A Model for Precise and Uniform Pelvic- and Limb-Sparing Abdominal Irradiation to Study the Radiation-Induced Gastrointestinal Syndrome in Mice Using Small Animal Irradiation Systems

Abstract

Background and purpose: Currently, no readily available mitigators exist for acute abdominal radiation injury. Here, we present an animal model for precise and homogenous limb-sparing abdominal irradiation (LSAIR) to study the radiation-induced gastrointestinal syndrome (RIGS).

Materials and methods: The LSAIR technique was developed using the small animal radiation research platform (SARRP) with image guidance capabilities. We delivered LSAIR at doses between 14 and 18 Gy on 8- to 10-week-old male C57BL/6 mice. Histological analysis was performed to confirm that the observed mortality was due to acute abdominal radiation injury.

Results: A steep dose-response relationship was found for survival, with no deaths seen at doses below 16 Gy and 100% mortality at above 17 Gy. All deaths occurred between 6 and 10 days after irradiation, consistent with the onset of RIGS. This was further confirmed by histological analysis showing clear differences in the number of regenerative intestinal crypts between animals receiving sublethal (14 Gy) and 100% lethal (18 Gy) radiation.

Conclusion: The developed LSAIR technique provides uniform dose delivery with a clear dose response, consistent with acute abdominal radiation injury on histological examination. This model can provide a useful tool for researchers investigating the development of mitigators for accidental or clinical high-dose abdominal irradiation.

Keywords: abdominal irradiation; dose response; histological analysis; radiation-induced gastrointestinal syndrome; small animal radiation research.

Figure 1.

Small animal irradiator setup to perform abdominal irradiation experiments.

Figure 2.

Panel (A) shows the in-house designed custom abdominal irradiation collimator and panel (B) illustrates the resulting irradiation field at the radiation isocenter distance of 33 cm (corresponding to the midline level of the animals).

Figure 3.

Dose–response survival curves following limb-sparing abdominal irradiation (LSAIR)--induced radiation-induced gastrointestinal syndrome (RIGS)are shown as a Kaplan-Meier survival plot. No deaths were recorded at doses below 16 Gy, and no animals that received a dose of 17 Gy or higher survived past 10 days. Number of animals in each dose group— 14 Gy (n = 4), 15 Gy (n = 10), 15.5 Gy (n = 10), 16 Gy (n = 15), 16.5 Gy (n = 16), 17 Gy (n = 16), 17.5 Gy (n = 18), and 18 Gy (n = 22).

Figure 4.

The mortality data are plotted as black dots with uncertainty bars representing 95% confidence intervals (CIs) for the logistic model fit (A) and the probit model fit (B). The CIs were calculated as the binomial 95% CIs based on the number of surviving and dying subjects, except for dose levels where there were consistently 0% or 100% mortality in repeated experiments. Thin blue lines represent 1000 bootstrap samples showing the variation in the dose–response curve from randomly sampling the data 1000 times within the presented 95% CIs.

Figure 5.

Histological analysis of jejunum of abdominally irradiated mice. Two mice per dose group were subjected to limb-sparing abdominal irradiation (LSAIR),and the jejunum was isolated and processed for histological analysis at day 4 postirradiation. Panel (A) shows a hematoxylin and eosin (H&E)-stained whole jejunum section from a 14 Gy irradiated mouse; right-hand panels show enlarged regions representing depleted or fully populated crypt areas, as indicated. Regenerative crypts (indicated by black arrows) were identified by their dark purple color, the presence of connected overlaying villi and underlying lamina propria. Panel (B) shows the corresponding images of a jejunum section from an 18 Gy irradiated mouse. Scale bars = 5000 μm.

Figure 6.

The dose-dependent limb-sparing abdominal irradiation (LSAIR) mortality rate is linked to the extent of intestinal cell damage. The number and density of regenerative crypts and length of fully populated crypt segments were quantified in the jejunum of animals receiving 14 Gy (n = 2, black bars) and 18 Gy (n = 2, gray bars) LSAIR. There was a significantly lower number of regenerative crypts per jejunal length in the jejunums of 18 Gy compared to 14 Gy irradiated mice. The data are shown as mean value with uncertainty bars representing 1 standard deviation; P values were generated using an unpaired t test comparing the difference between means in the 14-Gy and 18-Gy groups.