healthmedicinet

Animals

Male Sprague–Dawley rats, weighing 150–180 g (week 8), were initially housed in groups
of three in a temperature-controlled environment under a 12-h light/dark cycle, with
ad libitum access to food and water except during experimental procedures. All animal use procedures
were conducted according to the Regulations of Experimental Animal Administration
issued by the State Committee of Science and Technology of the People’s Republic of
China, with the approval of the Ethics Committee in The Experimental Animal Center
of the Second Xiangya Hospital.

Drug treatment and CUMS procedures

Rats were randomly assigned to six groups (?=?9): (1) control, (2) control?+?FO, (3) control?+?ser, (4) CUMS, (5) CUMS?+?FO,
(6) CUMS?+?ser. Fish oil (FO, EPA34 %, DHA24 %, Sheng Tianyu Biotechnology co., China)
and sertraline (ser, Eastbang Pharmaceuticals, China) were dissolved in sterile saline
containing 0.5 % Tween 21]. We use sertraline as positive control for behavior test of rats. The FO supplementary
groups received daily gavage of 1.5 g/kg fish oil 2 weeks before the CUMS procedures
started, until the end of the experiments. The sertraline groups received daily gavage
of 8 mg/kg sertraline at day 1 when the CUMS procedures started 22], while the control and CUMS groups received same volume of saline containing 0.5 %
Tween.

The CUMS procedures were performed as described previously with minor modifications
12]. This paradigm was designed to maximize unpredictability and mildness of the stress
intensity. The rats of CUMS groups were housed in a separate cage (cage size: 26?×?19?×?15 cm),
while others share one cage (three per cage, cage size: 90?×?45?×?25 cm). After acclimatized
to the laboratories for 5 days, and 2 weeks gavage of fish oil or saline respectively,
the CUMS groups received random unpredictable stress for 5 consecutive weeks (Fig. 1). Stress stimuli included: cage tilting for 24 h; damp bedding for 24 h; fasting
for 24 h; water deprivation for 24 h, finally with 1 h an empty bottle; light–dark-cycle
reversal (12 h/12 h), behavior restriction for 2 h; 30 min noise, 5 min tail pinch.
Rats received one of these stressors per day and same stressor was not applied in
2 consecutive days. The rats were weighed every 3 days and the dose was adjusted to
its weight gain. All animals were subjected to three behavioral tests to confirm induction
of depression-like behaviors in CUMS rats as described below.

Fig. 1. Timeline of experimental procedures

Sucrose preference test

Anhedonia, which is a central feature of depression, was defined as a reduction in
sucrose preference 23]. Briefly, rats were placed in individual cages for habituation to the sucrose solution
(1 %, w/v): two bottles of 1 % sucrose solution were placed in the cage for the first 32 h,
then after 16 h water deprivation, one bottle of 1 % sucrose solution was replaced
with water for 1 h. The rats were permitted free access to the two bottles. The volumes
of consumed sucrose solution and water were recorded. Completing this period, the
liquid consumption was measured and the sucrose liquid drinking volume, as a percentage
of the total liquid consumption, was calculated.

Open field test

For the open field test, spontaneous locomotor activity was measured as described
previously 22], with minor modifications. Reduced number of locomotor crossing and rearing reflected
the loss of interest, a hallmark of depression, in rodents 24]. Briefly, the test chamber consisted of a square arena (90 cm?×?90 cm?×?40 cm). The
floor was divided into 25 equal squares by black lines. At the beginning of the test,
the animal was placed in the center of the arena and allowed to freely explore for
5 min. The apparatus was cleaned with 75 % ethanol after each testing session to prevent
any odors deposited by the rats from influencing the following rat. The horizontal
locomotor activities (segments crossed with all four paws) and vertical exploratory
activities (standing on their hind paws) were scored.

Forced swimming test

FST is widely used for measurement of depression-like behavior in rodents. Briefly,
each rat was placed in a plastic drum (45 cm height, 25 cm diameter) containing approximately
35 cm of water (24?±?1 °C) for a 15-min pretest. Twenty-four hours later, the rat
was exposed to the same experimental conditions outlined above for a 5-min FST. Each
test session was videotaped and the duration of immobility, which defined as floating
passively and only making slight movements to keep the head above water, was scored
by an experienced observer blind to the experiment design.

Serum parameters

Upon completion of the experiments, all rats were weighed and blood was collected
from the anesthetized animals into blood collection tubes after a 12-h overnight fast.
After standing for 30 min, the serum was prepared by centrifugation of blood at 1000?×?g
for 10 min at 4 °C and stored at ?80 °C until analysis. Serum levels of triglyceride
(TG), total cholesterol (TCH), high density lipoprotein-cholesterol (HDL-c), low density
lipoprotein-cholesterol (LDL-c), free fatty acid (FFA) and glucose (GLU) were measured
by means of enzymatic methods, using assay kits (Sekisui Medic; Abbott Laboratories
or Beijing leadman biochemistry co., LTD). Enzyme-linked immunosorbent assays were
used to measure the serum levels of leptin (RD systems, USA), ghrelin (RayBiotech,
USA) and adiponectin (CUSABIO Biotech, Ltd).

Statistical analysis

Results were expressed as mean?±?SEM. Statistical analyses were performed with one-way
analysis of variance (ANOVA) for multiple comparisons followed by TUKEY for post-hoc
test. The prior level of significance was established at P??0.05.