Colored microspheres (Dye Trak; Triton Technology; San Diego, CA) were used to determine tissue blood flow as described previously.11 The polystyrene microspheres are coated with a single colored dye and are 15 ± 0.3 μm SD in diameter. Red-, yellow-, blue-, violet-, or white-colored microspheres were used. Depending on the different absorbance characteristics of each color, 6 to 15 million microspheres suspended in 2 to 5 mL of 0.9% saline solution containing 0.02% Tween 80 were used for each blood flow measurement. Prior to injection into the left ventricle through the pigtail catheter, the suspension of microspheres was thoroughly sonicated and vortexed. The sequence of the color of microspheres was randomly assigned, and microspheres were injected slowly and continuously so that systemic hemodynamics remained unchanged during injection at all time points. After injection, the catheter was flushed with 10 mL of saline solution. Starting 10 s before the injection of microspheres and continuing for 120 s after the injection was completed, two reference blood samples were withdrawn simultaneously from different lumina of the aortic catheter at a rate of 5 mL/min with a precision pump (AH 55–2226; Harvard Apparatus GmbH; March-Hugstetten, Germany). At the end of the experiments, the animals were killed using sodium pentobarbital and potassium chloride. The crural and costal portions of the diaphragm, intercostal, and psoas major muscles and adrenal glands were removed bilaterally. The tissues were carefully dissected free of adherent fat or connective tissue. While adrenal glands were analyzed as a whole, muscle tissue samples were cut into small pieces weighing approximately 2.5 mg (BP 310 S; Sartorius; Göttingen; Germany). The trapped colored microspheres in each tissue and reference blood samples were quantified by their dye content. After digestion of the tissue and blood samples with 4 mmol/L potassium hydroxide for at least 24 h at 70°C, the microspheres were harvested on a polyester filter (pore size, 8 μm; diameter, 25 mm; Nucleopore; Costar; Bodenheim, Germany). The microspheres were washed with 2% Tween 80 and then with ethanol. The dye was recovered from the microspheres by adding 200 μL of dimethylformamide. Then, the dye solution was separated from the microspheres by centrifugation at 3,000g for 10 min. Spectrophotometric analysis of mixed dye solutions was performed (spectrophotometer DU64; Beckmann; Düsseldorf, Germany; wave length range, 300 to 820 nm, with 1 nm optical band width). The complex spectra were transferred to a personal computer using software (Data-Leader; Beckmann; Düsseldorf, Germany), and the composite spectrum of each dye solution was resolved into spectra of single constituents using a matrix inversion software package (Dye-Trak; Triton Technologies; San Diego, CA). From the spectrophotometric data, tissue blood flow was calculated using the following equation,11:
where As is the absorbance of the tissue sample, Vref is the reference blood flow (milliliters per minute), Aref is the mean absorbance of both reference blood samples, and Ws is the weight of the tissue sample. For each organ, the respective median blood flow of all samples was calculated.