Real-Time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph...

The lymphatic vasculature, now often referred to as "the third circulation," is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca²⁺]i). This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca²⁺]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca²⁺]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca²⁺-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca²⁺]i and diameter measurements continuously in real time. The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca²⁺]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca²⁺]i measurements.