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Preclinical murine platform to evaluate therapeutic countermeasures against radiation-induced gastrointestinal syndrome - PubMed

  • ️Tue Jan 01 2019

Preclinical murine platform to evaluate therapeutic countermeasures against radiation-induced gastrointestinal syndrome

Paul B Romesser et al. Proc Natl Acad Sci U S A. 2019.

Abstract

Radiation-induced gastrointestinal syndrome (RIGS) is a limiting factor for therapeutic abdominopelvic radiation and is predicted to be a major source of morbidity in the event of a nuclear accident or radiological terrorism. In this study, we developed an in vivo mouse-modeling platform that enables spatial and temporal manipulation of potential RIGS targets in mice following whole-abdomen irradiation without the confounding effects of concomitant hematopoietic syndrome that occur following whole-body irradiation. We then tested the utility of this platform to explore the effects of transient Wnt pathway activation on intestinal regeneration and animal recovery following induction of RIGS. Our results demonstrate that intestinal epithelial suppression of adenomatous polyposis coli (Apc) mitigates RIGS lethality in vivo after lethal ionizing radiation injury-induced intestinal epithelial damage. These results highlight the potential of short-term Wnt agonism as a therapeutic target and establish a platform to evaluate other strategies to stimulate intestinal regeneration after ionizing radiation damage.

Keywords: Wnt signaling; intestinal regeneration; radiation enteritis; radiation mitigator; radiation-induced gastrointestinal syndrome.

Copyright © 2019 the Author(s). Published by PNAS.

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Conflict of interest statement

Conflict of interest statement: L.E.D. and S.W.L. are founders and scientific advisory board members of Mirimus, Inc., a company that incorporates animal modeling technologies similar to those presented in this work. P.B.R. has received an honorarium from Corning to discuss 3D cell culture techniques, has served as a consultant for AstraZeneca, and is a consultant for EMD Serono for work on radiation sensitizers.

Figures

Fig. 1.
Fig. 1.

Whole-abdomen irradiation permits escalating RT dose to the small intestine while minimizing RT dose to normal healthy bone marrow. (A) Anesthetized mice were aligned with laser coordinate positioning, a 25-mm diameter collimator was utilized to localize the radiation to the abdomen, treatment depth was verified, and fluoroscopic images were obtained to confirm mouse positioning relative to bony anatomy. (B) Whole-mouse microcomputerized topography scans were obtained to allow animal dosimetry and normal organ identification. Representative images of 5 replicates. (C) Small intestine, bone marrow, and heart dose–volume histograms were calculated. (D) RT-induced canities at 1 y after shApc induction and 14.5 Gy WAI.

Fig. 2.
Fig. 2.

Whole-abdominal irradiation elicits radiation-induced gastrointestinal syndrome (RIGS) but does not result in hematopoietic syndrome. (A) Notable differences in serial hematocrit and lymphocyte counts after whole-body and whole-abdominal irradiation. Five mice per cohort. (B) Appreciable differences in femur bone marrow cellularity between mice treated with whole-abdominal and whole-body irradiation at 7 d. Representative image of 5 biological replicates. (C) Kaplan–Meier survival after escalating dose of whole-abdomen irradiation (n ≥ 6 per group representing at ≥2 [range; 2 to 6] independent experiments depending on the dose). (D) Absolute change in weight at 8 d after escalating WAI dose (n ≥ 6 per group representing at ≥2 [range; 2 to 4] independent experiments depending on the dose). (E) Representative immunofluorescence images noted increased cleaved-Caspase 3 and decreased Ki67 positive cells in mice treated with lethal (14.5 Gy) and sublethal (12 Gy) WAI at 24 and 48 h, respectively. Error bars, mean ± 95% confidence interval (CI). ns, not significant. *P < 0.01. C, Log-rank comparison; D, two-sided t test.

Fig. 3.
Fig. 3.

Intestinal Wnt activation after whole-abdomen irradiation accelerates intestinal stem cell repopulation and promotes gut epithelial regeneration. (A) Experimental schematic. (B) Serial weight loss after escalating doses of whole-abdomen irradiation (n ≥ 3 per group). (C) Hematoxylin and eosin staining of shApc and shControl intestinal crypts at 6, 24, and 48 h after 14.5 Gy WAI and dox gavage. (D) Quantification of regenerating crypts per cross-sectional area at 6, 24, and 48 h after 14.5 Gy WAI and dox gavage (n ≥ 3 per group). Error bars, mean ± 95% confidence interval (CI).

Fig. 4.
Fig. 4.

Wnt activation after lethal whole-abdomen irradiation stimulates intestinal regeneration. (A) Serial immunofluorescence images (GFP, Ki67, DAPI) of shApc and shControl mice after 14.5 Gy WAI and dox gavage (representative images shown). (B) Quantification of Ki67+ IHC cells per cross-sectional area at 6, 24, and 48 h after WAI and dox gavage (n ≥ 4 per group). (C–G) qRT-PCR analysis of gene expression in bulk intestinal isolates following 14.5 WAI and dox gavage. Markers of transgene induction (GFP) (C), Wnt activation Myc (D) and Axin2 (E), and stem cells Lgr5 (F) and Ascl2 (G) are shown for shApc and shControl mice at 6, 24, and 48 h after WAI and dox gavage. Relative gene expression is normalized to unirradiated dox naïve shControl mice. Plots represent the mean ± SEM and are based on 3 biological and 3 technical replicates for each condition at each timepoint. (H) Serial Lgr5 ISH images after 14.5 Gy WAI and dox gavage.

Fig. 5.
Fig. 5.

Wnt activation after lethal whole-abdomen irradiation mitigates radiation-induced gastrointestinal lethality. (A) Kaplan–Meier survival curve comparing shControl and shApc hairpin induction after 14.5 Gy WAI (n ≥ 2 per group, with ≥2 experimental replicates). (B) Fourteen-day mortality with escalating WAI RT dose in shControl and shApc mice (n ≥ 3 per group). A, Log-rank comparison.

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