Use of a single-pass in situ perfused rat hindlimb to study tissue distribution kinetics. Method development and...

A single-pass in situ rat hindlimb preparation has been developed as an alternative to the isolated perfused preparation to study tissue drug distribution kinetics with minimal disturbance of the animal physiology. Evans blue (EB), a vascular marker, was administered (in saline or plasma) as a bolus into the femoral artery, and the total outflow blood was collected at timed intervals from the corresponding femoral vein. Donor blood was infused through the jugular vein at a rate that matched the loss. No changes in the blood flow or development of edema were observed. The outflow profile was characterized using statistical moments. Regardless of the injected solution, the estimated vascular volume (10% of the hindlimb wet weight) was higher than that reported for the isolated rat hindlimb (4.1%) but closer to the in vivo blood volume (6.8-8.1% of body weight for 250-g rat) and sum of vascular volumes of its composite tissues (6.9% of tissue weights). The large normalized variance (CV(2)) of the outflow (2.2+/-0.1) confirms the known heterogeneity of the hindlimb. Entrapment in the limb (approximately 4%) and escape to the rest of the body could explain the incomplete recovery (79-89%) of the dye.