The Relation Between Substance P Expression In The Dorsal Vagal Complex And Gastric Ulceration During The Restraint Water-immersion Stress In The Rat

INTRODUCTION: The rat undergoing restraint water-immersion stress(RWIS)is widely used to study the stress-induced gastric ulcer and to find the appropriate medical treatment which can cure this disease. Many evidence indicated that the gastric ulcer induced by the RWIS was mostly due to the hyperactivity of parasympathetic nerves that control the stomach. The vagal parasympathetic neurons innervating the stomach are largely located in the dorsal motor nucleus of the vagus (DMV) and partly in the nucleus ambiguus (NA).Moreover, nucleus of solitary tract (NTS) receives a part of afferent information of stomach. DMV, NTS and area postrema (AP) constitute the dorsal vagal complex (DVC) because of their near position, complicated neuronal contact and close functional correlation.Substance P (SP) is one of the most abundant neuropeptide in the central nervous system .It has been proposed to function in the central and peripheral nervous systems as a neurotransmitter or neuromodulator. SP has been implicated in a variety of physiological and pathophysiological processes including stress regulation, as well as affective and anxiety-related behaviour. Consistent with these functions, SP and its preferred neurokinin 1 (NK1) receptor has been found within brain areas known to be involved in the regulation of stress and anxiety responses. Aversive and stressful stimuli have been shown repeatedly to change SP content in brain tissue, as well as NK1 receptor binding. It is evident that SP in the brain modulates physiological and behavioural stress responses. In conscious rats, SP administered centrally induces a pattern of cardiovascular and behavioural responses which closely resemble the responses to stressful stimuli. Further evidence corroborating the role of SP in stress mechanisms come from the studies on the neuronal activation in brain areas known to be implicated in the modulation of stress reactions in response to various aversive stimuli. Pharmacological blockade or gene knockout of NK1 receptors has been found to attenuate stress-induced Fos expression (as marker for neuronal activation) in brain areas. It is suggested that the expression of SP which is widely distributed in DVC may affect gastric motility. But, what changes of SP and NK1 receptors expression occur in the DVC during the RWIS? To date, there has been no report about the temporal-spatial expression pattern of SP and NK1 in the rat DVC and NA during the RWIS. This study primarily probed into the effects of different RWIS duration on the SP and NK1 expression in DVC. More recently it has been demonstrated that emotional stressors increase SP efflux in hypothalamus and amygdala and that the magnitude of this effect depends on the severity of stressor. Depending on the brain area, an increase in intracerebral SP concentration (mimicked by SP microinjection) produces mainly anxiogenic-like responses in various behavioural tasks. Based on findings that, changes of SP and NK1 expression occur in hypothalamus(mostly focus on hypothalamic paraventricular nucleus-PVN, entopeduncular nucleus-EP, supraoptic nucleus-SON, dopamine cells -A13) and amygdala (mostly focus on medial amygdaloid nucleus, anterior part-MeA) during the RWIS were also detected in this study to investigate the influences of RWIS on them.METHODS: Male Wistar rats were randomly devided into five groups, 0 min (control) and 30, 60, 120 and 180 min for RWIS(the restraint water-immersion stress), ten rats in each group. At the end of RWIS, the rats were anesthetized with pentobarbital and perfused through the ascending aorta with saline followed by paraformaldehyde in phosphate buffered saline (PBS; pH 7.4). After perfusion, the brains were removed, postfixed overnight in 4% paraformaldehyde and transferred to sucrose for 24 h. Frozen sections were cut at 30μm into containing PBS (pH 7.4) throughout the length of the dorsal vagal complex (DVC) and the hypothalamus . After rinsing in PBS, brains were treated with an immunoperoxidase technique: sections were respectively incubated with different dilutions of primary rabbit anti-SP and anti-NK1. Subsequently, the sections were incubated with the biotinylated goat anti-rabbit IgG and then with streptavidin-biotin-horseradish peroxidase complex at room temperature. The sections were submitted to a diaminobenzidine reaction, yielding a brown deposit. Sections were mounted on gelatin-coated glass slides, dehydrated in a series of alcohols, cleared in xylene, and coverslipped. The specificity of the immunostaining was verified by incubation of the brain sections with normal goat serum, which produced no staining. The intensity of SP and NK1 expression was expressed by the integrated optical density (IOD) and the average integrated optical density(AIOD) which were counted using Image-Pro Plus 6.0.RESULTS: Obviously, the gastric erosion tended to be aggravated with the prolonging of RWIS.And in this duration,resumptively, SP expressions was the weakest at 30 min of the stress in the dorsal vagal complex (DVC) and hypothalamus, subsequently increased gradually to that of control group following the prolonging of RWIS. SP expression in NTS and DMV at 30 min of the stress decreased distinctly, compared with that of control group and 180min group. But in SubP and AP there were not significant changes in RWIS duration. In hypothalamus,SP expression of EP and PVN reduced during the early RWIS and then increased rapidly. The changes of NK1 expression in DVC in stress time were similar with that of SP expression. At the same time, in hypothalamus, there is a striking dissimilarity between the changes of SP and NK1 expression. NK1 expressions were the weakest at 30 min of the stress in A13 area and at 60min in MeA, subsequently increased gradually to that of control group following the prolonging of RWIS. In addition, we found that the locations of SP-positive neurons and NK1- positive neurons were different in hypothalamus.CONCLUSION: These results suggest that the early RWIS affect SP expression of NTS ,DMV, EP, PVN, and NK1 expression of NTS ,DMV, MeA, A13 area, and has no notable effect on the SP expression of AP.,SubP, MeA, SON, and NK1 expression of AP, SubP, SON, PVN, EP. So that, the changes of SP and NK1 expression in these areas including NTS, DMV, MeA, A13 and PVN may play a part in stress-induced gastric ulcer during the early RWIS.