Effects of dose rates on radiation-induced replenishment of intestinal stem cells determined by Lgr5 lineage tracing

摘要

Lgr5-EGFP-IRES-CreERT2 knock-in mice (B6.129P2-Lgr5tm1(cre/ERT2)Cle/J; JAX mice #008875) and ROSA26-LSL-LacZ mice (B6.129S4-Gt (ROSA)26Sortm1Sor/J; JAX mice #003474) were purchased from the Jackson Laboratory (ME, USA). The mice were bred in a conventional clean-room facility at the Central Research Institute of Electric Power Industry (CRIEPI) under conditions of controlled temperature (24 ± 2°C) and humidity (45% ± 5%), with a 12-h light–dark cycle and ad libitum access to γ-sterilized food (CLEA Japan, Tokyo, Japan) and filter-sterilized deionized water. All animal experiments were approved by the Animal Research and Ethics Committee at the CRIEPI and were performed in accordance with the guidelines for animal care in Japan. To irradiate mice with high-dose-rate (30 Gy/h) X-rays, an MBR-320R generator (Hitachi Ltd, Tokyo, Japan) installed in a conventional clean-room facility at the CRIEPI was operated at 260 kV, with a 4.5-mA tube current and a 0.5-mm Al + 0.3-mm Cu filter. 137Cs γ-rays (0.003 Gy/h) were used for long-term low-dose-rate irradiation at the low-dose-rate irradiation facility at the CRIEPI. The dose of irradiation was determined using a photoluminescence glass dosimeter GD-351 (Asahi Techno Glass Corporation, Tokyo, Japan). Mice were subjected to low-dose-rate γ-rays for ～2 weeks (the cumulative dose was 1 Gy). Control mice were sham-irradiated and handled in the same manner as the test animals. Detailed methods for the lineage tracing of Lgr5+ cells have been described previously [31]. In brief, in order to stain LacZ-labeled crypts, 4-hydroxytamoxifen (4OHT, Sigma, St Louis, MO, USA) at 10 mg/ml (3 mg/40 g body weight) was intraperitoneally injected into Lgr5-EGFP-IRES-CreERT2 × ROSA26-LSL-LacZ mice. One month after 4OHT administration, the mice were irradiated with X-rays or γ-rays. The mice were sacrificed 2 weeks after irradiation, and the intestinal LacZ+ crypts were counted as described [31]. In brief, the isolated distal colon was washed with phosphate-buffered saline (PBS), opened longitudinally, and placed on filter paper. The tissue samples were then fixed and stained to assess β-galactosidase activity [39]. The samples were then embedded in paraffin, sectioned horizontally, and counterstained with eosin (Sakura Finetek, Tokyo, Japan). Fully LacZ-labeled crypts were counted as LacZ+ crypts, and the percentage of LacZ+ crypts (%LacZ crypts) was calculated as follows: We used ImageJ version 1.46r (National Institutes of Health) to determine the LacZ+ area in each crypt. We first split the images of the crypt sections stained with X-gal (blue) and eosin (pink) into three channels (RGB). We used the red and blue channels to determine the areas of the LacZ+ cells and whole crypts. The area was measured after determining the shapes of crypts or LacZ+ cells. Tissue samples of the whole distal colon were harvested, cut into 5-mm pieces, and immediately frozen in liquid nitrogen. The frozen pieces were pulverized using a TK-AM5-S bullet mill (Tokken Inc., Chiba, Japan). Total RNA was extracted, purified using an Ambion RNAqueous-4PCR kit (Thermo Fisher Scientific Inc., MA, USA), and reverse-transcribed into cDNA using Invitrogen SuperScript II RNase-H RT enzyme (Thermo Fisher Scientific Inc., MA, USA). Primers and TaqMan probes for the Lgr5 gene (Assay ID: Mm00438890_m1) were purchased from Life Technologies. Gene expression levels were determined using an Applied Biosystems PRISM 7900HT analyzer (Thermo Fisher Scientific Inc.) and normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (Assay ID: Mm99999915_g1). Student's t-tests were used to compare the frequencies of LacZ+ crypts following 4OHT injection into mice of different ages (1 and 4 weeks old) and to evaluate the radiation-induced replenishment of colonic Lgr5+ stem cells. Tukey–Kramer multiple comparison tests were used to evaluate gene expression. A schematic diagram of the experiment is shown in Fig.?1. We administered 4OHT to Lgr5-EGFP-IRES-CreERT2 × ROSA-LSL-LacZ mice and then exposed the mice to radiation at different dose rates (0.003 or 30 Gy/h). After radiation exposure, staining and sectioning, LacZ-labeled crypts were counted. Note that not all colonic crypts of 4OHT-treated Lgr5-EGFP-IRES-CreERT2 × ROSA-LSL-LacZ mice were labeled (Fig.?1) [31, 40]. Labeling efficiency in the crypts was evaluated by determining the decrease in the number of labeled Lgr5+ stem cells during cell replacement. The efficiency of LacZ-labeling upon 4OHT injection in mice of different ages has not been previously investigated. Therefore, we first determined the percentage of LacZ+ crypts in 4OHT-treated infant (1-week-old) and young adult (4-week-old) mice. Prior to 2 weeks after 4OHT injection, the percentage of LacZ-labeled colonic cry