FIG. 3.

Protein diffusivity and blood perfusion through the de-endothelialized coronary artery vessel system. Protein diffusion was assessed via selective catheterization of the coronary ostia and coronary perfusion with Alexa Fluor 555-conjugated BSA (66 kDa) at a constant flow rate of 0.1 mL min⁻¹, while fluorescence images of representative targeted vessels and surrounding tissue were taken at defined time points. Analysis of protein concentrations—proportional to the fluorescence intensity (FI)—showed steadily increasing protein diffusion into the circumjacent tissue as a function of time and distance to the targeted vessel, with no gross perfusate leaks and sharply defined vessel borders indicating proper vessel sealing. Additionally, perfusion with citrated whole blood of selectively catheterized tissue flaps showed patency for cellular blood components with no erythrocyte extravasation of main and dependent vessels into the circumjacent tissue and drainage of the blood components down to the capillary system and along the greater vessel openings at the edge of the tissue flaps. (A) Representative bright field as well as fluorescent images of targeted coronary vessel and circumjacent tissue at 1 and 45 min of coronary perfusion with fluorescent-conjugated BSA (red), respectively. White rectangle, region of interest (ROI) for protein diffusivity analysis. (B) Representative fluorescent images of ROI plotted over time show the qualitative increase of fluorescent-conjugated BSA concentrations in the tissue surrounding the vessel. (C) and (D) representative plots of FI normalized to the midvessel intensity for 150-, 350-, and 550-μm vessel distance and 10, 20, and 50 min of perfusion, respectively, demonstrate an increase in tissue-conjugated BSA concentrations as a function of time and distance from the targeted vessel. Protein concentrations at 550 μm distance from the vessel wall reaches saturation at 50 min of perfusion. (n=4; mean±SD). (E) Macroscopic picture of catheterized and blood perfused tissue flap. (^#, 28G catheter in main coronary artery; *, capillaries; ⁺, erythrocyte accumulation and extravasation in capillaries). (F, G) Microscopic images before and after blood perfusion, showing the patent and integer vessel system with blood filled capillaries (*, capillaries). (See also Fig. 1B and Supplementary Movie SM2). Color images available online at www.liebertpub.com/tec