Study On Dynamic Changes Of Hippocampal Synapsin â… ,BDNF And Purarin Intervention In Vascular Dementia Rats

Objective:1.To observe the dynamic changes of SynapsinⅠand its phosphorylation in hippocampus formation and probing into the presynaptic mechanism of synaptic transmission disorder in vascular dementia(VD) rat.2.To observe the dynamic changes of brain-derived neurotrophic factor(BDNF) expression,and the relationship between synaptic transmission and BDNF.3.To explore the effects and protecting mechanisms of puerarin on neurons of VD rats.Methods:VD models,established by repeatedly clipping the common carotid arteries of the rat in combination with an intraperitoneal injection of sodium nitroprusside solution in anesthetized SD rats,were randomly divided into model group and puerarin treated group(both n=40),and another 40 condition-matched rats were selected as the sham-operated(SO) group in which the bilateral CCA were separated but with neither CCA occlusion nor intraperitoneal injection of sodium nitroprusside solution.Above 3 groups were further divided into 15d,1m,2m and 4m time point subgroups respectively after model-established operation.The puerarin treated group was stomachically treated with puerarin and the model group and SO groups given the same volume of normal solution as puerarin treated group. At different time point,Morris water maze(MWM) task was used as the judging criteria for spatial learning and memory ability.The changes of population spike(PS) of granule cellular layer in the dentate gyrus,which were induced by single pulse stimulation to perforate path fibers before and after high frequency stimulation(HFS) in vivo,were used to detect whether long term potention(LTP) was provoked.The microcosmic changes and the number of neurons in hippocampus was comparatively observed in HE stain slices through microscopy.The ultrastructural changes of hippocampal CA1 synapse were observed through transmission electron microscopy (TEM).Immunohistochemistry technique and images analysis were used to study the expression of synapsinⅠ,phosphorylated synapsinⅠand BDNF in hippocampal formation.Results:1.The escape latency(EL) became shortened following the increase of MWM training times.Compared with SO group,the EL of model group rats were significant longer(P＜0.01).The EL of puerarin treated group was significantly shorter than that of model group(P＜0.01,P＜0.05),but still longer than that of the SO group(P＜0.01,P＜0.05).There were no differences among the EL of 15d,1m, 2m and 4m time point subgroups of SO group(P＞0.05);but the EL of 2m and 4m subgroups were highly longer than that of 15d subgroup of model group and puerarin treated group(P＜0.01).2.After HFS,increased amplitude of PS was induced by single pulse stimulation and lasted for more than 60min in both SO group and puerarin treated group,but unchanged in model group.The relative amplitudes of PS after HFS were obviously reduced at 15d,1m,2m and 4m time points of model group compared with that of SO group(P＜0.01.P＜0.05),and the relative amplitudes of PS in puerarin-treated group were significantly lower at 15d and 1m time points than that of SO group(P＜0.01,P＜0.05),but much higher at 2m and 4m time points than that of model group(P＜0.01,P＜0.05).The LTP was successfully induced in about 90% of the rats in SO group,the inductivity of LTP in model group were significantly lower than that of SO group at different time points(P＜0.01),and also lower than that of puerarin treated group,especially in 2m and 4m time point subgroup(P＜0.01, P＜0.05).There were no differences of LTP inductivity among 15d,1m,2m and 4m time points within the SO group and model group(P＞0.05);but the LTP inductivity at 2m and 4m time points were higher than that at 15d and 1m time points within puerarin treated group(P＜0.01,P＜0.05).There were no significant differences of PS latency before and after HFS in each group,among different groups and different time point subgroups(P＞0.05).3.No significant pathological changes were revealedp under light microscope in SO group.In model group rats,the pyramidal neuroms and their apical dendrites were distinctly reduced,spreaded rare fraction and arranged sparsely and mussily in CA1 area of hippocampus,and these pathological changes were apparently amelioratedin in Pur-treated group.The numbers of neurons in model group were lower than that of SO group and Pur-treated group(P＜0.01),but there was no significant difference of neuron number between the latter two groups except their 15d and 1m subgroups(P＜0.01,P＜0.05).There was no differences of the neuron numbers among 15d,1m,2m and 4m time points within SO group and Pur-treated group(P＞0.05),but the numbers of neurons of 2m and 4m subgroups were significantly decreased than that of 15d and 1m subgroups within model group(P＜0.01,P＜0.05).4.There were no obviously pathological changes in SO group umder electron microscope.In model groups,there revealed edema in neurons and their ending, nucleus chromatin margination,locally widened perinuclear cisterna,mitochondria swelling,mitochondrial crista chaos and rupturing,electron density dense;decreased postsynaptic density,synaptic circa membrane ambiguity and fusion,reduced synaptic vesicles and vesicle cluster.Above pathological changes became gradually severe along with as the post-operation time prolonged.These pathological changes were apparently ameliorated in Pur-treated group,especially in 2m and 4m subgroups.5.Immunohistochemistry and image analysis results.Compared with SO group,the expression of SynapsinⅠin CA1 region significantly reduced in model group(P＜0.01.P＜0.05),but there were no significant changes in molecular layer of DG region(P＞0.05);the expression of SynapsinⅠin hippocampus significantly increased in Pue-treated group compared with model group(P＜0.01,P＜0.05);the expression of SynapsinⅠin hippocampus significantly increased in Pue-treated group compared with SO group,in adition to 2m and 4m subgroup(P＞0.05),the remains had statistical significant(P＜0.01,P＜0.05).After each time point compared,the sham-operated group had no significant difference(P＞0.05);the average expression of SynapsinⅠin hippocampus CA1 gradually reduced in model groups,but there were no significant changes in molecular layer of DG;In addition to 2m and 4m subgroups had no significant difference(P＞0.05),other groups were significantly different(P＜0.05,P＜0.01).The expression of SynapsinⅠin Pue-treated group rats hippocampal were inconsistent with the prolongation of the treatment,the average absorbance value of the overall trend is gradually increasing.Statistically significant difference between the treatment group(P＜0.01,P＜0.05).6.Immunohistochemistry and image analysis(1)Compared with SO,The number of P-SynapsinⅠpositive neurons in DG and CA1decreased in model group rats hippocampus(P＜0.05,P＜0.01);Compared with model groups,the number of P-SynapsinⅠpositive neurons significantly increased in Pur-treatment groups.In addition to 15 d,the remains were significantly different(P＜0.05,P＜0.01).But this figure was still below the SO group(P＜0.05,P＜0.01).After each time point compared,the sham-operated group had no significant difference(P＞0.05);The number of P-SynapsinⅠpositive neurons was gradually reduced in model group, there were difference between 15d and 1m,2m,4m subgroups(P＜0.05,P＜0.01);With the extended group of drug treatment,The number of P-SynapsinⅠpositive neurons decreased gradually,there were difference between 15d and 1m,2m,4m subgroups in DG area(P＜0.05,P＜0.01).the remains had no significant difference(P＞0.05). (2)Compared with SO group,the average absorbance value of DG and CA1 P-SynapsinⅠpositive neurons were decreased in 15d and 1m model group rats hippocampus(P＜0.01).But increased in CA1 and no changed in DG in 2m and 4m model groups rats hippocampus(P＜0.01);compared with SO and model group,the average absorbance value of DG and CA1 P-SynapsinⅠpositive neurons decreased in Pur-treatment group rat hippocampus(P＜0.05,P＜0.01).Each time point,there were no significant changes in the SO group(P＞0.05);the average absorbance of BDNF immunoreactive neurons gradually reduced in model groups and Pur-treatment groups,In addition to 2m and 4m subgroups had no significant difference(P＞0.05),other groups were significantly different(P＜0.05,P＜0.01).7.Immunohistochemistry and image analysis,(1) Compared with the sham-operated group,BDNF-positive neurons in the model group rat hippocampus decreased significantly(P＜0.01);Pur-treated group BDNF immunoreactive neurons in rat hippocampal volume models increased significantly than model groups(P＜0.05. P＜0.01);In addition to 15d and 1m group,the DG district of drug group had no stastistical significance compared with the SO,other groups were significantly different(P＜0.05,P＜0.01).After each time point,the hippocampal BDNF immunoreactive nerve cells of SO group had no significant changes(P＞0.05);In model group,BDNF immunoreactive neurons decreased gradually In addition to 2m and 4m subgroups(P＞0.05).The remaining groups were significant(P＜0.05);In drug groups,BDNF immunoreactive neurons in CA1 cells gradually increased.In addition to 2 m and 4 m outside the difference was not significant(P＞0.05).The remaining groups were significantly different(P＜0.05,P＜0.01).But drug group DG BDNF immunoreactive nerve cells had no significant changes(P＞0.05). (2)Compared with the sham-operated group,the average absorbance value of model group hippocampal BDNF immunoreactive neurons were significantly lower than in the sham-operated group(P＜0.05,P＜0.01);the average absorbance value of drugs group hippocampal BDNF immunoreactive neurons were significant lower than model group and SO(P＜0.05,P＜0.01).Each time point compared,the sham-operated group had no significant difference(P＞0.05);the average absorbance of BDNF immunoreactive neurons gradually reduced in model groups and drug groups,In addition to 2m and 4m subgroups had no significant difference(P＞0.05), other groups were significantly different(P＜0.05,P＜0.01).8.Correlation analysis shows that:(1)there were negative correlation between the EL and the number of CA1 pyramidal cells(r=-0.961,p=0.0386), LTP(-0.962.p=0.0384) and the number of BDNF-positive cells(r=-0.968,P＜p=0.0315) in model group.(2) there were high correlationl between LTP and the average A of SynapsinⅠ(r=-0.953,p=0.0465)and P-synapsinⅠpositive cells(r=-0.960,p=0.04) and the number of BDNF-positive cells(r=0.968,p=0.0315) in model group rat DG district.(3)there was positive correlation between the expression of BDNF and P-synapsinⅠ(r=0.988,p=0.0121) in model group rat CA1. (4)there was high correlation between the average A of BDNF-positive cells in CA1 and EL(r=-0.994.p=0.0063),the number of pyramidal cells in CA1(r =0.971,p=0.0276) in model group.Conclusion:(1) Severe damages of spatial learning and memory ability and lowered incidence rate of long term potentiation induction from the dentate gyrus of hippocampus existed and lasted for a long time in vascular dementia rats.(2) The number of CA1 pyramidal cells and synapses significantly reduced,and persistenly existed for a long time.SynapsinⅠand its phosphorylation levels of expression depressed,and then presynaptic transmission mechanism damaged.That may be one of the synaptic transmission mechanism in VD rats(3) Early after reperfusion of brain ischemic,BDNF had a profective effect to neurons,but its protection weakened at later stage.That played an important role in the course of occurance and development of VD rats.(4) Puerarin can reduce the loss of hippocampal pyramidal cells and dendritic, lower synaptic damage,increase the number of vesicles,increase the levels of synapsinⅠand its phosphorylation,improve synaptic transmission efficiency and increase the expression of BDNF.So Pue had obvious role in brain protection.