Behavior test
Behavior test included open filed test and recognition memory test. First day, the experimental apparatus of open filed test was a black open-field box (100cm ×100cm×50cm), positioned in a dimly illuminated room. Each rat were allowed to freely explore 10 min. During the test session, moving distance, time in center area and number of times to enter center area were measured automatically by a computer-based system[24].
Second day, each rat was placed in the black open-field box containing two identical objects and allowed to explore two identical objects (A1 and A2) for 10 minutes. The object with most object interactions were defined as similar object. After 5 minutes or 24h, similar object was replaced with a novel object and the rat were allowed to explore for 10 minutes. A discrimination index was calculated as the difference in number exploring the novel and familiar object, expressed as the ratio of the total number spent exploring both objects. (i.e., [Number Novel / Number Novel + Number Familiar] × 100%). In addition, all object combinations and locations were used in a balanced manner to reduce potential bias due to preferences for specific locations or objects[25].
Transmission Electron Microscopy
Transmission Electron microscopy was performed to observed synaptic ultrastructure in the hippocampus[26]. The sample (hippocampal rat brain tissue) were blocked with fresh TEM fixative for 2 hours and were dehydrated in graded ethanol solutions (30% to 100%). Brain tissues were stained with resin penetration and embedding as well as keep in 37℃oven overnight, then moved into 65℃ oven to polymerize for 2 day. Images were captured with a transmission electron microscope (Hitachi, Japan).
Golgi Staining
Golgi staining was performed as previously described. Briefly, the fresh whole brain was obtained and immediately fixed with 4% paraformaldehyde for 48h. Then, the sample was placed in to a 45ml EP tube containing Golgi-cox staining solution for 14 days. After treated with distilled water, 80% glacial acetic acid and 30% sucrose, respectively, the tissue were cut into 100 microns and dry in the dark overnight. The sections were slides with concentrated ammonia water and hardening fixing solution for 15 min, then washing with distilled water for 3 min, dry and seal the section with glycerin gelatin. Images of brain tissue by panoramic scanning with digital slice scanner[27].
Statistics
Data in this experiment are presented as the mean ± SEM. Data sets were compared with two-way ANOVA followed by Tukey’s post hoc analysis. Post-hoc analyses were performed only when ANOVA yielded a significant main effect or a significant interaction between the two factors. Results were considered to be significant at p < 0.05.
Results
Neurobehavioral assessment of VD model
A neurological evaluation was performed 7 day after 2-VO and scored on a 5-point scale. As shown in Fig. 2, rats in the control group had no neurological deficit. The results also showed that: the grade score were significantly higher in the VD group when compared with C group (P<0.001; Fig. 2). In addition, compared with the VD group, the rats in Exe-VD group significantly decrease the grade score (P<0.05; Fig. 2). Thus, we concluded that vascular dementia was successfully developed in our experiment undergoing the 2-VO procedure.