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 in vivo radiation models induce a high mortality early during the days following high dose radiation and therefore do not allow the study of intermediate and late phases of mucosal repair. The results presented in this study demonstrate that elemental liquid diet feeding markedly improved survival after high dose abdominal radiation. Importantly, we report a novel model of radiation of a specific small intestinal segment that induces localized complete crypt ablation with minimal adverse effects on the health of mice, thus permitting long-term studies of ISCs, crypt regeneration and mucosal healing after radiation-induced crypt ablation in the small intestinal epithelium.
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 
. Also germ-free mice, but not conventional mice, showed significantly decreased mortality and improved body weight loss after TBI when supplemented from 5-weeks after birth with a liquid diet containing all known essential nutrients required by mice, in addition to normal chow, and maintained on this supplemental diet until the end of the experiment 
. This is consistent with our findings that liquid diet feeding significantly promoted survival and accelerated normalization of body weight after abdominal radiation, although in our study mice were fed solely with liquid diet from the day prior to radiation until 7 days post-radiation, followed by a return to normal chow until the end of the study at 21 days post-abdominal radiation. Interestingly the microcolony assay results indicate that liquid diet did not impact crypt survival and regenerating microcolony formation during the earliest phases of crypt regeneration after abdominal radiation. Thus the beneficial effects of liquid diet appear not to originate from an impact on early regenerating ISC/crypts. However, our results in the abdominal radiation model demonstrate that liquid diet feeding promoted histological normalization of the intestinal epithelial architecture at day 21 after abdominal radiation when compared with littermates fed normal chow. Further studies will be required to define the mechanisms underlying the beneficial effect of liquid diet on epithelial normalization. Since liquid diet had no detectable impact on early phases of crypt regeneration and since mice were back on normal chow at day 7 after abdominal radiation, it seems possible that these mechanisms may involve protective effects of liquid diet on ISC/intestinal epithelial cells at times later than 4 days post-radiation assessed here, or effects on cell types other than ISCs or intestinal epithelial cells, such as resident immune cells or other cell types present in the intestinal epithelial cell microenvironment. We did not investigate specific effects of particular components of the liquid diet since it was shown in Walburg et al. 1966 
that none of the major groups of essential nutrients contained in a liquid diet was capable alone of modifying the mortality pattern after TBI radiation. One difference in the nutrient composition between liquid diet and normal chow used in the present study is that liquid diet contains vitamin C, while vitamin C is undetectable in chow diet. A recent study reported that a 3 day or 12 hour pre-treatment with vitamin C markedly improved survival of mice after 14 Gy TBI 
. Estimated daily intake of vitamin C in the liquid diet fed animals in the present study approximates the vitamin C supplementation in the study of Yamamoto et al. 2010 
. However in that study, vitamin C pre-treatment only had a beneficial effect if mice were subjected to BM transplantation 
. Therefore, while we cannot exclude a role of vitamin C in the beneficial effects of liquid diet feeding observed in our study, it seems unlikely that these beneficial effects are solely due to vitamin C since mice in the current study did not require BM transplantation.
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 
. In murine models, survival time after radiation has been shown to be strain-dependent with influence of both the genetic background and the resident microbiota 
. Also comparative studies become even more complicated when the field of radiation is considered, i.e.
abdominal radiation. Indeed traditional views in the field consider that morbidity in abdominally-radiated animals is typically due to GI failure 
. However, more recent findings suggest that abdominal radiation can also result in BM failure-induced death 
. In TBI murine models, timing of morbidity is usually used as sole indicator distinguishing GI failure from BM failure 
. Therefore, a recent report recommends that the dose-dependent mode of animal death after TBI or abdominal radiation should be constructed for each strain and for each experimental system based on autopsy examination 
. The segment radiation model reported in the present study has the advantage that it minimally affects the health of the mice and permits specific evaluation of the impact of high dose radiation on the intestine. Other groups have used segment radiation in mice 
. However, to our knowledge, these studies did not combine the segment radiation procedure with a liquid diet treatment. As a consequence, these models only allowed short-term studies 
or presented a high rate of mortality 
. This is consistent with our findings that no mice survived the segment radiation method when fed normal chow. Experience with surgical models such as proximal bowel resection or ileo-cecal resection has been that post-surgical ileus leads to impaction of solid chow. In these models, liquid diet feeding before and after surgery dramatically improved survival 
. Since radiation is known to alter gut motility 
, and the segment radiation method involves bowel handling and radiation, we believe that ileus was the likely cause of death/need for euthanasia in segment-radiated mice fed with normal chow. We did not study tissue histology in these animals due to the limited number studied under this protocol. We cannot exclude that fibrosis contributed to high mortality since adhesions were noted in segment-radiated mice fed with normal chow at autopsy.
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 vs. radiated and injured intestine of the same animal, with both regions of the intestine subjected to the same systemic environment. Also, the segment radiation model should prove valuable for assessing impact of nutritional or pharmacologic interventions on both the intestinal segment injured by radiation and the normal intestine distant from the site of radiation exposure. This has significant advantages for defining interventions that may specifically promote healing of radiated intestine. Furthermore the segment radiation model should prove useful to study impact of genetic modifications on responses of the intestine to radiation in mutant mice where phenotypic effects of the mutation on body size and growth, immune system or other aspects of physiology might preclude their ability to survive high dose TBI or abdominal radiation.
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.