Highly reproducible rat arterial injury model of neointimal hyperplasia

Abstract

Models of arterial injury in rodents have been invaluable to our current understanding of vessel restenosis and play a continuing role in the development of endovascular interventions for cardiovascular disease. Mechanical distention of the vessel wall and denudation of the vessel endothelium are the two major modes of vessel injury observed in most clinical pathologies and are critical to the reproducible modelling of progressive neointimal hyperplasia. The current models which have dominated this research area are the mouse wire carotid or femoral injury and the rat carotid balloon injury. While these elicit simultaneous distension of the vessel wall and denudation of the luminal endothelium, each model carries limitations that need to be addressed using a complementary injury model. Wire injuries in mice are highly technical and procedurally challenging due to small vessel diameters, while rat balloon injuries require permanent blood vessel ligation and disruption of native blood flow. Complementary models of vascular injury with reproducibility, convenience, and increased physiological relevance to the pathophysiology of endovascular injury would allow for improved studies of neointimal hyperplasia in both basic and translational research. In this study, we developed a new surgical model that elicits vessel distention and endothelial denudation injury using sequential steps using microforceps and a standard needle catheter inserted via arteriotomy into a rat common carotid artery, without requiring permanent ligation of branching arteries. After 2 weeks post-injury this model elicits highly reproducible neointimal hyperplasia and rates of re-endothelialisation similar to current wire and balloon injury models. Furthermore, evaluation of the smooth muscle cell phenotype profile, inflammatory response and extracellular matrix within the developing neointima, showed that our model replicated the vessel remodelling outcomes critical to restenosis and those becoming increasingly focused upon in the development of new anti-restenosis therapies.

Fig 1. 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.

Fig 2. 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.

Fig 3. 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.

Fig 4. 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.

Fig 5. 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.