In The Process Of Vascular Dementia Occurs, Tau Protein Affect Vesicle Transporter Subtypes Of Correlation Analysis

BackgroundsPart 1 The making of vascular dementia rat modelChronic cerebral ischemia (CCI) is associated with cognitive decline in aging, vascular dementia and Alzheimer's disease. Substantial evidence has shown that Chronic cerebral ischemia may cause cognitive impairment, but the underlying neurobiological mechanism is poorly understood so far. In the present study, we used a rat model of chronic cerebral ischemia by permanent bilateral common carotid artery occlusion (BCCAO) to investigate the cognitive deficits induced by chronic cerebral ischemia. We found that BCCAO rats exhibited spatial learning and memory impairments dysfunction 3 month after BCCAO.Objective:In this study, we assessed the change of spatial learning and memory in ischemia-induced VaD animals.MethodsAnimalAll protocols were in accordance with the Guidance for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China and approved by the Society for shanghai SCXK 2012-0002.Chronic cerebral ischemia (CCI) was induced in adult male Sprague-Dawley rats through bilateral common carotid artery occlusion (BCCAO) as described previously, twenty-seven carotids-occluded rats were assigned to one of the three groups randomly (n=9):(1) sham (2) control (3) VaD group. Control rats received the same surgical procedures without carotid artery ligation.Assessment of learning and memory by Morris water maze (MWM)From day 93 (three month) to 98 post surgery spatial learning and memory abilities of rats were evaluated with MWM (training trials from day 93 to day 97 and probe trials on day 98).Laboratory staff performing behavioral assessments had no knowledge of the group assignments at the time of testing. The test includes 2 phases:the training trial and the probe trial.Statistics analysisData were expressed as mean ± SEM and analyzed with SPSS. The escape latency and travel distance in the MWM were analyzed with two-way analysis of variance (ANOVA) and the Bonferroni post hoc test. The remaining data were expressed as x±s, the average number of each group was compared with the t' test. Significance was assumed with a criterion of P< 0.05.ResultsSpatial learning memory and cognitive impairment in CCI-induced VaD ratsThe escape latency was progressively shorter in each group in a day-dependent manner. Day 1 test showed no significant differences of the escape latency among the three groups (p>0.05). On day 2, while the animals in the VaD group did not show significant difference in finding the platform compared with that on day 1, the animals in the sham and control group displayed shorter latency when compared with VaD rats. On day 3, all groups showed significantly shortened latency to reach the platform when compared to day 1 and day 2 (p< 0.05). On day 4, group comparisons revealed that animals in the sham group as well as the control group displayed shorter escape latency when compared with the VaD group (p<0.05). Similar to day 4, the group differences of latency on day 5 were significant (p<0.05), whereas the difference between day 4 and day 5 was not significant. The percentage of searching time in the sham group was notably higher compared with the VaD group (p<0.05). Similar results were also observed in the traveled distance in probe trail among the groups. The percentage of traveled distance in the target quadrant in the VaD group was significantly lower than the sham group (p<0.01).T-maze:A trial was composed of an "information run" and a "test run." For the information run, each rat was placed in the starting box of the stem arm, with one branch arm blocked by a guillotine door; the rat was rewarded for entering either branch arm. Subsequently, for the test run, the rat was returned to the starting box, with both branch arms opened; the rat was rewarded for entering branch arm that was opened on that particular trial. The rat was only rewarded if it entered the branch arm that was not chosen in the information run (correct choice). At the end of the training trial, for a total of 10 trials per session, the rats demonstrating>90% correct choices were selected. Seven days after completing the training trials, following the information run, each rat performed the test run after several delay times (-1 or 7day s). Ten trials were performed for each delay time. The number of errors per test run was recorded. The delay achieved in a set number of testing sessions can be used as an index of cognitive ability. Between trials the T-maze was wiped with alcohol to remove any olfactory cues. The behavioral experimenter was blind to the animals'group.Conclusion:1.In this study, we observed severe impairment of cognition performance in VaD animals. We found that, during the training trail in the Morris water maze assessment, the latency time in the disease model significantly extended after ischemia compared with sham and control-operated animals, suggesting an impaired cognition after the injury.The searching time, which is direct evidence of impaired spatial memory, has to be observed in VaD animals in probe trail. Searching distance in target quadrant, a parameter more closely related to spatial learning, significantly decreased in VaD animals, suggesting impaired ability of spatial learning and memory in these animals.2.T-maze:we examined the working memory function of all rats in the delayed alternation task using a T-maze apparatus. Results did not detect the significant differences in the performance between VD rats and sham rats under no-delay (-ldays) condition (P>0.05). However, the performance of VD rats markedly decreased compared to sham and control rats under 7-day-delay condition (P< 0.001),Part 2 The expression fo tau protein, PSD-95, vGluTs,vAChTand vGAT in CCI induced Vascular DementiaTau has been found in additional subcellular compartments, including the synapse, where it may directly influence neuronal communication. More research suggested that tau may play an important role within both the pre and postsynapse and that its synaptic distribution may change during disease. There are several potential mechanisms by which tau could affect synaptic function and neuronal excitability. (1)Tau may directly influence synaptic function since, it has been shown to be localized within both pre-and post-synaptic compartments. (2)Tau binds to a post-synaptic protein complex which includes PSD-95, the scaffold for synaptic NMDA receptors and a critical regulator of synaptic plasticity. However, the molecular mechanism is not fully understood.In the present study, we employed BCCAO rats to examine the cognitive function, tau protein, vGluTs,vAChTand vGAT activation and sought to determine the correlates of cognitive deficits induced by chronic cerebral global ischemia.2.Methods2.1 Animal and experimental proceduresCarotids-occluded rats were assigned to one of the three groups randomly (n=14):(1) sham group (2) CCI control (3) VD-3m group:after artery occlusion three month. Sham-operated rats (n=14) received the same surgical procedures without carotid artery ligation. All the rats were allowed to recover from surgery for 7 days.2.2 Western blottingHippocampus were removed respectively and kept in-80? until use. The quantification of immunoblotting was analyzed using the Quantity One program (Bio-Rad), normalizing against GAPDH expression in each sample as an internal control.2.3 Immunohistochemistry analysisThree months after global ischemia, the animals were decapitated and their brains were rapidly removed. Sagittal slices of approximately 1 mm were obtained with a surgical blade and CA1-3 were dissected from each slice using a dissecting microscope. Under the microscope, the cells (positive+negative) in 4 randomly selected fields (x 200) were counted. The positive ratio was calculated as percentage of positive cells against the all cells counted.3. STATISTICS ANALYSISData were expressed as mean ± SEM and analyzed with SPSS. The escape latency and travel distance in the MWM were analyzed with two-way analysis of variance (ANOVA) and the Bonferroni post hoc test. Other data were analyzed with one-way ANOVA with multiple comparisons in Student-Newman-Keuls (SNK) test. Significance was assumed with a criterion of P<0.05.4.RESULTS4.1 Hyperphosphorylation of tau increased in CCI-induced VD rats Compared with the level in sham group (set as 100%), the level of Tauser396 increasedand Tauser404 increased in VD-3m groups.4.2 PSD-95 reduced in CCI-induced VD rat brainIn our research, the level of PSD-95 reduced in hippocampus in VD-lm group, and decreased by 28% in in VD-3m group.4.3 vGluT1, vGluT3 and vAChT levels reduced in hippocampus of VaD ratsWestern blotting analyses were used to compare the total amount of vGluTs in hippocampus in VaD group, control group and sham group. Results showed no difference of vGluTs between the sham and controls. In contrast, the total amount of vGluT1 in the VaD animals decreased (p<0.01). Similar to vGluT1, the total amount of vGluT3 in hippocampus decreased (p<0.01) compared to the sham groups. On the contrary, no difference of vGluT2 was observed between the VaD group and the sham or control animals. The total amount of vAChT in VaD group decreased (p<0.01). No difference of vGAT was observed between the sham, control and VaD animals4.4 vGluT1 and vGluT3-positive neurons reduced in CA1-3 regions in CCI-induced VaD ratsCompared to sham rats, the vGluT1- positive neurons number in CA1 regions were significantly reduced in the VaD rats (p<0.01) after ischemia. Similar results were observed in both CA2 regions (p<0.01) and CA3 regions (p<0.01). In the same way, vGluT3 positive-neurons number in CA1 field decreased significantly compared with sham groups (p<0.001); Smaller but still significant differences of vGlut3-positive neurons were observed in CA2 regions (p<0.01) and CA3 regions (p<0.01). The number of vGluT2-positive neurons in 3 subregions is similar among all groups.4.5 vAChT-positive neurons reduced in CA1-3 regions in CCI-induced VaD rats The number of vAChT-positive neuron number was significantly reduced in the all 3 subregion of hippocampus in the VaD animals compared with the sham (or control group)(p<0.01); whereas the vGAT-positive neuron number was no significantly changed in the hippocampus of VaD rats.Conclution1. The excessive phosphorylation of tau protein on the postsynaptic membrane, PSD-95 protein levels drop and vesicle transporter levels decreased may be the possible mechanism in the CCI-induced Vascular Dementia.2.The levels of vGluT1 and vGluT3 significantly decreased in hippocampus at three months post artery occlusion. However, the level of vGluT2 did not change significantly. Our results suggest that vGluT1 and vGluT3 is more sensitive than vGluT2 in response to chronic cerebral ischemia damage and injury in rat model.The vAChT level significant decreased in the Hippocampi of BCCAO rats.vGluT1 and vGluT3-positive neuron in CA1-3 fields significantly reduced in chronic cerebral ischemia rats. Meanwhile, the transmitter positive neuron of vGluT1 and vGluT3 in CA1 region significantly decreased compared with CA2 or CA3 fields. These results are in line with the protein levels. The same results was observed in vAChT-positive neuron in hippocampal sub-regions.