A Modified Technique for Arteriovenous Fistula Construction in Rabbits

Summary

The present protocol proposes the creation of an arteriovenous fistula in rabbits using a modified no-touch technique. The technique involves the side-to-side anastomosis of the common carotid artery and external jugular vein without the dissection of the perivenous tissues or cutting off the artery.

Abstract

Juxta-anastomotic stenosis is a challenging problem that often causes non-maturation and decreases the patency of an arteriovenous fistula (AVF). Injury to the veins and arteries during the operation and hemodynamic changes can lead to intimal hyperplasia, leading to juxta-anastomotic stenosis. To reduce injury to the veins and arteries during the operation, this study proposes a new modified no-touch technique (MNTT) for AVF construction that can decrease the rate of juxta-anastomotic stenosis and improve the AVF patency. To unravel the hemodynamic changes and mechanisms of the MNTT, this study presented an AVF procedure using this technique. Although this procedure is technically challenging, 94.4% procedural success was achieved after adequate training. Ultimately, 13 out of 34 rabbits had a functional AVF 4 weeks after the surgery, leading to a 38.2% AVF patency rate. However, at 4 weeks, the survival rate was 86.1%. Ultrasonography showed active blood flow through AVF anastomosis. Furthermore, the spiral laminar flow was observed in the vein and artery near the anastomosis, suggesting that this technique may improve the hemodynamics of the AVF. On histological observation, significant venous intimal hyperplasia was observed at the AVF anastomosis, whereas no significant intimal hyperplasia was observed at the proximal external jugular vein (EJV) of the anastomosis. This technique will improve the understanding of the mechanisms underlying the use of MNTT for AVF construction and provide technical support for the further optimization of the surgical approach in AVF construction.

Introduction

The construction of an arteriovenous fistula (AVF) is widely used in clinical practice for patients undergoing maintenance hemodialysis (MHD), and it has higher patency and fewer complications than an arteriovenous graft (AVG) or tunneled cuffed catheter (TCC)^1,2. Although AVF is the preferred mode of vascular access, it is not perfect and has inherent limitations. The 1 year primary AVF patency rates are only 60%-65%, with many failures occurring in the near anastomotic region^3,4,5.

Vessels undergo different degrees of damage during the traditional surgical approach, which ultimately affects the maturation of the AVF. New surgical modalities, such as the no-touch technique (NTT) (Supplementary Figure 1) proposed by Hörer et al.⁶ and radial artery excursion and reimplantation (RADAR) proposed by Sadaghianloo et al.^7,8 and Bai et al.⁹, were designed to decrease the rate of juxta-anastomotic stenosis and to improve the fistula patency by modifying the surgical technique. Although the effect of RADAR was better than that of NTT, inflow arterial stenosis was observed to be more prominent with RADAR. To further reduce injury to the veins and arteries during the operation, in 2021, a new modified no-touch technique (MNTT) was proposed to create a radio-cephalic AVF by preserving the perivenous tissue around the cephalic vein without cutting the radial artery (Supplementary Figure 1 and Supplementary Figure 2). The preliminary results showed increased primary patency, decreased juxta-anastomotic stenosis, and no arterial stenosis^10,11.

Considering the current lack of animal models of AVF using MNTT, and to further explore the mechanism of MNTT in AVF surgery, this study introduces a common carotid artery (CCA)-external jugular vein (EJV) AVF procedure using MNTT.

Protocol

The experimental procedures using laboratory animals were approved by the Experimental Animal Welfare Ethics Committee of Nanjing Medical University. New Zealand rabbits aged 10 months (of both sexes; body weight, 3.18 ± 0.24 kg) were used for this study. The animals were obtained from a commercial source (see Table of Materials).

1. Animal preparation

1. Anesthetize the rabbits using a mixed intravenous injection of tiletamine hydrochloride and zolazepam hydrochloride (3 mg/kg) into the marginal ear vein and an intramuscular injection of sumianxin II (0.02 mL/kg) (see Table of Materials) into the hindlimb muscle.
   NOTE: After about 1-3 min, the anesthetic effect stabilizes. Before proceeding, the level of anesthesia should be checked by pinching the skin behind the neck and observing the corneal reflex. Tiletamine hydrochloride, zolazepam hydrochloride (0.5 mg/kg), and sumianxin II (0.01 mL/kg) could be added during the operation if needed.
2. Lay the rabbit on a fixed table (see Table of Materials) in a supine position, and tie the limbs and incisors with binds.
3. Shave the neck and upper chest using an electric razor, and remove the hair by animal depilatory cream (see Table of Materials).
4. Maintain sterile conditions during the surgery by autoclaving the surgical equipment and cleaning the surgery area with povidone-iodine solution.

2. Skin incision

1. Position the rabbit with its head toward the surgeon.
2. Make a longitudinal incision of ~3 cm between the mandible and the sternoclavicular joint using surgical scissors or a scalpel blade.

3. Preparation of the external jugular vein (EJV)

1. Expose the incision, and identify the right EJV. Ensure that the EJV and its perivascular tissues are clearly visible and not dissected.
   NOTE: The EJV shows an inverted "Y" pattern, and the branch close to the medial neck needs to be anastomosed.
2. Make a tunnel that a vascular clamp can be passed through (see Table of Materials) along the direction perpendicular to the EJV. Ensure that the distances between the openings on both sides of the tunnel and the EJV are >1 cm.
3. Place a vascular clamp along the tunnel.
4. Make another tunnel (same as in step 3.2) at the distal EJV using the same method.
   NOTE: Ensure the distance between the two tunnels is ≥2 cm.
5. Apply a 4-0 suture (see Table of Materials) and a vascular clamp along the tunnel to control the blood flow (Figure 1A).

4. Dissecting and preparing the common carotid artery (CCA)

1. Use forceps (see Table of Materials) to explore the CCA lateral to the trachea and medial to the sternocleidomastoid muscle.
   NOTE: The CCA has a pulsatile sensation and runs parallel to the cervical nerve.
2. Bluntly dissect the CCA to a length of about 2 cm.
   NOTE: Avoid injury to the vagus nerve and its branches with a deep arterial course.
3. Place a 4-0 suture thread around the CCA to control the blood flow when needed.
4. Apply vascular clamps (see Table of Materials) as distally and proximally as possible (Figure 1B).

5. Preparing the anastomosis

1. For phlebotomy and anastomosis, use microscissors (see Table of Materials) to dissect the internal part of the EJV (4 mm long) free from the surrounding tissues.
2. Make a 4 mm long longitudinal incision with microscissors in the middle of the vein. Rinse the vein with heparin solution (100 IU/mL) to prevent thrombosis.
3. Make a longitudinal incision measuring approximately 4 mm in the anterior wall of the artery using a sharp blade and microscissors. Rinse the artery with 100 IU/mL heparin solution until the vessel is clear of blood.

6. Side-to-side anastomosis

1. Pull the EJV and CCA closely together, as close as possible.
2. Apply Kunlin's technique¹² for the side-to-side anastomosis of the CCA and EJV using 8-0 nonabsorbable sutures (see Table of Materials). Suture the posterior wall of the vessel first (Figure 1C), followed by the anterior wall of the vessel.
   NOTE: Since the EJV wall in a rabbit is thin, care should be taken during the surgery to prevent damage to the vessels that could later compromise the patency of the anastomosis. During the process of vascular anastomosis, heparin solution (100 IU/mL) must be used repeatedly to rinse the lumen to prevent thrombosis.

7. Vascular clamp removal and ligation of the vein

1. Remove the distal vascular clamp of the CCA, the proximal vascular clamp of the EJV, and the proximal vascular clamp of the CCA in turn. Observe active blood flow through the anastomosis.
2. Ligate the distal end of the EJV using the 4-0 suture that was placed earlier. Remove the distal vascular clamp of the EJV.
3. Remove the suture thread that was placed around the CCA (Figure 1D).

8. Skin closure and postoperative care

1. After ensuring no significant bleeding in the surgical field, close the skin of the neck using interrupted sutures (4-0).
2. Place the rabbit in a cage until it completely recovers. Typically, this takes 30-45 min.
   NOTE: In case of incomplete or delayed recovery, care should be taken to ensure that the rabbit does not experience hemodynamic shock due to bleeding in the surgical area. If required, administer Sumianxin II (0.01 mL/kg) post-surgery.

Results

The outcome of the successful application of this technique is a patent AVF in the rabbit neck. This study used the following criteria to evaluate the success: (1) when the vascular anastomosis is completed, the venous tremor of the AVF can be touched, and the vascular murmur can be heard; (2) 4 weeks after the AVF is established, the active blood flow through the internal fistula anastomosis can be measured by color Doppler ultrasound; (3) 4 weeks after the AVF is established, hematoxylin-eosin (H&E) staining shows ...

Discussion

Currently, several animal models are available for AVF. Among them, pigs, sheep, and dogs are mostly used as large animal models^13,14,15. The small animal models used include rabbits, rats, and mice^16,17,18. New Zealand rabbits were used in this study. New Zealand rabbits have abundant perivenous tissues around the EJV, which makes them...

Materials

Name	Company	Catalog Number	Comments
Animal Depilatory	Fuzhou Feijing Biotechnology Co., Ltd.	PH1877
Curved hemostatic forceps	Xinhua Surgical Instrument Co., Ltd.	ZH131R/RN
Dissecting forceps	Xinhua Surgical Instrument Co., Ltd.	ZDO25R/RN
electrical razor	Shenbao Technology Co., Ltd	PGC-660
Fixed Table	Zhenhua Biomedical Instrument Co., Ltd	ZH-DSB019
Halsey needleholder	Xinhua Surgical Instrument Co., Ltd.	ZM208R/RN
Heparin Dodium Injection	Jiangsu Wanbang Biochemical Pharmaceutical Group Co., Ltd.	H32020612
Medical gauze dressing	Nanchang Kangjie medical hygiene products Co., Ltd	20172640135
Micro forceops	Xinhua Surgical Instrument Co., Ltd.	ZD275RN/T
Micro needle holder forceps	Xinhua Surgical Instrument Co., Ltd.	ZF2618RB/T
Micro scissors	Xinhua Surgical Instrument Co., Ltd.	ZF022T
Non-silk sutures 4-0	Kollsut Medical Instrument Co., Ltd.	NMB020RRCN26C075-1
Non-absorbable sutures 8-0 (double needle)	Yangzhou Yuankang Medical Instrument Co., Ltd.	10299023602
Povidone iodine solution	Shanghai Likang Disinfection High-tech Co., Ltd.	310512
Rinse needle	Jiangsu Tonghui Medical Instrument Co., Ltd	20180039
scalpel handle	Shanghai Medical Instrument (Group) Co., Ltd. Surgical Instruments Factory	J11030
Sharp blade	Suzhou Medical Products Factory Co., Ltd.	TY21232001
Sodium Chloride Injection  (100 mL)	Guangdong Otsuka Pharmaceutical Co., Ltd.	B21K0904
Sugical Scissors	Xinhua Surgical Instrument Co., Ltd.	ZC120R/RN
Sumianxin II	Jilin Dunhua Shengda Animal Pharmaceutical Co., Ltd.	20180801
Syringe with needle（5 mL)	BD medical devices (Shanghai) Co., Ltd	2006116
Tiletamine Hydrochloride and Zolazepam Hydrochloride for Injection	Virbac Pet Health, France	83888204
Triangle needle	Hangzhou Huawei medical supplies Co., Ltd	7X17
Vascular clamp	Xinhua Surgical Instrument Co., Ltd.	ZF220RN
New Zealand rabbits	Suzhou Huqiao Biological Co., Ltd.	SCXK2020-0001