| Objectives : As an acquired syndrome of intelligent impairment, Vascular dementia (VD), which is a kind of cerebral dysfunction caused by various kinds cerebral vessel diseases, demonstrats mainly as learning and memory dysfunction. With the increase of the proportion of the elderly in the population and that of the curing rate of cerebral vascular disease, the morbidity of VD mounts continuously and hence brings about heavy loads to the society and family. However both the pathogenesis of VD and its specific treatment remain unknown up to now. It is therefore significant to study the pathophysiological mechanism of VD and to find some effective treatments of VD.Learning is a acquirement of experiences and skills. Memory is conservation and playbacks of experience and skills. As two advanced functions of human brains, learning and memory are two closely associated neurobiology procedures despite their differences. It is now well known that the hippocampus is considered as a important structure associated with learning and memory. The pathological and structure changes of neurons in the hippocampus have great effects upon the forming and maintance of learning and memory. It is generally agreed that the central muscarinic cholinergic system, which be composed by neurons synthesize acetylcholine, plays an important role in learning and memory processes. Cholinergic neurons of medial septal nucleus and Broca diagonal Band nucleus of basal forebrain release acetylcholines (ACh) to hippocampus, which are main sources of cholinergic fiber in hippocampus, participate in the form of learning and memory. Cholineacetyltransferase(CHAT) is the rate-limiting enzyme in the process of the synthesized acetylcholine. If cerebral tissue is suffered from ischemia and anoxia, the distribution and concentration of CHAT will be affected so that the concentration of ACh will change.dl-3n-butylphthalide(NBP), a yellow compound isolated from the seed of celery, is a new drug of Class I of P.R.China have never reported abroad up to now. In China, FengYipu has made lots of related researches which proves it as an anticerebral- ischemic drug which has multitarget effects upon, the prevention of the cerebral ischemic damage, the improvement of both chondriosome function and the level of NO and PGI2 of brain blood vessel endothelium, the restraints of glumatic acid release, the degrades of the intracellular concentration of calcium and the level of arAChidonic acid, the restraints of free radicle and improves the activity of antioxidase, the lightenments of cerebral edema and the reduction of area of cerebral infarction. In a word, it is proved that NBP can increase cerebral blood flow and improve brain cell energy metabolism, afunction and dysmnesia. As an anticerebral-ischemic new drug, it has been generally used to treated the cerebrovascular disease (CVD). However there is no scientific report the drug in VD at present.Based on what has been mentioned above, The VD mice models were established in the experiment, the changes of cholinergic pathogenesis and CHAT of VD in hippocampus were observed, and the pharmaco-cholinergic mechanism of NBP improving learning and memory function in VD would be discusssed.Methods:1) The VD model of mice were established for ischemia (20 min)- reperfusion (10 min) thrice by ligating the bilateral common carotid arteries. The changes of behavior were observed through the step-down avoidance test and water maze test. The hippocampal pathologic changes in the mice with VD was observed through HE staining.2) The changes of CHAT protein expression in hippocampus of ACh group of mice were measured with immunohistochemistry.Result:1. The evaluation of behavior of VD model and its pathologic features of the hippocampus 56 mice were divided into the control group, sham- operated group, VD model group, NBP treating group and NBP precaution treating group. The mice were subjected for ischemia (20 min)-reperfusion (10 min) thrice by ligating the bilateral common carotid arteries to establish the VD model. Beginning on day 29 and 30 day after operation, the functions of learning and memory of eACh mouse in eACh group were tested through step-down test and water maze test. The hippocampal pathologic changes in the mice with VD was observed through HE staining.1.1 The test of learning of miceOn day 29 after operation, the learning of every group were tested by step-down test and water maze test respectively. Response time (sec), error times (number/5min) in step-down test, and swimming time (sec) and error times (number/3min) in water maze test were used to evaluate the AChivements of learning.The results of step-down test revealed that:⑴The response time in learning phase of VD model group (89.41±12.08)s prolonged distinctly (P<0.01) compared with the response time of sham-operated group(48.76±9.24)s.⑵The response time of both the NBP treating group (59.68±10.27)s and the NBP precaution treating group (57.23±10.05)s shortened distinctly (P<0.01), compared with VD model group.⑶The error times in learning phase of VD control group (3.74±0.42) increased notably (P<0.01), compared with the error time of sham-operated group (1.23±0.27).⑷The error times of both the NBP treating group (1.52±038) and the NBP precaution treating group (1.48±0.31) decreased notably (P<0.01), compared with VD model group.⑸The learning of sham-operated group, NBP treating group and NBP precaution treating group had no distinct difference (P>0.05).The results of water maze test revealed that:⑴The swimming time in learning phase of the VD model group (145.80±13.29)s prolonged significantly(P<0.01), compared with sham-operated group (87.32±13.46)s.⑵The swimming time of both the NBP treating group (101.89±18.38)s and the NBP precaution treating group (99.18±11.25)s shortened distinctly (P<0.01), compared with VD model group.⑶The error times in learning phase of the VD model group (34.60±5.92) increased notably (P<0.01), compared with and sham-operated group (16.88±4.19).⑷The error times of both the NBP treating group (18.71±4.35) and the NBP precaution treating group (17.29±4.82) decreased remarkably (P<0.01), compared with VD model group.⑸The learning of the sham-operated group, the NBP treating group and the NBP precaution treating group had no distinct difference (P>0.05).The results above suggest that the learning ability of VD mice reduced and NBP may improve their learning.1.2 The test of memory of miceOn day 30 after the operations, the memory of the mice in every group was tested by step-down test and water maze test. The results of latency time (sec) and error times (number / 5 min) in step-down test and that of swimming time (sec) and error times (number / 3 min) are the memory.The results of step-down test revealed that:⑴The latency time in memory phase of VD model group (78.67±19.17)s shortened remarkably (P<0.01), compared with the latency time of sham-operated group (148.92±20.83)s.⑵The latency time of the NBP treating group (131.41±18.82)s and the NBP precaution treating group (128.57±18.29)s prolonged significantly (P<0.01), compared with VD model group.⑶The error times in memory phase of VD model group (1.72±0.13) increased notably (P<0.01), compared with the error time of sham-operated group (0.32±0.05).⑷The error times of both the NBP treating group (0.54±0.08) and the NBP precaution treating group (0.50±0.07) decreased notably (P<0.01), compared with VD model group.⑸The memory of sham-operated group, NBP treating group and NBP precaution treating group had no distinct difference (P>0.05).The results of water maze test revealed that:⑴The swimming time in memory phase of the VD model group (137.40±11.76)s prolonged remarkably (P<0.01), compared with sham-operated group (67.82±8.79)s.⑵The swimming time of both the NBP treating group (88.73±9.53)s and the NBP precaution treating group (83.87±8.62)s shortened distinctly (P<0.05), compared with VD model group.⑶The error times in memory phase of the VD model group (20.90±4.25) increased notably (P<0.05), compared with sham-operated group (9.57±2.91).⑷The error times of both the NBP treating group (11.17±3.04) and the NBP precaution treating group (10.27±2.83) decreased significantly (P<0.05), compared with VD model group.⑸The memory of sham-operated group, NBP treating group and NBP precaution treating group had no distinct difference (P>0.05).These results suggested that the memory of VD mice decreases and NBP may improve their memory.1.3. The pathologic changes of the hippocampus of the VD mice The observations under the light microscope show:⑴The arrangement of the pyramydal neurons in the hippocampal CA1 area in sham-operated group were tight and in order with the nucleus being large and round and its neucleolus being evident.⑵In the VD model group, the pyramydal neurons in the hippocampal CA1 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.⑶In the NBP treating group and the NBP precaution treating group, the condensed nucleus dense cytoplast and small body decreased apparently and there was no infiltration of inflammatory cells.2. The level of CHAT receptor in the hippocampal CA1 area of VD mice and the effect of NBP .Staining of Immunohistochemistry of CHAT.The mice were anaesthetized by 10% chloral hydrate solution and their brains were fixed up by 4% paraformaldehyde solution. The paraffin slices of hippocampus were made and CHAT was stained through immunohistochemistry. And then the statistic study of eACh group (surface density Sv showing their numbers) was conducted. The result revealed that:⑴The CHAT positive neurons of hippocampus CA1 area in VD model group (0.0823±0.0236) reduced distinctly (P<0.05), compared with sham-operated group (0.1178±0.0104).⑵The CHAT positive neurons of hippocampus CA1 area of the mice in both the NBP treating group (0.1097±0.0131) and the NBP precaution treating group (0.1116±0.0135) increased (P<0.05), compared with VD model group.⑶The NBP treating group, the NBP precaution treating group and the sham-operated group had no distinct difference (P>0.05).These suggested that the lower level CHAT of hippocampus CA1 area might participate in the pathogenesis of VD and NBP could prevent the level CHAT of hippocampus CA1 area of VD from decreasing and hence improve their learning and memory ability.Conclusion:1. The VD mice model which had the cognization obstacle 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 area in the VD mice model had the pathologic changes.2. The level of CHAT in VD mice hippocampus reduced, which was consistent with the decline of learning and memory. Those suggested that the reduction of the expression of CHAT might participate in the pathogenesis of VD.
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