| ObjectiveVascular dementia (VD), as a commom disease of people, has done harm to human's health. Patients with VD often have some functional impairments. The impairment of the ability of learning and memory is common. Hippocampus is the vital structure of closely related to learning and memory of CNS, and it is vulnerable to cerebral ischemia. The pathogenesis is mainly related to excitatory amino acid toxicity, calcium overload, apoptosis, reaction of astrocyte. Glutamic acid, an important excitabily neurotransmitter in CNS, is mainly distributed in the area of cerebral cortex, hippocampus etc, and has important effect upon learning, memory and synaptic plasticity. It participates in many physiological functions of cerebral cortex and hippocampus, such as learning, memory, motion and sensus. Glu neurotransmitter role achieves by excitatory amino acid receptors. It is now well known that the hippocampus is correlated with learning and memory. The pathological changes of neurons in the hippocampus have great effects upon the forming and maintance of learning and memory. Many results have confirmed that hippocampus long-term potentiation (LTP) is closely correlated with the process of learning and memory. It has been moreove proved that LTP as a synapse model of memory is produced and maintained with the participation of Glu and its receptor.Some researchers show that drugs can affect the expression of Glu receptors. The traditional Chinese medicine of reinforcing Qi and promoting blood circulation can improve the ability of learning and memory after cerebral ischemia. Buyanghuanwu decoction is one of reinforcing Qi and promoting blood circulation traditional Chinese medicines. The past investigation shows that Buyanghuanwu decoction can decrease blood glutinous degree, anti platelet aggregation, improve the level of NO and PGI2 of brain blood vessel endothelium, restraint glumatic acid release, degrade the intracellular concentration of calcium and the level of arachidonic acid, restraint free radicle and improves the activity of antioxidase. It is proved that buyanghuanwu decoction can increase cerebral blood flow and improve brain cell energy metabolism, afunction and dysmnesia. However the treatment function and mechanism of buyanghuanwu decoction on VD are not very clear. Based on what has been mentioned above, we attempt to study the therapeutic effect of buyanghuanwu decoction on VD and therefore explore the mechanism of action by observing the change of Glu receptors of VD.Methods1. The establishment of animal model Adult male SD rats were used. The VD models were produced for ischemia (5min)-reperfusion (60min) thrice by four vessel occlusion. The changes of behavior were observed through the water maze test.2. Detectation on changes of learning and memory in rats One hundred and forty-four adult male SD rats were randomly divided into 4 groups, 36 rats per group: sham-operated group, VD model group, buyanghuanwu decoction treatment group and nimodipine group. VD model were produced by four vessel occlusion in rats. Except for sham-operated group, the other groups were made VD by four vessel occlusion. VD rats were treated with intragastrical buyanghuanwu decoction suspension (50 g/kg/d) and nimodipine suspension (20 mg/kg/d) for 30 days. The Morris maze was used to detecte the changes of learning and memory in rats at different time: before operation, after operation 25 d～30 d.3. Tissue sample collection and treatment After operation 30 d, 12 rats of each group were anaesthetized by 1% Sodium Pentobarbitone solution and their brains were fixed up by 4% paraformaldehyde solution. The paraffin slices of hipocampus were made into 5μm thick. The rats were executed quickly through decollation and their hippocampuses were extracted in low temperature operation. They were conserved in -80 degrees. 4. Pathological observation The hippocampal pathologic changes in the rats with VD was observed with light microscope through HE staining.5. Glutamate receptor immunohistochemical staining Manual operation of the order in accordance with SP immunohistochemical staining, the paraffin slices of hipocampus were made and NMDAR1, NMDAR2A, NMDAR2B and AMPA-GluR1 were stained through immunohistochemistry. And then the statistic study of each group (surface density Sv showing their numbers) was conducted.6. Western blot of Glu receptors protein in hippocampus of VD Weigh hippocampus, extract total protein, BCA method of quantitative protein concentration. The level of NMDAR1, NMDAR2A, NMDAR2B and AMPA-GluR1 protein expression in hippocampus was examined through Western blot method.7. Real-time fluores quantitative PCR detection of hippocampal glutamate receptor mRNA expression in rat hippocampus Hippocampus were separated on ice quickly, the hippocampus total RNA was extracted by Trizol and reversed transcriptoion cDNA. The level of NMDAR1, NMDAR2A, NMDAR2B and AMPA-GluR1 mRNA expression in hippocampus was examined through PrimerScriptTM real-time quantitative PCR detection kit one-step method.Results1. The results of water maze test revealed that: (1) Before operation, the difference of the average escaping latency period (sec) and cross-platform times (number/60 sec) have no significance between every groups. (2) After operation 25 d～30 d, compared with sham-operated group, the average escaping latency period (sec) of VD model group prolonged significantly and cross-platform times (number/60 sec) shortened distinctly (P<0.05). (3) After operation 25 d～30 d, compared with VD model group, the average escaping latency period (sec) of both buyanghuanwu decoction treatment group and nimodipine group shortened distinctly and cross-platform times (number/60 sec) prolonged significantly (P<0.05). (4) After operation 25 d～30 d, the learning and memory results of sham-operated group, buyanghuanwu decoction treatment group and nimodipine group had no distinct difference (P>0.05). The results above suggest that the learning and memory ability of VD rats reduced and buyanghuanwu decoction may improve their learning and memory.2. The observations under the light microscope show: (1) The arrangement of the pyramydal neurons in the hippocampal CA1 region in sham-operated group were tight and in order with the nucleus being large and round and its neucleolus being evident. (2) In VD model group, the pyramydal neurons in the hippocampal CA1 region decreased and had no clear arrangement. There was empty dye area surrounding the neurons which had condensed nucleus dense cytoplast and small body. In addition, there was infiltration of inflammatory cells. (3) In buyanghuanwu decoction treatment group and nimodipine group, the condensed nucleus dense cytoplast and small body decreased apparently and there was no infiltration of inflammatory cells.3. The results of Staining of immunohistochemistry of Glu receptors in hippocampus CA1 region of rats revealed that: (1) Compared with sham-operated group, the NMDAR1, NMDAR2A, NMDAR2B and AMPA-GluR1 positive neurons of hippocampus CA1 region in VD model group separately reduced distinctly (P<0.05). (2) The NMDAR1, NMDAR2A, NMDAR2B and AMPA-GluR1 positive neurons of hippocampus CA1 region of the rats in both buyanghuanwu decoction treatment group and nimodipine group separately increased significantly (P<0.05), compared with VD model group. (3) Buyanghuanwu decoction treatment group, nimodipine group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower level NMDAR1, NMDAR2A, NMDAR2B and AMPA-GluR1 of hippocampus CA1 region might participate in the pathogenesis of VD and buyanghuanwu decoction could increase the level NMDAR1, NMDAR2A, NMDAR2B and AMPA-GluR1 of hippocampus CA1 region of VD and hence improve their learning and memory ability.4. The results of Western blot of Glu receptors protein in hippocampus of rats indicated that: (1) Compared with sham-operated group, the level of NMDAR1, NMDAR2A, NMDAR2B and AMPA-GluR1 protein expression in VD model group separately reduced notably (P<0.05). (2) The level of NMDAR1, NMDAR2A, NMDAR2B and AMPA-GluR1 protein expression in buyanghuanwu decoction treatment group and nimodipine group separately elevated notably (P<0.05), compared with VD model group; (3) buyanghuanwu decoction treatment group, nimodipine group and sham-operated group had no distinct difference (P>0.05). These suggested that the lower expression of NMDAR1, NMDAR2A, NMDAR2B and AMPA-GluR1 protein might participate in the pathogenesis of VD. but buyanghuanwu decoction could increase the expression of NMDAR1, NMDAR2A, NMDAR2B and AMPA-GluR1 protein and improve their learning ability and memory.5. Real-time fluorescence quantitative PCR detection of hippocampal glutamate receptor mRNA expression in rat hippocampus. The results indicated that: (1) Compared with sham-operated group, the level of NMDAR1, NMDAR2A, NMDAR2B and AMPA-GluR1 mRNA expression in VD model group separately reduced notably (P<0.05). (2) The level of NMDAR1, NMDAR2A, NMDAR2B and AMPA-GluR1 mRNA expression in buyanghuanwu decoction treatment group and nimodipine group separately elevated remarkably (P<0.05), compared with VD model group. (3) Buyanghuanwu decoction treatment group, nimodipine group and sham-operated group had no distinct difference (P>0.05). (4) The levels of Units housekeeping gene (β-actin) in the NMDAR1, NMDAR2A, NMDAR2B and AMPA-GluR1 expression in each groups also exist the same differences. These suggested that the lower expression of NMDAR1, NMDAR2A, NMDAR2B, and AMPA-GluR1 mRNA might participate in the pathogenesis of VD. but Buyang Huanwu Decoction could increase the expression of NMDAR1, NMDAR2A, NMDAR2B and AMPA-GluR1 mRNA and improve their learning ability and memory. Conclusions1. The VD rats model which had the cognization impairment in primary of damage of learning and memory was successfully established. The VD model is the perfect animal model and feasible to the further study of VD. The hippocampus CA1 region in the VD rats model had the pathologic changes.2. The level of NMDAR1, NMDAR2A and NMDAR2B protein and mRNA in VD rats hippocampus reduced, which was consistent with the decline of learning and memory. Those suggested that the reduction of the expression of NMDA receptors might participate in the pathogenesis of VD.3. The level of AMPA-GluR1 protein and mRNA in VD rats hippocampus reduced, which was consistent with the decline of learning and memory. Those suggested that the lower expression of AMPA-GluR1 might participate in the pathogenesis of VD.4. Buyanghuanwu decoction could increase the expression of NMDA receptors and AMPA receptor and ameliorate the pathologic changes in hippocampual CA1 region in the rats with VD. This was in accord to their better learnig and memory. All mentioned above suggest that the medicine may improve the pathogenesis of VD.
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