Fig. 1 Timeline in this experiment.
Treadmill exercise
Treadmill exercise was performed according to previous
literature[19]. Briefly, a treadmill exercise
apparatus for rat as used for chronic physical exercise. During the test
session, the rat were allocated in the test room for 30 min acclimation,
followed by normal exercise intervention. The speed of the treadmill
exercise was 15 m/min, and the animal performed exercise 30 min daily
for 4 weeks (5 days/week).
Repeated restraint stress protocol
Repeated restraint stress model was generated by physical restrain as
previous described[20]. The rat was restrained for
6 h daily (15:00–21:00) in a wire mesh restrainer for 21 days. After
restrain treatment, the rat were returned to their own cages. All the
procedures were performed after physical exercise.
Behavioral procedures
On the test day, rats were transferred to the testing room and
acclimated to the room conditions for at least 1 h. After each
individual test session, the apparatus was thoroughly cleaned with 70%
alcohol to eliminate the previously tested rat’s odor and trace.
Open field test was to evaluated anxiety-like
behavior[21]. Open field experiment box (100 cm ×
100 cm ×50 cm) were made out of plexiglass. Each animal was placed in
the open field box for freely exploring the arena during a single 5 min
session. During the test session, movement paths in open field
experiment box were measured automatically by digital camera.
Passive avoidance test is a common measure of learning
memory[22]. The apparatus consisted of a light
compartment and a dark compartment. In
acquisition trial, rats we placed
them in the light compartment and allowed them to freely explore it. As
expected, due to their natural aversion to the light and preference for
the dark, rats very quickly entered the dark chamber. The time of rat
from the light chamber to enter the dark compartment was noted. Upon
entry into the dark chamber, the guillotine door was closed. Then, rats
received a single mild electric footshock (0.5mA; 1s) for 30s and then
were returned to their homecage. 24 h later, we subjected rats to an
retention trial. The procedure was
similar to the acquisition trial except this time rats did not receive
the electrical foot shock, and we recorded the time to enter the dark
compartment (up to 300 s).
Immunofluorescence
Animals were sacrificed and perfused with ice-cold PBS, followed by
perfusion with 4% paraformaldehyde. The brain were removed rapidly,
immersed in a tube containing 4% paraformaldehyde for 12 hours at 4℃
and cryoprotected in PBS containing 20% and 30% sucrose, respectively.
They were then frozen on powdered dry ice and stored at - 80℃ until they
were processed. Serial coronal sections (40 μm) were cut through the
hippocampus on a cryostat. Brain sections were incubated at 37˚C for 30
min and rinsed in PBS for 10 min, incubated in 0.3% Triton X-100 for 10
min and 10% normal donkey serum for 2 hour. Then subsequently incubated
overnight at 4℃ with primary antibody, including rabbit monoclonal to
NeuN- neuronal marker. Next day, they were incubated in second antibody
(Goat Anti-Rabbit IgG) for 2 hour after washed by PBS. After rinsing
three times with PBS, the brain slices were attached to coated slide
glasses and evaluated for confocal fluorescence
microscopy[23].
Golgi staining
For the quantification of hippocampal morphological changes, Golgi
staining kit (FD Rapid Golgi Stain Kit PK-401, USA)was used to analyze
neuronal spine density following the manufacturer’s protocols. Briefly,
the whole brains were extracted and placed in in EP tube with Golgi-Cox
solution for 17 days in the dark. Then the brains were subsequently
frozen and coronal sections (60 µm) were obtained using a cryostat
(Leica, Nussloch, Germany). And the sections were incubated in the
staining solution for 15 min, dehydrated in ethanol solution (30%,
50%, 80%, and 100%). Finally, slides were cover-slipped with
Permount. Images of hippocampal pyramidal cells were obtained by
panoramic multi-layer scanning with digital slice scanner at a
magnification. To calculate the number of spine, a length of dendrite
(at least ≥10 µm long, 2nd-oder dendrites) was traced (at 1000×).
ELISA
The hippocampal brain tissue was lysed in RIPA buffer (Beyotime,
Shanghai, China) and processed in ultrasonication and was centrifuged
for 10 min to collect the supernatant. Hippocampal dopamine were
detected after purification using ELISA kits (Nanjingjiancheng, China).
Briefly, the wells of a 48-well flat-bottom high-affinity ELISA plates
were coated overnight and add prepared sample and standards.
Subsequently, the plate was washed five times in each well, and the
plate was incubated for 20 min in the dark at room temperature.
Finally,
the reaction was stopped and measure the OD values within 10 minutes.
Statistics
All data in this experiment are presented as the mean ± SEM. Data
analysis was performed by GraphPad Prism software. Data analysis was
performed by GraphPad Prism software. Student’s t-test was used to
compare means between two groups. A statistical significant level was
defined when P < 0.05.
Results
Stress-induced anxiety behavior can be rescued by treadmill
exercise
The open-field test showed that stress rat displayed decreased central
exploration time and central distance relative to the control group
(Fig. 2 A-B; p < 0.01, p < 0.001). Locomotor
activity, measured by total distance traveled, did not differ between
the stress and control groups (Fig. 2 C; p > 0.05). Then we
tested whether treadmill exercise could ameliorate stress related
behavior abnormality. The results demonstrated that significantly
elevated central exploration time and central distance in Exe-stress
group when compared with stress group (Fig. 2 A-B; p < 0.01, p
< 0.001). and there were no significantly different in total
distance between the stress and control groups (Fig. 2 C; p
> 0.05). These data demonstrate that stress rat develop an
anxiety-like phenotype after repeated restraint stress exposure, which
can be rescued by treadmill exercise.
Fig. 2. Treadmill exercise alleviated stress-induced anxiety behavior.###p<0.001 vs. control;***p<0.001 vs. stress.
Stress-induced working memory impairment was ameliorated by
treadmill exercise
Fig. 3 shows that the latency time of passive avoidance test in each
group. In the acquisition trail, there are no significant difference for
the four groups (Fig. 3, p > 0.05). In the retention trail:
when compare to C group, the time in stress group was significantly
shorter (Fig. 3, p < 0.001). As compare to stress group, the
time in Exe-stress groups were significantly longer (Fig. 3, p
< 0.001). The results indicated that stress-induced working
memory impairment was ameliorated by treadmill exercise.
Fig. 3. Treadmill exercise alleviated stress-induced working memory
impairment. ###p<0.001 vs. control;***p<0.001 vs. stress.
Treadmill exercise prevented stress-induced neuron loss
In order to examine the effect of exercise on hippocampal neuron in
stress rat, staining with NeuN was used to detect hippocampal neuronal
viability in each group (Fig. 4A). The number of NeuN cells in the
stress group was significantly increased compared with that observed in
the control group (Fig. 4B, p <0.01). But a greater
NeuN-positive cells was observed in the hippocampus after treadmill
exercise intervention (Fig. 4B, p <0.05). These results
suggest that stress-induced hippocampal neuron loss can be rescued by
treadmill exercise.