The overall goal of this procedure is to determine the effect of a pharmacological agent or disease state on pressure induced vascular tone in small resistance mesenteric arteries. This is accomplished by first isolating the mesenteric arcade from a murine model. The second step is to dissect the fourth order small resistance arteries.
The next steps are the cannulation, pressurizing, and incubation of the arterial preparation. In PSS, the final step is to assess the physiological integrity of the arterial preparation, which is demonstrated by spontaneous development of vascular tone and appropriate reversible response to a vasoconstrictor and a vasodilator. Ultimately, pressure induced constriction can be measured.
The implication of this technique extend towards therapy or diagnosis of hypertension, hypotension, or vascular dysfunction. Because this technique identifies key mechanisms underlying pressure induced changes in vascular tone, which is a key determinant of vascular resistance, perfusion, and blood pressure. This procedure would be demonstrated by Dr.Jja, who's a post-doctor fellow in our laboratory.
Begin this procedure by loading a glass cannula into a micro pipette holder and attaching the micro pipette holder to a perfusion chamber to prepare the perfusion chamber. Rinse it with Milli Q water, followed by dissection solution for five minutes each. Then load the chamber with two milliliters of dissection solution.
Next, carefully fill the entire cannula and the attached tubing without generating any bubbles. Prepare two sutures each with a half knot using blunt forceps under the dissection microscope. Use dissection forceps to load both cannulas with partially closed suture knots slightly away from the tip.
Next, perform a midline laparotomy on an anesthetized rat from the pelvis to the sternum by first making an incision in the skin subsequently make an incision in the underlying muscle layer. Care must be taken not to injure the intraabdominal organs. Tie both ends separately to prevent leak of chime and feces in order to avoid contamination of the extracellular bathing solution.
Afterward, cut the proximal end of the intestine close to the pori and the distal end, close to the ileocecal junction. Then make an incision on the mesentary at its base near the feeding vasculature. Next, transfer the entire small intestinal mesenteric bed to a 50 milliliter beaker containing ice cold dissection solution and incubate for five minutes.
Then rinse it with fresh dissection solution to remove the blood. Now pin down the proximal end of the intestine on the right hand side in a sard coated dish, extend the remaining intestine in a counterclockwise path. Pinning the segment down to spread the mesentary and exposing the blood vessels under a stereo zoom microscope.
Dissect out the third and fourth order small mesenteric arteries parallel to the small intestine by first removing all the covering fat near the second order branch. Then dissect out the vein and isolate the artery with a V-shaped branch point. Be careful not to puncture the selected segment.
Next, isolate a four to five millimeter section of the artery that is parallel to the small intestine. Cut all the fifth order branches embedding into the small intestine slightly away from the origin of branches and preserve a portion. These portions will be served as holding sites for transferring arterial segments to a perfusion chamber and subsequently guide their cannulation.
Then cut the arterial segments by making two incisions distal to the fifth order branches on each side of the artery and transfer them to the perfusion chamber. Cannulate one end of the vessels on a glass micro pipette by holding the tip of the arterial segment with dissection forceps. Slide the previously loaded partially closed suture onto the cannulated end and secure it.
Subsequently, attach a dissection solution loaded 10 milliliter syringe to the stop cock connected to this cannula. Gently raise the syringe. The gravitational force on the solution will remove the intravascular blood from the open end of the vessel.
After removing the intra arterial blood, close the stop cock. Now tie the distal end of the vessels onto a second glass cannula by carefully bringing the other cannula as close as possible to the untied end of the arterial segment. Slide the previous loaded, partially closed suture onto the cannulated end and secure it.
Care must be taken not to tug or pull on the arterial segments and make sure that stop Cox attached to both cannulas are closed. Transfer the perfusion chamber to the stage of the inverted microscope equipped with live video recording. Then connect the stop cock of cannula tied to the proximal end of the arterial segment to a servo controlled pressure regulating device.
Make sure the stop cock attached to the other cannula remains closed to maintain stable intraluminal pressure. Next, attach the vacuum tubing to the perfusion tubing and the suction port to the perfusion port of the chamber. Start perfusing the vessel with warm PSS through the single inline solution heater at two milliliters per minute.
Using a peristaltic pump, turn the vacuum on as well as the temperature of PSS in the chamber is close to 37 degrees Celsius. Slowly increase the intraluminal pressure from 20 to 100 millimeters of mercury. Check the vessel for any leaks by using the automatic pressure setting of the pressure regulator.
After that, assess the arterial segment for bends while maintaining the pressure at 100 millimeters of mercury. Using the screw lever, move the cannula to straighten the arterial segment and do not overstretch the arterial segments. Then reduce the pressure to 70 millimeters of mercury to mimic the in vivo pressure in the mesenteric arcade, and allow the arterial segment to stabilize and develop myogenic tone.
In this procedure, view the artery with a 10 x objective under a microscope equipped with a monochrome video charge. Couple device camera. Measure the luminal diameter using a video frame grabber and a realtime edge detection system.
Then record the vessel diameter and monitor the development of the myogenic tone. After that, reduce the pressure to 20 millimeters of mercury and allow the diameter to stabilize. Then increase the intraluminal pressure in incremental steps such as 20, 40, 60, 80, and 100, and allow the artery to achieve a stable diameter at each pressure.
Step next, repeat the pressure step response in calcium free PSS containing 0.39 millimolar EGTA and 0.1 millimolar sodium nitropress as indicated by the tracing, the diameter of the small mesenteric arteries from rats decreases spontaneously when pressurized at 70 millimeters of mercury, the addition of one micromolar acetylcholine increased the diameter to the near starting diameter. The addition of one micromolar phenylephrine to the tissue bath decreased the arterial diameter, and this figure shows that the incubation of the small mesenteric arteries in calcium free PSS increase the arterial diameter. The diameter of a single pressurized arterial segment in various perfuse eights is shown in this table.
Here the arterial diameter is recorded continuously while increasing intraluminal pressure incrementally in the presence of PSS and calcium free PSS. Here are the curves of the arterial diameter achieved at each pressure step and the bar graph of myogenic tone achieved at each pressure step is shown here Off. After watching this video, you should have a good understanding of how to assess myogenic behavior of small resistance arteries.
Following this technique, you can perform additional procedures such as endothelial disruption and arterial permeation. Both these procedures can a answer further questions regarding the role of endothelium and calcium in regulation of vascular tone of small resistance arteries.
