Fixation method influences FLASH skin sparing in an in vivo leg model

Abstract

Background and purpose: The FLASH effect, where ultra-high dose rate elicits a favourable normal tissue-sparing, has been shown in several preclinical studies. Study setup differences, for example fixation methods that affect blood flow, can influence radiation response but are unexplored for FLASH. This study compared FLASH's acute skin-sparing effect with two fixation methods: a glued fixation (no blood flow restriction) and taped fixation (slight blood flow restriction). Patient/material and methods: Female CDF1 mice were irradiated on their hind foot using a glue-fixation or tape-fixation method. Glue-fixated mice were only taped during the glueing procedure and had a 10-min unrestricted period afterwards before irradiation, while tape-fixated mice were taped shortly before and throughout irradiation. Mice received single-dose irradiation (19-58 Gy) with either conventional dose rate (CONV, protons 0.06 Gy/s, electrons 0.16 Gy/s) or FLASH (electrons, 223-233 Gy/s). Differences in skin toxicity were analysed.

Results: CONV-treated tape-fixated mice required a 16-17% higher dose to induce skin toxicity relative to glued mice for both protons and electrons. Meanwhile, the fixation method did not affect FLASH-treated mice. The resulting electron FLASH-sparing effect was reduced by 18% due to the shift in radiosensitivity for CONV-treated mice.

Interpretation: CONV-treated tape-fixated mice were more radioresistant than the glue-fixated mice, consistent with the expected response to mild hypoxia. FLASH-treated mice were unaffected. These findings demonstrate the impact of fixation and, in turn, oxygen level on the differential CONV versus FLASH skin response. The results highlight the importance of minimal systemic influence on animals during FLASH studies.

Figure 1

Target position example for a glued leg. The checkered template and ruler were used for water column depth determination.

Figure 2

Depictions of a tape-fixated mouse. (A) Target position example for a taped leg. The restrained mouse is in a jig with its right foot fixated. The checkered template was used for water column depth determination. (B) Plantar view of the fixated mouse. The loose tape around the foot forms a triangle with air between the tape and the toes.

Figure 3

Dose–response relationship for acute skin toxicity (Grade 3) for mice treated with CONV protons during glued (red) or taped (orange) fixation. Black lines span the 95% confidence interval for 50% toxicity.

Figure 4

Dose–response relationship for mice treated with conventional (CONV, red nuances) and FLASH (blue nuances) electron irradiation. Black lines span the 95% confidence interval for 50% toxicity. (A) Moderate acute skin toxicity equalling grade 2.5, (B) Severe acute skin toxicity equal grade 3.0, (C) Severe acute skin toxicity equal grade 3.5. The separate curves for glued mice can be found in Kristensen et al. [10]

Figure 5

Dose to elicit a response in 50% of mice (TD₅₀) in each acute skin grade for glued- and tape-fixated electron irradiated mice. Each dot represents the mean TD₅₀, with error bars being standard error of the mean (SEM). * highlights a significant difference between means (p < 0.05) and highlights no significant difference. (A) For mice treated with a conventional dose rate. (B) For mice treated with FLASH.