Animals

One hundred and four 11-week-old male C57/BL6 mice (Charles River Laboratories, Montreal, QC, Canada) weighing 25 to 28 g at the time of surgery were used in this study. Mice were single-housed in standard Plexiglass cages on a 12/12 h light/dark cycle with ad libitum access to food and water. Animals were allowed to acclimate to the housing facility for 2 weeks prior to surgery. Mice were behaviorally tested in different cohorts of 4 to 32 mice after surgery and/or chronic treatment, and some were perfused for postmortem histology (Table 1). Cohort 1 of PSD mice (n = 4) was used 4 days poststroke for histology. Another group of mice (n = 20) was equally divided into sham control and stroke and used 6-weeks postsurgery for histology studies.

Table 1 Mouse cohorts for behavioral testing Full size table

For the combined treatment study (cohort 2), a group of mice (n = 24) was randomly assigned to a either sham control (n = 12) surgery and [subjected to fixed wheels and sucrose 1% (vehicle)] or a stroke group (n = 12) subjected to voluntary exercise and 18 mg/kg/day FLX hydrochloride (Cedarlane, Hornby, ON, Canada), starting at 1 week postsurgery. Voluntary exercise constituted free access to running wheels in the home cages and the number of wheel rotations/day was monitored. Next, 2 separate cohorts (cohorts 3 and 4) were used for behavioral assays and histology after chronic monotherapy (for 3–4 weeks). These cohorts of mice (n = 32/cohort; 8/group) were randomly divided into 3 stroke groups and 1 sham control group. At 1-week postsurgery, the sham and PSD mice received sucrose 1% and fixed wheels (sham or vehicle groups, respectively), sucrose 1% and running wheels (RW) or FLX (80 mg/l in 1% sucrose, ad libitum to reach 18 mg/kg/day, p.o. [25]) and fixed wheels (FW). FLX was prepared in opaque bottles to protect it from light and liquid consumption was measured every day for the first week; every 2 days in the second week; and thereafter every 3 days to the end of treatment. For vehicle and FLX groups, the mean ± SD liquid consumption was 7.1 ± 0.4 and 6.1 ± 0.7 ml/day, respectively; and weight was 26.5 ± 0.3 and 27.3 ± 0.6 g at sacrifice. The automated wheel revolutions were assessed every 2 days and for the PSD/RW group the total distance was consistent between mice and was 72,660 ± 6280 m/mouse (mean ± SEM; n = 8) for 3 weeks. For the combination treatment group, liquid consumption was 5.8 ± 0.7 ml versus sham ctrl 6.1 ± 0.4 ml; rotations were 68340 ± 2070; and body weight was 25.8 ± 0.7 g versus sham ctrl 27.1 ± 0.4 g. The University of Ottawa Animal Care Committee approved all experimental procedures in accordance with guidelines established by the Canadian Council of Animal Care.

ET-1-Induced PSD Model

PSD was induced as described previously [19] by consecutive microinjections of 1 μl ET-1 (2 μg/μl = 800 pmol/μl) at 2 sites [19]: first, anterior–posterior, 2.0; medial–lateral, +0.5; dorsoventral, −2.4; second, anterior–posterior 1.5; medial–lateral +0.5; dorsoventral −2.6. Sham control mice underwent the same procedure except that the ET-1 was replaced with vehicle (sterile water). The mice were placed in a 37°C incubator to maintain body temperature until they regained mobility and were treated with 0.1 ml 2% transdermal bupivacaine (Chiron, Guelph, ON, Canada) as a topical anesthetic and 2 injections of buprenorphine (0.03 mg/kg, s.c.; Reckitt Benckiser Pharmaceuticals, Richmond, VA, USA) for pain management for 3 h postsurgery. For all cohorts, at 4 days poststroke, brains were visualized using a 7-T GE/Agilent magnetic resonance imager (University of Ottawa Preclinical Imaging Core Facility) in living animals, and the lesion area quantified using ImageJ as previously described [19].

Immunofluorescence

For immunofluorescence studies, mice were euthanized by Euthanyl (149.5 mg/kg i.p.; Bimeda-MTC Health, Cambridge, ON, Canada) and perfused by cardiac puncture with chilled phosphate-buffered saline (PBS) and then with 4% paraformaldehyde for fixation. Whole brains were isolated, cryo-protected overnight in 20% sucrose, and frozen at –80°C. Coronal brain slices (25-μm thickness) were prepared using the coordinates summarized in Table 2 . Slices were thaw-mounted on Superfrost slides (Thermo-Fisher, Waltham, MA, USA) and kept at –80°C. The sections were washed 3 times in PBS, blocked for 1 h in PBS with 1% bovine serum albumin, 10% donkey serum (NDS), 0.1% Triton X-100, followed by 24 h incubation (unless indicated otherwise) at 22°C with the primary antibodies listed in Table 3. The sections were then washed 3 times in PBS and incubated for 1 h in secondary antibodies at 22°C in blocking solution (see Table 3). Images were acquired with Axiovision imaging software on a Zeiss Axio Observer D1 microscope under 10× and 20× magnification (n = 4/group). The number of FosB/4’,6-diamidino-2-phenylindole-(DAPI)positive cells was counted on images taken under 20× magnification of 20-μm slices at coordinates indicated in Table 2 from 4 mice. A coding system was used so that the counter was blind to the treatment. Nuclear FosB+ staining marked by 4’,6-diamidino-2-phenylindole surrounded with GAD67 or CaMKIIα/VGluT3 was counted as FosB/GAD67+ or FosB-CaMKIIα/VGluT3+ cells. FosB+ cells were quantified per area/section and the mean values were then averaged (n = 4). For DR, FosB/tryptophan hydroxylase (TPH)+, FosB/VGluT3+, and FosB/GAD67+ cells were quantified at 4 different levels of middle dorsal raphe (DR)/mouse and averaged (n = 4).

Table 2 Coordinates relative to Bregma of regions assessed by immunofluorescence Full size table

Table 3 Primary and secondary antibodies for immunofluorescence staining Full size table

Behavioral Assays

Behavioral tests were performed following either at 1 week postsurgery or following 3 weeks of treatment (4 weeks poststroke). Mice were housed in 12h/12h light/dark conditions (lights on at 8:00 AM) and tests were performed beginning at 10:00 AM, after at least 1 h of habituation to the testing room. Testing was performed under white-light illumination or red light for the forced swim test (FST). For all cohorts, elevated plus maze (EPM) was done 1 week postsurgery (pretreatment) to verify the poststroke behavioral phenotype. Then, chronic treatment was begun and the mice were kept under treatment until the last test day and perfused. At 3 weeks posttreatment behavioral tests were done in the following order: open field (OF), FST, tail suspension (TS), novelty-suppressed feeding (NSF), and a second EPM test, according to the timeline (Fig. 1). Throughout the testing and behavioral analyses, a coding system was used so that the experimenters were blind to the treatment groups.

Fig. 1 Chronic fluoxetine (FLX), but not exercise, reverses behavioral phenotype in mice with poststroke depression (PSD). Timeline: mice were individually housed 14 days prior to surgery (Housing), microinjected with vehicle (sham ctrl) or endothelin 1 (ET-1; PSD) in the left medial prefrontal cortex (mPFC; surg, day 0); at 4 days poststroke, lesions were verified by magnetic resonance imaging (MRI), and at 7 days anxiety phenotype verified using elevated plus maze (EPM; 1 week); from 7 days onwards treatment was with FLX (PSD/FLX) or free running wheel (PSD/RW), with fixed wheel and vehicle as control (for PSD and sham ctrl) and compared with Sham-ctrl. Behavioral assays [EPM 4 weeks; open field (OF); forced swim test (FST); tail suspension (TS) test; novelty suppressed feeding test (NSF)] were done from days 28 to 40 followed by the Morris water maze (MWM; see Fig. 2) and perfusion (perfuse). (A) ET-1 lesion site visualized in vivo by MRI. Shown is a representative 7-Tesla MRI image done in an anaesthetized, living mouse at 4 days poststroke showing a 300-μm MRI section in which the lesion site is visualized and limited to the left mPFC [left (L); right (R)]. Infarct volumes obtained from in vivo MRI scanning were quantified at 2 different distances from Bregma, and showed low variability (n = 4 per experimental group). (B) EPM 1 week: prior to treatment at 7 days poststroke the 3 randomized stroke groups (PSD) compared with sham control showed anxiety phenotype with significantly reduced open-arm duration in EPM. (C) EPM 4 weeks: PSD reduced open-arm time compared with sham, indicative of an anxiety phenotype; this was reversed by chronic 3-week treatment with FLX (PSD/FLX) but not exercise (PSD/RW). As control for locomotion, total distance travelled did not differ between groups. (D) OF: PSD reduced large center duration and FLX or exercise reversed this anxiety phenotype, with no change in distance travelled. (E) NSF: PSD increased latency to feed in open field, and FLX but not exercise reversed this phenotype. As control for hunger, the latency to feed in home cage did not differ between groups. (F) FST: immobility time in PSD vs sham group was increased indicating behavioral despair, and this was reversed by FLX but not exercise. (G) TS: immobility increase in PSD was reversed by FLX but not exercise. Data represent mean ± SEM, n = 8 per group, *p <0.05, **p <0.01 Full size image

EPM

Mice were placed in the center of an EPM (20 cm high, ~ 6 cm wide, and ~75 cm long; Noldus, Wageningen, the Netherlands) with the head toward the closed arm and allowed to explore the maze for 10 min, videotaped, and the time spent in closed and open arms analyzed (Ethovision 10; Noldus).

OF Test

Mice were placed in a corner of the arena (45 cm long × 45 cm high) for 10 min at light levels of 300 lux, videotaped, and the time spent in the center (24 × 15 cm) and corners (squares with 10-cm sides) analyzed (Ethovision 10; Noldus).

FST

Each mouse was placed into clear plastic cylinder (22 cm diameter × 37 cm deep) filled with 4 l water (24°C), videotaped for 6 min under red lighting, and the duration of immobility time quantified using video-tracking software from Med Associates (Ethovision XT; Noldus).

TS Test

The tail of the mouse was taped to a horizontal bar in a TS box (Med Associates) for 6 min. Immobility duration was measured using an automated detection device (ENV-505TS Load-Cell Amplifier; Med 28 Associates, Fairfax, VT, USA) and quantified using Ethovision XT software. The lower threshold (3) was set and the force recorded below the threshold was considered immobility.

NSF Test

Mice deprived of food for 16 h were individually placed in an arena (45 cm long × 45 cm high; 100 lux) with a food pellet placed in the center. The latency for the mice to begin eating food was recorded manually and immediately after mice chewed the food or after 10 min had expired for the trial, the mice were removed from the arena. The mice were placed in their home cage and the latency to feed and amount of food consumed in 5 min was measured.

Morris Water Maze Test

A cued version of the MWM was used to evaluate spatial learning and memory in PSD and PSD treated mice [26]. The maze used was a circular pool (132 cm diameter) filled with nontoxic, white-colored water (24 ± 1°C). The platform diameter was 10 cm and was located 24 cm from the edge of the pool, hidden 1 cm beneath the surface of the water. Each 1-min trial was started by placing a randomly chosen mouse in 1 of 4 quadrant starting locations in the pool with its head outward. If the mouse found the platform within 60 s, it was left to stay on the platform for 5 s; if it did not find the platform, it was gently guided to the platform by the experimenter. Between the trials, all mice were placed back in their home cages while tucked in a towel in order to avoid direct contact with the experimenter. All trials were tracked using an overhead video camera and recorded automatically by an Ethovision digital tracking system (Noldus) to assess path line and latency to escape from the water. Four trials daily were conducted on each mouse with a 30-min intertrial interval. Trials ran for 10 days, at which time the sham control group consistently reached the platform. On the next day (probe day), the platform was removed and the amount of time the mouse spent in the quadrant where the platform used to be was measured.

Statistical Analyses

All analyses were done using SPSS (IBM, Armonk, NY, USA) and GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, CA, USA). Data are expressed as mean ± SEM. Data comparing the sham versus stroke mice or vehicle versus single treatment on one outcome measure were analyzed using an unpaired t test. Data comparing the immunopositive cell counts within 1 area were also analyzed using an unpaired t test. Statistical analyses were performed using 1-way analysis of variance when comparing data across different conditions. Post-hoc comparisons were made with Tukey’s multiple comparisons test.