Ethics statement
Experiments were performed in accordance to National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and approved by local government authorities (Istanbul Medipol University, Animal Research Ethics Committee). All animals were maintained under a constant 12-h light/dark cycle (lights on at 07:00 daily). Investigators were blinded for experimental groups at all stages of experiments and data analysis.
Experimental design and groups
The PDE10A inhibitor TAK-063 was purchased from MedKoo Biosciences (510331, North Carolina, USA) and suspended in a 1% (v/v) solution of dimethyl sulfoxide (DMSO) in tap water. Adult male C57BL/6j mice weighing 21–26 g were randomly assigned to one of three groups and administered with oral delivery of i) vehicle (100 µl water containing 1% DMSO), ii) 0.3 mg/kg TAK-063 (dissolved in 100 µl water containing 1% DMSO) or iii) 3 mg/kg TAK-063 (dissolved in 100 µl water containing 1% DMSO) at the reperfusion onset. In the first set; vehicle, low dose or high dose of TAK-063 was orally administered without any injury induction (n=4/ group). One hour after oral application of PDE10A inhibitor or vehicle, Western blotting analysis was carried out to assess the efficiency of PDE10A inhibition. In the second set, mice were subjected to 90 min of MCAO followed by 24 h reperfusion for the evaluation of neurological score, brain infarct volume, cerebral edema and serum IgG extravasation (n=7-8 per group). The third set of mice was subjected to 90 min of MCAO followed by 24 h reperfusion for the analysis of cerebral microcirculation by LSI (n=4-5 per group). The fourth set of mice was subjected to 30 min of MCAO and 72 h reperfusion for the analysis of disseminate neuronal injury in the striatum, cytokine/chemokine expression profiles, intracellular signal pathway analysis, and proteomic analysis (n=7-8 per group).
Middle cerebral artery occlusion (MCAO)
Mice were anesthetized with 1% isoflurane (30% O2, reminder N2O), and rectal temperature was controlled between 36.5 and 37.0 °C using a feedback-controlled heating system. During the experiments, cerebral blood flow (CBF) was monitored via laser Doppler flowmetry (LDF) using a flexible 0.5 mm fiber optic probe (Perimed, Sweden) which was attached with tissue adhesive to the intact skull overlying the MCA territory (2 mm posterior and 6 mm lateral from the bregma). Focal cerebral ischemia was induced using an intraluminal filament technique [11]. Briefly, after a midline neck incision, the left common and external carotid arteries were isolated and ligated. A microvascular clip (FE691; Aesculap, Tuttlingen, Germany) was temporarily placed on the internal carotid artery. A 7-0 silicon-coated nylon monofilament (701934PK5Re, Doccol, Massachusetts, USA) was inserted through a small incision into the common carotid artery and advanced 9 mm distal to the carotid bifurcation for MCAO. Reperfusion was initiated 30 or 90 min after onset of ischemia by gentle monofilament removal. Thereafter, mice were placed back into their home cages.
In mice subjected to 90 min MCAO, neurological deficits were evaluated 24 h after MCAO using the following 5-point scoring: 0 = normal function, 1 = flexion of torso and of the contralateral forelimb upon lifting of the animal by the tail, 2 = circling to the contralateral side but normal posture at rest, 3 = reclination to the contralateral side at rest, and 4 = absence of spontaneous motor activity. At 72 h (for 30 min MCAO) or 24 h (for 90 min MCAO) after reperfusion, mice were sacrificed under deep anesthesia (4% isoflurane with 30% O2, remainder N2O). Brains were removed, frozen on dry ice, and cut on a cryostat into coronal 18-μm sections, which were subsequently used for the analysis of disseminate neuronal injury, infarct volume and brain swelling and serum IgG extravasation. For 30 min MCAO, tissue samples obtained from the ipsilateral to the stroke were pooled for Western blots.
Analysis of infarct volume and brain swelling
For the evaluation of infarct volume and brain swelling, coronal brain sections were collected at four equidistant brain levels, 2 mm apart, from mice subjected to 90 min MCAO, which were stained with cresyl violet according to a standard protocol [19]. Within the sections, the border between infarcted and non-infarcted tissues was outlined using an image analysis system (Image J; National Institute of Health, Maryland, USA), and the infarct area was assessed by subtracting the area of the non-infarcted ipsilateral hemisphere from that of the contralateral side. Infarct volume was calculated by integration of these infarct areas. Edema was calculated as the volume difference between the ischemic and the non-ischemic hemisphere and expressed as mm3.
Analysis of serum IgG extravasation
With gentle stirring, brain sections from the bregma 0.0 mm level of mice subjected to 30 min MCAO followed by 72 h reperfusion were rinsed for 10 min at room temperature in 0.1 M PBS to remove intravascular IgG, and were fixed in 4% PFA [7]. Following the blocking of endogenous peroxidase with methanol/ 0.3% H2O2 and immersion in 0.1 M PBS containing 5% bovine serum albumin (BSA) and normal swine serum (1:1000), sections were incubated for 1 h in biotinylated horse anti-mouse IgG (BA-1300-2.2, Vectastain Elite; Vector Labs, California, USA), and stained with an avidin peroxidase kit (PK-7800; Vectastain Elite; Vector Labs) and DAB Substrate Kit (SK-4100; Vector Labs). For reasons of data comparability, all sections were processed in parallel. Sections were scanned and the integrated density of ipsilesional and contralesional hemisphere was quantified using he NIH ImageJ software. Afterward, density of ipsilesional tissue compared to contralesional tissue to determine BBB leakage. [19].
Laser Speckle Imaging (LSI)
To analyze the effect of low and high dose of TAK-063 on cerebral microcirculation, LSI was performed as described previously [20,19]. Briefly, C57BL/6 mice were subjected to 90 min MCAO as described above followed by the oral delivery of vehicle, low dose TAK-063 or high dose TAK-063 at the onset of reperfusion. Thereafter, cerebral microcirculation was recorded by Pericam PSI System (Perimed) for 90 min. To analyze CBF changes in the ischemic core and periphery, regions of interest (ROI) covering 1.0 mm x 5.5 mm (in lateral and rostrocaudal direction, respectively) were defined 1.5 and 2.5 mm lateral and 0.5 mm posterior to the bregma, in which mean CBF was calculated using a blood perfusion imaging software (PIMSoft; Perimed) [19]. Regional CBF was recorded throughout the 90 min observation period. From the measurements obtained, relative CBF changes (in %) at the end of the observation period compared to the beginning of the observation period were calculated.
Analysis of neuronal survival
Neuronal survival was evaluated as previously described [21]. Coronal brain sections were fixed in 4% paraformaldehyde (PFA)/ 0.1 M phosphate buffered saline (PBS), washed and immersed for 1 h in 0.1 M PBS containing 0.3% Triton X-100 (PBS-T)/ 10% normal goat serum. Sections were incubated overnight at 4 ºC with Cy3-conjugated monoclonal mouse anti-NeuN (MAB377C3; Merck, New Jersey, USA). The next day, sections were incubated with 4’,6-diamidino-2-phenylindole (DAPI). Sections were analyzed using a confocal laser scanning Zeiss LSM 780 microscope (Carl Zeiss, Jena, Germany). Nine different ROI in the striatum, each measuring 62,500 µm2, were evaluated. Mean numbers of NeuN cells were calculated in the ischemic and contralesional striatum. By dividing the results obtained from ischemic striatum by the results of non-ischemic striatum and multiplying by 100, the percentage of surviving neurons in the ischemic striatum was determined.
Analysis of neuronal injury
For the evaluation of DNA fragmentation, coronal brain sections at the level of the striatum from mice subjected to 30 min MCAO were fixed with 4% paraformaldehyde (PFA)/ 0.1 M phosphate buffered saline (PBS) and were labeled using a TUNEL assay kit (In Situ Cell Death Detection Kit; Roche, Switzerland). Sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) [21]. Stainings were analyzed by quantifying TUNEL (+) cells (which in 30 min MCAO are equivalent to neurons) in twelve adjacent ROI in the striatum, each measuring 62,500 µm2, under a confocal laser scanning Zeiss LSM 780 microscope (Carl Zeiss).
Western blot
For Western blot analysis, harvested tissue samples from the ischemic striatum belonging to the same group were pooled, homogenized, sonicated, and treated with protease/ phosphatase inhibitor cocktail (5872, Cell Signaling, Massachusetts, USA). Total protein content was evaluated using Qubit 3.0 Fluorometer (Q33216, Invitrogen, Life Technologies Corporation, California, USA) according to the manufacturer’s protocol. Equal amounts of protein (20 µg) were size-fractionated using 4-20% Mini-PROTEAN TGX (4561096, Bio-Rad, Life Sciences Research, California, USA) gel electrophoresis and then transferred to a PVDF membrane using the Trans-Blot Turbo Transfer System (1704155, Bio-Rad, Life Sciences Research). Thereafter, membranes were blocked in 5% nonfat milk in 50 mMol Tris-buffered saline (TBS) containing 0.1% Tween (TBS-T; blocking solution) for 1 h at room temperature, washed in 50 mMol TBS-T, and incubated overnight with monoclonal mouse PDE10A (sc-515023; Santa Cruz Biotechnology), polyclonal rabbit anti-phospho-Akt (9275, Cell Signaling), monoclonal rabbit anti-phospho-p44/42 MAPK (Erk1/2) (4370; Cell Signaling), polyclonal rabbit anti-phospho-PTEN (9551; Cell Signaling), polyclonal rabbit anti-phospho-GSK-3α/β (9331; Cell Signaling), polyclonal rabbit anti-phospho-mTOR (2971, Cell Signaling), polyclonal rabbit anti-MMP-9 (Ab38898, Abcam, Cambridge, UK), monoclonal rabbit anti-HIF-1α (36169; Cell Signaling), polyclonal rabbit anti-Bax (2772; Cell Signaling) or monoclonal rabbit anti-Bcl-xL (2764; Cell Signaling) antibody. On the next day, membranes were washed with 50 mM TBS-T and incubated with horseradish peroxidase-conjugated goat-anti-rabbit (sc-2004; Santa Cruz Biotechnology, Heidelberg, Germany) or goat anti-mouse (sc-2005; Santa Cruz Biotechnology) antibody (diluted 1:2500) for 1 h at room temperature. Blots were performed at least three times. Protein loading was controlled by stripping and re-probing with polyclonal rabbit anti-β-actin antibody (4967, Cell Signaling Technology). Blots were developed using Clarity Western ECL Substrate kit (1705060, Bio-Rad; Life Sciences Research) and visualized using the ChemiDoc MP System (1708280, Bio-Rad; Life Sciences Research). Protein levels were analyzed densitometrically using an image analysis system (Image J; National Institute of Health), corrected with values determined on β-actin blots and expressed as relative values compared with vehicle treated group.
Cytokine array
Tissue sections of the ipsilesional striatum were harvested, homogenized, sonicated, and treated with protease/ phosphatase inhibitor cocktail (5872, Cell Signaling). The expression of cytokines was analyzed by the Proteome Profiler Mouse Cytokine Array Panel A Kit (ARY006, R&D Systems, Boston, USA) from a total of 9 animals (n=3 for vehicle, n=3 for 0.3 mg/kg TAK-063, and n=3 for 3 mg/kg TAK-063), according to the manufacturer’s recommendation. Array panels were visualized using the ChemiDoc MP System (1708280, Bio-Rad). Protein levels were analyzed densitometrically using an image analysis system (Image J), corrected with values determined on positive controls and expressed as relative values compared with vehicle treated group.
Sample preparation for liquid chromatography tandem-mass spectrometry (LC-MS/MS)
Seventy-two hours after the onset of reperfusion, animals were sacrificed (n= 4-5 per group). Thereafter, brains were immediately removed, frozen on dry ice and stored at -80 °C. The brain tissues were taken from ipsilesional striatum and were homogenized in 50 mM ammonium bicarbonate and lysed by heating at 95 °C in protein extraction reagent kit (UPX Universal; Expedon, Heidelberg, Germany). Samples were incubated for an hour at 4 °C. After incubation step, samples were centrifuged at 14,000 G for 10 min. Then supernatants were collected. Protein concentrations were determined via Qubit 3.0 Fluorometer (Q33216, Invitrogen, Life Technologies) according to the manufacturer’s protocol. FASP (Filter Aided Sample Preparation) Protein Digestion Kit (ab270519, Abcam) was used for generating tryptic peptides according to the manufacturer's protocol [22]. A total of 50 µg protein samples were filtered using 6 M urea in a 30 kDa cut-off spin column. After this step, samples were alkylated with 10 mM iodooacetamide in the dark for 20 min at room temperature. Then samples were incubated overnight with MS grade trypsin protease (ratio 1:100, 90057, Thermo Scientific) at 37 °C. The following day, peptides were eluted from the columns and lyophilized. At the end of the lyophilization process, the peptides were suspended in 0.1% formic acid (1002642510, Merck) and diluted to 100 ng/µl before injecting to the LC-MS/MS system (ACQUITY UPLC M-Class coupled to a SYNAPT G2-Si high-definition mass spectrometer (Waters, Massachusetts, USA)).
LC-MS/MS analysis and data processing
LC-MS/MS and protein identification were performed with small modifications according to previously published protocols [23,24]. The samples were loaded onto the ACQUITY UPLC M-Class coupled to a SYNAPT G2-Si high-definition mass spectrometer (Waters). To equilibrate the columns, 97% of mobile phase (including 0.1% formic acid in UHPLC grade water) was used and column was heated to 55 °C. Ninety min gradient elution from the trap column ACQUITY UPLC M-Class Symmetry C18 trap column (180 µm x 20 mm; 186007496, Waters) to the analytic column (ACQUITY UPLC M-Class HSS T3 Column, 100Å, 1.8 µm, 75 µm X 250 mm,; 186007474, Waters) at 0.400 μl/min flow rate with a gradient from 4% to 40% hypergrade acetonitrile (100029, Merck) containing 0.1% formic acid (v/v) was used for the peptide separation. Positive ion modes of MS and MS/MS scans with 0.7 sec cycle time were performed sequentially. Ten volts was set as low collision energy and 30 V as high CE. Ion mobility separation (IMS) was used for the ion separation. A wave velocity was ramped from 1000 m/s to 55 m/s over the full IMS cycle. The release time for mobility trapping was set as 500 μs, trap height was set to 15 V. IMS wave delay was 1000 μs for the mobility separation after trap release [25]. Without any precursor ion preselection, all the ions within 50-1900 m/z range were fragmented in resolution mode. Additionally, 100 fmol/μl Glu-1-fibrinopeptide B was infused as lockmass reference with a 60 s interval. Progenesis-QI for proteomics software (Waters) was used for the identification and quantification of the peptides. Whole proteins were identified by at least 2 unique peptide sequences and then, the expression ratio of proteins was calculated.
Statistical analysis
Statistical analysis was performed using SPSS (version 15, SPSS Inc., Chicago USA) software. Data were evaluated by one-way ANOVA followed by LSD tests. Data are presented as mean ± S.D. values. Throughout the study, p values < 0.05 were considered as statistically significant.