| Objective：Vascular dementia (VD) is considered to result from ischemicinjury or sustained oligaemia to brain regions associated with cognitivefunction, memory and behavior. VD is the leading commonest form of seniledementia in our country. The aging of the population has led to an increasingprevalence of VD. However, the mechanisms underlying VD have not beenwell understood, and there is still no effective treatment for VD. Thereby, it isvery important to establish the good animal model for VD in order toinvestigate the mechanism and find effective drug for the prevention andtreatment of VD.Recent study showed that one important reason of VD was chronic andsustained oligaemia to brain regions which caused by carotid artery stenosis.Occluding blood flow within the bilateral common carotid arteries (two-vesselocclusion,2-VO) is the most commonly used method to mimic VD in VDstudy. Because of the compensation of cerebral arterial circle andestablishment of collateral circulation, however, it is sometimes hardly toachieve ideal cerebral ischemia enough to induce stable dementia. To producean ideal VD model with higher stability and lower mortality in experimentalanimals, modifications based on the principle of2-VO are necessary.Shuttle box test is commonly used to evaluate the conditioned learningand memory by testing active avoidance response of animals and can reflectthe degree of dementia. The hippocampus is considered to be an importantregion of the brain for learning and memory functioning and highly sensitiveto cerebral ischemia. Therefore, observation of morphological change in thehippocampus can further prove the model stability.Therefore, the present study was undertaken to evaluate the stability ofVD mice model established by modified2-VO method by observing dynamic changes in the learning and memory ability measured with shuttle box test andmorphological change of hippocampus. The results obtained provided newevidence for the study in the prevention and therapy of VD.1Grouping and method:One hundred and twenty-eight mice (28-32g) were randomly divided intothe following groups.①Normal control group (n=8)The mice were not subjected to any treatment and measured with shuttlebox test.②Sham group (n=24)Sham group was designed, in which all procedures in the model groupwere performed except occluding blood flow within the bilateral commoncarotid arteries and tail end depletion. Shuttle box test was performed at thetime points of15d,30d and45d (n=8in each time point) after the abovetreatments.③Model1group (n=24)The mice were subjected to occlusion of blood flow within the bilateralcommon carotid arteries for20min,3times, at10min intervals, and depletionfrom the tail end in a volume of0.3ml. Shuttle box test was performed at thetime points of15d,30d and45d (n=8in each time point) after the abovetreatments.④Model2group (n=24)Bilateral common carotid arteries of the mice were occluded for30min,3times, at10min intervals.0.3ml of blood was taken by the tail enddepletion method. Other procedures are same as the model1group.⑤Model3group (n=24)Left common carotid artery of the mice were permanently ligated, andright common carotid artery were occluded for30min,3times, at10minintervals. Other procedures are same as the model1group.⑥Model4group (n=24) Right common carotid artery of the mice were permanently ligated, andleft common carotid artery were occluded for30min,3times, at10minintervals. Other procedures are same as the model1group.2Establishment of VD modelsThe mice were anesthetized with10%chloral hydrate (350mg/kg, i.p.).Through a midline incision, the left and right common carotid arteries werecarefully separated from the cervical sympathetic and vagal nerves. In model1and2groups, bilateral common carotid arteries of the mice were occluded for20or30min,3times, at10min intervals. And at the same time, depletion wasperformed from the tail end in a volume of0.3ml. In model3and4groups,left or right common carotid artery was ligated with silk suture to permanentlyoccluding blood flow within it. And the contralateral common carotid arterywas occluded for30min,3times, at10min intervals. The mice in sham groupreceived all procedures in model group except occluding blood flow within thebilateral common carotid arteries and tail end depletion.3Shuttle box testThe apparatus was a shuttle box (44×28×23cm3) with the copperbottom. On the first day of testing, mouse was placed in the shuttle box andreceived10min acclimatization. The next day mouse was measured by shuttlebox test in the order of the first day. After a40s habituation period, the soundwas turned on as the conditioned stimulus. A1.0mA foot shock as theunconditioned stimulus was delivered if the mouse did not cross into the otherside within10s after the onset of the sound, and the foot shock was deliveredfor a maximum of10s. To avoid receiving a foot shock, the mouse had10s tomove to the opposite compartment, if the animal move to the other side, thenrecorded as an active avoidance response. During the foot shock was delivered,the animal move to the other side, recorded as a passive avoidance response.Each mouse was given60consecutive test cycles, each cycle interval of40s,10trials for a group, there was a total of6groups of circulation. Among each group of circulation, the mouse was given60s for rest. The shock time and/ornumber of the percentage of avoidances in each10trials were used foranalysis.4Pyramidal neurons DND in the CA1hippocampusMice were sampled for the observations at time point of24h after theshuttle box test in corresponding groups. The profile of pyramidal neuronsDND in the CA1hippocampus was evaluated under light microscope bydetermining the neuronal density (ND) and histological grade (HG). The NDwas determined by counting the number of surviving pyramidal neurons withintact cell membrane, full nucleus and clear nucleolus within1mm linearlength of the CA1hippocampus. The HG was divided into4grades accordingto the following standard: grade0, no neuron death; gradeⅠ, scattered singleneuron death; gradeⅡ, death of many neurons; grade Ⅲ, death of almostcomplete neurons.5Results5.1Results of shuttle box test5.1.1Behavioral change at the different time points in the same model①Model1: Compared to sham group, shock time was obviouslyincreased at15d and30d in the model group (P<0.05), while no significantchange at45d in the model group. Shock time was significantly increased at15d than that of45d (P<0.05), but without statistical difference between15dand30d in the model group. The results indicated that the conditionedlearning and memory ability obviously decreased at15d and subsequentlygradually recovered in this mouse model.②Model2: There were significantdifference in15d,30d and45d of the model group compared with shamgroup in the shock time (P<0.05), and no statistical change among15d,30dand45d in the model group, which suggested that the conditioned learningand memory ability obviously decreased and persisted longer time in thismouse model.③Model3: Shock time was obviously increased at15d,30d and45d in the model group compared to sham group (P<0.05), and withoutstatistical difference among15d,30d and45d in the model group. It wassuggested that the conditioned learning and memory ability obviouslydecreased and persisted longer time in this mouse model.④Model4:Compared to sham group, shock time was obviously increased at15d and30d in the model group (P<0.05), but no significant change at45d in the modelgroup. Shock time was significantly increased at15d and30d than that of45d (P<0.05), but without statistical difference between15d and30d in themodel group. The results indicated that the conditioned learning and memoryability obviously decreased at15d and30d, and subsequently graduallyrecovered in this mouse model.5.1.2Behavioral change at the different models in the same timeCompared to sham group, shock time was markedly increased at15d and30d in each model group (P<0.05), and no significant change among eachmodel group. The results indicated that the conditioned learning and memoryability obviously decreased at15d and30d in each mouse model. At the timeof45d, shock time was obviously increased in the model2and3groupscompared to sham group (P<0.05), but no significant change between the twogroups, which indicated that the conditioned learning and memory ability stilldecreased in these two mice models. However, shock time was withoutstatistical difference in the model1and4groups compared to sham group,which indicated that the conditioned learning and memory ability graduallyrecovered in these two mice models.5.2Results of morphological change of hippocampus5.2.1Morphological change of CA1hippocampus at the different timepoints in the same model①Model1: In normal and sham groups, pyramidal neurons in the CA1hippocampus were arranged in order with3~5cell layers, the outline of theneurons was intact, the nucleus was full, and the nucleolus was clear. Therewere obvious DND of pyramidal neurons in the model groups, and HDincreased and ND decreased significantly compared with sham group at15d, 30d and45d (P<0.05). There was no significant change of HD and NDamong15d,30d and45d in this model. It was suggested that the DND ofpyramidal neurons was obviously induced by this model, and the neurondamage was no statistical change among the three different time points.②Model2: It was showed that obvious DND of pyramidal neurons were foundin the model groups, and ND decreased significantly compared to sham groupat15d,30d and45d (P<0.05). The HD increased and ND decreasedsignificantly at45d compared with those of15d in this model (P<0.05).③Model3: Obvious destruction of pyramidal neurons was found in the modelgroups at15d,30d and45d, the value of ND was decreased, and HG wasincreased compared with those in the sham group (P<0.05). There was nosignificant change of HD and ND in model group among the three differenttime points.④Model4: The histological features in this model were similarwith those in model3.5.2.2Morphological change of CA1hippocampus at the different modelsin the same timeThere were obvious DND of pyramidal neurons in each model groups, andHD increased and ND decreased significantly compared with sham group(P<0.05), and the neurons damage was no statistical change in each modelgroups at15d and30d. Compared with model1group at45d, the NDdecreased significantly in model2and4groups (P<0.05). The resultsindicated that the neuron damage was more obvious in common carotid arteryocclusion30min model and left common carotid artery occlusion model thanthat of common carotid artery occlusion20min model at45d. Meanwhile,compared to model3group, the ND decreased significantly in model2groupat45d (P<0.05). It was suggested that DND of pyramidal neurons wasobviously induced by the common carotid artery occlusion30min model thanthat of left common carotid artery occlusion model at45d.6Conclusions⑴All four different VD mice models which established by modified2-VO method could decrease the conditioned learning and memory ability and causepyramidal neurons DND in the CA1hippocampus.⑵Model1(bilateral common carotid arteries occlusion20min+tail enddepletion) and model4(right common carotid artery ligation+left commoncarotid artery occlusion) may be reliable for short term observation of activeavoidance response, whereas model2(bilateral common carotid arteriesocclusion30min+tail end depletion) and model3(left common carotidartery ligation+right common carotid artery occlusion) may be reliable forlong term observation of active avoidance response. |
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