healthmedicinet

Animals and treatments

Adult 13-week-old Sprague–Dawley (SD) male rats weighing 230–250 g were purchased
from the Experimental Animal Center of Chongqing Medical University. The Laboratory
Animal Management Committee of Chongqing Medical University authorized the experimental
protocol. All animal procedures were carried out according to the guidelines of the
China Animal Protection Law and were approved of by the Institutional Ethics Committee
of Chongqing Medical University [Permit No. SCXK (Chongqing) 2007–0001] and the State
Science and Technology Commission of China.

The rats were group housed (five per cage) in quiet in a room that was maintained
at 21–22 °C with 50 % relative humidity, with 12/12-h light/dark cycles. They were
allowed free access to food and water and a 1-week adaptive feed before the baseline
SPT and OFT characteristics were measured. Then, 98 rats with similar baseline characteristics
were selected and randomized into five groups: normal, sham, FCIR, CUMS, and F/C.
Rats in the FCIR group underwent an FCIR operation. The rats in CUMS group were single-housed
and exposed to CUMS for 4 weeks. Rats in F/C group underwent FCIR surgery and were
then single-house while experiencing CUMS for 4 weeks. The model was created successfully
in 75 rats: 10 in the normal group, 10 in the sham group, 18 in the FCIR group, 20
in the CUMS group, and 17 in the F/C group. Eight rats were excluded because of they
had no neurological symptoms according to the mNNS and fifteen rats died during this
research.

Focal cerebral ischemia and reperfusion (FCIR) surgery

Focal cerebral ischemia surgery was performed according to the intraluminal occlusion
technique described previously by Koizumi et al. 6]. Briefly, rats were anesthetized using 3.5 % chloral hydrate (350 mg/kg). Then, the
right common carotid artery (CCA), the right external carotid artery (ECA), and the
internal carotid artery (ICA) were surgically exposed via a midline incision. The
CCA was ligated distally and the ECA was ligated proximally to the bifurcation of
the ICA and the ECA. A nylon filament (diameter 0.24–0.26 mm) was gently advanced
from the ECA into the lumen of the ICA. After 2 h of ischemia the nylon filament was
carefully removed to establish reperfusion. The body temperature was maintained at
37 °C using a heating pad during the surgery. In sham group, the embolus was inserted
to a distance of 12 mm and was pulled out immediately.

The neurological function of the rats was evaluated on the first day after surgery
using the mNSS. The mNSS is a composite of motor (muscle status, abnormal movement),
sensory (visual, tactile, and proprioceptive), beam balance, and reflex (pinna, corneal
and startle reflexes) tests, and is graded on a scale of 0–18 (normal, 0; maximum
deficit, 18). Two observers blinded to the experimental groupings performed all tests.
The mNSS of all rats was 0 before surgery, and any rats with a score of 0 on the first
day after surgery were excluded from the experiment.

Chronic unpredictable mild stress (CUMS)

The CUMS procedure was performed as described previously 7], 8]. However, some adjustments were made to increase the unpredictability. The rats were
subjected to CUMS for 28 days, including ice water swimming at 4 °C for 5 min, hot
water swimming at 45 °C for 5 min, shaking once per second for 5 min, restricted access
to water for 3-h following 20-h of water deprivation, tail pinch for 1 min, restricted
access to food for 3-h following 20-h of food deprivation, cage tilt at 45° for 24 h,
housing in a wet cage for 24-h (containing 100 g of sawdust in 200 ml water), shocking
the feet at 50 mV (three times/min each for 10 s for 10 min), stroboscopic lighting
for 36-h (300 flashes/min), intermittent white noise (150 dB once per min for 20 s
for a total of 100 min), and an uncomfortable smell (prednisone) for 12 h. Rats were
exposed to one of these 12 stimuli randomly per day, and the same stressor was not
used on consecutive days.

Behavioral tests

The rats were weighed on days 1, 29, and 57 of the study. The SPT and OFT were carried
out in sequence on day 1 to obtain baseline measurements. These tests were repeated
on days 22 (3rd week), 29 (4th week), and 57 (8th week) after surgery.

Sucrose preference test (SPT)

Rats were allowed to choose between two bottles after 20-h of food and water deprivation
(from 12.00 to 8.00): one contained 1 % sucrose water and the other had normal drinking
water. The consumption of water and sucrose solution was estimated by weighing the
bottles after 2 h. The data recorded from two bottles were analyzed for its sugar
consumption ratio as its changes in flavor preference. The sucrose preference (SP) = sucrose
intake water (g)/total intake water (g) × 100 %.

Open field test (OFT)

The experimental device consisted of an open field reaction tank and an automatic
data acquisition and processing system (Shanghai Xin-ruan Information Technology Co.,
China). The tank was sized 100 × 100 × 40 cm size, and the bottom of the box was divided
into 25 equilateral squares sized 4 × 4 cm. A digital camera was placed above the
box that could cover the entire field. The box was cleaned thoroughly before each
animal was tested. The animal was replaced in its home cage immediately after the
test.

The rats was placed in the open field reaction tank for 5 min, and its’ movement recorded
using a digital camera. The number of spontaneous moves (SM), the percentage of duration
time spent in the center square (duration = time spent in the center square (s)/total
time (s) × 100 %), and the number of rearings (rearing) were analyzed automatically
using sports track in XR-Xmaze supermaze software (Shanghai Xin-ruan Information Technology
Co.).

Statistical analysis

Data are expressed as mean ± standard error of the mean (SEM), and statistical differences
between groups were compared using two-way analysis of variance (ANOVA) with time
and groups as between subject factors. A value of P  0.05 was considered statistically
significant. All statistical analyses were performed using SPSS, version 17.0 (Chicago,
IL, USA).