Effects Of Fornix Transaction On The Expression Of CRH And AVP In The Paraventricular Nucleus Of The Hypothalamus In Rats

Corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) are the two major regulatory peptides for the secretion and/or synthesis of adrenocorticotropin (ACTH). CRH and AVP colocalize in the parvocellular neurons in the paraventricular nucleus of the hypothalamus (PVN) both in humans and rodents. These peptides colocalize in the same secretory granule at the nerve ending of the median eminence, and after co-released into the portal circulation, exert Synergistic effects on ACTH secretion rom the anterior pituitary.CRH and AVP regulative system may help organism to deal with various stress incident. The most feature of stress is a physiological and psychological reaction that activate the hypothalamic- pituitary-drenocortical axis (HPA axis). CRH plays a significant role to the HPA axis after stress, which controls the level of the HPA axis. AVP can compensate for the regulatory activity of CRH thereby providing the stimulus for increased releasing of ACTH. Hippocampus is not only an area of sensitivity of excitable damage, but also a high feedback regulation center of the HPA axis. However, the mechanism that the hippocampus regulates the hypothalamus has not been well elucidated. In this study we performed the transection of the fornix and observed the effects of fornix transection on the expression of CRH and AVP in the hypothalamus in rats, and discussed whether the hippocampus plays an inhibitory role to the HPA axis via the fornix in order to provide riching theoretical and morphological evidence for studying the mechanism of the hippocampus regulating the hypothalamus.Methods1. Experimental animals and groupsAdult Wistar male rats obtained from experimental animals center of China Medical University, were used. The rats were divided into fornix transection 0d, 4d, 7d, 10d groups, sham-operationed 0d, 4d, 7d, 10d groups and the control group, each group comprised 10 animals.2. Set up the fornix transaction modelRats were deeply anesthetized by intraperitioneal injection of pentobarbital sodium. Each animal was positioned in a stereotaxic instrument, and exposed the skull: anteroposterior (AP) -1.5 mm to bregma, dorsoventral (DV) -6 mm (FT) or -2mm (sham-operated) and mediolateral (ML)±1 mm 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. After all surgeries no animals exhibited seizure activity during the postoperative survival period. Antibiotics were used to anti-infection.3. Identification of modelUsing the method of Karnovsky to stain the hippocampus CA1 cholinergic nerve fibers, each group was chose 3～4 sections random, then observed by Olympus light microscope.4. Immunohistochemistry methods (SABC)Choosing hypothalamic sections of control group, sham-operationed 0d, 4d, 7d, 10d groups and fornix transection 0d, 4d, 7d, 10d groups, made rabbit-anti-rat CRH( 1:500) and AVP(l:1000) immunohistochemical stain, then observed by Olympus light microscope. Used the substitution method to made negative control experiments.5. Western blotting methods Using each group rats hypothalamus, tissue was homogenated and centrifuged. Samples were prestained and detected protein concentration. Samples were electrophoresed polyacrylamide gels, and transferred onto membranes. The membranes were blocked, then overnight at 4 C with one of two polyclonal anti-CRH and anti-AVP antibodies. After washing, membranes were incubated with HRP-linked antibody 2h. Membranes were stained by DAB and detected.6. Analysis of images and treatment of statistics2 visual fields were chose in each section of each group. we analyzed the result of expression of CRH and AVP using Motic Images Advanced 3.2 system and measured IOD. After scanned results of western blotting, we analyzed gray scale. These data were analyzed by SPSS13.0 for windows, and presented by x±s, and analyzed the data by analysis of variance in each group. A p < 0.05 was taken as statistically significant.Results1. Identification of modelThere are riching cholinergic nerve fibers in the hippocampus CA1 pyramidal layer of the control group. Fibers are uniformity and interlace into net. End buttons can be seen in the termination. Fibers in the hippocampus CA1 pyramidal layer of the fornix transection groups spread derangement and degrade density.2. The results of CRH immunohistochemistryThe expression of CRH in the control group is brown-yellow en plaque, and is located in cytoplasm around nucleus, extend to dendrites. Sham-operationed groups is similar to the control group. The expression of CRH in the fornix transection groups is not change on 4d and enhanced on 7d and more significant enhanced on 10d. The number of cells is increase and the volume of the cells is larger.3. The results of AVP immunohistochemistryThe expression of AVP in the control group is brown-yellow granularis, and is located in cytoplasm around nucleus, extend to axon. Sham-operationed groups is similar to the control group. The expression of AVP in the fornix transection groups is not change on 4d and enhanced on 7d and more significant enhanced on 10d. The number of cells is increase.4. The results of western blottingThe results of western blotting show that the protein concentration of CRH in the hypothalamic paraventricular nucleus (PVN) are increased on 7d in the fornix transection groups. There are more significant inceased on 10d than that on 7d (p< 0.05). There were only a little expression of CRH in the hypothalamus in sham-operated groups and the control group. The change of sham-operationed 0d, 4d, 7d, 10d groups are not obvious.ConclusionThe fornix transection can increase the expression of CRH and AVP, and enhance the activity of HPA axis and degrade the inhibitory effects of hippocampus on HPA axis. This experiment reveals that it is via the fornix that the hippocampus plays an inhibitory role to the HPA axis.