The Role Of Parabrachial Nucleus In Rat Restraint Water-immersion Stress

The rat model of restraint water-immersion stress(RWIS)is a compound stress model in which the limbs and heads of rats are fixed on a special hard plate and then immersed in cold water(water temperature 21±1℃)to produce serious physiological and psychological stimulation.This leads to emotional changes such as fear,anger and uneasiness in the body in a short period of time,accompanied by gastrointestinal disorders such as increased gastrointestinal activity,decreased gastric mucosal blood flow and increased gastric acid secretion,resulting in gastric mucosal damage.Stress gastric ulcer is mainly a disease caused by acute stress,which is easy to occur in people with strong sensitivity and anxiety,so this model is widely used to study the mechanism of stress gastric mucosal injury.Parabrachial nucleus(PBN)has long been considered as a sensory relay,which plays an important role in maintaining balance and promoting survival in the central nervous system.There are extensive fibrous connections between the parabrachial nucleus and the medullary vagal complex,hypothalamus,thalamus and cerebral cortex.The parabrachial nucleus receives visceral sensory information from the nucleus of the solitary tract(NTS),which is further integrated and transmitted to the cerebral cortical cortex via the thalamus.At the same time,parabrachial approval receives signals from the forebrain,such as nociceptive stimulation information,visceral information and hot and cold thermo sensory information,which has a fiber projection relationship with the paraventricular nucleus of the hypothalamus.It participates in the regulation of water-salt balance,thermoregulation,sleep awakening and gastrointestinal motility.The previous results of the research group showed that under restraint water-immersion stress,the parasympathetic nervous system(PNS)was mainly involved in the regulation of gastric function,and the paraventricular nucleus,nucleus of solitary tract,dorsal nucleus of vagus nerve and supraoptic nucleus were involved in the process of gastric dysfunction induced by restraint water-immersion stress,but whether PBN was involved in the process of RWIS and the role of PBN in stress-induced gastric mucosal injury.It has not been reported which types of neurons in PBN are activated in the RWIS process.In view of the above questions,an experiment is designed and the following results are obtained:1.The experimental rats were randomly divided into three groups:control group(RWIS 0 h),1h stress group(RWIS 1 h)and 3 h stress group(RWIS 3 h).Immunohistochemical staining and Western blotting were used to detect the expression of c-Fos protein,a specific marker of neuronal activation,and GFAP(GFAP),a specific marker of astrocyte,in the medial parabrachial nucleus(MPB),lateral parabrachial nucleus(LPB)and nucleus KF in different periods of RWIS.The results showed that compared with the control group,the expression of c-Fos protein and GFAP protein per unit area(0.01mm~2)of PBN in 1 h stress group(RWIS 1 h)and 3 h stress group(RWIS3 h)was significantly increased,among which the expression of(LPB)in lateral parabrachial nucleus was the most,and there was significant difference.These results suggest that neurons and astrocytes in the PBN are involved in the RWIS process.2.The experimental rats were randomly divided into three groups:normal saline group(saline+CNO),no-load virus group(AAV-Ca MKIIa-MCS-m Cherry+CNO),h M4D virus group(AAV-Ca MKIIa-h M4D(Gi)-m Cherry+CNO).Immunofluorescence staining,Western blotting and chemical genetics were used to detect the effects of localized injection of h M4D virus on the expression of c-Fos and GFAP in PBN and brainstem NTS,as well as gastric mucosal injury in rats after localized injection of h M4D virus into the parabrachial nucleus.The results showed that compared with the normal saline group,the expression of c-Fos and GFAP protein in the parabrachial nucleus decreased significantly in the h M4D virus group,the expression of Claudin-1 and Occludin in the gastric wall increased significantly,the expression of PCNA in the gastric wall decreased significantly,and the gastric mucosal injury index decreased significantly,indicating that h M4D virus inhibited the activity of the parabrachial nucleus and improved the gastric mucosal injury induced by Restraint Water-Immersion Stress.3.The experimental rats were randomly divided into two groups:normal saline group(saline injection)and administration group(injection inhibitor L-NAME).Through immunohistochemical staining and Western blotting analysis,it was observed that restraint-immersion stress had an effect on the expression of NOS(NOS)in MPB,LPB and KF in the brain regions of PBN,and the effect of NOS inhibitor L-NAME on gastric mucosal injury in rats after injection of NOS into PBN.It was found that restraint-immersion stress significantly increased the expression of NOS in PBN,and compared with the normal saline group,the gastric mucosal injury index and the degree of gastric mucosal injury in L-NAME group were significantly lower than those in saline group.The expression of occlusal protein Occludin and closure protein Claudin-1 increased,while the expression of PCNA in gastric wall decreased.The results showed that nitric oxide neurons in PBN participated in the process of restraint-immersion stress.The inhibition of NOS synthesis in PBN by L-NAME improved gastric mucosal injury induced by restraint-immersion stress and had a protective effect on stress-induced gastric mucosal injury.