Role of sensory afferent neurons in hypertonic damage and restitution of the rat gastric mucosa

GASTROENTEROLOGY 1996;111:1474–1483

Role of Sensory Afferent Neurons in Hypertonic Damage and Restitution of the Rat Gastric Mucosa JON ERIK GRØNBECH and ERIC R. LACY Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina

Background & Aims: Gastric mucosal hyperemia is a protective response mediated at least in part by the response of sensory afferent neurons to hydrogen ions. The aim of this study was to determine if other pathways to the hyperemic response are present and if these neurons have an effect exclusive of hyperemia on mucosal protection and repair. Methods: Rat sensory afferent neurons were ablated by capsaicin treatment. Chambered stomachs were damaged by hypertonic saline followed by either acidic or neutral isotonic saline. Blood flow was measured by laser Doppler velocimetry, and mucosal morphology was quantitatively evaluated by microscopy. Results: Mucosal damage alone evoked a strong hyperemic response in both control and ablated rats. Ablated rats lost gastric protection despite this hyperemic response. Acid exposure after damage sustained the hyperemic response. Rapid epithelial restitution occurred faster (even over hemorrhagic lesions) in control rats. Conclusions: The hyperemic response to mucosal damage alone is not mediated by sensory neurons. Protection of the stomach by sensory afferent neurons occurs by mechanisms also unrelated to their elicitation of hyperemia. Restitution during acid challenge is enhanced by the sustained hyperemic response mediated through sensory afferent neurons.

G

astric mucosal hyperemia is an important defense mechanism when the stomach is challenged luminally. Mucosal sensory afferent neurons initiate the protective hyperemia in response to increased H/ back-diffusion caused by disruption of the mucosal barrier.1 Although convincing evidence has been provided with regard to the role of these neurons for protective vasodilation during H/ back-diffusion, conflicting results exist whether this pathway may also be involved in mucosal hyperemia caused by superficial mucosal damage alone in the absence of H/ back-diffusion.2,3 One experimental approach has been a selective functional and morphological destruction of sensory afferent neurons by subcutaneous administration of capsaicin in the rat.4 After 1–2 weeks, this treatment produces reduced tolerance against a variety of noxious stimuli in the gastric mucosa,5,6 but it is not clear from these studies / 5e14$$0035

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whether ablation of sensory neurons renders the mucosa more susceptible to damage by injurious agents alone or whether they leave the mucosa more susceptible to acid attack after injury. Furthermore, none of these studies included detailed morphological analyses, and therefore it is not known whether ablation of sensory neurons also influences rapid epithelial restitution, a first-line protective mechanism of the stomach mucosa.7 – 9 The aims of this study were to evaluate whether ablation of sensory neurons influences the gastric hyperemic response to a superficial mucosal injury independently of the stimulatory effects of acid back-diffusion, renders the mucosa more susceptible to injury that does not involve acid back-diffusion, and influences mucosal restitution after damage. To achieve these goals, rats with capsaicinablated sensory neurons and control rats had the gastric mucosa damaged with hypertonic saline followed by exposure to neutral or acidic saline. Mucosal blood flow responses were measured during mucosal exposure to acid alone, during hypertonic damage alone, and after damage both in the presence and absence of mucosal exposure to acidic saline. Stomachs were fixed and sectioned, and quantitative microscopy was performed to evaluate damage and restitution.

Materials and Methods Animal Preparation Male Sprague–Dawley rats (300–330 g; Charles River Breeding Laboratories, Wilmington, DE) were kept on standard laboratory chow and a 12-hour light/dark cycle. Capsaicin (8-methyl-N-vanillyl-6-nonanamide; Sigma Chemical Co., St. Louis, MO) was used to produce functional ablation of capsaicin-sensitive sensory neurons.1,4 The capsaicin solution was prepared to a concentration of 40 mmol/L by dissolving it in 10% ethanol (100%), 10% Tween 80, and 80% saline vol/ vol/vol. Twelve to 16 days before the surgical part of the experimental protocol, the rats were given a total dose of 125 mg/kg capsaicin subcutaneously under ether anesthesia. Three q 1996 by the American Gastroenterological Association 0016-5085/96/$3.00

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injections of capsaicin were given during a 2-day period: 25 mg/kg in the morning and 50 mg/kg in the afternoon on day 1 and 50 mg/kg on day 2. Control rats were given subcutaneous vehicle for the capsaicin solution on the same schedule as experimental rats. On the day of the surgery, effectiveness of sensory neuron ablation was assessed by topical instillation of one drop of capsaicin (0.1 mg/mL) onto the eye of the rat. Vehicle-treated rats reacted by wiping the eye with the paw, whereas capsaicin-treated rats did not. The eye was immediately rinsed with water. The rats were deprived of food but not water for 16–20 hours before surgical manipulation. After induction of anesthesia with 50 mg/kg pentobarbital (Nembutal; Abbot Laboratories, Chicago, IL) intraperitoneally, the animals underwent tracheotomy and a catheter (PE-50) was introduced in the left carotid artery and connected to a Statham P23DC transducer (Statham Laboratories, Hato Rey, Puerto Rico), allowing continuous measurements of mean arterial blood pressure, which was displayed on a Grass Model 1 polygraph (Grass Instruments Co., Quincy, MA). Another catheter (PE-10) was placed in the right femoral vein and, by a constant rate pump (model 600-900, Harvard Infusion Pump; Harvard Apparatus, Harvard, MA), used for continuous infusion of 10 mLrkg01rh01 isotonic saline and 15 mgrkg01rh01 pentobarbital to maintain stable hydration status and anesthesia. The animals were placed on a heating pad, and rectal temperature was kept between 377C and 387C. After laparotomy, the esophagus was exposed and divided between ligatures and the pylorus was also ligated; in both instances, care was taken to avoid damage of extragastric nerves and blood vessels. The forestomach and the anterior two thirds of the glandular stomach were opened, and the stomach was

pulled up through an oval hole in an overlying Plexiglas plate, unfolded with the mucosal side up, and pinned along the edges. A 25-mm diameter cylinder was then clamped onto the mucosa forming a chamber.10 The chamber was thereafter filled with 10 mL of isotonic saline, and the animals were allowed to stabilize for 30 minutes before baseline recordings were collected.

Chamber Solutions Three types of solutions were made up in batches and used throughout the experiments; isotonic saline (154 mmol/ L NaCl) had the pH changed to 6.4 or to 1.0 by adding 0.05N NaOH or 0.5N HCl, respectively. Hypertonic NaCl of either 1 mol/L or 2 mol/L was prepared using deionized water; the pH of these solutions is about 6.4. A small day-to-day variation in the pH of isotonic saline with pH adjusted to 6.4 and 1.0 as well as of hypertonic NaCl was noted; therefore, after the end of each experiment, pH of fresh solutions filled into the chamber as well as of solutions removed from the chamber were measured by a pH-meter (Beckman 40 pH Meter; Beckman Instruments Inc., Pulleron, CA). The variance of pH appear in Tables 1 and 2 (see Results). All solutions were prewarmed to 377C before use.

Blood Flow Measurements Gastric blood flow was measured with the laser Doppler velocimetry technique using the Periflux PF 2B instrument (Perimed, Stockholm, Sweden) as previously described.11 The PF 308 standard probe was used, and the probe was fastened to a micromanipulator and thereafter lowered until it lightly contacted the mucosal surface with care being taken to avoid pressure on the mucosa. This arrangement allowed recordings

Table 1. Change in pH of Gastric Chamber Solution After Mucosal Damage in Control and Ablated Rats Postdamage during exposure to neutral saline or acidic saline pH after 30 min in the chamber

Groups 2 mol/L NaCl Neutral saline Control Ablated Acidic saline Control Ablated 1 mol/L NaCl Acidic saline Control Ablated

n

pH before instillation into the chamber at 0 and 30 min

Period 1 (0–30 min)

Period 2 (30–60 min)

7 7

6.87 { 0.13 6.53 { 0.21

7.50 { 0.03a 7.33 { 0.06a

7.43 { 0.03a 7.37 { 0.04a

8 8

0.97 { 0.05 1.02 { 0.02

1.13 { 0.04a 1.11 { 0.03a

1.11 { 0.05a 1.12 { 0.03a

9 9

0.96 { 0.02 0.93 { 0.02

1.12 { 0.01a 1.09 { 0.01a

1.10 { 0.01a 1.09 { 0.02a

NOTE. Results are expressed as mean { SEM; n Å number of rats. Functional ablation of sensory afferent neurons was performed by treatment with capsaicin (125 mg/kg subcutaneously) 2 weeks before the experiments. Control rats were injected with vehicle. Baseline conditions were the same in all groups, and the data are given in the text. The gastric mucosa was exposed to neutral saline or acidic saline for two periods of 30 minutes. a Significant difference compared with pH of saline instilled into the chamber.

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Table 2. Change in pH of Gastric Chamber Solution During Exposure to Acidic Saline Alone in Control Rats and Rats With Ablated Sensory Neurons During exposure to acidic saline alone pH after 30 min in the chamber

Groups

n

pH before instillation into the chamber at 0 and 30 min

Period 1 (0–30 min)

Period 2 (30–60 min)

Control Ablated

8 8

0.89 { 0.04 0.91 { 0.02

0.99 { 0.04a 0.99 { 0.03a

0.99 { 0.03a 0.98 { 0.02a

NOTE. Values are expressed as mean { SEM; n Å number of rats. See legend for Table 1. Baseline conditions were the same in all groups, and the data are given in the text. The gastric mucosa was exposed to acid for two periods of 30 minutes. a Significant difference compared with pH of saline instilled into the chamber.

from the same spot of the mucosa throughout the entire experiment. The recordings were displayed on a servograph. Baseline recordings of gastric blood flow were collected for 15 minutes by calculating the mean blood flow for this period. All later changes in blood flow caused by change of the solution in the chamber were calculated as percent of the baseline recording.

Microscopy At the end of each experiment, the stomach was gently removed from the chamber and immersed in half-strength Ito Fixative (2% glutaralaldehyde, 2.5% paraformalaldehyde, and 0.25% picric acid in 0.1 mol/L Na cacodylate buffer). Whole stomachs remained in Ito’s fixative for about 24 hours and were then rinsed several times and kept at 47C in 0.1 mol/L Na cacodylate buffer. Trimming of the tissue was performed in a standardized way after completion of all of the experiments and by a person unaware of the experimental group to which the tissue belonged. Strips of fundus (2–3mm wide) were cut with its long axis parallel with and 4–5 mm distal to the limiting ridge. Six wedges of tissue from the strips were randomly chosen and postfixed for 1 hour in 1% OsO4 in 0.1 mol/L Na cacodylate buffer or in the same osmium tetroxide solution to which 1% potassium ferrocyanide had been added. Subsequently, tissue from both procedures was dehydrated in graded concentrations of ethanol and embedded in Epon-Araldite (Tousimis Research Corp., Rockville, MD) after soaking in propylene oxide. Sections (0.5-mm thick) were mounted on glass slides, stained with alkalinized toluidine blue, and studied with an Olympus BHTU light microscope (Olympus Corp., Lake Success, NY). Slides were randomly numbered to hide the identity of their experimental group not only from the person cutting the tissue but also from the investigator reading the slides. Morphometry was accomplished using a modification of the technique by Lacy and Ito.8 An ocular micrometer was aligned parallel to and just above the mucosal surface at a magnification of 2001 in the light microscope. The length of the superficial mucosal epithelium covering nonhemorrhagic area in each of the following categories was recorded: in situ damage in which the cells showed extensive vacuolization, necrotic tissue in which lysed

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cells remained adherent to the basal lamina, denuded basal lamina in which the necrotic tissue had sloughed off and the basal lamina had no cells covering it, and restituted epithelium in which epithelial cells were covering the basal lamina but their morphology was either squamous or low cuboidal as previously described.8 In addition, the length of tissue with hemorrhagic lesions was recorded separately from the categories of restituting mucosal surface described above.

Experimental Protocol Rats with intact sensory afferent neurons were studied in parallel with rats that had undergone ablation of sensory afferent neurons by pretreatment with capsaicin as explained above. Exposure to acid alone. In 8 rats pretreated with capsaicin and 8 pretreated with vehicle only (control rats), the stomach was mounted in the ex vivo chamber. At the end of the stabilization period, the chamber was emptied by gentle aspiration and refilled with 10 mL isotonic saline, pH É 6.4 (neutral saline), for a 30-minute baseline period. The neutral saline was aspirated and the mucosa exposed to acidic saline (pH É 1.0) in two periods of 30-minutes each for the remaining 1 hour of the experiment. The initial acidic saline was replaced with fresh acidic saline 30 minutes after the initial mucosal exposure to this compound. Hypertonic saline damage without subsequent acid exposure. Thirteen rats with ablated sensory neurons and 13

control rats were used. After the baseline period (30 minutes), the isotonic saline at neutral pH was withdrawn from the chamber and replaced by 10 mL of high-molarity saline (2 mol/L NaCl), which was used to quickly flush the chamber. This solution was withdrawn and the chamber refilled with 10 mL fresh high-molarity saline. After 10 minutes, the highmolarity saline solution was removed and 6 rats in each group were killed and the stomachs fixed to obtain information about the effects of ablation of sensory neurons on early mucosal damage. In the remaining 7 rats in each group, the chamber was refilled with 10 mL isotonic neutral saline. The mucosa was exposed to this solution in two periods of 30 minutes each for the remaining 1 hour of the experiment. Fresh neutral

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saline replaced the initial saline 30 minutes after the removal of the high-molarity saline. Hypertonic saline damage with subsequent acid exposure. Eight rats with ablated sensory neurons and 8 control

rats were subjected to the protocol described above except the mucosa was exposed to acidic saline for 60 minutes (two periods for 30 minutes each) after hypertonic injury. Mucosal damage with low-molarity saline. In 15 ablated rats and 15 control rats, the experiments were performed identical to the experiments described above except that the molarity of the damaging solution was reduced to 1 mol/L NaCl. Six rats in each group were used to obtain information about early mucosal damage. The remaining 9 rats had the mucosa exposed to acidic saline after exposure to 1 mol/L NaCl. To determine the possible influence of the experimental manipulation on stomach morphology, microscopic analyses of stomachs from 12 additional rats were performed. Six rats with capsaicin pretreatment and 6 with vehicle pretreatment were fitted with catheters, and their stomachs were mounted in the ex vivo chamber and exposed to isotonic neutral saline for a few seconds.

Statistics Two-way analysis of variance for repeated measurements was performed for testing changes in mean aortic pressure, gastric mucosal blood flow, and change in H/ concentration in the chamber solution. Significant interaction effects were further explored according to the method described by Winer.12 When justified by preceding analysis of variance, contrast tests (Newman–Keuls test) were used to calculate probabilities within and between groups. The Mann–Whitney U test (twotailed) for unpaired data was used to test differences in morphology. Probabilities were regarded as significant when õ0.05. Interaction effects were, however, also further explored when õ0.1. Data are expressed as means { SEM.

Results pH of the Ex Vivo Chamber Solution Because baseline conditions were the same in all groups, we analyzed the change in acidity of the chamber solution during this 30-minute period together. The pH of isotonic saline instilled into the chamber of control rats (6.57 { 0.07; n Å 44) was not significantly different from the pH of instilled saline into the chamber of ablated rats (6.37 { 0.11; n Å 44). During the 30-minute baseline period, the pH of the chamber solution decreased to 3.22 { 0.08 in control rats (P õ 0.001) and 2.66 { 0.09 in ablated rats (P õ 0.001). The decrease in pH of the chamber solution during baseline conditions was significantly more pronounced in ablated than in control rats (P õ 0.001). The pH of the 2 mol/L NaCl solution instilled into / 5e14$$0035

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the chamber of control rats (6.54 { 0.07) was similar to that for ablated rats (6.37 { 0.06). After 10 minutes, pH remained unchanged in ablated rats (6.39 { 0.08) and slightly increased (6.85 { 0.04; P õ 0.06) in control rats. The 1 mol/L NaCl solution had a pH of 6.39 { 0.07 in control rats and 6.32 { 0.06 in ablated rats. After 10 minutes in the chamber, the pH of this solution was not significantly changed (5.95 { 0.20) in control rats but had decreased to 4.04 { 0.29 in ablated rats (P õ 0.01). After mucosal exposure to 2 mol/L NaCl and subsequent exposure to neutral saline at pH É 6.4, a significant alkalinization of the chamber solution was observed both in control and ablated rats (Table 1). In rats with the stomach mucosa exposed to acidic saline (pH É 1.0) after mucosal damage by both 2 and 1 mol/L NaCl, a substantial and similar decrease in acidity of the chamber solution was observed both in control and ablated rats (Table 1). Even in the rats with mucosal exposure to acidic saline at pH É 1.0 alone, a significant decrease in the acidity of the chamber solution was found both in control and ablated rats (Table 2). Mean Aortic Blood Pressure Table 3 summarizes the changes in blood pressure throughout the experiment. Mucosal exposure to acidic saline alone did not cause any significant change in systemic blood pressure either in control rats or in rats with ablated sensory neurons. Exposure to hypertonic saline was associated with a significant increase in blood pressure both in control and ablated rats with the mucosa exposed to either 2 mol/L NaCl or 1 mol/L NaCl for 10 minutes and thereafter to acidic saline. During mucosal exposure to acidic saline, this elevation of blood pressure was sustained for 30 minutes after mucosal damage with 2 mol/L NaCl and throughout the entire experiment after damage by 1 mol/L NaCl. Gastric Mucosal Blood Flow Effects of acid alone. Exposure to isotonic acidic

saline only caused an immediate increase in mucosal blood flow of 30%–40% in control rats (P õ 0.005), whereas no significant change was observed in capsaicintreated rats (Figure 1). This elevated level of blood flow was sustained for the 1-hour duration of the experiment (P õ 0.005 for all time points). This was confirmed between groups of comparison showing that flow in control rats was clearly higher than in ablated rats during exposure to acidic saline (P õ 0.005 for all time points). WBS-Gastro

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Table 3. Effects of Luminal Acid and Hypertonic Injury on Mean Aortic Pressure in Rats After Sensory Neuron Ablation Postdamage exposure to neutral or acidic saline Groups Acid alone Control Ablated 2 mol/L NaCl Neutral saline Control Ablated Acidic saline Control Ablated 1 mol/L NaCl Acidic saline Control Ablated

n

Baseline

8 8

111 { 7 110 { 4

7 7

109 { 4 95 { 7

8 8

9 9

Damage

15 min

30 min

60 min

117 { 6 112 { 5

120 { 4 113 { 4

121 { 5 111 { 4

115 { 3 106 { 7

110 { 4 107 { 7

110 { 4 104 { 7

104 { 4 96 { 7

110 { 4 92 { 4b

119 { 3a 99 { 4a,b

118 { 3a 103 { 5a,b

117 { 3a 108 { 5a

110 { 3 106 { 5a

101 { 3 103 { 5

113 { 3a 111 { 6a

118 { 4a 112 { 6a

117 { 3a 113 { 5a

114 { 4a 113 { 4a

NOTE. Values are expressed as mean { SEM (in mm Hg); n Å number of rats. The gastric mucosa was either exposed to acid alone (isotonic saline, pH É 1.0) or damaged by exposure to 2 or 1 mol/L NaCl and thereafter exposed either to neutral saline (pH É 6.4) or acidic saline (pH É 1.0) for 60 minutes. a Significant difference compared with baseline. b Significant difference between control and ablated rats.

Effects of hypertonic damage. Both of the hypertonic damaging agents, 2 mol/L NaCl as well as 1 mol/L NaCl, caused an immediate and marked increase in gastric blood flow both in control rats (P õ 0.025) and ablated rats (P õ 0.001) (Figure 2). The increase as measured at the end of the 10-minute damage period was approximately 75% above baseline. Effects of acidic saline after hypertonic injury. Control. Replacement of the 2 mol/L damaging solution

with isotonic saline at neutral pH resulted in an immediate decrease of blood flow to baseline levels. By the end

of the experiment, these values had continued to decrease about 25% below baseline values (P õ 0.05). Postdamage exposure of the mucosa to acid evoked a sustained elevation of blood flow compared with baseline levels (Figure 2A). In the 2 mol/L NaCl group, this elevation of blood flow was statistically significant for the first 30 minutes after damage (P õ 0.05); in the 1 mol/L NaCl group, the hyperemia was evident also at the end of the experiment (P õ 0.001). Between-group comparison showed that blood flow during subsequent acid exposure was slightly higher in the 1 mol/L NaCl group than in the 2 mol/L NaCl group (P õ 0.05). Both groups showed clearly higher blood flow compared with animals not exposed to acid after hypertonic damage (P õ 0.001 and P õ 0.005, respectively). Ablated. Gastric mucosal blood flow decreased to the baseline level after hypertonic injury in ablated rats irrespective of mucosal exposure to acid or neutral saline (Figure 2B). Microscopic Analyses

Figure 1. Change in gastric blood flow in chambered rat stomachs exposed to acidic saline alone (pH É 1.0) for 60 minutes. Results are expressed as mean values { SEM; n Å 8 for both control rats and rats with ablated sensory neurons by pretreatment with capsaicin (125 mg/kg subcutaneously) 2 weeks before the experiments were performed. h, Control; m, ablated.

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The gastric mucosa of control or ablated rats exposed to acidic saline alone did not show any changes in the surface or the glandular epithelium (data not shown). The distribution of damaged and restituted surface epithelium as well as lesions immediately after and 60 minutes after exposure to hypertonic saline are shown below. WBS-Gastro

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Figure 3. Distribution of different stages of superficial damage, restitution, and lesions after gastric mucosal exposure to 2 mol/L NaCl in (A ) control rats and (B ) rats with ablated sensory neurons. Mean values { SEM. n Å 6 for both groups immediately (0 minutes) after exposure to 2 mol/L NaCl, n Å 7 for both groups after hypertonic damage and subsequent exposure to neutral saline for 60 minutes, and n Å 8 for both groups after hypertonic damage and subsequent exposure to acidic saline for 60 minutes. †Significant difference between control and ablated rats. *Significant difference compared with immediately (0 minutes) after mucosal damage.

Figure 2. Change in gastric blood flow in chambered rat stomachs exposed to 2 mol/L NaCl for 10 minutes and subsequent exposure to neutral saline (pH É 6.4; n Å 7) or acidic saline (pH É 1.0; n Å 8) for 60 minutes and stomachs exposed to 1 mol/L NaCl and subsequently to acidic saline (n Å 9) for 60 minutes. (A ) Control and (B ) ablated.

NaCl (2 mol/L): 0 minutes after injury. Mucosal exposure to 2 mol/L NaCl in control rats caused mucosal damage of about 90% of the surface. The damage ranges from vacuolization of the surface epithelium (in situ damage) to complete exfoliation of the cells, leaving the basal lamina denuded. However, no hemorrhagic lesions were observed. Ablated rats had hemorrhagic lesions from the mucosal surface extending to 10%–25% of its depth. These lesions comprised about 35% of the surface area (Figure 3). Other categories of damage were comparable between the two groups. NaCl (2 mol/L): 60 minutes after injury. In control rats after subsequent exposure to isotonic neutral saline after injury, about 60% of the surface epithelium appeared normal. During this time period, the amount of necrotic tissue and denuded surface were significantly

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less than at 0 minutes, indicating restitution had occurred. A significant increase of normal and restituted epithelium occurred in ablated animals after mucosal exposure to neutral saline for 60 minutes. Remarkably, even a reduction of the hemorrhagic lesion area occurred compared with 0 minutes in ablated rats. Higher-power magnification showed that some epithelial restitution had occurred over these superficial lesions (Figure 4). Mucosal exposure to acidic saline in control rats resulted in less than half the amount of normal epithelium and increases in each category of damaged mucosa compared with the corresponding neutral saline group. However, acidic saline for 60 minutes after mucosal damage produced a significant increase in the amount of normal epithelium compared with 0 minutes. Exposure to acid also affected restitution in ablated rats (Figure 3). Hemorrhagic lesions constituted more than one third of the total mucosal surface area, which was more than double that of the neutral saline group. NaCl (1 mol/L): 0 minutes after injury. Instillation of 1 mol/L NaCl into the chamber of control rats caused less severe damage to the mucosal surface produced by 2 mol/L NaCl (Figure 5). Damage to the ablated rats showed more lesions and more necrotic surface than control rats (Figure 5). WBS-Gastro

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NaCl (1 mol/L): 60 minutes after injury. Sixty minutes after damage and mucosal exposure to acidic saline, control rats had more normal and restituted epithelium but also more denuded surface than immediately after damage at 0 minutes. Ablated rats also had more normal epithelium at 60 minutes than immediately after exposure to 1 mol/L NaCl and more denuded surface, whereas the lesion area remained unchanged.

Discussion The present study shows that ablation of sensory neurons of the gastric mucosa did not diminish its hyper-

Figure 4. (A ) Low-power light micrograph showing full-thickness gastric mucosa exposed 60 minutes earlier to 2 mol/L NaCl followed by isotonic neutral saline. Lighter areas adjacent to lumen are hemorrhagic areas (arrows ) as shown in greater detail in B (original magnification 2001). (B ) High-power light micrograph of upper gastric mucosa showing flattened epithelial cells covering a hemorrhagic lesion indicated by extensive extravasated red blood cells in lamina propria (arrows ). Note the marked hemostasis in the vessels around the deeper gastric glands (arrowheads ) (original magnification 4001).

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emic response to injury in the absence of acid. However, ablation of sensory neurons did reduce protection of the mucosa under such conditions. Therefore, it seems that sensory neurons confer protection against injury via mechanisms that are unrelated to gastric blood flow responses initiated by superficial damage of the mucosa. On the other hand, mucosal hyperemia in response to acid challenge was abolished by ablation of sensory neurons. Repair of hemorrhagic lesions in ablated rats, which was substantial in a neutral environment, did not occur during acid challenge. These findings therefore emphasize the role of sensory neurons, most likely mediated via increased gastric blood flow, in rapid gastric mucosal repair. Weakened mucosal defense after functional ablation of sensory neurons has been reported to occur using experimental models involving acid challenge of the mucosa alone such as pylorus ligation and acid distention of the stomach5 or after challenge with 15% ethanol in relatively strong acid.1 In other models, impaired defense after ablation of sensory neurons was found 30 minutes after topical ethanol challenge, 6 hours after indomethacin given intraperitoneally, 18 hours after cysteamine subcutaneously, and 20 minutes after arterial infusion of platelet-activating factor.6,13 Because even the shortest time frame after administration of the noxious agent in these models is sufficient for a substantial decrease of

Figure 5. Morphology after mucosal exposure to 1 mol/L NaCl. Results are expressed as mean values { SEM. n Å 6 for both groups immediately (0 minutes) after hypertonic damage, and n Å 9 for both groups after damage and subsequent exposure to acidic saline for 60 minutes.

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gastric luminal pH, as shown by our findings with regard to luminal pH during the 30-minute baseline period, and because acid was not removed from the stomach before mucosal challenge, it is almost certain that the stomach contained a substantial amount of acid in these models. Therefore, it remains uncertain whether sensory neurons afforded a genuine protection against the noxious substances alone or to what extent loss of protection was caused by rendering the damaged mucosa more susceptible to acid attack. The present study shows that ablation of sensory neurons results in substantially decreased protection as evaluated immediately after 10 minutes of exposure to hypertonic saline. The pH of this damaging hypertonic solution is about 6.4 and remained so during the 10 minutes of exposure, at least after challenge by 2 mol/L NaCl. We therefore conclude that sensory neurons afford protection against hypertonic saline that are unrelated to hydrogen back-diffusion. It is well established that increased gastric blood flow in response to increased H/ back-diffusion after mucosal damage represents an important defense mechanism against further damage caused by acid back-diffusion.14 – 17 Furthermore, there is also good evidence to support the view that sensory afferent neurons are instrumental in mediation of this hyperemic response to acid back-diffusion.1,18 – 20 Increased gastric blood flow caused by hypertonic saline alone, without acid, has also been shown to represent an important defense mechanism because impairment of this hyperemic response to topical 2 mol/L NaCl by nicotine21 and indomethacin3 resulted in significantly increased mucosal damage. The mechanisms behind the hyperemic response to hypertonic saline alone are not established. For example, Matsumoto et al. reported that gastric hyperemia caused by 1 mol/L NaCl was attenuated by functional ablation of sensory neurons.2 On the other hand, Endoh et al. showed that pretreatment of rats with capsaicin did not influence gastric hyperemia caused by intragastric instillation of 2 mol/L NaCl.3 They also showed that gastric hyperemia in response to 2 mol/L NaCl was unaffected by H1-receptor blockade and blockade of nitric oxide but was completely abolished by pretreatment with indomethacin, suggesting an important role of prostaglandins. Our findings of a similar and marked increase in gastric blood flow in response to topical application of hypertonic saline in both ablated and control rats are in agreement with those of Endoh et al. Because lesion areas were much larger immediately after mucosal exposure to both 1 and 2 mol/L NaCl in ablated than in control rats, we conclude that increased protection against this type of injury in the presence of intact sensory neurons / 5e14$$0035

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cannot be explained by stimulation of gastric blood flow by these neurons. In search of possible mechanisms for the observed decreased mucosal tolerance against hypertonic saline alone in ablated rats, there are several factors that must be considered. Studies with the hydrogen gas clearance method did not show any differences in basal gastric blood flow between such rats and rats with intact sensory neurons.1,3 Investigations using the laser Doppler method, modified to yield semiquantitative measure of gastric blood flow, even showed slightly increased blood flow in adult rats pretreated with capsaicin in a similar way as in the present study.22 Gastric release of the prostaglandins E2 and I2 was unaffected after pretreatment with capsaicin.6 Furthermore, inhibition of prostaglandin release with indomethacin failed to alter the protective effect of acute intragastric administration of capsaicin against ethanol injury, and capsaicin given intragastrically alone did not change the mucosal ex vivo formation of prostaglandins.23 Decreased basal gastric blood flow and changes in eicosanoid product generation in the gastric mucosa therefore seem to be unlikely explanations for decreased protection against nonacid-related damage in ablated rats. An additional protective mechanism that involves the influence of sensory neurons on mucosal fluid secretion and thus dilution of noxious luminal agents has recently been proposed. Hatakeyama et al. reported that stimulation of sensory neurons with intraluminal capsaicin greatly increased gastric fluid volume, an effect that was completely abolished by ablation of sensory neurons.24 These findings bear relevance to the results of the present study. There is a strong possibility that the increased acidity of the chamber solution during baseline conditions and during exposure to 1 mol/L NaCl, as observed in ablated rats in our study, could be explained by this mechanism rather than changed acid secretion in rats with defunctionalized sensory neurons. Mast cells are closely apposed to and interact with sensory neurons in the rat gastric mucosa,11,25 and involvement of this cell type in mucosal protection should therefore be considered. Recently, Eliakim et al. reported that mast cell stabilization with ketotifen completely attenuated the deleterious effect of sensory neuron ablation in acetic acid–induced colitis in rats.26 The protection provided by ketotifen in capsaicin-treated rats was accompanied by a significant decrease in mucosal leukotriene B4 generation and NO synthase activity. It is reasonable to assume that similar mechanisms related to changed interaction between mast cells and sensory neurons could operate in the stomach as well. Hypotheses WBS-Gastro

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about mechanisms for the protective ability provided by sensory neurons that cannot be explained by increased gastric blood flow seem at present to be promising along the lines mentioned above. In contrast to our findings with regard to similar blood flow responses caused by hypertonic saline alone both in control and ablated rats, we found that mucosal exposure to acid after mucosal barrier disruption was associated with a sustained hyperemia in control rats but not in rats with ablated sensory neurons, confirming the original observation by Holzer et al.1 We even found that the significant hyperemia caused by exposure of the normal mucosa to acid alone was completely attenuated by ablation of sensory neurons. In control rats, restitution of nonhemorrhagic damage during exposure to acidic saline was reduced compared with rats with mucosal exposure to neutral isotonic saline, which is in agreement with previous observations.27 However, it should be emphasized that, even during exposure to acidic saline, restitution was substantial in control rats. In ablated rats with mucosal exposure to acidic saline, the hemorrhagic lesions did not diminish either after damage with 1 or 2 mol/L NaCl and restitution remained the same as time 0. Our microscopic evaluation of the mucosa from ablated rats exposed to neutral isotonic saline for 1 hour after damage showed that many of the superficial hemorrhagic lesion areas showed epithelial cell migration across the lesions. In addition, previously static vessels were open, indicating a reperfusion of the area. These observations are novel and extend the knowledge about the restitution process. This phenomenon has been believed to occur primarily after superficial damage confined to the surface epithelium only and not the type of damage involving hemostasis and blood extravasation as observed in our study.7 – 9,15,16,28 It must be noted that control and ablated rats in our study cannot be directly compared with regard to mucosal repair because there was a difference in initial damage by hypertonic saline. However, our data show a large difference in restitution, particularly over hemorrhagic lesions in ablated rats exposed to neutral saline as compared with ablated rats exposed to acid. Because ablated rats lacked a mucosal hyperemic response to acid and pH of luminal saline was the same as in control rats, it is reasonable to assume that a main function of the hyperemic response to acid back-diffusion is to provide conditions that support restitution over hemorrhagic lesions. In summary, the present study provides evidence that the immediate hyperemic response to mucosal injury in the absence of acid is not mediated by sensory neurons. It therefore appears that protection of the gastric mucosa, / 5e14$$0035

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mediated by sensory neurons under such conditions, is via mechanisms other than gastric blood flow responses. Sensory neurons do account for mucosal hyperemia in response to luminal acid both in the normal and newly damaged mucosa, and efficient repair of the damaged mucosa by restitution during acid challenge is dependent on intact sensory afferent neurons and a sustained increased blood flow.

References 1. Holzer P, Livingston EH, Guth PH. Sensory neurons signal for an increase in rat gastric mucosal blood flow in the face of pending acid injury. Gastroenterology 1991;101:416–423. 2. Matsumoto J, Takeuchi K, Ueshima K, Okabe S. Role of capsaicin-sensitive afferent neurons in mucosal blood flow response of rat stomach induced by mild irritants. Dig Dis Sci 1992;37: 1336–1344. 3. Endoh K, Kao J, Domek MJ, Leung FW. Mechanism of gastric hyperemia induced by intragastric hypertonic saline in rats. Gastroenterology 1993;104:114–121. 4. Holzer P. Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacol Rev 1991;43: 143–201. 5. Szolcsa´nyi J, Bartho´ L. Impaired defense mechanism to peptic ulcer in the capsaicin-desensitized rat. In: Mo´zsik Gy, Ha¨nninen O, Ja´vor T, eds. Adv Physiol Sci. Volume 15. Gastrointestinal defense mechanisms. Oxford and Budapest: Pergamon Press and Akade´miai Klado´, 1981:39–51. 6. Holzer P, Sametz W. Gastric mucosal protection against ulcerogenic factors in the rat mediated by capsaicin-sensitive afferent neurons. Gastroenterology 1986;91:975–981. 7. Svanes K, Ito S, Takeuchi K, Silen W. Restitution of the surface epithelium of the in vitro frog gastric mucosa after damage with hyperosmolar sodium chloride. Morphologic and physiologic characteristics. Gastroenterology 1982;82:1409–1426. 8. Lacy ER, Ito S. Rapid epithelial restitution of the rat gastric mucosa after ethanol injury. Lab Invest 1984;51:573–583. 9. Ito S, Lacy ER. Morphology of rat gastric mucosal damage, defense, and restitution in the presence of luminal ethanol. Gastroenterology 1985;88:250–260. 10. Mersereau WA, Hinchey EJ. Effect of acidity on gastric ulceration induced by haemorrhage in the rat, utilizing a gastric chamber technique. Gastroenterology 1973;64:1130–1135. 11. Grønbech JE, Lacy ER. Substance P attenuates gastric mucosal hyperaemia after stimulation of sensory neurons in the rat stomach. Gastroenterology 1994;106:440–449. 12. Winer BJ. Statistical principles in experimental design. 2nd ed. Tokyo: McGraw Hill Kogakusha Ltd., 1971:514–603. 13. Esplugues JV, Whittle BJR, Moncada S. Loccal opoid-sensitive afferent sensory neurones in the modulation of gastric damage induced by Paf. Br J Pharmacol 1989;97:579–585. 14. Ritchie WP. Acute gastric mucosal damage induced by bile salts, acid, and ischemia. Gastroenterology 1975;68:699–707. 15. Grønbech JE, Grong K, Varhaug JE, Lekven J, Svanes K. Gastric epithelial restitution at low luminal pH during influence of pentagastrin or cimetidine in the cat. Gastroenterology 1987;93:753– 764. 16. Grønbech JE, Matre K, Stangeland L, Svanes K, Varhaug JE. Gastric mucosal repair in the cat: Role of the hyperemic response to mucosal damage. Gastroenterology 1988;95:311–320. 17. Guttu K, Sørbye H, Gislason H, Svanes K, Grønbech JE. Role of bicarbonate in blood flow–mediated protection and repair of

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damaged gastric mucosa in the cat. Gastroenterology 1994;107: 149–159. Holzer P, Livingston EH, Saria A, Guth PH. Sensory neurons mediate protective vasodilatation in rat gastric mucosa. Am J Physiol 1991;260:G363–G370. Holzer P, Lippe IT. Gastric mucosal hyperemia due to acid back diffusion depends on splanchnic nerve activity. Am J Physiol 1992;262:G505–G509. Li DS, Raybould HE, Quintero E, Guth PH. Calcitonin gene-related peptide mediates the gastric hyperemic response to acid backdiffusion. Gastroenterology 1992;102:1124–1128. Endoh K, Kauffman GI, Leung FW. Mechanism of aggravation of mucosal injury by intravenous nicotine in rat stomach. Am J Physiol 1991;261:G1037–G1042. Hottenstein OD, Pawlik WW, Remak G, Jacobson ED. Capsaicinsensitive nerves modulate resting blood flow and vascular tone in rat gut. Naunyn Schmiedebergs Arch Pharmacol 1991;343: 179–184. Holzer P, Pabst MA, Lippe IT, Peskar BM, Peskar BA, Livingston EH, Guth PH. Afferent nerve-mediated protection against deep mucosal damage in the rat stomach. Gastroenterology 1990; 98:838–848. Hatakeyama Y, Matsuo M, Tomoi M, Ohtsuka M, Shimomura K. Luminal dilution caused by certain mild irritants and capsaicin contributes to their gastric mucosal protection. Am J Physiol 1995;268:G200–G206.

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25. Stead RH, Tomioka M, Quinonez G, Simon GT, Felton SY, Bienenstock J. Intestinal mucosal mast cells in normal and nematodeinfected rat intestines are in intimate contact with peptidergic nerves. Proc Natl Acad Sci USA 1987;84:2975–2979. 26. Eliakim R, Karmeli F, Okon E, Rachmilewitz D. Ketotifen ameliorates capsaicin-augmented acetic acid–induced colitis. Dig Dis Sci 1995;40:503–509. 27. Rutten MJ, Ito S. Luminal acid effects on reconstitution of damaged guinea pig gastric mucosa in vitro. In: Allen A, Flemstro¨m G, Garner A, Silen W, Turnberg LA, eds. Mechanisms of mucosal protection in the upper gastrointestinal tract. New York: Raven, 1984:41–47. 28. Dzienis H, Grønbech JE, Varhaug JE, Lekven J, Svanes K. Regional blood flow and acid secretion associated with damage and restitution of the gastric surface epithelium in cats. Eur Surg Res 1987;19:98–112. Received November 1, 1995. Accepted September 3, 1996. Address requests for reprints to: Jon Erik Grønbech, M.D., Department of Surgery, University Hospital of Trondheim, N-7006 Trondheim, Norway. Fax: (47) 73-99-74-28. Supported by the Norwegian Research Council for Science and the Humanities (344.92/1029), the American Scandinavian Foundation, and grant R01-DK39074 from the National Institutes of Health (to E.R.L.). Dr. Grønbech is an international Fogarty Fellow (F05 TW074754).

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