Comparative Studies On C-Fos Expression In The Different Brain Regions And During The Different Durations Of Restraint Water-immersion Stress In The Rat

Restraint water-immersion stress (RWIS), considered to be a mixture of physical and psychological stressors, induces vagally-mediated gastric hypercontractility, hypersecretion of gastric acid and acute gastric erosions within a few hours. It is widely used to study the pathogenesis of stress-induced gastric lesions and to filtrate the medicine that can cure this disease. Convergent studies suggest that the gastric dysfunctions induced by RWIS were mainly due to neuronal activities in the brain and mediated by the parasympathetic fibers in the vagus. 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, the nucleus of the solitary tract (NTS) receives a part of the afferent information from the stomach. The area postrema (AP) is the chemoreceptor trigger zone of the vomitive reflex and also receives inputs from the vagal sensory fibres of the stomach. The adjacent DMV, NTS and AP have complicated neuronal contact and close correlation in function, so that they constitute the dorsal vagal complex (DVC). Thus, the DVC and NA are the primary nerve centres of regulating gastric functions. However, stimulation of above nuclei respectively all resulted in the inhibition of gastric motility in anesthetic rats. Whether the neurons of the DMV, NTS, AP and NA are excited, and characterization of the temporal-spatial pattern of neuronal activities in these four nuclei under the RWIS, have not been reported to date.The limbic forebrain is the higher brain centre regulating visceral functions and is responsible for forming emotional reactions to the stress. Previous studies about neuronal activities of the forebrain mainly focused on the medial prefrontal cortex (mPFC) and the hypothalamic paraventricular nucleus (PVN), and didn't compare the intensity of neuronal activities in different nuclei.It is well known that stress can activate hypothalamus-pituitary-adrenocortical (HPA) axis. However, RWIS mainly induced the hyperactivity of the parasympathetic nervous system. So, the activity pattern of HPA axis under the RWIS needs to be determined.Then, in the present study, we used the c-Fos expression as a marker of the activation of neurons, investigated the temporal-spatial pattern of neuronal activities in the different brain regions of the rats induced by different durations of RWIS, consequently, probed into the central mechanism of gastric dysfunction induced by RWIS and provided an important foundation for centrally preventing and curing the stress gastric lesions and improving gastroenteritic motivity. Male Wistar rats were randomly divided into 5 groups designated according to their duration of RWIS, respectively 0, 30, 60, 120 and 180 min. The rats stressed for 0 min were considered as a control group. Additionally, to determine the intrinsic kinetics of c-Fos expression, another 5 rats were given RWIS for 30 min and then replaced in their home cages for 30 min before sacrifice, comparing with the rats stressed for 30 min or 60 min. At the end of RWIS, the rat was anesthetized with pentobarbital sodium,then the body temperature was measured and the stomach was removed. The stomach was fixed with 1% formalin and the gastric lesions were evaluation. After the stomach was removed, the rat was perfused transcardially with phosphate-buffered saline (PBS, pH 7.4) followed by freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffer (4℃). Afterward, the brain, pituitary and adrenal gland were removed immediately, post-fixed in the same fixative for 4 h and cryoprotected overnight in 20% sucrose in 0.1 M PBS at 4℃until sectioning. Immunohistochemical detection of c-Fos protein was used to map brain neuronal pathways activated by RWIS. The number and integrated optical density (IOD) of Fos-IR neurons in 0.01 mm2 denote the intensity of c-Fos expression. Additionally, for the basal level of c-Fos expression in different nuclei was very diverse in the control group, the relative intensity of c-Fos expression can better reflect the differences of c-Fos expression among the different nuclei. Then, the multiples of controls were counted, that is, the quantum of c-Fos expression of each rat was divided by the mean values of the control group.Different durations of RWIS significantly affected the c-Fos expression in the DVC and NA of the rats. Resumptively, c-Fos expression in the DVC and NA peaked at 60 min of the stress, subsequently decreased gradually with increasing durations of RWIS. Interestingly, the most intense c-Fos expression was observed in the DMV during the stress, next to the NA, NTS and AP. The peak of c-Fos expression in the caudal DMV appeared at 120 min of the stress, slower than that in the rostral and intermediate DMV (60 min). In group 120, Fos-IR neurons in the caudal DMV were significantly more than that in the rostral and intermediate DMV. The c-Fos expression in the intermediate and caudal NTS was significantly more intense than that in the rostral NTS. These results indicate that the neuronal hyperactivity of the DVC and NA, the primary center controlling gastric functions, especially the DMV and NA, may play important roles in the gastric dysfunctions induced by the RWIS. The patterns of c-Fos expression in the different zones of the DMV and NTS may reflect their different roles in modulating gastric functions.Different durations of RWIS significantly affected the c-Fos expression in the different forebrain regions of the rats. The c-Fos expression in the midline cortical areas peaked at 60 min of the stress, subsequently decreased gradually. The c-Fos expression in the septum area and amygdala tended to a peak at 120 min of stress. The c-Fos expression in most nuclei of the hypothalamus, especially the SON and PVN, was significantly greater in rats stressed for 30 min than in controls and then maintained at a higher level. However, a striking new finding in the present study is that the most intense expression of c-Fos was always observed in the supraoptic nucleus (SON) during the RWIS, next in the hypothalamic paraventricular nucleus (PVN), posterior cortical amygdaloid nucleus (PCoA), central amygdaloid nucleus (CeA) and medial prefrontal cortex (mPFC). Moreover, the body temperature was reduced to the lowest degree after 60 min of the RWIS and the gastric lesions tended to gradually aggravate with the prolonging of RWIS duration. These data strongly suggest that these nuclei participate in the organismal response to the RWIS in different degree, and may be involved in the hypothermia and gastric lesions induced by the RWIS.The c-Fos expression in three organs of the HPA axis induced by RWIS was not parallel. Remarkably more Fos-IR neurons in the PVN and adenohypophysis, especially in the intermediate part, were observed in the rats of four stress groups than those of control group. Nevertheless, no prominent differences of c-Fos expression were observed in each of the PVN and adenohypophysis among the four stress groups. No c-Fos expression was observed in the zona fasciculata of the adrenal cortex of the control rats and the stress rats. Robust c-Fos expression in the PVN and adenohypophysis induced by the RWIS suggests the PVN and adenohypophysis participate in the organismal response to the RWIS, and c-Fos in the two regions may be involved in regulating the synthesis of corticotropin releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) during stress response.Moreover, to investigate the roles of body restraint and cold-water immersion in the development of gastric ulcers induced by restraint water-immersion stress and the relationship between hypothermia and stressed gastric ulcers, the effects of restraint water-immersion and water-immersion alone under different water temperatures on gastric ulcers and body temperature were studied in rats. Restraint plus water-immersion and water-immersion alone under the same water temperature resulted in the same degree of stomach erosions and hypothermia. It appears that cold-water immersion does play an important role on gastric ulceration induced by the restraint water-immersion procedure while body restraint does't. The lower water temperature, the lower body temperature and the severer gastric ulcers are during the experiment. It is concluded that hypothermia induced by cold water exposure is an inducement of gastric ulcers.In conclusion, the present study provided a neuroanatomical evidence for the RWIS inducing the hyperactivity of the parasympathetic nervous system, and uncovered the central mechanism of the gastric lesions induced by the RWIS. In the primary center controlling gastric functions, RWIS induced the most intense c-Fos expression in theDMV and NA, which suggests the neuronal hyperactivity of the DMV and NA may be one of the primary central mechanisms of the gastric dysfunctions induced by the RWIS. In the higher brain centre modulating visceral functions, RWIS induced the most intense expression of c-Fos in the SON and more intense expression of c-Fos in the PVN, PCoA, CeA and mPFC, which suggests the neuronal hyperactivity of these above nuclei may be one of the higher central mechanisms of the gastric dysfunctions induced by the RWIS. Robust c-Fos expression in the PVN and adenohypophysis induced by the RWIS suggests the PVN and adenohypophysis participate in the organismal response to the RWIS.