*4.4. Measurement of Lymphatic Vessels Contractility*

Afferent extra-renal lymphatic collecting vessels were isolated by microdissection and lymphangions mounted on glass pipets in vessel perfusion chambers as reported [17]. A digital image capture system (IonOptix) was used to record pre-valve intraluminal diameters. Vessels were warmed to 37 ◦C, pressurized to 0.5 mmHg using a column of Krebs buffer, and allowed to equilibrate (20–60 min) before incrementally increasing the intraluminal pressure to 2.5 mmHg. Vessels that failed to contract spontaneously were excluded from further study. For high-sodium environment studies, the vessels were exposed to a modified high-sodium Krebs buffer (see below). Some vessels were also challenged with increasing concentrations of furosemide (10-7-10-3M, Hospira). For each experimental condition, lumen diameters were allowed to plateau (20–40 min) before moving to the next condition. Single vessels were exposed to 1 to 3 compounds over the course of each experiment. We found no difference in response or viability based on the order of compound administration. As previously reported [17,36], the amplitude of contraction was measured as the difference between the end diastolic diameter and end systolic diameter (EDD-ESD). The ejection fraction was calculated as (EDD2-ESD2)/EDD2.
