Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Summary

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Abstract

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

Introduction

The murine aortovenous fistula (AVF) puncture model between the aorta and the inferior vena cava (IVC) is now an established technique.¹ In this model, both walls of the infra-renal aorta are punctured with a 25 G needle, exiting into the adjoining infra-renal vena cava; the anterior aortic entrance hole is repaired with simple compression, and does not require suture repair. Serial follow-up examination by high-resolution Doppler ultrasound and histological analysis shows the AVF to have a maturation phase and then a failing phase, recapitulating the known pathophysiology of human AVF.²

To explore mechanisms that modulate AVF maturation, improved methods for delivery of therapeutic agents to the maturing AVF endothelium are needed. Delivery of therapeutic agents to vessels can be either via endovascular delivery to the lumen, or via external delivery to the adventitia. One example of external delivery is the commonly used adventitial application of Pluronic gel. This copolymer is thermo-reversible and transformed from liquid to solid gel when warmed to body temperature. Prior studies have shown sustained drug delivery is achieved when drug mixed in pluronic gel is applied topically in vivo.^3,4 Adventitial application of viral vectors or siRNA with Pluronic gel has been reported to be effective as a perivascular delivery system.^5,6 We have also reported that treatment of explanted human saphenous veins with adventitial stimulation by peptides resulted in phosphorylation of endothelial receptor proteins.⁷

On the other hand, investigators have also used intraluminal delivery of both viral and nonviral vectors in canine^8-10 and rabbit^11,12 models of vein grafts. In these reports, gene transfer was performed ex vivo after vein harvest. Eslami et al. reported endovascular viral gene delivery to carotid veins in situ without creating a bypass.¹³ Gloverman et al. reported intraluminal and adventitial delivery of naked DNA in rat femoral artery-superficial epigastric vein fistulae.¹⁴ The Mayo group reported adventitial drug delivery in mouse carotid artery-jugular vein fistulae.^15,16 However, these previously reported models required a sutured anastomosis to create the AVF. In this report, intraluminal drug delivery with simultaneous AVF creation in mice is described, using a suture-less model of AVF creation. By using this modified murine AVF model a simple method for intraluminal drug delivery to the venous limb of the fistula can be performed.

Protocol

Approval by the appropriate Institutional Animal Care and Use Committee is obtained.

1. Anesthesia and Pre-operative Procedures

1. Anesthetize male C57Bl/6 ice, aged 8 weeks, with vaporized 3% isoflurane and 0.8 L/min oxygen administered into an acrylic induction chamber.
2. Confirm adequate anesthesia by lack of reaction to toe pinch. Position the mouse supine on the operation table and position a silicone mask to deliver vaporized 2 - 3% isoflurane by continuous inhalation.
3. Remove ventral hair from the neck to lower abdomen using a chemical depilatory cream.
4. Perform Doppler ultrasound examination prior to AVF surgery to record baseline characteristics of arterial and venous flow and vessel diameter at the areas of interest.^1,2
5. Attach a 1 ml syringe to a 25 G needle and load the syringe with the desired drug. Bend the needle to a 60 degree angle approximately 4 mm from the needle tip. Grasp the 25 G needle with a curved needle holder.

2. Operative Procedures

1. Prepare the incision site with a topical antiseptic and apply a surgical drape. Use sterile gloves and instruments to maintain aseptic technique throughout the surgery.
2. Make a midline abdominal incision with a scalpel extending from the level of the lower liver edge to just above the pubis.
3. Insert a retractor and eviscerate all bowels from the abdominal cavity toward the right side. Wrap the bowels in gauze soaked with saline. Dissect the membrane connecting the retro-peritoneum and lower colon to obtain full view of the aorta and IVC.
4. Dissect the infra-renal aorta and IVC from surrounding tissues, preparing for proximal and distal clamping.
5. Place a single microsurgery clip across both the proximal aorta and the proximal IVC at the level just below the left renal vein. Place a second microsurgery clip across both the distal aorta and the distal IVC.
6. Grasp the connective tissue surrounding the aorta and rotate medially so that the dorsal surface of the aorta is slightly exposed for the arterial puncture, as described previously.¹
7. Quickly expose the puncture site. The puncture site will be at the caudal aspect of the vessels, approximately three quarters of the distance from the left renal vein to the aortic bifurcation. Keeping the aorta in a rotated position with the left hand, dissect the left lateral margin of the aorta so that there is ample exposure to allow puncture with the right hand. Be careful not to dissect between the aorta and the IVC.
8. Maintain the aorta in a rotated position and puncture the aorta through into the IVC using a 25 G needle with an attached 1 ml syringe containing drug solution. (Figure 1A)
9. Infuse the drug solution (100 - 200 µl) using the left hand. The needle can be seen through the dilated and thin IVC wall when the transparent drug solution displaces the venous blood out of the IVC (Figure 1B,C). Remain still and maintain the needle in position for 15 min.
10. Remove the distal microsurgery clip to de-clamp only the distal aorta and the distal IVC.
11. Remove the needle and then cover the puncture site of the aorta by pulling up adjacent retro-peritoneal tissue.
12. Remove the proximal microsurgery clip to de-clamp the proximal aorta and the proximal IVC. Upon de-clamping, arterial blood is observed flowing into the IVC instead of dark venous blood flow. Keep covering the puncture hole for 1 min.
13. After confirmation of hemostasis by observation for 30 sec without compression, return the bowels into their natural position and close the abdomen with a running suture according to your approved animal protocol. 

3. Post-operative Procedures

1. After closure of the abdomen, discontinue anesthesia. Apply post-operative care including analgesia and wound care in accordance with instructions recommended by the Institutional Animal Care and Use Committee. For analgesia we use buprenorphine at 0.1 mg/kg intrasmuscularly every 12 hr for 24 hr following the surgical procedures.
2. On the first day after operation, perform Doppler ultrasound to confirm patency of the AVF. In addition, measure other vessel and flow characteristics serially and compare for changes from pre-operative baseline values.^1,2

Figure 1. (A) Operative Photo Showing Intraluminal Delivery during AVF Surgery. Clamp the proximal and distal aorta, as well as the IVC by applying microsurgery clips. Puncture the aorta through into the IVC using a 25 G needle with attached syringe containing drug solution. (B) A Higher Power Picture (4X magnification) of the Punctured IVC before Infusion. The needle tip is obscured by dark colored venous blood. Yellow arrowheads denote the wall-to-wall diameter of the IVC. (C) A Higher Power Picture (4X magnification) of the Punctured IVC after Infusion. The needle tip (black arrow) can be seen through the gently distended and thin IVC wall (yellow arrowheads) as the transparent drug solution displaces the venous blood.

Results

In a series of 33 mice, survival on the first post-operative day was 97.0%; AVF patency, as determined by ultrasound, was 84.9%.

Gene transduction efficiency of this endovascular delivery route with the traditional external route was compared. For intraluminal delivery (ILD), immediately after the puncture, 200 µl of Adenovirus-GFP (Ad-GFP) vector solution (1 × 10⁹ PFU/ml) was infused into the IVC via t...

Discussion

This modification of the murine AVF model incorporates intraluminal drug delivery to the venous endothelium at the time of AVF creation. An AVF was created by puncturing the infra-renal aorta with a 25 G needle and extending the puncture through the opposite aortic wall into the IVC, followed by injection of drug solution through the same needle. The solution is maintained intra-caval, i.e., in the venous limb of AVF, until de-clamping. What distinguishes this model from other murine AVF models^17-19

Materials

Name	Company	Catalog Number	Comments
Pluronic F-127	Sigma-Aldrich	P2443-250G	Used as 30% solution in d-water
GFP antibody	NOVUS BIOLOGICALS INC	NB100-1770
Ad-CMV-GFP	VECTOR BIOLABS	1060
0.9% Sodium Chloride Irrigation, USP	Baxter	2F7122
BD PrecisionGlide Needle 25 G x 5/8	BD	305122
BD 1 ml Syringe Tuberculin Slip Tip	BD	309659
Scalpel	Surgical Design Inc	22079707
6-0 ETHILON P-1 11 mm 3/8c Reverse Cutting	ETHICON INC	697G
Vevo 770 ultrasound machine	Visualsonics	20 - 60 Mhz scan head; RMV-704
Vascular clamp	Roboz Surgical Instrument Co.	RS-5424
Clamp applying forceps	Roboz Surgical Instrument Co.	RS-5410