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The effect of the autonomic nervous system on cell proliferation of the gastric mucosa in stress ulcer formation
Journal of the Autonomic Nervous System, 43 (1993) 179-188
179
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1838/93/$06.00 JANS 01390
The effect of the autonomic nervous system on cell proliferation of the gastric mucosa in stress ulcer formation Junichiro Hashiguchi, Masahiro Ito and Ichiro Sekine Department of Pathology, Atomic Disease Institute, Nagasaki University School of Medicine, Nagasaki, Japan (Received 6 October 1992) (Revision received 25 December 1992) (Accepted 28 December 1992)
Key words: Stress ulcer; Cell proliferation; Spontaneously hypertensive rat; Autonomic nervous system Abstract The role of the autonomic nervous system in cell proliferation of the gastric mucosa during restraint water immersion stress was investigated utilizing the spontaneously hypertensive rat (SHR) and its progenitor, the Wistar-Kyoto rat (WKY). Three hours of water-immersion restraint induced gastric lesions more frequently in WKY than in SHR, although there were few visual lesions in either strain in two hours of stress. The number of 5-bromo-2'-deoxyuridine (BrdU) positive cells in the corpus ventriculi mucosa markedly decreased in the WKY, but did not change in the SHR after two hours of water-immersion restraint stress. The acetylcholine content in the mucosa significantly decreased and the density of acetylcholinesterase (ACh-E) containing fiber of mucosa remarkably increased in only WKY after two hours stress exposure. On the other hand, there was no significant difference in either the responsiveness or the content of norepinephrine and epinephrine, which indicates sympathetic nervous function, after stress exposure in WKY and SHR. Similarly, the response of serum gastrin was not significantly different between the two strains. These results strongly suggest that the parasympathetic nerve plays an essential role in cell proliferation of the gastric mucosa in the pathogenesis of stress ulcer.
Introduction
The pathogenesis of stress ulcer has been discussed mainly in view of aggressive and defensive factors [23]. Recently, investigations have focused on the detailed analysis of defensive factors in the cell proliferation of the gastric mucosa. With regard to the cell kinetics in the defensive factors, the combination of increased cell loss and inhibition of cell proliferation may play an important
Correspondence to: J. Hashiguchi, Department of Pathology, Atomic Disease Institute, Nagasaki University School of Medicine, Sakamoto, Nagasaki 852, Japan.
role in the process of stress ulcer [6,13,14]. It has been reported that the inhibition of cell proliferation occurred in the early stage before the completion of macroscopic stress ulcer after waterimmersion restraint [11,14]. It seems that cell proliferation is controlled by a regulatory mechanism mediated by the nervous system a n d / o r humoral substances [8], but details remain to be established. Spontaneously hypertensive rats (SHR) were bred from normotensive Wistar-Kyoto rats (WKY) through selective sib-breeding and developed as a model of genetic hypertension with an autonomic disorder. This strain also shows significant resistance to stress ulcer [1,19], and the inhibition
180 mechanism of acid secretion and gastric motility by sympathetic facilitation was elucidated in the hypertensive rats [7]. SHR and WKY are therefore considered to be a useful tool for the analysis of the autonomic disorder in stress formation. Since Rokitansky first proposed it in 1842 [21], a large number of investigations have been made into the association between the autonomic nervous system and the pathogenesis of stress ulcer. In this study, we attempted to investigate the effect of the autonomic nervous system mainly on cell proliferation of the gastric mucosa in an early stress condition by the comparative study of the mucosa of corpus ventriculi in WKY and SHR.
Materials and Methods The animals were handled according to the guidelines of the N.I.H. Animal Research Committee (Bethesda, MD). Eighteen-week-old male SHR and WKY rats were purchased from Charles River Japan, Inc. (Atsugi, Japan). The rats were housed in the Laboratory Animal Center for Biomedical Research, Nagasaki University. The rats in stress groups were placed in a restraint cage and immersed up to the xyphoid process in water at 23°C as described previously by TakagiOkabe [24]. In order to exclude the influence of food intake and circadian rhythm, the rats were sacrificed under diethyl ether anesthesia between 11:00 a.m. and 2:00 p.m. after a 15-h fast, and the stomachs were promptly removed.
Experiment I: Severity of the gastric lesion (ulcer indices) in the SHR and WKY A total of 24 rats were used in this experiment. After 2 or 3 h of restraint water immersion stress, the animals were sacrificed and the resected stomachs were opened along the major curvature. Gastric mucosal lesions were recognized as linear breaks (erosions) at the mucosal surface of the corpus ventriculi. The extent of the lesions (ulcer index) was expressed as the sum of the area (mm 2) of the mucosal breaks.
Experiment II: Height of the gastric mucosa In this experiment, the gastric mucosal height was measured in the non-stressed and stressed
condition in both strains. The 24 rats were divided into four groups as follows: (1) WKY control group (n = 6); (2) SHR control group (n = 6); (3) WKY stress group (n --- 6); and (4) SHR stress group (n---6). Restraint water immersion stress was performed for 2 h in groups 3 and 4. The division into four groups was the same in the following experiments from (III-VI). The resected stomach was opened along the major curvature, and the anterior and posterior wall of the corpus ventriculi was cross-sectioned parallel to the minor curvature. Each preparation was embedded in paraffin and cut in 3-1zm strips for staining with hematoxyline and eosin (HE). In the anterior and posterior section, the mucosal thickness of the cardia, angle and pylorus side (six preparations per stomach) was carefully measured by micrometer.
Experiment III: Width of the proliferative zone and number of labeled cells using 5-bromo-2'-deoxyuridine (BrdU) BrdU (Sigma, St. Louis, MO) 100 mg/kg body weight in 1 ml of phosphate-buffered saline (PBS) solution was injected intraperitoneally 2 h before sacrifice in each group of rats, and then in the stress groups the treated rats underwent restraint water immersion for 2 h. Using the same procedure as that described in experiment II), the preparation of sections was performed by fixation in 70% ethanol, embedding in paraffin and cutting in 3-/zm strips. After deparaffinization, sections were treated with 2 N HC1 for 30 rain at room temperature and neutralized with 0.1 M Na2B40 7. In order to inhibit endogenous peroxidase activity, the preparation was incubated in 5% normal horse serum after incubation in 0.3% H 2 0 2 for 30 min to avoid non-specific reaction. The first antibody used was 1:50 diluted antiBrdU monoclonal antibody (Becton Dickinson, San Jose, CA) in PBS (0.1 M, pH 7.4) incubated overnight at 4°C. Then, after incubation with 1 : 200 diluted biotinylated anti-mouse IgG for 2 h at room temperature, the sections were incubated with avidine biotin peroxidase (Vector, Burlingame, CA) for 30 rain. Finally, the BrdU incorporation was localized by incubation for 2
181
min with 3,3'-diamino-benzidine-tetrahydro chloride (DAB). After the measurement of the width of the proliferative zone, six sites per one strip of proliferative zone were photographed 200 × magnification. The number of BrdU positive cells per one visual field was counted using the Image Visual Analyzer System (IVAS-2000, Carl Zeiss, FRG). This analyzer system distinguishes intensity of staining on a scale of 0: (black) to 255: (white). Using this scale, positive staining for BrdU has an intensity level of 40-100, and the detection of positively stained cells is easy and impartial.
Experiment I~." Measurement of acetylcholine and catecholamine content in the gastric mucosa The stomachs were promptly removed from each group of rats under diethylether anesthesia and opened along the major curvature. The mucosal surface of the opened stomach was rinsed with freezing spray while turned upwards. The mucosa was scraped off with a scalpel. Acethylmm 8"
p < 0.01
choline and catecholamine contents in the gastric mucosa were determined using high performance liquid chromatography with the electrochemical detector (HPLC-ED) system described elsewhere [22].
Experiment V." Measurement of enzymehistochemistry for acetylcholinesterase activity Tissue preparation was performed by the method described in (experiment II). The sections were immersed in 4% paraformaldehyde for 1 h and then in 15% sucrose for 6 h, and 10-p~m cryostat sections were prepared. These sections were pretreated with 10 -5 M-tetraisopropyl pyrophosphoramide (iso-OMPA, Sigma, St. Louis, MO) for 30 min at room temperature to inhibit pseudocholinesterase activity, then washed with 0.1 M maleate buffer (pH 6.0) and incubated for 30 min at 37°C in a solution containing acetylcholine iodide as a substrate, according to Karnovsky and Roots [10]. Then the density of ACh-E containing fiber was determined as total length per visual field by the Image Visual Analyzer System (IVAS-2000). Experiment VI: Measurement of serum gastrin Blood was collected from each group of rats by heart puncture under diethylether anesthesia, and the serum was separated by centrifugation at 4°C. Gastrin was determined by double antibody radioimmunoassay (RIA) using a gastrin 1-125 kit (Dinabot, Tokyo, Japan).
7"
6" 5" 4"
Statistical evaluation of data All data were expressed as mean + S.E.M. (standard error of the mean). Student's t-test (two-tailed) was used to determine statistically significant differences between experimental groups of data.
3" NS 2" 1 0 2hrs 3 hrs stress
WKY
2 hrs 3 hrs stress
Results
SHR
Fig. 1. Changes in ulcer index of W K Y and SHR after 2 or 3 h restraint water immersion stress. Each group consists of 6 rats. The ulcer indices after 3 h of stress were 0,12 + 0.08 mm 2 in SHR and 5.19+1.83 mm 2 in W K Y ( P < 0.01). The ulcer indices after 2 h stress were 0.06+_0.05 mm z in SHR and 0.09 +_0.07 mm 2 in WKY.
Experiment I: Severity of the gastric lesion (ulcer indices) in the SHR and WKY The ulcer indices were 0.12 +_0.08 mm e in SHR and 5.19_+1.83 mm 2 in WKY after 3 h stress (P < 0.01). After 2 h stress, the lesions
182
were very slight if present at all, and significant difference was not observed in ulcer indices between SHR and WKY (see Fig. l). Based on these results, 2 h stress was applied to the following experiments II to VI.
Experiment 11: Height of the gastric mucosa and width of the proliferative zone in BrdU labeling There were no significant differences among the four groups in either height or width of proliferative zone of the gastric mucosa (see Table I).
Experiment III: Number of BrdU positive cells after 2 h stress There was no significant difference between the anterior wall and posterior wall in any of the
?i
TABLE I
Height and width of the proliferative zone of the gastric mucosa Group
n
height (mm)
width of P.Z. (mm)
WKY-control SHR-control WKY-stress SHR-stress
6 6 6 6
0.518 ± 0.047 0.515±0.011 0.526 ± 0.048 0.538± 0.040
0.121± 0.011 0.131±0.012 0.112± 0.008 0.129± 0.005
No significant difference in either mucosal height or width of, proliferative zone is observed in the four groups (each group,
n = 6).
groups. With regard to mean value in WKY, the number of BrdU positive cells decreased from 29.9 + 2 . 1 / F (control) to 17.4 + 4 . 2 / F (stress group) ( P < 0.001). In SHR, however, this num-
¸¸ i ¸
Fig. 2. Photomicrographs of the gastric oxyntic mucosa immunostained for BrdU with monoclonal antibody and standard avid±n-biotin technique 2 h after BrdU injection. The preparation is described in the Materials and Methods section. In the WKY stress group (W-S), the fewest BrdU positive cells were observed among WKY control (W-C), SHR control (S-C), WKY stress (W-S) and SHR stress (S-S).
183
ber did not change between the control group (21.7 +_ 0 . 7 / F ) and the stress group (22.1 +_ 3 . 7 / F ) (see Figs. 2, 3).
Experiment I1<"Parasympathetic nervous function Acethylcholine content of the gastric mucosa. The content decreased from 35.7 + 10.1 n g / g tissue in the control to 15.0 _+ 5.1 n g / g tissue in the stress group ( P < 0.01) in the WKY, but there was no significant change between the control group (29.0 +_ 8.4 n g / g tissue) and the stress group (31.3 _+ 20.7 n g / g tissue) in S H R (see Fig. 4). Density of ACh-E containing fiber The density of A C h - E containing fiber increased from 5.78 + 0.70 m m / m m 2 in the control group to 7.99 +_ 0.58 m m / m m z in the stress group ( P < 0.05) in WKY, but there was no significant change between the control group (5.80 + 0.28 m m / m m 2) and the stress group (5.26 -+ 0.30 m m / m m 2) in S H R (see Figs. 5, 6).
Experiment V." Catecholamine content of the gastric mucosa after 2 h stress With regard to norepinephrine content, there was a significant difference ( P < 0.01) between
n mollgtissue 50 t
] p < 0.01
NS
I
40
30
20
10
0 cont
stress
WKY
cont stress SHR
Fig. 4. Changes in acethycholine content (nmol/g tissue) in the gastric mucosa of WKY and SHR after stress, Each group consists of 6 rats. In the WKY, acethycholine content decreased from 35.7_+10.1 (nmol/g tissue) in the control to
15.0+5.1 (nmol/g tissue) in the stress group, but there was no significant difference between the control (29.0_+8.4 nmol/g tissue) and stress (31.3_+20.7 nmol/g tissue) groups in the SHR.
40p< 0.001
[
NS
30
20'
10"
cont
stress
WKY
eont stress SHR
Fig. 3. Changes in the number of BrdU positive cells (IF) in the gastric mucosa of WKY and SHR after stress. Each group consists of 6 rats. In the WKY, BrdU positive cells decreased from 29.9+ 2.1/F (control) to 17.4+ 4.2/F (stress) (P < 0.001). In the SHR, however, there was no significant difference between the control and stress groups.
W K Y (327.0 + 69.0 n m o l / g tissue) and S H R (774.1 -+203.1 n m o l / g tissue) in the control groups, but there was no significant difference between the control and stress groups in either strain. The epinephrine content in the control groups was WKY; 15.6 +_ 12.4 n m o l / g tissue and SHR; 16.3 + 16.5 nrnol/g tissue (N.S. vs. W K Y control). There were significant increases in the stress groups in both strains (WKY; 53.2 _+ 12.4 n m o l / g tissue, vs. W K Y control, P < 0.05, SHR; 57.0 _+ 29.4 n m o l / g tissue, vs. S H R control, P < 0.05) (see Table II).
Experiment VI: Serum gastrin after 2 h stress S H R (202.2_+51.4 n g / m l ) showed slightly higher than WKY(168.2 _+ 25. 1 n g / m l ) , but not significantly different value in the control state. After stress exposure, the levels of serum gastrin tended to increase but did not significantly change in either W K Y (184.0 -+ 35.9 n g / m l ) or S H R (204.2 -+ 8.2 n g / m l ) (see Fig. 7).
184 : ~:
~ i
¸:III~: :
Fig. 5. Photomicrographs of gastric oxyntic mucosa stained for demonstration of ACh-E containing fiber by the Karnovsky-Roots method. In the WKY-stress group (W-S), the highest density was observed among WKY control (W-C), SHR control (S-C), WKY stress (W-S) and SHR stress (S-S).
Discussion The life span of epithelial cells of the gastric mucosa is composed of four stages: i.e. proliferation, migration, differentiation and exfoliation [2]. In general, the cell cycle of rat epithelial cells is so rapid that the changes in the generative cell closely reflect those of the epithelial cell. Cell proliferation plays an essential role in the pathogenesis of ulcer formation and repair, especially stress-induced ulcer. Because water-immersion restraint induced gastric lesions frequently in WKY but less frequently in SHR after 3 h of stress, the strain differences in sensitivity to stress are thought to be very useful in investigating the effect of the autonomic nervous system on cell proliferation of
the gastric mucosa in the early stage before the completion of macroscopic stress ulcer. The inhibition of mucosal cell proliferation is observed in the early period of stress before the appearance of visible lesions. Therefore the regulatory mechanism of cell proliferation is very important. Cell proliferation has been investigated by autoradiography or BrdU techniques that label the nuclei of proliferating cells during the synthesis of new DNA. The proliferation is apparently controlled by a regulatory mechanism mediated by the nervous system a n d / o r humoral substances, but details remain to be established in stress exposure. In our experiment, the cell proliferation of corpus ventriculi was investigated with reference to the influence of the autonomic nervous system under 2 h of stress before the
185 pg/mi 300'
mmJmrn
10
200.
100 ¸
o
o ¢ont
stress
WKY
eont
stress SHR
Fig. 6. Changes in acetylcholinesterase containing fiber. Each group consists of 6 rats. The density increased from 5.78 + 0.7 m m / m m 2 in the control group to 7.99+0.58 m m / m m 2 in the stress group ( P < 0.05) in the WKY, but there was no significant difference between the control (5.80-+0.28 m m / m m 2) and stress groups (5.26 +_0.30 r a m / r a m 2) in the SHR.
TABLE II
Norepinephrine (ng / g tissue) and epinephrine content (ng / g tissue) of the gastric mucosa Group
n
Norepinephrine (nmol/g)
Epinephrine (nmol/g)
WKY-control SHR-control WKY-stress SHR-stress
6 6 6 6
327.8_+ 69,0 774.1 _+203.1 a 376.0+ 34.4 b 601.6_+114.8 c,d
15.6_+ 12.4 16.3_+ 6.5 e 53.2+--26.1f 57.0_+29.4 g,h
There was no significant difference in norepinephrine content between the control and stress groups in either strain (each group, n = 6). There was a significant increase in epinephrine content between the control and stress groups in both strains (each group, n = 6). Values are mean +- S.E. a p < 0.01 (vs. WKY control). b N.S. (vs. WKY control). c p < 0.01 (vs. WKY stress). d N.S. (vs. SHR control). N.S. (vs. WKY control). f P < 0.05 (vs. WKY control). g P < 0.05 (vs. SHR control). h N.S. (vs. WKY stress).
¢ont
stress
WKY
cont
stress
SHR
Fig. 7. Serum Gastrin ( p g / m l ) and Serum Corticosterone (ng/ml). There were no significant differences in the concentration of gastrin between SHR and WKY, or between the control and stress groups.
completion of visible ulcerations in WKY and SHR. We chose a stress duration of 2 h for BrdU evaluation because any duration longer than 2 h may produce overt lesions and result in the observation of repair ability rather than the inhibition of cell proliferation in ulcer formation. There is no remarkable difference in the height of the gastric mucosa and the thickness of the proliferative zone between WKY and SHR. Even under stress conditions, they do not exhibit any significant change. These results indicate that the number of BrdU positive cells per visual field is synonymous with the labeling index (labeling c e l l / n u m b e r of proliferative zone) in flash labeling. The inhibition of proliferation after short-time water-immersion restraint has been demonstrated in the Wistar strain rat by Kuwayama and Eastwood [14]. In our experiment, WKY, which is susceptible to stress ulcer, also displayed the inhibition o f proliferation of the gastric mucosa. On the contrary, SHR, which is resistant to ulcer formation, did not show inhibition of cell proliferation under stress. A remarkable change was observed in WKY as compared to SHR: under
186
the stress condition, a decrease in acetylcholine content and increase in ACh-E activity was observed in the gastric mucosa. Although the vagus is not entirely cholinergic [4], acetylcholine is the most important chemical mediator of the vagus. In this study, the acetylcholine content of the gastric mucosa decreased in WKY but not in SHR after stress load. A decrease in acetylcholine content of the gastric mucosa indicates two possibilities. One is a decrease in the production of acetylcholine, the other is the consumption of acetylcholine. ACh-E is localized on the postsynaptic membrane of the effector cells in the gastric mucosa [17]. The density of ACh-E-containing fibers closely reflects the ACh-E activity histopathologically. Therefore, the decrease in acetylcholine content and increase in ACh-E of the gastric mucosa support the hypothesis that the facilitation of the vagus occurred after 2 h of stress in WKY, resulting in the consumption of acetylcholine. Ultimately, the facilitation of the vagus may be closely related to the inhibition of cell proliferation in our experiment. In 1966, Pearl reported that the chronic stimulation of the hypothalamus caused hyperplasia of mucous neck cells, parietal cells and chief cells, but that vagotomy diminished this effect and revealed the trophic effect of the vagus [20]. Recently, however, there have been many reports that, both experimentally and by the investigation of human materials, vagotomy exerts a stimulatory effect on cell proliferation [5,15]. These reports are closely consistent with the result of the present study that the facilitation of the vagus had an inhibitory effect on cell proliferation. Gastrin is a strong acid secretagogue and plays an important role in the elicitation of cell proliferation in the gastric mucosa [9]. The cholinergic neuron is one of many factors involved in the regulatory mechanism of gastrin secretion. The results of this study suggest that gastrin is not related to the inhibition in the early stage of stress in WKY, since a significant decrease in gastrin was not observed in this strain. With regard to the sympathetic nervous system, on the other hand, it has been reported that the chemical or surgical resection of sympathetic
nerves results in the inhibition of cell proliferation of the gastric mucosa [3,12,16]. Furthermore, Tutton reported that a-receptor stimulation by norepinephrine or /3-receptor blockade by propranolol had a stimulatory effect on cell proliferation of the gut mucosa, but that /3-receptor stimulation by epinephrine and a-receptor blockade by phentolamine had an inhibitory effect on cell proliferation [25]. In the present experiment, the concentration of norepinephrine remained the same even after 2 h of stress in both rat strains, although it was higher in SHR than in WKY in both stress and control groups. This result is closely consistent with the report by Shichijo et al. [22] that the norepinephrine value in the gastric mucosa of SHR in both controls and 7 h restraint water immersion stress remained higher than that of WKY. Norepinephrine has been shown to regulate parasympathetic nerve-effector transmission presynaptically by reducing ACh release in the digestive tract [18]. With regard to parasympathetic nervous function in cell proliferation, the advantage of the norepinephrine concentration in SHR might reduce ACh release and inhibition of cell proliferation. Epinephrine, which is mainly derived from the adrenal gland, showed a significant increase under stress in both WKY and SHR, but there was no difference between control WKY and SHR, or between stressed WKY and SHR, and the response to stress showed the same range in the two strains, suggesting that sympathetic response to short-time stress did not differ between WKY and SHR. Although many factors such as microcirculation and acid secretion participate in the mechanism of cell proliferation in the gastric mucosa, we focused mainly on the autonomic nervous system for the evaluation of stress ulcer resistance in SHR in this study. The present study showed that the cell proliferation in the gastric mucosa was inhibited after short-time stress in rats with vagal facilitation (WKY) but not in those without it (SHR). In the latter, the vagal function might be tonically suppressed by sympathetic facilitation. The autonomic nervous system
187
is intimately involved in cell proliferation during stress, and the parasympathetic nerve in particular plays an essential role. The inhibition of cell proliferation may result in mucosal damage after prolonged stress. In conclusion, our results strongly suggest that the parasympathetic nerve is the foremost factor in the inhibition of cell proliferation in the pathogenesis of stress ulcer.
References 1 Athey, G.H. and Iams S.G., Cold-restraint induced gastric lesions in normotensive and spontaneously hypertensive rats, Life Sci., 28 (1981) 889-894. 2 Eastwood, G.L., Gastrointestinal epithelial renewal, Gastroenterology, 72 (1977) 962-975. 3 Furness, J.B. and Costa, M., The adrenergic innervation of gastrointestinal tract, Rev. Physiol. Biochem. Exp. Pharmacol., 69 (1974) 1-51. 4 Grossman, M.I., Neural and hormonal regulation of gastrointestinal function, an overview, Ann. Rev. Physiol., 41 (1979) 27. 5 Hakanson, R., Vallgren, S., Ekelund, M., Rehfeld, J.F. and Sundler, F., The vagus exert trophic control of the stomach in the rat, Gastroenterology, 86 (1984) 28-32. 6 Harding, R.K. and Morris, G.P., Cell loss from normal and stressed gastric mucosae of the rat, Gastroenterology, 72 (1977) 857-863. 7 Ito, M., Shichijo, K., Hsu, C.T., Sekine, I., Ozaki, M. and Tuchiya, K., The role. of the sympathetic nervous system in stress-induced ulcer in stroke-prone spontaneously hypertensive rats (SHRSP). In M. Yoshikawa (Ed.), New Trends in Autonomic Nervous System Research, Elsevier, Amsterdam, 1991, pp. 257-258. 8 Johnson, L.R., Regulation of gastrointestinal growth. In Johnson, L.R. (Ed.), Physiology of the Gastrointestinal Tract, 2nd Edn., Raven Press, New York, 1987, pp. 301325. 9 Johnson, L.R. and Guthrie, P.D., Mucosal DNA synthesis: A short term index of the trophic action of gastrin, Gastroenterology, 67 (1974) 453-459. 10 Karnovsky, M.J. and Roots, L., A direct coloring thiocholine method for cholinesterases, J. Histochem. Cytochem., 12 (1964) 219-221. 11 Kim, Y., Kerr, R. and Lipkin, M., Cell proliferation during the development of stress erosions in mouse stomach, Nature, 215 (1967) 1180-1181.
12 Klein, R.M., Analysis of intestinal cell proliferation after guanethidine-induced sympathectomy. I. Stathmokinetic, labeling index, mitotic index, and cellular migration studies, Cell Tiss. Res., 195 (1978) 239-250. 13 Kuwayama, H., Takeuchi, K. and Kohashi, E., Effects of water-immersion restraint stress on rat gastric epithelial cell loss and migration, J. Clin. Gastroenterol. (Suppl), (1988) $78-$83. 14 Kuwayama, H. and Eastwood, G.L., Effects of water immersion restraint stress and chronic indomethacin ingestion on gastric antral and fundic epithelial proliferation, Gastroenterology, 88 (1985) 362-365. 15 Ley, R., Willems, G. and Vansteenkiste, Y., Influence of vagotomy on parietal cell kinetics in the rat gastric mucosa, Gastroenterology, 65 (1973) 764-772. 16 Musso, F., Lachet, J.J., Cruz, A.R. and Goncalves, R.P., Effect of denervation on the mitotic index of the intestinal epithelium of the rat, Cell Tiss. Res., 163 (1975) 395-402. 17 Nakamura, M., Oda, M., Yonei, Y., Tsukada, N., Watanabe, N., Komatsu, H. and Tuchiya, M., Demonstration of the localization of muscarinic acetylcholine receptors in the gastric mucosa-light and electron microscopic autoradiographic studies useing 3H-quinuclidinyl benzilate, Acta Histochem. Cytochem., 17 (1984) 297-309. 18 Osterlitz, H.W. and Watt, A.J., Kinetic parameters of norcotic agonists and antagonists, with particular reference to N-allylnoroxymorpone, Br. J. Pharmacol., 33 (1968) 266-276. 19 Pare, W.P. and Schimmel, G.T., Stress ulcer in normotensive and spontaneously hypertensive rats, Physiol. Behav., 36 (1986) 699-705. 20 Pearl, J.M., Ritchie, W.P., Gilsdorf, R.B., Delaney, J.P. and Leonard, A.S., Hypothalamic stimulation and feline gastric mucosal cellular populations, Factors in the etiology of the stress ulcer, J.A.M.A., 195 (1966) 281-284. 21 Rokitansky, C., Handbuch der speziellen pathologischen Anatomie. Vol. 3, Mosler, Wien, 1842. 22 Shichijo, K., Ito, M. and Sekine, I., The mechanism of low susceptibility to stress in gastric lesions of spontaneously hypertensive rats. Life Sci., 49 (1991) 2023-2029. 23 Sun, D.C.H., Etiology and pathology of peptic ulcer. In H.C. Bockus (Ed.), Gastroenterology Vol. I, W.B. Saunders Company, Philadelphia, London, Toronto, 1974, pp. 579-610. 24 Takagi, K. and Okabe, S., The effect of drugs on the production and recovery of the stress ulcer, Jpn. J. Pharmacol., 18 (1968) 9-18. 25 Tutton, P.J.M., The influence of adrenoreceptor activity on crypt cell proliferation in the rat jejunum, Cell Tiss. Kinet., 7 (1974) 125-136.
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© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1838/93/$06.00 JANS 01390
The effect of the autonomic nervous system on cell proliferation of the gastric mucosa in stress ulcer formation Junichiro Hashiguchi, Masahiro Ito and Ichiro Sekine Department of Pathology, Atomic Disease Institute, Nagasaki University School of Medicine, Nagasaki, Japan (Received 6 October 1992) (Revision received 25 December 1992) (Accepted 28 December 1992)
Key words: Stress ulcer; Cell proliferation; Spontaneously hypertensive rat; Autonomic nervous system Abstract The role of the autonomic nervous system in cell proliferation of the gastric mucosa during restraint water immersion stress was investigated utilizing the spontaneously hypertensive rat (SHR) and its progenitor, the Wistar-Kyoto rat (WKY). Three hours of water-immersion restraint induced gastric lesions more frequently in WKY than in SHR, although there were few visual lesions in either strain in two hours of stress. The number of 5-bromo-2'-deoxyuridine (BrdU) positive cells in the corpus ventriculi mucosa markedly decreased in the WKY, but did not change in the SHR after two hours of water-immersion restraint stress. The acetylcholine content in the mucosa significantly decreased and the density of acetylcholinesterase (ACh-E) containing fiber of mucosa remarkably increased in only WKY after two hours stress exposure. On the other hand, there was no significant difference in either the responsiveness or the content of norepinephrine and epinephrine, which indicates sympathetic nervous function, after stress exposure in WKY and SHR. Similarly, the response of serum gastrin was not significantly different between the two strains. These results strongly suggest that the parasympathetic nerve plays an essential role in cell proliferation of the gastric mucosa in the pathogenesis of stress ulcer.
Introduction
The pathogenesis of stress ulcer has been discussed mainly in view of aggressive and defensive factors [23]. Recently, investigations have focused on the detailed analysis of defensive factors in the cell proliferation of the gastric mucosa. With regard to the cell kinetics in the defensive factors, the combination of increased cell loss and inhibition of cell proliferation may play an important
Correspondence to: J. Hashiguchi, Department of Pathology, Atomic Disease Institute, Nagasaki University School of Medicine, Sakamoto, Nagasaki 852, Japan.
role in the process of stress ulcer [6,13,14]. It has been reported that the inhibition of cell proliferation occurred in the early stage before the completion of macroscopic stress ulcer after waterimmersion restraint [11,14]. It seems that cell proliferation is controlled by a regulatory mechanism mediated by the nervous system a n d / o r humoral substances [8], but details remain to be established. Spontaneously hypertensive rats (SHR) were bred from normotensive Wistar-Kyoto rats (WKY) through selective sib-breeding and developed as a model of genetic hypertension with an autonomic disorder. This strain also shows significant resistance to stress ulcer [1,19], and the inhibition
180 mechanism of acid secretion and gastric motility by sympathetic facilitation was elucidated in the hypertensive rats [7]. SHR and WKY are therefore considered to be a useful tool for the analysis of the autonomic disorder in stress formation. Since Rokitansky first proposed it in 1842 [21], a large number of investigations have been made into the association between the autonomic nervous system and the pathogenesis of stress ulcer. In this study, we attempted to investigate the effect of the autonomic nervous system mainly on cell proliferation of the gastric mucosa in an early stress condition by the comparative study of the mucosa of corpus ventriculi in WKY and SHR.
Materials and Methods The animals were handled according to the guidelines of the N.I.H. Animal Research Committee (Bethesda, MD). Eighteen-week-old male SHR and WKY rats were purchased from Charles River Japan, Inc. (Atsugi, Japan). The rats were housed in the Laboratory Animal Center for Biomedical Research, Nagasaki University. The rats in stress groups were placed in a restraint cage and immersed up to the xyphoid process in water at 23°C as described previously by TakagiOkabe [24]. In order to exclude the influence of food intake and circadian rhythm, the rats were sacrificed under diethyl ether anesthesia between 11:00 a.m. and 2:00 p.m. after a 15-h fast, and the stomachs were promptly removed.
Experiment I: Severity of the gastric lesion (ulcer indices) in the SHR and WKY A total of 24 rats were used in this experiment. After 2 or 3 h of restraint water immersion stress, the animals were sacrificed and the resected stomachs were opened along the major curvature. Gastric mucosal lesions were recognized as linear breaks (erosions) at the mucosal surface of the corpus ventriculi. The extent of the lesions (ulcer index) was expressed as the sum of the area (mm 2) of the mucosal breaks.
Experiment II: Height of the gastric mucosa In this experiment, the gastric mucosal height was measured in the non-stressed and stressed
condition in both strains. The 24 rats were divided into four groups as follows: (1) WKY control group (n = 6); (2) SHR control group (n = 6); (3) WKY stress group (n --- 6); and (4) SHR stress group (n---6). Restraint water immersion stress was performed for 2 h in groups 3 and 4. The division into four groups was the same in the following experiments from (III-VI). The resected stomach was opened along the major curvature, and the anterior and posterior wall of the corpus ventriculi was cross-sectioned parallel to the minor curvature. Each preparation was embedded in paraffin and cut in 3-1zm strips for staining with hematoxyline and eosin (HE). In the anterior and posterior section, the mucosal thickness of the cardia, angle and pylorus side (six preparations per stomach) was carefully measured by micrometer.
Experiment III: Width of the proliferative zone and number of labeled cells using 5-bromo-2'-deoxyuridine (BrdU) BrdU (Sigma, St. Louis, MO) 100 mg/kg body weight in 1 ml of phosphate-buffered saline (PBS) solution was injected intraperitoneally 2 h before sacrifice in each group of rats, and then in the stress groups the treated rats underwent restraint water immersion for 2 h. Using the same procedure as that described in experiment II), the preparation of sections was performed by fixation in 70% ethanol, embedding in paraffin and cutting in 3-/zm strips. After deparaffinization, sections were treated with 2 N HC1 for 30 rain at room temperature and neutralized with 0.1 M Na2B40 7. In order to inhibit endogenous peroxidase activity, the preparation was incubated in 5% normal horse serum after incubation in 0.3% H 2 0 2 for 30 min to avoid non-specific reaction. The first antibody used was 1:50 diluted antiBrdU monoclonal antibody (Becton Dickinson, San Jose, CA) in PBS (0.1 M, pH 7.4) incubated overnight at 4°C. Then, after incubation with 1 : 200 diluted biotinylated anti-mouse IgG for 2 h at room temperature, the sections were incubated with avidine biotin peroxidase (Vector, Burlingame, CA) for 30 rain. Finally, the BrdU incorporation was localized by incubation for 2
181
min with 3,3'-diamino-benzidine-tetrahydro chloride (DAB). After the measurement of the width of the proliferative zone, six sites per one strip of proliferative zone were photographed 200 × magnification. The number of BrdU positive cells per one visual field was counted using the Image Visual Analyzer System (IVAS-2000, Carl Zeiss, FRG). This analyzer system distinguishes intensity of staining on a scale of 0: (black) to 255: (white). Using this scale, positive staining for BrdU has an intensity level of 40-100, and the detection of positively stained cells is easy and impartial.
Experiment I~." Measurement of acetylcholine and catecholamine content in the gastric mucosa The stomachs were promptly removed from each group of rats under diethylether anesthesia and opened along the major curvature. The mucosal surface of the opened stomach was rinsed with freezing spray while turned upwards. The mucosa was scraped off with a scalpel. Acethylmm 8"
p < 0.01
choline and catecholamine contents in the gastric mucosa were determined using high performance liquid chromatography with the electrochemical detector (HPLC-ED) system described elsewhere [22].
Experiment V." Measurement of enzymehistochemistry for acetylcholinesterase activity Tissue preparation was performed by the method described in (experiment II). The sections were immersed in 4% paraformaldehyde for 1 h and then in 15% sucrose for 6 h, and 10-p~m cryostat sections were prepared. These sections were pretreated with 10 -5 M-tetraisopropyl pyrophosphoramide (iso-OMPA, Sigma, St. Louis, MO) for 30 min at room temperature to inhibit pseudocholinesterase activity, then washed with 0.1 M maleate buffer (pH 6.0) and incubated for 30 min at 37°C in a solution containing acetylcholine iodide as a substrate, according to Karnovsky and Roots [10]. Then the density of ACh-E containing fiber was determined as total length per visual field by the Image Visual Analyzer System (IVAS-2000). Experiment VI: Measurement of serum gastrin Blood was collected from each group of rats by heart puncture under diethylether anesthesia, and the serum was separated by centrifugation at 4°C. Gastrin was determined by double antibody radioimmunoassay (RIA) using a gastrin 1-125 kit (Dinabot, Tokyo, Japan).
7"
6" 5" 4"
Statistical evaluation of data All data were expressed as mean + S.E.M. (standard error of the mean). Student's t-test (two-tailed) was used to determine statistically significant differences between experimental groups of data.
3" NS 2" 1 0 2hrs 3 hrs stress
WKY
2 hrs 3 hrs stress
Results
SHR
Fig. 1. Changes in ulcer index of W K Y and SHR after 2 or 3 h restraint water immersion stress. Each group consists of 6 rats. The ulcer indices after 3 h of stress were 0,12 + 0.08 mm 2 in SHR and 5.19+1.83 mm 2 in W K Y ( P < 0.01). The ulcer indices after 2 h stress were 0.06+_0.05 mm z in SHR and 0.09 +_0.07 mm 2 in WKY.
Experiment I: Severity of the gastric lesion (ulcer indices) in the SHR and WKY The ulcer indices were 0.12 +_0.08 mm e in SHR and 5.19_+1.83 mm 2 in WKY after 3 h stress (P < 0.01). After 2 h stress, the lesions
182
were very slight if present at all, and significant difference was not observed in ulcer indices between SHR and WKY (see Fig. l). Based on these results, 2 h stress was applied to the following experiments II to VI.
Experiment 11: Height of the gastric mucosa and width of the proliferative zone in BrdU labeling There were no significant differences among the four groups in either height or width of proliferative zone of the gastric mucosa (see Table I).
Experiment III: Number of BrdU positive cells after 2 h stress There was no significant difference between the anterior wall and posterior wall in any of the
?i
TABLE I
Height and width of the proliferative zone of the gastric mucosa Group
n
height (mm)
width of P.Z. (mm)
WKY-control SHR-control WKY-stress SHR-stress
6 6 6 6
0.518 ± 0.047 0.515±0.011 0.526 ± 0.048 0.538± 0.040
0.121± 0.011 0.131±0.012 0.112± 0.008 0.129± 0.005
No significant difference in either mucosal height or width of, proliferative zone is observed in the four groups (each group,
n = 6).
groups. With regard to mean value in WKY, the number of BrdU positive cells decreased from 29.9 + 2 . 1 / F (control) to 17.4 + 4 . 2 / F (stress group) ( P < 0.001). In SHR, however, this num-
¸¸ i ¸
Fig. 2. Photomicrographs of the gastric oxyntic mucosa immunostained for BrdU with monoclonal antibody and standard avid±n-biotin technique 2 h after BrdU injection. The preparation is described in the Materials and Methods section. In the WKY stress group (W-S), the fewest BrdU positive cells were observed among WKY control (W-C), SHR control (S-C), WKY stress (W-S) and SHR stress (S-S).
183
ber did not change between the control group (21.7 +_ 0 . 7 / F ) and the stress group (22.1 +_ 3 . 7 / F ) (see Figs. 2, 3).
Experiment I1<"Parasympathetic nervous function Acethylcholine content of the gastric mucosa. The content decreased from 35.7 + 10.1 n g / g tissue in the control to 15.0 _+ 5.1 n g / g tissue in the stress group ( P < 0.01) in the WKY, but there was no significant change between the control group (29.0 +_ 8.4 n g / g tissue) and the stress group (31.3 _+ 20.7 n g / g tissue) in S H R (see Fig. 4). Density of ACh-E containing fiber The density of A C h - E containing fiber increased from 5.78 + 0.70 m m / m m 2 in the control group to 7.99 +_ 0.58 m m / m m z in the stress group ( P < 0.05) in WKY, but there was no significant change between the control group (5.80 + 0.28 m m / m m 2) and the stress group (5.26 -+ 0.30 m m / m m 2) in S H R (see Figs. 5, 6).
Experiment V." Catecholamine content of the gastric mucosa after 2 h stress With regard to norepinephrine content, there was a significant difference ( P < 0.01) between
n mollgtissue 50 t
] p < 0.01
NS
I
40
30
20
10
0 cont
stress
WKY
cont stress SHR
Fig. 4. Changes in acethycholine content (nmol/g tissue) in the gastric mucosa of WKY and SHR after stress, Each group consists of 6 rats. In the WKY, acethycholine content decreased from 35.7_+10.1 (nmol/g tissue) in the control to
15.0+5.1 (nmol/g tissue) in the stress group, but there was no significant difference between the control (29.0_+8.4 nmol/g tissue) and stress (31.3_+20.7 nmol/g tissue) groups in the SHR.
40p< 0.001
[
NS
30
20'
10"
cont
stress
WKY
eont stress SHR
Fig. 3. Changes in the number of BrdU positive cells (IF) in the gastric mucosa of WKY and SHR after stress. Each group consists of 6 rats. In the WKY, BrdU positive cells decreased from 29.9+ 2.1/F (control) to 17.4+ 4.2/F (stress) (P < 0.001). In the SHR, however, there was no significant difference between the control and stress groups.
W K Y (327.0 + 69.0 n m o l / g tissue) and S H R (774.1 -+203.1 n m o l / g tissue) in the control groups, but there was no significant difference between the control and stress groups in either strain. The epinephrine content in the control groups was WKY; 15.6 +_ 12.4 n m o l / g tissue and SHR; 16.3 + 16.5 nrnol/g tissue (N.S. vs. W K Y control). There were significant increases in the stress groups in both strains (WKY; 53.2 _+ 12.4 n m o l / g tissue, vs. W K Y control, P < 0.05, SHR; 57.0 _+ 29.4 n m o l / g tissue, vs. S H R control, P < 0.05) (see Table II).
Experiment VI: Serum gastrin after 2 h stress S H R (202.2_+51.4 n g / m l ) showed slightly higher than WKY(168.2 _+ 25. 1 n g / m l ) , but not significantly different value in the control state. After stress exposure, the levels of serum gastrin tended to increase but did not significantly change in either W K Y (184.0 -+ 35.9 n g / m l ) or S H R (204.2 -+ 8.2 n g / m l ) (see Fig. 7).
184 : ~:
~ i
¸:III~: :
Fig. 5. Photomicrographs of gastric oxyntic mucosa stained for demonstration of ACh-E containing fiber by the Karnovsky-Roots method. In the WKY-stress group (W-S), the highest density was observed among WKY control (W-C), SHR control (S-C), WKY stress (W-S) and SHR stress (S-S).
Discussion The life span of epithelial cells of the gastric mucosa is composed of four stages: i.e. proliferation, migration, differentiation and exfoliation [2]. In general, the cell cycle of rat epithelial cells is so rapid that the changes in the generative cell closely reflect those of the epithelial cell. Cell proliferation plays an essential role in the pathogenesis of ulcer formation and repair, especially stress-induced ulcer. Because water-immersion restraint induced gastric lesions frequently in WKY but less frequently in SHR after 3 h of stress, the strain differences in sensitivity to stress are thought to be very useful in investigating the effect of the autonomic nervous system on cell proliferation of
the gastric mucosa in the early stage before the completion of macroscopic stress ulcer. The inhibition of mucosal cell proliferation is observed in the early period of stress before the appearance of visible lesions. Therefore the regulatory mechanism of cell proliferation is very important. Cell proliferation has been investigated by autoradiography or BrdU techniques that label the nuclei of proliferating cells during the synthesis of new DNA. The proliferation is apparently controlled by a regulatory mechanism mediated by the nervous system a n d / o r humoral substances, but details remain to be established in stress exposure. In our experiment, the cell proliferation of corpus ventriculi was investigated with reference to the influence of the autonomic nervous system under 2 h of stress before the
185 pg/mi 300'
mmJmrn
10
200.
100 ¸
o
o ¢ont
stress
WKY
eont
stress SHR
Fig. 6. Changes in acetylcholinesterase containing fiber. Each group consists of 6 rats. The density increased from 5.78 + 0.7 m m / m m 2 in the control group to 7.99+0.58 m m / m m 2 in the stress group ( P < 0.05) in the WKY, but there was no significant difference between the control (5.80-+0.28 m m / m m 2) and stress groups (5.26 +_0.30 r a m / r a m 2) in the SHR.
TABLE II
Norepinephrine (ng / g tissue) and epinephrine content (ng / g tissue) of the gastric mucosa Group
n
Norepinephrine (nmol/g)
Epinephrine (nmol/g)
WKY-control SHR-control WKY-stress SHR-stress
6 6 6 6
327.8_+ 69,0 774.1 _+203.1 a 376.0+ 34.4 b 601.6_+114.8 c,d
15.6_+ 12.4 16.3_+ 6.5 e 53.2+--26.1f 57.0_+29.4 g,h
There was no significant difference in norepinephrine content between the control and stress groups in either strain (each group, n = 6). There was a significant increase in epinephrine content between the control and stress groups in both strains (each group, n = 6). Values are mean +- S.E. a p < 0.01 (vs. WKY control). b N.S. (vs. WKY control). c p < 0.01 (vs. WKY stress). d N.S. (vs. SHR control). N.S. (vs. WKY control). f P < 0.05 (vs. WKY control). g P < 0.05 (vs. SHR control). h N.S. (vs. WKY stress).
¢ont
stress
WKY
cont
stress
SHR
Fig. 7. Serum Gastrin ( p g / m l ) and Serum Corticosterone (ng/ml). There were no significant differences in the concentration of gastrin between SHR and WKY, or between the control and stress groups.
completion of visible ulcerations in WKY and SHR. We chose a stress duration of 2 h for BrdU evaluation because any duration longer than 2 h may produce overt lesions and result in the observation of repair ability rather than the inhibition of cell proliferation in ulcer formation. There is no remarkable difference in the height of the gastric mucosa and the thickness of the proliferative zone between WKY and SHR. Even under stress conditions, they do not exhibit any significant change. These results indicate that the number of BrdU positive cells per visual field is synonymous with the labeling index (labeling c e l l / n u m b e r of proliferative zone) in flash labeling. The inhibition of proliferation after short-time water-immersion restraint has been demonstrated in the Wistar strain rat by Kuwayama and Eastwood [14]. In our experiment, WKY, which is susceptible to stress ulcer, also displayed the inhibition o f proliferation of the gastric mucosa. On the contrary, SHR, which is resistant to ulcer formation, did not show inhibition of cell proliferation under stress. A remarkable change was observed in WKY as compared to SHR: under
186
the stress condition, a decrease in acetylcholine content and increase in ACh-E activity was observed in the gastric mucosa. Although the vagus is not entirely cholinergic [4], acetylcholine is the most important chemical mediator of the vagus. In this study, the acetylcholine content of the gastric mucosa decreased in WKY but not in SHR after stress load. A decrease in acetylcholine content of the gastric mucosa indicates two possibilities. One is a decrease in the production of acetylcholine, the other is the consumption of acetylcholine. ACh-E is localized on the postsynaptic membrane of the effector cells in the gastric mucosa [17]. The density of ACh-E-containing fibers closely reflects the ACh-E activity histopathologically. Therefore, the decrease in acetylcholine content and increase in ACh-E of the gastric mucosa support the hypothesis that the facilitation of the vagus occurred after 2 h of stress in WKY, resulting in the consumption of acetylcholine. Ultimately, the facilitation of the vagus may be closely related to the inhibition of cell proliferation in our experiment. In 1966, Pearl reported that the chronic stimulation of the hypothalamus caused hyperplasia of mucous neck cells, parietal cells and chief cells, but that vagotomy diminished this effect and revealed the trophic effect of the vagus [20]. Recently, however, there have been many reports that, both experimentally and by the investigation of human materials, vagotomy exerts a stimulatory effect on cell proliferation [5,15]. These reports are closely consistent with the result of the present study that the facilitation of the vagus had an inhibitory effect on cell proliferation. Gastrin is a strong acid secretagogue and plays an important role in the elicitation of cell proliferation in the gastric mucosa [9]. The cholinergic neuron is one of many factors involved in the regulatory mechanism of gastrin secretion. The results of this study suggest that gastrin is not related to the inhibition in the early stage of stress in WKY, since a significant decrease in gastrin was not observed in this strain. With regard to the sympathetic nervous system, on the other hand, it has been reported that the chemical or surgical resection of sympathetic
nerves results in the inhibition of cell proliferation of the gastric mucosa [3,12,16]. Furthermore, Tutton reported that a-receptor stimulation by norepinephrine or /3-receptor blockade by propranolol had a stimulatory effect on cell proliferation of the gut mucosa, but that /3-receptor stimulation by epinephrine and a-receptor blockade by phentolamine had an inhibitory effect on cell proliferation [25]. In the present experiment, the concentration of norepinephrine remained the same even after 2 h of stress in both rat strains, although it was higher in SHR than in WKY in both stress and control groups. This result is closely consistent with the report by Shichijo et al. [22] that the norepinephrine value in the gastric mucosa of SHR in both controls and 7 h restraint water immersion stress remained higher than that of WKY. Norepinephrine has been shown to regulate parasympathetic nerve-effector transmission presynaptically by reducing ACh release in the digestive tract [18]. With regard to parasympathetic nervous function in cell proliferation, the advantage of the norepinephrine concentration in SHR might reduce ACh release and inhibition of cell proliferation. Epinephrine, which is mainly derived from the adrenal gland, showed a significant increase under stress in both WKY and SHR, but there was no difference between control WKY and SHR, or between stressed WKY and SHR, and the response to stress showed the same range in the two strains, suggesting that sympathetic response to short-time stress did not differ between WKY and SHR. Although many factors such as microcirculation and acid secretion participate in the mechanism of cell proliferation in the gastric mucosa, we focused mainly on the autonomic nervous system for the evaluation of stress ulcer resistance in SHR in this study. The present study showed that the cell proliferation in the gastric mucosa was inhibited after short-time stress in rats with vagal facilitation (WKY) but not in those without it (SHR). In the latter, the vagal function might be tonically suppressed by sympathetic facilitation. The autonomic nervous system
187
is intimately involved in cell proliferation during stress, and the parasympathetic nerve in particular plays an essential role. The inhibition of cell proliferation may result in mucosal damage after prolonged stress. In conclusion, our results strongly suggest that the parasympathetic nerve is the foremost factor in the inhibition of cell proliferation in the pathogenesis of stress ulcer.
References 1 Athey, G.H. and Iams S.G., Cold-restraint induced gastric lesions in normotensive and spontaneously hypertensive rats, Life Sci., 28 (1981) 889-894. 2 Eastwood, G.L., Gastrointestinal epithelial renewal, Gastroenterology, 72 (1977) 962-975. 3 Furness, J.B. and Costa, M., The adrenergic innervation of gastrointestinal tract, Rev. Physiol. Biochem. Exp. Pharmacol., 69 (1974) 1-51. 4 Grossman, M.I., Neural and hormonal regulation of gastrointestinal function, an overview, Ann. Rev. Physiol., 41 (1979) 27. 5 Hakanson, R., Vallgren, S., Ekelund, M., Rehfeld, J.F. and Sundler, F., The vagus exert trophic control of the stomach in the rat, Gastroenterology, 86 (1984) 28-32. 6 Harding, R.K. and Morris, G.P., Cell loss from normal and stressed gastric mucosae of the rat, Gastroenterology, 72 (1977) 857-863. 7 Ito, M., Shichijo, K., Hsu, C.T., Sekine, I., Ozaki, M. and Tuchiya, K., The role. of the sympathetic nervous system in stress-induced ulcer in stroke-prone spontaneously hypertensive rats (SHRSP). In M. Yoshikawa (Ed.), New Trends in Autonomic Nervous System Research, Elsevier, Amsterdam, 1991, pp. 257-258. 8 Johnson, L.R., Regulation of gastrointestinal growth. In Johnson, L.R. (Ed.), Physiology of the Gastrointestinal Tract, 2nd Edn., Raven Press, New York, 1987, pp. 301325. 9 Johnson, L.R. and Guthrie, P.D., Mucosal DNA synthesis: A short term index of the trophic action of gastrin, Gastroenterology, 67 (1974) 453-459. 10 Karnovsky, M.J. and Roots, L., A direct coloring thiocholine method for cholinesterases, J. Histochem. Cytochem., 12 (1964) 219-221. 11 Kim, Y., Kerr, R. and Lipkin, M., Cell proliferation during the development of stress erosions in mouse stomach, Nature, 215 (1967) 1180-1181.
12 Klein, R.M., Analysis of intestinal cell proliferation after guanethidine-induced sympathectomy. I. Stathmokinetic, labeling index, mitotic index, and cellular migration studies, Cell Tiss. Res., 195 (1978) 239-250. 13 Kuwayama, H., Takeuchi, K. and Kohashi, E., Effects of water-immersion restraint stress on rat gastric epithelial cell loss and migration, J. Clin. Gastroenterol. (Suppl), (1988) $78-$83. 14 Kuwayama, H. and Eastwood, G.L., Effects of water immersion restraint stress and chronic indomethacin ingestion on gastric antral and fundic epithelial proliferation, Gastroenterology, 88 (1985) 362-365. 15 Ley, R., Willems, G. and Vansteenkiste, Y., Influence of vagotomy on parietal cell kinetics in the rat gastric mucosa, Gastroenterology, 65 (1973) 764-772. 16 Musso, F., Lachet, J.J., Cruz, A.R. and Goncalves, R.P., Effect of denervation on the mitotic index of the intestinal epithelium of the rat, Cell Tiss. Res., 163 (1975) 395-402. 17 Nakamura, M., Oda, M., Yonei, Y., Tsukada, N., Watanabe, N., Komatsu, H. and Tuchiya, M., Demonstration of the localization of muscarinic acetylcholine receptors in the gastric mucosa-light and electron microscopic autoradiographic studies useing 3H-quinuclidinyl benzilate, Acta Histochem. Cytochem., 17 (1984) 297-309. 18 Osterlitz, H.W. and Watt, A.J., Kinetic parameters of norcotic agonists and antagonists, with particular reference to N-allylnoroxymorpone, Br. J. Pharmacol., 33 (1968) 266-276. 19 Pare, W.P. and Schimmel, G.T., Stress ulcer in normotensive and spontaneously hypertensive rats, Physiol. Behav., 36 (1986) 699-705. 20 Pearl, J.M., Ritchie, W.P., Gilsdorf, R.B., Delaney, J.P. and Leonard, A.S., Hypothalamic stimulation and feline gastric mucosal cellular populations, Factors in the etiology of the stress ulcer, J.A.M.A., 195 (1966) 281-284. 21 Rokitansky, C., Handbuch der speziellen pathologischen Anatomie. Vol. 3, Mosler, Wien, 1842. 22 Shichijo, K., Ito, M. and Sekine, I., The mechanism of low susceptibility to stress in gastric lesions of spontaneously hypertensive rats. Life Sci., 49 (1991) 2023-2029. 23 Sun, D.C.H., Etiology and pathology of peptic ulcer. In H.C. Bockus (Ed.), Gastroenterology Vol. I, W.B. Saunders Company, Philadelphia, London, Toronto, 1974, pp. 579-610. 24 Takagi, K. and Okabe, S., The effect of drugs on the production and recovery of the stress ulcer, Jpn. J. Pharmacol., 18 (1968) 9-18. 25 Tutton, P.J.M., The influence of adrenoreceptor activity on crypt cell proliferation in the rat jejunum, Cell Tiss. Kinet., 7 (1974) 125-136.