Myoview 2

The endothelial function of PAs was assessed by pressure myography as described previously by our lab [2 (link),9 ,29 (link),30 (link)]. A 5 × 3 mm section of the brain containing the middle cerebral artery (MCA) was dissected to isolate the arterioles. The pia with the MCA was separated from the brain, and the PAs branching off the MCA were used for experiments. A total of n=15–16 rats were used to complete dilation and myogenic tone experiments; each arteriole was used for only one concentration-response curve. Isolated arterioles were cannulated using two glass micropipettes in a custom-made cannulation chamber. PAs were equilibrated in physiological salt solution (PSS) containing 140mmol/L NaCl, 5mmol/L KCl, 1.8mmol/L CaCl₂, 1mmol/L MgCl₂, 10mmol/L HEPES, and 10mmol/L glucose. A servo-null system was used to pressurize the arterioles, and a leak test was performed before each experiment. Arterioles were pressurized to 60mmHg [2 (link),20 (link),29 (link)] until the development of stable myogenic tone (% tone = [1-(active lumen diameter/passive lumen diameter)] × 100. Arterioles that generated less than 20% myogenic tone were discarded. The diameter of the arterioles was recorded using MyoView 2.0 software (Danish Myo Technology, Aarhus, Denmark).

A 5 x 3 x 3 mm (length x width x depth) segment of brain tissue containing the MCA was removed and placed in Ca²⁺-free PSS at 4°C, with 1% bovine serum albumin (BSA) + 10μM diltiazem + 10μM SNP. The pia with the MCA was gently separated from the tissue, and PAs branching from the MCA were transferred to a custom-made cannulation chamber using a glass micropipette (Wiretrol, Drummond Scientific Company, Broomall, PA). PAs were cannulated between two glass pipettes, bathed in warm (37°C) PSS containing 1.8mM Ca²⁺, and pressurized to 60mmHg until spontaneous myogenic tone developed [34 ]. PA outer and lumen diameters were constantly tracked and recorded using MyoView 2.0 software (Danish Myo Technology, Aarhus, Denmark).