The overall goal of this procedure is to implant a sheer stress modifier around one of the two common carotid arteries in a mouse model of atherosclerosis. This is accomplished by first cutting the two longitudinal halves of the sheer stress modifier out of polyether ketone molding. Next, the neck of the experimental mouse is opened and one of the two common carotid arteries is exposed.
Two cuff half shells are then placed around the two common carotid arteries and the half shells are interconnected, using a surgical thread to form the conical inner lumen of the functional cuff. Finally, the animal's neck is closed. Ultimately, results can be obtained that show altered flow parameters and while shear stress, which finally causes the formation of atherosclerotic plaques upstream and downstream of the cuff.
Though this method can provide insight into the formation of atherosclerotic plaques. It can also be applied to other questions regarding endothelial dysfunction and vascular inflammation. The sheer stress modifier consists of two longitudinal halves of a cylinder with a cone-shaped lumen.
The half shells are made of thermoplastic polyether ketone, and each cast contains half shells of different sizes, ranging from 150 micrometers to 300 micrometers. To prepare the shear stress modifier working under a surgical microscope, hold the cuff half shell with blunt forceps and use a sharp scalpel to gently cut it off from the cast. Place the half shell precursor on an even non slippery plate.
Fix it with blunt forceps and cut it exactly along the preformed incision. In order to remove the closed end gently remove any remaining sharp edges. Store the cuff half shells classified according to size in 70%ethanol.
For the cuff implanting procedure. Begin with 10 week old APOE knockout mice that weigh at least 20 grams. Wear a surgical gown, mask, and cap and sterile gloves.
Sterilize the instruments for 30 seconds in a bead instrument sterilizer, and place them on a sterile opaque sheet. Anesthetize the mouse with 3%iso fluorine, and check the depth of anesthesia by performing a toe pinch. Remove the hair between the mandible and the sternum by either applying a detory agent or by using a fine electric shaver.
Place the mouse on a heated surgical plate in the supine position. Then apply is a V to prevent the eyes from drying. If using isof fluorine, place the rodent space in a mask and administer 2%ISO fluorine.
Tape down the floor and hind paws and fix the face mask. Have ready previously prepared cuff, half shells of different sizes, and a 2.5 centimeter long piece of six oh silk suture for interconnecting the half shells at the site of carotid occlusion. Visual demonstration of this method is critical as the steps showing the dissection of the carotid artery at the defined position helps to minimize tissue manipulation.
Besides, I would like to point out that the correct placing end fit of the cuff is the most important step in order to generate reproducible results. In order to ensure this, we have defined a precise anatomic position as the implantation site. In addition, the correct fit of the cuff is facilitated by a groove on the outer surface of the cuff, half shells, which serves as a guide for the interconnecting threat.
Disinfect the skin with a liberal amount of Betadine. Use small, sharp scissors to open the skin and the underlying fascia of the neck with a four to five millimeter medial incision starting from the top of the sternum, dilate the opening shift the right parotid gland aside and insert expanders to expose the surgical area. Then bluntly dissect inwards just left of the trachea where the right sternal mastoid muscle crosses the right omohyoid muscle to locate the pulsating right common carotid artery.
Using very fine angled or curved forceps. Dissect the right common carotid artery by gently removing the surrounding connective tissue. Separate the carotid artery from the vagus nerve, the white stringy object running directly along the carotid artery.
As this step is necessary to completely expose the vessel, take care not to harm either the vagus nerve or a branch of the right internal jugular vein, which is also in close connection to the carotid. In order to choose the right cuff size, compare the diameter of the exposed carotid artery with the inner diameter of the cuff, half shells, the largest width of the cuff's lumen should meet the outer diameter of the carotid. Next, carefully put the tip of the forceps under the carotid artery.
Open the forceps. Thread the piece of six oh silk suture under the artery and form a loop between the loop and the carotid place, one cuff half shell beneath the carotid, the side of largest narrowing has to be downstream. Place the second cuff, half shell within the loop on top of the carotid artery.
Gently tighten the suture loop and fasten the thread. By doing this, the functioning sheer stress modifier is formed. For the precise fitting of the cuff, it is essential that the suture is running exactly within the preformed groove on the outer surface of the cuff.
The conical constriction of the artery should now be clearly visible. The cuff should always be placed around one of the two common carotid arteries of an animal, and the contralateral side serves as a control. Here, the conical shape of the inner lumen is visible.
This conical shape is essential for establishing the three regions with distinct flow dynamics. Move the right parotid gland back in its original position. Approximate and close the skin using either a small amount of six oh proline sutures or wound clips.
Inject a single dose of five milligrams per kilogram of carprofen subcutaneously to provide prophylactic pain treatment and place the mouse in a warming chamber until it recovers. If the animal is experiencing distress the following day, administer a second dose of analgesia. Typical shear stress patterns induced by the cast calculated from doppler measurements and the corresponding flow velocities based on phase contrast velocity.
MR.Imaging are shown here when the cuff is implanted in a P OE knockout, mice fed a western type diet. The altered flow dynamics provoke sheer stress-induced atherosclerotic plaque deposition upstream of the conical constriction. Low laminar shear stress leads to massive development of atherosclerotic plaques of a more vulnerable phenotype characterized by lipid cores close to the central lumen covered only by a thin fibrous cap.
The conical inner lumen of the cuff leads to an increase in flow velocity. Nearly no plaque deposition is observed in this area directly downstream of the site of the bottleneck. The immediate broadening of the artery results in an area of vortices and oscillatory flow parameters, which causes less extended plaque development of a more stable phenotype.
After watching this video, you should have a good understanding of how to properly place a shear stress modifier around the common carted artery of a mouse.
