Comprehensive analysis of keloid vasculature by tissue clearing and 3D imaging

Keloids are a complex type of scar tissue formed by exaggerated wound healing, characterised by the overgrowth of thick fibrous tissue beyond the original wound boundary. While the crucial relationship between keloid formation and the vascular system has been highlighted, conflicting findings have been reported regarding the characterisation of keloid vasculature. Here, we successfully characterised the detailed three-dimensional (3D) structure of vasculature in keloid tissues using tissue clearing methods combined with 3D imaging. First, we compared two optical tissue clearing methods, the clear, unobstructed brain imaging cocktails and computational analysis and immunolabelling-enabled 3D imaging of solvent-cleared organ protocols, and found the latter to provide greater transparency of keloid scars. We then conducted a detailed 3D vascular analysis using light sheet and confocal fluorescence microscopy. In normal skin, capillary loops and the superficial vascular plexus were located in the papillary layer and at the boundary between the papillary and reticular layers, respectively. However, the density of these vessels was higher in keloid scars than in normal skin. The reticular layer of normal skin exhibited fewer blood vessels. In contrast, keloid scars exhibited significantly thickened dermal reticular layers, with the upper reticular layer showing significantly greater vascularisation. The lower reticular layer of keloid scars also exhibited vertically aligned blood vessels, although their density was lower than in the upper reticular layer. These results indicate that excessive vascularisation is predominantly induced in the papillary and upper reticular layers of keloid scars, which might contribute to keloid pathogenesis. The technique described here has the potential to serve as a crucial template for future pathological analyses of abnormal scars.