A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

This video demonstrates a partial carotid ligation surgery in which the external carotid artery, the internal carotid artery and the occipital artery are ligated in an anesthetized mouse to induce disturbed flow conditions and subsequent rapid atherosclerosis development. The success of the surgery is verified by ultrasound, then ligated and controlled carotids are taken from the euthanized mouse and high purity endothelial RNA is obtained. Hi, my name is Doug Na, from the laboratory of hon Jo at the Biomedical Engineering and Division of Cardiology at Georgia Tech and Emory University.

Hi, my name is Amir Revan. Also from the JO Lab. I'll be demonstrating the ultrasound verification of ligation And I'm CHI also from the jaw lab.

I will demonstrate the internal RNA preparation Today. We'll show you a procedure for partial carotid ligation, ultrasound, verification of the ligation and a method for intimate RNA isolation. We use this procedure in our laboratory to study the effects of sheer stress on atherosclerosis.

So let's get started. Before beginning this procedure, verified the depth of ketamine and xylazine induced anesthesia. In an eight week old mouse by toe pinch, place the mouse on a surgical stage in the subline position, tape the four paws down to the stage, palms up.

Then tape the hind paws, soles down. Use a gauze as a cushion for the mouse's neck. Next, apply a liberal amount of depilatory agent to the animal's neck between the mandible and the sternum.

Gently massage the area until all the hair is removed. Using a cotton applicator, clean off the depilatory agent. Then apply a liberal amount of Betadine using a small pair of sharp scissors.

Make a four to six millimeter vertical incision in the middle of the animal's neck, cutting both the skin and the underlying fascia. Using dissecting forceps, gently move the left carotid gland from the midline to the left of the animal. Bluntly, dissect the tissue using dissecting forceps.

To reveal the pulsating common carotid. Follow the common carotid coddly. To locate the bifurcation.

Carefully dissect the bifurcation to expose the following. Four distal branches of the left common carotid artery, external carotid artery, internal carotid artery, occipital artery, and superior thyroid artery. These vessels, especially superior thyroid, can be very fragile.

Pass one set of forceps underneath the external carotid above the superior thyroid and grab a one inch long piece of six oh silk suture from another pair of forceps. Gently pull the suture to pass it underneath the artery. Tie off the external carotid firmly avoiding any surrounding tissue.

Using the same technique, tie off the internal carotid and occipital arteries with a single knot. After verifying the position of sutures and patency of superior thyroid artery by visual inspection, move back the parotid gland, approximate the skin and use tissue men to close the skin. Following the surgical procedure, place the mouse in a warming chamber until it recovers.

Typically 30 to 60 minutes. The mice can then be moved to their regular housing area. Place some food at the bottom of the cage as the mice may feel uncomfortable reaching up for food during the first day.

A single dose of buprenorphine should be administered the next day for pain relief. One day post ligation. Prepare the isof fluorine anesthetized animal for ultrasound by placing it on the imaging stage belly side up.

Tape the arms and legs to the ECG sensor. Apply echo gel to the animal's neck. The animal's body temperature should be monitored throughout the procedure.

Turn on the computer and the imaging software. Select the appropriate user and echo probe. Set the ultrasound to the bee mode.

Then with the knob of the probe pointing toward the nose of the mouse, lower the probe onto the mouse's neck until an image is obtained. Manipulate the probe stage right and left until the trachea can be identified. Then find the left common carotid artery and tilt the imaging stage so that an angle is created between the imaging plane and the carotid.

Switch the ultrasound from B mode to pulse wave doppler keeping the probe in the middle of the left common carotid. Use the angle correction knob to adjust for the angle such that the angle correction line is parallel to the common carotid artery. Take an image by pressing the frame store button.

The velocity profile of the blood in the common carotid will be documented. A reversal of the velocity profile during diastole is expected. Repeat the proceeding steps on the right common carotid for comparison.

The velocity will be higher and no reversal seen in the right common carotid. Once satisfactory images have been obtained, turn off the anesthesia and wipe the echo gel off the mouse. Free the mouse from tape restraints.

Place the animal in a warmed recovery chamber. Recovery from inhaled isof fluorine usually occurs in less than five minutes. Tape the pause of a carbon dioxide euthanized mouse to a paper towel using scissors.

Cut the skin of the mouse from the abdomen to the top of the thorax. Then using a sharp pair of scissors, open the abdominal wall below the rib cage using forceps. Lift the sternum and cut the diaphragm.

Cut away the rib cage to expose the heart. Then cut the vein of CVA next, using a butterfly perfusion kit, pressure perfuse for two to three minutes with normal saline containing 10 units per milliliter heparin through the left ventricle until the lungs and liver become pale. Following the saline perfusion, cut the skin of the neck and remove all the fat muscles and connective tissues until the carotid arteries are exposed.

Now place the mouse under a dissecting microscope using a 29 gauge needle punctual hole right below the ligation sites in the left carotid for the second perfusion. Pressure perfuse again for about one minute through the left ventricle, making sure the left carotid is well perfused following the second perfusion. Use fine tip forceps and small spring scissors to carefully remove the peri advent tissues surrounding the carotids.

Be careful not to squeeze or stretch the carotids during this cleaning step now between the aortic arch and the ligation points above the carotid bifurcation. Cut the left carotid artery then between the right subclavian and artery branching point and the carotid bifurcation. Cut the right carotid artery.

Transfer the carotids to a 35 millimeter culture dish containing ice cold HBSS. Carefully remove any remaining peri adventitial tissue. Insert an insulin syringe with a 29 gauge needle into one end of a carotid and grasping the carotid and the tip of the needle with forceps.

Quickly and carefully. Flush each carotid with 150 microliters of cazo lysis buffer collecting the buffer in a 1.5 milliliter tube labeled intima eit. Finally, rinse each carotid once in HBSS.

Then place it in a 1.5 milliliter micro fuge tube labeled media plus adventitia and snap. Freeze it in liquid nitrogen. RNA can then be extracted from the frozen samples using gin's.

Micro RZ mini kit. Ultrasound examination of a mouse was carried out one day following partial ligation of the left carotid artery as shown in this figure, successful partial ligation reduces blood flow velocity and reverses flow in the left common carotid artery during diastole partial ligation of APOE knockout mice on a high fat diet induces robust atherosclerosis in the left common carotid artery, but not in the contralateral right carotid by two weeks. Atherosclerosis is shown using oil red o staining for lipids and hemat toin counterstain for nuclei.

RNA collected from the carotid intima contains endothelial marker genes such as pcam one medial smooth muscle cell marker. Genes such as alpha SMA are absent in the RNA collected from the remainder of the artery marked m plus A for media plus adventitia smooth muscle marker is present while endothelial marker is absent. We've just shown you how to perform a partial carotid ligation.

Use ultrasound to verify the ligation and also a method for intimal RNA isolation When doing this procedure as essential to properly identify the branches of the common correct artery and assured that the superior thyroid artery reman pattern while other the branches are tightly ligated. It is important to verify the ligation result using ultrasound during, in a simple preparation, pay attention so that the flushed iLet does not come into contact with any other part of the artery except the lumen. So that said, thank you for watching and good luck with your experiments.