The Astrocytes In The Periaqueductal Gray Involved In Water-immersion Restraint Stress In Rats

Restraint water immersion stress (RWIS) model is regarded as a strong compositestressor. The limbs of rats are to fix, and put into cold water (water temperature21±1℃), enforced to accept the physical and psychological stimulation. This model maylead to the gastrointestinal disorders, and bring about the acute injury of gastricmucosa, which have been widely used to study the pathogenesis of stress-inducedgastric lesion. The data of previous studies showed that the gastric lesion induced byRWIS mainly relies on the hyperactivities of the parasympathetic nervous system(PNS). Vagus afferent nerve which comes from the gastrointestinal tract and otherorgans convey sensory information to the brain stem. The secondary neuron in thesolitary nucleus (NTS) can integrate and relay information to other related nuclei, forexample the nuclear arms. through the glutamate neurotransmitter. Signals from thePara brachial nucleus is passed to the PAG via the hypothalamus and amygdala.Previous studies reported the plastic change of NO positive neurons of PAG in thestress-induced hypertension rats may be mediated by Ach in the rostral ventrolateralmedulla(RVLM)，which revealed the role of RVLM and the PAG in the regulation ofcardiovascular activity.PAG not only can receive the input signal of the parabrachial next to the nucleus, thehypothalamus, the ental nucleus of the amygdala and anterior cingulate cortex (ACC),the insula (INS)and the medial prefrontal cortex (PFC), but the information in PAGmay come from the anterior cingulate cortex (ACC)and transmit to the dorsal nucleusof the vagus nerve (DMV)which plays an important role in the regulation ofgastrointestinal function.ERK1and ERK2are related protein-serine/threonine kinases that participate in theRas-Raf-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including cell adhesion, cell cycleprogression, the inflammatory and stress response.The ERK1/2catalyzedphosphorylation of nuclear transcription factors including those of Ets, Elk, and c-Fosrepresents an important function and requires the translocation of ERK1/2into thenucleus by active. Whether the ERK pathway are involved in RWIS has not beenreported. From the above we can propose some questions:If so:1. Whether astrocyte in the periaqueductal gray (PAG) take part in the injury ofgastric mucosa caused by RWIS?2. If the astrocyte is involved in RWIS, what is the relationship between the neuronand the astrocyte in PAG?3. Whether ERK1/2signal pathway in the periaqueductal gray (PAG) is involved ingastric mucosal injury caused by RWIS?So, in order to resolve these questions, we designed the following experiments:1. The experimental rats were randomized into control group (RWIS0h) and stressgroup (RWIS0.5,1,2,3, and5h). c-Fos protein and glial fibers acidic protein (GFAP)were used to characterize neurons and astrocytes respectively. In this study,wedetected the c-Fos protein and GFAP protein expression in the periaqueductal gray atdifferent period of RWIS with Immunohistochemistry staining and Western blottingtechnique. we explored whether the neurons and the astrocytes in PAG took part inRWIS and the variation at different periods.2. The experimental rats were randomized into saline control group, L-AA treatmentgroup and ASO administration group. We examined whether ASO influenced theGFAP protein expression when the c-Fos protein expression was inhibited Meanwhile,we examined whether L-AA influenced the c-Fos protein expressionwhen GFAPprotein expression was inhibited in the ASO treatment group.3. The experimental rats were randomized into control group (RWIS0h) and stressgroup (RWIS0.5,1,3,5h). We detected the expression of p-ERK1/2protein at thedifferent period of RWIS by the immunohistochemistry staining and the Westernblotting technique. We wander to understand whether ERK1/2signal pathway is involved in gastric mucosa injury caused by RWIS? The experimental rats wererandomized into normal saline control group and PD98059dosage group. We studythe influence of c-Fos、GFAP expression and Ulcer Index when p-ERK1/2wasinhibited in PD98059treatment group.The experimental outcome as following:1. Compared to control group, the c-Fos protein and GFAP protein in the per unit areaof periaqueductal gray (PAG) were significantly increased in RWIS stress group.These results illustrated that the neurons and astrocytes in the periaqueductal gray(PAG) involved in RWIS.2. We found that the astrocyte inhibitors L-AA not only inhibited astrocyte activationc,but also significantly reduced the c-Fos protein expression. And the c-Fos proteininhibitor could both remarkably reduced the number of activated neurons andastrocytes. These results indicated that there is an interactional relationship betweenneurons and astrocytes.3. These experimental outcomes indicated that the astrocyte cell in the PAG involvedin Gastric mucosal injury Compared to control group, p-ERK1/2protein expressionwas significantly increased in RWIS stress of the group, and ERK1/2protein inhibitorcould remarkably inhibited c-Fos and GFAP protein expression. These evidencesillustrated that ERK1/2signaling pathway involved in RWIS stress.