Expression Changes Of Nuclear Receptor MR And GR In The Central Amygdaloid Nucleus Of PTSD-like Rats

ObjectivePost-traumatic stress disorder (PTSD) is an anxiety syndrome or the continued state of mental and physical symptoms caused by terror, tragedy or enormous suffering due to disasters, wars, terrorist incidents, traffic accidents and abuse. Along with wars, violence, natural disasters and major traffic accidents have increased, the incidence of PTSD are increasingly high. Currently, PTSD with the characteristics of high incidence, long course, difficult to cure and seriously impact on people's physical and mental health, has attracted a lot of attention.Clinical studies found that blood glucocorticoid (GC) concentration of patients with PTSD is very low. Corticosterone plays in the role of gene transcription regulation mainly by combining with two nuclear receptors mineralocorticoid receptor (MR) and glucocorticoid receptor (GR). Amygdala is the target of GC. Central amygdaloid nucleus (CeA) and medial amygdaloid nucleus (MeA) have MR and GR expression. And the expression of MR was significantly weaker than GR. With the existence of MR and GR, it is and suggested that CeA and MeA are able to adjust the basal and stress level of GC. This regulatory role is substantially different from that of hippocampus and frontal cortex with the function of feedback inhibition. Now it is believed that GR in the amygdala plays in the role of positive feedback to stress regulation. Therefore, Studied the expression changes of MR and GR in CeA of PTSD-like rats to provide with experimental data for further research in the pathogenesis of PTSD.Methods1. Laboratory animals and groups80 adult male Wistar rats (provided by the Experimental Animal Center of China Medical University), 150-- 180g weight," keeping in laboratory conditions, were randomly divided into four groups. Single prolonged stress (SPS) is not performed in the control group and the rest three groups are undisturbedly raised for respectively 24h, 7d and 14d after SPS stimulation. PTSD-type symptoms can be seen in the rats group 7 days after SPS stimulus.2. PTSD-like rat modelIt's determined on the International Scientific Conference on PTSD conducted by Japan's Ministry of Education in 2005 that PTSD-like rat model can be established by giving rats SPS stimulus followed by 7 days without any interferences. SPS stimulus refers to the following steps: (A) immobilization for 2h; (B) immediately a forced swim in 25°C water for 20 minutes in a plastic tub (40cm H); (C) 15 min recuperation, then exposure to ether until the loss of consciousness.3. Immunohistochemical detection of MR and GR expression in CeAThe rats in the control group and other three groups of 24h, 7d, 14d after SPS stimulus were anesthetized and perfusion fixed. Rats brains were taken and fixed in Holt's solution to sink. Frozen sections of 14μm thickness were produced and stained with MR, GR immunohistochemical SABC staining. Then sections were observed with light microscope (OLYMPUS, BX60, Japan) and photographed. And image analysis and statistical analysis were performed later.4. Western Blotting Detection of MR and GR expression in CeAFresh CeA tissues in rats brain of control group and other three groups of 24h, 7d, 14d after SPS stimulus were taken. MR and GR expression were detected with Western Blotting method. And image analysis and statistical analysis were performed later. Results1. The immunohistochemical results(1) The immunohistochemical results of MR:Positive neurons can be seen with microscope. The strongest expression of MR is in the control group which locates mainly in the perikaryon and less in the processes. Compared with the control group, MR expression in the group of 24h after SPS stimulus is significantly lower. The number of positive neurons is decreased. Compared with the group of 24h after SPS stimulus, MR expression in the group of 7d after SPS stimulus is significantly increased. The number of positive neurons is increased. And some long processes with MR positive performance can be seen which bulge from the perikaryon. Compared with the group of 7d after SPS stimulus, the number of MR positive neurons in the group of 14d after SPS stimulus is increased. However, the average strength of positive is a little downward. And there is no MR positive expression in the processes.(2) The immunohistochemical results of GR:Positive neurons can be seen with microscope. The strongest expression of GR is in the control group which locates mainly in the nucleus. Many strong positive nuclei can be seen. Compared with the control group, GR expression in the group of 24h after SPS stimulus is significantly lower. The number of positive nuclei is decreased. Compared with the group of 24h after SPS stimulus, GR expression in the group of 7d after SPS stimulus is significantly increased. The number of positive nuclei is increased. Compared with the group of 7d after SPS stimulus, the number of GR positive nuclei in the group of 14d after SPS stimulus is increased. However, the average strength of positive is not increased apparently.2. Western Blotting results(1) Western Blotting results of MR showed that, MR expression in CeA of rats is decreased 24h after the SPS stimulus, and rapidly rebound 7d and 14d. (2) Western Blotting results of GR showed that, GR expression in CeA of rats is decreased 24h after the SPS stimulus, and gradually rebound 7d and 14d.3. Image analysis and statistical analysisExpression of MR is significantly reduced in the 24 hours after SPS stimulus, quickly picked up in 7 days and to be stable in 14 days, but still lower than normal state (P<0.01); Expression of GR is sharply reduced in the 24 hours after SPS stimulus, gradually picked up in 7 days and 14 days, but still lower than normal state (P<0.01); The MR/GR ratio in CeA is significantly up-regulated in the 24 hours after SPS stimulus, and show a gradually downward trend in 7 days and 14 days which is getting closer to normal conditions.Conclusion1. In CeA of PTSD-like rats, expression changes of minerolocorticoid receptor & glucocorticoid receptor and MR/GR ratio imbalance may be one of the most important ?factors which lead to HPA axis regulation disorder and PTSD symptoms.2. In CeA of PTSD-like rats, neuronal processes are prolonged in length and increased in number, which can provide morphological basis for relationship of the central amygdaloid nucleus with enhanced terrorist-related memory in PTSD.