Introduction 31 47 7 12 13 24 29 38 41 45 46 28 12 32 16 18 21 28 35 15 23 5 33 2 37 8 11 14 27 8 10 8 in vitro 43 17 8 10 The objective of this study was to develop an experimental mucositis mouse model to characterize DOX-induced intestinal damage and subsequent repair. In addition, we aimed to correlate the alterations in morphology, epithelial homeostasis, and gene expression with changes in BMP4 and TCF4 expression. This, in order to gain insight into possible modulation of the epithelial-mesenchymal cross-talk and progenitor compartment during chemotherapy-induced intestinal damage and regeneration. Materials and methods Animals Animal experiments were performed with permission of the Animal Ethics Committee of the Erasmus MC-Sophia. Upon arrival at our institute, 10-week-old male BALB/c mice (Harlan, Horst, The Netherlands) were housed individually during the whole experiment in micro-isolator cages under specific pathogen-free conditions with free access to a standard palletized diet (Hope farms, Woerden, The Netherlands) and water. After 1 week of adjustment to the new environment, the mice were divided into three groups and injected intravenously with doxorubicin (DOX) (Doxorubicin, Pharma Chemie, Haarlem, The Netherlands) on two subsequent days. At day −1 and 0, the first group of mice was injected with a low dose of DOX of 6 and 4 mg/kg (low dose) respectively, a second group was injected with a medium dose of 8 and 5 mg/kg (medium dose) and a third group was injected with a high dose of 10 and 6 mg/kg (high dose). Controls were given equivalent volumes of 0.9% NaCl. Mice in the low- and high-dose group were sacrificed at day 1, 2, 3 and 7 after the final DOX injection; mice in the medium dose group were only sacrificed at day 3 and 7. One hour before sacrifice, the mice were injected with 120 μl 10 mg/ml 5- Bromo-2’deoxyUridine (BrdU) (Sigma-Aldrich, Zwijndrecht, The Netherlands), an uridine analog, to locate the proliferating cells. Per time point 4–6 DOX-treated animals and 2–4 control animals were sacrificed. Segments of mid-jejunum were collected and either processed immediately for histological analyses or snap-frozen in liquid nitrogen for storage at −80°C and subsequent protein isolation. Histochemistry 45 45 34 49 Crypt and -villus length Longitudinal sections of crypts and their corresponding villi were selected so that the base (marked by Paneth cells), middle and top of the crypt were all in the plane of section and thus well orientated. The depth of ten crypts and the length of ten villi were measured on three slides per animal, four animals per time point, with the use of a Nikon Eclipse E800 microscope and IM 500 software. Protein dot blotting 46 2 49 125 125 46 Western-blot analysis The same protein homogenate was used as described for protein dot blot analysis. Twenty μg of protein was loaded per lane and run on a 12.5% SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes (Protran BA83, 0.2 μm) and blocked for 1 h at room temperature in blocking buffer as described above. The blots were incubated overnight at 4°C with primary antibodies diluted in blocking buffer: mouse monoclonal anti-human PCNA, clone PC10 (1:250, Novo Castra Laboratories, Newcastle upon Tyne, UK) and mouse monoclonal anti-human BMP4, clone 3H2 (1:100), (Novocastra Laboratories, Newcastle upon Tyne, UK), rabbit polyclonal anti-human cleaved Caspase-3 antibody (1:1000, Cell Signaling, Beverly, MA), and mouse monoclonal anti-human TCF-4, clone 6H5-3 (1:250, Upstate, Waltham, MA). After washing with PBS, 0.2% Tween-20 blots bound antibodies were revealed using HRP conjugated goat anti-mouse or rabbit anti-goat secondary antibodies (1:1000) and SuperSignal West Femto Luminol Enhancer kit (Pierce, Rockford, IL). The signal was detected and quantified by the ChemiGenius gel documentation system (Syngene, Cambridge, UK) and the expression of the specific proteins analyzed was expressed per 20 μg protein of tissue. Average expression levels of PCNA, BMP4, TCF4, and caspase-3 in the mid-jejunum were calculated per mouse, followed by calculation of the mean expression of these specific proteins per time point studied. Subsequently, the average expression of PCNA, BMP4, TCF4, and caspase-3 in control mice was set at 100%. Statistical analysis p Results Dose-response analysis of DOX-induced mucositis in an experimental mouse model To optimize the dose of DOX necessary to induce severe intestinal damage (i.e., villus atrophy, crypt loss and flattening of the epithelial cells) a dose-response curve was performed. Thereto, mice were divided in three treatment groups: low dose, medium dose, and high dose (see Materials and methods, section Animals for details). In the high-dose treatment group, two of the six mice died at day 4. Necropsy showed an excess of fluid in the abdominal cavity of unknown source. Because of the elapsed time after death, morphological evaluation could not be performed. 1 1 1 1 1 1 Quantitative analysis of crypt and villus length 1 1 1 Effects of high-dose DOX treatment on enterocyte-specific gene expression 42 2 2 2 2 Effects of DOX treatment on goblet cell-specific gene expression 6 22 3 3 3 3 3 3 Effect of high-dose DOX treatment on epithelial proliferation 4 4 Fig. 1 A B C D E E A B * P  Fig. 2 A B C D E F * P bars  Fig. 3 A E A B C D E B C D E F * P bars  Fig. 4 A B C D E F G * P bars  4 40 4 4 4 Effect of high-dose DOX treatment on enterocyte apoptosis 4 5 26 5 Fig. 5 4 A B * P bars  Effect of high-dose DOX treatment on TCF-4 protein expression 1 19 17 4 6 Fig. 6 4 * P bars  Effect of high-dose DOX treatment on expression of Bone morphogene 4 (BMP4) 8 10 8 27 4 7 P 7 Fig. 7 4 A P C B * P ** P bars  Discussion This study revealed that DOX, in a dose of 10 and 6 mg/kg induced severe morphological damage to the small intestine of mice within 3 days, which was almost completely regenerated by day 7. Moreover, it revealed that the intestine was virtually not or much less affected by lower doses of DOX. Mucositis induced by the chosen dose of DOX was characterized by an increasing degree of intestinal morphological damage at day 1 and 2, which correlated with a significant increase in both apoptosis and proliferation. During this phase of epithelial hyper-proliferation, the epithelial cells lost their highly differentiated status as measured by a significant down-regulation of epithelial-specific SI at days 2–3. The decreased expression of TFF3 at days 1–2 could be caused by a decrease in goblet cell differentiation, but on the other hand, could also be the result of increased TFF3 secretion. At day 3, the time-point when intestinal damage was most severe, the morphology was characterized by severe villus atrophy, a significant rise in crypt length, epithelial flattening, crypt loss, inhibition of proliferation and impaired epithelial differentiation. During morphological regeneration, at day 7, proliferation started to return to control level, and SI and TFF3 expression levels were normalized again. et al. 12 9 11 34 38 44 46 48 12 45 46 48 6 22 30 42 45 45 48 34 45 45 46 20 35 36 8 10 8 10 8 8 10 3 10 39 25 39 In conclusion, high-dose DOX induces severe damage to the epithelium, which closely resembles damage induced by MTX, indicating that general mechanisms of damage and repair are involved. We show that signaling pathways involving BMP4 and TCF4 and thus epithelial-mesenchymal cross-talk are modulated by DOX-induced damage in such a way that homeostasis of the progenitor compartment is restored by initially inducing cell proliferation and inhibiting differentiation and subsequently inducing differentiation, inhibiting proliferation and promoting crypt fission. Understanding these mechanisms is essential to develop clinical strategies to prevent chemotherapy-induced mucositis.