A 0.025 mL aliquot of PMMTM resuspended in methanol, as above, was loaded onto a 1.49 cm2 quartz punch along with a duplicate and blank. Total OC/EC were calculated from the resulting spectra, as HDAC inhibitor previously described . IT was performed as previously described . Briefly, extracted PMMTM samples were resuspended in sterile saline (Normosol®-R, Hospira, Lake Forest, IL, USA) with 5% fetal bovine serum via sonication for 30 seconds. Rats were briefly
anesthetized (isoflurane gas) and instilled with 0.3 mL of vehicle or vehicle with 300 μg of PMMTM. Twenty-four hours following instillation, mesenteric and coronary arterioles were isolated or intravital microscopy was performed. Intravital microscopy was performed as previously described . Briefly, rats were anesthetized by an i.p. injection of Inactin (100 mg/kg) and maintained at 37ºC. The trachea was intubated to ensure a patent airway, and the right carotid artery was cannulated to measure arterial pressure. The right spinotrapezius muscle was exteriorized for microscopic observation over a clear pedestal, leaving all feed arteries and innervations
intact. The tissue bath was continuously superfused with an electrolyte solution ([in mm] 119 NaCl, 25 NaHCO3, 6 KCl, and 3.6 CaCl2, pH 7.4, 290 mOsm), warmed to 35ºC, and equilibrated with 95% N2, 5% CO2 with a superfusion flow rate of 4–6 mL/min. The preparation was then transferred to the stage of an Olympus intravital microscope coupled to a CCD camera and was observed under a 20× water immersion objective (final image magnification DAPT was
743×). Greater than three images Reverse transcriptase were digitally captured via DP controller (Olympus, Center Valley, PA, USA) during a baseline period and immediately following each experimental period. Arteriolar diameters from each digital image were measured with Microsuite analysis software (Olympus). Steady-state arteriolar diameters were averaged per experimental period to reduce sampling variability . Coronary arterioles were isolated as previously described [26, 27]. Arterioles from the mesentery were also removed in a similar manner. Briefly, the heart or the mesentery was removed from isoflurane anesthetized animals and placed into a silastic-coated dish containing chilled (4°C) PSS (in mm; 129.8 NaCl, 5.4 KCl, 1.1 NaH2PO4, 1.7 MgCl2, 19.0 NaHCO3, 1.8 CaCl2, and 5.5 glucose, pH 7.4, 290 mOsm). The heart was flushed of excess blood and the LAD artery was located. Arterioles ≤170 μm, which corresponded to third to fourth order arterioles in the heart or fourth and fifth order arterioles in the mesentery, were isolated and transferred to a vessel chamber containing fresh PSS oxygenated with normoxic gas (21% O2–5% CO2–74% N2), cannulated with glass micropipettes, and secured with nylon suture (10–0 ophthalmic; Alcon, Hemel Hempstead, UK).