生体内でウサギの一般的な頚動脈内皮細胞への遺伝子導入

The overall goal of this surgical procedure is to introduce a transgene into the endothelial cells of rabbit carotid arteries. This method helps answer questions in vascular biology and gene therapy about the roles of arterial wall transgene products and the roles of DNA-regulatory sequences in controlling transgene expression. This technique allows the efficient expression of transgenes within a focal segment of the vascular endothelium, allowing the site-specific study of the biological roles of endothelial-expressed genes.

Generally, individuals new to this method will struggle as new investigators often puncture back wall of the artery and do not tie off all of the small side branches. After confirming a lack of response to toe pinch, apply ointment to the animal's eyes and shave both ears and the left-rear middle toe. Insert a 24 gauge IV catheter into the left ear vein and apply a fentanyl patch to the right ear.

Shave the anterior neck from the sternal notch to the edge of the mandible. Place the rabbit in the supine position on the operating table with a rolled neck support towel just below the animal's head. Gently extend the rabbit's neck until it is straight and approximately horizontal.

To begin the surgery, use electrocautery to cut the skin approximately seven to nine centimeters along the midline. Clamp the tissue open with towel clamps and make a short lateral cut through the fascia with the electrocautery. Insert large scissors into the cut to bluntly dissect the fascia along the midline and cut through the dissected fascia with the electrocautery device.

Beginning on the right side, use small dissecting scissors to dissect between the V-shaped sternocephalic muscles and the sternohyoid muscle over the trachea to expose the common carotid arteries. Dissect the right common carotid artery from the surrounding tissues from the base of the neck caudally to the crossing of the pharyngeal nerve cranially and use surgical silicon loops to aid in the retraction of the artery. Ligate any branches coming off of each side of the common carotid artery with 5-0 silk sutures.

Then cut each branch distally one to two millimeters away from the sutures to free a four to five centimeter segment of the carotid. Next, move a surgical microscope into position and drape a sterile towel over the microscope to allow manipulation of the microscope without contaminating the sterile field. Place two silk ties, each with a loose overhand knot, around the midportion of the mobilized carotid segment.

Place a vascular clip at the cranial end of the isolated artery segment to allow the artery to fill, then place a vascular clip at the caudal end of the artery. Using a 19 gauge needle bent just above the bevel at an approximately 80-degree angle, puncture the artery just cranial to the caudal vascular clip, taking care not to puncture the back or side walls of the vessel. Advance the needle tip into the lumen, withdrawing the tip twice to make sure the arteriotomy completely traverses the artery wall before carefully withdrawing the needle completely.

Next, bend an IV catheter about four millimeters from the tip to an approximately 75-degree angle and loosely attach the IV catheter to a syringe filled with sterile tissue culture medium. Insert the catheter into the artery up to the bend point and fill the artery with 250 microliters of medium, then gently press with a gloved finger along the artery from just caudal to the cranial vascular clip to the arteriotomy to flush out the medium. After a second wash as just demonstrated, tighten the two silk sutures around the catheter tip to seal the lumen and attach a syringe containing the virus solution to the catheter.

Gently depress the syringe plunger to infuse 250 microliters of the virus until the artery is distended to its physiological caliber and gently lay the syringe on a nest of gauze for support. Place a 7-0 polypropylene suture in the common carotid adventitia just caudal to the cranial vascular clip to mark the cranial boundary of gene transduction. After 20 minutes, replace the virus-containing syringe with an empty syringe and gently aspirate the virus-containing solution until the vessel collapses.

Then remove the syringe, silk ties, and catheter. Using 7-0 polypropylene suture, make a loose X-pattern across the arteriotomy. Very briefly release the cranial vascular clip to flush the artery of any air or residual virus and tighten the suture.

Release the cranial and caudal vascular clips using light pressure and gauze to stop any bleeding and close the midline fascia with a continuous suture, then close the skin with an intradermal pattern with buried knots at both ends. To harvest the transduced carotid arteries at the appropriate experimental endpoint, use 3-0 silk suture to ligate the common carotid artery cranially and caudally to the segment infused with the vector. The 7-0 suture placed in the adventitia marks the cranial end and the arteriotomy marks the caudal end.

Excise the carotid segment between the ligations and flush the lumen with saline. Then trim away any excess adventitia and cut the transduced carotid segment into smaller pieces for the appropriate experimental endpoint analyses. En face images of the lumenal surfaces of rabbit common carotid arteries infused with adenovirus expressing beta-galactosidase demonstrate a robust endothelial staining with X-gal.

Axial images of the lumenal surfaces of intact carotid rings reveal X-gal staining primarily on the lumenal surface. X-gal staining is also observed primarily in the endothelial cells of axially-opened vessels, although a small amount of staining is found in the adventitial layer. Analysis of RNA extracted from the vessel segments reveals substantial over-expression of transgenes relative to their endogenous levels, with considerable intra-and inter-artery variability in transduction efficiency and transgene expression observed among arteries transduced by experienced operators.

Once mastered, the rabbit common carotid artery gene transfer surgical procedure can be completed in less than two hours. Following this procedure, RT-PCR, immunohistochemistry, Western blotting, or other methods can be performed to answer questions about the role of DNA-regulatory sequences in altering transgene expression and the role of transgene products within the artery wall. After its development, this technique paved the way for the study of gene therapy in treatment of vascular diseases such as arterial atherosclerosis and of vein graft disease.

Don't forget proper bio-safety precautions should always be taken when working with viral vectors and sharp objects.