| ObjectivePTSD(post-traumatic stress disorder) is a kind of abnormal psychic reaction when organisms suffered serious stimulate factors. Such as trauma. It refers to the sudden, threatening or catastrophic life event led to delayed and individual long-term persistence of mental disorders. The clinical manifestations to re-experience the trauma characteristics, and with emotional irritability and evasive behavior .At the present,it is concerned very much because of a high incidence and the complex pathogenesis. PTSD patients show reduced hippocampal volume, a relatively low level of foundation of corticosterone in the blood and the lower response on the stress . These data imply that PTSD patients hypothalamus - pituitary - abnormal axis function is adrenal. It had also reported that patients with mental illnesses in glucocorticoid negative feedback function was abnormal. There were some reasearches which found the feedback of GC in patients with severe depression was cut down, but it was strengthened in PTSD patients . These data suggests that the HPA-axis is abnormal in PTSD.In the process of feedback adjustment GC may be adjusted by Minealocorticoid Receptor (MR) and glucocorticoid Receptor (GR). In the HPA axis, these two receptors play an important role in the negative feedback effects on GC . Both receptors have different affinity with corticosterone. MR is a high-affinity and low capacity of the GC receptor system, and GR for the low affinity and high capacity GC receptor system. In addition, the distribution of two receptors in the body are different. MR mainly distribute in the limbic system of brain: such as the hippocampus, amygdala and the frontal cortex cells, and GR widely distribute in all brain regions . It is speculated that two receptors may exercise different functions in the PTSD . Studies found the importance medium of MR and GR in the mental disorder. These receptors can regulate memory, behavior, fear and fear related sys . MR is an important controlling factor in inhibiting HPA-axis. When the body under stress, the combination of MR with corticosterone is gradually replaced by the GR. Therefore, the increased MR capacity can reduce the neuroendocrine stress response .For example, antidepressant drugs can increase the expression of hippocampal MR and reduce the basis of HPA axis activity and stress-induced activity, blocking MR and GR to induce the anxiety and panic in the body. The fear will enhance MR by antagonist . These studies showed that the changes of hippocampal MR and GR could adjust mental disorder diseases and emotional changes. On the other hand, too much or chronic deficiency of cortisol hormone can induce changes of hippocampal neurons. It suggested that cortisol hormone receptor is an important factor on damaging hippocampus. It was reported that the application of MR ligand can treat synaptic function of the degradation and loss. . So the effect of MR and GR on hippocampal neurons in PTSD suggests that MR and GR are important factors to change the structure and function of the hippocampus.In addition, the clinical examination also found that PTSD in patients with low levels of glucocorticoids in the blood but the corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) instead of increased concentration . In HPA-axis hypothalamus as an important hub location, which determines the blood corticosterone change, but also accepts with the negative feedback inhibition of hippocampus. So the change of hippocampus function must have an impact on the change in the hypothalamus. And the change in the concentration of cortisol in the blood may also affect the hypothalamic change. From the regulation of cortisol in the hypothalamus, it suggests that MR is not directly involved in the HPA axis function in the hypothalamus. Therefore it considers that MR in the central nervous system has a dual role in the hippocampus, MR in the hypothalamus regulate cortisol circadian changes in the basis of level, but it only affects on the salt ion absorption and regulation of blood pressure . GR as of the implementation on glucocorticoid effect, not only in the body of the energy metabolism, but also for a wide range of gene transcription regulation, and directly receive hormone feedback regulation. In addition, when the body is stimulated by stress, the neuron in PVN (Paraventricular nucleus) secret a variety of of hormones encourage adrenocorticotropic hormone (ACTH), which is the most common corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP). These two hormones in the blood furtherly regulate the level of steroid hormones.To sum up, and the hippocampus is related to the structure of memory, and full of MR and GR, MR and GR as a steroid hormone receptors, they can regulated with steroid hormone levels in PTSD, at the same time, they also affect the role of the hippocampus affecting on the hypothalamus. Thus, both the hippocampus and the hypothalamus are closely related to the pathogenesis of PTSD. In addition, it was reported in the literature that single prolongned stress (SPS) method can now better reflect the changes in hormone levels of PTSD, it is an animal model of an ideal method to produce PTSD, and the method has been recognized by international. In this study, using SPS methods to stimulate rats to have PTSD-like change. And used immunohistochemical techniques, immune double technical and Western blotting technology to research the expressions of MR and GR in the hippocampus and hypothalamus in each time after SPS stimulation, and the expressions of hypothalamic CRH and AVP ,inoder to approach the mutual impact of HPA and reveal the mechanism of PTSD and clinical studies provide the basis for performance.Methods1. Model building and groupingUsed the SPS model of PTSD which had been established by international science conference that convoked in Japan in 2005. It is carried out by the following consecutive steps : Immobilization, 2h; Forced swimming, 20min; Ether anesthesia; Undisturbed. The rats were randomly divided into four groups: control group and SPS after 24 h, 7d, 14d.2. Immunohistochemic staining of hippocampal MR, GR and hypothalamic MR, GR, CRH and AVPTaking out the brain of the rats respectively from control group, SPS-24h, SPS-7d and SPS-14d which had been fixed and dipped down in 40% sucrose. Made them into frozen sections, ascerted the locum of hippocampus and hypothalamus. And then stained by SABC mathed with anti-MR (1:300), anti-GR (1:800), anti-CRH(1:500) and anti-AVP (1:1 000). And DAB coloration, neutral gummi mounting the result was observed by light microscope. (OLYMPUS, BX60, Japan), taking photograph as well as undertaking image analysis .3. Western Blotting detecting of hippocampal MR, GR and hypothalamic MR, GR, CRHThe fresh hippocampal and hypothalamic tissue were respectively 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), anti-GR antibody (1:1000) and anti-CRH antibody (1:800) after block. MR, GR and CRH were incubated overnight at 4℃, then, they were incubated in IgG with HRP marked 2h, DAB coloration, scanning band to calculate the optical density values.4. Double-label Immunofluorescent methods and Confocal microscopy of MR and GRThe rats of each group were perfused fixed in 4% paraformaldehyde, drawing the materials making frozen slices. After washing with PBS, the slices were pretreated with blocking solution consisting of 5 % BSA for 20 min at room temperature to block the nonspecific reaction. Then they were incubated with anti-GR (1:800) for 24h at 4℃. After rinsing with PBS, they were incubated with FITC-labeded sheep anti-mouse IgG for 2 hours in RT. Then they incubated with non-labeded normal rabbit serum for 2 hours in RT after rinsing with PBS. After incubated in non-labeded Protein A for 2h in RT, they were incubated in anti-MR for 24h in 4℃.And then incubated with alexa 546-labeded Protein A for 2h in RT. After washing thoroughly with PBS, the coverslips were mounted on glass slides and sealed using Anti-fade. Cells were observed with a Confocal laser scanning microscope, and photograghed.Results一,The result of immunohistochemistry1. The expression of MR in hippocampusMRs were found distributed in various regions of the hippocampus under lens. Hippocampal CA1 region were taken and observed under the high-power lens. The visible expression of MR in neuron showed different distribution with the functional status of the cells. Some distributed in the nucleus, some visible expression in the cytoplasm, and in the 24 h, 7, 14 days after the SPS, MR expression has continued to decrease.2. The expression of GR in hippocampusThe expression products of GR mainly distributed in the CA1 region of the hippocampus under low lens. Its main focus on the distribution of nuclei but relatively small in the cytoplasm. The positive expression of GR in hippocampal CA1 neurons decreased and it's processes shortened after SPS-24h. However the expression of GR gradually increased, and gradually extended processes after SPS-7d and 14d.3. The expression of MR in HypothalamusThe expression product of MR was observed in small cell neurons and large neurons in PVN under low lens. Under high lens, the expression product of MR mainly concentrated in cytoplasma. But there were no obviously difference among control, 24h, 7d and 14d after SPS. 4. The expression of GR in HypothalamusThe expression product of GR was mainly observed in small cell neurons under low lens. Under high lens, the expression product of GR mainly concentrated in nucleus, less in cytoplasm. After SPS, we found that the expression product of GR in neuron attenuated in 24h. But in the 7th, the positive expression of GR in neurons increased and higher than normal. While in the 14th it lower back near normal control group.5. The expression of CRH in HypothalamusUnder low magnification microscope CRH in PVN distributed in the cytoplasm of neurons. Intracytoplasmic CRH within the rat upward trend and were higher than normal control group when dealed with the SPS, 24 h, 7d. And they resumed when 14d.6. The expression of AVP in HypothalamusThe positive expression products of AVP could be seen that they under high magnification, distributed mainly in the cell body and processes the nucleus in the hypothalamic paraventricular. After SPS, the expression of AVP is in the same with CRH. It showed a rising trend in 24h and 7d. Ther were obvious beaded kind of the positive in processes on 7 d. On 14 d after the expression of AVP gradually resume.二,Western blotting results1. The results of MR in hippocampusWestern blotting detection found after SPS , the expression of rat hippocampal MR gradually continued downward in 24 h, 7d, 14d.2. The results of GR in hippocampusWestern blotting detection found after SPS,the expression in rat hippocampal GR reduced in 24 h,and continued gradual recovery in 7 d, 14d.3. The results of MR in hypothalamus Western blotting detection found after SPS, rat hypothalamal MR expression did not change significantly at 24 h, 7 d, 14 d.4. The results of GR in hypothalamusWestern blotting showed that the expression of GR decreased at 24 h after SPS. But it was higher than normal at 7 d, and reduced to near normal when 14 d.5. The results of CRH in hypothalamusWestern blotting detection found CRH in the hypothalamus of rats gradually increased after SPS. It reached the maximum at 7 d, while it callbacked at 14 d.三,The Immunolocalization of MR and GR in hippocampal CA1 subregionSlices were observed with a Confocal laser scanning microscope. MR-Alexa excitation wavelength was 543 nm for red light, GR-FITC excitation wavelength was 488 nm for the green light, for the integration of the two yellow or orange light. MR of control group distributed in the cytoplasm and nucleus, the GR distributed mainly in the nucleus, a few found in the cytoplasm; In SPS-24h, MR were mainly in the cytoplasm and reduced in nucleus, but GR had no obvious change; In SPS-7d, MR was in the further reduction of nuclear expression, while the expression of GR moved to the cytoplasm from the nucleus, and occasionally found in the nucleus; In SPS-14d, MR is still concentrated in the cytoplasm, and the GR transfered from the nucleus into the cytoplasm again. With double excitation wavelength, it could be seen at the same time both fluorescence which were integrated to yellow or orange light. The integration of the control group mainly distributed in the nucleus, SPS-24 h group nucleus fusion-weakened when 7 d, more than two-fusion seen in the cytoplasm, and their re-emergence of the nucleus in SPS-14 d.Conclusion1. Glucocorticoid receptor (MR) in hippocampal CA1 subregion of PTSD showed the gradual downward trend. It suggests that hippocampal MR closely related with the lower level of glucocorticoid (GC) in blood of PTSD. It confirms the hippocampus has negative feedback inhibition to the hypothalamus. It also shows that MR is related to the fear of PTSD rats.2. Glucocorticoid receptor (GR) in hippocampal CA1 subregion showed a decrease in the resumption of the phenomenon suggests that GR may be related to changes in the GC level of PTSD.3. The MR / GR ratio in hippocampal CA1 subregion decreased suggests the MR / GR ratio is related with hippocampal injury in PTSD.4. MR in hypothalamus may not be relevant with steroid hormone levels of PTSD.5. The expression of GR in hypothalamus was lower at frist, and then returned sequentially. It suggests that the level of GR in hypothalamus is effected by hippocampus, and then regulate the changes of CRH and AVP to effect the changes in the level of steroid hormones.6. Hypothalamic CRH and AVP display at the same trend of increasing, suggesting that CRH in coordination with AVP regulate steroid hormone levels in PTSD, and they are related to the clinical symptoms about the spirit in PTSD.7. The plasma cortisol concentration change in PTSD rats can chang the distribution of MR and GR in hippocampal CA1 subregion neurons, resulting in damage to the hippocampus and functional lower.
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