Construction and Evaluation of End-to-side Anastomosis Model of Autologous Arteriovenous Fistula in Mice

doi:10.12300/j.issn.1674-5817.2023.093

Abstract

Abstract:

Objective To establish an animal model of autologous arteriovenous fistula in mice and evaluate its effect. Methods The left external jugular vein and common carotid artery of 10 8-week-old male C57BL/6 mice were separated by end-to-side anastomosis of external jugular vein and common carotid artery after anesthesia, and the right jugular vein was exposed without suture as a control, so as to establish an animal model of internal arteriovenous fistula. Doppler ultrasound, HE and Masson staining and immunohistochemical staining were used to observe the hemodynamics, intimal hyperplasia and protein expression of smooth muscle cell proliferation in the outflow vein of the internal arteriovenous fistula and the contralateral control vein, and to evaluate the effect of model construction. Results A total of 10 mice were selected for this study, and 9 mice were successfully modeled, with a success rate of 90%. Ultrasound examinations were performed on the day of surgery, 7 and 14 days after surgery, respectively. The results showed that the flow velocity near the anastomosis was linearly correlated with the diameter of the tube. The higher the flow velocity, the larger the diameter of the tube. There was a positive correlation between peak velocity and lumen diameter (P=0.000 6, R²=0.831 7). After surgery 14 days, HE staining results showed that after autologous arteriovenous fistula molding, the average lumen area of outflow segment vein was significantly decreased (P < 0.000 1), the intima area was significantly increased (P < 0.000 1), the intimal area was significantly increased (P < 0.000 1). On the surgical side of arteriovenous fistula, collagen deposition was significantly increased, and the proportion of Masson-positive regions was significantly increased (P < 0.000 1). Immunohistochemical staining showed that the proportion of collagen 1 positive areas on the surgical side of arteriovenous fistula was significantly upregulated (P < 0.000 1), and α-smooth muscle actin (α-SMA) , proliferating cell nuclear antigen (PCNA) positive cells increased significantly (P < 0.000 1), indicating an increase in local cell proliferation level. Conclusion The established mouse autologous arteriovenous fistula model has the advantages of high success rate, good stability and low cost. The model provides a good carrier for exploring the biological mechanism of intimal hyperplasia in arteriovenous fistulas.

Key words: Arteriovenous fistula, Animal model, Uremia, Hemodialysis, Intimal hyperplasia, Mice

CLC Number:

Q95-33

Xin LIU, Shaobo SHI, Cui ZHANG, Bo YANG, Chuan QU. Construction and Evaluation of End-to-side Anastomosis Model of Autologous Arteriovenous Fistula in Mice[J]. Laboratory Animal and Comparative Medicine, 2023, 43(6): 595-603.

Figures/Tables 6

Figure 1 Operation diagram of autologous arteriovenous fistula in miceNote：The red blood vessel represents the common carotid artery and the blue blood vessel represents the external jugular vein. A， The external jugular vein and common carotid artery were separated， and incisions were made； B， Arteriovenous end-to-side anastomosis.

Figure 2 Procedures of end-to-side anastomosis of external jugular vein and common carotid artery in miceNote：A， The external jugular vein and the common carotid artery were separated， the distal end of the external jugular vein was ligated， and the proximal end of the external jugular vein as well as the proximal and distal end of the common carotid artery were closed with a microvascular clamp； B， Sutured the anterior wall of the anastomosis continuously at 0 and 12 points of the anastomosis； C， Lifted the inverted vein and sutured the posterior wall of the anastomosis continuously； D， After opening the vascular clamp， it could be observed that the end of the vein was full. Check for any blood seepage.

Figure 3 Noninvasive Doppler ultrasound assessment in mice autologous arteriovenous fistula （AVF） modelNote：A, Anastomotic ultrasound flow rate after surgery 14 days; B, The anastomotic flow rate was monitored on the day after surgery and 7 days and 14 days after surgery (n=9); C, The relationship between anastomotic flow velocity and outflow diameter of arteriovenous fistula after surgery 14 days (n=9).

Figure 4 Analysis of vascular intima in mice autologous arteriovenous fistula （AVF） modelNote：A， the representative pictures of HE sections of the outflow veins on the control side and the AVF side at 14 days after surgery， the scale = 250 μm； B， the lumen area on the AVF side was significantly smaller than that on the control side at 14 days after surgery （n=5）； C， the intimal area on the AVF side was significantly larger than that on the control side at 14 days after surgery （n=5）； D， the intima-media ratio on the AVF side was significantly higher than that on the control side at 14 days after surgery （n=5）； E， the intimal cell density on the AVF side was significantly higher than that on the control side at 14 days after surgery （n=5）.

Figure 5 Analysis of collagen deposition in mice autologous arteriovenous fistula （AVF） modelNote： A. Masson staining and representative images of Collagen 1 immunohistochemical detection of outflow veins on the control side and AVF side at 14 days after surgery （scale = 250 μm）； B， the proportion of MASSON-positive areas on AVF side at 14 days after surgery was significantly higher than that on the control side （n=5）； C， the proportion of Collagen 1 positive areas on the AVF side was significantly higher than that on the control side at 14 days after surgery （n=5）.

Figure 6 α-SMA and PCNA analysis in mice autologous arteriovenous fistula （AVF） modelNote： The expression of α-smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) in the outflow vein on the control side and AVF side at 14 days after surgery was detected by immunohistochemistry, scale =250 μm; B, the proportion of α-SMA positive cells on AVF side was significantly up-regulated at 14 days after surgery (n=5); C, the proportion of PCNA positive cells on AVF side increased significantly at 14 days after surgery (n=5).

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