Papaverine hydrochloride

Mice were deeply anesthetized with 3-fold higher dose of anesthetic (see above). To induce vasodilation, animals were first perfused through the right heart ventricle with the 37^° C warm phosphate buffered saline (PBS with Ca²⁺ and Mg²⁺, Lonza) containing papaverine hydrochloride (4μg/ml, Sigma), adenosine (1mg/ml, Roth) and heparin (50IU/ml, Ratiopharm), followed by infusion of fixative 4% parafolmaldehyde (PFA, Sigma) in PBS. Contrast solution, containing 50% of Bi₃OCl and 5% bovine skin gelatin (both from Sigma) was prepared ahead and pre-warmed to 42⁰C. Mice were first perfused with PBS to remove excess fixative followed by slow infusion of 700μl of the contrast solution, during 2 min. with 120mm Hg pressure. Mice were chilled immediately and immersed in 2% of PFA overnight. For the CT scan (TriFoil imaging eXplore CT 120), the body of the animal was placed into an imaging chamber and positioned with the hind limb region in the center of the field of view. A 360° scan consisting of 1200 single views with a 1 second delay between two views (100 ms, 80 kV, 32 mA) resulted in a voxel size of 50x50x50 µm³. Data were visualized using Osirix viewer software.

L-(−)-Noradrenaline (+)-bitartarate monohydrate (Cat# A9512), serotonin creatinine sulphate monohydrate (Cat# H-7752), papaverine hydrochloride (Cat# P3510), acetylcholine chloride (Cat# A6625), lucigenin (Cat# 2315-97-1) and luminol (Cat# 521-31-3) were purchased from Sigma-Aldrich, Germany. Potassium chloride (Cat# 2538810) and hydrogen peroxide (Cat# 1-08597-1000) were supplied from Merck, Germany. Doxycycline hyclate was kindly given by Eastpharma, Turkey. RPMI 1640 (Cat# 21875-034) and L-Glutamin (Cat# 25030-024) were purchased from Gibco, UK. Penicillin/streptomycin (Cat# 9.A-4061) was supplied from PAA Cell Culture Company, USA. Hepes buffer (Cat# LA-0010E) was obtained from BioWhittaker, Belgium. All drugs were dissolved in distilled water daily. Then, further dilutions of doxycycline were prepared with distilled water. Other drugs were diluted with % 0.9 NaCl from their stock solutions for cumulative concentrations.

Cerebrovascular anatomy of the α5 KO and Ctrl mice was measured by intravenous injection of a combination of two carbon ink dyes as previously described.^{23 (link)} Briefly, papaverine hydrochloride (50 mg/kg; Sigma, St. Louis, MO, USA) was injected intravenously for vessel dilation just prior to the injection of carbon ink. A mixture of ink 1 (Fount India, Pelikan, Germany) and ink 2 (Super Black, Speedball, USA) was intravenously injected at a ratio of 1:9, respectively. After 10 min, the brain was removed and fixed in 4% paraformaldehyde prior to images being taken with a Moticam 2 (Motic, British Columbia, Canada) camera and software. Points of anastomoses between the MCA and the anterior cerebral artery (ACA) were determined and the total area of the MCA and ACA was measured using Image J software (NIH).