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Method Article

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

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DOI:

10.3791/56982

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May 6th, 2018

 ,  , 
1Department of Medicine, University of Washington

In This Article

Summary

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

Abstract

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

Introduction

The goal of this method is to introduce a transgene into the endothelium of rabbit common carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product both in normal arteries and in rabbit models of human arterial disease. Overexpression of the transgene in disease models can reveal whether the transgene (and its protein product) show promise as therapeutic agents1,2,3,4. Inclusion of cis-acting regulatory elements in the transgene expression cassette enables assessment of the activity of these elements in arterial endothelium in vivo5,6. Knowledge of the activity of specific cis-acting regulatory elements can be used to design more-active expression cassettes and to probe mechanisms of gene regulation in large artery endothelium in vivo7.

Rabbits are a valuable model for various aspects of human vascular physiology and disease. Rabbits share many vascular features with humans. For example, baseline hematological values, hemostatic regulation, and vascular longitudinal tension are similar between rabbits and humans8. Rabbit models of vascular diseases replicate key features of many human diseases including: aneurysms (similar geometric and flow characteristics)9, vasospasm (similar response to endovascular treatment)10,11, and atherosclerosis (intimal plaques with similar features including a core rich in lipid, macrophages, and smooth muscle cells in a fibrous cap)12,13. Accordingly, rabbit models have been developed for many vascular diseases such as thrombosis, vasospasm, aneurysm, diabetes, vascular graft stenosis, and atherosclerosis8,13,14,15,16.

For researchers choosing among animal models of vascular physiology and disease, the rabbit has several advantages. Compared to rodents, the larger vessels of rabbits allow easier surgical manipulation, use of endovascular devices, and a larger amount of tissue for quantitative measurements. Rabbits are much closer phylogenetically to primates than are rodents17, and the greater genetic diversity of outbred rabbits better approximates the genetic variability of humans. Genetic diversity is particularly important for preclinical studies, which-by their nature-aim to develop therapies that can be applied to the genetically diverse human population. As with many if not all other model species, rabbit genes are easily cloned or synthesized because the rabbit genome has been sequenced with high coverage (7.48x) [http://rohsdb.cmb.usc.edu/GBshape/cgi-bin/hgGateway?db=oryCun2]. Compared to other large animal models (such as dogs, pigs, or sheep), rabbits are relatively inexpensive to purchase and house and they are easier to breed and handle. Specific vascular disease models in rabbits each have their own advantages and shortcomings as models of human disease that are beyond the scope of this manuscript8,12,18. An investigator should review these advantages and shortcomings to determine if the rabbit is the best model for answering a specific experimental question.

Introduction of deoxyribonucleic acid (DNA) regulatory sequences into endothelial cells in vivo enables investigation of the activity of these sequences in a complex physiologic environment. In vitro studies in transfected endothelial cells can be useful for the initial assessment of DNA regulatory sequences; however, expression levels in tissue culture models are sometimes not reproduced when the studies are repeated in vivo5,19,20. In vitro systems can also be useful for exploring basic pathways of protein signaling and endothelial physiology as well as communication between cultured vascular cells; however, more-complex pathways or regulatory networks that are influenced by complex populations of neighboring vascular cells or the immune system are best studied in an in vivo system6,20. The method described herein provides a platform for exploring regulation of transgene expression in the endothelium within the context of an intact vessel, with or without disease. The in vivo system also permits investigation of physiological and pathological cellular crosstalk and identification of contributions of the immune system to regulation of gene expression6.

Germ-line transgenesis (especially in mice) is an alternative approach for directing transgene expression to endothelial cells. This approach can provide life-long transgene expression, with endothelial targeting mediated by specific promoter or regulatory regions21,22. However, the generation of transgenic mice is time-consuming and expensive, several transgenic lines must be often tested to ensure targeting of the transgene to the desired cell type and achievement of adequate transgene expression levels, and experimental results in murine systems can be strain-dependent. Murine transgenic models with endothelial-targeted transgenes have many advantages: there is no need to perform surgery on every experimental animal in order to achieve transgenesis, experimental mice can be bred with numerous other available transgenic mice in order to test genetic and phenotypic interactions, and there is a wide selection of antibodies that react with murine proteins, facilitating characterization of phenotypes. However, targeting of transgenes to the endothelium via the germ line typically results in transgene expression throughout the vasculature,22 making it difficult to determine the site at which the transgene product is acting. This is especially true when the transgene product is secreted, because a transgene product secreted by endothelial cells throughout the vasculature could have biological activity at any number of sites within an animal. Although the method described in this manuscript requires technical expertise and specialized facilities, it can be less time consuming and less expensive than developing an endothelial-specific transgenic mouse line. It allows for the assessment of the function of a protein selectively in endothelial cells of a segment of large artery, and it permits use of the contralateral common carotid as a paired control (eliminating systemic factors that can vary among experimental animals-for example, blood pressure or cholesterol levels-as uncontrolled variables).

Gene therapy is a promising approach for the treatment of vascular diseases, particularly chronic diseases, because a single application can provide sustained or possibly life-long expression of a therapeutic gene23. The therapeutic promise of gene therapy has been explored in animal models of somatic gene transfer, often targeting the liver24,25, which is a relatively easy target because many blood-borne viral vectors are hepatotropic. However, to have an effect on vascular disease, gene therapy targeted to the liver must achieve systemic overexpression of proteins. This typically requires large doses of vector, which can be toxic or even fatal26. Moreover, increased systemic levels of a protein raise the risk of off-target side effects, which could complicate or even obscure interpretation of experimental results. Local gene therapy targeting vascular endothelium as described in this manuscript could avoid systemic side effects because the infused vector is not widely disseminated beyond the transduced arterial segment, and local vascular effects can be achieved without changes in systemic plasma levels of protein.27 In addition, a far lower amount of vector is needed to transduce an arterial segment than is needed to achieve robust hepatic transduction. Transgene expression from the liver has been reported to decline over time, probably due to cell turnover, requiring repeated dosing if high-level transgene expression is to be maintained.28 In contrast, the low turnover rate of the endothelium provides stable expression for at least 48 weeks in chow-fed rabbits and for at least 24 weeks in atherosclerotic lesions of cholesterol-fed rabbits.1,27

To determine if this method of gene transfer to rabbit common carotid endothelium is appropriate, the advantages and disadvantages (Table 1) should be considered in the context of the specific research goals. Advantages of this method include: outbred rabbits are better representative of human genetic diversity than are inbred mice (important for preclinical work); rabbits provide larger vessels for easier manipulation and more tissue for analysis; the method can achieve endothelium-targeted transgene expression far more quickly than does germ-line endothelial targeting in transgenic mice; vector dose can be easily adjusted to model variable levels of transgene expression; processes specific to large-artery endothelium can be investigated; and local vascular transgenesis allows the opposite carotid in the same animal to be used as a control, eliminating systemic factors as uncontrolled variables. Disadvantages include: special facilities and expertise are required; fewer genetically modified backgrounds on which to experiment are available in rabbits than in mice; and there is a less extensive selection of antibodies to rabbit versus mouse proteins (for immunodetection of transgene protein and other antigens that may be important in interpreting experimental results).

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Protocol

All methods described here were approved by the University of Washington Office of Animal Welfare and associated Institutional Animal Care and Use Committee (IACUC), and were completed in accordance and compliance with all relevant regulatory and institutional guidelines.

Note: Gene transfer to rabbit common carotid arteries is performed on rabbits by a surgeon with the aid of an anesthesiologist or assistant.

1. Gene transfer to rabbit common carotid arteries: Pre-operation

  1. Anesthetize the rabbit. Weigh the rabbit. Combine 30 mg/kg ketamine and 2 mg/kg xylazine into a syringe. Inject ketamine/xylazine intramuscular (IM) in the paraspinous muscles to induce anesthesia.
  2. While waiting for rabbit to become anesthetized, prepare the preparation room and operating room (OR) tables.
    1. In the OR preparation room: place the ophthalmic ointment and the fentanyl patch within reach of the preparation table; set up hair clippers and a vacuum for shaving the rabbit's neck and ear; set aside an alcohol prep pad, a 24G x ¾" catheter, an injection port, and surgical tape to secure the intravenous line (IV) in the rabbit's ear vein.
    2. In the OR: set up the monitoring equipment and position the probes [electrocardiogram (EKG), oxygen saturation (SpO2), temperature] on the OR table; prepare oxygen and isoflurane; tie gauze strip to the face mask to secure it to the rabbit and place the mask on the head end of table.
      NOTE: The gauze strips tied on the face mask should have 2 tails of about 45 cm each.
    3. Turn on the warming water blanket on the OR table. On top of water blanket, position a rolled towel for neck support and a dispersive electrode plate (later placed under rabbit's back).
    4. Set up the OR IV pump with 100 mL of a saline IV bag with an 18-19G needle attached and set the flow rate to 10 mL/h per kg.
    5. Combine 1 mL of lidocaine HCl (2% stock solution) and 1 mL of bupivicaine HCl (0.5% stock solution) in a syringe, to be used as a local analgesic.
  3. When rabbit is fully anesthetized, prepare the rabbit for surgery.
    ​NOTE: Check for the lack of a pedal reflex (pinch a toe; look for limb withdrawal) to ensure anesthetic depth, and continue to monitor the pedal reflex throughout the surgery.
    1. Apply ophthalmic ointment to eyes. Remove stool from the rabbit's rectum by pressing with gloved fingers on the lower anterior abdomen (above rectum) and moving fingers towards the anus. This will allow for later placement of a rectal temperature probe.
    2. Shave the anterior neck from the sternal notch to the edge of the mandible while using a vacuum to keep the area clean. Also shave both ears for the IV placement and the fentanyl patch attachment, and shave a left rear middle toe for the pulse-oximetry probe.
      NOTE: The protocol assumes that the surgeon is on the rabbit's right side for the entire procedure. If the OR setup places the surgeon on the opposite side, reverse sides in this step to keep the wires and IV opposite of the surgeon. The sides of future steps should also be switched as needed.
    3. Wipe the left ear with an alcohol prep pad, place an 24G IV catheter into the left ear vein, cap with the injection port, and tape the catheter/port to the rabbit's ear to secure it. Apply a fentanyl patch (25 µg/h) to the rabbit's right ear.
    4. Transport the rabbit into the OR and place supine on the operating table with a rolled neck support towel under the rabbit's neck just below the head. Gently extend the rabbit's neck until it is straight and approximately horizontal.
    5. Hook the rabbit up to the face mask with O2 on at 1 L/min, and start isoflurane administration. Secure the mask by wrapping the ends of the gauze strip tied to the mask around the neck support towel under the rabbit. Adjust isoflurane (typically 1 - 2%) as needed (based on heart rate, respiratory rate, and pedal reflex) to maintain proper anesthesia for the remainder of the procedure.
    6. Ensure that the dispersive electrode plate is centered under the rabbit's back. Place the temperature and pulse-oximeter probes and apply the EKG leads to the rabbit.
    7. Hook up IV saline to the catheter port in the rabbit's ear, starting the saline pump at 10 mL/h per kg.
      NOTE: After 1 h, the saline pump can be reduced to 5 mL/h per kg.
    8. Restrain the rabbit's front legs by loosely tying them to the table. Optionally, place a small plastic table over the rabbit to protect the rabbit from being subjected to chest/abdominal pressure from the surgeon leaning on rabbit's chest/abdomen.
      NOTE: The small table may help prevent forced regurgitation of abdominal contents.
    9. Inject 2 mL of previously prepared lidocaine (2% stock)/bupivacaine (0.5% stock) (50/50 mix) subcutaneously along the planned neck incision line for local anesthesia.
    10. Let the assistant prepare the surgical site with 3 alternating scrubs of chlorhexidine and isopropanol, and then a spray with betadine. Scrub, gown, and glove, following proper aseptic technique.
      NOTE: The surgeon should be wearing 2x surgical loupes to aid with the first half of the surgery. During the survival surgery it is vital to maintain sterility of the surgeon and the surgical field. Only the assistant should handle any non-sterile items, and sterilized materials must be aseptically passed to the surgeon or placed on the draped instrument table. Sterile towels can be used by the surgeon to maintain sterility while manipulating non-sterile equipment such as the microscope.

2. Survival Surgery (Gene Transfer)

  1. Prepare instruments and sterile field.
    1. Let the assistant open the sterile drape pack containing a table drape, one paper drape for the rabbit, and several towels. Aseptically transfer the items to the surgeon.
    2. Drape the instrument table. Place the 6 sterile towels and the paper drape onto the draped table.
    3. With 4 towels, drape the rabbit's neck, leaving only the surgical site exposed (approximately 4 cm x 10 cm). Lay the paper drape over the rabbit.
    4. Let the assistant open the sterilized instrument pack and place aseptically onto the instrument table.
    5. Let the assistant open the following equipment and aseptically place onto the instrument table or hand to the surgeon: three 1-mL syringes (and one needle if the syringes are not already loaded with needles), one 20 mL syringe, one 21G needle, one 19G needle, 3-0 polyglycolic acid (PGA) suture, 5-0 PGA suture, 7-0 polypropylene suture, and two 24G IV-catheters.
    6. Secure the electrocautery cable to the rabbit drape using the 7.25" Kantrowitz Forceps and then drop the plug end off of the table. Let the assistant connect the cable to the electrosurgery unit and turn the power on.
    7. Fill a 20 mL syringe with sterile saline, drawn from an IV bag or vial held by the assistant. Use this saline as needed to keep exposed tissue moist during the procedure.
    8. Prepare one 1 mL syringe with 1 mL of Dulbecco's Modified Eagle Medium (DMEM, for washing the artery). For each carotid artery that will be transduced, 0.35 mL of diluted virus are needed. If the same virus is used for both sides, prepare one 1 mL syringe with virus diluted in DMEM to a volume of 0.7 mL. If different sides are receiving different viruses [for example a "Null" virus control on one side], prepare two 1 mL syringes, each with 0.35 mL of virus solution. For helper-dependent adenovirus, the virus concentration is 2 x 1011 viral particles (vp)/mL.
      NOTE: Let the assistant prepare the virus. The surgeon draws up DMEM and virus suspension into sterile syringes.
    9. Cut a hole in the drape. The size of the hole should be the same size as the surgical site on the rabbit's neck that is framed by the towels in step 2.1.3. Clamp the corners of the hole in the paper drape to the underlying towels that are draped over the rabbit's neck with the towel clamps.
  2. Isolate the common carotid arteries.
    ​NOTE: 2x surgical loupes should be used for this part.
    1. Cut the skin with an electrocautery along the midline extending approximately 7 - 9 cm cranially toward the mandible and clamp the skin open with towel clamps.
    2. Make a short lateral cut through the fascia with the electrocautery at the caudal end. Then insert the large scissors into the cut and bluntly dissect the fascia along entire midline. Cut the dissected fascia down the midline with electrocautery.
    3. With small dissecting scissors, dissect between the V-shaped muscles (sternocephalic muscle) and the sternohyoid muscle over the trachea, to expose the common carotid arteries, starting on the right side.
    4. Dissect the right common carotid artery free from surrounding tissues, dividing all branches, from base of neck caudally to the crossing of the pharyngeal nerve cranially. Use surgical silicon loops to aid in retracting the artery during dissection.
      NOTE: Care should be taken to not disturb either the vagus nerve that runs parallel to the common carotid, or the smaller nerves that cross over the common carotid.
    5. Ligate any large branches coming off the common carotid artery (1 - 2 per side) with 5-0 silk sutures prior to cutting the branch distally to free a 4 - 5 cm segment of the carotid. Cut ligated branches 1 - 2 mm away from the tie so that the tie does not slip off.
    6. Repeat dissection on the left side (steps 2.2.3 - 2.2.5).
  3. Infuse vectors into common carotid arteries.
    ​NOTE: A surgical microscope (16X) is used as needed for performing arteriotomy, infusing vector/control solution, and arteriotomy repair.
    1. Let the assistant remove the surgical loupes from the surgeon and move the surgical microscope into position. Drape a sterile towel over the microscope to allow manipulation of the microscope without contaminating the sterile field.
    2. Inject heparin [150 international units (IU)/kg] in the IV catheter and flush with 10 mL of saline.
    3. Returning to the isolated right common carotid, put two silk ties around the mid-portion of the mobilized carotid segment and tie a single overhand knot on each - without tightening them.
    4. Bend the 19G needle just above the bevel to approximately 80° using the large needle driver (do not bend the bevel; Figure 1).
    5. Clamp the artery at each end of the isolated segment with vascular clips, placing the cranial clip first to allow for artery filling, and then placing the caudal clip.
    6. Puncture the common carotid artery with the bent 19G needle just cranial to the caudal vascular clip, taking great care to not puncture the back or side walls. Advance the needle tip into the lumen and withdraw it twice to make sure arteriotomy completely traverses the artery wall. Then carefully withdraw the needle.
      Notes: It is important to create an arteriotomy that penetrates all layers of the artery wall cleanly and does not dissect the artery wall (by passing axially through the adventitia and media). To accomplish this, the carotid should be punctured with the needle tip positioned as close as possible to a 90° angle against the artery wall (Figure 1). Carotid puncture at a 90° angle is aided by grabbing the artery by the adventitia with fine forceps and gently lifting the artery surface while pressing the tip of needle just caudal to the lift point. This maneuver also reduces the risk of hitting the back wall (Figure 1).
    7. Unfold and bunch-up several gauze pads into a nest on which to lay the syringe used for the infusions. Place the nest on the rabbit's chest caudal to the incision.
    8. Put an IV-catheter on the syringe that contains DMEM-only (not too tight) 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 in the artery up to the bend point and wash out all blood from the artery with DMEM (~ 0.25 mL x 2). For each wash, fill the artery with DMEM, and then remove residual DMEM from the vessel lumen by gently pressing on the artery with a gloved finger, beginning just caudal to the cranial vascular clip. While maintaining gentle pressure, slide the finger from cranial to caudal. The luminal contents will wash out via the arteriotomy.
    10. Keep the catheter in the artery and exchange the DMEM syringe for the syringe containing virus solution. Make sure that no air enters the catheter.
    11. Slide the silk ties down the artery so that they surround the artery at the location of the catheter tip, but do not tighten them.
    12. Infuse 0.03 mL of the virus solution to push the remaining DMEM out of the catheter and then empty the artery. Collapse it by again removing all fluid from the lumen with a finger, cranial to caudal (as in step 2.3.9).
    13. Tighten the two ties around catheter tip to seal the lumen. Infuse the virus solution (~ 0.25 mL; 2 x 1011 vp/mL for adenovirus) by pressing the syringe plunger gently until the artery is distended to physiological caliber.
      NOTE: It is important that the artery expands to physiological caliber and remains distended during virus infusion. If not, the degree of gene transfer will drop significantly.
    14. Gently lay the syringe down on the nest of the gauze.
    15. Place a 7-0 polypropylene suture in the common carotid adventitia just caudal of the cranial vascular clip to mark the cranial boundary of gene transduction
    16. After the virus-containing solution has been in the artery lumen for 20 min, remove the virus-containing syringe and replace it with an empty syringe. Aspirate the virus-containing solution gently until the vessel collapses and remove the syringe. Cut or undo the silk ties and gently remove the catheter.
      NOTE: Cutting the ties very short aids in removing them. Remove the catheter carefully to avoid causing damage to the carotid endothelium.
    17. Using the surgical microscope, close the arteriotomy with 7-0 polypropylene using an X-pattern (Figure 2).
      1. Make a first pass entering at the bottom-right of the arteriotomy and exiting the vessel at the bottom-left. Then cross the opening and make a second pass from top-right to top-left.
      2. Before tying down the suture, flush out the artery by very briefly releasing the cranial vascular clip. Blood will flow out of the arteriotomy when the clip is released, removing air and residual virus from the lumen.
      3. Pull the suture to gently close the arteriotomy and tie a suture with 2 square knots.
        NOTE: Pulling the suture too tight will cause bunching of the tissue that will disturb flow, increasing the thrombosis risk and altering flow which can be an important uncontrolled variable in disease models.
    18. Release the cranial vascular clip, and then the caudal vascular clip using light pressure with gauze to stop any bleeding. If bleeding persists, continue pressure for 1 - 2 min. If the arteriotomy was properly closed, the bleeding always stops within this time frame.
    19. Let the assistant inject buprenorphine [0.02 mg/kg; subcutaneous (SQ)] at about this time to provide postoperative analgesia until fentanyl plasma levels become therapeutic.
      ​NOTE: A second buprenorphine injection (0.02 mg/kg; SQ) may be needed 6 h after the first injection to maintain analgesia until fentanyl plasma levels become therapeutic.
    20. Repeat virus infusion protocol on left side following steps 2.3.2 - 2.3.19.
  4. Wound closure
    1. Use a 5-0 PGA suture to close the midline fascia with a continuous suture.
    2. With a 3-0 PGA suture, close the skin with an intradermal pattern using a buried knot at both ends.
  5. Post-operative recovery and cleanup.
    ​NOTE: The rabbit must be monitored continuously for proper oxygenation and body temperature while recovering from anesthesia. Recovery should take place in a calm, quiet environment.
    1. Turn off isoflurane and oxygen flow and remove the face mask from rabbit.
    2. Unhook IV fluids but leave the IV port in place for emergency IV access.
    3. Take the rabbit to the recovery area and lay on its side in a cage with a warming water blanket turned on (or optionally a Bair Hugger heater).
    4. Give O2 by mask until the SpO2 is stable.
    5. Let the rabbit recover in a cage, flipping it to the other side every 15 min, until the rabbit can sit up on its hind legs and move around.
    6. When the rabbit is mobile, remove the IV from its ear before returning to the cage.
      Note: Do not return the rabbit to the company of other animals until it is fully recovered from anesthesia.
  6. Dispose of anything that had contact with the vector or was in the operative field, following appropriate biohazard and sharps waste protocols.

3. Gene transfer to rabbit common carotid arteries: Post-operative Care

  1. Assess the rabbit's overall condition daily after surgery, checking for wound healing, evidence of infection, appetite, respiration, and signs of pain.
    1. Check the rabbit's ear position (ears down can be a sign of pain or distress, especially when coupled with an untidy appearance). Check that the rabbit remains mobile and active. Check the rabbit for normal respiration without stridor. Check that the wound is clean, dry and intact. Check the rabbit for a normal body temperature. Consult veterinary services if the rabbit is not mobile and active, with normal body temperature, a tidy appearance, a clean wound, and normal respiration.
    2. Check for normal food intake and evidence of fresh stool and urine.
      NOTE: Food intake may be decreased following surgery, but should return to normal within 2 days; provide supplemental food as needed.
  2. Remove the fentanyl patch on post-operative day 3.
    NOTE: Buprenorphine can be administered as needed for post-operative pain that is not managed by the fentanyl patch, or in case the fentanyl patch is removed early.

4. Terminal Harvest Surgery: Pre-operation

  1. Anesthetize the rabbit. See sections 1.3 and 1.3.5 regarding achievement and maintenance of anesthesia.
    1. Weigh the rabbit. Combine 30 mg/kg ketamine and 2 mg/kg xylazine into a syringe. Inject ketamine/xylazine IM in the paraspinous muscles to induce anesthesia.
  2. Prepare the preparation room and OR tables while waiting for the rabbit to become anesthetized.
    1. In the OR preparation room: setup hair clippers and a vacuum for shaving the rabbit's neck and ear.
    2. In the operating room: setup monitoring equipment and position the probes (SpO2, temperature) on the OR table; prepare oxygen and isoflurane; tie a gauze strip to the face mask to secure it to the rabbit and place the mask on the head end of table.
      ​NOTE: The gauze strips tied on the facemask should have 2 tails of about 45 cm each.
    3. Turn on warming water blanket on the OR table. On top of the water blanket, position a rolled towel for neck support and a dispersive electrode plate (later placed under rabbit's back).
    4. Combine 1 mL of lidocaine HCl (2% stock) and 1 mL of bupivicaine HCl (0.5% stock) (50/50 mix) in a syringe as a local analgesic.
  3. When the rabbit is fully anesthetized, prepare the rabbit for surgery.
    1. Remove stool from the rectum, as described in 1.3.1, to allow for later placement of a temperature probe.
    2. Shave the rabbit from the sternal notch to the angle of the mandible while using a vacuum to keep the area clean. Also shave the left rear middle toe for the pulse-oximetry probe.
      NOTE: The protocol assumes that the surgeon will be on the rabbit's right side. If the OR setup will place surgeon on the opposite side, reverse sides in this step to keep the wires opposite of the surgeon. The sides of future steps should also be switched as needed.
    3. Transport the rabbit into the OR and place supine on the operating table with a rolled neck support towel under the rabbit's neck just below the head. Gently extend the rabbit's neck until it is straight and approximately horizontal.
    4. Hook the rabbit up to face mask with O2 on at 1 L/min, and start isoflurane administration. Secure the mask by wrapping the ends of the gauze strip tied to the mask around the neck support towel under the rabbit. Adjust isoflurane (typically 1 - 2%) as needed to maintain proper anesthesia for the remainder of the procedure.
    5. Ensure the dispersive electrode plate is centered under rabbit's back. Place temperature and pulse-oximeter probes.
    6. Restrain the rabbit's front legs by loosely tying them to the table.
      NOTE: Optionally, place a small plastic table over rabbit to protect the rabbit from being subjected to chest/abdominal pressure from the surgeon leaning on rabbit's chest/abdomen. The goal here is to prevent forced regurgitation of abdominal contents.
    7. Inject 2 mL of lidocaine (2% stock solution)/bupivacaine (0.5% stock solution) (50/50 mix; from step 2.4) subcutaneously along the planned neck incision line for local anesthesia.
    8. Spray the surgical site with betadine. Put on clean gloves for surgical procedure.

5. Terminal Surgery (Vessel Harvest)

  1. Prepare instruments and surgical field.
    1. Open a clean drape pack containing a table drape and one paper drape for the rabbit. Drape the instrument table.
    2. Open the instrument pack, place onto the instrument table, and arrange instruments. Place the following equipment onto the instrument table: one 20 mL syringe, and one 21G needle.
    3. Secure the electrocautery cable to the rabbit drape using the 7.25" Kantrowitz Forceps. Connect the plug to the electrosurgery unit and turn it on.
    4. Fill a 20 mL syringe with sterile saline. Use this saline as needed to keep the exposed tissue moist during the procedure.
    5. Place the paper drape over the rabbit and cut a hole in the drape over the surgical site on rabbit's neck. Clamp the corners of hole in place to the rabbit's skin with the towel clamps.
  2. Isolate common carotid arteries.
    ​NOTE: 2x surgical loupes should be used for this part.
    1. Cut the skin with an electrocautery along the midline extending approximately 7 - 9 cm cranially toward the mandible and clamp skin open with towel clamps.
      NOTE: If harvest is only a few days after previous surgery, electrocautery is not needed. Just cut the sutures and gently pull open the incision from the previous surgery.
    2. Make a short lateral cut through the fascia with an electrocautery at the caudal end. Then insert large scissors into the cut and bluntly dissect the fascia along the entire midline. Cut the dissected fascia down the midline with the electrocautery.
    3. With small dissecting scissors, dissect between the V-shaped muscles (sternocephalic muscle) and the sternohyoid muscle over the trachea, to isolate the common carotid arteries, starting on the right side.
    4. Dissect the right artery free from surrounding tissues from base of neck caudally to the crossing of the pharyngeal nerve cranially. Use surgical silicon loops to aid in retracting the artery during dissection.
    5. Repeat dissection on the left side (steps 5.2.3 - 5.2.4).
    6. With a 3-0 silk suture, ligate the common carotid artery cranial to the segment that was infused with the vector (use adventitial suture placed at first surgery to locate the cranial extent of vector infusion. Then ligate the carotid caudal to the repaired arteriotomy.
    7. Excise the carotid segment between the ligations, and flush the lumen with saline. Trim away excess adventitial tissue from the vessel and cut the carotid segment into smaller pieces for the different endpoint analyses (histology, DNA, ribonucleic acid (RNA), protein, explant culture, etc.).
    8. Inject 1 mL of Beuthanasia IV to euthanize, and confirm euthanasia of the rabbit.
  3. Dispose of anything that had contact with vector or was in the operative field, following appropriate biohazard and sharps waste protocols.

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Results

To implement this method with confidence, preliminary experiments are necessary to establish that the operator achieves efficient and reproducible gene transfer, with transgene expression primarily in luminal endothelial cells. In our experience, this is most easily assessed using a vector that expresses β-galactosidase. 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) staining of common carotid segments removed 3 days after vector infusion, as well as measurement of β-galactosida...

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Discussion

Certain aspects of surgical technique merit particular attention. Full exposure and mobilization of the common carotid artery via careful dissection will facilitate gene transfer and arteriotomy repair. However, during the dissection, direct manipulation of the carotid artery should be minimized to prevent vasospasm. In addition, any bleeding adjacent to the artery should be stopped by applying light pressure with gauze and extravasated blood should be cleaned up immediately by rinsing the area with normal saline. I...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank AdVec, Inc. for permission to use HDAd reagents, Julia Feyk for administrative assistance, and the Department of Comparative Medicine veterinary services for surgical advice and support. This work was supported by HL114541 and the John L. Locke, Jr. Charitable Trust.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Disposables
3mL syringe with 24G needleBecton Dickinson3095712x for gene transfer surgery; 3x for harvest surgery
1mL syringe with 27G needleBecton Dickinson3096236x for gene transfer surgery; 1x for harvest surgery
20mL syringe, luer lockNipro Medical CorpJD+20L
Catheters, 24G x 3/4"Terumo Medical ProductsSROX2419V
19G needleBecton Dickinson305187Gene transfer surgery only
21G needleBecton Dickinson305165For 20 mL syringe of saline
Gauze 4" x 4"Dynarex3242~10-15 per surgery
3-0 silk sutureCovidien Ltd.S-244
5-0 silk sutureCovidien Ltd.S-182Gene transfer surgery only
7-0 polypropylene sutureCP Medical8648PGene transfer surgery only
5-0 polyglycolic acid sutureCP Medical421AGene transfer surgery only
3-0 polyglycolic acid sutureCP Medical398AGene transfer surgery only
Alcohol swabsCovidien Ltd.6818For placement of I.V. line
Catheter plugVetoquinol411498Gene transfer surgery only
Ketamine HCl, 100 mg/mLVedco Inc.05098916106
Xylazine, 100 mg/mLAkorn Inc.4821
Lidocaine HCl, 2%Pfizer00409427702
Bupivacaine HCl, 0.5%Pfizer00409161050
Beuthanasia D-SpecialIntervet Inc.NDC 00061047305Harvest surgery only
Buprenorphine HCl, 0.3 mg/mL Patterson Veterinary12496075705Gene transfer surgery only
Saline IV bag, 0.9% sodium chlorideBaxter2B13092x for gene transfer surgery; can use vial of sterile saline in place of one
Heparin  (5000 U/mL)APP PharmaceuticalsNDC 63323-047-10Gene transfer surgery only
Fentanyl patch, 25 mcg/hr Apotex Corp.NDC 60505-7006-2Gene transfer surgery only
IsofluraneMultiple vendorsCatalog number not available
Gene transfer vectorDilute 350 µL per artery; 2 x 1011 vp/mL for adenovirus; gene transfer surgery only
Surgical Instruments
Metzenbaum needle holder 7" straightRobozRS-7900Gene transfer surgery only
Operating scissors 6.5" straight blunt/bluntRobozRS-6828
Needle holder /w suture scissorsMiltex8-14-IMCGene transfer surgery only
Castroviejo scissorsRobozRS-5658
Castroviejo needle holder, 5.75" straight with lockRobozRS-6412Gene transfer surgery only
Stevens scissors 4.25" curved blunt/bluntRobozRS-5943
Alm retractor 4" 4X4 5mm blunt prongsRobozRS-65142x
Backhaus towel clamp 3.5"Roboz4x
Micro clip setting forceps 4.75"RobozRS-6496Gene transfer surgery only
Micro vascular clips, 11 mmRoboz2x for gene transfer surgery only
Surg-I-LoopScanlan International1001-81M5 cm length
Bonaccolto forceps, 4” (10 cm) long longitudinal serrations, cross serrated tip, 1.2mm tip widthRobozRS-5210
Dumont #3 forceps Inox tip size .17 X .10mmRobozRS-5042
Graefe forceps, 4” (10 cm) long serrated straight, 0.8mm tipRobozRS-5280
Halstead mosquito forceps,  5" straight, 1.3mm tipsRobozRS-71102x
Halstead mosquito forceps,  5" curved, 1.3mm tipsRobozRS-7111
Jacobson mosquito forceps 5" curved extra delicate, 0.9 mm tipsRobozRS-7117
Kantrowitz forceps, 7.25" 90 degree delicate, 1.7 mm tipsRobozRS-7305
Tissue forceps 5", 1X2 teeth, 2 mm tip widthRobozRS-8162
Allis-Baby forceps, 12 cm, 4x5 teeth, 3 mm tip widthFine Science Tools11092-122x
Adson forceps, 12 cm, serrated, straightFine Science Tools11006-12
Veterinary electrosurgery handpiece and electrodeMACAN ManufacturingHPAC-1; R-F11
Surgical Suite Equipment
Circulating warm water blanket and pumpMultiple vendorsCatalog number not available
Forced air warming unit3MBair Hugger Model 505Gene transfer surgery only
IV infusion pumpHeskaVet IV 2.2Gene transfer surgery only
Isoflurane vaporizer and scavengerMultiple vendorsCatalog number not available
Veterinary multi-parameter monitorSurgivetSurgivet Advisor
Veterinary electrosurgery unitMACAN ManufacturingMV-9
Surgical microscopeD.F. VasconcellosM900Needs ~16x magnification

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