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.
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.
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 region3,4,5.
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 stenosis10,11.
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.
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
2. Skin incision
3. Preparation of the external jugular vein (EJV)
4. Dissecting and preparing the common carotid artery (CCA)
5. Preparing the anastomosis
6. Side-to-side anastomosis
7. Vascular clamp removal and ligation of the vein
8. Skin closure and postoperative care
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 significant venous intimal hyperplasia at the AVF anastomosis.
In all, 36 healthy New Zealand rabbits were included in this study. In total, 34 rabbits had an immediately successful AVF using the MNTT. Three rabbits had significant postoperative bleeding, and one died because of blood loss. The remaining two rabbits required compression hemostasis to arrest the bleeding. Additionally, four rabbits died after surgery, with common symptoms including sneezing, coughing, runny nose, anorexia, and diarrhea. Finally, 31 rabbits survived, and 13 had a functional AVF 4 weeks after surgery. The survival rate was 86.1% (Figure 2).
The AVF was assessed using color Doppler ultrasonography (CDU) 4 weeks after surgery to confirm patency, defined as active blood flow through the AVF anastomosis (Figure 3). Moreover, the spiral laminar flow was observed at both the vein and artery near the anastomosis (Figure 3). In terms of the ultrasound parameters between the AVF and normal vessels on the contralateral neck, there were significant differences in the diameter and PSV of the EJV and the diameter of the CCA (Table 1).
The AVF was obtained at 4 weeks after surgery and made into sections. H&E staining was performed on all the obtained sections. Significant venous intimal hyperplasia was observed at the site of the AVF anastomosis (Figure 4), whereas no significant intimal hyperplasia was observed at the proximal EJV of the anastomosis (Figure 4).
Figure 1: The CCA-EJV AVF created in rabbits using the MNTT. (A) Two tunnels were made along the direction perpendicular to the EJV. (B) The CCA was mobilized. (C) Using Kunlin's technique, a side-to-side anastomosis of the CCA and EJV was performed. (D) The distal end of the EJV was ligated, and patent blood flow was visible through the proximal end. Abbreviations: CCA = common carotid artery; EJV = external jugular vein; AVF = arteriovenous fistula; MNTT = modified no-touch technique. Please click here to view a larger version of this figure.
Figure 2: Survival curves for the rabbits. One rabbit died immediately after surgery because of blood loss. The remaining four rabbits died on the 3rd, 7th, 10th, and 26th days after surgery. Finally, 31 rabbits were alive at 4 weeks after surgery. Please click here to view a larger version of this figure.
Figure 3: The CDU evaluation atlas of the AVF. (A) The CCA showed unidirectional low-resistance blood flow spectra, the loss of normal triphasic blood flow, widened systolic peaks, and abundant diastolic blood flow. (B) The EJV showed artery-like low-resistance blood flow spectra, with an increased PSV and widened spectra. (C) Active blood flow through the AVF anastomosis. (D) Spiral laminar flow was observed in the EJV outflow tracts. Abbreviations: AVF = arteriovenous fistula; CCA = common carotid artery; EJV = external jugular vein; PSV = peak systolic velocity. Please click here to view a larger version of this figure.
Figure 4: Observation of the AVF morphology of rabbits at 4 weeks after surgery (H&E staining). (A) No significant intimal hyperplasia of the CCA with a patent AVF was observed. (B) The elastic membrane of the EJV at the anastomosis site with a patent AVF was severely disrupted, with significant intimal hyperplasia. Thick hyperplastic fibrous tissues were clearly visible on the inner side of the elastic membranes, with reduced and fragmented elastic fibers. (C) The proximal EJV of the anastomosis had no significant intimal hyperplasia with a patent AVF. This showed intact elastic membranes and slender, wavy elastic fibers. Abbreviations: AVF = arteriovenous fistula; CCA = common carotid artery; EJV = external jugular vein. Magnification: 200x. Please click here to view a larger version of this figure.
Group | External jugular vein | Common carotid artery | ||
Diameter (mm) | PSV (cm/s) | Diameter (mm) | PSV (cm/s) | |
AVF | 7.21 ± 1.55 | 79.64 ± 39.31 | 3.06 ± 0.32 | 59.38 ± 32.25 |
Normal vessel | 3.13 ± 0.66 | 9.21 ± 2.77 | 2.17 ± 0.41 | 39.02 ± 11.56 |
t | 5.413 | 3.996 | 3.779 | 1.329 |
P | 0.001 | 0.004 | 0.005 | 0.22 |
Table 1: Comparison of ultrasound parameters between the AVF and normal vessels on the contralateral neck in rabbits (n = 5). Abbreviation: PSV = peak systolic velocity. t-test is used for data analysis. When the P value is <0.05, the comparison between the two groups is statistically significant.
Supplementary Figure 1: Schematic diagram of the vascular anastomosis modes in AVF surgery. (A) Traditional AVF surgery. (B) An AVF created using the NTT. (C) An AVF created using the MNTT. Abbreviations: AVF = arteriovenous fistula; NTT = no-touch technique; MNTT = modified no-touch technique. Please click here to download this File.
Supplementary Figure 2: Functional end-to-side anastomosis AVF with the MNTT. Abbreviations: AVF = arteriovenous fistula; MNTT = modified no-touch technique. Please click here to download this File.
Supplementary Figure 3: Rabbit model of AVF created using the conventional technique. (A) The EJV was dissected from the perivenous tissue. (B) The EJV and CCA were pulled together. (C) Using Kunlin's technique, a side-to-side anastomosis of the CCA and EJV was conducted. (D) The distal end of the EJV was ligated, and patent blood flow visibly passed through the proximal end. Abbreviations: CCA = common carotid artery; EJV = external jugular vein; AVF = arteriovenous fistula. Please click here to download this File.
Currently, several animal models are available for AVF. Among them, pigs, sheep, and dogs are mostly used as large animal models13,14,15. The small animal models used include rabbits, rats, and mice16,17,18. New Zealand rabbits were used in this study. New Zealand rabbits have abundant perivenous tissues around the EJV, which makes them conducive for the study of the MNTT method. The advantages of using New Zealand rabbits include the simple surgical operation, convenient feeding, and low cost. However, large animal models have benefits over rabbit models when studying hemodynamics.
This study proposed a unique CCA-EJV AVF procedure using MNTT without the dissection of the perivenous tissue or cutting off the artery. Functional end-to-side anastomosis19,20 for AVF creation was accomplished by side-to-side arteriovenous anastomosis followed by the ligation of the distal EJV. Compared to conventional techniques (Supplementary Figure 3), AVF creation with MNTT more adequately preserved the perivenous tissue. During arteriovenous vascular anastomosis, due to the preservation of the perivenous tissue, the venous wall could be more fully exposed by pulling the perivenous tissue, which was conducive to vascular anastomosis.
In ultrasonography, spiral laminar flow was observed in the vein and artery near the anastomosis, indicating that the MNTT may have more favorable hemodynamics, which may account for the excellent rates of patency and maturation21,22. On histological observation, significant venous intimal hyperplasia was observed at the AVF anastomosis, whereas no significant intimal hyperplasia was present at the proximal EJV of the anastomosis. This finding is probably related to the improvement of the juxta-anastomotic stenosis by this surgical technique or the spiral laminar flow.
Common problems encountered and suggestions
Given the thin wall of the EJV, gentle operation is necessary when anastomosing blood vessels to prevent damage to the EJV. Since the tissue around the EJV is preserved, it can be pulled during anastomosis to unfold the vessel and make it more conducive for suturing. However, the preservation of the tissue surrounding the EJV is bothersome. After venotomy, given the blood outflow from the venous vessels, the blood vessels collapse and cause EJV retraction. During EJV anastomosis, microvascular tweezers must be used to pull the surrounding tissues of the EJV and fully expose the vein wall. Additionally, if the distance between arteries and veins is long, the CCA should be allowed a free sufficient length to ensure that the two are close to each other and, thus, to facilitate the anastomosis. An 8-0 sterile vascular suture was used for the vascular anastomosis to reduce damage to the vessels.
Technical limitations
The preparation of the vein still requires tunneling and clamping along the tunnel, and this maneuver can cause venous injury. Prior to performing an arterial and venous side-to-side anastomosis, vascular injury may result from pulling the artery and vein. Since the patency rate of 38.2% was lower than that of other AVF models23,24, further improving the care and detection of rabbits after AVF surgery is necessary.
Applications of the technique
To further study the mechanisms of the MNTT and related hemodynamics, pathological, molecular, and genomic studies are needed to validate this technique.
Conclusion
A CCA-EJV AVF was successfully created in this study using the MNTT method. The operation was simple, with good reproducibility and a high success rate, indicating that this technique has the potential to be ideal for further studies on the application of MNTT in AVF surgery.
The authors have nothing to disclose.
This study was supported by grants from the Suzhou Science and Technology Plan Project (SYS2020077), Suzhou High-tech Zone Medical and Health Science and Technology Plan Project (2020z001), Suzhou Science and Technology Development Plan Project-Medical and Health Science and Technology Innovation (SYK2021030), Nanjing Medical University Science and Technology Development Fund-General Project (NMUB20210253), Suzhou Science and Technology Bureau of the application of the basic research project (No.SYSD2019205, No.SYS2020119), Jiangsu Province Traditional Chinese Medicine Science and Technology Development Plan Project (No.MS2021098), the Ministry of Education Industry-University Cooperation Collaborative Education Project (No. 202102242003), the Sixth "333 High-level Talent Cultivation" Project in Jiangsu Province, Suzhou Science and Technology City Hospital 2022 Hospital-level Pre-research Fund Project (SZKJCYY2022014), and Suzhou "KeJiaoXingWei" Youth Science and Technology Project (KJXW2022086).
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 |