This method can answer key questions about the pathogenesis of vein graft failure. For example, it could identify mediators of graft failure and test strategies aimed at preventing graft failure. The main advantage of this technique is its ability to achieve durable, stable transgene expression in the vein graft endothelium.
After anesthetizing a rabbit according to the text protocol, shave the animal from the sternal notch to the edge of the mandible. Upon completion of the surgical setup use electrocautery to cut the skin along the midline between the sternal notch and the mandible, approximately seven to nine centimeters in length. Then attach towel clamps to hold open the incision.
Bluntly dissect under the fascia along the entire midline. Then use electrocautery to cut through the dissected fascial layer along the midline. Next, beginning on the right side, dissect between the sternal hyoid muscle overlying the trachea and the sternoephalic muscle to expose the common carotid artery.
Then carefully dissect a four to five centimeter segment of the common carotid artery and its larger branches free from surrounding tissues. Use 5-0 silk sutures to ligate the larger branches of the common carotid artery before cutting each branch distal to the suture. After repeating the dissection on the left common carotid, use tissue holding forceps to temporarily close the subcutaneous tissue layer.
Then dissect the superficial fascia from the skin on the right side until the external jugular vein is exposed. Dissect a four to five centimeter segment of the external jugular vein and free its branches from the surrounding tissue. Use 5-0 silk sutures to ligate the branches, then cut the branches.
Next ligate the isolated vein at both ends, cut the cranial end, and flush the lumen with heparinized saline. To carry out vein grafting, use a vascular clip to clamp the right common carotid artery at the cranial end of the isolated segment. Then place a vascular clip on the caudal end.
Now perform a caudal arteriotomy just cranial to the caudal clamp equal in length to the diameter of the cranial end of the vein segment. Then insert an IV catheter connected to a syringe of heparinized normal saline through the caudal arteriotomy and flush the carotid artery lumen. The fluid will exit via the arteriotomy.
Returning to the jugular vein, excise the vein and lay it next to the carotic artery. Using single stitches of 7-0 polypropylene suture the caudal end of the caudal arteriotomy to the cranial end of the vein segment. Then place a second stitch 180 degrees from the first stitch to suture the cranial edge of the caudal arteriotomy to the cranial end of the vein segment.
Place a third stitch at a point equidistant from the first two stitches on the lateral side of the anastomosis. Then place a fourth stitch on the medial side of the anastomosis equidistant from the first two stitches and across from the third stitch. Place four additional stitches between each of stitches one through four.
Perform the cranial arteriotomy on the caudal side of the distal clip. The distance between the cranial end of the cranial arteriotomy and the caudal end of the caudal arteriotomy should be the same as the length of the vein segment. The length of the cranial arteriotomy is equal to the diameter of the caudal end of the vein segment.
With the vein extended cranially from the anastomosis along the carotid, ensure the vein is not twisted. Then use heparinized normal saline to flush the carotid artery and vein via the cranial arteriotomy. Next place a first stitch to suture the caudal end of the cranial arteriotomy to the caudal end of the vein segment.
Then place a second stitch 180 degrees from the first stitch to suture the cranial edge of the cranial arteriotomy to the caudal end of the vein segment. Complete the anastomosis as demonstrated earlier in the video. Temporarily release the cranial clip to allow the vein graft to fill with blood and apply gentle pressure with dry gauze to stop any bleeding.
Repeat this step until the anastomosis no longer bleeds. Then remove both the cranial and caudal clips. Use gauze to stop any additional bleeding at the anastomoses.
Use a 3-0 silk suture to ligate both ends of the common carotid artery segment between the anastomoses. Then divide the carotid halfway between the ligatures. Brisk pulsations should be seen in the vein graft.
Repeat the vein graft placement on the left side. With a 5-0 PGA suture, close the subcutaneous tissue with a continuous pattern. Then use a 3-0 PGA suture to close the skin with a intradermal pattern and bury the knots on both ends.
Carry out post operative care and transcutaneous ultrasound according to the text protocol. To perform gene transfer surgery, carefully isolate the right vein graft including 1.5 to two centimeters of the carotid artery adjacent to the graft on both the cranial and caudal sides. Repeat the dissection on the left side in the same manner.
Use normal saline to fill the surgical wound cavity on the right side to enable the transmission of sound waves. Connect a two millimeter perivascular flow probe to a volume flow meter and immerse the probe in the saline in the wound cavity. Set the flow rate to zero.
Place the flow probe around the carotid artery either caudal or cranial to the vein graft. Then use an electronic data acquisition system to record data from the flow probe. After injecting heparin into the rabbit via the the IV catheter and flushing it with saline, use a vascular clip to clamp the common carotid artery at the cranial end of the vein graft.
And then apply a second clip to the carotid approximately 10 millimeters caudal to the caudal anastomosis to leave room for the arteriotomy. Then place two silk ties loosely around the carotid artery just caudal to the vein graft. Next using a previously prepared bent 21 gauge needle, puncture the carotid caudal to the graft and just cranial to the caudal vascular clip.
Advance the needle into the lumen back and forth twice to ensure that the lumen is clear. Then carefully withdraw the needle. After preparing a syringe and IV catheter with DMEM according to the text protocol, insert it into the common carotid arteriotomy up to the bend point and wash the vein graft lumen by filling it with 0.5 milliliters of DMEM two times.
Remove the DMEM from the graft by using a gloved finger to lightly press at the cranial end of the graft. Then carefully slide the finger along the graft toward the caudal end of the graft to flush the luminal contents out via the arteriotomy. Remove the DMEM syringe from the catheter leaving the catheter in the vessel.
Connect the syringe containing the virus solution making sure that no air enters the catheter. Move the loosely knotted silk ties down the artery until they are around the catheter tip but do not tighten them. Infuse 0.03 milliliters of virus solution to push the remaining DMEM out of the catheter.
Then using a finger, remove all fluid from the vein graft lumen pressing gently from cranial to caudal. Tighten the two ties around the artery and catheter tip to seal the lumen. Then infuse the virus solution until the vein is distended.
Gently lay the syringe down on a nest of gauze. After 20 minutes, use an empty syringe to gently aspirate the virus containing solution from the graft until the vessel collapses. Remove the syringe leaving the catheter in place.
Then cut and untie the silk sutures and withdraw the catheter from the vessel. Now use 7-0 polypropylene suture in an X pattern to close the arteriotomy. Then gently pull the suture ends and tie them together with two square knots.
After restoring blood flow in the right vein graft as described in the text protocol, repeat the virus infusion on the left side. To harvest the transduced grafts, dissect free the vein grafts and common carotid arteries and make flow measurements on both the left and right sides as demonstrated earlier. Use 3-0 silk suture to ligate the right common carotid artery cranial to the graft.
Then use 3-0 silk suture to ligate the carotid caudal to the graft. Excise the right vein graft by dividing the adjacent carotid artery between each of the ligations and the adjacent anastomosis. Then remove the vein graft from the rabbit and use saline to flush the lumen.
The first milestone that a new operator must achieve is consistent vein graft patency after both the initial vein grafting surgery and the subsequent delayed transduction surgery. When a vein graft is patent, the ultrasound exam will reveal a large blood vessel with brisk caudal to cranial blood flow. The grafts were harvested three days after the transduction, cut into sections and stained with X-gal.
En-face imaging of the luminal surfaces showed vein grafts with efficient transduction and poor transduction. As shown in this plot, the levels of beta galactosidase mRNA in vein grafts transduced by the new operator, were not significantly different from the levels of beta galactosidase mRNA in vein grafts transduced by an experienced operator. When performing this protocol, it is important to remember to reverse the vein graft cranial to caudal before contracting the anastomosis.
This is necessary to avoid obstruction of blood flow by venous bolus. Once mastered these surgeries can be completed in about three hours for the vein grafting procedure, two hours for the vein graft gene transfer procedure and 45 minutes for the harvest procedure. Don't forget that because this protocol requires working with viral vectors and sharp objects, proper biosafety and sharps procedures must be followed.
