A new arteriovenous fistula model to study the development of neointimal hyperplasia

Abstract

This study describes an alternative arteriovenous fistula (AVF) model in the rat in which the animals develop significant neointimal hyperplasia (NIH) not only at the distal anastomotic site, but also throughout the fistula body. This aortocaval fistula was established by anastomosing the distal end of the renal vein to the abdominal aorta after unilateral nephrectomy. The increased hemodynamic stress resulting from exposing the renal vein to the arterial circulation induced venous NIH as early as 7 days after surgery. This experimental AVF was characterized by the early lack of endothelium, the accumulation of proliferating vascular smooth muscle cells and the neovascularization of the fistula adventitia. In summary, we have described an informative animal model to study the pathobiology of NIH in native AVF.

Fig. 1

a Diagrammatic representation of the AVF. Fistulae were created in rats by anastomosing the left renal vein to the abdominal aorta after unilateral nephrectomy. In this model, blood circulates from the aorta to the renal vein and then to the inferior vena cava. b Photograph of the aortocaval fistula after anastomosis. The arrow identifies the ligature at the distal renal vein to restrict blood flow. c Imaging of the AVF using ultrasound biomicroscopy. The AVF appears inside the box.

Fig. 2

Histological characterization of the rat AVF. a–l Histological changes in sham-operated renal veins and AVFs. a, d The typical histology of the ligated renal vein (sham-operated rats). The successive microphotographs show the temporal histological changes in the fistula wall at 6 h (b, e), 3 days (c, f), 7 days (g, j), 14 days (h, k) and 30 days (i, l) following surgery. All specimens were formalin-fixed and paraffin-embedded. Cross-sections were stained with hematoxylin and eosin. The boxes in a–c and g–i indicate the highly magnified area in the corresponding picture below. Neointimal lesions appear between arrowheads. T = Thrombus associated with a necrotic neointima. m The fistula was divided into three sections for histomorphometric analysis (A, B and C). Arrows represent the direction of the blood flow. n, o AVF neointimal area (n) and vascular wall thickness (o) at different postsurgical times in the three sections of the AVF. Neointimal development was similar throughout the AVF venous limb. Each point represents the mean ± SEM. The number of animals per group appears at the top of each bar in n.

Fig. 3

Immunohistochemical analyses of the rat AVF using antibodies specific for endothelial cells and SMCs. a–h Serial sections from sham-operated veins (a, e) and AVFs harvested at day 1 (b, f), 7 (c, g) and 14 (d, h) were stained for endothelial cells with an anti-vWF antibody (a–d) and for SMCs with an anti-SMA antibody (e–h). Note the absence of endothelium early after fistula creation. i Quantification of the reendothelialized luminal area at different time points after surgery. The reendothelialized area was determined by measuring the luminal area positive for vWF. j Accumulation of neointimal cells in the fistula wall over time. Each bar represents the mean ± SEM of values from 4–8 animals. The number of animals in each group is the same as shown in figure 2p. p values were calculated using one-way ANOVA and the Tukey-Kramer post hoc test.

Fig. 4

Proliferating neointimal cells in the venous limb of the fistula. a AVF neointima stained for BrdU, a marker for proliferating cells. BrdU, which is a thymidine analog that is selectively incorporated into the DNA of proliferating cells, was administered to the rats between day 14 and 21 after fistula creation. BrdU-positive nuclei appear as brown spots. The neointima (N) is marked between arrowheads. b High-power view of the neointimal area within the rectangle in a. c An AVF feeding artery stained for BrdU. No BrdU-positive cells were seen in the feeding artery.

Fig. 5

Neoangiogenesis in the AVF vascular wall. a, b Low- (a) and high-power (b) views of a representative AVF at day 14. The section was stained with hematoxylin and eosin and shows marked neovascularization around the fistula wall. The rectangle in a indicates the magnified area in b. c Quantification of adventitial blood vessels in the venous limb of the fistula at different times following surgery. Each bar represents the mean ± SEM of values from 4–8 animals. The number of animals in each group is the same as shown in figure 2p. p values were calculated using one-way ANOVA and the Tukey-Kramer post hoc test. d, e Immunohistochemistry to demonstrate the presence of endothelial cells (e) and SMCs (d) in the neovasculature around the fistula wall. A = Arteriole; V = small vein; C = capillary. f Linear regression analysis comparing neointimal area versus the number of microvessels per section. A strong relationship was observed between adventitial neovascularization and the development of NIH.