Imaging Ca²⁺ Signals in Small Pulmonary Veins at Physiological Intraluminal Pressures

In this protocol, we present a novel technique for recording and analyzing Ca²⁺ signals in intrapulmonary veins (small pulmonary veins or PVs) at physiological intraluminal pressures. The technique involves isolating small PVs, incubating them with a Ca²⁺ indicator, cannulating and pressurizing them, confocal imaging of Ca²⁺ signals, and data analysis.

Calcium signals regulate blood vessel function, varying in source and role. We aim to understand their specific functions in the vascular wall and identify abnormalities linked to disease. Most lung blood vessel studies focus on arteries or capillaries, not pulmonary veins.

No studies have examined calcium signals in pulmonary veins under normal intraluminal pressures despite their physiological relevance. Pulmonary veins undergo pressure change during the cardio cycles, but their impact on calcium signals was unstudied. Our protocol enables acquisition and automatic analysis of calcium signals in small pulmonary veins at normal pressure.

Our protocol enables future studies on how intraluminal pressure affects calcium signals in small pulmonary veins, advancing understanding of their regulation and role in health and disease. To begin, clean the dissection tools and dishes in 100%ethanol, then wash them in deionized water. With a pair of scissors.

Open the thoracic cavity of a euthanized mouse. Use forceps to carefully remove the heart and lungs from the thoracic cavity with minimal touching of the lungs. Next, place the tissue on a seal guard coated plate containing cold HEPES buffered physiological salt solution.

Pin down the heart and lungs using dissection pins so that the large pulmonary veins and pulmonary arteries are clearly visible, and the left lobe of the lung is slightly stretched. Using the large pulmonary veins as reference points, carefully remove the tissue surrounding the small inter-pulmonary veins with the help of fine scissors. Then gently isolate the small pulmonary veins from the surrounding tissue.

Next, prepare a stock concentration of 2.5 millimolar Fluo-4 AM with DMSO. Using the stock solution, prepare the loading solution. Place the small pulmonary veins in a 1.5 milliliter tube with the loading solution.

Cover the tube with aluminum foil, incubated in a water bath at 37 degrees Celsius for one hour. To cannulate the small pulmonary veins, remove a vein from the washing solution. Place it in a prepared pressure myography chamber.

With a pair of fine-tipped forceps, carefully cannulate one end of the small pulmonary vein onto one of the cannulas, then use a nylon thread microfilament to tie a knot around the cannulated end of the small pulmonary vein and the tip of the cannula to secure it. Push the physiological salt solution gently through the cannula to remove the blood inside the pulmonary vein. Use nylon thread to tie off the other end with a glass cannula using micro filaments.

For calcium imaging, first attach a servo pressure controller to a tubing containing HEPES buffered physiological salt solution. Use the controller to pressurize the small pulmonary vein from one end. Now connect a peristaltic pump to an inlet and an outlet and an outlet and superfuse the solution.

After the equilibration period, use the 40x water dipping objective and a spinning disc confocal imaging system to acquire calcium images for 1, 000 frames. To perform image analysis, launch the custom designed Spark and software. Use the five by five filter and median filter to smooth the images, followed by a frame outlining a flat region of the pulmonary vein with multiple cells for Event Auto Detection.

Click on view and Event Auto Detection. Set the event threshold at an amplitude of 1.3F over F0, the tolerance to 20%the scan box to seven by seven pixels, and the running average to seven images. Click on Start Search or the eye icon.

Find the events table by clicking on small events table under the view menu, then calculate the events per micrometer square by dividing the number of detected calcium ion signals by the area of the selected frame. The number of spontaneously occurring calcium signals in small pulmonary veins at five millimeters of mercury intraluminal pressure was 0.73 plus or minus 0.2 events per minute per square micrometer under basal conditions. The addition of ryanodine drastically reduced calcium signaling activity.