Animals

A total of 142 male BALB/c mice weighing 22–30 g were obtained from the National Animal Center (Taipei, Taiwan) and maintained in the animal center of Chi Mei Medical Center (Tainan, Taiwan). The animals were housed 3–4 mice per cage on a 12/12-hr light/dark cycle with ad libitum access to food and water except during behavioral tests. Mice were introduced to the experiment room at least 1 h before the behavioral tests. This project was approved by the Institutional Animal Care and Use Committee of Chi Mei Medical Center (No. 105111531). All of the animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Treatment schedule

This study was conducted according to the experimental protocols described in Fig. 5.

Figure 5 Timeline of experimental procedures. Full size image

To investigate the effect of telmisartan on depression-like behavior in UCMS mice, mice were randomly divided into five groups (n = 8): control group, UCMS model group, UCMS + telmisartan (1 mg.kg−1) group26, UCMS + losartan (1 mg.kg−1) group, UCMS + fluoxetine (20 mg.kg−1) group46.

To investigate the role of telmisartan in PPARδ antagonist treated UCMS mice, mice were randomly divided into six groups (n = 8): control group, control group + GSK0660 (10 mg.kg−1), UCMS model group, UCMS + telmisartan (1 mg.kg−1) group, UCMS + GSK0660 (10 mg.kg−1) + telmisartan (1 mg.kg−1) group, and UCMS + GSK0660 (10 mg.kg−1).

To investigate the role of the role of telmisartan in PPARδ konckdown mice mice were randomly divided into 10 groups (n = 8): control, control + scramble, control + scramble + telmisartan, control + PPARδ shRNA, control + PPARδ shRNA + telmisartan, UCMS, UCMS + scramble, UCMS + scramble + telmisartan, UCMS + PPARδ shRNA, UCMS + PPARδ shRNA + telmisartan.

After an adaptation period (week 0 to week 1), mice were exposed to UCMS for 5 weeks (week 1 to week 6). Telmisartan, losartan and fluoxetine was administered 30 min by intragastric gavage before behavioral test and/or chronically 30 min before UCMS procedure to stressed as well as to unstressed control mice; while GSK0660 was administered by intraperitoneal injection, 30 min before drug treatment. All the animals were treated with respective drugs from week 1 to week 6. Behavioral testing was done in independent groups of mice in the week 7; all mice were subjected to one test daily, always in the same sequence. Blood pressure was measured every week during the experiment. Finally, all mice were sacrificed by cervical dislocation, and each hippocampus was removed, immediately frozen in liquid nitrogen, and kept at −80 °C for protein assays.

The establishment of the depression-like mouse model

The UCMS model was used to explore depressive-like behaviors in mice as described previously47,48. Experimental mice (n = 8 per group) were exposed to unpredictable mild stressors randomly every day in one week. The stressors applied included the following: water deprivation (24 h), food deprivation (24 h), reversed light/dark cycle (24 h), overnight illumination (12 h), soiled cage (12 h), and cage tilt (18 h, 45°). Each stressor was randomly assigned two or three times over a 5-week period. Stressors continued to be applied during the testing phase, except on testing days to avoid effects of acute stress. The non-stressed control mice were housed in groups (3–4 per cage), and the stressed mice were singly housed49. At least 12 h of rest was provided between a stressor and a test50. All of the procedures were organized in a random in order to ensure the unpredictable characteristic of the experiment.

Behavioral testing

OPT

Mice were placed in an open field area made of a 70 × 70 × 40 cm wooden box and equipped with an infrared floor to measure locomotor activity. The arena was subdivided into a central and a peripheral zone. Mice were placed in the open field boxes for 5 min under normal light conditions, and the locomotor activity of mice was automatically scored with a camera connected to a computerized system (Viewpoint, Lyon, France). Individual animals were gently placed in the same corner of the apparatus in all trials. Time stay in central, rearing (number of times the mice stood on their hind legs), grooming (total seconds of the mice spent licking or scratching itself) and excretion were observed51.

SPT

SPT is widely used to measure the anhedonic response, which was defined as a reduction in sucrose preference relative to baseline levels52,53. The mice were exposed to bottles, the one containing 1% sucrose and the other containing tap water for 24 h. After the deprivation of food and water overnight24, mice were used to receive the bottle of 1% (w/v) sucrose or the bottle of tap water for 1 hour. Then, the sucrose preference was evaluated according to the formula: sucrose preference = [sucrose intake/(sucrose intake + water intake)] × 100, as described previously53.

Blood pressure measurement

To investigate the possible effect of telmisartan on blood pressure in depression-like mice, the systolic blood pressures were measured in mice received telmisartan treatment and others using the tail-cuff method by a sphygmomanometer without animal heating (Muromachi Kikai Co., Ltd., Tokyo, Japan). The blood pressure of mice under anesthesia was measured at 15-min intervals. Each value was calculated as the average of 3 measurements.

Intracerebroventricular (ICV) injection

Mice were held in a towel with the dummy cannula to inject the testing agent as described previously54. Mice were anesthetized with a mixture of isoflurane in oxygen (2%) and placed in a Kopf stereotaxic instrument equipped with blunt ear bars. A dummy cannula was placed into the guide55. Mice were allowed to recover for 7 days.

The infusion cannula (22 gauge), attached to PE-10 tubing, was inserted into the guide cannula and extended 0.5 mm beyond the guide. A 10.0-μl Hamilton syringe was used to manually deliver saline or drugs over a two-minute period56. The infusion cannula was kept in place for an additional 1 min following infusion.

Moreover, the solution containing shRNA specific to PPARδ (Gene ID 25682) with an expression vector (pCMV6-Entry) was administered via ICV injection into mice using 25 μl of the prepared solution (0.12 μg.μl−1), while mice receiving a similar injection of an empty vector at the same volume were used as a control.

Cell Cultures

Rat-derived hippocampus H19-7 cell line cells (CRL-2526; American Type Culture Collection, Manassas, VA) were maintained at 37 °C and 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, South Logan, UT, USA) with 4 mM l-glutamine that was adjusted with sodium bicarbonate (1.5 g/L), glucose (4.5 g/L), G418 (200 μg/mL), and puromycin (1 μg/mL) and supplemented with 10% fetal bovine serum57. Cells (1 × 106) were plated on 60-mm culture dishes, and at 80% confluence, they were differentiated by culturing for 6–7 days in DMEM containing 2% fetal bovine serum. Medium was changed every other day.

Western Blotting Analysis

Western blotting analysis was performed as previous58. Total protein lysates from mouse hippocampus or cells were extracted in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris [pH 7.5] and 5 mM ethylenediaminetetraacetic acid), containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich, MO, USA). The protein concentration was determined with the BCA assay kit (Pierce Biotechnology, Rockford, IL, USA). The following primary antibodies were used at 4 °C overnight: anti-PPARδ (1:1000) (Abcam, Cambridge, UK); anti-5-HTT (1:1000) (Merck Millipore, Darmstadt, Germany); anti-β actin (1:5000) (Merck Millipore) was used as an internal control. The next day, the blots were incubated with a 1/5000 dilution of horseradish peroxidase-conjugated secondary antibodies at 25 °C for 1 h. Protein bands were visualized using the enhanced chemiluminescence kit (PerkinElmer, Boston, MA, USA). The optical densities of the bands were determined using software (Gel-Pro Analyzer version 4.0 software; Media Cybernetics Inc., Silver Spring, MD, USA).

Statistical analysis

Data were expressed as the mean-standard error of the mean. Statistical analyses one-way ANOVA was used to investigate the differences between groups with pharmacological treatments. Among multiple groups were analyzed by two-way ANOVA with “stress” and “drugs” are the factors to evaluate data in the knockdown experiments. If an interaction and/or main effect were observed, pairwise comparisons following ANOVA were made using Bonferroni post-hoc test. Data sets of two sample groups were analyzed using independent Student’s t-tests. All analyses were carried out by SPSS, version 21. Statistical differences were accepted at p < 0.05.