Mouse vein graft hemodynamic manipulations to enhance experimental utility

Abstract

Mouse models serve as a tool to study vein graft failure. However, in wild-type mice, there is limited intimal hyperplasia, hampering efforts to identify anti-intimal hyperplasia therapies. Furthermore, vein graft wall remodeling has not been well quantified in mice. We hypothesized that simple hemodynamic manipulations can reproducibly augment intimal hyperplasia and remodeling end points in mouse vein grafts, thereby enhancing their experimental utility. Mouse inferior vena cava-to-carotid interposition isografts were completed using an anastomotic cuff technique. Three flow restriction manipulations were executed by ligating outflow carotid branches, creating an outflow common carotid stenosis, and constructing a midgraft stenosis. Flowmetry and ultrasonography were used perioperatively and at day 28. All ligation strategies decreased the graft flow rate and wall shear stress. Morphometry showed that intimal thickness increased by 26% via carotid branch ligation and by 80% via common carotid stenosis. Despite similar mean flow rates and shear stresses among the three manipulations, the flow waveform amplitudes were lowest with common carotid stenosis. The disordered flow of the midgraft stenosis yielded poststenotic dilatation. The creation of an outflow common carotid stenosis generates clinically relevant (poor runoff) vein graft low wall shear stress and offers a technically flexible method for enhancing the intimal hyperplasia response. Midgraft stenosis exhibits poststenotic positive wall remodeling. These reproducible approaches offer novel strategies for increasing the utility of mouse vein graft models.

Figure 1

Hemodynamically modified cuff technique mouse vein graft models. A: Normal-flow model. B: Outflow carotid artery (CA) restriction by ligating the internal CA plus the occipital artery. C: Outflow common CA 33-gauge focal stenosis model. D: Midgraft 32-gauge focal stenosis model. The horizontal planes in the schemata show the locations of histologic sections for Masson's trichrome staining and morphologic analysis. Micrography was performed after each model was completed. An electron microscopy mesh was used in micrography for the midgraft stenosis model (D) for accurate vessel scaling. Representative color and pulsed wave Doppler sonograms of mouse vein grafts at day 4 are shown. Pulsed wave Doppler images were captured 900 μm from the distal cuff or midgraft ligature. Red arrows depict blood flow direction; green arrows, ligation sites; white arrows, cuffs; and yellow arrows, blood flow turbulence. The image colors represent the direction and magnitude of the Doppler signal frequency shift. Representative Masson's trichrome staining of the serial sections for that portion of the vein graft are shown. Red staining depicts cytoplasm and muscle fibers; green, collagen; and black, cell nuclei. Three black lines delineate the boundaries of different layers of vein graft wall (from the lumen to the outside): lumen boundary, internal elastic lamina, and outside boundary. Between the lumen boundary and the internal elastic lamina = intima; and between the internal elastic lamina and the outside boundary = M+A. Scale bars = 200 μm.

Figure 2

Vein graft shrinkage factor calculation. A: Schematic drawing of an in vivo/in vitro vein graft labeled with lengths and diameters. B: Vein graft SF_L results calculated by micrography (at days 0 and 28) and by B-mode ultrasonography (at days 4 and 28). Error bars represent SEM.

Figure 3

Flowmetry comparison of all four mouse vein graft models. A: Mean blood flow rate measured before (baseline) and after (day 0 graft) hemodynamic manipulation and at harvest (day 28 graft). B: Mean wall shear stress comparison: acute (day 0) shear stress calculated via micrography of in vivo vein grafts and day 28 shear stress calculated via adjusted in vitro histologic analysis. Values are shown as mean ± SEM (error bars). *P < 0.01 versus baseline of each group; ^†P < 0.05 versus day 0 graft of each group; and ^‡P < 0.01 versus the normal-flow group at the same portion of vein graft.

Figure 4

Vein graft blood flow rate waveforms. A: Representative waveforms of each group: baseline graft waveforms (before flow restriction), acute waveforms (day 0 graft after flow restriction; no restriction for the normal-flow group), and harvest waveforms (day 28). B: Comparisons of mean flow waveform amplitudes. Values are shown as mean ± SEM (error bars). *P < 0.01 versus baseline of each group; ^†P < 0.01 versus day 0 graft of each group; ^‡P < 0.05 versus the normal-flow group at the same time point; and ^§P < 0.05.

Figure 5

Morphologic analysis of mouse vein graft wall adaptation. The mean of any measurement from proximal/distal 400- and 600-μm sections or proximal/distal midgraft 400- and 600-μm sections (to the stenosis site) in the midgraft stenosis model are shown. A: Intimal thickness. B: M+A thickness. C: Intima (I)/(M+A) thickness ratio. D: Concentric lumen area. E: RI. Values are shown as mean ± SEM (error bars). *P < 0.05 versus the normal-flow group at the same portion of vein graft; ^†P < 0.05 versus outflow 33-gauge common carotid stenosis group at the same portion of vein graft; ^‡P < 0.05; and ^§P = 0.09 versus the distal midgraft of the midgraft stenosis model.

Figure 6

Morphologic analysis of the midgraft 32-gauge focal stenosis model. A: Radii of different vein graft layers corresponding to the axial position relative to the focal stenosis site. Values are shown as mean ± SEM (error bars). *P < 0.05 and ^†P = 0.051 versus the counterpart at the proximal vein graft. B: Representative Masson's trichrome staining of the longitudinal serial cross sections. Scale bars = 200 μm.

Figure 7

Intimal hyperplasia and wall remodeling took place in a failed 8-month-old human vein graft (originally from a uniform-sized anterior branch of the saphenous vein). A–V: Masson's trichrome staining of longitudinal serial (5-mm interval) cross sections. Intimal hyperplasia (all A–V images) and wall remodeling [negative remodeling, A–I (RI = 0.63); positive remodeling, J–P (RI = 1.28); and compensatory remodeling, Q–V (RI = 0.86)] can be found. Scale bars = 1 mm. W: Angiogram of the failing graft. Yellow arrows depict two stenosis sites.