動静脈瘻の障害の新規マウスモデル：詳細の外科的手順

The overall goal of this procedure is to create an in vivo animal model that mimics the pathophysiology of arteriovenous fistula failure. This method can be used to unravel the pathophysiology of arteriovenous fistula failure and can help researchers from all over the world to develop new therapeutic strategies to prevent vascular access related complications. The main advantage of this technique is that it incorporates a veinous end to arterial site anastomosis that closely resembles the clinical situation that is utilized in humans.

The procedure will be demonstrated by Chun Yu Wong, an excellent PhD student from my laboratory. After anesthetizing a one to three month old mouse with isoflurane, shave the hair off the ventral side of the neck and the inner region of the upper left leg. Use tape to clean up the loose hairs.

Next, apply opthalmic ointment to both eyes then prepare a 37 degree Celsius heating blanket with a fixed nose ventilation mask and secure the head of the animal in position with adhesive tape. To the mask, send 1.5%to 2%isoflurane at a rate of 0.3 liters air per minute along with 100%oxygen at a rate of 0.2 liters per minute. Pinch the skin between the toes to assess the depth of anesthesia.

Adjust the concentration of isoflurane if needed. Then, fix the mouse on the blanket in a supine position by taping down it's limbs. Now subcutaneouly inject a bolus of buprenorphine in a half milliliter of 0.9%sodium chloride into the left flank.

Complete the preparation by cleaning the exposed skin for incision using 0.5%chlorhexidine. Under a dissection microscope start with a longitudinal incision along the midline of the neck, make it about 1.5 centimeters long exposing the salivary glands. Move the right salivary gland cranially until it is partially outside of the wound to uncover the right jugular vein.

Next, find the dorsomedial branch of the vein and bluntly dissect it from the perivascular tissue by spreading the tissue just alongside the blood vessel. Then, place a loop of 10/0 suture around the dissected vein and add a loose knot. The next major step is to remove the sternocleidomastoid muscle.

Begin by using a pair of forceps to bluntly dissect around the right sternocleidomastoid muscle. Then, ligate the muscle with two 6/0 suture loops and excise it using a cauterizer. Moving on, dissect and prepare the common carotid artery.

Once identified, place a 6/0 suture around it and then, place vascular clamps as far distally and proximally as possible on to the artery. Then, with microscissors, make a one millimeter longitudinal incision into the artery. Once incised, rinse the artery with heparin solution to remove the blood then measure and adjust the length of the incision to match the width of the vascular clamp.

Now position a thread around the vein and attach a vessel clamp proximally. Apply gentle traction caudally using a hemostat and ligate the vein as distally as possible using the already positioned 10/0 suture. Once ligated, cut the vein just proximal of the ligation to access the lumin.

Gently dilate the lumin with the vascular forceps and rise the vein out with heparin and proceed with the anastomosis. To start the anastomosis connect the vein to the artery with an interrupted 10/0 suture, using two square knots at the 12:00 position followed by a suture at the 6:00 position. Make sure there is no torsion of the vein.

Next, position a suture thread around the vein and apply gentle, lateral traction. Then, add three or four more interrupted sutures at the visible ventral side of the anastomosis to complete the connection. In order to create a patent arteriovenous fistula it is of utmost importance to prevent torsion of the vein and to prevent needle placement through both the anterior and posterior vessel wall during the suturing procedure.

Now, rotate the heating blanket 180 degrees and focus on the left upper leg. Identify the femoral bundle by looking for a vascular structure that runs longitudinally in the medial part of the upper leg. It can be seen through the skin.

Make an approximately one centimeter incision over the femoral vein in a lengthwise manner and carefully dissect the perivascular tissue of the femoral vein. Then, inject a dose of heparin into the femoral vein. Now, return to the neck area and remove the suture thread that was placed around the vein, follow this by placing a 6/0 suture thread below the carotid artery over the vein and again below the carotid artery.

Now, remove the veinous vascular clamp. Next, twist the vascular clamps and apply traction to the suture thread to turn the half completed arteriovenous fistula 180 degrees around the carotid artery in a clockwise manner. Complete the anastomosis using interrupted sutures as previously demonstrated.

Adequate of the posterior wall of the anastomosis is crucial in order to complete the second part. The use of a strategically placed suture thread eases this portion of the procedure. Now, remove the suture thread and remove the twist in the arteriovenous fistula.

Following this, remove the distal vascular clamp and then remove the proximal vascular clamp. Assess the anastomosis by gently occluding the veinous outflow tract with vascular forceps. If it is patent, then the pre-occluding veinous section will expand in a pulsatile fashion.

Patency can also be confirmed with near infrared fluoroscopy. A failure can lead to an occlusion caused by too narrow of an anastomotic area, torsion of the vessels, inadequate heparin dose, or an accidental suture placement that connects the front side of the anastomosis to the back side. To proceed with the surgery, relocate the salivary gland to it's original anatomical position and close up the two skin incisions using uninterrupted 6/0 suture.

Allow the animal to recover in a darkened, heated cage after administration of 0.5 milliliters of 0.9%sodium chloride injected subcutaneously. Allow the animal to fully recover in a dark cage under a heat lamp. At a histological level, vascular remodeling in the AVF can be investigated elegantly using this model.

Vascular remodeling in AVF occurs as a result of the increase in blood flow and pressure. In mice, this results in an increase in circumference and luminal area during the first two weeks after surgery. Immunostainin of the veinous outflow tract shows that the cellular compartment of the intima mainly consists of alpha smooth muscle actin positive cells.

Two weeks ends up being the optimal time to harvest the AVF as after four weeks, approximately 50%of the AVFs are occluded. After watching this video you should have a good understanding of how to perform the surgical procdure and what the crucial steps are in this animal model. Once mastered this procedure can be performed in one hour.

This technique offters new opportunities for researchers in the field of arteriovenous fistula failure to explore its pathophysiology. This model will also help to develop new therapeutic strategies to reduce the morbidity of patients with end stage renal disease that requite chronic hemodialysis.