Temporal Changes in Sparing and Enhancing Dose Protraction Effects of Ionizing Irradiation for Aortic Damage in Wild-Type Mice

Abstract

In medical and occupational settings, ionizing irradiation of the circulatory system occurs at various dose rates. We previously found sparing and enhancing dose protraction effects for aortic changes in wild-type mice at 6 months after starting irradiation with 5 Gy of photons. Here, we further analyzed changes at 12 months after stating irradiation. Irrespective of irradiation regimens, irradiation little affected left ventricular function, heart weight, and kidney weight. Irradiation caused structural disorganizations and intima-media thickening in the aorta, along with concurrent elevations of markers for proinflammation, macrophage, profibrosis, and fibrosis, and reductions in markers for vascular functionality and cell adhesion in the aortic endothelium. These changes were qualitatively similar but quantitatively less at 12 months than at 6 months. The magnitude of such changes at 12 months was not smaller in 25 fractions (Frs) but was smaller in 100 Frs and chronic exposure than acute exposure. The magnitude at 6 and 12 months was greater in 25 Frs, smaller in 100 Frs, and much smaller in chronic exposure than acute exposure. These findings suggest that dose protraction changes aortic damage, in a fashion that depends on post-irradiation time and is not a simple function of dose rate.

Keywords: C57BL6/J; aortic damage; enhancing dose protraction effect; fibrosis; inflammation; intima-media thickening; ionizing irradiation; sparing dose protraction effect; temporal change.

Figure 1

Morphological alterations in the aortic endothelium. Quantitative analysis for (A) the number of crests/field, (B) percentage of mice with detachment, and (C) percentage of mice with large detachment (8–10 mice/group analyzed, Welch’s t-test for A, Fisher’s exact test for B,C). Frs, fractions. **, p < 0.001. *, 0.001 ≤ p < 0.05. #, 0.05 ≤ p < 0.1 (marginally significant). ns, p ≥ 0.1 (nonsignificant). See footnote in Supplementary Materials for an outline of statistical comparisons. The data in Figure 1A–C for 8 groups at 6 mos ASI were taken from the 2021 Cancers paper [8].

Figure 2

Molecular alterations in the aorta. Representative merged images (all at 12 mos ASI) for double immunofluorescence of CD31 with (A) eNOS, (B) VE-cadherin, (C) TNF-α, (D) CD68, (E) F4/80, (F) CD3 or (G) TGF-β1, with cell nuclei counterstained with DAPI. (A,C–G) X-rays in 25 Frs. (B) Acute X-rays. Upper panels, 0 Gy. Lower panels, 5 Gy. Boxed areas in the left panels (tiled images) are presented at higher magnification in the right panels. Scale bars are as indicated.

Figure 3

Molecular alterations in the aorta. Quantitative analysis of immunofluorescence for (A) CD31 negativity, (B) DAPI negativity, (C) eNOS, (D) VE-cadherin, (E) TNF-α, (F) CD68, (G) F4/80, (H) CD3, (I) TGF-β1, and (J) IMT (8–10 mice/group analyzed, Welch’s t-test or Wald test). **, p < 0.001. *, 0.001 ≤ p < 0.05. #, 0.05 ≤ p < 0.1 (marginally significant). ns, p ≥ 0.1 (nonsignificant). See footnote in Supplementary Materials for an outline of statistical comparisons. AU, arbitrary unit. Frs, fractions. The data in Figure 3A–J for 8 groups at 6 mos ASI were taken from the 2021 Cancers paper [8]. Representative images are presented in Figure 2.

Figure 4

Fibrotic alterations in the aorta. (A). Representative images for Masson’s trichrome staining (at 12 mos ASI with 0 Gy or 5 Gy of X-rays in 25 Frs). Scale bars are as indicated. Quantitative analysis for (B) intensity of aniline blue (9–10 mice/group analyzed, Welch’s t-test). AU, arbitrary unit. Frs, fractions. **, p < 0.001. *, 0.001 ≤ p < 0.05. #, 0.05 ≤ p < 0.1 (marginally significant). ns, p ≥ 0.1 (nonsignificant). See footnote in Supplementary Materials for an outline of statistical comparisons. The data in Figure 4 (B,C) for 8 groups at 6 mos ASI were taken from the 2021 Cancers paper [8].