| Background and purposeWith the acceleration of population aging, vascular dementia (VD) has become the second cause of dementia. Due to such characteristics of VD as higher prevalence and disability rate, longer course of disease and higher cost of treatment, it will bring a huge burden to the patient's family and society. Therefore, the treatment for VD attracts more and more people's attention.Memantine is a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists. The current studies show that it is effective to VD, but its mechanism to improve learning and memory in VD remains unclear.cAMP responsive element binding protein (CREB) is a eukaryotic transcription factors existing in the nucleus, and it is also a "molecular switch" in the process of long-term memory formation. The release of cytochrome C from mitochondrion into cytoplasm eventually leads to apoptosis. However, in VD rat model, whether the expression changes of CREB and cytochrome C exist is unknown. Additionally, it is still unknown whether memantine improves learning and memory function by means of influencing the expression of CREB and the release of cytochrome C. So we establish the vascular dementia rat model and then to observe the expression changes of the CREB and cytochrome C before and after memantine treatment, aiming to explore the possible mechanism of memantine in the treatment of VD.MethodsThe first part:we applied the permanent bilateral common carotid artery ligation method to prepare VD models with SD rats, then the rats were randomly divided into there groups (the sham-operated group, the model group, and the memantine group). The sham-operated group and the model group were given normal saline, once a day, for 8 weeks. The memantine group were given memantine after operation with 3mg/kg (dissolved in normal saline) one time a day by intragastric administration, for 8 weeks. In order to observe the differences between behaviors of rats, we tested their learning and memory performances with Y type maze experiments after the intragastric administration. The higher the learning grade was, the worse the learning ability was; The higher the memory grade was, the better the memory ability was.The second part:we used HE and Nissl staining to observe the pathological changes in rat hippocampus in each group and applied SP immunohistochemical technique to observe the expression changes of CREB as well as cytochrome C. We used Qwin550CW image processing and analysising system to measure the positive material's average gray value, then compared the changes of CREB and cytochrome C in each group, and judged whether there were differences between them. We also used TUNEL method to observe the apoptotic cells and counted the percentage of apoptotic cells under the light microscope.Results1 Y maze test:In the sham-operated group, the learning grades were lowest, while the memory grades were highest; In the model group, the learning grades were highest, while the memory grades were lowest, showing that learning and memory performance decreased significantly compared with the sham-operated group(P<0.01). Compared to model group, the memantine group were better in learning and memory capability (P<0.01).2 The HE staining:The sham-operated group showed that the neurons arrangement were uniform and tidy, the nuclei were large and round, and nucleoluses were clear; The model group demonstrated that neurons arranged loosely, their structures were unclear, nuclears were condensative; The memantine group showed that neurons arranged tightly, the nuclei were round, nucleoluses were clear, in addition, a few condensation and degeneration neurons were also observed.3 Nissl staining: In the sham-operated group, the cells'structure were integral, with many dark blue granular Nissl bodys filling in the cytoplasm; In the model group, cells were corrugativus and deformed, the coloration of Nissl bodys were weak; In the memantine group, neurons were clear, structures were complete, Nissl bodys in cytoplasmic were abundant.4 The expression of CREB in hippocampus by SP immunohistochemistry: CREB positive neurons were brownishly yellow, located in the cytoplasm and nucleus. In the sham-operated group, there were a large number of positive neurons. A large number of brown-yellow granules were seen in both the the nuclei and the cytoplasms, stained the most darkly; In model group, the number of positive neurons were reduced significantly and dyeing lighter; Compared with model group, the number of positive neurons of the memantine group were increased. After counting the average gray value of CREB-positive neurons, we found the average gray value in the model group was significantly lower than the sham-operated group (P<0.01). While the average gray value in the memantine group was increased, there was a significant difference compared with the model group (P<0.01).5 The expression of cytochrome C by SP immunohistochemistry:The cells with brown-yellow granules in the cytoplasm were defined as the positive cells. The sham-operated group was colored the most lightly; In the model group, we detected a large number of positive cells; In the memantine group showed a weak or moderate intensity staining. The positive reaction average gray value of cytochrome C in model group was higher than the sham operated group (P<0.01), while the memantine group was lower than the model group (P<0.01).6 Apoptotic cell by TUNEL:In the sham-operated group, only a small number of apoptotic cells were seen, and the nuclear shapes were irregular; In the model group, we observed a large number of apoptotic cells; In the memantine group, apoptotic cells were significantly reduced compared with the model group. The percentage of apoptotic cell showed that the percentage in model group was increased compared with the sham operated group (P<0.01), and in the memantine group it was significantly lower than that in the model group (P<0.01).Conclusions1 It might be related to the damages of learning and memory ability that the expression of CREB was decreased and the release of cytochrome C from mitochondria to cytoplasm was increased in the hippocampus of VD rat.2 Memantine might improve learning and memory funtion in VD rats by inhibiting the NMDA receptor, upregulating the expression of CREB and inhibiting the release of cytochrome C.
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