The authors have declared that no competing interests exist.
Conceived and designed the experiments: LVL PKL SJH MAH. Performed the experiments: LVL REB JJD. Analyzed the data: LVL REB PKL SJH MAH. Wrote the paper: LVL PKL SJH REB MAH.
In C57Bl/6J mice, a predetermined small intestinal segment was exteriorized and exposed to 14Gy-radiation, while a lead shield protected the rest of the body from radiation. Sham controls had segment exteriorization but no radiation. Results were compared to C57Bl/6J mice given 14 Gy-abdominal radiation. Effects of elemental liquid diet feeding from the day prior to radiation until day 7 post-radiation were assessed in both models. Body weight and a custom-developed health score was assessed every day until day 21 post-radiation. Intestine was assessed histologically.
At day 3 after segment radiation, complete loss of crypts occurred in the targeted segment, while adjacent and remaining intestine in segment-radiated mice, and entire intestine of sham controls, showed no detectable epithelial damage. Liquid diet feeding was required for survival of mice after segment radiation. Liquid diet significantly improved survival, body weight recovery and normalization of intestinal epithelium after abdominal radiation. Mice given segment radiation combined with liquid diet feeding showed minimal body weight loss, increased food intake and enhanced health score.
The segment radiation method provides a useful model to study ISC/crypt loss and long-term crypt regeneration and epithelial repair, and may be valuable for future application to ISC transplantation or to genetic mutants that would not otherwise survive radiation doses that lead to complete crypt loss. Liquid diet is a simple intervention that improves survival and facilitates long-term studies of intestine in mice after high dose abdominal or segment radiation.
Increasing attention has been given to the impact of radiation on the gastrointestinal (GI) tract due to concerns about exposure to radiation after an accident or due to terrorist activity and a need for medical countermeasures
Recent identification of ISC biomarkers
In the present work, we developed and validated a method of radiation localized to an intestinal segment that induces complete crypt loss within the targeted segment but minimally impairs the health of wild type mice. We also compared chow feeding
All experiments were performed on adult male or female C57BL/6J mice (6–10 week old) which were obtained from The Jackson Laboratory (Bar Harbor, ME). All animal experiments were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University of North Carolina School of Medicine Institutional Animal Care and Use Committee (IACUC protocol number 10–197) and included criteria for euthanasia to minimize suffering.
A custom-designed lead shield with an opening allowing the isolation and subsequent radiation of a specific intestinal segment was purchased from Nuclead Co. Inc. and Sharp Manufacturing Inc. (West Bridgewater, MA) (
Composition | Daily intake |
||||
Normal Chow | Liquid Diet | Normal Chow | Liquid Diet | ||
Prolab Isopro RMH 3000 | Nutren 1.0 | Prolab Isopro RMH 3000 | Nutren 1.0 | ||
|
22.5 | 4 |
|
1.125 | 0.72 |
|
56 | 12.72 |
|
2.8 | 2.290 |
|
11.8 | 3.8 |
|
0.59 | 0.684 |
|
0.26 | 0.088 |
|
13 | 15.768 |
|
0.91 | 0.124 |
|
45.5 | 22.32 |
|
29 | 3.2 |
|
145 | 57.6 |
|
NA | 0.14 |
|
NA | 2.52 |
|
1 | 0.067 |
|
50 | 12.024 |
|
380 | 12 |
|
1.9 | 0.216 |
|
2.4 | 0.268 |
|
12 | 4.824 |
|
75 | 28 |
|
0.375 | 0.504 |
|
1.9 | 0.05 |
|
9.5 | 0.9 |
|
10 | 2 |
|
0.05 | 0.036 |
|
14 | 2.4 |
|
0.07 | 0.043 |
|
63 | 28 |
|
0.315 | 0.504 |
|
7.6 | 4 |
|
0.038 | 0.072 |
|
1.2 | 0.54 |
|
6 | 9.72 |
|
75 | 8 |
|
0.375 | 0.144 |
|
0.38 | 0.4 |
|
1.9 | 7.2 |
|
13 | 14 |
|
0.065 | 0.252 |
|
1.19 | 0.067 |
|
59.5 | 12.024 |
|
0.98 | 0.1 |
|
4.9 | 1.8 |
|
0.24 | 0.027 |
|
12 | 4.824 |
|
120 | 14 |
|
0.6 | 0.252 |
|
0.21 | 0.04 |
|
1.05 | 0.72 |
|
12 | 1.4 |
|
0.06 | 0.025 |
|
96 | 2.72 |
|
0.48 | 0.049 |
|
1.4 | 0.04 |
|
7 | 0.72 |
|
NA | 0.12 |
|
NA | 2.16 |
|
0.44 | 0.12 |
|
22 | 21.6 |
|
NA | 80 |
|
NA | 1.44 |
|
0.02 | 0.008 |
|
1 | 1.44 |
|
1600 | 452 |
|
8 | 8.136 |
|
0.26 | NA |
|
13 | NA |
|
16 | NA |
|
0.08 | NA |
|
0.27 | NA |
|
1.35 | NA |
|
2.6 | NA |
|
13 | NA |
Daily intake was estimated for 5 grams of normal chow and 18 ml of liquid diet eaten/drunk per day.
The segment radiation model was compared to abdominal radiation. The abdominal radiation model was also used to study the impact of liquid diet
To assess the health of the mice, four objective criteria were defined (
Criterion | Score | |
|
active | 1 |
stationary | 0 | |
|
normal | 1 |
hunched | 0 | |
|
not dehydrated | 1 |
dehydrated | 0 | |
|
smooth | 1 |
rough | 0 |
In order to evaluate the effects of radiation on health of mice, four objective criteria were used: activity (
Mice were euthanized with a lethal dose of Nembutal (150 µg/g body weight). The entire small intestine was collected on ice, flushed with ice cold 1X phosphate buffered saline (PBS, 0.137 M NaCl, 3 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). The small intestine was divided into 3 segments corresponding to duodenum, jejunum and ileum. Tissues were opened longitudinally and fixed in in fresh 4% paraformaldehyde (PFA) in 1X PBS overnight at 4°C. Tissues were then rinsed in 1X PBS and cryoprotected by sequential incubations in 10% sucrose and 30% sucrose overnight at 4°C. Tissues were embedded in Optimal Cutting Temperature medium (OCT) as Swiss Rolls
To assess whether liquid diet impacted survival of ISCs and formation of regenerating crypts, a subset of abdominally-radiated mice given normal chow or liquid diet were euthanized at 4 days after irradiation, a time known to be associated with formation of regenerating microcolonies following 14 Gy radiation
Data were expressed as the mean ± SEM. Mann-Whitney test, paired t-test, one way ANOVA or two-way ANOVA were performed to compare different groups as indicated in the results or figure legends. A p-value of less than 0.05 was considered statistically significant.
We first sought to assess whether the segment radiation method led to damage specifically in the radiated segment. Three independent mice were subjected to segment radiation and compared with sham controls. Intestinal tissues were collected at day 3 post-radiation, time associated with complete crypt ablation in jejunum after 14 Gy abdominal radiation
A. Illustrative photograph of H&E-stained Swiss-rolled jejunum from sham controls shows that no damage was induced by the segment exteriorization procedure (n = 3). Scale Bar: 500 µm. B. The schematic represents Swiss-rolled jejunum (
To validate that the segment radiation method allows long-term post-radiation studies, we then monitored mice for 21 days after 14 Gy abdominal or segment radiation. Mice were fed either with normal chow for all 21 days of the study or with elemental liquid diet from the day prior to radiation and the 7 consecutive days followed by return to normal chow for the rest of the study.
Survival was calculated as the percentage of mice that did not require euthanasia due a loss of body weight greater than 25% or an overall health score of 0. After segment radiation, 100% mice survived when they were placed on liquid diet, while none survived beyond day 7 when fed with normal chow (
Graphs show percent survival at different times after abdominal or segment radiation in mice given normal chow or liquid diet (from the day prior to radiation until day 7 post-radiation). 52.9% of the mice fed with normal chow and abdominally irradiated survived for 21 days post-radiation while none survived when fed with normal chow and locally radiated on a determined intestinal segment. Liquid diet treatment significantly improved survival after both local and abdominal radiation (n≥6: Abdominal Radiation-Normal Chow n = 17; Abdominal Radiation-Liquid Diet n = 14; Segment Radiation-Normal Chow n = 6; Segment Radiation-Liquid Diet n = 14; Mann-Whitney;
In all groups, radiation induced body weight loss which peaked at day 6 post-radiation. Between days 2 and 6 post-radiation, mice radiated on a targeted segment and fed with liquid diet exhibited minimal body weight loss when compared to abdominally-radiated mice fed with either normal chow or liquid diet (7.9% ±1.7 (segment radiation) vs. 24.0% ±1.3 (abdominal radiation- normal chow) and 22.7% ±2.0 (abdominal radiation- liquid diet) body weight loss at day 6 post-radiation) (
Graphs show percent body weight loss (mean ± SEM) at different times after irradiation in abdominally-radiated mice given normal chow or liquid diet and in segment-radiated mice given liquid diet. Mice locally radiated on an intestinal segment and fed with liquid diet exhibited minimal body weight loss and rapid body weight recovery after radiation compared with mice abdominally radiated. Liquid diet significantly improved body weight recovery of mice treated with abdominal radiation (n≥9: Abdominal Radiation-Normal Chow n = 12; Abdominal Radiation-Liquid Diet n = 9; Segment Radiation-Liquid Diet n = 9; Two Way ANOVA;
To assess the health of mice after radiation, we evaluated activity, posture, dehydration and pelage of the mice (
A. Graphs show health scores (mean ± SEM) of abdominally-radiated mice given normal chow or liquid diet and segment-radiated mice given liquid diet at different times after irradiation. Mice locally radiated on a targeted intestinal segment and fed with liquid diet exhibited significantly improved health score between day 4 and day 8 post-radiation as compared with abdominally radiated mice. Liquid diet tends to slightly improve health score between day 4 and day 8 post abdominal radiation (n≥8: Abdominal Radiation-Normal Chow n = 11; Abdominal Radiation-Liquid Diet n = 8; Segment Radiation-Liquid Diet n = 8; ANOVA;
Intestines of abdominally-radiated mice given normal chow or liquid diet (from the day prior to radiation until day 7 post-radiation) were studied at day 21 to assess mucosal healing and epithelial normalization. Mice given liquid diet showed normal crypt-villus architecture throughout duodenum, jejunum and ileum (
Normal Chow | Liquid Diet | |||||||
Normal Ranges | mean | +/− | SEM | mean | +/− | SEM | p-value | |
|
2.6∶ 10.1 | 0.63 | +/− | 0.17 | 0.95 | +/− | 0.43 | 0.51 |
|
1.3∶ 8.4 | 0.30 | +/− | 0.11 | 0.53 | +/− | 0.33 | 0.59 |
|
0.4∶ 2.0 | 0.30 | +/− | 0.09 | 0.50 | +/− | 0.10 | 0.20 |
|
0.0∶ 0.3 | 0.13 | +/− | 0.04 | 0.13 | +/− | 0.03 | 1.00 |
|
0.0∶ 99.9 | 38.67 | +/− | 11.23 | 37.47 | +/− | 9.56 | 0.93 |
|
0.0∶ 99.9 | 49.40 | +/− | 13.29 | 53.97 | +/− | 8.89 | 0.73 |
|
0.0∶ 99.9 | 11.93 | +/− | 3.13 | 8.57 | +/− | 2.28 | 0.32 |
|
32.8∶ 48.0 | 30.58 | +/− | 2.96 | 34.78 | +/− | 1.80 | 0.27 |
|
42.3∶ 55.9 | 40.55 | +/− | 0.32 | 40.28 | +/− | 0.18 | 0.48 |
|
6.5∶ 10.1 | 7.53 | +/− | 0.69 | 8.63 | +/− | 0.46 | 0.24 |
|
10.1∶ 16.1 | 12.23 | +/− | 1.00 | 13.60 | +/− | 0.61 | 0.29 |
|
13.7∶ 18.1 | 16.25 | +/− | 0.18 | 15.80 | +/− | 0.20 | 0.15 |
|
29.5∶ 35.1 | 40.18 | +/− | 0.61 | 39.20 | +/− | 0.35 | 0.22 |
|
0.0∶ 99.9 | 19.28 | +/− | 0.41 | 19.33 | +/− | 0.24 | 0.92 |
|
0.0∶ 99.9 | 6.10 | +/− | 0.07 | 5.98 | +/− | 0.02 | 0.15 |
|
780∶ 1540 | 645.75 | +/− | 109.03 | 636.50 | +/− | 28.90 | 0.94 |
Complete blood count performed at day 4 following abdominal radiation showed no difference between mice fed with normal chow or liquid diet (Abdominal Radiation-Normal Chow n = 4; Abdominal Radiation-Liquid Diet n = 4; t-test).
H&E staining of Swiss-rolled intestines at day 21 post-abdominal radiation showed enlarged crypts as well as blunted villi in mice fed with normal chow and overall more abnormal mucosal epithelium when compared with mice fed with liquid diet (n = 3). Scale Bar: 500 µm.
In segment-radiated mice, the radiated intestinal segment was still identifiable by H&E staining at day 21 post-radiation and did not exhibit complete normalization of architecture of the mucosa and bowel wall as compared with non-irradiated tissue adjacent to the radiated segment (
A–C. H&E staining (
Recent identification of ISC biomarkers offers new opportunities to investigate the roles of ISCs and functionally relevant pathways during crypt regeneration and mucosal repair after radiation. Conventional
There is very limited information about the impact of nutrition and hydration on survival or mucosal healing after irradiation. However, liquid diet composed of equal volumes of evaporated milk and water with additional glucose has been shown to slightly improve survival of mice after lethal irradiation of the head
A recent report issued following a workshop of the Centers for Medical Coutermeasures against Radiation (CMCRs) stressed the importance of the animal models used for the identification and the assessment of the effects of pharmacological agents on radiation-induced intestinal damage and subsequent repair
The present study is the first comprehensive documentation of the segment radiation technique combined with liquid diet in a mouse model that allows long-term studies of intestinal responses to high dose radiation. It is also the first demonstration of the value of a liquid diet treatment in both the abdominal and segment radiation procedures. Under the conditions we described, the segment radiation/liquid diet protocol allows analyses of ISC-mediated crypt regeneration in a context of healthy surrounding organs, including a grossly healthy, adjacent GI tract. More importantly, the segment radiation method allows analyses of non-irradiated and irradiated intestine of the same mouse. This provides a useful model for direct comparisons of crypt homeostasis and ISCs in uninjured
Intestinal fibrosis is a long-term potential complication of TBI or abdominal radiation exposure that can lead to bowel dysfunction and potentially bowel obstruction or intestinal failure. Analyses of intestinal fibrosis after high dose TBI or abdominal radiation in rodent models can be problematic due to the high morbidity in the early period after high dose radiation or due to obstruction or motility disorders in fibrotic intestine. Interestingly, in the segment radiation model, at 21 days post-radiation, we observed increased collagen deposition within the submucosa and serosa of the irradiated intestinal segment concomitant with a thickening of the muscularis, both features of fibrosis. This localized fibrosis within the specific radiated intestinal segment could prove very valuable for studying the mechanisms of radiation-induced fibrosis or testing anti-fibrotic therapies. It is perhaps surprising that the abdominally-radiated mice did not exhibit fibrosis at 21 days post-radiation. However this may reflect the time point selected and more studies and time points are needed to compare fibrosis in the abdominal and segment radiation models.
In summary, this work demonstrates that liquid diet feeding represents a simple and low cost intervention which promotes survival and allows long-term studies of intestinal epithelial repair in murine models after high dose abdominal radiation. Importantly the high dose segment radiation model induces complete localized ISC/crypt loss while minimally affecting the health of the mice, and may be extremely useful for the study of genetically engineered mice with defects in mucosal repair or physiological impairments that may otherwise preclude survival after high doses TBI or abdominal irradiation, as well as for testing therapeutic strategies to activate ISCs and promote crypt regeneration. This radiation model could also be of value for future application to ISC transplantation.
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The authors wish to thank: Douglas von Allmen and Richard von Furstenberg, for useful input on design of the custom made shield, and Kirk McNaughton and J. Ashley Ezzell for expert input on histology. Useful discussions with Scott Magness, Shengli Ding and Agostina Santoro are gratefully acknowledged.