Figure 5
Abdominal FLASH irradiation produces less apoptosis and DNA damage in crypt base columnar cells (CBCs) than CONV irradiation in non-tumor bearing and tumor bearing mice. (a) Representative TUNEL stained images of crypts from non-tumor bearing mice at the indicated time points after sham or sublethal 14 Gy abdominal irradiation. (b) Quantification of the average number of TUNEL + CBCs per crypt in the jejunum at 4 and 24 h after 14 Gy abdominal irradiation. (c) Representative cleaved caspase-3 IHC images of crypts from non-tumor bearing mice at the indicated time points after sham or sublethal 14 Gy abdominal irradiation. (d) Quantification of the average number of cleaved caspase-3 + CBCs per crypt in the jejunum at 4 and 24 h after 14 Gy abdominal irradiation. CBC cells were identified based on location at the + 1 to + 3 position wedged between Paneth cells. Apoptosis in CBCs by TUNEL and cleaved caspase-3 was less at 24 h after FLASH vs. CONV irradiation. Scale bar shows 25 μm. Crypts in 3 circumferences per mouse were analyzed, n = 4 mice per group. (e) Representative γ-H2AX (red), lysozyme (green, a marker of Paneth cells) and DAPI (blue) stained images of crypts from ID8 ovarian tumor bearing mice at the indicated time points after sham or sublethal 14 Gy abdominal irradiation. (f) Quantification of the average number of γ-H2AX + foci per CBC cell located in between lysozyme + Paneth cells in the jejunum at 4 h, 12 h and 24 h after 14 Gy abdominal irradiation. n = 4 mice per group. An average of 10 CBC cells per mouse were quantified for γ-H2AX foci. At 4 and 12 h after irradiation, γ-H2AX foci in CBCs were fewer after FLASH than CONV (p < 0.05 at 12 h), and resolved in both groups by 24 h. ns no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001; Unirradiated control versus CONV or FLASH compared by one-way ANOVA followed by Tukey’s multiple comparisons test. Error bars represent standard deviation of the mean.
