| Objective: Vascular dementia (VD) is a chronic brain syndrome characterized with progressive cognitive handicap due to brain tissue damage by various cerebrovascular factors such as brain ischemia, brain hemorrhage and so on. VD is one of the important causes of senile dementia. In some western countries, VD is the second cause of senile dementia, and it is the major factor in many Asian countries. In recent years, with the aging of population, the incidence of VD is increasing year by year, and seriously threatening the health and life quality of elderly people.It is very important to establish stable and reproducible animal model for studying the mechanisms, prevention and therapy and curative effect evaluation of VD. At present, rats and mice are commonly used in VD models. The rat VD models include vessel occlusion VD model, removal cerebral cortex VD model, hypertension VD model, spontaneous VD model and intravenous injection magnetic iron oxide VD model and so on. Most mouse VD models are incomplete brain ischemia/reperfusion, in which the bilateral common carotid artery occlusion induced-ischemia/reperfusion accompanied with tail end depletion method is classical. Although many studies of VD animal models have made a great progress, there are still a lot of problems, such as the disunity of standard, poor repeatability, complex operation of individual method, low animal survival rate, the many differences from the clinical patients and difficulty for long-term observation, etc. Reliable VD models should be consistent with the pathological damage of the clinical patients theoretically, and accompanied with significant intelligence disturbances of the model animals. At the same time, the VD model should be easy to operate, good repeatability, easy to various electrophysiology and intelligent test. But it is regret that there is no VD model consistent with the above standards at present.Eight-arm maze test was used to evaluate the spatial learning and memory ability of experimental animal according to observing the length of path and the times of typical fault during finding food, which could objectively reflect the degree of dementia. In addition, besides behavioral changes, morphological changes are also important for evaluating VD model. As an important part of participating learning and memory function, the hippocampus is very sensitive for brain ischemia. Repeated and severe brain ischemic insult could induce the damage of pyramidal neurons in CA1 hippocampus. Therefore, morphological changes of hippocampal neurons could help to judge the success of VD models.Therefore, the present study was undertaken to establish four VD models of mice based on the previous experiments. The efficacy of the VD models was evaluated by eight-arm maze test which indicates the spatial learning and memory ability, and morphological changes of the CA1 hippocampus of mice by thionin staining. The above results might provide new experimental evidence for the study of VD.Experimental protocol and methods:One hundred and fifty adult male kunming mice, 34±2.5 g in weight, were randomly assigned to five groups (n=30 in each group):(1) Sham group: the bilateral common carotid arteries were exposed, but without blocking the blood flow.(2) Bilateral occlusion 20 min group: bilateral common carotid arteries were occluded for 20 min, 3 times, at 10 min intervals, accompanied with tail end depletion of 0.3 ml.(3) Bilateral occlusion 30 min group: bilateral common carotid arteries were occluded for 30 min, 3 times, at 10 min intervals, accompanied with tail end depletion of 0.3 ml.(4) Right ligation + left occlusion group: right common carotid artery was permanently ligated using silk thread. At the same time, left common carotid artery was occluded for 30 min, 3 times, at 10 min intervals. (5) Left ligation + right occlusion group: left common carotid artery was permanently ligated using silk thread. At the same time, right common carotid arteries was occluded for 30 min, 3 times, at 10 min intervals.The animals in each group were further divided into 15 d, 30 d and 45 d subgroups according to the time of reperfusion. There were 10 mice in each group.The mice were used for eight-arm maze test on 15 d, 30 d and 45 d, respectively. The length of path (path) and the times of typical fault (typical fault) during finding food were recorded for continuous 10 d to reflect the spatial learning and memory ability. Then, 6 mice were sacrificed randomly by decapitation. And the slices were stained with thionin to observe the delayed neuronal death (DND) of the pyramidal neurons of the CA1 hippocampus. Under light microscope, the histological grade (HG) of the CA1 region in the hippocampus was analyzed for the evaluation according to methods described by Kitagawa and Kato. Briefly, the histological changes of the CA1 hippocampus were divided into 4 grades: grade 0, no cell death; grade ?, scattered cell death; gradeП, mass cell death; gradeШ, almost complete cell death. The neuronal density (ND) of the CA1 hippocampus was determined by counting the number of surviving pyramidal neurons with intact outline, full nucleus and clear nucleolus within 1 mm linear length of the CA1.Results:1 Changes of the spatial learning and memory ability of mice1.1 Differences of the spatial learning and memory ability of mice of four VD models at the same time1.1.1 Differences of the spatial learning and memory ability of mice of four VD models on 15 dCompared with the sham group, there were no significant differences in the path and the typical fault of mice in bilateral occlusion 20 min, bilateral occlusion 30 min and left ligation + right occlusion groups (P>0.05). However, the path was significantly prolonged and the typical fault was significantly increased in right ligation + left occlusion group compared with the sham group (P<0.05), which indicated that the mice presented obvious learning and memory disability.1.1.2 Differences of the spatial learning and memory ability of mice of four VD models on 30 dThe results showed that the path and the typical fault of mice in the bilateral occlusion 20 min group had no significant changes compared with the sham group (P>0.05). While the path and the typical fault of mice were both significantly increased in bilateral occlusion 30 min, right ligation + left occlusion and left ligation + right occlusion groups (P<0.05). And the mice in right ligation + left occlusion group presented the worst spatial learning and memory ability.1.1.3 Differences of the spatial learning and memory ability of mice of four VD models on 45 dCompared with the sham group, the path and the typical fault of mice in four VD models presented significant increase during finding food (P<0.05). The results of the right ligation + left occlusion group were statistically significant compared with three other VD models (P<0.05).1.2 Differences of the spatial learning and memory ability of mice of the VD model at different timeThe path and the typical fault in the sham group on 15 d, 30 d and 45 d had no significant difference (P>0.05). Therefore, a random sham group was chosen to statistics.1.2.1 Bilateral occlusion 20 min groupCompared with the sham group, the path and the typical fault of mice in 15 d and 30 d groups increased without statistical significance (P>0.05). However, the path and the typical fault obviously increased in 45 d group compared with the sham group.1.2.2 Bilateral occlusion 30 min groupThe results showed that the path and the typical fault of mice in 15 d group increased without statistical significance compared with the sham group (P>0.05). While the path and the typical fault all presented significant increase in 30 d and 45 d groups compared with the sham group (P<0.05).1.2.3 Right ligation + left occlusion groupCompared with the sham group, there were significant increase in the path and the typical fault in 15 d, 30 d and 45 d groups (P<0.05), especially the 30 d and 45 d groups.1.2.4 Left ligation + right occlusion groupCompared with the sham group, there were no significant differences in the path and the typical fault in 15 d group (P>0.05). However, both the path and the typical fault were significantly increased in 30 d and 45 d groups compared with the sham group (P<0.05).2 Evaluation of morphology in the CA1 hippocampus2.1 Histological changes of the CA1 hippocampus of mice of four VD models at the same time2.1.1 Histological changes of the CA1 hippocampus of mice of four VD models in 15 d groupIn sham group, the pyramidal neurons in the CA1 hippocampus of mice were arranged in order with 3~5 cell layers, the outline of the neurons was intact, the nucleus was full, and the nucleolus was clear. The mice underwent bilateral occlusion 20 min, bilateral occlusion 30 min and left ligation + right occlusion groups had no significant neuronal damage in the CA1 hippocampus. However, there was a segment loss of the pyramidal neurons in CA1 hippocampus of mice in right ligation + left occlusion group. HG was significantly higher (P<0.05) and ND was significantly lower than those in sham group (P<0.05).2.1.2 Histological changes of the CA1 hippocampus of mice of four VD models in 30 d groupThere was no obvious DND in CA1 hippocampus in the bilateral occlusion 20 min group. Compared with the sham group, the HG slightly increased and ND slightly reduced without statistical significance (P>0.05). While there was obvious DND of the pyramidal neurons in bilateral occlusion 30 min, right ligation + left occlusion and left ligation + right occlusion groups. Compared with the sham group, HG was significantly increased (P<0.05) and ND was significantly decreased (P<0.05). And the much severe lesion of the neurons presented in right ligation + left occlusion group.2.1.3 Histological changes of the CA1 hippocampus of mice of four VD models in 45 d groupThe results showed that the CA1 hippocampus of the four VD models all exhibited obvious destruction. Compared with the sham group, HG was significantly increased (P<0.05) and ND was significantly decreased (P<0.05), especially the right ligation + left occlusion group.2.2 Histological changes of the CA1 hippocampus of mice of the VD model at different timeThe HG and ND of the CA1 hippocampus of mice in the sham group in 15 d, 30 d and 45 d groups had no significant difference (P>0.05). Therefore, a random sham group was chosen to statistics.2.2.1 Bilateral occlusion 20 min groupThere were no significant changes of HG and ND in 15 d and 30 d groups (P>0.05). However, there was obvious DND of the pyramidal neurons in 45 d group. Compared with the sham group, HG was obviously increased and ND was obviously reduced (P<0.05).2.2.2 Bilateral occlusion 30 min groupThe results showed that there was rarely a loss of pyramidal neurons in the CA1 hippocampus in 15 d group. The CA1 hippocampus presented serious damage in 30 d and 45 d groups, which was represented by the increase in HG (P<0.05) and decrease in ND (P<0.05) compared with the sham group.2.2.3 Right ligation + left occlusion groupThe CA1 hippocampus in four VD models presented obvious DND in 15 d, 30 d and 45 d groups. Compared with the sham group, HG was significantly increased (P<0.05) and ND was significantly decreased (P<0.05). Moreover, the changes of HG and ND in 30 d and 45 d groups were more obvious.2.2.4 Left ligation + right occlusion groupThe results of thionin staining showed that the CA1 hippocampus of mice in 15 d group had no significant DND, and HG and ND had no significant difference compared with the sham group (P>0.05). In 30 d and 45 d groups, there was obvious DND of the CA1 hippocampus, and HG was significantly increased (P<0.05), ND was significantly decreased (P<0.05) compared with the sham group.Conclusions:In the four kinds of VD models established in the present study, the mice presented spatial learning and memory disability and the delayed neuronal death of the CA1 hippocampus at different time, especially the right ligation + left occlusion group. |
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