Anesthetic oxygen use and sex are critical factors in the FLASH sparing effect

Abstract

Introduction: Ultra-high dose-rate (UHDR) radiation has been reported to spare normal tissue compared to conventional dose-rate (CDR) radiation. However, reproducibility of the FLASH effect remains challenging due to varying dose ranges, radiation beam structure, and in-vivo endpoints. A better understanding of these inconsistencies may shed light on the mechanism of FLASH sparing. Here, we evaluate whether sex and/or use of 100% oxygen as carrier gas during irradiation contribute to the variability of the FLASH effect.

Methods: C57BL/6 mice (24 male, 24 female) were anesthetized using isoflurane mixed with either room air or 100% oxygen. Subsequently, the mice received 27 Gy of either 9 MeV electron UHDR or CDR to a 1.6 cm² diameter area of the right leg skin using the Mobetron linear accelerator. The primary post-radiation endpoint was time to full thickness skin ulceration. In a separate cohort of mice (4 male, 4 female) skin oxygenation was measured using PdG4 Oxyphor under identical anesthesia conditions.

Results: In the UHDR group, time to ulceration was significantly shorter in mice that received 100% oxygen compared to room air, and amongst them female mice ulcerated sooner compared to males. However, no significant difference was observed between male and female UHDR mice that received room air. Oxygen measurements showed significantly higher tissue oxygenation using 100% oxygen as the anesthesia carrier gas compared to room air, and female mice showed higher levels of tissue oxygenation compared to males under 100% oxygen.

Conclusion: The FLASH sparing effect is significantly reduced using oxygen during anesthesia compared to room air. The FLASH sparing was significantly lower in female mice compared to males. Both tissue oxygenation and sex are likely sources of variability in UHDR studies. These results suggest an oxygen-based mechanism for FLASH, as well as a key role for sex in the FLASH skin sparing effect.

Figure 1.

A visual demonstration of the study design. In study 1, forty-eight C57BL/6 mice (24 male, 24 female) were anesthetized using isoflurane delivered in 100% oxygen or room air and received 27 Gy of radiation to right leg skin using a Mobetron linear accelerator. Time to skin ulceration was measured as the primary endpoint. In study 2, eight C57BL/6 mice (4 male, 4 male) received subcutaneous injections of PdG4 Oxyphor and were anesthetized similar to study 1. An excitor-detector fiber-optic pair was used to read tissue oxygenation levels at the injection site.

Figure 2.

Kaplan-Meier curves demonstrating time to skin ulceration in leg skin of UHDR and CDR irradiated mice under room air and 100% oxygen conditions. Animals were removed from the study when a full thickness ulcer developed. Mice that did not show lesions after 20 days post-irradiation were censored for survival analysis and monitored for an additional 10 days to ensure no lesions developed. UHDR room air mice (median = >20 days) developed ulcers significantly later than both UHDR 100% oxygen (median = 12 days) and CDR room air mice (median = 15.5 days). UHDR 100% oxygen mice did not differ significantly from CDR 100% oxygen mice (median = 12 days). CDR room air and CDR 100% oxygen mice did not differ from each other in time to ulceration. * = p < 0.05, ns = not statistically significant.

Figure 3.

Kaplan-Meier curves demonstrating sex differences in time to skin ulceration in leg skin of UHDR and CDR irradiated mice under room air and 100% oxygen conditions. (A) Male and female UHDR room air mice did not differ in time to skin ulceration. However, female mice in UHDR 100% oxygen developed ulcers earlier compared to male mice in the same group. (B) No significant sex differences were seen between male and female CDR mice in neither room air nor 100% oxygen conditions. * = p < 0.05, ns = not statistically significant

Figure 4.

Direct tissue pO2 measurements under room air and 100% oxygen conditions using PdG4 Oxyphor. Mice that received 100% oxygen had significantly higher tissue pO2 levels compared to room air mice. The female mice that received 100% oxygen (mean = 56 mmHg, SD = 11) had higher tissue pO2 levels compared to males that received 100% oxygen (mean = 36 mmHg, SD = 7). * = p < 0.05, ns = not statistically significant.