4.1
Mice body weight
Body weight of mice in the experiment 1 was shown in Figure 1. Through the experimental duration of the experiment 1, although there were differences between total 750 μg DEPs group and control group, the differences were below 10%.
Body weight of mice in the experiment 2 was shown in Figure 2. There were no difference between total 300 μg DEPs group, total 100 μg DEPs group and control group through the experimental duration.
4.2
Acquisition phase of Morris water maze test
In the experiment 1, escape latency, distance moved, cumulative distance and swimming velocity in acquisition phase were shown in Table 4 and Figure 3 to 4.
The median escape latency in total 750 μg DEPs group was longer than that in the control group in the fourth day of acquisition phase which was close to significance (45.1 seconds vs. 17.7 seconds, P=0.059). Median distance mice moved in total 750 μg DEPs group was also longer than that in the control group in the fourth day of
acquisition phase (464 cm vs. 325 cm, P=0.156). Median cumulative distance from the center of platform quadrant was significant longer in total 750 μg DEPs group than in control group in the fourth day of acquisition phase (11.5 m vs. 4.02 m, P<0.05). These differences were under no clear difference in median swimming velocity of mice between total 750 μg DEPs group and the control group (10.4 cm/s vs. 16.6 cm/s, P=0.227).
In the experiment 2, escape latency, distance moved, cumulative distance and
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swimming velocity in acquisition phase were shown in Table 5 and Figure 5 to 6.
The median escape latency in total 300 μg DEPs group was longer than that in total 100 μg DEPs group and control group in the fourth day of acquisition phase but without significance (40.7 seconds, 31.8 seconds vs. 30.4 seconds, P=0.550). Median distance mice moved in total 300 μg DEPs group was also longer than that in total 100 μg DEPs group and control group in the fourth day of acquisition phase (370 cm, 268 cm vs. 304 cm, P=0.088). Median cumulative distance from the center of platform in total 300 μg DEPs group was also longer than that in total 100 μg DEPs group and control group in the fourth day of acquisition phase (9.05 m, 6.88 m vs. 5.94 m, P=0.600). These were under no clear difference in median swimming velocity of mice between total 300 μg DEPs group, total 100 μg DEPs group and control group (11.5 cm/s, 9.16 cm/s vs. 12.2 cm/s, P=0.361).
We observed that swimming velocity in near half of mice was below 10 cm/s in the experiment 2 in all groups. Mice with swimming velocity below 10 cm/s may consider as failure in the Morris water maze test[75]. We then excluded mice with mean
swimming velocity below 10 cm/s in acquisition phase and the results were presented in Table 6 and Figure 7 to 8.
The exclusion revealed significant differences in the escape latency and cumulative distance. The median escape latency in total 300 μg DEPs group was significantly longer than that in total 100 μg DEPs group and control group in the fourth day of acquisition phase (35.4 seconds, 23.2 seconds vs. 14.2 seconds, P<0.05). Post hoc test showed that the significant difference was between total 300 μg DEPs group and control group. Median distance mice moved in total 300 μg DEPs group was still longer than that in total 100 μg DEPs group and control group in the fourth day of acquisition phase (440 cm, 237 cm vs. 252 cm, P=0.081). Median cumulative distance from the center of
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platform in total 300 μg DEPs group was also significant longer than that in total 100 μg DEPs group and control group in the fourth day of acquisition phase (8.64 m, 4.29 m vs.
2.18 m, P<0.05). Swimming velocity was still comparable with higher mean and median after exclusion in the fourth day of acquisition phase (13.2 cm/s, 10.9 cm/s vs.
15.6 cm/s, P=0.201).
4.3
Probe test of Morris water maze test
In the experiment 1, percentage of time spent in target area, area crossing, average proximity and swimming velocity in probe test were shown in Table 7 and Figure 9 to 10.
The median percentage of time spent in platform quadrant for total 750 μg DEPs group was 25.4%. The median percentage of that for the control group was 26.1%.
(P=0.958). The difference was not obvious. Median times mice crossed the quadrant area previously with platform in total 750 μg DEPs group was slightly lower than that in the control group (3.25 times vs. 4.88 times, P=0.343). Median average distance from the center of platform quadrant (average proximity) was almost the same in total 750 μg DEPs group and control group (41.8 cm vs. 43.5 cm, P=0.564).The median swimming velocity of total 750 μg DEPs group and control group were comparable in probe test (12.4 cm/s vs. 15.0 cm/s, P=0.189).
In the experiment 2, percentage of time spent in target area, area crossing, average proximity and swimming velocity in probe test were shown in Table 8 and Figure 11 to 12.
The median percentage of time spent in platform quadrant for total 300 and 100 μg DEPs group were 40.9% and 31.4% and the median percentage of that for control group was 24.2% (P=0.437). The median percentage of time spent in platform area (platform
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area plus 5 cm around the platform) for total 300 μg DEPs group, total 100 μg DEPs group and control group were also showed no difference (3.22%, 1.52% vs. 2.03%, P=0.724). Median times mice crossed the quadrant previously with platform (quadrant area crossing) and the area previously around platform (platform area plus 5 cm around the platform, platform area crossing) were not significantly different between total 300 μg DEPs group, total 100 μg DEPs group and control group (3.00 times, 2.63 times vs.
3.00 times, P=0.981 and 2.25 times, 0.88 times vs. 1.00 times, P=0.520). Median average distance from the center of platform (average proximity) showed no difference in total 300 μg DEPs group, total 100 μg DEPs group and control group (36.7 cm, 39.0 cm vs. 41.9 cm, P=0.652). The median swimming velocity of total 300 μg DEPs group, total 100 μg DEPs group and control group were comparable in probe test (10.4 cm/s, 8.82 cm/s vs. 10.0 cm/s, P=0.839)..
As mentioned above, we also tried to exclude mice with swimming velocity below 10 cm/s in probe test and the results were presented in Table 9 and Figure 13 to 14.
However, most results showed similar pattern after exclusion except slight difference in median times mice crossed the area previously around platform (platform area plus 5 cm around the platform, platform area crossing) with P=0.098 (total 300 μg DEPs group: 4.13 times, total 100 μg DEPs group: 5.00 times and control group: 3.50 times).
4.4
Histopathology
In the experiment 2, we performed a histopathological examination in multiple brain regions, including olfactory bulb, frontal cortex, hippocampus and cerebellum, and the results were shown in Figure 15 to 20. However, we found no significant
difference in these regions. Each mice in total 300 μg DEPs group, in total 100 μg DEPs group and in control group were within normal limit.
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