Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

The overall goal of this procedure is to isolate cross sections of bovine mesenteric arteries and veins in a multim graph to permit the screening of experimental compounds for their vaso activity. This is accomplished by first collecting a mesenteric tissue sample from the animal. In the second step, the sample is cleaned and the vessels are sectioned.

Next, the sections are mounted and equilibrated on the myo graft. In the final step, the isolated vessels are exposed to the compounds of interest. Ultimately, this artery and vein bioassay can be used to evaluate the vaso activity of the compounds and the effects they have on blood flow within bovine mesenteric blood vessels.

The main advantage of this technique compared to existing methods like measuring blood flow in vivo, is that with this technique, tissue from a single animal can be exposed to multiple compounds simultaneously. Although this method can provide insight in a very specific animal syndromes that can cause vasoconstriction such as fescue, toxicosis, or Ergotism, it can also be applied to other scenarios that may be of interest to the researchers, such as pharmaceuticals that are being developed or natural products. Additionally, it could also be applied to not just cattle vasculature, but also vasculature from really any other mammalian species.

After removing the blood vessels from the mesenteric tissue, use a pair of forceps to place the tissue sample on a cutting surface and partially submerge it in ice cold crebs henzel hens aite buffer. Then using number five jeweler's, forceps and noise, Iris scissors carefully dissect away the fat and connective tissues surrounding the vessels. After separating out the artery and vein, identify the vessel opening at one end of the section and carefully grasp the fascia surrounding the vessel with the forceps.

Then slide the tip of the scissors underneath the raised fascia to make a cut with the scissors parallel to the vessel. After the initial incision, cut down either side of the vessel to further detach the fat and connective tissues, and to clean the vessel. Place the cleaned blood vessels in a fresh tube of Krebs Henzel light buffer, and store the tube at four degrees Celsius for up to 24 hours.

When ready, use a tissue slicer to obtain consistent cross sections of the vessel of interest into the desired number of two millimeter sections, and then examine each section at a 12.5 x magnification to ensure that each sample has no abnormalities, branches, valves, or superficial damage acquired during the dissection and cleaning process. Store the acceptable sliced vessel sections submerged in Krebs Henzel light buffer at four degrees Celsius for up to 30 minutes. Then gently insert the supports through the lumen of each sample, and use the micro positioner on the myo graft to gradually increase the tension to mount the vessel onto the myo graft.

Taking care not to stretch the vessels above a two to three gram reading. Once all the chambers are covered, vacuum the buffer from each chamber and refill them with five milliliters of fresh buffer. After all the vessel sections have equilibrated for 1.5 hours to achieve a stable resting tension of one gram at 500 microliters of 1.32 molar potassium chloride to a 0.12 molar final concentration to the 5.5 milliliter solution, bathing the vessel.

Do not adjust the tension manually, as the maximal response to the potassium chloride will be used to normalize the treatment data, replace the solution with fresh Krebs Henzel light buffer in 15 minute intervals until the tension has returned to the baseline value of one gram. Once the tension has returned to baseline, add the standards in 25 microliter aliquots to each chamber according to the experimental protocol. When the last standard has been added, start a nine minute timer.

At the end of the incubation, remove the treatment containing buffer from the chambers and add five milliliters of fresh buffer to each chamber. Then start a 2.5 minute timer after completion, repeat this 2.5 minute rinse. A second time at the end of the second rinse, aspirate the chambers.

Then after adding fresh buffer once more, start a one minute timer to count down to the commencement of the second standard edition. As observed in the nine minute incubation periods illustrated here, there are incremental increases in the response to the standards that with the increasing concentrations of the treatment, the potassium chloride addition at the conclusion of the experiment is essential as it confirms the viability of the tissue. This bioassay can be used to evaluate the vaso activity of the experimental compounds on the blood vessels that supply nutrients to the small intestine, as well as on the blood vessels that carry the absorbed nutrients away.

For example, in this experiment, there was no difference observed in the arterial or venous contractile responses after five hydroxy tryptamine treatment. Conversely, the contractile response of the mesenteric vein to the increasing concentrations of norepinephrine was greater than that of the mesenteric artery. Following this procedure, the method could be further modified to include agonists or antagonists in the buffer that were of specific interest to the researcher.

This would allow the researcher to better understand the involvement of very specific receptors and the mechanisms behind the vascular responses observed during the myo experiment. After watching this video, you should have a good understanding on how to collect, process and isolate blood vessels. For a my experiment.