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Background & Aims: Intestinal crypts have a remarkable capacity to regenerate after injury from loss of crypt base columnar (CBC) stem cells. After injury, facultative stem cells (FSCs) are activated to replenish the epithelium and replace lost CBCs. Our aim was to assess the role of insulin-like growth factor-1 (IGF-1) to activate FSCs for crypt repair. Methods: The intestinal regenerative response was measured after whole body 12-Gy γ-irradiation of adult mice. IGF-1 signaling or its downstream effector mammalian target of rapamycin complex 1 (mTORC1) was inhibited by administering BMS-754807 or rapamycin, respectively. Mice with inducible Rptor gene deletion were studied to test the role of mTORC1 signaling in the intestinal epithelium. FSC activation post-irradiation was measured by lineage tracing. Results: We observed a coordinate increase in growth factor expression, including IGF-1, at 2 days post-irradiation, followed by a surge in mTORC1 activity during the regenerative phase of crypt repair at day 4. IGF-1 was localized to pericryptal mesenchymal cells, and IGF-1 receptor was broadly expressed in crypt progenitor cells. Inhibition of IGF-1 signaling via BMS-754807 treatment impaired crypt regeneration after 12-Gy irradiation, with no effect on homeostasis. Similarly, rapamycin inhibition of mTORC1 during the growth factor surge blunted the regenerative response. Analysis of Villin-CreERT2;Rptorfl/fl mice showed that epithelial mTORC1 signaling was essential for crypt regeneration. Lineage tracing from Bmi1-marked cells showed that rapamycin blocked FSC activation post-irradiation. Conclusions: Our study shows that IGF-1 signaling through mTORC1 drives crypt regeneration. We propose that IGF-1 release from pericryptal cells stimulates mTORC1 in FSCs to regenerate lost CBCs.

When radiotherapy is applied to the abdomen or pelvis, normal tissue toxicity in the gastrointestinal (GI) tract is considered a major dose-limiting factor. Proton beam therapy has a specific advantage in terms of reduced doses to normal tissues. This study investigated the fundamental differences between proton- and X-ray-induced intestinal injuries in mouse models. C57BL/6J mice were irradiated with 6-MV X-rays or 230-MeV protons and were sacrificed after 84 h. The number of surviving crypts per circumference of the jejunum was identified using Hematoxylin and Eosin staining. Diverse intestinal stem cell (ISC) populations and apoptotic cells were analyzed using immunohistochemistry (IHC) and a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay, respectively. The crypt microcolony assay revealed a radiation-dose-dependent decrease in the number of regenerative crypts in the mouse jejunum; proton irradiation was more effective than X-ray irradiation with a relative biological effectiveness of 1.14. The jejunum is the most sensitive to radiations, followed by the ileum and the colon. Both types of radiation therapy decreased the number of radiosensitive, active cycling ISC populations. However, a higher number of radioresistant, reserve ISC populations and Paneth cells were eradicated by proton irradiation than X-ray irradiation, as shown in the IHC analyses. The TUNEL assay revealed that proton irradiation was more effective in enhancing apoptotic cell death than X-ray irradiation. This study conducted a detailed analysis on the effects of proton irradiation versus X-ray irradiation on intestinal crypt regeneration in mouse models. Our findings revealed that proton irradiation has a direct effect on ISC populations, which may result in an increase in the risk of GI toxicity during proton beam therapy.

ABSTRACT Protection of hematopoietic, immunological, and gastrointestinal injuries from deleterious effects of ionizing radiation is prime rational for developing radioprotector. The objective of this study, therefore, was to evaluate the radioprotective potential of melatonin against damaging effects of radiation-induced hematopoietic, immunological, and gastrointestinal injuries in mice. C57BL/6 male mice were intraperitoneally administered with melatonin (50-150 mg/kg) 30 min prior to whole-body radiation exposure of 5 and 7.5 Gy using 60Co-teletherapy unit. Thirty-day survival against 7.5 Gy was monitored. Melatonin (100 mg/kg) pretreatment showed 100% survival against 7.5 Gy radiation dose. Melatonin pretreatment expanded femoral HPSCs, and inhibited spleenocyte DNA strands breaks and apoptosis in irradiated mice. At this time, it also protected radiation-induced loss of T cell sub-populations in spleen. In addition, melatonin pretreatment enhanced crypts regeneration and increased villi number and length in irradiated mice. Translocation of gut bacteria to spleen, liver and kidney were controlled in irradiated mice pretreated with melatonin. Radiation-induced gastrointestinal DNA strand breaks, lipid peroxidation, and expression of proapoptotic-p53, Bax, and antiapoptotic-Bcl-xL proteins were reversed in melatonin pretreated mice. This increase of Bcl-xL was associated with the decrease of Bax/Bcl-xL ratio. ABTS and DPPH radical assays revealed that melatonin treatment alleviated total antioxidant capacity in hematopoietic and gastrointestinal tissues. Present study demonstrated that melatonin pretreatment was able to prevent hematopoietic, immunological, and gastrointestinal radiation-induced injury, therefore, overcoming lethality in mice. These results suggest potential of melatonin in developing radioprotector for protection of bone marrow, spleen, and gastrointestine in planned radiation exposure scenarios including radiotherapy. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 501-518, 2017. [ABSTRACT FROM AUTHOR]

Background Severity of villous atrophy in celiac disease (CeD) is the cumulative effect of enterocyte loss and cell regeneration. Gluten-free diet has been shown to benefit even in patients having a positive anti-tissue transglutaminase (tTG) antibody titre and mild enteropathy. Aim We explored the balance between mucosal apoptotic enterocyte loss and cell regeneration in mild and advanced enteropathies. Methods Duodenal biopsies from patients with mild enteropathy (Marsh grade 0 and 1) ( n = 26), advanced enteropathy (Marsh grade ≥2) ( n = 41) and control biopsies ( n = 12) were subjected to immunohistochemical staining for end-apoptotic markers (M30, H2AX); markers of cell death (perforin, annexin V); and cell proliferation (Ki67). Composite H -scores based on the intensity and distribution of markers were compared. Results End-apoptotic markers and marker of cell death (perforin) were significantly up-regulated in both mild and advanced enteropathies, in comparison to controls; without any difference between mild and advanced enteropathies. Ki67 labelling index was significantly higher in crypts of mild enteropathy, in comparison to controls, suggesting maintained regenerative activity in the former. Conclusions Even in patients with mild enteropathy, the rate of apoptosis is similar to those with advanced enteropathy. These findings suggest the necessity of reviewing the existing practice of not treating patients with mild enteropathy. [ABSTRACT FROM AUTHOR]

Background & Aims: Intestinal crypts have a remarkable capacity to regenerate after injury from loss of crypt base columnar (CBC) stem cells. After injury, facultative stem cells (FSCs) are activated to replenish the epithelium and replace lost CBCs. Our aim was to assess the role of insulin-like growth factor-1 (IGF-1) to activate FSCs for crypt repair., Methods: The intestinal regenerative response was measured after whole body 12-Gy γ-irradiation of adult mice. IGF-1 signaling or its downstream effector mammalian target of rapamycin complex 1 (mTORC1) was inhibited by administering BMS-754807 or rapamycin, respectively. Mice with inducible Rptor gene deletion were studied to test the role of mTORC1 signaling in the intestinal epithelium. FSC activation post-irradiation was measured by lineage tracing., Results: We observed a coordinate increase in growth factor expression, including IGF-1, at 2 days post-irradiation, followed by a surge in mTORC1 activity during the regenerative phase of crypt repair at day 4. IGF-1 was localized to pericryptal mesenchymal cells, and IGF-1 receptor was broadly expressed in crypt progenitor cells. Inhibition of IGF-1 signaling via BMS-754807 treatment impaired crypt regeneration after 12-Gy irradiation, with no effect on homeostasis. Similarly, rapamycin inhibition of mTORC1 during the growth factor surge blunted the regenerative response. Analysis of Villin-CreER T2 ;Rptor fl/fl mice showed that epithelial mTORC1 signaling was essential for crypt regeneration. Lineage tracing from Bmi1-marked cells showed that rapamycin blocked FSC activation post-irradiation., Conclusions: Our study shows that IGF-1 signaling through mTORC1 drives crypt regeneration. We propose that IGF-1 release from pericryptal cells stimulates mTORC1 in FSCs to regenerate lost CBCs., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

When radiotherapy is applied to the abdomen or pelvis, normal tissue toxicity in the gastrointestinal (GI) tract is considered a major dose-limiting factor. Proton beam therapy has a specific advantage in terms of reduced doses to normal tissues. This study investigated the fundamental differences between proton- and X-ray-induced intestinal injuries in mouse models. C57BL/6J mice were irradiated with 6-MV X-rays or 230-MeV protons and were sacrificed after 84 h. The number of surviving crypts per circumference of the jejunum was identified using Hematoxylin and Eosin staining. Diverse intestinal stem cell (ISC) populations and apoptotic cells were analyzed using immunohistochemistry (IHC) and a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay, respectively. The crypt microcolony assay revealed a radiation-dose-dependent decrease in the number of regenerative crypts in the mouse jejunum; proton irradiation was more effective than X-ray irradiation with a relative biological effectiveness of 1.14. The jejunum is the most sensitive to radiations, followed by the ileum and the colon. Both types of radiation therapy decreased the number of radiosensitive, active cycling ISC populations. However, a higher number of radioresistant, reserve ISC populations and Paneth cells were eradicated by proton irradiation than X-ray irradiation, as shown in the IHC analyses. The TUNEL assay revealed that proton irradiation was more effective in enhancing apoptotic cell death than X-ray irradiation. This study conducted a detailed analysis on the effects of proton irradiation versus X-ray irradiation on intestinal crypt regeneration in mouse models. Our findings revealed that proton irradiation has a direct effect on ISC populations, which may result in an increase in the risk of GI toxicity during proton beam therapy. [ABSTRACT FROM AUTHOR]

Introduction Dose-volume effects imply that the tolerance of healthy organs increases when the irradiated volume decreases. These effects are important for uniform and non-uniform dose delivery in acute reacting organs like the intestinal tract, considering optimization constraints in human radiotherapy treatment planning. It is expected that other mammals, such as rodents, could exhibit similar response to radiation as humans and aid in understanding such effects. We investigate this effect in rodent jejunum. Materials and methods Mice (NIH strain & female) were sedated and had a section of jejunum extracted and irradiated. A Perspex jig allowed immobilization of the mice during irradiation. Mice were divided into groups where some received whole-body exposures, while others received a circular 5 mm or 1 × 1 cm 2 field of 6 MV X-rays to the jejunum. This was done at dose levels of 8–20 Gy. Eighty-four hours later the mice were euthanized and the irradiated sections of jejunum were removed. Cross-sectional cuts of the sections were made and set onto glass slides. The number of regenerated crypts per circumference were counted and compared. Results Crypt regeneration decreased with increasing dose levels. Significant differences in the number of regenerated crypts were found in the 5 mm field compared to the whole-body and 1 × 1 cm 2 field. The recovered dose (Dose1-Dose2) expressed at the level of 50 regenerated crypts, was 3.55 Gy. These results demonstrated the presence of a critical local migration distance for cells in the regeneration process. Conclusion The 5 mm field irradiations appeared to have had a greater regeneration capacity compared to the larger fields. This suggests that a non-local repair mechanism, acting from the surrounding non-irradiated tissue, may aid in the repair of small irradiated volumes. However the 1 × 1 cm 2 field results suggests that repair capacity is greatly reduced beyond a certain critical distance. [ABSTRACT FROM AUTHOR]

With the tamoxifen-inducible CreER T2 system, genetic recombination can be temporally controlled in a cell-type-specific manner in intact animals, permitting dissection of the molecular underpinnings of mammalian physiology. Here we present a significant drawback to CreER T2 technology for analysis of intestinal stem cells. Using the intestine-specific Villin-CreER T2 mouse strain, we observed delayed intestinal regeneration post irradiation. Villin-CreER T2 activation was associated with DNA damage and cryptic loxP site cleavage. Analysis of stem cell-specific CreER T2 strains showed that the genome toxicity impairs function of crypt base columnar stem cells, resulting in loss of organoid initiating activity. Importantly, the stem cell impairment is short-lived, with return to normal by 7 days post tamoxifen treatment. Our findings demonstrate that mouse genetic experiments that utilize CreER T2 should consider the confounding effects of enhanced stem cell sensitivity to genome toxicity resulting from CreER T2 activation., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Protection of hematopoietic, immunological, and gastrointestinal injuries from deleterious effects of ionizing radiation is prime rational for developing radioprotector. The objective of this study, therefore, was to evaluate the radioprotective potential of melatonin against damaging effects of radiation-induced hematopoietic, immunological, and gastrointestinal injuries in mice. C57BL/6 male mice were intraperitoneally administered with melatonin (50-150 mg/kg) 30 min prior to whole-body radiation exposure of 5 and 7.5 Gy using 60 Co-teletherapy unit. Thirty-day survival against 7.5 Gy was monitored. Melatonin (100 mg/kg) pretreatment showed 100% survival against 7.5 Gy radiation dose. Melatonin pretreatment expanded femoral HPSCs, and inhibited spleenocyte DNA strands breaks and apoptosis in irradiated mice. At this time, it also protected radiation-induced loss of T cell sub-populations in spleen. In addition, melatonin pretreatment enhanced crypts regeneration and increased villi number and length in irradiated mice. Translocation of gut bacteria to spleen, liver and kidney were controlled in irradiated mice pretreated with melatonin. Radiation-induced gastrointestinal DNA strand breaks, lipid peroxidation, and expression of proapoptotic-p53, Bax, and antiapoptotic-Bcl-xL proteins were reversed in melatonin pretreated mice. This increase of Bcl-xL was associated with the decrease of Bax/Bcl-xL ratio. ABTS and DPPH radical assays revealed that melatonin treatment alleviated total antioxidant capacity in hematopoietic and gastrointestinal tissues. Present study demonstrated that melatonin pretreatment was able to prevent hematopoietic, immunological, and gastrointestinal radiation-induced injury, therefore, overcoming lethality in mice. These results suggest potential of melatonin in developing radioprotector for protection of bone marrow, spleen, and gastrointestine in planned radiation exposure scenarios including radiotherapy. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 501-518, 2017., (© 2016 Wiley Periodicals, Inc.)