A mouse model of stenosis distal to an arteriovenous fistula recapitulates human central venous stenosis

Abstract

Objective: Central venous stenosis (CVS) is a major cause of arteriovenous fistula (AVF) failure. However, central veins are relatively inaccessible to study with conventional Doppler ultrasound methods. To understand mechanisms underlying AVF failure owing to CVS, an animal model was established that creates a stenosis distal to an AVF. We hypothesized that this mouse model will show comparable morphology and physiology to human CVS.

Methods: An aortocaval fistula was created between the distal aorta and inferior vena cava (IVC); a stenosis was then created distal to the fistula by partial IVC ligation. Sham-operated animals, AVF without venous stenosis, and venous stenosis without AVF were used as controls. Physiologic properties of the IVC, both upstream and downstream of the stenosis, or the corresponding sites in models without stenosis, were assessed with ultrasound examination on days 0 to 21. The spectral broadening index was measured to assess the degree of disturbed shear stress. The IVC was harvested at day 21 and specimens were analyzed with immunofluorescence.

Results: The IVC diameter of mice with an AVF and stenosis showed increased upstream (P = .013), but decreased downstream diameter (P = .001) compared with mice with an AVF but without a stenosis, at all postoperative times (days 3-21). IVC wall thickness increased in mice with an AVF, compared with IVC without an AVF (upstream of stenosis: 13.9 μm vs 11.0 μm vs 4.5 μm vs 3.9 μm; P = .020; downstream of stenosis: 6.0 μm vs 6.6 μm vs μm 4.5 μm vs 3.8 μm; P = .002; AVF with stenosis, AVF, stenosis, sham, respectively). AVF patency significantly decreased in mice with an AVF and stenosis by day 21 (50% vs 90%; P = .048). The IVC of mice with AVF and stenosis showed a venous waveform with pulsatility as well as enhanced velocity at and downstream of the stenosis; similar waveforms were observed in a human case of CVS. Downstream to the stenosis, the spectral broadening index was significantly higher compared with mice with AVF alone (1.06 vs 0.78; P = .011; day 21), and there was a trend towards less immunoreactivity of both Krüppel-like factor 2 and phosphorylated-endothelial nitric oxide synthase compared with mice with an AVF alone.

Conclusions: Partial IVC ligation distal to a mouse aortocaval fistula alters the fistula diameter and wall thickness, decreases patency, and increases distal disturbed flow compared with fistulae without a distal stenosis. Our mouse model of stenosis distal to an AVF may be a faithful representation of human CVS that shows similar morphology and physiology, including disturbed shear stress.

Keywords: Arteriovenous fistula; Central venous stenosis; Disturbed flow; Shear stress; Spectral broadening index.

Graphical abstract

Fig 1

The stenosis model with an arteriovenous fistula (AVF). A, Representative postprocedural photograph of the mouse stenosis model with an AVF. The aorta and IVC are outlined with interrupted white lines. Numbered circles show the location of where ultrasound measurements were obtained: ①, infrarenal aorta; ②, upstream (2 mm) of the stenosis; ③, downstream (2 mm) of the stenosis. B, Representative Doppler waveforms of the mouse infrarenal aorta with a patent AVF (top) and an occluded AVF (bottom). The end-diastolic velocity (EDV) is increased in the presence of a patent fistula, whereas the EDV is diminished in the presence of an occluded fistula. C, Representative Doppler waveform of the IVC distal to an AVF. The spectral window is the clear black zone between the spectral line and the baseline. Spectral broadening is the widening of the spectral line. Maximum velocity = Spectral window + Spectral broadening. D, Calculation formulae for data acquired from ultrasound measurements. Blood viscosity, η, was assumed to be constant at 0.035 poise. E, Flow chart showing the experimental design of this study and the number of animals used in each analysis. Ao, Aorta; IF, immunofluorescence; IHC, immunohistochemistry; IVC, inferior vena cava; LIV, left iliolumbar vein; LRV, left renal vein; PSV, peak systolic velocity; r, radius (in centimeters); RIV, right iliolumbar vein; TAMV, time-averaged maximum velocity; U/S, ultrasound.

Fig 2

Morphology of the mouse aorta and inferior vena cava (IVC). A-D, Line graphs showing diameter of the (A) infrarenal aorta (P < .001; analysis of variance [ANOVA]). ∗P < .0001 (post hoc, AVF with stenosis vs sham, day 21). B, Inferior vena cava (IVC), upstream, P < .0001 (ANOVA). ∗P = .0130 (post hoc, arteriovenous fistula [AVF] with stenosis vs AVF, day 21). C, IVC, stenosis (P < .0001, ANOVA). D, IVC, downstream (P < .0001; ANOVA). ∗P = .0010 (post hoc, AVF with stenosis vs AVF, day 21; n = 5-8). E, Representative photomicrographs of Van Gieson staining showing wall thickness of the infrarenal aorta (top row) and IVC, upstream (middle row) and downstream (bottom row) of stenosis, at day 21. Scale bar = 20 μm. F-H, Bar graphs showing (F) aortic medial thickness (P = .9006, ANOVA). G, IVC upstream wall thickness (P = .0201, ANOVA). ∗P = .0442 (post hoc, AVF with stenosis vs sham). H, IVC downstream wall thickness (P = .0024, ANOVA). ∗P = .0160 (post hoc, AVF with stenosis vs sham). ∗∗P = .0038 (post hoc, AVF vs sham; n = 4-5). I, Line graph showing cumulative patency rate up to day 21 (P = .0481, log-rank). AVF with stenosis vs AVF (n = 10-20). J, Line graph showing overall survival up to day 21 (P = .3940, log-rank). AVF with stenosis vs AVF (n = 10-20).

Fig 3

Physiologic properties of the mouse aorta. A, Representative Doppler waveforms of the infrarenal aorta at postoperative day 3. B, Line graph showing peak systolic velocity (PSV) of the aorta, P < .0001 (analysis of variance [ANOVA]). ∗P = .0008 (post hoc, arteriovenous fistula [AVF] with stenosis vs sham, day 21; n = 5-8). C, Line graph showing end-diastolic velocity (EDV) of the aorta (P < .0001, ANOVA). ∗P = .0048 (post hoc, AVF with stenosis vs sham, day 21; n = 5-8). D, Line graph showing blood flow of the aorta (P < .0001, ANOVA). ∗P = .0021 (post hoc, AVF with stenosis vs sham, day 21; n = 5-8). E, Line graph showing resistance index of the aorta (P < .0001, ANOVA). ∗P = .0010 (post hoc, AVF with stenosis vs sham, day 21; n = 5-8). F, Line graph showing shear stress of the aorta (P < .0001, ANOVA). ∗P = .0167 (post hoc, AVF with stenosis vs sham, day 21; n = 5-8).

Fig 4

Physiologic properties of the mouse inferior vena cava (IVC). A, Representative Doppler waveforms of the IVC at postoperative day 3. In the longitudinal view (left most column), red arrowheads indicate the stenosis; yellow arrowheads indicate site of insonation 2 mm upstream; green arrowhead indicates site of insonation 2 mm downstream of the stenosis. Scale bar = 1 mm. B-D, Line graph showing time-averaged maximum velocity (TAMV) of the IVC (B) downstream (P < .0001; analysis of variance [ANOVA]). ∗P < .0001 (post hoc, arteriovenous fistula [AVF] with stenosis vs AVF at day 21). C, Stenosis (P < .0001, ANOVA). ∗P < .0001 (post hoc, AVF with stenosis vs AVF at day 21). D, Upstream (P < .0001, ANOVA; P = .9887, post hoc; AVF with stenosis vs AVF at day 21; n = 4-8). E-G, Line graph showing spectral broadening of the IVC (E) downstream (P < .0001, ANOVA). ∗P < .0001 (post hoc, AVF with stenosis vs AVF at day 21). F, Stenosis (P < .0001, ANOVA). ∗P < .0001 (post hoc, AVF with stenosis vs AVF at day 21). G, Upstream (P < .0001, ANOVA; P = .9412, post hoc; AVF with stenosis vs AVF at day 21; n = 4-8). H-J, Line graph showing spectral broadening index of the IVC in mice with AVF (H) downstream (P < .0052, ANOVA). ∗P = .0108 (post hoc, AVF with stenosis vs AVF at day 21). I, Stenosis (P = .0224, ANOVA). ∗P = .0588 (post hoc, AVF with stenosis vs AVF at day 21). J, Upstream (P = .3219, ANOVA; n = 4-8).

Fig 5

Expression of mechanosensitive proteins in the mouse inferior vena cava (IVC). A, Representative immunofluorescence photomicrographs of IVC sections at day 21 with anti-von Willebrand factor (vWF), anti-Krüppel-like factor 2 (Klf2) (upper panels), and anti-phosphorylated endothelial nitric oxide synthase (p-eNOS) (lower panels) antibodies in arteriovenous fistula (AVF) vs AVF with stenosis, upstream of stenosis. Scale bar = 20 μm. B, Bar graphs showing quantification of Klf2 (top) and p-eNOS (bottom) positive signal in the endothelium, upstream; Klf2 vWF: P = .6102 (t-test), p-eNOS vWF: P = .3843 (t-test); n = 4. C, Representative immunofluorescence photomicrographs of IVC sections at day 21 with anti-vWF, anti-Klf2 (upper panels) and anti-p-eNOS (lower panels) antibodies in AVF vs AVF with stenosis, downstream of stenosis. Scale bar = 20 μm. D, Bar graphs showing quantification of Klf2 (top) and p-eNOS (bottom) positive signal in the endothelium, downstream; vWF Klf2: P = .2243 (t-test), vWF p-eNOS: P = .0905 (t-test); n = 4. L, Lumen.

Fig 6

Central stenosis in the outflow of an arteriovenous fistula (AVF) in a human case. A, Venogram showing central venous stenosis (CVS) in a human patient. A severe stenosis is visible in the proximal right subclavian vein (red arrowhead). B, Venogram (left column) and Doppler waveforms (right columns) before (top row) and after (bottom row) percutaneous angioplasty of the stenosis in the same case. Doppler waveforms were obtained at the stenosis (red arrowhead) as well as upstream (yellow arrowhead) and downstream (green arrowhead) to the stenosis. BrV, Brachial vein; CV, cephalic vein; SCV, subclavian vein.

Supplementary Fig

Fistula wall composition at postoperative day 21. A, Representative immunofluorescence (IF) photomicrographs of inferior vena cava (IVC) at day 21 with anti-von Willebrand factor (vWF) and anti-α-smooth muscle actin (α-SMA) antibodies, upstream of the stenosis. Scale bar = 20 μm. B, Bar graphs showing number of cells in the intima and media per area; P = .3951 (t-test); n = 3. C, Representative IF photomicrographs of IVC at day 21 with anti-CD68 (top row) or anti-CD3 (bottom row) and anti-α-SMA antibodies, upstream of the stenosis. Scale bar = 20 μm. D, Bar graphs showing number of CD68⁺ or CD3⁺ cells in the IVC wall; CD68; P = .9725; CD3; P = .5942 (t-test); n = 3. (E) Representative photomicrographs of Masson’s trichrome staining of the IVC, upstream of the stenosis, at day 21. Scale bar = 20 μm. F, Bar graphs showing % collagen area in the IVC wall; P = .4986; n = 4. AVF, Arteriovenous fistula; HPF, high-power field; L, lumen.