Experimental ulcers alter voltage-sensitive sodium currents in rat gastric sensory neurons

GASTROENTEROLOGY 2 0 0 2 ; 1 2 2 : 3 9 4 - 4 0 5

Experimental Ulcers Alter Voltage-Sensitive Sodium Currents in Rat Gastric Sensory Neurons K. BIELEFELDT,* N. OZAKI/r and G. F. GEBHARTt Departments of *Internal Medicine and ~Pharmacology, College of Medicine, The University of Iowa, Iowa City, Iowa

Background &Aims: Voltage-dependent Na + currents are important determinants of excitability. We hypothesized that gastric inflammation alters Na + current properties in primary sensory neurons. Methods: The stomach was surgically exposed in rats to inject the retrograde tracer l.l'-dioctadecyl-3,3,3,'3-tetramethylindocarbocyanine methanesulfonate and saline (control) or 20% acetic acid (ulcer group) into the gastric wall. Nodose or thoracic dorsal root ganglia (DRG) were harvested after 7 days to culture neurons and record Na + currents using patch clamp techniques. Results: There were no lesions in the control and 3 - 1 ulcers in the ulcer group. Na + currents recovered significantly more rapidly from inactivation in nodose and DRG neurons obtained from animals in the ulcer group compared with controls. This was partially a result of an increase in the relative contribution of the tetrodotoxin-resistant to the peak sodium current. In addition, the recovery kinetics of the tetrodotoxin-sensitive current were faster. In DRG neurons, gastric inflammation shifted the voltagedependence of activation of the tetrodotoxin-resistant current to more hyperpolarized potentials. Conclusions: Gastric injury alters the properties of Na + currents in gastric sensory neurons. This may enhance excitability, thereby contributing to the development of dyspeptic symptoms.

isceral sensation plays a central role in the physiology and pathophysiology of autonomic functions. In the upper gastrointestinal tract, vagal and spinal visceral afferent nerves provide information to the central nervous system that lead to a variety of consciously perceived and unconscious inputs. Alterations in visceral sensation may be involved in the pathogenesis of a wide spectrum of diseases. ~-~ They contribute to pain and discomfort experienced by patients with neoplastic or acute and chronic inflammatory diseases of the gastrointestinal tract. In addition to these organic diseases, visceral pain is the leading symptom in functional diseases, such as noncardiac chest pain, nonulcer dyspepsia, and irritable bowel syndrome. 4 6 Several animal models of visceral hyperalgesia have been established to systematically study factors involved

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in its development and maintenance, v,8 These and other disease models rely on the induction of peripheral sensitization by inflammatory processes, which alter the response to stimulation. The animals demonstrate behavioral and neurophysiological changes consistent with hyperalgesia" a lower threshold for responses to noxious visceral stimuli and an increase in the duration or intensity of responses triggered by noxious stimuli, v-9 Moreover, drugs that are used clinically for the treatment of acute and chronic pain syndromes are also efficacious in these animal models, further supporting the validity of this experimental approach, lo,1 Peripheral sensitization of visceral afferent fibers is likely associated with an increase in membrane excitability. Because voltage-sensitive sodium channels are responsible for the rapid upstroke of the action potential in neurons, they are clearly critical determinants of membrane excitability. 12'13 England et al. 14 found an increase in the number of tetrodotoxin (TTX) binding sites, an indirect measure of the number of sodium channels, in neuromas from patients treated for chronic neuropathic pain, consistent with an important role of voltage-sensitive sodium channels in the pathogenesis of chronic pain syndromes. Moreover, animal experiments have shown changes in sodium currents after surgical compression injury or complete transection of the sciatic nerve) 5-19 However, it is not known whether changes in sodium channel expression or function contribute to chronic pain syndromes, such as visceral hyperalgesia, that are not associated with traumatic nerve damage. W e have recently shown that induction of experimental ulcers by acetic acid (HAc) injection into the gastric wall results in visceral hyperalgesia in rats. 2° W e hypothesized that this experimental model of ulcer disease sensitizes gastric sensory neurons by altering voltageAbbreviations used in this paper: Dil, 1.1'-dioctadecyl-3,3,3,'3-tetramethylindocarbocyanine methanesulfonate; DRG, dorsal root ganglia; HAc, acetic acid; TI'X, tetrodotoxino © 2002 by the American Gastroenterological Association 0016-5085/02/$35.00 doi:10.1053/gast.2002.31026

February 2 0 0 2

sensitive s o d i u m currents, t h e r e b y c o n t r i b u t i n g to the d e v e l o p m e n t of gastric hyperalgesia.

M a t e r i a l s and M e t h o d s Experimental Animals Male Sprague-Dawley rats (ages 2-3 months; Harlan, Indianapolis, IN) were used for the experiments. The animals were housed under a 12-hour light and dark cycle with free access to water and food. Animal handling followed the guidelines of the American Physiological Society. The experimental protocol was approved by the Animal Care and Use Committee of The University of Iowa. We used a previously established model to induced gastric ulcerations in rats. 21-23 Food but not water was withheld for 12 hours before surgery. Animals were anesthetized with pentobarbital (45-50 mg/kg intraperitoneally). The stomach was exposed through a midline incision, and 5 btL of 20% HAc (vol/vol in sterile saline) was injected into 10 sites in the submucosal layer of the stomach in the glandular portion of the dorsal and ventral stomach wall with care taken not to disturb the vascular supply. Saline injection alone served as control. To label gastric sensory neurons, 16 DL of the dicarbocyanine dye DiI (1. l'-dioctadecyl-3,3,3,' 3-tetramethylindocarbocyanine methanesulfonate; 25 mg in 0.5 mL methanol) was injected (2 btL each in 8 sites) into the stomach wall with a 30-gauge needle. DiI is incorporated into the lipid bilayer of nerve processes close to the site of injection and is transported to the cell body without transfer to adjacent ceils. For experiments with nodose neurons, the injection of DiI was performed during the same surgical procedure in which HAc or saline was injected into the stomach wall. The epigastric incision was closed, and rats were allowed to recover for 7 days. Preliminary experiments showed minimal or no labeling of neurons from the T9 and T10 dorsal root ganglia (DRG) 7 days after simultaneous injection of DiI and HAc. Therefore, we performed a laparotomy for DiI injection 7 days before the injection of saline or HAc into the gastric wall in animals used to harvest DRG.

Histologic Grading of Gastric Inflammation To document the induction of inflammation, the stomach was removed 7 days after injection of saline or HAc, rinsed in normal saline solution, and fixed in formalin. The tissue was embedded in paraffin and stained with hematoxylin and eosin. A blinded investigator quantified the degree of inflammation using a validated scoring system with 0 = no inflammation, 1 = mild inflammation, 2 = intermediate inflammation, 3 = high grade inflammation, 4 = transmural infiltration with wall thickening. 24

Cell Dissociation and Culture Rats were anesthetized and decapitated, and the nodose ganglia were quickly removed under a dissection microscope. For D R G neuron culture, rats were anesthetized with

AFFERENT NEURONS AND GASTRIC INJURY

395

sodium pentobarbital (45-50 mg/kg, intraperitoneally) and the T9 and T10 D R G were removed, stripped of their connective tissue capsules, transferred into ice-cold culture media, and minced with microscissors. The tissue was minced with a surgical blade and incubated for 50 minutes in modified Leibowitz L-15 medium (Life Technologies, Grand Island, N Y ) containing collagenase (type 1A, 1 mg/mL), trypsin (type III, 1 mg/mL), and deoxyribonuclease (type IV, 0.1 mg/mL) at 37°C. The enzymatic digestion was terminated by adding soybean trypsin inhibitor (2 mg/mL), 3 mmol/L CaC12, and bovine serum albumin (1 mg/mL). After gentle trituration, the tissue fragments were centrifuged at 800 rpm for 5 minutes and then resuspended in modified L-15 medium with 5% rat serum and 2% chick embryo extract (Life Technologies). The cells were plated on poly-L-lysine-coated glass coverslips and incubated for 3 to 24 hours at 37°C before the electrophysiological studies. At that time, the nerve cell bodies were round and did not have processes, reducing the potential for spaceclamp problems during electrophysiological experiments.

Electrophysiological Recordings The cells attached to the coverslips were transferred into a 0.5-mL recording chamber on the stage of an inverted microscope. Only DiI-labeled cells, identified by their redorange color under Hoffman contrast optics (400×) in fluorescent light with a rhodamine filter (excitation wavelength --546 nm and barrier filter at 580 nm), were studied. Sodium currents were recorded using the whole cell patch-clamp technique with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) interfaced with a personal computer. The patch pipettes were pulled from thin-walled borosilicate glass (TW150-4; World Precision Instruments, Sarasota, FL) with tip resistances of 1-3 M ~ after fire-polishing. Current recordings were filtered at 2 kHz and digitized at 10 kHz using a Digidata 1200 interface (Axon Instruments). The series resistance and whole cell capacitance were compensated by more than 80%. The passive membrane properties were checked repeatedly during the course of the experiments. Cells were used for data analysis only if these properties remained stable. The leak current and residual capacitative transients were digitally subtracted using the p/n protocol for leak subtraction with p being the test pulse and n = 4. All experiments were performed at room temperature (21°C).

Solutions and Chemicals To measure sodium currents, the pipette solution contained 100 mmol/L CsAspartate, 20 mmol/L CsC1, 2.3 mmol/L CaCl2, 4.8 mmol/L MgCI> 10 mmol/L ethylene glycol-bis(~-aminoethyl ether)-N.N,N',N'-tetraacetic acid, 10 mmol/L HEPES, 4 mmol/L Mg-adenosine triphosphate, and 0.5 mmol/L Na-guanosine triphosphate buffered to p H 7.2 with CsOH. The extracellular solution was composed of 20 mmol/L NaC1, 120 nmol/L choline chloride, 10 mmol/L tetraethylammonium chloride, 3 mmol/L MgCI2, 10 mmol/L HEPES, 100 ~mol/L CdCle, and 3.5 mmol/L glucose buffered at p H 7.3 with tetraethylammonium-OH. All chemicals were

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analysis grade and were obtained from Sigma Chemical (St. Louis, MO).

Data Analysis The software package pCLAMP6.0 (Axon Instruments) was used for data acquisition and analysis. TTX-sensitive currents were isolated by digital subtraction of current tracings before and after the administration of the sodium channel blocker. All data are expressed as mean + SEM. Curve fits were obtained using the curve fitting routine of the Origin software package (Microcal, Northampton, MA). The 2-tailed t test for unpaired variables was used to discern significant difference after intervention. Statistical significance was determined at P < 0.05.

Results Sodium Currents in Gastric Sensory Neurons In an initial series of experiments, we randomly chose 5 coverslips to determine the percentage of DiIlabeled neurons. Based on the orange-red fluorescence when visualized with rhodamine filter under UV light, a total of 2.7% + 0.3% (total number of cells counted: 430) and 0.6% + 0.2% (total number of cells counted: 1278) were identified as gastric sensory neurons for no-

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dose and T9, T10 D R G neurons, respectively. In ceils from control animals, depolarization from - 7 0 to 10 m V triggered rapidly activating and inactivating inward currents with peak amplitudes of - 2 4 7 4 + 290 pA (n = 64) and - 4 6 3 5 + 949 pA (n = 21) in nodose and D R G neurons, respectively. Prior experiments 25-3° have shown that these neurons express sodium channels that differ in their sensitivity to TTX. Consistent with these reports, 1 I~mol/L T T X decreased the peak sodium current in gastric sensory neurons to - 1 1 9 9 --- 269 pA and - 1 2 9 9 + 261 for nodose and D R G neurons, respectively (P < 0.001 compared with baseline). As shown in Figure 1A and C, the TTX-sensitive current activated and inactivated rapidly. In contrast, TTX-resistant currents had a slow onset of activation and inactivation (Figure 1B and D). Moreover, TTX-sensitive and -resistant currents could also easily be distinguished based on their voltage-dependence of activation and inactivation (Figure 2). The TTX-sensitive sodium current activated and inactivated at voltages that were significantly more negative compared with the TTX-resistant sodium current. Compared with nodose neurons, both TTX-sensitive and T T X resistant current in D R G neurons activated at signifi-

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Figure 1. Voltage-sensitive sodium currents recorded from gastric sensory neurons. Representative current tracings obtained from (A and B) nodose and (C and D) DRG neurons by depolarizations from 70 mV to various test potentials between - 6 0 mV and 40 mV are superimposed. The upper panel shows (A and C) I-i-X-sensitive; the Iowerpanel shows (B and D) R-X-resistant current.

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Figure 2. Voltage-dependence of sodium currents recorded from gastric sensory neurons• The voltage dependence of activation and inactivation was determined for (A and B) nodose and (C and D) T9, TIO DRG neurons. Sodium currents were differentiated based on their pharmacologic properties as TTX-sensitive (open symbols) and "FrX-resistant (closed symbols) currents. To determine the voltage dependence of activation, celts were stepped from - 7 0 mV to various test potentials between - 6 0 mV and 40 mV. The results were converted into normalized conductance {C - (I / Imax)/(V - Vreversal)} and fitted by the Boltzmann equation (broken lines) for (A) nodose and (C) DRG neurons. To determine the voltage-dependence of inactivation, cells were held at potentials between - 1 1 0 mV and 20 mV for 750 ms before stepping to a test potential of 10 mV. The results were normalized and fitted by the Boltzmann equation (broken lines) for (B) nodose and (D) DRG neurons• See text for details.

cantly more depolarized potentials (voltage of half-activation for TTX-sensitive current: - 1 8 . 8 -+ 1.8 m V vs. - 8 . 4 + 2.0 m V [P < 0.01]; voltage of half-activation for TTX-resistant current: - 1 0 . 8 ± 2.7 m V vs. 2.1 --1.2 m V [P < 0.01]; values for nodose neurons are given first). In contrast, the voltage-dependence of inactivation did not differ significantly between nodose and D R G neurons (voltage of half-inactivation for TTX-sensitive current: - 6 0 . 6 +-- 2.0 m V vs. - 5 7 , 3 +-- 1.4 mV; voltage of half-inactivation for TTX-resistant current: - 1 8 . 5 --0.8 m V vs. - 2 3 . 8 +- 1.6 mY; values for nodose neurons are given first). W e also examined the recovery from inactivation with a 2-pulse protocol. Cells were stepped from - 7 0 m V to 10 m V for 15 ms and repolarized to - 7 0 m V for 2 ms to 200 ms before a second test pulse to 10 mV. As shown in Figure 3, the recovery of the TTX-resistant sodium current was significantly faster than that of the T T X sensitive sodium current. The data were well fitted by a

single exponential. Although the recovery of the T T X resistant current was slower in nodose than D R G neurons (time constants: 1.46 --- 0.1 ms vs. 1.07 + 0.1 ms for nodose and D R G neurons, respectively; P < 0.01), the TTX-sensitive current recovered significantly faster in nodose neurons (time constants: 20.2 + 3.4 ms vs. 53.6 +- 4.7 ms for nodose and D R G neurons, respectively; P < 0.01). HAc-lnduced

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All animals treated with H A c or vehicle injections into the gastric wall recovered from surgery. Although none of the 8 control animals showed evidence of gastric ulceration, all of the HAc-treated rats (n = 7) had a thickened gastric wall and macroscopic evidence of ulceration. A visual analysis revealed 3 -+ 1 ulcers in the glandular portion of the stomach. Histologic sections of formalin-fixed tissue confirmed the macroscopic impression (Figure 4A). There was thickening of all layers of

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GASTROENTEROLOGY Vol. 122, No. 2

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the gastric wall with inflammatory infiltrate as reported in the literature. 21-23 The inflammatory infiltrate was primarily located in the submucosa, which was edematous and showed signs of intramural hemorrhage (Figure 4B). Similarly, the muscularis propria showed signs of injury or frank necrosis in about one third of the sections. The basal third of the mucosa was edematous with a mixed inflammatory infiltrate. In contrast, control animals showed minimal or no inflammation (Figure 4C) with a mean histologic score of 0.3 ± 0.3 compared with 3.6 -+ 0.2 in HAc-treated animals (P < 0.01). Gastric Sodium

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In neurons from HAc-treated rats, sodium current triggered by depolarization from - 7 0 m V to 10 m V increased from - 2 7 7 2 + 317 pA in control conditions (n = 64) to - 3 9 5 2 ± 514 pA in ceils from HAc-treated animals (n = 48; P < 0.01). However, labeled cells from HAc-treated animals were larger compared with control neurons as judged by the whole cell capacitance (43.0 ± 2.8 pF vs. 51.9 + 2.8 pF for the control and ulcer group, respectively; P < 0.05). To account for this apparent difference in cell size, we calculated the peak current density, which did not differ significantly between the 2 groups (67.5 ± 8.4 pA/pF vs. 78.5 + 9.1 pA/pF for the control and ulcer group, respectively). W e next determined whether gastric inflammation altered the pharma-

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Figure 3. Time course of recovery from inactivation. To determine the recovery from inactivation, cells were depolarized from - 7 0 mV to 10 mV for 15 ms, followed by a second test pulse to 10 mV after repolarization to - 7 0 mV for 2 to 200 ms. The upperpanel shows superimposed sample traces obtained during the second test pulse from cells expressing (A) TTX-sensitive and (B) TTX-resistant sodium current (calibration bars 1 nA and 5 ms, respectively). The time course of recovery is summarized in the lower panel for TTX-sensitive (open symbols) and TTX-resistant (closed symbols) currents. The experimental data were fitted to a single exponential (broken lines) for (C) nodose and (D) DRG neurons.

cologic properties of sodium currents in nodose neurons. Although the peak TTX-sensitive current did not differ significantly between groups ( - 2 0 3 2 ± 276 pA vs. - 2 4 1 9 ± 384 pA for the control and ulcer group, respectively), the peak amplitude of the TTX-resistant current was significantly higher in cells from HActreated animals compared with saline controls ( - 1 1 9 9 ± 269 pA vs. - 2 5 2 5 ± 482 pA; for the control and ulcer group, respectively; P < 0.01). Gastric inflammation shifted the voltage-dependence of activation for the TTX-sensitive current slightly to the left (voltage of half-activation - 1 8 . 8 ± 1.8 m V vs. -23.6 ± 1.7 mV; P = 0.06; Figure 5A). There was no change in the voltage-dependence of inactivation (Figure 5B). Voltage-dependence of activation and inactivation for the TTX-resistant current remained unchanged (Figure 5C and D). As described above, the TTX-resistant current recovers more rapidly from inactivation than the TTX-sensitive current. As HAc-treatment led to an increase in the TTX-resistant current, we noted a more rapid recovery from inactivation in ceils from HAc-treated rats compared with controls (Figure 6A). The experimental data were fitted with a double exponential. The fast and slow time constants differed between control and H A c neurons (4.6 - 0.6 ms vs. 2.3 --- 0.4 ms and 159.6 + 31.3 ms vs. 53.2 +- 12.1 ms for the control and ulcer group,

February 2002

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respectively; P < 0.01). To identify whether the kinetic characteristics of TTX-sensitive or TTX-resistant currents were altered, we analyzed the recovery from inactivation for these pharmacologically distinct currents. The experimental data were fitted with a single exponential. Although induction of experimental ulcers did not affect the recovery kinetics of TTX-resistant sodium currents (time constants: 1.46 + 0.1 ms vs. 1.66 +- 0.1 ms for the control and ulcer group, respectively; Figure 6C), HAc treatment accelerated the recovery of the TTX-sensitive current (Figure 6B). This was caused by differences at 2 ms with a recovery (3 + 1% vs. 15 + 5%; for the control and ulcer group; P < 0.05), whereas the time course of recovery was similar between the groups (time constants: 20.3 + 3.4 ms vs. 15.1 + 2.3 ms for the control and ulcer group, respectively).

Gastric Inflammation and Voltage-Sensitive Sodium Currents in DRG Neurons

Figure 4. Histologic changes after submucosal injection of HAc or saline. (A) Microscopic confirmation of an ulcer in the glandular stomach induced by HAc-injection. (B) Representative section of the glandular stomach in ulcer-free areas after HAc-injection showing thickening of the gastric wall, inflammatory infiltrate in submucosa and muscularis, and partial necrosis of the muscularis. In contrast, (C) saline injection did not lead to significant inflammation.

In contrast to the results in nodose neurons, we did not observe a significant difference in peak sodium current between D R G neurons from control and H A c treated rats ( - 4 6 3 5 + 949 p A v s . - 4 1 1 6 ~ 886 for control and ulcer group, respectively). Similarly, there were no apparent differences in cell size based on whole cell capacitance measurements (56.0 + 2.6 pF vs. 58.6 + 4 pF for the control and ulcer group, respectively), resulting in similar current densities of 84.4 + 18.2 and 75.6 -+ 17.9 pA/pF for the control (n = 21) and ulcer group (n = 18), respectively. As described above, we next determined whether gastric inflammation altered the pharmacologic properties of sodium currents in D R G neurons. Although the peak TTX-sensitive current tended to decrease ( - 4 2 0 6 + 932 pA vs. - 3 3 0 2 + 974 pA for the control and ulcer group, respectively; P = 0.1), the TTX-resistant current remained unchanged ( - 1299 + 261 vs. - 1379 --- 340 pA for the control and ulcer group, respectively). Consequently, the relative contribution of the TTX-resistant current to the peak current amplitude rose from 35% + 5% to 53% + 8% (P = 0.06; for the control and ulcer group, respectively). Gastric inflammation did not affect the voltage-dependence of activation and inactivation of the TTX-sensitive current (Figure 7A and B). In contrast, the voltagedependence of activation for the TTX-resistant current shifted by about 10 m V to the left (voltage of halfactivation 2.1 --- 1.2 m V vs. - 7 . 7 + 2.1 mV; P = 0.01; Figure 7C), whereas the voltage-dependence of inactivation remained unchanged (Figure 7D). The increased contribution of the TTX-resistant current to the peak sodium current after ulcer induction resulted in a more rapid recovery from inactivation (Fig-

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Figure 8. Effect of gastric inflammation on the time course of recovery from inactivation of in T9, TIO DRG neurons. To determine the recovery from inactivation, cells were depolarized from - 7 0 mV to 10 mV for 15 ms, followed by a second test pulse to 10 mV after repolarization to - 7 0 mV for 2 to 200 ms. The peak current recorded during the second test pulse was normalized and plotted as (A) a function of time for control conditions (closed symbols; n = 21) and cells obtained from HAc-treated animals (n = 18; open symbols). The results were fitted by a double exponential (broken line). The recovery kinetics were analyzed for the (/3) TTXsensitive and (C) the TTX-resisrant currents and fitted with a single exponential.

402

BIELEFELDT ET AL.

sitive and TTX-resistant currents were altered, we analyzed the recovery from inactivation for these pharmacologically distinct currents. The experimental data were fitted with a single exponential. Although induction of experimental ulcers did not affect the recovery kinetics of TTX-resistant sodium currents (time constants: 1.1 + 0.1 ms vs. 1.4 + 0.2 ms for the control and ulcer group, respectively; Figure 8C), HAc-treatment resulted in a more rapid recovery from inactivation of TTX-sensitive currents (time constants: 53.6 -+ 4.7 ms vs. 29.3 + 4.6 ms for the control and ulcer group, respectively; P < 0.01; Figure 8B).

Discussion The key results of this study show that gastric inflammation induced by HAc-injection into the stomach wall alters the properties of primary afferent neurons by accelerating the recovery from inactivation of voltagedependent sodium currents. This is partially caused by an increased contribution of the TTX-resistant sodium current to the peak sodium current. In addition, the recovery kinetics of the TTX-sensitive sodium current are changed. Together with a left-shift of the voltagedependence of activation of TTX-resistant current in D R G neurons, these changes may enhance excitability, thereby contributing to peripheral sensitization and the development of dyspeptic symptoms. H A c injection into the gastric wall induced significant inflammation and ulceration in rats as had previously been reported. 21-23 In preliminary experiments, we noted a behavioral correlate of hyperalgesia in rats who had undergone HAc-treatment. Compared with control animals, these rats exhibited an enhanced visceromotor response to gastric distension with a shift in the stimulus response curve to the left, consistent with the development of gastric hyperalgesia. 2o In contrast, surgery with submucosal injection of saline did not lead to inflammation or signs of hyperalgesia. Based on these findings, we determined whether the experimental injury caused changes in the properties of sensory neurons, which may contribute to peripheral sensitization. The stomach has a dual afferent innervation with vagal and splanchnic fibers. Mechanosensitive vagal afferents are typically activated by low intensity stimulation. 31-33 In contrast, splanchnic afferents contain both low and high threshold fibers. 34 It is generally accepted that these high threshold fibers are important in mediating acute pain. 54,35 However, vagal and splanchnic afferents can be sensitized in vivo, 32 suggesting that both pathways may play a role in the pathogenesis of pain and other symp-

GASTROENTEROLOGY Vol. 122, No. 2

toms associated with inflammation. Moreover, noxious distension of the stomach leads to an expression of c-los in brainstem neurons where vagal afferents terminate, but not the thoracic spinal c o r d ) 6 Gastric acid challenge induced c-fos transcription in the nucleus of the solitary tract and the area postrema. 37 Because gastric acidity is known to contribute to dyspeptic symptoms, vagal afferents may be involved in chemonociception. Moreover, the majority ofvagal sensory neurons express message for the VR1 receptor, which responds to noxious heat stimulation and is often used as a surrogate marker for nociceptive neurons. 38 W e therefore determined the effect of gastric inflammation on nodose and T9, T10 D R G neurons. To identify gastric sensory neurons, we injected the retrograde tracer DiI into the gastric wall at the time of surgery. About 3% of nodose neurons were clearly identified as gastric sensory neurons that expressed both TTX-sensitive and TTX-resistant sodium currents. In contrast, less than 1% o f T 9 and T10 D R G neurons were similarly labeled. To prevent leakage of the retrograde label, we separately injected DiI and H A c into the gastric wall, potentially resulting in an incomplete overlap between the site of acute injury and the labeled neurons. However, we noted a diffuse thickening of the gastric wall with a significant inflammatory infiltrate even in areas without frank ulceration, indicating that the majority of labeled neurons were affected .by the inflammation. As previously reported, 25-3° we typically recorded a mixture of both TTX-sensitive and T T X resistant currents in both nodose and D R G neurons. Consistent with prior investigations, 25-3° the 2 pharmacologically distinct currents differed in their voltagedependence of activation and inactivation as well as in recovery kinetics. Gastric inflammation accelerated recovery from inactivation in nodose and D R G neurons. This was at least partly caused by the relative increase in the TTX-resisrant sodium current, as it recovers more rapidly compared with the TTX-sensitive current. In addition, the properties of the TTX-sensitive current were changed with a faster recovery from inactivation in neurons from animals with experimental ulcers. W a x m a n et al. 39 recently summarized similar changes in sodium current properties after injury to the sciatic nerve. Interestingly, the sodium channel isoform Navl.3, which is normally not expressed in peripheral sensory neurons, can be detected after nerve injury. 15,4° Compared with other T T X sensitive sodium channels, Nay1.3 recovers more rapidly from inactivation. 4° Although we noted qualitatively comparable changes in spinal and vagal neurons, several

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differences in the effects of gastric inflammation on sodium currents became apparent. This may at least partially be a result of differences in the expression of sodium channel isoforms. 25,26 Moreover, the proliferation and survival of nodose and D R G neurons depends on different trophic signals, 41,42 indicating that receptors for growth factors and cytokines or other signaling molecules may not be not similarly expressed in both nodose and D R G neurons. Additional experiments with molecular and immunohistochemical techniques are required to determine differences in the signaling pathways during inflammation between vagal and spinal neurons. Such studies may also enable us to assess the effects of gastric inflammation on the expression of the various sodium channel subunits and isoforms in these neurons. As mentioned above, we also noted a relative increase in TTX-resistant sodium current. Several lines of evidence suggest that this current plays a role in nociception. The channel underlying this TTX-resistant current, Navl.8, was initially cloned using complementary D N A from D R G neurons. 43 Based on significant structural homology, a second TTX-resistant sodium channel, Nay1.9, has been identified. 44 Both are essentially exclusively expressed in sensory neurons where it is primarily found in small diameter neurons that have been implicated in nociception. 43,44 Inflammatory mediators that are involved in acute pain syndromes rapidly increase the TTX-resisrant sodium current. 45,46 Both knockout experiments as well as the use of antisense oligonucleotides in rodents to decrease the expression of this sodium channel have shown a blunted response in some models of chronic pain. 47,48 Finally, carrageenan-induced inflammation of the hindpaw caused an upregulation of T T X resistant current in rat D R G neurons, further supporting the role of this ion channel in nociception. 19 In contrast to inflammation, axotomy typically down-regulates sodium currents 15,18 and decreases excitability. 49 This may be a result of a loss of target-derived trophic factors, which are important in maintaining normal neuron function. Consistent with this hypothesis, nerve growth factor rescues the decrease in TTX-resistant sodium current seen after axotomy. 17,5°,51 As nerve growth factor is upregulated during inflammation, 5e,53 it is possible that this important neurotrophin contributes to the changes observed after gastric inflammation. Although we noted an increase in the relative contribution of the TTX-resistant sodium current, we did not attempt to determine whether this was caused by an up-regulation of Navl.8 and/or Navl.9. The relative importance of Navl.9 in action potential generation remains unclear. Interestingly, Akopian et al. 47 did not

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record any TTX-resistant current in Na~l.8 knockout mice. Moreover, a recent study indicates that the sodium channel encoded by Navl.9 undergoes profound slow inactivation with less than 5% of the channels being functionally available around the resting membrane potential. 54 Prolonged hyperpolarization to - 1 2 0 mV was required to allow recovery from slow inactivation of this second TTX-resistant sodium current. Based on these results, it is unlikely that an increase in Navl.9 expression contributed to the enhanced TTX-resistant sodium current seen after gastric injury. In addition to changes in the relative contribution of the TTX-resistant sodium current to the peak sodium current, we noted a significant left-shift in the voltagedependence of activation of the TTX-resistant sodium current in D R G neurons obtained from animals in the ulcer group. Similar results had previously been described after chronic constriction injury of the sciatic nerve. 16 An activation at less depolarized potentials is certainly consistent with an increase in excitability. Gold et al. 45,46 reported that prostaglandin E2, adenosine and serotonin, similarly shifted the voltage-dependence of activation of TTX-resistant sodium currents. However, these effects rapidly reversed after removal of the hyperalgesic agent, suggesting that different mechanisms of action are responsible for changes in current properties that persist for hours after cell dissociation and culture. Additional studies are needed to determine whether prolonged exposure to these or other mediators lead to changes in the expression of sodium channel subunits and/or associated modulatory proteins. In conclusion, we have shown that gastric inflammation alters voltage-sensitive sodium currents in nodose and T9, T10 D R G neurons. The accelerated recovery from inactivation and the change in the properties of TTX-resistant current are both consistent with an increase in cellular excitability and may contribute to peripheral sensitization after gastric injury. References 1. Bueno L, Fioramonti J, Delvaux M, Frexinos J. Mediators and pharmacology of visceral sensitivity: from basic to clinical investigations. Gastroenterology 1 9 9 7 ; 1 1 2 : 1 7 1 4 - 1 7 4 3 . 2. Gebhart GF. Pathobiology of visceral pain: molecular mechanisms and therapeutic implications. Am J Physiol 2 0 0 0 ; 2 7 8 : G 8 3 4 838. 3. Mayer EA, Gebhart GF. Basic and clinical aspects of visceral hyperalgesia. Gastroenterology 1 9 9 4 ; 1 0 7 : 2 7 1 - 2 9 3 . 4. Lembo T, Munakata J, Naiiboff B, Fullerton S, Mayer EA. Sigmoid afferent mechanisms in patients with irritable bowel syndrome. Dig Dis Sci 1 9 9 7 ; 4 2 : 1 1 1 2 - 1 1 2 0 . 5. Agreus L, Talley NJ. Dyspepsia: current understanding and management. Annu Rev Med 1 9 9 8 ; 4 9 : 4 7 5 - 4 9 3 . 6. Horwitz BJ, Fisher RS. The irritable bowel syndrome. N Engl J Med 2001;334:1846 -1850.

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Received July 20, 2001. Accepted October 8, 2001. Address requests for reprints to: Klaus Bielefeldt, M.D., Ph.D., University of Iowa, Department of Internal Medicine, 200 Hawkins Drive, 4614 JCP, Iowa City, Iowa 52242. e-mail: [email protected]; fax: (319) 353 6399. The study was supported by grants from the National Institutes of Health (DK01548 and NS 35790).