Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

The overall goal of this procedure is to implant a gene eluding stent in the common carotid artery of a laboratory rat and to determine the extent and location of ensuing transgene expression over time. To do this, an arter otomy is made in the external carotid branch to gain vascular access to the common carotid artery. An inflated two French Fogarty catheter is passed across the common carotid artery to denude the endothelium and injure the medial layer.

An add Luke eluding stent is then mounted on an angioplasty catheter inserted into the mid segment of the balloon injured common carotid artery and deployed finally to determine arterial luciferase expression. Luciferian co-formulated with onic gel is applied to the perivascular region and bioluminescence imaging is performed. Ultimately, results can be obtained that allow comparison of different gene eluding stent formulations in regards to the extent and duration of vascular tissue transduction.

The advantage of preventing instant restenosis and stent thrombosis using gene therapy is that it's the most direct way to monitor exogenous gene expression from vial vectors and stented arteries. While our strategy for tethering adenoviral vectors to stents is completely understandable as text nuance of the surgical procedure for stent implantation in rats is clear. When demonstrated, visually Prepare, add Luke eluding stents as described in the accompanying written document.

Store the virus derivate stents in sterile PBS at four degrees Celsius for no longer than 24 hours prior to use. After anesthetizing male Sprague Dolly rats determine the depth of anesthesia by paw pinch response and muscle tous shave the neck and upper chest region. Apply ophthalmic vet ointment to prevent dryness of the sclera, administer antibiotic analgesic and saline.

Next, catheterize the tail vein with a 24 gauge catheter and administer heparin aseptically. Prepare the neck and upper chest region with povidone and ethanol. Then place the animal supine with the head pointing to the operator.

Then place sterile drapes using a scalpel. Perform a midline incision through the skin and neck fascia. Use blunt dissection techniques to isolate the left external carotid Artery.

The most critical aspect of this procedure is preparing the artery for our ectomy. To do that, isolate the external carotid artery and clean it of all fashion. Tie off the external carotid artery at the most distal approachable site.

Then apply a sliding temporary ligature of four oh silk to the origin of the internal carotid artery. Next, using spring scissors, make a two millimeter arterio incision in the left external carotid artery through the incision. Insert a two French fogerty catheter into the common carotid artery.

Inflate the tip of the catheter with saline. Then to denude the endothelium, pass the inflated balloon three times from the aortic arch to the carotid bifurcation. Slide a piece of tubing over the fogerty catheter and into the common carotid artery.

Withdraw the Fogarty catheter mount and crimp and add Luke Derivate stent over the balloon of a 1.5 millimeter diameter angioplasty catheter. Insert the stent through the Teflon tubing and advance it into the mid segment of the common carotid artery. Avoid rubbing the stent against the tube or vessel wall.

To deploy the stent, connect the angioplasty catheter to an in deflator pressure gauge and apply 12 atmospheres pressure for 30 seconds. Withdraw the angioplasty catheter. Next, tie off the external carotid artery proximal to the Arter Otomy site, and release the temporary ligature on the internal carotid artery.

Repair the operative wound in layers with running four oh Vicryl sutures and staple the skin. Place the animal on a warming pad for recovery until it is ambulatory. Then return it to its isolated cage.

While no signs of pain or discomfort are typically exhibited by the postoperative animals beyond the first 12 hours after the procedure, consider extension of meloxicam therapy for 72 hours at time. Points ranging from one day to three weeks after stent deployment. Prepare for bioluminescence imaging by opening living image version 4.2 or higher software for the ivus spectrum apparatus.

Initialization of the imaging hardware takes about 10 minutes. Next, prepare a mixture of 50 milligrams per milliliter Lucifer in PBS and 25%onic F1 27 in PBS one to four, volume to volume, and store it on ice. The solution should be viscous.

After anesthetizing the rat with isof fluorine and checking the depth of anesthesia, remove the surgical staples aseptically. Prepare the site and reopen the operative wound. Administer three milligrams per kilogram of meloxicam subcutaneously using blunt dissection, regain access to the left common carotid artery and separate it from the vagus nerve and adjacent connective tissue.

Using a pipette, apply 200 microliters of the chilled luciferian onic mixture directly to the exposed segment of the common carotid artery. Upon contact with the tissue at 37 degrees Celsius, the solution should gel. Next, place the animal in the supine position in the imaging chamber of the ivus spectrum apparatus, and place a face mask to maintain isof fluorine anesthesia.

Then in the acquisition control panel window of the software, choose position B, which corresponds to a 6.6 centimeter camera to object distance from the field of view dropdown menu, select binning and set the binning factor to medium. Then in the subject height box, type in 2.5 centimeters in the exposure time box. Choose minutes as a unit of time and choose a numerical value of two.

Adjust the position of the animal in the chamber such that the neck and the upper chest of the rat are completely confined to the square projected on the bottom of the chamber. Three minutes after the application of the Luciferian onic gel, click the acquire button on the screen to start the image acquisition. Acquisition time can vary from one to six minutes depending on the anticipated signal strength.

After acquiring the image, use a syringe to wash the gel off with saline and dab the peri arterial space with sterile gauze and cotton applicators. Close the wound with Vicryl sutures and staple the skin. Then allow the animal to recover and return it to its cage.

As before, repeat the imaging procedure at later time. Points to study the time course of arterial expression brought about by the stent immobilized adenoviral vectors for systemic administration. Prepare a solution of 50 milligrams per milliliter Lucifer in PBS after anesthetizing and preparing the animal.

As before, catheterize the tail vein with a 24 gauge catheter and secure it by placing a piece of surgical tape between the tail and the catheter. Then turn the tapes edges up and wind them around the tails several times. Inject one milliliter of the solution through the catheter over a ten second interval one minute after injection.

Start image acquisition. As before, note that an injection rate faster than 10 seconds can provoke seizure activity and respiratory arrest to examine the potential effects related to the use of different hydrolyzed cross linkers. On arterial transduction, animals were implanted with Luke eluding stents and imaged one and eight days after stent deployment.

As can be seen here one day after stent implantation, animals treated with rapidly hydrolyzed crosslinker formulated add, Luke stents typically exhibit the highest luminescence signal rats receiving intermediately hydrolyzed crosslinker had the next highest luminescence signal, followed by slowly hydrolyzed crosslinker formulated stents. No perceptible signal was observed in the group of rats implanted with stents prepared using non hydrolyzed crosslinker tethered add Luke. This underscores the importance of unimpeded vector release from the stent for effective transduction of vascular tissue.

By day eight, the average intensity of luciferase expression with RHC formulated stents dropped several fold. When the day one and day eight intensities were individually plotted on a logarithmic scale, a 1.4 fold increase was seen with intermediately hydrolyzed crosslinker formulated stents and a 1.8 fold increase was seen with slowly hydrolyzed crosslinker formulated stents. This finding is consistent with the hypothesis of the more durable vascular transduction with gene eluding stents exhibiting a slower kinetics of vector release from the stent surface.

After watching this video, you should have a good understanding of how to implant an add Luke alluding stent into the back carotid artery and to image the ensuing vascular expression of luciferase when mastered the stent. Deployment surgery can take 20 to 25 minutes while the imaging procedure can take 10 to 15 minutes. We use the same technique to study the antiotic effects of gene therapy formulations and non-genetic drugs by determining the extent of arterial obstruction.

Two weeks after stent deployment.