Animals and Irradiation

All animal procedures for this study were approved by the Institutional Animal Care and Use Committee (IACUC) at Duke University. The p53^FL/FL and VECre mice have been described elsewhere (12). Experiments were performed with both male and female mice that were between 8 and 12 weeks old and were on mixed genetic backgrounds. Age-matched, littermate controls that retained one allele of wild-type p53 were utilized to minimize the effect of genetic background. Therefore, potential genetic modifiers of the response to radiation were randomly distributed among the experimental and control groups.

Total-abdominal irradiation (TAI) was performed using a small-field biological irradiator, the X-RAD 225Cx (Precision X-Ray Inc., North Branford, CT). Mice were irradiated with parallel-opposed anterior and posterior fields, which encompassed the small and large intestines, using a collimating cone that produces a square radiation field of 40 mm at treatment isocenter. The average dose rate was 298.8 cGy/min at target depth with a 225 kVp, 13 mA beam and a 0.3-mm copper filter. The dose rate was measured with an ion chamber by members of the Radiation Safety Division at Duke University.

GI Epithelium Permeability Assay

Fluorescein isothiocyanate (FITC)-dextran (4 kD; Sigma-Aldrich^® LLC, St. Louis, MO) was dissolved in sterile water at 40 mg/ml. Mice were gavaged before irradiation or at various time points postirradi-ation with 0.6 mg/g body weight of the FITC-dextran solution 4 h before sacrifice when serum was collected. The levels of FITC-dextran in serum were quantified following methods described elsewhere (13).

Histology

To prepare paraffin-embedded tissues, tissue specimens were fixed in 10% neutralized formalin overnight and preserved in 70% ethanol. The small intestines were bundled for embedding to obtain the ideal orientation of the crypts (11). To prepare frozen tissues, tissue specimens were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 2 h at 4°C and transferred to 30% sucrose in PBS overnight at 4°C. The small intestines were bundled and immersed in OCT compound (Sakura Finetek), snap frozen in dry ice/isopentane slurry and stored at –80°C. Immunofluorescent staining for wheat germ agglutinin (WGA), Griffonia simplicifolia isolectin B₄ (GS-IB₄) and 2-nitroimidazole EF5 were performed following methods described elsewhere (12). Images were quantified, using MATLAB^® (MathWorks^® Inc., Natick, MA), by a single observer (SH) blinded to genotypes and radiation treatment. The radiation injury score was assessed by a veterinary pathologist (LB) blinded to genotypes and radiation treatment according to a published scoring system (14, 15).

Isolation of Intestinal Epithelial Cells

The intestinal epithelium was isolated according to a method described elsewhere (6). Small intestines were cut longitudinally and washed with ice-cold PBS without Mg²+^/Ca²⁺ (PBS⁰) and then incubated in PBS⁰ supplemented with 2 mM EDTA, 1 mM DTT, 50 mM gentamicin (Sigma-Aldrich) and 1× penicillin/streptomycin antibiotics (Gibco^®, Grand Island, NY) at 4°C for 60 min on a rotating platform. The intestines were then gently transferred to another tube containing cold PBS⁰ supplemented with 2 mM EDTA and 1 mM DTT and shaken vigorously to detach the epithelial cells. The muscularis layers were then removed and the cells were centrifuged to obtain an epithelial isolate. The cell fraction was then washed one more time in cold PBS⁰. Cell pellet was snap frozen in liquid nitrogen and stored at –80°C.

Quantitative Reverse Transcription-PCR

Mouse small intestinal epithelial cell frozen pellets were thawed and resuspended in 1 ml of TRI Reagent (Invitrogen^™, Grand Island, NY). Total RNA was extracted and quantified. cDNA was generated from 1 µg of RNA using iScript^™ cDNA Synthesis Kit (Bio-Rad^® Laboratories Inc., Hercules, CA). Relative expression levels were determined using qPCR assays performed on the QuantStudio^™ 6 Flex Real-Time PCR System with PowerUp^™ SYBR^® Green Master Mix and specific primer sets (16) or TaqMan^™ Fast Advanced Master Mix and specific Taqman probes (Thermo Fisher Scientific^™ Inc., Waltham, MA). Target gene quantification levels were normalized to the housekeeper gene, Gapdh.

Mdr1 (Abcb1b): Taqman probe Mm00440736_m1;

Tff3: Taqman probe Mm00495590_m1;

Gapdh: Taqman probe Mm99999915_g1;

Tjp1/ZO-1: 5 ′ -CCACCTCTGTCCAGCTCTTC-3 ′; 5 ′ -CACCG GAGTGATGGTTTTCT-3 ′

Claudin-2: 5 ′ -GTCATCGCCCATCAGAAGAT-3 ′; 5 ′ -ACTGTTG GACAGGGAACCAG-3 ′.

Computed Tomography (CT) Scan

To assess the effects of radiation on the GI tract we used an in-house dual source micro-CT system specifically developed for dynamic and spectral applications (17, 18). The system incorporates two imaging chains capable of independently triggered acquisitions. The system contains two G-297 X-ray tubes (Varian Medical Systems, Palo Alto, CA) with 0.3/0.8-mm focal spot size, two Epsilon high-frequency X-ray generators (EMD Technologies; Saint-Eustache, Canada), and two CCD-based detectors with a Gd₂O₂S phosphor (XDI-VHR 2 Photonic Science, East Sussex, UK) with 22-µm pixels, which we bin to 88 µm. Dual-energy acquisition is controlled via custom sequencing applications written in LabVIEW (National Instruments^™, Austin, TX).

A total of 360 projection images per imaging chain were acquired. Dual-energy settings were configured as follows: 1. 40 kVp, 250 mA, 16 ms per exposure; and 2. 80 kVp, 160 mA, 10 ms per exposure. The radiation dose for each micro-CT involving 720 projections with the described exposure parameters is approximately 245 mGy.

To assess the vascular permeability, mice were administered gold nanoparticles (AuNps) via retro-orbital injection ( www.nanoprobes.com) at a dose of 0.004 ml/g. As a result of radiation damage to the endothelial cells in the GI tract, the gold nanoparticles accumulated via the enhanced permeability and retention effect (19). Three days later the mice were re-injected with a liposomal iodinated contrast agent Lip-I (20) (dose 0.012 ml/g). The use of both nanoparticle agents for this application has been described elsewhere (21) and enables the visualization of both vascular permeability in the damaged GI tract via AuNps (which extravasate from vessels and accumulate in the injured tissue) and the vessels (via Lip-I which remains within the vessels). Micro-CT imaging resulted in two separate 3D datasets for each scan, which were acquired at the two different energies with a voxel size of 88 µm. The size of the reconstructed volume was 512 µm³ voxels spanning 45 mm³.

For dual-energy micro-CT, material decomposition of iodine and gold was performed after reconstruction and Hounsfield unit (HU) conversion, as described elsewhere (22, 23). Briefly, image-based decomposition used paired temporal reconstructions at two different kVps (40 kVp, 80 kVp) and solved a linear system at each voxel,

where C is the least-squares solution for the concentrations of iodine and gold in mg/ml in the voxel under consideration, A is a constant sensitivity matrix measured in Hounsfield units per contrast agent concentration (HU/mg/ml) for iodine and gold at 40 and 80 kVp, respectively, and b is the intensity of the voxel at the two energies, in Hounsfield units.

After the solution of the matrix equation was determined, a non-negativity constraint was enforced by setting voxels with negative concentrations of both materials to zero. Voxels with a negative concentration of one material and a positive concentration of the other material were orthogonally projected onto the subspace of positive concentrations. Values for the coefficients of the sensitivity matrix at each energy were determined empirically using a calibration phantom.

Statistical Analysis

For all experiments, measurements are presented as mean ± SEM. Each data point represents one mouse. Two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test was performed to examine the interaction between genotype and radiation treatment. Two-tailed Student's t test (parametric) or Mann-Whitney test (nonparametric) was performed to compare the data between two groups. If the normality test failed, data were log-transformed prior to applying statistical tests. For the GI radiation injury study, Kaplan–Meier estimate was performed followed by the log-rank test. We considered the evidence against a null hypothesis to be significant if the unadjusted P value for the corresponding test was less than 0.05. GraphPad Prism version 8 (GraphPad Software Inc., La Jolla, CA) was used to perform these statistical analyses.

Deletion of p53 Specifically in Endothelial Cells Promotes Delayed, but not Acute, Radiation-Induced Intestinal Injury

To sensitize endothelial cells to radiation, we deleted the floxed p53 allele (p53^FL) specifically in endothelial cells using VECre (12). VECre; p53^FL/FL mice, in which both alleles of p53 are deleted in endothelial cells, as well as their VECre; p53^{FL/ +} littermates, which retain one allele of p53 in endothelial cells, received 15 Gy TAI. After irradiation, the percentage of mice that became moribund within 10 days as a result of the acute GI radiation syndrome (11) was not significantly different between the VECre; p53^FL/FL cohort (35%, 7 out 20) and the VECre; p53^{FL/ +} cohort (45%, 10 out of 22) (Fig. 1A). One hallmark of the acute GI radiation syndrome is breakdown of the GI mucosal barrier due to extensive crypt destruction (11). Therefore, we examined the integrity of the GI mucosal barrier by delivering FITC-dextran solution through oral gavage and then detected the level of FITC-dextran in serum 4 h later (13). Compared to nonirradiated mice, the concentration of FITC-dextran in serum was highly elevated 5 days after 15 Gy TAI in both VECre; p53^FL/FL and VECre; p53^{FL/ +} mice (Fig. 1B). Together, these results suggest that deletion of p53 in endothelial cells does not promote acute GI radiation injury.

FIG. 1

Deletion of p53 in endothelial cells sensitizes mice to delayed, but not acute, radiation-induced GI injury. Panel A: Kaplan-Meier survival analysis of VECre; p53^FL/FL mice (p53 deleted in endothelial cells) and VECre; p53^{FL/ +} mice (p53 retained in endothelial cells) after a single dose of 15 Gy TAI. Panel B: Quantification of FITC-dextran in serum collected from VECre; p53^{FL/ +} (blue) and VECre; p53^FL/FL (red) mice at various time points after 15 Gy TAI. Mice were given FITC-dextran via oral gavage 4 h before sacrifice. Serum was collected from each mouse and the concentration of FITC-dextran in serum was quantified. *P < 0.05 by Student's t test compared to nonirradiated mice. Each dot represents the averaged data from one mouse. Panel C: Change in body weight of VECre; p53^{FL/ +} and VECre; p53^FL/FL mice after 15 Gy TAI. *P < 0.05 by two-way ANOVA with Bonferroni post hoc test. Panel D: The expression of tight junction genes Tjp1 (ZO-1) and Claudin-2 mRNA in intestinal epithelial cells from unirradiated mice or at day 30 after 15 Gy TAI. P value was calculated by two-way ANOVA with Bonferroni post hoc test. *P < 0.05. Each dot represents one mouse.

Although deletion of p53 in endothelial cells did not sensitize mice to the acute GI radiation syndrome, VECre; p53^FL/FL mice that survived acute radiation injury became moribund starting at approximately one month after 15 Gy TAI (Fig. 1A) and showed a significant decrease in body weight (Fig. 1C). In contrast to the mice that developed acute GI radiation injury, both VECre; p53^FL/FL and VECre; p53^{FL/ +} mice, at 30 days after 15 Gy TAI, did not exhibit a significant increase in the concentration of FITC-dextran in serum (Fig. 1B), suggesting that intestinal crypts were not extensively depleted. To validate the results of the FITC-dextran assay, we isolated intestinal epithelial cells from VECre; p53^FL/FL and VECre; p53^{FL/ +} mice at 0 and 30 days after 15 Gy TAI to examine mRNA expression of tight junction genes Tjp1 (ZO-1) and Claudin-2 (16). Tjp1 (ZO-1) is a tight junction protein expressed ubiquitously in all tight junctions that binds directly to the cytoplasmic tails of claudins and occludin (24, 25). It has been shown that the mRNA and protein of Tjp1 (ZO-1) is markedly decreased in the small intestine at days 1 and 7 after 13 Gy TAI (26). However, our results indicated that Tjp1 mRNA in the intestinal epithelium was not significantly decreased in either VECre; p53^FL/FL or VECre; p53^{FL/ +} mice 30 days after 15 Gy TAI compared to nonirradiated controls (Fig. 1D). In contrast, we unexpectedly found that intestinal epithelial cells collected from VECre; p53^{FL/ +} mice and VECre; p53^FL/FL mice 30 days after 15 Gy TAI significantly overexpressed Claudin-2. Of note, Claudin-2 overexpression was substantially greater in irradiated VECre; p53^FL/FL mice compared to irradiated VECre; p53^{FL/ +} mice (P = 0.01 by two-way ANOVA) (Fig. 1D). Claudin-2 is a pore-forming tight-junction protein that is highly upregulated in patients with inflammatory bowel diseases including ulcerative colitis and Crohn's disease (27–30). Collectively, our findings indicate that sensitization of endothelial cells to radiation exacerbates delayed, but not acute, intestinal injury.

Delayed Radiation-Induced Intestinal Injury is Characterized by Increased Vascular Permeability and Tissue Hypoxia

Examination of the gross histology indicated that, compared to the small intestines from irradiated VECre; p53^{FL/ +} mice, the small intestines from irradiated VECre; p53^FL/FL mice that developed delayed intestinal injury were shorter and hemorrhagic (Fig. 2A). To quantify the severity of tissue injury, we examined intestinal tissue sections to calculate the radiation injury score according to a published scoring system (14, 15). This analysis revealed that irradiated VECre; p53^FL/FL mice that succumbed to delayed intestinal injury (32 to 38 days after 15 Gy) showed a significantly higher injury score compared to irradiated VECre; p53^{FL/ +} mice that were sacrificed at approximately the same time period (32 to 73 days after 15 Gy) (Fig. 2B and  Supplementary Table S1 (258_rare-192-03-02_s01.pdf);  http://dx.doi.org/10.1667/RR15371.1.S1). The small intestines of VECre; p53^FL/FL mice that developed delayed intestinal injury 32 days after irradiation exhibited hemorrhage of blood vessels with secondary epithelial damage and inflammation. Throughout the sections the lamina propria and submucosa were moderately expanded by edema and hemorrhage, which was characterized by a pale eosinophilic material mixed with small scattered aggregates of extravasated red blood cells (Fig. 2C).

FIG. 2

Histological examination of delayed radiation-induced intestinal injury. Panel A: Gross histology of small intestines collected from VECre; p53^FL/FL and VECre; p53^{FL/ +} mice at day 32 after 15 Gy TAI. Panel B: Quantification of tissue injury score of the small intestine. Tissues were collected from irradiated VECre; p53^FL/FL mice that succumbed to delayed intestinal injury and irradiated VECre; p53^{FL/ +} mice that were sacrificed at approximately the same time frame after 15 Gy TAI. Nonirradiated VECre; p53^FL/FL and VECre; p53^{FL/ +} mice were used as controls. *P < 0.05 by Mann-Whitney test. Each dot represents one mouse. Panel C: Representative H&E-stained sections of the small intestines collected from VECre; p53^{FL/ +} and VECre; p53^FL/FL unirradiated mice as well as at days 3 and 32 after 15 Gy TAI. Notably, at day 3 after 15 Gy TAI both VECre; p53^FL/FL and VECre; p53^{FL/ +} mice showed almost complete depletion of intestinal crypts. In contrast, at day 32 after 15 Gy TAI the VECre; p53^FL/FL mice that developed delayed intestinal injury still retained some intestinal crypts even in regions where vascular injury was present. Scale bar: 100 µm.

We next examined vascular injury of the small intestines collected from VECre; p53^FL/FL and VECre; p53^FL/ + littermates 30 days after 15 Gy TAI by staining with GS-IB₄ to label endothelial cells and an antibody against EF5 to identify regions of hypoxia (12). We found that while irradiated VECre; p53^{FL/ +} mice retained vasculature within the villi and lacked regions of hypoxia, irradiated VECre; p53^FL/FL mice exhibited destruction of GS-IB₄ ⁺ microvessels in the villi and displayed focal lesions of tissue hypoxia (Fig. 3A). Quantification of tissue sections indicated that, compared to irradiated VECre; p53^{FL/ +} mice, irradiated VECre; p53^FL/FL mice showed a decrease in the percentage of GS-IB₄ ⁺ fraction area (P = 0.057) and a significant increase in EF5⁺ fraction area per high-power field. We also assessed mRNA expression of hypoxia-responsive genes Tff3 (31) and Mdr1 (Abcb1) (32) in intestinal epithelial cells of VECre; p53^FL/FL and VECre; p53^{FL/ +} mice at day 30 after 15 Gy TAI. Our results showed that, compared to nonirradiated mice, the expression of Mdr1 was modestly increased in irradiated VECre; p53^{FL/ +} mice, but it was highly elevated in a subset of irradiated VECre; p53^FL/FL mice (Fig. 3D).

FIG. 3

Delayed radiation-induced intestinal injury is characterized by microvessel damage and tissue hypoxia. Panel A: Representative tissue sections of one VECre; p53^{FL/ +} mouse and two VECre; p53^FL/FL mice at day 30 after 15 Gy TAI. The small intestines of irradiated VECre; p53^FL/FL mice showed a decrease in GS-IB4⁺ endothelial cells within the lamina propria of the villi. In addition, the small intestines of irradiated VECre; p53^FL/FL mice, but not the VECre; p53^{FL/ +} mouse, exhibited areas of hypoxia as shown by EF5 staining. Note that in irradiated VECre; p53^FL/FL mice, even in regions with loss of microvessels in the villi and associated hypoxia, the adjacent crypts (yellow arrows) are largely intact. Scale bar: 100 µm. Panels B and C: Quantification of the area stained positive for GS-IB4 or EF5 per high-power field 30 days after 15 Gy TAI. P value was calculated using Student's t test. Each dot represents one mouse. Panel D: The expression of hypoxia-responsive genes Tff3 and Mdr1 (Abcb1) in intestinal epithelial cells from unirradiated mice or at day 30 after 15 Gy TAI. *P < 0.05 (two-way ANOVA with Bonferroni post hoc test).

To quantify vascular permeability of the small intestine in vivo, we utilized dual-energy-micro-CT imaging to detect the leakage of gold nanoparticles from blood vessels (21). Our previously published studies using a mouse model of radiation-induced cardiac injury demonstrated accumulation of gold in the myocardium where endothelial cells were damaged (21). Dual-energy-micro-CT was performed using VECre; p53^FL/FL and VECre; p53^{FL/ +} mice that were not irradiated or irradiated at day 28 after 15 Gy TAI. These mice were injected intravenously with a PEGylated gold nanoparticles three days before imaging and a blood-pool Lip-I immediately before imaging to label the blood pool (21). Compared to VECre; p53^{FL/ +} mice and VECre; p53^FL/FL mice at day 28 after 15 Gy TAI had a marked accumulation of gold nanoparticles along the irradiated GI tract (Fig. 4A). Quantification of gold signals also indicated a significant increase in the amount and concentration of gold in the small intestine of VECre; p53^FL/FL mice at day 28 after 15 Gy TAI (Fig. 4B and C). Collectively, our results demonstrate that radiosensitization of endothelial cells promotes delayed intestinal radiation injury that is manifested by increased vascular permeability and tissue hypoxia.

FIG. 4

Assessing vascular permeability of the small intestine in vivo using dual-energy-micro-CT. Panel A: VECre; p53^{FL/ +} and VECre; p53^FL/FL mice were intravenously injected with gold nanoparticle (AuNp; green) contrast agent once on day 0. Three days later mice were injected with iodinated nanoparticle (Lip-I; red). The use of both types of nanoparticles enables the measurement of vascular permeability in the irradiated intestines (via AuNp, which extravasated after a few days and accumulated in the injured tissue), and the measurement of vascularity (via Lip-I which remains within the blood vessels). Accumulation of AuNp along the small intestine is indicated by yellow arrows. Panels B and C: Quantification of the mean gold mass and gold concentration in the small intestine at days 0 and 28 after 15 Gy TAI. *P < 0.05 (Student's t test). Each dot represents one mouse.