LTP And ACP Expression Change Complication The Hippocampus Neuron Apoptosis In PTSD-Like Rats

ObjectivePost-traumatic stress disorder(PTSD)involves the development of long-lasting symptoms following exposure to a life threatening experience to which the immediate reaction includes intense fear,helplessness,or horror.The resulting symptoms include persistent and spontaneous recollection of the traumatic event,psychic numbing, avoidance of trauma associated stimuli and increased arousal,selective forgetting to wound experience along with the natural disaster,the significant traffic accident,the war,the social violence and the accident,the PTSD formation rate is much more higher. Nowadays PTSD characteristics with high incidence rate,the long illness course,with difficulty cures and so on,having pay much more attention as the seriously physical and moral affects on people,hippocampus is the important brain area for learning and memory and play a role in the nutrition and function,so damaged by stress selectively. the decrease on hippocampus volume was reported by research.Wether the apoptosis of neuron and the decrease of neuron account contribution to the volume decline,and how the function of learning and memory should change.It is reported that the activity of Acid phosphatase can indicate the degree of neuron damage in hippocampus.Long term potentiation is the accepted electrophysiology matrix to show the function of learning and memory.So we study the change of LTP and ACP to supply some experiment evidence to reveal the mechanical. Methods1.AnimalsMale Wistar male rats(220～250g)were used for all experiments.(the animal center of china medical university supply,housed under the laboratory condition)300 rats were allocation to five groups random as a number of 60.all the groups were prepared for Annexin V-F1TC/PI,TEM,ACP Enzym EM,LTP.Each group were allocation to six subgroups as 6 h;12 h;1 d;7 d;14 d after SPS and normal as a number of 10.2.Experimental procedure(SPS)Follow an adaptation period of 5～6 days,experimental procedures were undertaken.All stressed rats had similar single prolonged stress procedure on the first day.The single session of prolonged stress consisted of:restraint for 2 h,followed by forced swim for 20min(24℃)followed by ether anaesthesia,and then remained without interference in their home cages and be killed 7 days later.(The SPS mode is refer to the 28th Annual Meeting of the Japan neuroscience Society.)3.Flow cytometry detect the neuronal in hippocampus.Prepare the single cell suspension of rat hippocampus of normal 6 h,12 h,1 d,7 d, 14 d after SPS use usual method,add in FITC and PI,flow cytometry detect and statistical analysis.4.TEM sample prepare of hippocampus neuronalPrepare TEM sample of rat hippocampus of normal 6 h,12 h,1 d,7 d,14 d after SPS use usual TEM sample prepare method,located in CA3 area and obverse under the TEM(JEOL 1200EX)80KVT.5.ACP enzyme histochemistry and image analysisPrepare rat brain of normal 6 h,12 h,1 d,7 d,14 d after SPS use usual method. fixed to the Holt's until down,14μm freezing slices,incubation for 90 minutes use Gomori'S method observe by microscopy(OLYMPUS BX80 Japan),take photo image and statistical analysis.6.Electron microscopy and enzyme histochemistryPrepare rat brain of normal 6 h,12 h,1 d,7 d,14 d after SPS use usual method. fixed to the Holt'suntil down,40μm freezing slices,incubation for 30 minutes at 37℃use Gomori'S method.TEM sample prepared.Locate in CA3 area and obverse under the TEM(JEOL 1200EX)80KVT and take photo.7.Electrophysiology measureUse rat of normal 6 h,12 h,1 d,7 d,14 d after SPS;narcosis and implantation electrode to note the singal and statistical analysis.Results1.Flow cytometry detect resultsThe rate of apoptosis of 6 h after the SPS increased compared to the control group as well as the 12 h after the SPS,especially the group 1 d after SPS.On the other hand, the rate of apoptosis of 7 d after the SPS decreased compared to the group 1 d after SPS, the 14 d after SPS decreased heavily.2.TEM resultsPart of hippocampus neuron show karyopyknosis in the group of 12 h after SPS, some of the nucleus showed significant margination of the chromatin and crescent shape.1 d after SPS show crescent shape;apparant wrinkled nuclear membrane and splitted nuclear.3.ACP enzyme histochemistry resultsUnder the low time of mirror the entire hippocampus presents the ACP positive reaction,most obvious in the CA3 area,under the high power mirror the ACP positive reaction product assumes the sepia the lead sulfide pellet uniform distribution at the perinuclear,extending breaks out.The ACP expression strengthens in the 6 h group after SPS compare to the control group as well as the 12 h after SPS,Much more obvious in the group 1 d after SPS.The ACP expression decrease in the 7 d group after SPS compare to the 1 d after SPS group,especially in the group 14 d after SPS.4.ACP electron microscopy and enzyme histochemistry resutosUnder the electron microscope,in the normal control group neuron,the ACP enzyme positive reaction product precipitates the obvious circular for the high electron density black lead phosphate to dissolve the enzyme body(Spherical Lysosome SLY), distribution in the Golgi complex.The swallows strengthens in the group 6 h after PTSD.The Lysosome have becomes line shape(NLT)from circular;arrive the highest strengthens in the group 1 d after PTSD,the line shape Lysosome is obvious (NematoLysosome NLY).The 7 d After PTSD group obvious striation and kinds of shape Lysosome distribution in the cycytoplasm.5.The elelectrophysiology resultsThe PS average value after HFS has a drop compare to the normal rat 6 h after SPS.Further reduce on 12 h after SPS,achieve lowest point on 1d after SPS,starting to rise on 7 d after SPS,rise obviously when on 14 d after SPS,but lower than the normal rat.6.The image analysis and statistics analysisThe rate of apoptosis of 6h after the SPS increased compared to the control group as well as the 12 h after the SPS,especially the group 1 d after SPS.On the other hand, the rate of apoptosis of 7d after the SPS decreased compared to the group 1 d after SPS, the 14d after SPS decreased heavily(P＜0.05)The ACP expression strengthens in the 6h group after SPS compare to the control group as well as the 12 h after SPS,Much more obvious in the group 1 d after SPS.The ACP expression decrease in the 7 d group after SPS compare to the 1 d after SPS group,especially in the group 14 d after SPS(P＜0.05).The PS average value after HFS has a drop compare to the normal rat 6h after SPS.Further reduce on 12 h after SPS,achieve lowest point on 1 d after SPS,starting to rise on 7 d after SPS,rise obviously when on 14 d after SPS,but lower than the normal rat(P＜0.01)Conclusion1.PTSD like rat hippocampus neuron appears apoptosis result in the decrease of neuron account.And the ACP increase to be advantageous to clean up the apoptosis product,from this may explain the hippocampus atrophy and volume reduces.2.PTSD like rat hippocampus neuron LTP suppression prompts the complication memory abnormal mechanism by PTSD.