This method can be used to study coronary microcirculation in living murine heart tissue and vascular-tree components by ex vivo monitoring of the arterial perfusion pressure and flow. This method provides a useful platform for studying the function of pericytes and reagents microcirculation within the heart, by so we tend measuring the vascular diameter and arteriole luminal pressure. To prepare a cannula for the experiment, use a micropipette puller to produce two cannulae with long thin tips, from a single clean borosilicate glass tube.
Use a fine pair of scissors and a dissecting microscope to cut the tip of each cannula to a final tip diameter of 100 to 150 micrometers, and fire polish the tips to slightly round the sharp edges. Use a platinum wire positioned on the side of the shaft, approximately two millimeters from the tip to bend the cannula along the shaft to an about 45-degree angle, and insert the open end of the cannula into the cannula holder. Then, tighten the fitting, and use a five milliliter syringe to flush the cannula and tubing with Tyrode's solution.
Mount the cannula holder onto micro-manipulation of a pressure myograph chamber. To collect a mouse heart for the experiment, first enter a pair in it inject 500 microliters of heparin into an anesthetized eight to 16-week-old C 57 black six mouse. After 10 minutes, place the mouse onto a heated bed in the supine position, and stabilize the paws with labeling tape.
Use forceps and surgical scissors to make an incision in the abdominal skin, above the diaphragm and cut the diaphragm and the sternum to allow dissection of the heart from the thoracic cavity. Place the heart into ice cold Tyrode's solution containing 30 millimolar BDM and remove any connective tissues from the heart as necessary. Then place the heart into a pre-chilled dissecting chamber, filled with fresh Tyrode's solution supplemented with 30 millimolar BDM.
To prepare the septal artery for cannulation, turn on the servo pomp, and set the servo controller pressure to flow mode. In the heart onto the PDMS, taking care to avoid damage to the middle area of the tissue and place the tissue under a dissecting microscope. Remove one or both atria, and cut open the right ventricle.
Remove the right ventricular free wall and expose the septal artery. Use a 30 micrometer diameter nylon thread to tie a loose knot around the septal artery. Use fine scissors to remove the left ventricular free wall and transfer the papillary muscle preparation to the experimental chamber.
Use a micro manipulator to insert a cannula into the septal artery and secure the cannula with the suture. Then use pins to secure the papillary muscle to the chamber floor with the tip of the cannula parallel to the arterial wall, so that a clear view of the vasculature can be obtained and use the syringe to gradually flush the solution through the cannula. To stabilize the tissue preparation, turn on a peristaltic pump and continuously super fuse the preparation with pre-gassed physiological saline solution at a three to four milliliters per minute flow rate, turn on the temperature controller for the superfusion solution, and adjust the temperature of the bath superfusate to 35 to 37 degrees Celsius.
Connect the cannula to the servo pump and adjust the flow to set the pressure to approximately 10 millimeters of mercury. Let the superfusion run for about 10 minutes, then increase the flow of the luminal solution to set the artery pressure to approximately 60 millimeters of mercury and allow the sample to stabilize for 30 to 60 minutes with monitoring. To load the sample with wheat-germ agglutinin, perfuse the preparation with five milliliters of Tyrode's solution, supplemented with 100 micrograms of Alexa Fluor-488 Conjugated Wheat-Germ Agglutinin.
After 30 minutes, change the perfuse aid back to normal Tyrode's solution and turn on the confocal microscope system. Use the microscope at a lower power of magnification, to locate the septal artery in transmitted light mode, and begin imaging with this spinning disc confocal. Select a 40X objective magnification, and the 488 nanometer excitation laser, and adjust the laser intensity and the sampling rate.
To define the imaging range, set the top and bottom imaging positions for the microscope and set the step size and Z-stack imaging parameters. Then, select a folder for storing the image and click run to begin recording the images. When a fluorescence vascular marker is perfused into the vascular lumen, it's possible to visualize whole vascular trees, using high-speed confocal microscopy.
Further magnification enables imaging of the capillary in detail. When pinacidil and ATP sensitive potassium channel agonist is served from the lumen, the diameter of the arterials increases. When the vasoconstrictor endothelin one is applied from the lumen.
The diameter of the arterial decreases. When the flow set is constant, the luminal pressure increases. In these images of papillary tissue from an NG two DS red expressing transgenic mouse, both the capillary and pericytes can be visualized within the papillary muscle tissue, under conditions that better mimic the physiology of live animals.
It's important that no bubbles are present within the tubing at any point during the procedure. Combining this method with optical imaging techniques, the preparation can be used to study microcirculation in the heart and intercellular communications between different cell types, using fluorescence tech transgenic mice.
