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.