Development of neointimal hyperplasia and smooth muscle cell phenotype switching at 2 weeks post-injury.

A) Representative photos of H&E staining depicting neointimal area and SMC-α, PCNA, and DAPI triple immunostaining showing switch of SMC phenotype in control vessels versus injured vessels; solid line indicates the original lumen and dashed line shows the lining of the neointima; n = 3 control, n = 11 injured B) Quantification of H&E staining showing vessel occlusion percentage and C) immunostaining showing total SMC-α+ neointimal area and D) PCNA/SMC-α ratio indicating proportions of contractile and synthetic smooth muscle cell phenotypes in the neointima; n = 3 control, n = 11 injured. Data presented as average ± SEM; scale bar = 300μm, inset = 50μm; *p<0.05, **p<0.01.</p

Surgical equipment and setup for performing microforcep and rat carotid injury.

1) Surgical microscope 2) vessel clamps and 22G needle catheter 3) No.22 scalpel blade 4) sterilised gauze 5) surgical forceps and 6) microscissors. 7) Microforceps for performing vessel distention 8) sterilised gauze tips 9) 9–0 silk suture for arteriotomy closure and 10) 3–0 silk suture to close up neck incision. (TIF)</p

Neointimal remodelling at 2 weeks post-injury.

A) Representative photos of collagen deposition (red) using picrosirius red (PSR) and quantification represented as a percentage of the neointimal area stained red (arrows); n = 3 control, n = 10 injured B) Representative photos and quantification of proteoglycans (blue) using Alcian Blue and quantification represented as a percentage of the neointimal area stained blue (arrows); n = 3 control, n = 13 injured. Data presented as average ± SEM; scale bar = 300μm, inset = 50μm; *p<0.05.</p

Schematic diagram of histopathology and immunohistochemistry staining analysis.

Explanted common carotid arterial segments were sectioned along their length in 5μm sections. Three sections were placed on each slide, and for each stain, three equidistant slides were selected at the proximal, middle, and distal ends. These slides were averaged to create a single data point for the sample. The total samples were then averaged and presented with ± as the standard error of the mean (SEM). (TIF)</p

Neointimal hyperplasia immunostaining of M1 macrophages.

A) Representative co-staining using iNOS (M1) and CD68 (macrophages), scalebar = 300μm; inset: individual channels of iNOS and CD68, scalebar = 100μm. B-C) Secondary antibody (no primary) control images of AlexaFluor488 (AF488—green) and AlexaFluor594 (AF594) secondary antibodies showing negative staining in the neointima but auto-fluorescence of elastic laminae, scalebar = 100μm. (TIF)</p

Activation of vascular inflammation at 2 weeks post-injury.

A) Representative photos of MHC Class II (MHCII), CD68, and DAPI triple immunostains labelling M1 macrophages in control versus injured vessels. Co-localisation of MHCII and CD68 appear yellow in the merged images and indicate M1 macrophages. Insets show individual channels of MHCII (red) and CD68 (green). Quantification of total macrophages represented as total CD68 staining area as percentage of the vessel cross section; n = 3 control, n = 9 injured. Similar representative imaging and quantification of inflammatory cytokine expression in the neointima for B) IL-1β and C) TNF-α; n = 3 control, n = 9 injured. Data presented as average ± SEM; scale bar = 300μm, inset 50μm; *p<0.05.</p

Surgery records across 48 rat procedures performed in 4 separate batches.

Surgery records across 48 rat procedures performed in 4 separate batches.</p

Microforcep and catheter injury causes near complete and sustained denudation of the vessel endothelium.

A) En-face imaging of CD31+ stained carotid arteries and quantification of CD31+ coverage of DAPI counterstained vessels immediately following injury. Denuded endothelium folded into the distal side of the artery (*); n = 3 control, n = 3 injured B) Cross-sectional CD31+staining of injured vessels 2 weeks post-injury and quantification of CD31+ lumen coverage. Inset showing magnified endothelium with concentric staining in controls and incomplete staining in injured vessels (arrows); n = 4 control, n = 16 injured. Data presented as average ± SEM; scale bar = 0.5mm, inset = 100μm; ***p<0.01.</p

Surgical procedure of the combined microforcep and catheter rat injury model.

Beginning with 1) arteriotomy of the common carotid artery 2) insertion and expansion of microforceps to mechanically distend the vessel wall, followed by 3) insertion of needle catheter to denuded the vessel endothelium after which 4) the arteriotomy is sutured closed and blood flow re-established. Rat schematic created with BioRender.com.</p

Raw Data.

PDF file containing all raw data presented throughout the manuscript. (PDF)</p