Changes Of Expression Of Mineralocorticoid Receptor And Glucocorticoid Receptor In The Rat Hippocampal Neurons Induced By Fornix Transection

ObjectiveHypothalamic-pituitary-adrenal axis as a hub for the neuroendocrine-immune network to maintain a balance between the environment and in vivo regulation of the body's aging process plays an important role. Glucocorticoid effects on the pituitary gland and hypothalamus constitute the major sources of negative feedback regulation of HPA axis. However, a great deal of existing data shows that the structure over the hypothalamic, especially hippocampus is involved in the regulation of the HPA axis, but is of great significance to the negative feedback.Current research generally believe hippocampus inhibites the function of paraventricular nucleus in the hypothalamus by the mineralocorticoid receptor and glucocorticoid receptor. Under resting conditions, glucocorticoid with the combination of MR in the hippocampus participate basal level of participation of negative feedback regulation of HPA axis. Under conditions of stress, a high level of stress hormone with the combination of GR in the hippocampus inhibited the HPA axis over-reaction or make the HPA axis stress to return to base level. The two receptors MR and GR belong to the nuclear receptor family which is the protein binding with special hormone and a kind of ligand-dependent transcription factors. Through gene expression induced or inhibited cell proliferation, differentiation and death , resulting in a variety of biological effects. But from the hippocampus to the paraventricular nucleus have no direct neural projection, and indirect path to go through the fornix Fornix transaction can also have an impact on existing research reported in the paraventricular nucleus CRH and AVP levels, This led us to speculate hippocampus inhibition of the hypothalamic paraventricular nucleus whether media the fornix.In this experiment, the fornix was transected to set animal models. We employed the methods of immunohistochemistry, western blotting and analysis of images to study the expression of MR and GR of the rat hippocampus neuron after fornix transaction. We wish our study may provide experimental evidence for the mechanism the hippocampal MR and GR participate the regulation of HPA axis.Methods一,Experimental animal and groupAdult healthy male Wistar rats 90, weighing 200-220g were provided by experimental animal center in China Medical University, Rats were housed under laboratory condition and were divided into 0d, 4d, 7d, 10d for immunohistochemistry and western blotting. Normal group (n=10).二,To set animal modelRats were anesthetized by pentobarbital. Each animal was positioned in a stereotaxic instrument, and the scalp was cut and retracted to expose the skull by home-made double edged knife. Anteroposterior (AP)-1.5 mm to bregma, dorsoventral (DV)-6 mm (FT) or -2mm (sham-operated) and mediolateral (ML)±1mm to transect the fornix. The blade was lowered over the course of 1 min and left in the brain for 5 min. At the completion of all surgeries, the skin was sutured. Routine breeding , antibiotic control infection.三,Identification of rat model fornix transactionThe rats of the normal group and fornix transaction group hippocampus cholinergic fibers were stained for AchE.四,Immunohistochemical stainingThe rats of each group were perfused fixed in 4% paraformaldehyde, drawing the materials making frozen sections for immunohistochemistry with SABC method (MR working concentration of 1:200,GR working concentration of 1:500) , DAB coloration, neutral gummi mounting the result was observed by light microscope.(OLYMPUS, BX60, Japan) , taking photograph as well, as undertaking image analysis.五,Western BlottingThe fresh hippocampal tissue was homogenated ultrasonicated and then high-speed centrifuged in low tempreture. The supernatant albumen aquired was then operated though electrophoresis and trarsmembrane. Add anti-MR antibody (1:500) and anti-GR antibody (1:1000) after block. MR and GR were incubated overnight at 4℃, then, they were incubated in IgG with HRP marked 2h, DAB coloration, scanning band to calculate the gray scale of MR, GR.Results一,The results of rat hippocampal fiber AchE stainingNormal hippocampal cholinergic fibers are rich, interwoven into uniform fiber network. Fornix transaction group loss of a large number of AchE-positive fibers. To observe the pyramidal layer CA1 area of the hippocampus, we can see the Fine fibers also lower densities disarranged. It suggests the fornix transaction model was built successfully.二,The results of MR, GR Immunohistochemical staining reactionMR, GR immunoreactive product were brown expression in the cytoplasm and nucleus in normal group, MR mainly located in the hippocampal CA1,CA2,CA3 pyramidal cell layer and dentate gyrus granule cells. GR mainly Located in the hippocampal CA1, CA2 pyramidal cell layer, and dentate gyrus granule cells. The pyramidal cells expressing MR and GR are abundant, dark-dyeing and lined up in order, the expression of MR,GR in each sham-operation is resemble to normal group. 0,4 days fornix transaction group compared with normal group and corresponding sham-operation group MR. GR-immunoreactive cell number and morphology were not altered, 7,10 days fornix transaction group the hippocampal CA1 area MR, GR-immunoreactive cells were fewer smaller cells. light stain . 10 days fornix transaction group than seven days fornix transection group fewer positive cells in the immune response, more superficial staining.三,The results of MR, GR Western BlottingWestern Blotting detection discover the expression of hippocampus MR,GR on 7,. 10d fornix transaction group compared with the normal group, sham-operation group reduced also 10 days less than seven days of expression. 0, 4 days fornix transaction group compared with normal group and corresponding sham-operation group have no significant difference, the expression of MR, GR in each sham-operation is resemble to normal group.四,The results of analysis of images7, 10d fornix transaction group compared with the normal group , sham-operation group reduced also 10 days less than seven days of expression (p<0.05) . 0,4 days fornix transaction group compared with normal group and corresponding sham-operation group have no significant difference (p>0.05) , the expression of MR,GR in each sham-operation is resemble to normal group (p>0.05) . . the expression of MR,GR on 7,10d fornix transaction group grow downwards than normal and sham-operation (p<0.05) ,the expression on10d is less than 7d (p<0.05), 0,4d fornix transaction group have no change than normal and sham-operation (p> 0.05) , the expression of MR,GR in each sham-operation is resemble to normal group (p>0.05) .ConclusionsThe expression of MR, GR of hippocampal neurons occurs down regulation after fornix transection. We suppose that may be the inhibition of hippocampus to HPA axis is attenuated after fornix transaction...