bilateral) of FG-labeled primary afferent neurons supplying the fundus, corpus, and pyloric antrum. C: size distribution of FG-labeled neurons in dorsal root ganglia innervating the fundus, corpus, and pyloric antrum of the stomach.

Fiber Sample

A total of 997 afferent fibers were identified by electrical stimulation of the left greater splanchnic nerve. Fifty-five percent (n = 544) of the fibers had C-fiber (mean CV = 1.2 ± 0.02 m/s; range 0.4-2.4 m/s), and forty-five percent (n = 451) had Adelta -fiber (mean CV = 7.8 ± 0.2 m/s; range 2.5-21.7 m/s) CVs; two fibers had faster CVs (32.6 and 40.7 m/s). Of 997 fibers, 31 (3%) responded to gastric distension; 6 (19%) were C-fibers (mean CV = 1.2 ± 0.3 m/s; range 0.7-2.3 m/s) and 25 (81%) were Adelta -fibers (mean CV = 7.6 ± 0.9 m/s; range 2.6-16.3 m/s). Twenty-eight fibers had some resting activity; three fibers were not spontaneously active. The resting activity of 25 fibers was <= 1 imp/s (mean = 0.5 ± 0.1 imp/s, range: 0.01-2.9 imp/s; n = 28).

Response to GD

Responses to phasic GD typically (30 of 31 fibers) exhibited an initial dynamic response followed by a slowly adapting response during maintained GD. Slow adaptation to a tonic discharge was generally observed at all intensities of GD; an example is given in Fig. 2A. One fiber gave a nonadapting, sustained response during GD. After termination of GD, some fibers gave evidence of a period of poststimulus inhibition of spontaneous activity (Fig. 2B). The frequency of discharge fell below the resting level of activity after termination of GD in 9 of 31 fibers for a mean duration of 55.1 ± 14.9 s. Other fibers exhibited afterdischarge at rates greater than resting. Although we did not follow the duration of all afterdischarges, afterdischarges in 9 of 31 fibers continued for 4 s to >219 s after termination of 60-mmHg GD (see, e.g., Fig. 2C). No fibers gave on-off type, rapidly adapting responses. The characteristics of gastric splanchnic afferent fibers are summarized in Table 1, in which they are contrasted with a group of gastric vagal afferent fibers studied under similar conditions (50).


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Fig. 2.   Response patterns of splanchnic nerve afferent fibers to gastric distension (GD). In each record, the response is illustrated as a peristimulus time histogram (1-s bin width) and the intragastric pressure is illustrated below each histogram. A: example of slowly adapting response to phasic GD, observed in 30 of 31 fibers studied. B: example of poststimulus inhibition. C: example of afterdischarge.


                              
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Table 1.   Characteristics of afferent fiber responses to 60-mmHg gastric distension

Reproducibility of Responses

Six fibers were tested for response to repetitive GD at 60 mmHg for 30 s. Four of the six fibers studied showed modest adaptation to repeated GD. Overall, the mean response of the six fibers after the 10th distension was 79.8 ± 9.0% of the response to the 1st distension (paired t-test, P > 0.05). Although not statistically significant, there is a clear indication that response magnitude to GD decreases at this interstimulus interval. Figure 3 shows examples of responses to 10 successive gastric distensions at 4-min intervals.


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Fig. 3.   Responses to repeated GD (60 mmHg for 30 s every 4 min). Responses are illustrated as peristimulus time histograms (1-s bin width); phasic distending pressure is presented below. The top example gave reproducible, nonadapting responses to repeated GD. Responses to GD of the bottom example decreased at the 10th trial to 64% of the initial response, typical of 4 of 6 fibers studied.

Stimulus-Response Functions

Responses to graded GD were studied in all 31 fibers. Fibers generally gave monotonic increases in firing with increasing distending pressure (5 to 60 or 80 mmHg). Extrapolation of the linear portion of individual SRFs revealed that gastric splanchnic nerve afferent fibers exhibit a wide range of thresholds for response to GD (0-53 mmHg). We identified 24 fibers as having low thresholds for response. Most (13) responded to the lowest intensity of GD tested (5 mmHg), and 23 of 24 responded to 10-mmHg GD. The extrapolated mean response threshold for these 24 fibers was 1.4 ± 1.5 mmHg. Seven of the thirty-one fibers only responded to distending pressures >= 30 mmHg and were thus classed as high threshold (HT). Consistent with previous findings in the rat pelvic nerve (62, 63), low-threshold (LT) gastric splanchnic nerve afferent fibers encoded distending stimuli throughout the range of distending pressures tested and gave, as a group, greater-magnitude responses to all pressures tested. Examples are illustrated in Fig. 4, and data are summarized in Fig. 5.


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Fig. 4.   Responses of splanchnic nerve afferent fibers to graded intensities (5-60 mmHg) of gastric distension. Responses of a low-threshold (LT) and a high-threshold (HT) fiber are illustrated as peristimulus time histograms (1-s bin width); intragastric pressure is presented below. Insets for each fiber reproduce an action potential and oscillographic trace for the response to distension identified by the shaded box.



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Fig. 5.   Individual stimulus-response functions (SRFs) of 31 fibers that responded to graded GD (30 s). Inset: mean SRFs for the 24 LT (A) and 7 HT (B) fibers. *Fiber responding with >15 impulses/s at 60- and 80-mmHg distension.

Chemo- and Thermosensitivity

The effects of nutrients, irritants, and/or thermal stimuli were tested on 18 of the 31 mechanosensitive fibers. Fifteen of the fibers had low thresholds for response to GD (11 Adelta -fibers and 4 C-fibers), and three fibers (2 Adelta and 1 C) had a high threshold for response to GD.

Chemosensitivity. The effect of instillation of 20% glucose (3 tests), 300 mM L-arginine (2 tests), or 40 mM potassium oleate (1 test) into the stomach on spontaneous activity and responses to graded GD (5-80 mmHg) was tested on three mechanosensitive splanchnic nerve afferent fibers. Neither spontaneous activity nor mechanosensitivity in this small sample of fibers was affected.

The effect of instillation of 12% glycerol into the stomach on spontaneous activity and responses to graded GD (5-80 mmHg) was tested on two fibers. Glycerol remained in the stomach for the duration of the experiment. The mean resting activity of one fiber increased slightly (from 0.3 to 0.5 imp/s) after intragastric instillation of glycerol. Both fibers, however, were sensitized, as evidenced by increased magnitudes of response to graded GD 15, 60, and 120 min after intragastric instillation of glycerol. In the example given in Fig. 6A, response magnitude increased progressively over time.


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Fig. 6.   Effects of intragastric instillation of glycerol (A) or inflammatory soup (IS; B) on responses to graded intensities of GD. Responses are illustrated as peristimulus time histograms (1-s bin width) before (control) and at varying times after intragastric instillation of the irritant. Glycerol and IS remained in the stomach for the duration of the experiments. The shaded boxes illustrate response magnitude at 40 mmHg as % of the respective control response.

The effect of IS on spontaneous activity and responses to graded GD was tested on four fibers. IS remained in the stomach for the duration of the experiments. The spontaneous activities of fibers were unaffected after intragastric instillation of IS, but two of four fibers exhibited sensitization of responses to graded GD after intragastric instillation of IS. Response magnitude to GD was increased and response threshold decreased after IS treatment (see example in Fig. 6B).

Thermosensitivity. The effect of intragastric perfusion of hot and/or cold saline was tested. Cold was tested first, and none of eight fibers responded. Four fibers were also tested for responses to a second cold stimulus 10 min after the first cold stimulus; none of these fibers responded on the second trial. The responses of five fibers to graded intensities of GD were tested 10 min after cold stimulation. The fibers showed moderate desensitization or no change after cold stimulation.

Four of twelve mechanosensitive afferent fibers responded to gastric instillation of heated saline. Spontaneous activity in these four fibers increased from a mean of 0.3 ± 0.1 imp/s to a mean maximum of 8.0 ± 4.4 imp/s during intragastric perfusion of heated saline; the estimated mean response threshold was 47.2 ± 1.8°C (n = 4; range 44-52°C), which, because the thermistor monitored intraluminal temperature, is likely greater than the response threshold at the receptive ending. An example of response to heat is given in Fig. 7A. Two fibers were also tested for possible sensitization of response to a second heat stimulus 10 min after the first stimulus. In neither instance was the response to the second heat stimulus different from the response to the first heat stimulus.


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Fig. 7.   Effects of heat stimulation. Responses are illustrated as peristimulus time histograms (1-s bin width) during (A) and before (control) and 10 min after intragastric instillation of heated saline (B). Spontaneous activity 10 min after heat stimulation is twice control, and response magnitude is increased a mean 172% (20-80 mmHg) above control.

The responses of 10 fibers (4 heat sensitive and 6 heat insensitive) to graded intensities of GD were tested 10 min after heat stimulation. Only heat-sensitive (2 of 4) fibers exhibited sensitization to GD after heat stimulation. Figure 7B shows sensitization of responses of a fiber to GD 10 min after heat stimulation. The six heat-insensitive fibers showed moderate desensitization or no change in response to GD after heat stimulation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study found that gastric splanchnic nerve afferent fibers innervating the stomach of the rat exhibit a range of response thresholds to constant-pressure GD. A proportion of the fibers studied responded only to distending pressures >= 30 mmHg, an intensity we consider to be in the noxious range, suggesting that acute gastric pain is likely signaled by activation of these fibers. In support of this suggestion, visceromotor responses to GD in unanesthetized rats are apparent at distending pressures >= 30 mmHg (48). In contrast, a previous study of gastric vagal afferent fibers revealed no HT fibers in that sample (50). Together, these studies support clinical evidence that acute gastric pain is conveyed to the central nervous system by gastric splanchnic afferent fibers. Furthermore, exposure of the luminal surface of the stomach to irritant chemicals or prior heat stimulation led to sensitization of responses to gastric distension. Accordingly, gastric splanchnic afferent fibers have the ability to sensitize and thus contribute to altered sensations from the stomach that characterize disorders such as functional dyspepsia.

Gastric Innervation

Although the splanchnic nerve innervation of the stomach has been examined previously, we anticipated a low yield of mechanosensitive fibers in the electrophysiological experiments and wanted to target in rats from our vendor the most appropriate dorsal roots for study. Retrograde tracing studies (8, 15, 23, 24, 33, 46, 67) document that most splanchnic nerve afferent fibers from the stomach enter the spinal cord through the thoracolumbar (T3-L3) dorsal roots, but there is apparent variation between and among strains of rats. For example, in an unspecified strain of albino rat, cells retrogradely labeled from the stomach were observed bilaterally in spinal ganglia T4-L1, being most numerous in ganglia T8-T10 (46), whereas in male Sprague-Dawley rats labeled cells were found in DRG bilaterally from T6 to L1, with the greatest numbers in T11 (23, 24). Similarly, minor differences have been reported for the pelvic nerve innervation in two strains of rats (57). The present results in male Sprague-Dawley rats, revealing the largest input from the stomach in the T9 and T10 dorsal roots, are largely consistent with the horseradish peroxidase studies of Neuhuber and Niederle (46) and differ slightly from results in male Sprague-Dawley rats reported by others (23, 24, 67). Similarly, the cell body diameter determined in the present study (principally 30-40 µm) is consistent with other reports.

We injected tracer into restricted areas of the stomach to determine whether the splanchnic nerve innervation of the rat stomach might be organized viscerotopically. We found that the afferent innervation of the fundus, corpus, and pyloric antrum was similar in distribution in thoracolumbar DRG. Moreover, labeled cells from all stomach areas were similarly most numerous in ganglia T8-T10.

Response Thresholds

Recent studies provide evidence for the existence of separate populations of LT and HT mechanosensitive afferent fibers in the viscera (e.g., esophagus, gallbladder, colon, urinary bladder, ureter, uterus; for review, see Ref. 65). Cervero (10) first described two populations of afferent fibers in the splanchnic nerve innervating the gallbladder of the ferret. Most of the fibers responded to low intensities (2-5 mmHg) of gallbladder distension, but about one-third responded to distending pressures >20 mmHg, which was interpreted as evidence for their role in nociception. In the splanchnic nerves supplying the esophagus of the opossum (60, 61), about two-thirds of the fibers were LT and one-third had high thresholds for response. Pan and Longhurst (55) described LT and HT splanchnic nerve C-fibers innervating the gastrointestinal tract of the cat. The proportion of HT fibers typically reported is 20-30% of the sample studied and was 22% (7 of 31) of the sample reported here. Given their high thresholds for response (>= 30 mmHg), these fibers are likely important to acute stomach pain (i.e., are nociceptors). LT fibers, on the other hand, likely mediate events that are not sensed or play a role in nonpainful sensations that arise from the stomach.

Receptor Location

In a previous study of gastric vagal afferent fibers (50), we identified receptive fields of fibers. We were unable to similarly determine fiber receptive fields in the present study. Rats were suspended from thoracic and lumbar spinal clamps to stabilize the preparation for recording from short-length T9 and T10 dorsal roots. Recordings were disrupted and fibers lost when we attempted to access and probe the stomach surfaces to search for receptive fields. Accordingly, we cannot be certain that HT fibers encountered here did not have mucosal or distant (e.g., serosal or mesenteric) receptive fields. For several reasons, we believe that receptive fields of the HT fibers studied here were in the muscle layers.

Responses of splanchnic nerve afferent fibers innervating the stomach to controlled, phasic GD were characteristically dynamic and slowly adapting. Similar slowly adapting responses to sustained GD have been reported for gastric vagal afferent fibers (3, 6, 17, 18, 25, 35, 36, 40, 47, 50-54, 72, 73) and for visceral nerves innervating other hollow organs (see Refs. 64 and 65 for review). Such responses typically encode distending pressure; are commonly associated with receptors variously termed stretch, in-series, or tension receptors; and are located in hollow organ muscle (see Refs. 11 and 64 for review). Gastric mucosal afferent fibers, in contrast, are rapidly adapting to a mechanical stimulus, giving an on-off response when a steady mechanical stimulus is applied and terminated but no response when the stimulus is maintained (41). Similarly, on-off responses have been described for gastric serosal receptors (4, 14) and in the gastrointestinal tract and pacinian corpuscles in the peritoneum (43, 58). No fibers in the present study gave on-off, rapidly adapting responses suggestive of sensitivity to movement of fluid across the mucosa.

Erroneously high thresholds for response could arise when distending at a site distant from the receptor in the viscus. For example, using two balloons placed in the cat colon, Jänig and Koltzenburg (39) demonstrated that thresholds for response to distension are high when the balloon distended is distant from the receptive ending. It is unlikely in the present study that we would have preferentially excited one and not all branches of an afferent fiber because we employed fluid, not balloon, distension that completely filled the nonspherical stomach. Rapid fluid filling of the stomach, however, could have indirectly activated distant serosal or mesenteric mechanosensitive receptors by increasing tension in stomach muscle and/or distorting the mesentery. Although mesenteric serosal afferent fibers can be slowly adapting in response to distension (20), changes in mesenteric tension during distension do not closely correlate with the magnitude of distension (20, 58). Other investigators describe serosal afferent fibers as rapidly adapting in response to intestinal distension or localized pressure on receptive spots and desensitizing to repetitive stimulation (4). In a recent study of colonic mucosal, serosal, and muscle receptive fields (43), neither mucosal nor serosal afferent fibers responded to stretch; only afferent fibers with endings in muscle gave sustained, slowly adapting responses to stretch. Finally, luminal challenge with thermal and chemical stimuli led to sensitization of responses to distension, further arguing against a distant site of activation. Accordingly, despite our inability to locate the receptive spots/fields of the fibers studied here, we believe that the receptive endings were in stomach muscle.

Sensitization

Sensitization of responses to gastric distension was apparent after intragastric instillation of chemical irritants (glycerol or IS) or heated saline. Six of ten fibers exposed to these stimuli showed sensitization to subsequent gastric distension. Glycerol is an irritant that promotes a bowel movement when infused rectally (59) and induces high-amplitude colonic contractions in humans (31). In animals, intracolonic instillation of glycerol stimulates colonic motility and induces contractions (74). Glycerol sensitized responses to GD of both fibers tested; spontaneous activity was not appreciably changed, but response magnitude increased. Given its irritant nature, glycerol may induce contractions in the stomach or alter compliance of the organ (which we did not monitor). Because spontaneous activity was not affected, however, and response magnitudes progressively increased over time to high-intensity distension, it seems unlikely that a decrease in gastric compliance would explain the outcome observed.

Intragastric instillation of IS increased the magnitude of response of two of four fibers to GD throughout the range of distending pressures tested and also reduced the thresholds for response of both fibers. We showed previously (69) that this cocktail of inflammatory mediators, developed by Handwerker and Reeh (30) to mimic the key mediators released or synthesized at the site of an acute somatic inflammation, sensitizes pelvic nerve afferent fibers to colonic distension. Luminal application of IS is not associated with obvious macroscopic damage to the stomach and thus represents a relatively mild insult.

A number of studies have reported that mechanosensitive visceral afferent fibers are responsive to and/or sensitized by chemicals, principally algesic or irritant chemical. It has long been known that mechanosensitive gastric mucosal afferent fibers in the vagus nerve are often also sensitive to chemical stimuli (37). Others have since documented that mechanosensitive afferent fibers innervating colon or urinary bladder muscle also respond and/or sensitize to chemical stimuli (e.g., bradykinin, capsaicin, acetic acid, mustard oil, xylene, IS; Refs. 19, 27, 32, 38, 43, 62, 63, 66, 69-71). In the upper gastrointestinal tract, Longhurst extensively studied the chemosensitivity (serotonin, histamine, bradykinin) of afferent fibers innervating the stomach, proximal small intestine, and mesentery in the cat, many of which are also mechanosensitive (e.g., Refs. 21, 26, 56; see Ref. 42 for overview). Adelson et al. (1, 2), employing an in vitro preparation of rat mesenteric nerve, reported H2O2- and bradykinin-sensitive splanchnic afferent C-fiber units. Recently, Brunsden and Grundy (9) examined the effects of inflammatory mediators on rat jejunum afferent fibers in vitro, reporting that afferent discharges induced by bradykinin were augmented by histamine, adenosine, and PGE2. The IS employed here is a cocktail of similar inflammatory mediators.

Because mechanosensitive gastrointestinal afferents sensitize after insult or even after a noninjurious stimulus, they can contribute to central hyperexcitability and to visceral hyperalgesia. There are a number of clinical conditions, categorized as functional bowel disorders (e.g., nonulcer or functional dyspepsia), that are characterized by discomfort and pain in the absence of tissue inflammation or apparent pathology (44, 45). These disorders are complex and involve both peripheral and central contributions. It is apparent that a significant component of the discomfort and pain associated with such functional bowel disorders is associated with altered sensory input and/or altered integration in the central nervous system. Improved knowledge of adequate stimuli for receptors in the gastrointestinal tract and their basic physiology will clarify the extent to which the peripheral sensory component contributes to the altered sensations and pain that characterize functional bowel disorders.


    ACKNOWLEDGEMENTS

We thank Sherry Kardos and Kathy Walters for their technical assistance, Michael Burcham for preparation of the figures, and Susan Birely for secretarial assistance. Drs. Su Xin, J. N. Sengupta, and Klaus Bielefeldt provided valuable suggestions and helpful discussions.


    FOOTNOTES

This work was supported by National Institute of Neurological Disorders and Stroke awards NS-35790 and NS-19912. N. Ozaki is an exchange scientist from the Department of Anatomy, Fukushima Medical University, Fukushima, Japan.

Address for reprint requests and other correspondence: G. F. Gebhart, Dept. of Pharmacology, Bowen Science Bldg., Univ. of Iowa, College of Medicine, Iowa City, IA 52242 (E-mail: gf-gebhart{at}uiowa.edu).

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 7 June 2001; accepted in final form 16 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 281(6):G1449-G1459
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