Histological characterization and quantification of late damage in mice after proton and photon irradiation

Purpose: Radiotherapy is a widely used cancer treatment, and radiation-induced fibrosis is a frequent late effect that can significantly reduce patients’ quality of life. Many approaches for evaluating and grading radiation late damage, such as fibrosis, are based on semi-quantitative methods. This study aimed to characterize the histopathological changes associated with late radiation damage in mice after exposure to proton and photon irradiation, and to evaluate the applicability of stereological methods for quantitative assessment of these changes. Materials and Methods: A mouse leg model was used to evaluate and compare the potential radiation-induced functionality impairments with histopathological changes. Mice (n = 32) were subjected to a single high dose of photon (n = 18) or proton (n = 14) irradiation on the right foot, while the left, unirradiated leg served as a control. Late damage was assessed using a leg contracture assay, while histopathological changes were quantified using stereological point counting. Results: Proton- and photon-irradiated legs histologically showed a dose-dependent increase in connective tissue and epidermal thickness and reduced adipose tissue. Adipose tissue was replaced with connective tissue, adnexal structures disappeared, and the epidermis was altered. An association was found between leg contracture in the living mice and histopathological connective tissue changes, suggesting that fibrosis contributes to impaired joint mobility. However, discrepancies between histological findings and the leg contracture assay indicate that factors other than connective tissue changes, such as tendon damage and experimental uncertainties, influence joint movement. Conclusions: This study provides a quantitative approach for associating radiation effects in normal tissue with histopathological changes, offering a valuable model for investigating late radiation-induced damage. The study highlights the need for larger studies to fully elucidate the late side effects of proton and photon irradiation.