3153 - Development of Computational Immunohistochemical Stained Tissue Analysis to Quantitatively Investigate Intracellular Radiochemical Oxygen Depletion in FLASH Irradiated Murine Intestine

05:00pm - 06:00pm PT

Hall F

Screen: 11

POSTER

Presenter(s)

Rodney Wiersma, PhD - University of California Los Angeles, Los Angeles, CA

N. Curtis¹, W. Zou², I. I. Verginadis¹, M. M. Kim³, C. J. Koch¹, and R. D. Wiersma⁴; ¹Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA, ²Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, ³Department of Radiation Oncology, Division of Medical Physics, Hospital of the University of Pennsylvania, Philadelphia, PA, ⁴Department of Radiation Oncology, UCLA, Los Angeles, CA

Purpose/Objective(s):

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.

Materials/Methods:

Murine subjects were administered EF5 intravenously to label hypoxic regions and then irradiated with either FLASH or standard dose-rate radiation. Jejunal tissues were harvested, rapidly frozen, sectioned, and immunohistochemically stained for ?-H2AX (a marker of double-strand DNA breaks), total DNA content, and EF5 binding (indicative of hypoxia). An in-house programming environment algorithm was developed to generate nuclear masks from digitized tissue images by determining the centroids of individual nuclei. 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:

Preliminary analysis indicates that FLASH irradiation is associated with a reduction in ?-H2AX signal compared to standard dose-rate irradiation. Moreover, our computational pipeline shows an inverse correlation between hypoxia (EF5 binding) and DNA damage (?-H2AX expression) in the FLASH group that was not consistently observed in the standard group, suggesting that ROD may mitigate DNA damage in hypoxic regions of tissue.

Conclusion:

The computational immunohistochemical stained tissue analysis method was shown to accurately identify all nuclei for their respective stains and preliminary data identifies a relationship between DNA damage and hypoxia content. 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.