The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The overall goal of this procedure is to surgically isolate, remove, and doubly cannulate individual resistance arterials. For study in an ex vivo setting, this is accomplished by first identifying and surgically isolating a micro vessels in an anesthetized animal. After transferring the micro vessels to a vessel chamber, the proximal and distal ends of the micro vessels are both cannulated.

Finally, the micro vessels is exposed to pharmacological or physiological challenges and experimental interventions to assess mechanisms underlying function or dysfunction that are intrinsic to the vessel itself. Ultimately, the results show the patterns and contributing mechanisms underlying vascular reactivity in an isolated setting through appropriately targeted stimuli and interventions. Generally, individuals new to this method will struggle because this preparation requires a development of not only excellent surgical skills to be performed under a microscope, but also the patients and fine motor control required to doubly cannulate an isolated micro vessels with a diameter that is frequently much less than a hundred microns.

This must be done with an absolute minimum of trauma to the vessel, or the entire procedure will result only in an unresponsive vessel demonstrating the procedure will be Joshua Butcher, a graduate student from my laboratory Prior to carrying out isolated micro vessels.Preparation. Begin by bending previously prepared glass micro pipettes to an appropriate configuration by heating them over a butane flame. Then use fine forceps to break the tips to an appropriate diameter.

Place two micro pipettes in opposition into the pipette holders for the micro vessels chamber, orienting them so that the tips are in the same vertical and horizontal plane within the vessel chamber. Next, prepare a physiological salt solution or PSS and check that the pH is set to appropriate levels for the specific experimental conditions. Using a hemo, cytometer or microscope stage micrometer calibrate the digital video calipers that will be used to measure vessel diameter.

Use eight oh or smaller ophthalmic suture to prepare single tip loops for attaching the micro vessels to the pipettes. Store the loops in a Petri dish on a small strip of tape wrapped around a microscope slide on the day of the experiment. Turn on the equilibration gasses in the vessel chamber and in the PERFUSE eight and SUSE eight reservoirs.

Fill all the reservoirs, tubes and chambers with the PSS solution. If needed, use a syringe to gently push the PSS all the way through the pipette to ensure that it is unblocked and completely full. Keep the inflow pressure within reasonable limits to avoid damaging the pressure transducers.

After anesthetizing the animal and exposing the micro vessels, remove the micro vessels by using fine forceps to grasp the exterior side and cutting along the length of the vessel. Take extreme care not to tug or pull the vessel. Next, place the vessel in the filled water bath and using a pair of fine forceps and a modest perfusion rate, hold each side of the proximal vessel lumen wall and cannulate the proximal end on the inflow pipette.

Then slip the vessel on the cannula tips and advance the vessel on the tip to the point where the T loops can secure the vessel. Put in place the T loop for the distal end of the vessel and cannulate it on the outflow pipette, securing it with the T loops. Adjust the cannula until there is minimal distortion in the vessel and the vessel approximates the in vivo length.

Place a small bubbling stone delivering the appropriate gas mixture into the bath and place. Plastic wrap or a glass cover over the chamber to avoid splashing the microscope lens. Periodically clamp at the outflow tubing to determine if the vessel is developing rusting tone.

Check the vessels for leaks by increasing the pressure in the vessel and clamping the outflow followed by the inflow lines. If the intraluminal pressure is stable, then there are no discernible leaks. If the vessel has a small side branch, that leaks tie it off with a single loop of 10 o ophthalmic suture after cannulation of the micro vessels, equilibration and validation, begin the experiment to determine initial control responses.

For example, for a physiological stimulus such as myogenic activation or pressure induced constriction, randomly alter the intraluminal pressure over a desired range and allow the vessel time to respond to challenge with phenylephrine at the drug at varying concentrations in a random order. After an appropriate intervention time, initiate a second round of data collection by either performing additional interventions or by washing out the initial solution or removing the physiological challenge and verifying that the vessel has returned to equilibrium. Once the vessel loses active tone, terminate the experiment or if desired, collect data on the mechanics of the vessel wall.

When the experiment is complete, flush all lines and chambers with PSS and copious amounts of distilled or deionized water, then allow them to fully dry. Shown here is a cannulated micro vessel with both inflow and outflow pipettes, as well as loops at high magnification. The inner diameter of hundred 12 microns as determined using digital calipers is evident here.

A cannulated micro vessels is shown following equilibration. Here is the same vessel following dilation in response to challenge with acetylcholine. And finally, this is the constricted vessel after challenge with phenylephrine.

These graphs summarize the responses of an isolated micro vessels to challenges with hypoxia, increasing concentrations of acetylcholine or changes in intravascular pressure under control conditions. Then in the presence of calcium free PSS Once mastered, this technique can be done in approximately one hour if it is performed properly, and the vessel can easily remain viable for many hours in the chamber with a maintenance of appropriate temperature gas and SUSE eight or perfuse eight conditions.