Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The overall goal of this procedure is to provide an in vivo platform to study the molecular underpinnings of vascular diseases in a rat. The left side carotid is exposed and temporarily cut off from the bloodstream. The second step is to insert a balloon catheter into the lumen and perform a balloon injury.

Next, a test reagent is perfused into the injured vessel lumen. The final step is to ligate the external carotid artery and return the flow of blood. Ultimately, the injured artery is collected two weeks later and used to investigate morphological and cellular responses to the injury and the relative effect of the test agent.

This method can help answer key questions regarding the molecular mechanisms of smooth muscle remodeling, which occurs during cardiovascular diseases such as restenosis, atherosclerosis, and hypertension. Generally, individuals new to this method will struggle because they will find difficulties in locating the interlock carotid artery. And in inserting balloon catheter into artery lumen, Be sure to use sterile technique.

All surgical instruments should be autoclave or sterilized by dry beads, and the saline solution should be sterilized by filtration. After anesthetizing the rat and verifying a surgical plane of anesthesia by a toe pinch, inject three milliliters of saline subcutaneously for hydration. Then apply ophthalmic ointment by a cotton swab to both eyes.

Now, place the rat in a supine position over a warm heat pad on the surgical platform there. Remove the hair from the ventral neck region using hair removal ointment completely Wipe off the ointment after 30 seconds and swap the skin clean with alternating applications of iodine and 70%ethanol. Now Don protective equipment.

Ready the tools and drape the rat. Then proceed with the surgery Every 15 minutes. During the surgical procedure, recheck the animal's response to a toe pinch.

If there is any response, administer a 10%dose of anesthetic cocktail. Begin the surgery with the dissection of the common carotid artery. First, use a scalpel to make an incision through the neck.

Then bluntly, dissect the connective tissue from the skin, being careful to avoid puncturing the underlying tissues. Second, dissect the muscle layer along the left side of the trachea to visualize the left common carotid artery and the vagus nerve attached to it. Then carefully separate the nerve from the artery so as not to damage or stretch the nerve.

Now, continue the dissection distally to the arterial bifurcation. Then continue along the two branches until one and a half to two centimeters of the artery have been isolated. Prepare to make the balloon injury by first ligating the ECA at the proximal side of the superior thyroid branch.

Next, ligate the occipital branch of the ECA. This is very close to the bifurcation of the ECA and ICA. Then clip on the proximal end of the CCA and the distal end of the IC.A blood flow should now be stopped permanently through ligation and temporarily through clipping.

Thus, the luminal contents in the bifurcation area have been isolated. Now using small micro scissors, make an artery otomy incision on the ECA close to the distal suture knot as needed, clean the blood with saline and cotton swabs. Make sure you have isolated a long portion of the external carted artery because the balloon catheter will enlarge the incision when you push it forward and pull it back.

The next step is to insert an uninflated two F balloon catheter into the ECA lumen. Advance the catheter proximally to the CCA lumen until its tip reaches the position of the clip. Then connect the balloon inflator to a female lure lock on a three-way stop cock and connect a male lure lock of the three-way stop cock to the balloon catheter.

Now using about one and a half atmospheres of pressure, slowly inflate the balloon, distending the carotid artery to about one and a half times its initial diameter. Once inflated, gently pull the balloon back while rotating it to the bifurcation. Then deflate the balloon and advance it back to its proximal end.

Repeat this inflation tugging deflation process two more times. Then withdraw the balloon catheter from the artery lumen. For this demonstration.

Lentiviral particles encoding short hairpin RNA toim one are tested using an intravascular over the needle catheter with a syringe aspirate 30 microliters of the testing reagents. Then insert the catheter into the ECA incision and advance it into the CCA. To secure the catheter, use an knotted loop of suture on the EC to close the incision temporarily.

Next, inject the testing reagent and let it incubate in the CCA lumen for 30 minutes. During this time, do not let the tissues dry out. Never leave the animal alone during the 30 minute incubation period.

Make sure the rat is sedated and the incubating solution remains in the lumen without any leakage. After the incubation, aspirate the solution out of the vessel. Loosen the knot and remove the catheter.

Then tie off the ECA with a piece of suture proximal to the incision and as close as possible to the bifurcation because the injury may cause a leak or puncture blood vessels. Remove the clip on the ICA inspect for any damage and staunch any bleeding. Then remove the clip on the CCA and repeat the process with all bleeding cared for.

Remove the clamps, other surgical instruments and excess suture. Then close the wound with skin sutures. Follow with the gentle scrub of antiseptic bactericide or ide.

Agent for hydration. Administer three milliliters of sterile saline subcutaneously. Now, observe the rat on a heat pad, administer a dose of buprenorphine intramuscularly and until it is awakened ambulant, keep the animal's eyes and mouth moist.

When it is fully recovered. Return the rat to the animal room and house it individually. Two weeks post-injury, carotid arteries were cross sectioned and stained with h and d Normally, vessel walls contain four layers of elastic lamina, which appear as pink lines in tissue subjected to the balloon injury.

There is obvious neointima formation and adventitia and media thickening. The thickness of the neointima is of equal size or greater than the thickness of the media in the same artery stem one S-H-R-N-A was used to test the role of STEM one in neointima formation First DPI and stim one immunofluorescence were checked to confirm the knockdown of stem one expression in the experimental condition. H and d standing then revealed that stem one expression decrease the neointima of the injured vessel compared to the control.

It is important for surgeons to be cautious and not overinflate. The balloon Improper ballooning causes eccentric as opposed to concentric neointima formation. Excessive balloon damage can result in a rupture of the vessel wall resulting in a robust thrombus formation both in the lumen and on the outer surface of the artery.

Once mastered, this technique can be done in one and a half hours if it is performed properly. The use of this in vivo technique has really allowed the researchers study in cardiovascular diseases to explore the molecular mechanisms involved in vascular smooth muscle remodeling, and determine the suitability of novel therapeutic targets for controlling neointimal hyperplasia. In an in vivo se.