This procedure uses doll salt sensitive and salt resistant rats to compare the reactivity of mesenteric resistance arteries under phenylephrine tension to extracellular calcium. This is accomplished by first isolating the mesenteric arcade from animals and dissecting out the resistance artery branches.Next. The first of two stainless steel wires is threaded through a short segment of the dissected artery and the vessel is mounted into the graph chamber.
Then the second stainless steel wire is threaded through the vessel lumen parallel to the first wire. Next, the mounted vessel is stretched to a normalized internal circumference. The final step of the procedure is to reactivate the vessel by challenging it with repeated applications of phenylephrine and relaxing it with cumulative applications of calcium.
Ultimately, the results show changes in tension in the vessel, which can be converted into a calcium response curve and used to determine EC 50 values. For comparison. We first had the idea for using this method when we discovered that the nev network of rat Masonary batteries expressed a gpro copper calcium sens receptor.
That can be studied in the same way as bitter AIC receptors, which are activated by agonist. Generally, individuals new to this method may struggle because the endothelium and smooth muscle layers is small. Vessels are easily damaged.
If this occurs, the mechanical properties of these vessels may be compromised. To begin, prepare the work area and surgical instruments. Place an anesthetized animal on its back and wipe the abdomen with alcohol.
After performing a midline laparotomy, use scissors to remove about 85 centimeters of intestine, including the superior mesenteric artery. Cut the proximal end close to the pylori and the distal end near the ileocecal junction. Place the excised section into a coated Petri dish containing physiological salt solution or PSS at room temperature.
Pin down the proximal end of the intestine to the right and then pin the remainder of the intestine in a counterclockwise direction. Next, dissect away a segment of the mesenteric artery containing branch two and three and a piece of the intestine from the proximal end. Identify the V-shaped branch point of the mesenteric artery and dissect it away from the vein.
Use forceps to gently pull away adipose and connective tissue to avoid direct contact with the artery. To begin mounting the vessel cut two artery segments about two millimeters long. The tip of a four centimeter 40 micrometer tungsten free stainless steel wire may be used to open the lumen of the artery.
Then use fine forceps to insert the wire into the lumen of each artery. Taking care not to damage the endothelium, fill the myo graft chamber with PSS at 37 degrees Celsius. Use forceps to transfer the threaded vessel segment into the chamber.
Next, transfer the excised proximal vessel segment to the myo graph chamber and pull the end along the wire to feed it into the vessel. Avoid stretching the vessel. Secure the near end of the wire counterclockwise under the near fixing screw on the right hand jaw connected to the micrometer.
Catch the free end of the wire with forceps and secure it clockwise under the far fixing screw on the right hand jaw, ensure that the vessel segment along the wire is situated in the gap between the jaws without making any contact with the jaw itself. Screw the jaws apart. Align the second wire parallel with the vessel and insert it into the far end of the lumen.
Gently feed the wire through the lumen of the vessel segment in one motion using the already mounted wire as a guide. Hold the wire about one centimeter from the vessel to avoid stretching it during the maneuver and avoid touching the endothelium. Screw the jaws together and ensure that the second mounting wire moves underneath the first one secured on the right hand.
Jaw secure the near end of the second wire in a clockwise direction under the near fixing screw of the left hand jaw connected to the transducer. Secure the far end of the wire under the fixing screw on the left hand jaw and tighten to stretch the wire. Once mounting is complete, allow 30 minutes for equilibration at 37 degrees Celsius.
After equilibration, the motor is reset from the mounting menu and the normalization procedure, which stretches the vessel to a normalized internal circumference is initiated. To begin open the normalization menu and set the target transmural pressure to 13.3 kilopascals. Next, set the duration of each of the normalization steps to 60 seconds.
The IC one to IC 100 ratio should equal 0.9 and the IP calibration should be set to two star delta. Measure the length of the mounted mesenteric artery using the microscope eyepiece readings when the hair lines are over the far and neurons of the vessel segment. After normalization, the vessels are reactivated by performing a standard start consisting of a series of stimulations and washout periods.
First, replace the myo chamber cover and start aeration with 95%air and 5%carbon dioxide. Fill the chamber with fresh PSS containing 100 micromolar ascorbic acid. Contract the vessel with five micromolar fennel, rine, or PE for five minutes and wash four times with PSS buffer After the last wash, refill the chamber with PSS and wait three minutes before refilling the chamber with PSS and contracting with PE.As shown previously, the artery is now ready for the experiment.
Measure the relaxation of contracted vessels by cumulatively adding increasing concentrations of calcium chloride. This table shows that the basal tensions following phenylephrine application varied between wistar DS or doll salt sensitive and DR or doll salt resistant rats. This trace from a wistar rat shows relaxation of PE contracted arteries by cumulative additions of calcium relaxation with severely compromised in tissues from DS rats compared to Wistar rats and compared to Dr.Rats as seen here, the differences in calcium sensitivity can be seen in the calcium response curves and again when calculating the EC 50 values.
Finally, DS rats show a reduction in the expression of the calcium sensing receptor in arteries compared to Dr.Rats Once mastered. This technique can be done in two hours if performed properly. After watching this video, you should have a good understanding of how to compare vascular reactivity of small me enteric attri to exogenous compound under isometric conditions.
