Objective To determine microvascular hematocrit (HMV) and permeability-surface area product (PS)

Objective To determine microvascular hematocrit (HMV) and permeability-surface area product (PS) in skeletal muscle of obese Zucker rats (OZR) and evaluate the impact of increased microvascular perfusion heterogeneity on mass transport/exchange. in OZR HA-1077 HA-1077 2HCl 2HCl and normalized following treatment with all three agents. PS was reduced in OZR and was not impacted by intervention. Conclusions Increased microvascular perfusion heterogeneity in OZR reduces HMV in muscle vascular networks and increases its variability potentially contributing to premature muscle fatigue. While targeted interventions can ameliorate this the reduced microvascular surface area is not acutely reversible. and were housed in the animal care facility at the West Virginia University Health Sciences Center. All protocols received prior IACUC approval. At ��17 weeks of age rats were anesthetized with injections of sodium pentobarbital (50 mg/kg i.p.) and received tracheal intubation to facilitate maintenance of a patent airway. In all rats a carotid artery and an external jugular vein were cannulated for determination of arterial pressure and for infusion of supplemental anesthetic and pharmacological agents as necessary. Any animal in which mean arterial pressure was found to be below 85 mmHg or where MAP had decreased by more than 15% from that following equilibration (without any pharmacological intervention) was not used in the present study. Blood samples were drawn from the venous cannula for determination of glucose and insulin concentrations (Millipore Billerica MA) as well as cholesterol/triglyceride levels (Wako Diagnostics Richmond VA) and nitrotyrosine (Oxis International. Foster City CA). Blood gases were determined using a Corning RapidLab 248 Blood Gas Analyzer (Siemens Medical Solutions Malvern PA). Unless otherwise noted all drugs and chemicals were purchased from Sigma-Aldrich (St. Louis MO). Preparation of In Situ Cremaster Muscle In one cohort of rats (LZR n=6; OZR HA-1077 2HCl n=14) an cremaster muscle was prepared for study using intravital microscopy as described previously (21). After completion of the cremaster muscle preparation the tissue was continuously superfused with physiological salt solution (PSS) equilibrated with a 5% CO2-95% N2 gas mixture and maintained at 35��C as it flowed over the muscle. The ionic composition of the PSS was as follows (mM): NaCl 119.0 KCl 4.7 CaCl2 1.6 NaH2PO4 1.18 MgSO4 1.17 NaHCO3 24.0 HA-1077 2HCl and disodium EDTA 0.03. Succinylcholine chloride (0.1 mM) was added to the superfusion solution to prevent spontaneous contractions of the cremaster muscle. Capillary tube hematocrit (HT) was determined by counting the number of erythrocytes within a measured capillary YAF1 segment from still images with final HT measures representing the mean of multiple HA-1077 2HCl determinations made during data collection periods. The calculation of HT used the following equation (8 9 cremaster muscle with the anti-oxidant TEMPOL (10-3 M) combined with the TxA2 receptor antagonist SQ-29548 (10-4 M) and/or the ��1/��2 adrenergic receptor antagonist phentolamine (10-5 M); within the superfusate solution. No cremaster muscle was exposed to all interventions in order to avoid compromising data quality owing to experiments of excessive duration. In addition to the collection of responses under control conditions individual cremaster preparations were exposed to a maximum of three interventions each separated by ��30 minutes of washout. Treatment or washout effectiveness was verified by determining abolition or recovery of mechanical responses following challenge with appropriate agonists (e.g. the ��1 adrenergic agonist phenylephrine the stable TxA2 mimetic U-46619 and the endothelium dependent dilator agonist acetylcholine). Maximum experimental duration from preparation to termination was approximately four hours after which time all animals were humanely euthanized by an intravenous overdose of anesthetic followed by a bilateral pneumothoracotomy. Preparation of In Situ Blood Perfused Hindlimb In a separate set of LZR (n=6) and OZR (n=24) the left hindlimb of each animal was isolated (13) with minor modifications. Heparin (500 IU/kg) was infused via the jugular vein to prevent blood coagulation. Subsequently an angiocatheter was inserted into the femoral artery proximal to the origin of the gastrocnemius muscle to allow for bolus tracer injection. Additionally a small shunt was placed in the femoral vein draining the gastrocnemius muscle that allowed for diversion of.