Quantitative assessment of the age of fibrotic lesions using polarized light microscopy and digital image analysis

Reliable histologic methods for gauging the maturity of fibrotic lesions are limited, making interventions in the healing process difficult to assess. As collagen ages there is enhanced birefringence due to increased molecular and fibrillar organization. The purpose of this study was to develop a microscopal technique to quantify this process and to determine its ability to distinguish scars of varying ages. Fibrosis in the rat gracilis muscle was studied 5 to 63 days after superficial injury. Sections were stained with picrosirius red and illuminated with monochromatic, polarized light. The microscope fields were digitized using a computer-video system yielding an image in which noncollagenous material was dark (gray level 0) and collagen was depicted by grey levels 1 to 255. In the fibrosis model used, the collagen area fraction plateaued at 80% by day 21. The median collagen grey level increased progressively as the scar aged. It is concluded that this histologic, nondestructive technique can reliably quantify age-related optical properties of fibrotic collagen and that this could be used to determine the maturity of fibrotic lesions.