| Objective:To observe rats’ behavioral,Hippocampal CA1region morphology and the protein and gene expressions of CREB changes in vascular dementia model. To explore the relationship between the injury of Hippocampal CA1region neurons and VD, and the intervention mechanism of method of Modified Shuyu Pill in treating change vascular dementia model.Methods:Forty SPF male Sprague-Dawley mice were randomly divided into four groups:Control group,Sham-operated group,The vascular dementia model was produced by2-VO ischemia/reperfusion and tail bloodletting legal system. vascular dementia model is building3days,The four groups rats above mentioned were treated by saline, Modified Shuyu Pill lavage intervention for8weeks respectively,. and then observing the changes of rats’ behavioral by Morris water maze experiment, with HE staining Hippocampal CA1region of brain tissue morphology change was observed by light microscopy;The protein and mRNA expressions of CREB were measured by real-time polymerase chain reaction(RT-PCR) and immunofluorescence respectively.Results:1.The ability of learning and memory tests:in the trial of6days of escape navigation test, the average escape latency of each group showed a decreasing trend; compared with the model group, the escape latency time of the Modified Shuyu Pill obviously shorten (P<0.01), the escape latency time comparison between Normal group and Sham-operated group no significant difference (P>0.05); In space exploration test:crossed the original platform of the former platform quadrant activities was observed, compared with the model group,the crossed platform numbers of the Modified Shuyu Pill increased significantly(P<0.01), between Sham-operated group and Normal group which was no statistically significant difference (P>0.05).2. Rat Hippocampal CA1region of brain tissue HE staining:in the Hippocampal CA1region, the model group rats’cone cells of structure failure, scattered, cell body swelling, membrane dissolved, deep nuclei, cytoplasm pyknosis, Cell count decreased significantly; compared with model group, cone cells damage in the Modified Shuyu Pill group,the number of neuronal cone cells significantly increased and level of clarity, structure, hierarchy arrangement is neat, clear rules of the nucleus, nucleolus, rich, is a small amount of nuclear chromatin pyknosis degeneration of neurons;between Sham-operated group and Normal group which was no statistically significant difference.3. In paraffin section of brain tissue by Immunofluorescence technique detecting rat hippocampal CA1region after CREB protein expression:compared with the model group, the fluorescence intensity of the CREB positive cells in rat Hippocampal CA1region of brain tissue was obviously less then Modified Shuyu Pill group,and fluorescence intensity increased, cytoplasm staining deep, the difference are significantly (P<0.01),between Normal group and Sham-operated group no significant difference (P>0.05).4. Determination of the expression of CREB gene in rat Hippocampal CA1region:each group of rats CREB mRNA expression in Hippocampal CA1region, Modified Shuyu Pill group then model group increasedsignificantly, with obviously difference (P<0.01).between Sham-operated group and Normal group rats CREB mRNA expression in Hippocampal CA1region was no significant difference (P>0.05).Conclusion:1.By2-VO ischemia/reperfusion and tail bloodletting legal system to build the VD model was stable and reliable, good repeatability.2. Hippocampal neurons loss and structural damage may be the Ischemia hypoxia resulted in the morphological basis of the VD model to learning and memory dysfunction.3. Modified Shuyu Pill has the effect of Jian-pi-bu-shen,Tian-jing-yi-sui,Huo-xue-tong-luo,Hua-tan-kai-qiao;that can significantly improve the VD rats learning and memory ability,and increase CREB mRNA and protein expression, reduce pathological changes in hippocampal CA1region, which may be of the important therapeutic mechanisms to vascular dementia. |
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