Evaluation of synthetic vascular grafts in a mouse carotid grafting model

<div><p>Current animal models for the evaluation of synthetic grafts are lacking many of the molecular tools and transgenic studies available to other branches of biology. A mouse model of vascular grafting would allow for the study of molecular mechanisms of graft failure, including in the context of clinically relevant disease states. In this study, we comprehensively characterise a sutureless grafting model which facilitates the evaluation of synthetic grafts in the mouse carotid artery. Using conduits electrospun from polycaprolactone (PCL) we show the gradual development of a significant neointima within 28 days, found to be greatest at the anastomoses. Histological analysis showed temporal increases in smooth muscle cell and collagen content within the neointima, demonstrating its maturation. Endothelialisation of the PCL grafts, assessed by scanning electron microscopy (SEM) analysis and CD31 staining, was near complete within 28 days, together replicating two critical aspects of graft performance. To further demonstrate the potential of this mouse model, we used longitudinal non-invasive tracking of bone-marrow mononuclear cells from a transgenic mouse strain with a dual reporter construct encoding both luciferase and green fluorescent protein (GFP). This enabled characterisation of mononuclear cell homing and engraftment to PCL using bioluminescence imaging and histological staining over time (7, 14 and 28 days). We observed peak luminescence at 7 days post-graft implantation that persisted until sacrifice at 28 days. Collectively, we have established and characterised a high-throughput model of grafting that allows for the evaluation of key clinical drivers of graft performance.</p></div

Neointimal area.

<p>A: Distribution of NH throughout the graft. Neointimal area represented as a percentage of total luminal area as defined by the inner graft wall. Data expressed as mean ± SEM and analysed using two-way ANOVA, n = 7 animals/timepoint. B: Representative images of H&E staining of cross sections. Black dotted lines indicate the graft wall. Scale bar = 100 μm.</p

Cell tracking.

<p>A: Schematic of experimental design. Bone marrow mono-nuclear cells are isolated from FVB-L2G mice and injected into FVB-N mice that received that carotid grafting. B: Representative IVIS images of carotid grafted mice with BM-MNC injection. Warm colours denote higher signal and cold colours denote lower signal. C: Representative images of cross sections with eGFP staining in green and nucleus in blue. Scale bar = 100 μm. D: Quantification of eGFP positive cells cells. Data expressed as mean ± SEM and analysed using one-way ANOVA, n = 3 animals/timepoint.</p

Endothelialisation.

<p>A: Scanning electron microscopy images of the luminal side of the graft. Scale bar = 500 μm. Inset scale bar = 50 μm. B: Quantification of CD31 coverage represented as a percentage of total luminal circumference. Data expressed as mean ± SEM and the mid section of the grafts were analysed using one-way ANOVA, n = 7 animals/timepoint. C: Representative images of cross sections with CD31 staining in red, nucleus in blue. White dotted lines indicate the graft wall. Scale bar = 100 μm.</p

Characterisation of electrospun PCL graft.

<p>A: Macroscopic image of PCL graft. B: Scanning electron microscopy images of a transverse cross section. Scale bar = 200 μm C: Scanning electron microscopy images of electrospun fibres on the luminal side. Scale bar = 10 μm. D: Histogram of fibre diameter distribution.</p

Mouse carotid grafting procedure with corresponding schematic diagram (adapted from [7]).

<p>A: Carotid artery was isolated. B: Double ligation at the mid-point. C: Cuffs were placed on each end of the ligation. D, E: Clamps were applied and artery segment was everted over the cuff and fixed in place with suture. F: Graft was secured to each ends of the cuffs. G: Clamps were removed and blood flow confirmed.</p