Technical Aspects of the Mouse Aortocaval Fistula

Summary

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Abstract

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

Introduction

The most common option chosen for renal replacement therapy in patients with end stage renal disease is hemodialysis. Access for hemodialysis is performed by arteriovenous fistula (AVF), arteriovenous graft, or temporary catheter placement. Although AVF are the preferred mode of access, AVF are still far from perfect. One-year primary AVF patency rates are only 60-65%, with many failures due to neointimal hyperplasia. The molecular biology and physiology of normal AVF formation and maladaptive formation remains poorly characterized. As such, much is to be gained in improving AVF outcomes with the accompanying gain in understanding of the regulation of fistula formation.^1-3

Exposure of a vein to arterial flow is commonly studied in the context of vein grafts that are direct models of surgical bypass.^{4, 5} Although vein grafts and AVF similarly expose a vein to an arterial environment, there are several critical differences between them. First, the resistance of the distal runoff bed is much lower in AVF, as the AVF drains to the right atrium whereas the vein graft drains to the often diseased distal small arteries, resulting in increased blood flow and shear stress in the AVF. Second, the blood in the venous limb of the AVF can be a mixture of venous and arterial blood leading to lower oxygenation compared to the purely arterial content in the vein graft. Third, the vein in the AVF is not removed from its bed, leading to preservation of its vaso venorum and nervous innervations, compared to the complete transposition of the vein graft into the arterial bed. Lastly, the number of anastomoses is usually two in vein grafts whereas an AVF typically has only one. In this study, we introduce a mouse model of aortocaval fistula that can be used for the study of AVF.

Protocol

1. Anesthesia and Pre-operative Procedures

1. Induction of general anesthesia
   Approval by the appropriate Institutional Animal Care and Use Committee is obtained. Male C57/black mice, aged 10 weeks, are anesthetized. Deliver 4% isoflurane with 0.8 L/min oxygen to a Plexiglas box via an isoflurane vaporizer and place the mouse inside. Alternatively, if preferred by the surgeon, intraperitoneal anesthesia can be used.
2. Preserving anesthesia
   After confirmation of anesthesia by lack of reaction to toe pinch, the mouse is put on the operation table in a supine position with a silicone mask. Isoflurane is decreased to 2% to maintain appropriate anesthesia level.
3. Hair removal
   Ventral hair is removed from the neck to lower abdomen using a hair remover. Hair remover is preferred because the hair that is left using a shaver may interfere with the Doppler ultrasound.

2. Preoperative Ultrasound

1. Objectives of the ultrasound
   Doppler ultrasound examination is performed prior to AVF surgery so as to record the base level findings that can be compared with post-operative findings to see anatomical and physiological changes.
2. Points of observation
   The jugular vein, supra- and infra-renal inferior vena cava (IVC), and abdominal aorta are examined using both pulse Doppler and B-mode.
3. Measurements
   Using the B-mode, diameter of each vasculature is measured. In addition, maximum velocity (peak systolic velocity in arteries) and waveforms are recorded in each of the measuring points described above.

3. Operative Procedures

1. Mouse Preparation
   Prepare the incision site with a topical antiseptic and continue proper aseptic technique during the surgery including using a surgical drape, sterile gloves and instruments, etc.
2. Laparotomy
   Midline abdominal incision is made from lower edge of the liver to just above the pubis.
3. Exposure of the aorta and IVC.
   A retractor is inserted and all of the bowels are pulled out of the abdominal cavity to the right side and kept wrapped in gauze soaked with saline. Dissect the membrane that connects the retroperitoneum and lower colon to get full view of the aorta and IVC. Apply pressure on the bladder if it is dilated and gets in way.
4. Exposure of the puncture site
   Puncture site will be between half to three quarters of the distance from left renal vein to aortic bifurcation. (Figure 1) Dissect the left margin of the aorta so that it can be punctured. Keep in mind that the aorta is positioned about 45 degrees behind the IVC and it must be dissected posteriorly in addition to the left. (Figure 2) The most important point of this surgery is not to dissect between the aorta and IVC. Keep far away from them.
5. Exposure of the clamp site of the aorta
   Dissect the infrarenal aorta from surrounding tissues. Be careful not to injure the IVC on the left and left renal veins with its tributaries on the right.
6. Clamping of the aorta
   Clamp the proximal aorta by applying microsurgery clips.
7. Rotation of the aorta
   By grasping the connective tissue surrounding the aorta, rotate the aorta so that the puncture can be made from slightly dorsal side of the aorta (Figure 2).
8. Puncture of the aorta through to IVC
   Keeping the aorta in a rotated position, puncture the aorta through to IVC using a 25 G needle. (Figure 2) If successful, the needle can be seen through the thin IVC wall.
9. Hemostasis
   By pulling the retroperitoneal tissues, including the muscles, wrap the puncture site of the aorta. Do not compress the aorta against the IVC, as it may clot the AVF (Figure 3).
10. De-clamping the aorta
    After confirmation of the primary hemostasis, de-clamp the aorta. Additional bleeding may occur but it can be stopped with further compression described above. Upon de-clamping, arterial blood can be seen flowing into the IVC instead of dark venous blood flow.
11. Closure of the abdomen
    Return the bowels into its natural position and close the abdomen.

4. Post-operative Procedures

1. Post-operative care
   After closure of the abdomen, isoflurane is stopped. Post-operative care including analgesia and wound care is applied in accordance with the instructions recommended by 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. Post-operative ultrasound
   On the first day after operation, Doppler ultrasound is performed to confirm the AVF. In addition, other variables are measured to check the changes from pre-operative values.

Results

After practice, survival on the first post-operative day is typically over 95%; at post-operative day 7, survival was 91.3%. Figures 1 and 2 show the ideal puncture site in coronal and cross-sectional views; placement of the puncture site is critical to obtaining good survival without excessive hemorrhage. Intra-operative deaths and early post-operative deaths are typically due to hemorrhage. This tends to occur with excessive dissection between the aorta and the IVC, or from inadequate hemostatic pressu...

Discussion

There are several reports that have described AVF in rats, including femoral AVF,^{6, 7} carotid to jugular AVF⁸ and aorto-caval AVF.^{9, 10} The rat is several times larger than the mouse, allowing easier surgery in the rat compared to the mouse. Nevertheless, in order to take advantage of mouse genetic strains and variants the procedure must be extended to include mouse models. Several previously published reports introduced the AVF model in mice, but these reports have generally been in the ...

Materials

Name	Company	Catalog Number	Comments
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
25 Gauge Needle	Becton Dickinson	BD PrecisionGlide Needle 25G x 7/8 #305124