Author: Najah Curtis, Michele M. Kim, Cameron Koch, Ioannis I Verginadis, Rodney D. Wiersma, Jennifer Wei Zou 👨🔬
Affiliation: Department of Radiation Oncology, University of Pennsylvania, University of Pennsylvania, UCLA 🌍
Purpose: FLASH has been shown to have increased normal tissue sparing compared to standard dose rate radiation and, separately, has been shown to rapidly consume oxygen intercellularly in vivo through radiochemical oxygen depletion (ROD). To address these findings, we have developed an algorithm to measure immunohistochemical stained tissues for the purpose of investigating ROD as a potential mechanism behind the FLASH effect.
Methods: Mice were pre-injected intravenously with EF5, irradiated with FLASH or standard radiation, then euthanized for tissue collection of their jejunum. The tissue was frozen then sectioned, stained, and imaged for use in the program. These tissue sections were stained for gamma-H2AX (identifying double strand DNA breaks), total DNA content, and hypoxia content (EF5 binding). The in-house developed MATLAB program creates a mask of the input images that were used to find the central point of each nucleus (centroid). The centroids were then dilated to 11x11 pixel squares that encompassed the entire nucleus. These squares were correlated back to the original images and allowed the program to calculate the average signal intensity of each nucleus for each stain.
Results: Using this program, investigation of the tissue sections’ preliminary images revealed reduced DNA damage in the FLASH compared to the standard dose rate irradiated tissue. Furthermore, there was an inverse correlation between hypoxia and DNA damage that was observed for FLASH irradiated tissue that was not consistently observed for the standard tissue.
Conclusion: This program can be used to accurately identify all nuclei for their respective stains. Using the output file, the relationship between DNA damage and hypoxia content has been preliminarily identified. Current results suggest FLASH could provide increased DNA protection, especially in normal tissue with increased hypoxia content. Therefore, this supports the hypothesis that the ROD occurring at FLASH dose rates contributes to decreased DNA damage.