Measurement of Cytosolic Ca²⁺ in Isolated Contractile Lymphatics

This experimental system determines whether changes in intracellular calcium relate to changes in the lymphatic contractile pattern caused by elevated luminal pressure. First excise a contractile lymphatic vessel from the rat mesentary and cannulate it on both ends to tightly control the luminal pressure. Next, load the vessel with the ratio metric calcium sensing dye fira 2:00 AM which enables the measurement of changes in cytosolic calcium using fluorescent microscopy with alternating three 40 and three 80 nanometer excitation.

Record changes in lymphatic vessel diameter and cytosolic calcium levels while the vessel is subjected to luminal pressure. Step protocol also determine the lymphatic diameter from the time-lapse images obtained. The results indicate a temporal relationship between transient increases in cytosolic calcium and phasic contractions of lymphatic vessels, and a correlation between calcium levels and myogenic constriction.

The main vantage of this technique over existing methods like intravital microscopy, is that extrinsic influence on lymphatic pumping, including the rate of inflammation, neurogenic and endrin controls and luminal pressure and flow are all entirely controlled, allowing a precise study of the signaling pathways that determine the lymphatic contractile cycle. Visual demonstration of this method is critical as the isolation and cannulation steps are difficult to learn. The lymphatic vessels are extremely delicate and poor manipulation can cause them to lose their contractile function.

Mount resistant matched micro pipettes in an isolated vessel chamber. Fill the chamber and micro pipettes with albumin physiological sort solution or A PSS ensuring no air bubbles in the system. Then prepare overhand knots with ophthalmic sutures and place one on each micro pipette.

Now anesthetize the animal with an intramuscular injection of ketamine. Xylazine sterilize the abdominal area with 70%alcohol, perform a midline laparotomy, exteriorize, and ex size the small intestine and mesentary. Place the tissue in ice cold.

A PSS proceed to pin a section of mesentary in a dissection chamber filled with ice cold A PSS under a stereo microscope. Select vessels with only one valve to ensure optimal pressure control in the entire segment. Carefully dissect the collecting lymphatic vessel from surrounding adipose and connective tissue.

Now transfer the lymphatic sample to the prepared vessel chamber containing A PSS. Mount the vessel onto the two resistance matched glass micro pipettes by tying with the ophthalmic sutures. Transfer the chamber to an inverted fluorescent microscope system.

Attach the tubing originating from the micro pipettes to the servo feedback pump system. Then connect the chamber to the heating unit and set the bath to 37 degrees Celsius. View the lymphatic directly through the cover.

Slip in the base of the chamber and acquire rapid time lapse image sets to load the lymphatic vessel with calcium sensing dye. Exchange the bath to A PSS containing two micromolar URA 2:00 AM and 0.2%onic acid. After 30 minutes, replace the A PSS solution and equilibrate for 20 minutes to allow reestablishment of spontaneous contractions.

Set a data acquisition protocol that alternatively illuminates at three 40 and three 80 nanometer wavelengths for durations of 50 milliseconds each. Also set parameters to collect two minutes of data at a baseline intraluminal pressure of two centimeters water and during step increases. Now acquire ratio metric fewer two measurements in the isolated lymphatics.

Select a region of interest, including the lymphatic vessel and surrounding area to track cytosolic calcium concentrations during relaxation and contraction. Also determine the luminal diameter over time using regions of interest. That track movement of the vessel walls.

Initially set the intraluminal pressure at two centimeters of water for 45 to 60 minutes to allow the development of lymphatic spontaneous contractions. For this study, select vessels that meet two criteria within the e equilibration period. First, the vessel should develop spontaneous tone at two centimeters of water.

And second, the vessel should develop regular spontaneous contractions that are reasonably uniform across the length of the vessel. Record the rapid time-lapse images during the pressure step procedure. Now change the bath solution to a calcium free.

A PSS. Measure the maximal passive diameter to calculate tone and normalize data between lymphatics of different sizes. Also, calculate calcium fluorescence measurements at each luminal pressure.

Finally, determine the required lymphatic contractile parameters such as lymphatic luminal diameter measurements. This is a representative time-lapse video of purative intensity in an isolated lymphatic presented in heat map format. A plus of the 3 43 80 ratio and the lymphatic diameter over time shows that a transient increase in calcium precedes each phase it contraction of the lymphatic vessel.

This experiment evaluated whether the cytosolic calcium concentration during diastole is associated with the lymphatic tone and myogenic constriction caused by stretch when step increases in pressure are imposed. Each pressure step caused an elevated frequency of transient increases in the 3 43 80 ratio indicative of the frequency of transient increases in the cytosolic calcium concentration. There was also a gradual increase in the 3 43 80 ratio between transient, indicating a gradual elevation of cytosolic calcium between calcium transience.

Lymphatic diameter also initially increased with each step increase in luminal pressure for the upward pressure steps between four to 10 centimeters of water. The decrease in the end diastolic diameter following the initial widening of the vessel represents lymphatic myogenic constriction. A significant lymphatic myogenic constriction occurs after pressure step increases of eight and 10 centimeters of water.

Likewise, significant increases in the 3 43 80 ratio during diastole occur after upward pressure steps of six, eight, and 10 centimeters of water. Suggesting an association between cytosolic calcium during diastole and myogenic constriction Once mastered, this technique can be completed in four to six hours if it is performed properly. After watching this video, you should have a good understanding of how to isolate and cannulate lymphatic vessels that can be used for imaging studies to determine both function and changes in cytosolic calcium.