Isolated Arteriole Perfusion for Vascular Function

Adipose tissues were dissected, and resistance arterioles were isolated and cleaned of excess fat and connective tissues. Arterioles were then washed and prepared for measuring changes in the internal diameter in response to flow and acetylcholine (Ach) as previously described [26 (link),28 (link),29 (link)]. In summary, isolated arterioles were cannulated using glass micropipettes in an organ perfusion chamber, and both ends were secured using a 10-0 nylon Ethilon monofilament suture. Cannulated arterioles within the organ chamber were then placed on the stage of an inverted microscope attached to a video camera, a video monitor, and a video measuring device (model VIA-100; Boeckeler, Madison, WI, USA). The organ chamber was perfused with heated physiological salt solution (Krebs buffer) that contained the following components (mM): 4.4 KCL, 123 NaCl, 2.5 CaCl₂, 20 NaHCO₃, 1.2 MgSO₄, 1.2 KH₂PO₄, and 11 glucose. The pH of the solution was kept at 7.4 ± 0.05, and the temperature was maintained at 37 °C. The buffer was also supplied with air mixture of 21% O₂, 5% CO₂, and 74% N₂. Each end of the cannulated arteriole was connected via silicon tubes to a physiological buffer-containing reservoir, and the intraluminal pressure gradient (10–100 cm H₂O) was established by modifying the distance between both reservoirs in equal and opposite directions [30 (link)].