Change Of Apoptosis And LTP In The Amygdala Neurons Of PTSD 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 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 sensitive area response to stress which function is close with stress.Amygdala,one of the key regions in the limbic system of the brain,play an important role in PTSD.Chronic immobilization stress induces an enhancement of dendritic arborization of pyramidal and stellate neurons or neurons atrophy.The current studies of magnetic resonance imaging(MRI) showed that PTSD patients have smaller hippocampal size. Furthermore,other studies showed that the mechanism causing such smaller volume is apoptosis.The amygdala has many relations to the hippocampus.Parts of the functions of the hippocampus are depending on amygdala regulation.Studies of patients with PTSD have smaller amygdala volume.So,does the amygdala have apoptosis when the amygdala volume became smaller after PTSD? The current study has shown that the apoptosis-related genes Bcl-2 family play a crucial role in the control of apoptosis.Bax and Bcl-2 were considered one of the final pathway to regulate apoptosis,furthermore, when the ratio of Bax/Bcl-2 increased,the cells tend to apoptosis.Thus,put the two genes as one pair to study appear to be particularly important in the apoptosis.The role of lysosome is very important in apoptotic cells.TMP(Trimetaphosphatase) and ACP(Acid phosphatase) is a hallmark enzymes of lysosome,their activity changing directly reflect the functional state of lysosome and also reflect the state of cell apoptosis.PTSD exhibits four major types of characteristic symptoms:re-experiencing, avoidance,numbing,and hyperarousal.Amygdala is the key region of fear information and expression.At present,LTP is widely accepted neural basis of learning and memory. Formed in fear conditioned reflex process,the neural network(such as the amygdala) emerged LTP.It showed that LTP is an indispensable mechanisms of PTSD.AChE is an important hydrolytic enzyme of nervous system,and its main function is the hydrolyzes neurotransmitter(acetylcholine) of the synaptic cleft and keeps the nervous system's stability.Lack of the neurotransmitter acetylcholine can cause hypofunction of learning and memory.In addition,it was reported in the literature that single prolonged stress(SPS) method is an ideal animal model of PTSD,and the method has been recognized by international,in this study,we use the SPS model to research the expressions of apoptosis relate-gene and the activity change of TMP and ACP.We also detect the changes of LTP and ACHE.Our purpose is that 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: normal control group and SPS 1d,4d,7d,and 14d. 2.Immunohistochemistry,immunofluorescence,Western blotting,and RT-PCR for Bax and Bcl-2.Immunohistochemistry:took out the rats brain which had been fixed.Made them into paraffin sections,and then were stained by PV method.And DAB coloration,last, neutral gummi mounted.The result was observed by light microscope.Immunofluorescence:took out the rats brain which had been fixed and dipped down.Made them into paraffin sections.They were stained with double-label,and were mounted with glycerol-PBS.Western 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-Bax antibody (1:50),anti-Bcl-2 antibody(1:50) 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.3.TUNEL stainingTook out the brain of the rats respectively from each group,and made them into paraffin sections.Then stained,DAB coloration,neutral gummi mounted.The result was observed by light microscope.Took photograph as well as undertook image analysis.4.Annexin V-FITC/PI double-labeled flow cytometryDissected the basolateral amygdala and made them into single-cell suspension. Centrifuged it tree times,and then cleaned the supernatant.Cell pellets were resuspended in 200μl binding buffer,and then incubated with 10μl Annexin V-FITC and 5μl Propidium Iodide at room temperature for 15 min.Finally,added 300μl binding buffer into the incubation solution in 1h.5.Enzymohistochemistry of TMP,ACP and AChE.Frozen sections were washed in PBS,incubated at 37℃for 0.5h.Sulfide coloration,and then glycogelatin mounting.The results were observed by light microscope.6.Using TEM to observe the changes of amygdala neuronsConventional 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.7.LTPRats were decapitated and fixed on stereotaxis instrument.First,locate the Entorhinal cortex and amygdala;next,inset stimulating electrode and recording electrode;then,measure low frequency transduction and high frequency stimulation, last,calculate the ratio.Results1.The results of Bax and Bcl-2Bax peaked at SPS 4d,the expression of Bcl-2 at SPS 1d was most.The ratio of Bax/Bcl-2 peaked at SPS 4d and then gradually decreased.2.The results of TUNELThe TUNEL positive cells were detected in each SPS group.The TUNEL positive rate peaked at SPS 4d.3.The results of apoptosis rateAfter SPS,the apoptosis rate of amygdala neuronal was significantly increased and reached the peak at SPS 4d.At SPS 7d and 14d,apoptotic rate gradually decreased.4.The results of TMP,ACE AChE TMP and ACP activity was significantly enhanced compared with the normal control group.They peaked at SPS 4d,and then decreased.AChE activity early increased,and late decreased.Increased to the maximum at SPS 1d,and decreased to the minimum at SPS 7d,later,gradually resumed to normal.5.Morphologic changes by TEMAfter SPS,the amygdala neurons have characteristic morphologic changes of apoptosis.It shows that chromatin condensation,appearance of chromatin crescents, nucleus fragmentation and nucleolus disappearance.6.The results of LTPAfter high-frequency stimulation,compared to control groups:early amygdala activity gradually been contained,the most significant changes at SPS 1d,the enhancement rate of PS amplitude reduce most.Late amygdala activity increased,and the enhancement rate of PS amplitude raised to the highest at 7d,then,gradually decreased to normal.Conclusion1.PTSD made the rats amygdala have apoptosis.2.TMP and ACP of lysosome marker enzyme activity enhanced.It showed that lysosome play role in amygdala neuronal apoptosis products phagocytosis.3.The amygdala ultrastructure has changed at the PTSD acute phase.LTP was inhibited Fear reaction was reduced;LTP enhanced at SPS 7d and fear reaction enhanced;later, gradually decreased to normal.