A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Podsumowanie

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

Streszczenie

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

Wprowadzenie

Atherosclerosis is a chronic inflammatory disease in which lipid accumulation and inflammation in the blood vessel wall lead to narrowing of the vessel lumen, heart attacks, strokes, and loss of limbs^1,2. Percutaneous interventions (e.g., angioplasty and stenting) and medical therapy (e.g., statins and antiplatelet agents) are useful treatments for atherosclerosis; however, they are often ineffective in treating severe obstructive disease both in the coronary and peripheral circulations. Bypass grafting, using autogenous vein segments, remains a common procedure for treating patients with severe, diffuse coronary and peripheral vascular disease^3,4. However, vein grafts placed in both the coronary and peripheral circulations have poor long-term patency rates. In the coronary circulation, approximately 10-20% of vein grafts are occluded at 1 year and 50% are occluded by 10 years^5,6.In the peripheral circulation, vein graft failure rates are 30-50% at 5 years⁷.

Gene therapy is an attractive approach for the prevention of vein graft failure because it can deliver a therapeutic gene product precisely at the site of the disease. Accordingly, numerous preclinical studies have tested vein graft gene therapy^8,9. However, essentially all of these studies have examined the efficacy at early time points (2-12 weeks)^{10,11,12,13,14,15,16,17}. We are aware of no evidence that gene-therapy interventions can provide durable (years) protection against late vein graft failure that typically results from neointimal hyperplasia and atherosclerosis⁴. We developed a method that allows durable transgene expression in grafted veins, and thereby allows the testing of gene-therapy interventions at late as well as early time points. To achieve durable transgene expression, the method incorporates HDAd vectors and a delayed transduction strategy. HDAd vectors provide prolonged transgene expression because they lack viral genes, preventing the recognition (and rejection) of transduced cells by the immune system^18,19,20,21. Delayed transduction (performed 28 days after the graft placement) prevents the loss of transduced cells during the arterialization process that occurs early after the grafting²².

Other methods that achieve therapeutic transgene expression in the vein graft wall rely on the transduction of the vein graft at the time of the graft placement^{10,11,12,15,16,17}. When measured serially, transgene expression using this approach declines quickly after the transduction^22,23. Accordingly, studies using this approach have not examined the efficacy beyond 12 weeks after the vein grafting, with most not assessing efficacy beyond 4 weeks. In contrast, our method achieves vein graft transgene expression that persists stably for at least 24 weeks and-based on similar studies performed in arteries-likely continues far longer^22,24. We are aware of no other vein graft gene therapy intervention that achieves stable transgene expression of this duration.

We used a rabbit model to develop our method. Others have used rodents, rabbits, or larger animals to test vein graft gene therapy^{10,11,12,15,16,17,25,26}. Compared to rodent models, rabbits are more expensive and are subject to more stringent regulatory requirements. However, compared to larger animals (e.g., pigs and dogs), rabbits are far less expensive to purchase and house and much easier to handle. Moreover, rabbit vessels resemble human vessels physiologically²⁷, they are sufficiently large that they can be used for testing percutaneous interventions^28,29, and they provide sufficient tissue that multiple endpoints (e.g., histology, protein, RNA) can be examined using a single blood vessel specimen^22,30. In addition, when the rabbits with vein grafts are fed with a high-fat diet, they develop vein graft atherosclerosis^31,32, which is a common cause of coronary artery bypass vein graft failure^4,5. These atherosclerotic rabbit vein grafts can serve as a substrate for testing gene-therapy interventions delivered with this method. The provided protocol can help investigators to master the technical skills required to achieve durable transgene expression in rabbit vein grafts.

Protokół

All animal protocols and studies were approved by the University of Washington Office of Animal Welfare.

1. Pre-operation (for All Surgeries)

1. Anesthetize the rabbit with 30 mg/kg ketamine and 1.5 mg/kg xylazine by intramuscular injection (IM) in the paraspinous muscles.
   ​NOTE: Food and water are not restricted before surgery.
2. While waiting for sufficient depth of anesthesia, set up the tables in the preparation room and operating room (OR).
   1. Prepare the preparation room for shaving the rabbit's neck and the placement of the intravenous (IV) port in the ear (survival surgery only). Place the ophthalmic ointment, fentanyl patch (survival surgery), hair clippers, alcohol prep pad, 24-G x ¾" catheter, injection port, and surgical tape on the preparation table.
   2. In the OR, set up the monitoring equipment and associated probes (electrocardiogram (EKG), pulse oximetry (SpO₂), and temperature). Prepare the IV pump with a 100 mL saline IV bag with an 18-G or 19-G needle and set the flow rate as 10 mL/h/kg (survival surgery only). Set up the oxygen and isoflurane equipment with a nosecone (vein grafting or harvest surgeries).
      1. To help secure the nosecone to the rabbit, loop a gauze strip around the tube that supplies gas to the nosecone. This loop should surround the tube where it attaches to the nosecone. The free ends of the gauze strip should both be approximately 45 cm long. Place the nosecone (with attached gauze) on the head end of table.
   3. Turn on the circulating water and/or forced air warming blanket on the OR table. On the warming blanket, position a rolled towel as a neck support and place the electrocautery dispersive electrode plate.
   4. As a local analgesic, combine in a syringe 1 mL of 2% lidocaine HCl and 1 mL of 0.5% bupivacaine HCl. Dilute the virus to the desired concentration (here, use 2 x 10¹¹ viral particles/mL for HDAd) in sterile DMEM and keep on ice (gene transfer surgery only).
   5. After an adequate anesthetic depth is reached, prepare the rabbit for surgery.
      ​NOTE: Test for the lack of a pedal withdrawal reflex to ensure adequate anesthetic depth. Continue to monitor the pedal reflex throughout the surgery.
      1. Apply ophthalmic ointment to the rabbit's eyes.
      2. To allow for the placement of a rectal temperature probe, press with gloved fingers just above the rectum and move fingers towards the anus to remove stooling from the rabbit's rectum.
      3. Shave the rabbit from the sternal notch to the edge of mandible. Shave one ear for IV placement and the opposite ear for fentanyl patch administration (survival surgery only). Shave the left rear middle toes for the placement of the SpO₂ probe.
   6. For vector infusion surgery, intubate the rabbit. Use a 4 mm O.D./3 mm I.D. uncuffed endotracheal tube, threaded over an arthroscope. Place the rabbit in sternal recumbency and hyper-extend the neck. Hold the rabbit's mouth wide open and use the arthroscope to visualize the glottis and laryngeal folds. During the inspiration, gently insert the endotracheal tube through the larynx and into the trachea.
   7. For the survival surgeries, place and secure a 24-G IV catheter into the rabbit's left ear vein and cap it with an injection port. Apply a 25 µg/h fentanyl patch to the rabbit's right ear.
      NOTE: The protocol is for a surgeon positioned on the rabbit's right side. If the surgeon will be on the rabbit's left side, reverse the sides in this step to keep the wires and IV opposite of the surgeon. Switch the sides in future steps as needed.
   8. For the vein grafting and harvest surgeries, administer general anesthesia with a nosecone; for the vector infusion surgery, administer general anesthesia via an endotracheal tube.
      NOTE: Intubation can be used for all surgeries.  In our experience, however, the risks of complications from tracheal and laryngeal trauma during intubation can outweigh the benefits of intubation.  The surgery that includes gene transfer to a graft (particularly in cholesterol-fed rabbits) is the only surgery in which intubation seems to provide a net benefit.
   9. Carry the rabbit into the OR. Place the rabbit in a supine position on the operating table with the neck support towel positioned just below the rabbit's head. Gently extend the rabbit's neck until it is straight and approximately horizontal.
   10. Place the nosecone to the rabbit (vein grafting and harvest surgeries) or connect the endotracheal tube (vector infusion surgery) with O₂ at 1 L/min, and start isoflurane. If using a nosecone, secure it by wrapping the ends of the attached gauze strips around the rabbit's neck support towel. Adjust the isoflurane concentration as needed (typically 1-2%) to maintain a surgical level of anesthesia.
   11. Center the electrocautery dispersive electrode plate under the rabbit's back. Insert the rectal temperature probe and apply the SpO₂ probe and EKG leads to the rabbit.
   12. Connect the saline IV line to the catheter port in the rabbit's left ear and start the IV infusion pump at 10 mL/h/kg. After 1 h, reduce the saline infusion rate to 5 mL/h/kg.
   13. Loosely tie the rabbit's front legs to the operating table as a restraint.
       ​NOTE: Optionally, a small plastic table can be placed over the rabbit to prevent the surgeon from pressing on the rabbit's chest/abdomen, and potentially causing gastroesophageal reflux and aspiration.
   14. Inject lidocaine/bupivacaine (2 mL, 50/50 mix, step 1.2.4) subcutaneously (SQ) in the neck along the planned incision line, for local anesthesia.
   15. Have the assistant disinfect the surgical site with 3 alternating scrubs of chlorhexidine gluconate and isopropanol, then spray the site with povidone-iodine. Have the surgeon scrub, gown, and glove, following aseptic principles.
       1. Perform survival surgeries under aseptic conditions. Have the assistant handle any non-sterile items, and aseptically pass sterile materials to the surgeon or aseptically place them on the draped instrument table.
       2. Have the surgeon use sterile towels to aseptically manipulate non-sterile equipment such as the microscope. Have the surgeon wear 2x surgical loupes while performing the first half of the surgery.

2. Vein Graft Surgery (Survival)

1. Prepare the instruments and sterile field.
   1. Have the assistant open sterile packs (a table drape, a paper drape, and 6 towels) and aseptically transfer the contents to the surgeon.
   2. Drape the instrument table. Place the paper drape and sterile towels onto the draped instrument table. With 4 towels, drape the rabbit, leaving only the surgical site on the neck exposed. Lay the paper drape over the rabbit. One towel will be used later, and the last one is a backup.
   3. Have the assistant open the sterilized instrument pack and aseptically pass the instrument tray to the surgeon. Arrange the instruments on the instrument table.
   4. Have the assistant open the following equipment and aseptically either pass it to the surgeon or place it onto the instrument table: one 1-mL syringe, one 3-mL syringe, one 20-mL syringe, one 21 G needle, 3-0 polyglycolic acid (PGA) suture, 5-0 PGA suture, 7-0 polypropylene suture, and one 24 G IV catheter.
   5. Secure the electrocautery cable to the drape covering the rabbit, using a 7.25" Kantrowitz forceps. Drop the plug end of the cable over the edge of the operating table to be connected to the electrosurgery unit and turned on by the assistant.
   6. Fill a 20-mL syringe with sterile saline from a saline bag or vial held by the assistant. Use this saline as needed to prevent the dehydration of exposed tissue during the operation.
   7. Prepare 5 mL of heparinized physiologic saline solution by adding 0.1 mL of 5,000 IU/mL heparin to 5 mL of saline solution, i.e., 100 IU heparin/mL, in a small surgical bowl. Use this solution to flush the carotid artery and vein graft regularly during the grafting procedure.
   8. Cut a hole in the paper drape over the surgical site on the rabbit's neck. Use towel clamps to clamp the corners of the hole in place to the underlying towels.
2. Vascular dissection
   ​NOTE: Use 2x surgical loupes to aid with this part of the surgery.
   1. Cut the skin with electrocautery along the midline between the sternal notch and the mandible (approximately 7-9 cm in length) and clamp the rabbit's neck skin to the towels with the towel clamps. At the caudal end, make a short lateral cut through the fascia with electrocautery. Bluntly dissect under the fascia along the entire midline using large scissors. Cut through the dissected fascial layer along the midline with electrocautery.
   2. Start on the right side. Dissect between the sternohyoid muscle overlying the trachea and the V-shaped sternocephalic muscle, to expose the common carotid artery.
   3. Carefully dissect a 4-5 cm segment of the common carotid artery and its larger branches (typically 1-2 per side) free from surrounding tissues. Extend the dissection from the base of neck proximally to the crossing of the pharyngeal nerve distally. Use surgical silicon loops to aid in the retraction of the carotid artery during the dissection. Ligate the larger branches of the common carotid artery with 5-0 silk sutures before cutting each branch distally.
      ​NOTE: Be careful not to disturb the vagus nerve that parallels the common carotid artery, or the smaller nerves that cross over the artery.
   4. Repeat the dissection on the left side (step 2.2.3).
   5. Use tissue-holding forceps to temporarily close the subcutaneous tissue layer. Dissect the superficial fascia from the skin on the right side until the external jugular vein is exposed. Dissect a 4-5 cm segment of external jugular vein and free its branches from the surrounding tissue, ligating small branches with 5-0 silk sutures before cutting the branch.
   6. Repeat the dissection on the left side (step 2.2.5).
   7. Inject heparin (150 IU/kg) in the IV catheter and flush with 10 mL of saline.
   8. Measure a 3-cm segment of external jugular vein on the right side and ligate the caudal end of the segment with 5-0 silk suture. Allow the vein to fill with blood, then ligate the cranial end of the vein segment. Divide the cranial end of the external jugular vein segment, carefully flush the vessel with heparinized normal saline (100 U/mL) using a 3-mL syringe attached to a 24-G IV-catheter. Divide the vein segment at its caudal end. Place the vein segment in a standardized position, in which the caudal and cranial ends are unambiguously identifiable.
3. Vein grafting
   1. Let the assistant remove the surgical loupes from the surgeon and move the surgical microscope (25X) into position.
      NOTE: The surgeon should drape a sterile towel over the microscope. This allows the surgeon to manipulate the microscope while maintaining sterility.
   2. Clamp the right common carotid artery at each end of the isolated segment with mini-clamps, placing the cranial clamp first to allow for artery filling, then placing the caudal clamp.
   3. Cut a 1 × 2 cm piece of stiff paper (available from sterile suture packaging) and place it beneath the common carotid artery, to improve the exposure.
   4. Perform an arteriotomy just cranial to the caudal clamp (the caudal arteriotomy). The arteriotomy length should be equal to the diameter of the cranial end of the vein segment. Insert the syringe with the IV catheter through the caudal arteriotomy and flush the carotid artery lumen with heparinized normal saline.
   5. Suture the cranial end of the vein to the caudal arteriotomy, using 7-0 polypropylene suture with single stitches (Figure 1).
      1. Place the first stitch to suture the caudal end of the caudal arteriotomy to the cranial end of the vein segment. Place the second stitch 180° from the first stitch, to suture the cranial end of the caudal arteriotomy to the cranial end of the vein segment.
      2. Place the third stitch at a point equidistant from the first two stitches, on the lateral side of the anastomosis. Place the fourth stitch on the medial side of the anastomosis, equidistant from the first two stitches and across from the third stitch. Place 4 additional stitches (stitches 5-8) between each of stitches 1-4.
      3. Perform the cranial arteriotomy on the caudal side of the cranial 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.
   6. Extend the vein cranially from the anastomosis, laying it on top of the carotid, without introducing any twists. Flush the carotid artery and vein with heparinized normal saline via the cranial arteriotomy. The saline will flow through the artery, into the vein via the caudal anastomosis and will exit from the free and unsutured end of the vein. Flushing the vein also prevents it from twisting.
   7. Suture the caudal end of the vein to the cranial arteriotomy (Figure 1).
      1. Place a first stitch to suture the caudal end of the cranial arteriotomy to the caudal end of the vein segment. Place a second stitch to suture the cranial end of the cranial arteriotomy to the caudal end of the vein segment. Complete the anastomosis as described in step 2.3.5.2. Just before the last knot is tied on the cranial anastomosis, briefly open the clamp on the cranial end of the dissected common carotid artery segment, to allow back-bleeding that washes out the air in the carotid artery and grafted vein. Then, tighten the last knot.
   8. Release the cranial clamp to allow vein graft filling with blood and then release the caudal clamp. Brisk pulsations should be seen in the vein graft. Apply gentle pressure with dry gauze to stop any bleeding at the anastomoses.
   9. Ligate both ends of the common carotid artery segment that connects the anastomoses with a 3-0 silk suture and divide the carotid halfway between the ligatures (Figure 1). Apply several drops of papaverine (3.5 mg/mL) to the carotid artery adjacent to each anastomosis.
   10. Inject heparin (75 IU/kg) into the ear IV catheter and flush with 10 mL of saline. At this time, inject buprenorphine (SQ, 0.02 mg/kg) to provide postoperative analgesia until the fentanyl patch has provided analgesic plasma levels of fentanyl.
       NOTE: An additional buprenorphine injection (SQ, 0.02 mg/kg) may be necessary 6 h after the first injection to maintain analgesia until plasma fentanyl reaches a therapeutic concentration.
   11. Repeat grafting on the left side (steps 2.2.8, 2.3.2-2.3.9).
   12. Close the subcutaneous tissue with 5-0 PGA suture using a continuous pattern. Close the skin with 3-0 PGA suture using an intradermal pattern. Bury the knots on both ends.
4. Post-operative recovery, cleanup, and care
   ​NOTE: Allow the rabbit to recover from anesthesia in a calm, quiet environment. Monitor the rabbit continuously for proper oxygenation and body temperature until it has fully recovered.
   1. Turn off isoflurane and oxygen and disconnect the anesthesia machine from the rabbit. Disconnect the IV fluid tubing from the rabbit, but leave the IV port in the ear vein for emergent access.
   2. Transport the rabbit to a recovery cage and place it on its side. Provide thermal support with a warm water blanket (or forced-air warming). Give O₂ by nosecone until SpO₂ is stable.
   3. Until the rabbit can sit up and ambulate in its cage, flip the rabbit to its other side every 15 min. Then remove the ear IV cannula and return the rabbit to its cage. Return the rabbit to the company of other animals only after it is fully recovered from anesthesia.
   4. Dispose of any waste following appropriate protocols for biohazard and sharps waste disposal.
   5. Postoperatively, check the rabbit's health and surgical wound each day. Administer buprenorphine as need for pain not managed by the fentanyl patch. Remove the fentanyl patch on post-operative day 3.

3. Transcutaneous Ultrasound

1. To assess graft patency, perform ultrasound exam on non-anesthetized rabbit 5-7 days after the vein-graft surgery and gene-transfer surgery.
   1. Wrap the non-anesthetized rabbit securely in a blanket and place the rabbit supine in a veterinary V-trough with its neck extended. Shave the neck, or if needed, remove the hair with a depilatory agent. Apply ultrasound gel to the neck and perform an ultrasound exam.
      NOTE: An ultrasound exam is also performed on anesthetized rabbits at the time of both the gene transfer surgery and the terminal graft harvest surgery to examine graft patency and to measure graft lumen diameter. The exam is done just before the neck scrubbing and is performed in a similar manner as for the non-anesthetized rabbits.

4. Gene Transfer Surgery Performed ~ 28 Days After the Graft Placement (Survival Surgery)

1. Prepare the instruments and the sterile field.
   1. Follow steps 2.1.1-2.1.3. in vein graft surgery.
   2. Let the assistant open the following equipment and either aseptically pass it to the surgeon or place it onto the instrument table: six 1-mL syringes (with needle), one 3-mL syringe, one 20-mL syringe, one 21 G needle, one 19-G needle, 3-0 PGA suture, 5-0 PGA suture, 7-0 polypropylene suture, two 24 G IV catheters, and a sterile blood flow probe.
   3. Follow steps 2.1.5-2.1.6. in vein graft surgery.
   4. Prepare one 1-mL syringe with 1 mL DMEM for washing the artery and vein graft and prepare two 1-mL- syringes with virus solution (~ 0.5-0.7 mL virus solution/vein graft). Let the assistant thaw the virus and dilute it in DMEM. Prepare one 3-mL syringe with 0.5 mL of lidocaine/bupivacaine (50/50 mix, step 1.2.4).
   5. Follow step 2.1.8 in vein graft surgery.
2. Isolation of vein grafts and adjacent carotid arteries
   ​NOTE: 2x surgical loupes should be used for this part.
   1. Cut the skin with electrocautery along the midline between the sternal notch and the mandible (approximately 7-9 cm in length) and clamp the skin open with towel clamps. Apply 0.5 mL of lidocaine/bupivacaine (50/50 mix, step 1.2.4) to the subcutaneous tissue.
   2. At the caudal end, make a short lateral cut through the fascia with electrocautery. Bluntly dissect under the fascia along the entire midline. With electrocautery, cut through the dissected fascia along the midline.
   3. Start on the right side. Dissect between the sternohyoid muscle overlaying the trachea and the V-shaped sternocephalic muscle, to expose the vein graft. Carefully isolate the vein graft and 1.5-2.0 cm of the carotid artery adjacent to the graft on both the cranial and caudal sides.
   4. Repeat the dissection on the left side following step 4.2.3.
3. Measure the blood flow in the vein grafts.
   1. Fill the surgical wound cavity on the right side with normal saline to enable the transmission of sound waves. Immerse a 2-mm perivascular flow probe (connected to a volume-flow meter) in the saline in the wound cavity and set the flow rate to zero.
   2. Place the flow probe around the carotid artery caudal or cranial to the vein graft. Record data from the flow probe with an electronic data acquisition system.
   3. Repeat the flow measurement on the left side following step 4.3.2.
   4. Calculate the pulsatility based on peak systolic flow rate, minimum diastolic flow rate, and mean flow rate. Also calculate laminar shear stress, based on the flow rate and lumen diameter (measured with transcutaneous ultrasound).
4. Infuse vector solution into the vein grafts
   ​NOTE: A surgical microscope (16X) is used as needed for the puncturing, infusion, and repair of the carotid artery.
   1. Have the assistant remove the surgeon's surgical loupes and move the surgical microscope into position. Have the surgeon drape a sterile towel over the microscope. The sterile towel allows the surgeon to manipulate the microscope while maintaining sterility.
   2. Have the assistant inject heparin (150 IU/kg) in the IV catheter and flush with saline.
   3. Use a large needle driver to bend the 21 G needle to approximately 80° just above the bevel (do not bend the bevel; Figure 2A).
   4. Clamp the common carotid artery at each end of the vein graft with mini-clamps, placing the cranial clamp first to allow for artery filling, then placing the caudal clip. Place the caudal clip approximately 10 mm caudal to the anastomosis, to leave some room for the arteriotomy.
   5. Put two silk ties around the artery just caudal to the graft and tie a single overhand knot on each - without tightening them.
   6. Puncture the carotid caudal to the graft with the bent 21 G needle just cranial of the caudal vascular clip. Be careful to not puncture the back or side walls. Advance the needle into the lumen back-and-forth twice to ensure that the lumen is clear, then carefully withdraw the needle.
      Notes: Common carotid puncture can be aided by grasping the arterial adventitia with fine forceps and gently lifting the front wall while inserting the tip of the needle just caudal to the lift point (Figure 2A). This reduces the risk of hitting the back wall with the needle.
   7. With several unfolded gauze pads, create a nest to place the syringe used for infusions. Place this gauze nest caudal to the surgical site.
   8. Put a 24 G IV-catheter on the syringe with DMEM-only and tighten the catheter on the syringe just enough to prevent leakage (the syringe will be removed later in this surgery) and bend the catheter ~ 4 mm from the tip so that the bend holds at about 75° after it is released.
   9. Insert the IV catheter into the common carotid arteriotomy up to the bend point and wash the vein graft lumen twice with 0.5 mL of DMEM. For each repetition, fill the vein graft with DMEM. Then remove the DMEM from the graft by lightly pressing with a gloved finger at the cranial end of the graft. Carefully slide the finger toward the caudal end of the graft to flush the luminal contents out via the arteriotomy.
   10. 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.
   11. Infuse 0.03 mL of virus solution to push the remaining DMEM out of the catheter. Remove all fluid from the vein graft lumen with a finger, pressing gently from cranial to caudal.
   12. Tighten the two ties around the artery and catheter tip to seal the lumen. Infuse the virus solution until the vein is distended. Gently lay the syringe down on the nest of gauze.
       NOTE: It is vital that the vein graft expands to its pre-transduction caliber and remains inflated during the virus infusion. If not, the amount of gene transfer will be significantly decreased.
   13. Leave the virus-containing solution in the vein graft lumen for 20 min, and then replace the virus-containing syringe attached to the catheter with an empty syringe. Gently aspirate the virus-containing solution from the graft until the vessel collapses. Remove the syringe, leaving the catheter in place. Cut and untie the silk sutures and withdraw the catheter from the vessel. Remove the catheter from the carotid artery carefully to avoid damaging the endothelium.
   14. With the aid of the surgical microscope, close the arteriotomy with 7-0 polypropylene suture using an X-pattern (Figure 2B).
       1. Grasp the suture with a needle-holder, enter the vessel at the bottom-right of the arteriotomy, and exit the vessel at the bottom-left. Cross the opening outside the vessel and make the second pass from top-right to top-left.
       2. Before tightening the suture, briefly release the cranial vascular clip to flush air and residual virus from the vein graft and artery. Blood will flow out of the arteriotomy when the clamp is released.
       3. Close the arteriotomy by gently pulling the suture ends and tie them together with 2 square knots.
          NOTE: Pulling the suture too tight will cause bunching of the tissue which will disturb the flow, increase thrombosis risk, and potentially introduce uncontrolled variables.
   15. Release the cranial vascular clip, then the caudal clip. Stop any bleeding by using gauze to apply light pressure.
   16. Around this time, inject buprenorphine (SQ, 0.02 mg/kg) to provide postoperative analgesia until the fentanyl patch has provided analgesic fentanyl plasma levels.
       ​NOTE: An additional buprenorphine injection (SQ, 0.02 mg/kg) may be necessary 6 h after the first injection to maintain analgesia until plasma fentanyl reaches a therapeutic concentration.
   17. Repeat the virus infusion protocol on the left side, following steps 4.4.5-4.4.15.
5. Wound closure
   1. Close the subcutaneous tissue with 5-0 PGA suture, using a continuous pattern. Close the skin with 3-0 PGA suture using an intradermal pattern. Bury the knots on both ends.
6. Post-operative recovery, cleanup, and care
   1. Follow step 2.4.

5. Harvest Surgery (Terminal)

1. Prepare the instruments and the surgical site.
   1. Follow steps 2.1.1-2.1.3 in vein graft surgery.
   2. Let the assistant open and aseptically place on the instrument table (or pass to the surgeon): one 20-mL syringe, one 21G needle, 3-0 silk suture, and a sterile blood flow probe.
   3. Follow steps 2.1.5-2.1.6 and 2.1.8 vein graft surgery.
2. Isolation of common carotid arteries and vein grafts
   1. Cut the skin with electrocautery along the midline between the sternal notch and the mandible (approximately 7-9 cm in length) and clamp the rabbit's neck skin to the towels with towel clamps.
   2. At the caudal end, make a short lateral cut through the fascia with electrocautery. Bluntly dissect under the fascia along the entire midline. Cut through the dissected fascia along the midline with electrocautery.
   3. Start on the right side. Dissect between the sternohyoid muscle overlying the trachea and the V-shaped sternocephalic muscles to expose the common carotid and vein graft.
   4. Dissect the right vein graft and common carotid artery free from the surrounding tissues. Use surgical silicon loops for the retraction of the artery and the vein graft during the dissection.
   5. Repeat the dissection on the left side following steps 5.2.3-5.2.4.
3. Make flow measurements following steps 4.3.1-4.3.3 in gene transfer surgery.
4. Harvest of the vein grafts
   1. With 3-0 silk suture, ligate the right common carotid artery cranial to the graft. Then ligate the carotid caudal to the graft.
   2. Excise the right vein graft by dividing the adjacent carotid artery between each of the ligations and the adjacent anastomosis. Remove the vein graft from the rabbit and flush the lumen with saline.
   3. Trim the carotid artery and anastomoses from both ends of the vein graft. Trim away excess adventitial tissue from the vein graft and cut the graft into segments as needed for different endpoint analyses.
   4. Harvest the left vein graft by repeating steps 5.4.1-5.4.3.
5. Inject 1 mL of phenytoin/pentobarbital IV to euthanize the rabbit.
6. Dispose of any waste following appropriate protocols for biohazard and sharps waste disposal.

Wyniki

The technical proficiency of a new operator must be validated before the operator can use this method to generate experimental data. 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. Over 90% patency after each of the surgeries is desirable and achievable. The patency can be assessed non-invasively using transcutaneous ultrasound, which we typically perform on ...

Dyskusje

Critical steps in this protocol include the management of anesthesia, anticoagulation, surgical manipulation of the artery/grafted vein, and hemodynamic measurements of the grafted vein. Proper management of anesthesia is critical in this multiple survival surgery model that includes two relatively long operations (typically 3-3.5 h for bilateral vein grafting and 1.5-2.5 h for bilateral graft transduction). We have administered anesthesia both via a nosecone and by endotracheal intubation and found that the intubation i...

Materiały

Name	Company	Catalog Number	Comments
Disposables
3mL syringe with 24G needle	Becton Dickinson	309571	2x for vein graft surgery; 2x for gene transfer  surgery
1 mL syringe with 27G needle	Becton Dickinson	309623	2x for vein graft surgery, 5x for gene transfer surgery
19G needle	Becton Dickinson	305187	Gene transfer surgery
20 mL syringe, luer lock	Nipro Medical Corp	JD+20L
Catheters, 24Ga x 3/4”	Terumo Medical Products	SROX2419V
21G needle	Becton Dickinson	305165	Gene transfer surgery and for 20 mL syringe of saline
Gauze 4” x 4”	Dynarex	3242	~10-15 per surgery
3-0 silk suture	Covidien Ltd.	S-244
5-0 silk suture	Covidien Ltd.	S-182
7-0 polypropylene suture	CP Medical	8703P	Vein graft surgery
7-0 polypropylene suture	CP Medical	8648P	Gene transfer surgery
5-0 polyglycolic acid suture	CP Medical	421A
3-0 polyglycolic acid suture	CP Medical	398A
Alcohol swabs	Covidien Ltd.	6818	For the placement of I.V. line
Catheter plug	Vetoquinol	411498
Ketamine HCl, 100 mg/mL	Vedco Inc.	5098916106
Xylazine, 100 mg/mL	Akorn Inc.	4821
Lidocaine, 20 mg/mL	Pfizer	409427702
Marcaine 0.5%	Pfizer	409161050
Beuthanasia D-Special	Intervet Inc.	NDC 00061047305	Harvest surgery only
Buprenorphine HCl, 0.3 mg/mL	Patterson Veterinary	12496075705
Saline IV bag, 0.9% sodium chloride	Baxter	2B1309
Heparin  (5000 U/mL)	APP Pharmaceuticals	NDC 63323-047-10
Papaverine (3.5 mg/ml)	American Reagent Inc.	NDC 0517-4002-25	Diluted from 30mg/mL stock; Use 1 mL maximum
Fentanyl patch, 25 mcg/h	Apotex Corp.	NDC 60505-7006-2
Isoflurane	Multiple vendors	Catalog number not available
Viral vector			Gene transfer surgery only
Surgical Instruments
Metzenbaum needle holder 7" straight	Roboz	RS-7900
Operating scissors 6.5" straight blunt/blunt	Roboz	RS-6828
Needle holder /w suture scissors	Miltex	8-14-IMC
Castroviejo scissors	Roboz	RS-5658
Castroviejo needle holder, 5.75" straight with lock	Roboz	RS-6412
Stevens scissors 4.25" curved blunt/blunt	Roboz	RS-5943
Alm retractor 4" 4X4 5mm blunt prongs	Roboz	RS-6514	2x
Backhaus towel clamp 3.5"	Roboz		4x
Micro clip setting forceps 4.75"	Roboz	RS-6496
Micro vascular clips, 11 mm	Roboz
Surg-I-Loop	Scanlan International	1001-81M	5 cm length
Bonaccolto forceps, 4” (10 cm) long longitudinal serrations, cross serrated tip, 1.2mm tip width	Roboz	RS-5210
Dumont #3 forceps Inox tip size .17 × .10 mm	Roboz	RS-5042
Graefe forceps, 4” (10 cm) long serrated straight, 0.8 mm tip	Roboz	RS-5280
Halstead mosquito forceps,  5" straight, 1.3 mm tips	Roboz	RS-7110	2x
Halstead mosquito forceps,  5" curved, 1.3mm tips	Roboz	RS-7111
Jacobson mosquito forceps 5" curved extra delicate, 0.9 mm tips	Roboz	RS-7117
Kantrowitz forceps, 7.25" 90 degree delicate, 1.7 mm tips	Roboz	RS-7305
Tissue forceps 5", 1X2 teeth, 2 mm tip width	Roboz	RS-8162
Allis-Baby forceps, 12 cm, 4x5 teeth, 3 mm tip width	Fine Science Tools	11092-12	2x
Adson forceps, 12 cm, serrated, straight	Fine Science Tools	11006-12
Veterinary electrosurgery handpiece and electrode	MACAN Manufacturing	HPAC-1; R-F11
Surgical Suite Equipment
Circulating warm water blanket and pump	Multiple vendors	Catalog number not available
Bair hugger warming unit	3M	Model 505
IV infusion pump	Heska	Vet IV 2.2
Isoflurane vaporizer and scavenger	Multiple vendors	Catalog number not available
Veterinary multi-parameter monitor	Surgivet	Surgivet Advisor
Veterinary electrosurgery unit	MACAN Manufacturing	MV-9
Surgical microscope	D.F. Vasconcellos	M900	25X magnification for vein graft surgery; 16X magnification for gene transfer surgery