Longitudinal in vivo microcomputed tomography of mouse lungs: No evidence for radiotoxicity

Abstract

Before microcomputed tomography (micro-CT) can be exploited to its full potential for longitudinal monitoring of transgenic and experimental mouse models of lung diseases, radiotoxic side effects such as inflammation or fibrosis must be considered. We evaluated dose and potential radiotoxicity to the lungs for long-term respiratory-gated high-resolution micro-CT protocols. Free-breathing C57Bl/6 mice underwent four different retrospectively respiratory gated micro-CT imaging schedules of repeated scans during 5 or 12 wk, followed by ex vivo micro-CT and detailed histological and biochemical assessment of lung damage. Radiation exposure, dose, and absorbed dose were determined by ionization chamber, thermoluminescent dosimeter measurements and Monte Carlo calculations. Despite the relatively large radiation dose delivered per micro-CT acquisition, mice did not show any signs of radiation-induced lung damage or fibrosis when scanned weekly during 5 and up to 12 wk. Doubling the scanning frequency and once tripling the radiation dose as to mimic the instant repetition of a failed scan also stayed without detectable toxicity after 5 wk of scanning. Histological analyses confirmed the absence of radiotoxic damage to the lungs, thereby demonstrating that long-term monitoring of mouse lungs using high-resolution micro-CT is safe. This opens perspectives for longitudinal monitoring of (transgenic) mouse models of lung diseases and therapeutic response on an individual basis with high spatial and temporal resolution, without concerns for radiation toxicity that could potentially influence the readout of micro-CT-derived lung biomarkers. This work further supports the introduction of micro-CT for routine use in the preclinical pulmonary research field where postmortem histological approaches are still the gold standard.

Keywords: Monte Carlo; dosimetry; in vivo lung imaging; radiation safety.

Fig. 1.

Longitudinal lung microcomputed tomography (micro-CT) scanning protocols. Schematic outline of the study setup and four micro-CT scanning regimes used in this study. A total number of 32 mice were divided in the following experimental and control groups (n = 4/group): group E1, 1× micro-CT/wk during 5 wk; group E2, 2× micro-CT/wk during 5 wk; group E3, 2× micro-CT/wk during 5 wk, including 1× high-dose micro-CT scan in week 3; group E4, 1× micro-CT/wk during 12 wk; group C1, age-matched once-scanned controls, 1× micro-CT at 5 wk; group C2, age-matched never scanned controls, killed after 5 wk; group C3, age-matched once-scanned controls, 1× micro-CT at 12 wk; and group C4, age-matched never scanned controls, killed after 12 wk.

Fig. 2.

Dosimetry. Axial (left) and sagittal (right) reconstructed micro-CT images of a dead mouse carrying three thermoluminescent dosimeters at the level of the lungs (TLDs, top) and of the ionization chamber (bottom) used for measuring the radiation dose of a typical lung micro-CT scan as used in this study.

Fig. 3.

Monte Carlo simulations of tissue-specific absorbed dose. A: Monte Carlo simulation input and results according to method 2, illustrating reconstructed axial micro-CT images indicating the voxel used for which the absorbed skin dose was estimated (a), color-scaled results of the segmentation based on tissue density into bone (yellow), lung (purple), and soft tissues (orange) (b), and the absorbed dose into these tissues (c). B: Monte Carlo simulation input and results according to method 1, showing coronal (a and d), axial (b and e), and sagittal (c and f) slices of reconstructed anatomical end-expiratory micro-CT images (top) used as input for the estimation of the relative dose (in %), illustrated in the corresponding color-scaled slices (bottom). The lungs are delineated in pink.

Fig. 4.

In vivo and ex vivo lung micro-CT. Axial, end-expiratory lung micro-CT images acquired at the last in vivo imaging time point at 5 wk (groups E1, E2, E3, and C1) or 12 wk (groups E4 and C3) after baseline and corresponding ex vivo images for the same mouse, acquired after the last in vivo micro-CT scan.

Fig. 5.

Quantitative in vivo micro-CT results. Graphs of the aerated lung volume, lung tissue volume, total lung volume, and mean lung signal intensity (SI) quantified from the end-expiratory in vivo micro-CT data acquired at the last imaging time point of longitudinally scanned mice (for groups E1–E3 at 5 wk, for group E4 at 12 wk after baseline) vs. their respective once-scanned controls (group C1 at 5 wk or group C3 at 12 wk). Error bars indicate SD of 4 mice. NS, not significant.

Fig. 6.

Detailed histological assessment of potential radiation-induced lung damage. A: graphs of Ashcroft scores for lung fibrosis (left) and quantification of lung collagen content (right) after the last in vivo and ex vivo micro-CT scans at 5 wk (groups E1–E3 and C1–C2) and 12 wk (groups E4 and C3–C4). B: light microscopy images of representative (randomly selected) hematoxylin- and eosin-stained mouse lung tissue sections for a control mouse (C2) and longitudinally micro-CT-scanned mice after 5 wk (E2) and 12 wk (E4) at ×200 magnification. Scale bars measure 100 μm. Error bars indicate SD of 4 mice.