Arch Animal Care from the 5-HT7 Receptor site Massachusetts Common Hospital, Boston, MA. We
Arch Animal Care of the Massachusetts Common Hospital, Boston, MA. We studied 64 eight to ten week old male C57BL6 (WT) mice weighing 27 g and six BKS.Cg-Dock7m+/+Leprdb/J (db/db) mice weighing 60g on a C57BL6 background (Jackson Laboratory, Bar Harbor, ME). See Tables 1 and two for the numbers of mice studied in each and every group. Reagents We studied the thromboxane agonist U46619 (Cayman Chemical Company, Ann Arbor, MI) along with the non-selective NO synthase (NOS) inhibitor NG-nitro-L-arginine methylester (LNAME; Sigma-Aldrich, St. Louis, MO). Preparation of cell-free Hb solution Murine cell-free Hb option (4 g l-1, methemoglobin 2 ) for i.v. injection was prepared as previously described [28]. Measurements of plasma Hb and methemoglobin (metHb) concentration In a separate group of WT mice (n=6 per group), entire blood was heparinized and collected by means of cardiac puncture at baseline, and at 15 and 30 min after i.v. infusion of cell-free Hb. Plasma was obtained by centrifuging whole blood at 1699g for 8 min at four , and was stored at -20 . Cell-free Hb and metHb concentrations had been determined by the cyanmethemoglobin technique [29]. Absorption was measured at 540 nm and 630 nm with a spectrophotometer (Biomate three; Thermoelectron Corporation, Waltham, MA). Surgical preparation of anesthetized, open-chest mice Surgical preparation of animals plus the measurement of left lung pulmonary vascular resistance indexed to body weight (LPVRI) had been performed as described previously [30]. Briefly, mice were 5-LOX site anesthetized with an intraperitoneal (i.p.) injection of ketamine (120 mg g-1) and fentanyl (0.09 mg g-1). Following tracheostomy, pancuronium (two mg g-1) was injected i.p. to induce muscle relaxation and mice have been subjected to a median thoracotomy. Volume-controlled ventilation was offered at a respiratory price of one hundred breaths in-1, a tidal volume of ten ml g-1 and inspired O2 fraction (FIO2) of 1.0 (Mini Vent 845; Harvard Apparatus, Holliston, MA). Invasive hemodynamic measurements in anesthetized mice at thoracotomy For hemodynamic measurements, fluid-filled PE ten catheters have been inserted into the suitable carotid artery along with the most important pulmonary artery, and also a 0.five mm VB-HSE flow probe (Transonic Systems Inc., Ithaca, NY) was placed about the left pulmonary artery. Heart rate (HR), systemic arterial stress (SAP), pulmonary arterial pressure (PAP), and left pulmonary arterial blood flow (QLPA) were constantly measured and recorded. For some experiments the left atrial stress (LAP) was measured via a fluid-filled PE ten catheter placed inside the left atrium. Cardiac output (CO) was estimated by measuring lower thoracic aortic flow (QLTAF) with a flow probe. To estimate LPVRI, the inferior vena cava (IVC) was partially occludedNitric Oxide. Author manuscript; out there in PMC 2014 April 01.Beloiartsev et al.Pageto transiently cut down QLPA to 50 . LPVRI was calculated in the slope on the PAP/QLPA relationship.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptTotal systemic vascular resistance (TSVR) was estimated utilizing dynamic measurements of SAP and QLTAF. These measurements had been performed during partial occlusion in the IVC to transiently reduce QLTAF to 50 . TSVR was calculated in the slope of your SAP/QLTAF relationship. Just after getting hemodynamic measurements, arterial blood was sampled in the right carotid artery. Arterial blood gas tensions and pHa were measured making use of an ABL800 FLEX analyzer (Radiometer America Inc., Westlake, OH). Admi.