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

We study mechanisms of lymph vessel contractions, and lymph flow to better understand the underlying pathology of cancer related lymphedema, as well as the connection between the lymphatic system and hypertension. Our goal is to identify therapeutic targets within the lymphatic vasculature so that we can develop novel treatments for lymphatic diseases. Recent discoveries of different ion channels in lymphatic muscle and endothelial cells, which contribute to the underlying calcium dynamics have proved instrumental in moving the field forward.

Isolated vessels and intravital microscope have been crucial techniques for studying lymphatic vessel functions. More recently, there has been an upward trend of using organ on a chip and micro fabricated vessels to investigate many aspects of lymphatic biology. The very small tissue size challenges studying the lymphatic vasculature, requiring a lot of time, patience, and the development of new technologies.

The unavailability of lymphatic muscle cell specific markers is another hurdle for the whole scientific community. The advantage of this protocol is the ability to measure lymphatic contractions and absolute calcium concentrations within the same vessel. This provides additional insight into calcium signaling, mechanisms of contraction, as well as the ability to measure baseline comparisons between treatment groups, compared to other techniques that may be rely on relative changes in calcium or paralyzed vessels.

To begin, take Borosilicate glass micro pipettes acquired from a commercial source. Cut and polish the micro pipettes to a length of approximately one to two centimeters for use in the isolated vessel perfusion chamber, connect each mounted glass cannula in the vessel profusion chamber to independent pressure transducers aligned with independent gravity, fed pressure regulators and connected using polyethylene tubing, backfill the profusion chamber, glass micro pipettes, and the gravity fed pressure regulator along with the polyethylene tubing. With the physiological salt solution, then clamp the pressure to ensure the cannulas are not pressurized.

To begin, take a braided silk suture thread and separate it into a single filament. Use two Dumont 5 forceps with micro blunted tips and forceps to make a double loop around the tip of the other forceps, with the looped forceps, grab the loose end of the suture and carefully pull it through both loops. Employ Vannas spring scissors to trim excess sutures from either side.

After decapitating the euthanized rat, make a lengthwise cut along the midline of the abdominal wall. Then exteriorize the mesentery, snip the connection just below the pyloric sphincter, approximately two to three centimeters above the cecum as well as the connection to the rectum. Then rinse the dissected whole mesentery in 200 milliliters of ice cold physiological salt solution.

Subsequently transfer and secure them in a silicone lined Petri dish containing ice cold physiological salt solution using a stereo microscope, Dumont 5, inox fine forceps and Vannas spring scissors dissect second order mesenteric lymph vessels from surrounding fat and connective tissue. Now transfer the dissected limb vessel to the vessel profusion chamber for cannulation on glass cannulas. Slide a single prettied knot onto each glass cannula in preparation for securing the vessel onto the cannulas later.

Using a small piece of fat, orient the lymph vessels direction and cannulate the distal end onto P one. Slide the knot down the cannula and tighten it to secure the lymph vessel. Then pressurize the lymph vessel to four to five millimeters of mercury in the profusion chamber.

Place a bubbling stone with 7%carbon dioxide and 93%oxygen in the bath chamber to maintain physiological pH, connect the chamber to the temperature regulator and set it at 37 degrees Celsius. To begin, isolate and cannulate the mesenteric lymph vessels of the euthanized rat. Pressurize the lymph vessels in the profusion chamber and allow them to equilibrate and develop stable spontaneous contractions.

Then incubate the lymph vessels with Fura-2-acetoxymethyl ester and pluronic acid for 30 minutes in the dark. After the incubation, empty the complete bath volume with a negative pressure vacuum and refill it with a temperature matched reagent free physiological salt solution three times. Then incubate the lymph vessels in darkness for an additional 15 minutes to remove any excess indicator and allow for de-esterification transfer the chamber to an inverted fluorescent microscope with LED light 20 XS Fluor objective cell framing adapter, and camera for 15 hertz fluorescence capture.

Connect the microscope to a computer equipped with imaging software to record fluorescence and perform edge detection. Activate the LED light source and the fluorescent system interface. Now launch the software Ion wizard.

Under the file tab, select the option New. Then go to the Collect tab and select Experiment. Load the desired experimental template and click Okay.

Click the start button located at the bottom of the screen to initiate the experiment. Next, adjust the on-screen traces to display vessel diameter numerator 340 signal denominator 380 signal and ratio in descending order. To modify the Y-axis scale for better trace visualization, go to Traces.

Ensure that automatic limits is unchecked and select Edit User Limits. Select the parameter to adjust. Input the minimum and maximum values for the axis, and then select Okay to confirm.

Using the edge detection software, adjust the lighting so that the lymph vessel wall appears as dark lines. Select a region of interest or ROI free of fat and debris and ensure not to move this ROI, set the threshold such that the vessel wall edge is detected throughout the entire contraction cycle. After activating the photo multiplier tube using LED illuminators, alternate 340 and 380 nanometer wavelengths and 50 millisecond exposures to excite Fura 2, capture the emission spectra at 510 nanometers and 15 hertz across the entire imaging field.

To measure the signal to background ratio, first obtain the 340 and 380 fluorescence with the lymph vessel in the center of the field of view. Then move the field of view to the edge of the bath with no vessel to capture the background afterward, exchange the bath solution with a temperature matched reagent free physiological salt solution. To remove excess Fura 2 indicator record baseline Fura 2 fluorescent signal and spontaneous contractions for about 30 minutes.

Then record the cumulative concentration response of Nifedipine and obtain background measurements for each drug concentration. At the end of each experiment, wash the lymph vessels with temperature matched calcium free physiological salt solution to obtain the minimum Fura 2 fluorescent signal and the maximum diameter of the lymph vessels in the absence of calcium ions, exchange the bath solution with a temperature matched physiological salt solution containing 10 millimolar calcium ions and ionomycin to obtain the maximum Fura 2 fluorescent signal and the minimum diameter of the lymph vessels in conditions of saturating calcium ions. The parameters including calcium spike amplitude, baseline calcium, and peak calcium exhibited a concentration dependent reduction with the incremental addition of nifedipine to the perfusion chamber.

Concurrently contractile parameters such as contraction, amplitude, and calculated flow also demonstrated a stepwise decrease. Manhattan plots showed the individual lymph vessel responses for measures of rhythmicity including interval, contraction time and relaxation time.