Changes Of Ca²⁺ Cascades, COX And Caspase 9 In The Amygdala Of Post-traumatic Stress Disorder

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 severe stressor. Along with wars, violence, major traffic accidents and natural disasters have increased, the incidence of PTSD are increasingly high. Currently, PTSD with the characteristics of high incidence and prevalence, long course, difficult to be cured and seriously impact on people's physical and mental health. So they have attracted a lot of attention.In the acute phase of severe trauma stress, the limbic system is a sensitive area. Amygdala, one of the key regions in the limbic system of the brain, has been documented to have an important role in fear, rage and emotional memory. So amygdala may play an important role in PTSD.Amygdala is the key region of fear information and expression. Ca2+-CaM-CaMKIIa signal passage play an important role in plasticity of central nervous, learning and memory, mind and behavior and other kinds of cognition activities. The current studies showed that apoptosis took place in amygdala. Chondrosome play an important role in apoptosis. The role of chondrosome is very important in apoptotic cells. COX (cytochrome c oxidase) is a hallmark enzymes of chondrosome, their activity changing directly reflect the functional state of chondrosome and also reflect the state of cell apoptosis. Caspase 9 has many relations to promote apoptosis and play a crucial role in the control of apoptosis.Single-prolonged stress (SPS), an ideal animal model of PTSD, has been developed and employed for PTSD research, and the method has been recognized by international. In this study, we use the SPS model to research changes of Ca2+-CaM-CaMKIIa and expression of COX. We also detect the changes of caspase 9. So we explore the changes in fear learning and memory in amygdala of PTSD rat and provide the evidence to reveal the mechanism of PTSD.Methods1. Model building and groupingUse 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; exposed to ether vapor until loss of consciousness. The rats were randomly divided into five groups: control group and SPS 1d,4d,7d and 14d.2. Determination of free calcium contentRats were rapidly decapitated, and the brain were removed and immediately placed in a dish standing on crushed ice, then the basolateral amygdala was dissected out,106-107/ml cell suspension was prepared by routine method and loaded with 1 mmol/L Fura-2/AM for 35 min, then detected with spectrofluorometer.3. Immunohistochemistry, Western blotting and RT-PCR for CaM and CaMKIIaImmunohistochemistry:took out the rats brain which had been fixed and dipped down. Made them into frozen sections, and then were stained by PV method. And DAB coloration, neutral gummi mounted at last. The result was observed by light microscope.4. Western blotting and RT-PCR for CaM, CaMKIIa and Caspase-9Western blotting:the fresh amygdala was respectively homogenated ultrasonicated and then high-speed centrifuged in low temperature. The acquired supernatant albumen was then operated though electrophoresis and trarsmembrane. Add anti-CaM antibody (1:1000), anti-CaMKIIa antibody (1:2000) and anti-Caspase 9 (1:2000) and after block. They were incubated overnight at 4℃, then, they were incubated in IgG with HRP marked 2h, ECL, the optical density values were calculated.RT-PCR:dissected the fresh amygdala. Extracting the total RNA to reverse transcription and PCR amplification. Then, have gel electrophoresis and imaging. Last, analyzed integrated optical density.5. Enzymohistochemistry and using TEM to observe COXFrozen sections were washed in PBS, incubated at 37℃for 0.5h, and then glycogelatin mounting. The results were observed by light microscope.Conventional made the sample, Flip-embedded and finally polymerized in pure Epon 812. Basolateral amygdala was localized on semi-thin sections. Ultra-thin sections were cut on an ultramicrotome, and were observed with transmission electron microscopy.6. Immunofluorescence for caspase-9Immunofluorescence:took out the rats brain which had been fixed and dipped down. Made them into paraffin sections. They were stained with FITC, and were mounted with glycerol-PBS.Results1. The results of free calcium contentIntracelluloar free calcium in basolateral amygdala neurons was obviously higher at day 1 after SPS stimulation than that in control group and gradually reached to normal level at days 7 after SPS.2. The results of CaM and CaMKIIa.CaM peaked at SPS 4d and the expression of CaMKIIa at SPS Id was the least.3. The results of COXEvaluation of COX by enzymohistochemistry indicated a significant decrease in the SPS model groups compared with the normal control group. Tumescent mitochondria and the release of COX was found at SPS groups.4. The results of Caspase 9Evaluation of Caspase 9 content indicated a significant increase in the SPS model groups compared with the normal control group, and peaked at SPS 7d.Conclusion1. Post-traumatic stress disorder induces dysfunction of Ca2+-CaM-CaMKⅡαsignal passage in the amygdala.2. Post-traumatic stress disorder induces the release of COX from chondrosome to cytoplasm and upregulation of Caspase 9, which led to apoptosis of amygdala cells.