This work was supported by an unrestricted grant from the Novo Nordisk Center for Basic Metabolic Research and the Lundbeck Foundation.

All methods described were conducted in accordance with the guidelines of Danish legislation governing animal experimentation (1987). Studies were performed with the permission from the Danish Animal Experiments Inspectorate (2013-15-2934-00833) and the local ethical committee.
NOTE: Female C57BL/6J mice (~20&#8722;25 g) were obtained and housed eight per cage in standard 12 h light, 12 h dark cycle with free access to water and standard chow. Animals were left to acclimatize for one week before experiments began.
1. Induction of mucositis using 5-fluorouracil
<ol>
	<li>Obtain 5-fluorouracil (5-FU) in a 50 mg/mL solution.</li>
	<li>Weigh and record the body weight of the mice. From the body weight, calculate the amount of 5-FU for injection (e.g., 400 mg/kg).</li>
	<li>Prepare a 1 mL syringe connected to a 27 G x 30 mm needle and fill the syringe with the calculated amount of 5-FU for injection.</li>
	<li>Restrain the mouse by the scruff method. To do this, grab the base of the tail with one hand and place it on a toe-gripping surface, such as a wire bar lid. While positioning the tail with one hand, hold the scruff of the neck with the other hand.</li>
	<li>Firmly position the body of the mouse across one hand by extending the forefinger and thumb back as far as possible. Place the tail between the fingers of this same hand to secure the mouse.</li>
	<li>While maintaining the mouse in a firm but gentle grip, expose the ventral side of the mouse and insert the needle into the intraperitoneal cavity on the lower right or left quadrant of the abdomen. Aspirate to ensure proper placement and inject the 400 mg/kg 5-FU.</li>
</ol>
2. Tissue collection
<ol>
	<li>Anesthetize the mouse with an intraperitoneal injection of ketamine/xylazine (100/10 mg/kg) diluted in saline solution (0.9% NaCl). Monitor the depth of anaesthesia by observing the pinch withdrawal reflex. 
	NOTE: The anaesthesia is a non-recovery anaesthesia.</li>
	<li>Record the weight of the anesthetized mouse.</li>
	<li>Place the mouse in a supine position and perform a laparotomy to expose the abdominal cavity, followed by an incision of the chest cavity to introduce pneumothorax.</li>
	<li>Using scissors, sacrifice the animal by cutting open the diaphragm.</li>
	<li>Remove the small intestine. Cut superior to the pylorus, carefully retract the small intestine away from the carcass until the cecum is reached and make a cut just before the cecum.</li>
	<li>Using forceps, gently clamp the proximal lumen shut. Using a 1 mL syringe with a 25 G needle attached, flush the small intestine with saline to remove the feces.</li>
	<li>Gently remove the saline from the intestinal lumen with surgical instruments.</li>
	<li>Place the small intestine on clean blotting paper to carefully remove excess saline.</li>
	<li>Record the weight of the flushed tissue.</li>
	<li>Calculate the flushed small intestinal weight as a percentage of body weight.</li>
</ol>
3. Small intestine histology
<ol>
	<li>Tissue preparation
	<ol>
		<li>In a fume hood, use scissors to cut 3 cm sections of the flushed intestinal tissue from each mouse from the duodenum, jejunum and ileum and fixate sections in 10% formalin for 24 h at room temperature. 
		NOTE: The fixative volume should be 5&#8722;10 times of the tissue volume.</li>
		<li>Transfer the fixed tissue to a cutting board.</li>
		<li>Using a scalpel, trim the tissue to approximately 1 cm and transfer into an embedding cassette. 
		NOTE: The cassette should be labelled with the sample identification (ID) in pencil.</li>
		<li>Submerge the cassette in 70% ethanol and store at 4 &#176;C until further processing.</li>
	</ol>
	</li>
	<li>Tissue embedding in paraffin
	<ol>
		<li>Dehydrate the tissue by placing in ascending alcohol solutions. Place the cassette in 70% ethanol for 1 h, followed by 80% ethanol for 1 h, 95% ethanol for 1 h and 100% ethanol for 1.5 h. Transfer the cassette from 100% ethanol to xylene for 1.5 h.</li>
		<li>Immerse the cassette with paraffin wax heated between 58&#8722;60 &#176;C. Use forceps to position the tissue in a transverse orientation and leave the blocks to cool more than 24 h until solid.</li>
	</ol>
	</li>
	<li>Cutting tissue using a microtome
	<ol>
		<li>Place block, tissue face down on ice for 5 min. Using a microtome, trim the paraffin blocks at a thickness of 10&#8722;30 &#181;m to expose the tissue surface.</li>
		<li>Cut cross-sections of the tissue block between 3&#8722;5 &#181;m, making transverse sections from the duodenum, the jejunum and the ileum.</li>
		<li>Place paraffin ribbon in a water bath set between 40&#8722;45 &#176;C. Use forceps to separate the sections.</li>
		<li>Use microscope slides to pick up the sections from the water.</li>
		<li>Air dry the slides for 30 min before staining. 
		NOTE: For storage over a longer period of time, store slides in a fridge.</li>
	</ol>
	</li>
	<li>Hematoxylin-eosin staining of tissue
	<ol>
		<li>Place the microscope slides in a histology cradle. Deparaffinize the slides in a heating cabinet set at 60 &#176;C for 60 min. 
		NOTE: Slides directly from the fridge should be left to reach room temperature before placing them in a heating cabinet.</li>
		<li>In the fume hood, rehydrate the tissue in 3 changes of a clearing agent (Table of Materials), make 20 dips in the first clearing agent jar and then let them stand for 7&#8722;8 min in each of the two additional clearing agent jars. Transfer the slides to descending alcohol solutions, start with 3 changes of 99% ethanol, followed by 2 changes of 96% ethanol and 70% ethanol. Make a minimum of 20 dips in each jar. Transfer to running water and let the slides stand for 5 min.</li>
		<li>Immerse the slides in filtered Meyer&#8217;s hematoxylin for 1 min.</li>
		<li>Wash the samples in running tap water for 5 min.</li>
		<li>Immerse sections in the eosin stain for 1&#8722;2 min, then rinse in tap water.</li>
		<li>Dehydrate the tissue by placing in ascending alcohol solutions. Start with 70% ethanol, followed by 96% ethanol, 96% ethanol, and 4 times 99% ethanol. Make a minimum of 20 dips in each jar, and let the cradle stand in 99% ethanol until they are mounted.</li>
		<li>Mount coverslips by applying a small amount of the mounting medium to the surface of the slides. Put the coverslip on top of the mounting medium without creating bubbles under the coverslip. Let it dry for 24 h.</li>
		<li>Examine the tissue using a light microscope connected to a camera to obtain histological photos. Use a 10x objective to take snapshots until a full coverage of the tissue slide is reached.</li>
	</ol>
	</li>
</ol>
4. Measurement of crypt depth and/or villus height
<ol>
	<li>Download and install the analysis software (i.e., Zen Lite, Table of Materials).</li>
	<li>Open the image in the software and connect to the camera. Take snapshots in camera mode with 20x objective.</li>
	<li>In processing mode, open the snapshot.</li>
	<li>To measure the crypt depth: in the 2D view, select the line tool from the graphics tab. Choose a well-orientated crypt and start the line at the bottom of a villus and finish at the bottom of the crypt. Repeat this action for 20 well-orientated crypts (Supplementary Figure 1).</li>
	<li>To measure the villus height: in the 2D view, select the line tool from the graphics tab. Choose a well-orientated villus and start the line at the end of the luminal projection and finish at the start of the crypt. Repeat this action for 20 well-orientated villi (Supplementary Figure 1).</li>
	<li>Switch from 2D view to the Measure view to display the measurements.</li>
	<li>Export the measurements and calculate the average of the crypt depth and villus height.</li>
</ol>
5. BrdU quantification (proliferation) by immunohistochemistry
<ol>
	<li>Injection of the BrdU solution
	<ol>
		<li>Weigh and record the body weight of the mice.</li>
		<li>In a fume hood, prepare a representative amount of 0.5% w/v bromodeoxyuridine (BrdU) solution in phosphate buffered saline (PBS).</li>
		<li>In the fume hood, inject 50 mg/kg of BrdU by intraperitoneal injection. 
		NOTE: To ensure that each mouse is euthanized at exactly 150 min post BrdU injection, inject each individual mouse in succession with 10&#8722;20 min interval in between in order to properly perform the tissue collection.</li>
		<li>Record the time of injection.</li>
		<li>Wait 150 min, then anesthetize mice and collect the tissue as described in steps 2.1&#8722;2.7. Perform BrdU immunohistochemistry as described in section 5.2.</li>
	</ol>
	</li>
	<li>BrdU immunohistochemistry 
	<ol>
		<li>Prepare the tissue as described in steps 3.1.1&#8722;3.3.4.</li>
		<li>For antigen retrieval, place a slide cradle with the sections in a glass histology jar and fill it with the EDTA buffer (pH 9) in a microwave oven for 1 min at 750 W followed by 9 min at 350 W. Repeat cycle. 
		NOTE: Ensure that the tissue does not dry out, which might require adding more buffer to the jars between heating.</li>
		<li>Wash sections 2&#8722;3 times in tris-buffered saline and polysorbate 20 (TBS-T) buffer for 3 min each.</li>
		<li>Apply 5 drops of peroxide block (Table of Materials) for 10&#8722;15 min at room temperature to block endogenous peroxidase activity. Wash sections 2&#8722;3 times in TBS-T buffer for 3 min each.</li>
		<li>Apply 5 drops of rodent block buffer (Table of Materials) for 30 min at room temperature, to block non-specific binding. Wash sections 2&#8722;3 times in TBS-T buffer for 3 min each.</li>
		<li>Incubate the sections with a monoclonal mouse anti-BrdU antibody diluted 1:500 for 1 h at room temperature. Wash sections 2&#8722;3 times in TBS-T buffer for 3 min each.</li>
		<li>Visualize immunopositivity using 3,3&#8217;-diaminobenzidine (DAB) by applying 5 drops of horseradish peroxidase to each slide and incubate for 15 min at room temperature. Transfer the sections to a fume hood, add 150 &#181;L of DAB and incubate for 5 min. 
		NOTE: Always wear gloves when handling DAB and dispose of waste appropriately.</li>
		<li>Rinse sections in deionized water.</li>
		<li>Dehydrate the tissue and mount a coverslip as described in steps 3.4.6 and 3.4.7.</li>
	</ol>
	</li>
	<li>Visualization of the immunoreactions
	<ol>
		<li>Visualize the tissue sample with a light microscope connected to a camera.</li>
		<li>Use the analysis software to obtain microscope images using a 20x objective of all sections and save files in the .JPG format.</li>
		<li>Take a snapshot of a stage micrometer using a 20x objective, which will be used as a calibration tool in ImageJ software.</li>
	</ol>
	</li>
	<li>Measuring the area of BrdU immunoreactive cells 
	<ol>
		<li>Install ImageJ software (Table of Materials).</li>
		<li>Assigning scale to an image in ImageJ: Open the stage micrometer image using the ImageJ File | Open menu command. Select the straight-line selection tool.</li>
		<li>Select the straight tool and draw a straight line on the micrometer image to define a known distance.</li>
		<li>Select Set Scale under the Analyse menu.</li>
		<li>Enter a value in the Known Distance box and define the unit of length in the Unit of Length box. Select Global so that this calibration applies to all images that are opened in this ImageJ session.</li>
		<li>Open an image of interest by using the ImageJ File | Open menu command to measure the area of the BrdU immunoreactive cells per crypt.</li>
		<li>Set the color graphics to 8-bit under the Image/Type menu.</li>
		<li>Increase the contrast of the image under Process/Enhance contrast and set the saturated pixels to 0.4%.</li>
		<li>Apply a threshold to the image to segment the immunopositivity. Select Image/Adjust under menu, then threshold and select the color Red (dark area shown in red). Choose a threshold of around 10&#8722;20% by moving the threshold bar. 
		NOTE: Choose a threshold that includes all the BrdU positive cells with as little background as possible. The chosen percentage should apply to all sections.</li>
		<li>Choose a well-orientated crypt, i.e., a complete crypt from intact tissue, and select the free hand selection tool to mark the area around it.</li>
		<li>To measure the threshold area, select the Analyse tab, followed by Analyse particles. In the Analyse particle window, set Size to 20-infinity, click the box pixel units, set Circularity to 0.00&#8722;1.00, and set Show to Outlines. Click boxes Display Results, Summarize, and Include holes. 
		NOTE: The values for BrdU positive cells are automatically generated in a result window, showing the individual measurements for each BrdU positive cell. Furthermore, a summary window is automatically generated, showing the number of counts, total area, average size and percentage of area with BrdU positive cells.</li>
		<li>Repeat steps 5.4.10 and 5.4.11 to measure a new crypt. The results of all measured crypts will be displayed in the Summary window.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Here, we demonstrate a widely accessible method to study SI injury and regeneration in a mouse model. A wide variety of preclinical animal models of intestinal injury exist, but it is vital we understand that each model is unique and that the endpoints must be appropriate to answer the research question. This model is excellent to study adaptive response to injury, but the endpoints should be modified when using the model as a pre-clinical model of mucositis. However, translation from animal models to patients is challenging23. Our suggested endpoints of SI weight and proliferation should be limited to the study of adaptive response only. The study of endogenous factors often requires the use of transgenic mice, and even if small bowel resection is possible in mice1, this model could be an alternative to avoid post-operative mortality. When applying this model to transgenic mice, it is important to watch the mice carefully and monitor their weight every day. During this study, some of the mice experienced a weight loss of up to 30%, which is quite substantial. To avoid high mortality in sensitive phenotypes, we suggest performing pilot studies in transgenic mice, since dose adjusting might be necessary.
A critical step within the described method is the removal and weighing of the SI. It is important that the removal and handling be performed in the same manner and by the same researcher each time to avoid large inter-assay variations.
The consistency of crypt and villus selection is important to avoid variance and bias when measuring crypt depth and villus height. When embedding the tissue in paraffin, the intestines are positioned in an upright position to make transverse cuts, thus increasing the possibility for intact villi and crypts. After cutting of the tissue, a well-orientated crypt and/or villus is selected. Selection is based on the full visualization of the whole crypt and villus in the same plane and the presence of clear borders of cells within the crypt and villus. A limitation to this method is the somewhat subjective approach when selecting a well-orientated crypt since the selection of a well-orientated crypt and/or villi is made after cutting the tissue. A previous study24 has presented an alternative method to overcome this limitation, where they use microdissection. In this method, villi and crypts are selected while observing under a microscope, prior to the tissue being cut, thus making it possible to ensure that an intact crypt and villi are being dissected from the tissue.
In contrast to previous methods used to quantity BrdU positive cells25,26, this protocol describes the area of BrdU positive cells per crypt, which provides a fast way to quantitate proliferative cells within each crypt. This technique, however, may be somewhat restrictive since it requires a more profound knowledge of the software suggested for the measurement of this technique. A future application of this protocol could be to create a more automatic generated method to quantify and measure the BrdU positive cells.

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to disease or surgery1,2. After trauma, the gut undergoes a morphometric and functional adaptive response, characterized by crypt cell proliferation and increased nutrient absorption3. This step is critical in recovery, yet poorly understood. Experimental studies of the intestinal adaptive response have focused on the changes occurring after small bowel resection in mice, rats, and pigs, but understanding the molecular mechanism behind the adaptive response in other kinds of injuries (e.g., chemical or bacterial) is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Experimentally, different approaches have been used to describe the complex molecular and cellular index of small intestinal pathology, including histopathological scoring and measuring the outcome of injury. Despite this, what is absent from the literature is a detailed description of how to perform the procedures that are needed to obtain reproducible data. When identifying factors involved in adaptation, such as gut hormones, an easy, low cost, and reproducible animal model is warranted and here we suggest using a model of chemotherapy-induced intestinal mucositis (CIM).
One of the simplest and very informative endpoints of both injury and adaptation is to measure the mass of the small intestine (SI). We know that a hallmark of mucositis is apoptosis of enterocytes, time-dependent villus atrophy and reduced mitosis. Therefore, examining intestinal morphology is highly relevant in preclinical models4,5. In humans, a decline in plasma citrulline, a marker of functioning enterocytes, correlates with toxicity scores and inflammatory markers6 in addition to the absorptive capacity7, suggesting this amino acid is an excellent biomarker of mucositis. Citrulline can be measured in both mice and rats, and has shown excellent correlations with villus length8, crypt survival9, and radiation-induced mucositis10.
A major advantage of measuring plasma citrulline is the ability to collect repeated measurements from one animal. However, multiple blood sampling in mice is restricted to a total blood volume of 6 &#181;L/g/week and requires general anaesthesia. This unfortunately also limits the use of citrulline measurements in mice. Furthermore, the measurement of citrulline requires high-performance liquid chromatography11,12, which is costly and time-consuming. Recently, we showed that citrulline levels in mice correlate significantly with SI weight (p &#60; 0.001) (unpublished data), making citrulline a direct measurement reflecting enterocyte mass. A limitation to the measurement of SI weight is the necessity for the mice to be sacrificed and thus no repeated measurements within the same mouse are possible. Still the method provides the possibility to perform a variety of other tissue analyses directed to the research question, and these facts can conceivably make up for the additional use of animals. We, therefore, suggest using SI weight as an easy, low-cost, and fast biomarker of injury and adaptation in mice. To ensure reproducibility and acceptable analytic variation, the intestines should be carefully removed from the animal, flushed with saline, emptied and dried before weighing. In this article, we show exactly how this procedure is performed.
Another hallmark of mucositis is the loss of the proliferating cells in the crypts and a compensatory hyperproliferation during the regenerative period3. The cellular marker Ki67 has been frequently used to determine fast proliferative cells by means of immunohistochemistry13. Even though Ki67 is a simple marker of proliferation, it has a tendency for imprecision as Ki67 is present during all active phases of the cell cycle (G1, S, G2, and M)14. Specific labelling is essential to detect replicating cells, which is why we suggest in situ incorporation of 5-bromo-2&#39;-deoxyuridine (BrdU), a synthetic analogue of thymidine, as it is largely restricted to replicating cells in the S-phase15. BrdU is injected in the animals 150 minutes before sacrificing and cells can be subsequently detected with immunohistochemistry using BrdU specific antibodies. In this method article, we show exactly how to measure the area of BrdU immunopositive cells within a crypt using a free image software.
Morphologic and functional changes are often studied in 5-FU induced mucositis models, where the intestinal adaptation is assessed by villus height and crypt depth. During this study, we found that during the acute phase of mucositis, which is equal to the injury phase, proliferation measured by BrdU incorporation is not correlated with crypt depth. In contrast to this, crypt depth is significantly correlated with proliferation seen in the repair phase of mucositis, 3 to 5 days after induction. This suggests that the acute phase of mucositis is not measurable by crypt depth alone. We suggest that when using proliferation as an endpoint in the acute phase of mucositis mice, BrdU incorporation should preferably be used but when quantitating hyperproliferation in the later stage during the regenerative phase, crypt depth is a reasonable alternative to BrdU incorporation. The goal of this study was to describe this model in a way that it can be used by all researchers, both in the field of oncology but especially researchers not familiar with intestinal injury models.
The described model can be used to phenotype transgenic models according to the adaptive response using body weight, SI weight and crypt depth as endpoints. As an example, we show here how we used the model of 5-fluorouracil (5-FU) induced mucositis in a cellular knock out model with insufficient L-cell secretion16. Glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) are intestinal hormones co-secreted from the enteroendocrine L-cells in response to food intake17,18. GLP-2 is recognized as an important factor for intestinal healing, the regulation of mucosal apoptosis and the improvement of the barrier function of the SI19,20,21,22. Based on the literature, we hypothesized that endogenous hormones are essential for compensatory hyperproliferation occurring in the adaptive response after injury.

<table><tbody><tr><td>5-Fluorouracil</td><td>Hospira Nordic AB, Sweden</td><td>137853</td><td></td></tr><tr><td>Ketaminol&amp;reg;Vet</td><td>Merck, New Jersey, USA</td><td>511485</td><td></td></tr><tr><td>Rompun&amp;reg;Vet Xylazine</td><td>Rompunvet, Bayer, Leverkusen, Germany.</td><td>148999</td><td></td></tr><tr><td>10% nautral formalin buffer</td><td>Cell Path Ltd, Powys, United Kingdom</td><td>BAF-5000-08A</td><td></td></tr><tr><td>HistoClear</td><td>National Diagnostics, United Kingdom</td><td>HS-200</td><td></td></tr><tr><td>Pertex</td><td>HistoLab&amp;reg;, Sweden</td><td>840</td><td></td></tr><tr><td>BrdU</td><td>Sigma-Aldrich, Germany.</td><td>B5002</td><td></td></tr><tr><td>Tris/EDTA pH 9 buffer</td><td>Thermofisher scientific, Denmark</td><td>TA-125-PM4X</td><td></td></tr><tr><td>Peroxide Block</td><td>Ultravision Quanto Mouse on Mouse kit, Thermofisher Scientific, Denmark</td><td>TL-060-QHDM</td><td></td></tr><tr><td>Rodent Block buffer</td><td>Ultravision Quanto Mouse on Mouse kit, Thermofisher Scientific, Denmark</td><td>TL-060-QHDM</td><td></td></tr><tr><td>Monoclonal mouse anti-BrdU antibody</td><td>Thermofisher Scientific, Denmark.</td><td>MA1-81890</td><td></td></tr><tr><td>Lab Vision Antibody Diluent OP Quanto</td><td>Thermofisher Scientific, Denmark.</td><td>TA-125-ADQ</td><td></td></tr><tr><td>Horseradish peroxidase</td><td>Ultravision Quanto Mouse on Mouse kit, Thermofisher Scientific, Denmark</td><td>TL-060-QHDM</td><td></td></tr><tr><td>DAB Quanto Substrate</td><td>DAB Substrate Kit, Thermofisher Scientific, Denmark</td><td>TA-125-QHDX</td><td></td></tr><tr><td>DAB Quanto Chromogen</td><td>DAB Substrate Kit, Thermofisher Scientific, Denmark</td><td>TA-125-QHDX</td><td></td></tr><tr><td>Zen Lite Software (Blue edition)</td><td>Carl Zeiss A/S</td><td></td><td>https://www.zeiss.com/microscopy/int/products/microscope-software/zen-lite.html</td></tr><tr><td>ImageJ Software</td><td>LOCI, University of Wisconsin</td><td></td><td>https://imagej.nih.gov/ij/</td></tr></tbody></table>

important endpoints and proliferative markers to assess small intestinal injury and adaptation using a mouse model of chemotherapy-induced mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

In the first experiment, we induced mucositis in mice at day 0 and sacrificed a group of mice each day for 5 consecutive days. When measuring the SI weight, we found that this parameter decreased from day 2 until day 4 suggesting a loss in the enterocyte mass. We also found that at day 5, the SI weight was not significantly different from day 0 (untreated mice) (Figure 1). The proliferation measured by the incorporation of BrdU was almost abolished at day 1 and day 2, but at day 4 and day 5 there were approximately four-fold and five-fold increases in proliferation, respectively (Figure 2). This hyperproliferation was also illustrated when measuring the crypt depth (Figure 3). What is not illustrated by measuring crypt depth is the loss in proliferating cells at day 1 and day 2, where crypt depth was reduced by approximately 13% but was not significantly different from the healthy mice. We could show that, in the regenerative phase of mucositis there was a strong correlation between BrdU incorporation and crypt depth, but this did not count for the acute phase indicating that crypt depth as an endpoint might not be suitable in the acute phase of mucositis (Figure 4).
In the second study, we examined mucostis in our transgenic mouse model with insufficient L cell secretion. Mice with deficient GLP-1 and GLP-2 showed a severe loss of body weight (BW) and a decrease in SI weight in the recovery phase, which was highly significant compared to the wild type (WT) 5-FU mice (p &#60; 0.01) (Figure 5A,B). Furthermore, the transgenic mice failed to show compensatory hyperproliferation; crypts were significantly shorter than in both WT mice and healthy controls. Contrary to this the WT mice showed an increase in crypt depth as a sign of hyperproliferation (Figure 5C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59236/59236fig1.jpg" /> 
Figure 1: Small intestinal weight. Mice were sacrificed 1 to 5 days after 5-FU injection at day 0 and the intestinal weight was measured as described. Results are shown as mean &#177; standard error of the mean (SEM). n = 13. This figure has been modified from Hytting-Andreasen et al.16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59236/59236fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59236/59236fig2.jpg" /> 
Figure 2: BrdU immunopositive cells per crypt for the duodenum, jejunum, and ileum of the SI. Mice were sacrificed 1 to 5 days after 5-FU injection at day 0 and BrdU incorporation was quantified by immunohistochemistry as described. Results are shown as mean &#177; SEM. n = 13. *p &#60; 0.05, ***p &#60; 0.001 compared to day 0 (analysis of variance [ANOVA] followed by Dunnett multiple comparison test). ***p &#60; 0.001 compared to day 0 (ANOVA followed by Dunnett multiple comparison test). This figure has been modified from Hytting-Andreasen et al.16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59236/59236fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59236/59236fig3.jpg" /> 
Figure 3: Measurement of crypt depth. Mice were sacrificed 1 to 5 days after 5-FU injection at day 0 and crypt depth was measured as described. Results are shown as mean &#177; SEM. n = 13. *p &#60; 0.05, ***p &#60; 0.001 compared to day 0 (ANOVA followed by Dunnett multiple comparison test). This figure has been modified from Hytting-Andreasen et al.16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59236/59236fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59236/59236fig4.jpg" /> 
Figure 4: Correlation of crypt depth and BrdU. Crypt depth (in duodenum, jejunum, and ileum) is correlated to BrdU incorporation at each day of sacrifice using two-tailed Pearson correlation test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59236/59236fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59236/59236fig5.jpg" /> 
Figure 5: GLP-1 and GLP-2 deficient mice fail to regenerate after acute mucositis. (A) Change in BW (%), (B) SI weight (g), and (C) crypt depth in ileum (&#181;m). Results are shown as mean &#177; SEM. Tg = transgenic mice; WT = wild type mice; 5-FU = 5-fluorouracil. n = 4&#8722;8. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001 compared to healthy control (WT saline), a = p &#60; 0.05, aa = p &#60; 0.01, aaa = p &#60; 0.001 compared to WT 5-FU (two-way ANOVA followed by Bonferroni multiple comparison test [BW] or ANOVA followed by Dunnett multiple comparison test). This figure has been modified from Hytting-Andreasen et al.16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59236/59236fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Small intestinal tissue before the induction of mucositis, during the acute phase and the recovery phase of mucositis. (A) Haemotoxylin and eosin (H&#38;E) staining and (D) BrdU staining in untreated mice. The black dotted line in panel A exemplifies a well-orientated crypt, whereas the dotted green line demonstrates a well-orientated villus. (B) H&#38;E staining and (E) BrdU staining during the acute phase of mucositis. (C) H&#38;E staining and (F) BrdU staining during the recovery phase after the induction of mucositis. Scale bar = 100 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59236/SF1.pdf">Please click here to download this file.</a>

Watch this Scientific Journal Video about Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis at JoVE.c

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...

important-endpoints-proliferative-markers-to-assess-small-intestinal

Nanyang Technological University

Research

JoVE Journal

Cancer Research

5.8K Views.  University of Copenhagen. Dit model van chemotherapie-geïnduceerde mucositis kan worden gebruikt om belangrijke eindpunten en vermenigvuldigde markers van kleine darmletsel en compenserende hyperproliferatie bij muizen te bepalen. Deze methode is tijd- en kostenefficiënt en eenvoudig uit te voeren. Met een gestandaardiseerde methode als deze is het gemakkelijker om analytische eindpunten te vergelijken met weinig variaties.Voor 5-Florauracil of 5FU mucositis inductie, laad een 1-milliliter spuit uitgerust met...

Video: Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Medicine

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) face and is frequently associated with a poor clinical outcome. While, for instance, GvHD manifestations of the skin are usually responsive to established immune-suppressive therapies and are, hence, not taking a fatal course, the presence and the intensity of intestinal GvHD, especially of the mid-to-lower parts of the gut, strongly influence the outcome and overall survival of patients with acute GvHD. Therapeutic options are essentially limited to the classic immune-suppressive agents yielding only moderate disease-mitigating effects. Hence, detailed knowledge about the tissue-resident immune cascade, changes in the intestinal microbiota, and the stromal response prior, upon, and after intestinal GvHD onset are urgently needed to understand the events and mechanisms underlying its pathogenesis and to develop innovative therapeutic options. Murine models of GvHD are frequently employed to identify and functionally assess molecules and pathways putatively driving intestinal GvHD. However, means to specifically monitor and evaluate intestinal inflammation over time are essentially lacking since established scores to assess and grade acute GvHD are routinely comprised of various parameters which rather reflect&#160;systemic GvHD manifestations. The detailed evaluation of intestinal GvHD has been restricted to studies using euthanized mice, thereby essentially excluding longitudinal (i.e., kinetic) analyses of the colonic compartment under a given experimental condition (e.g., antibody-mediated blockade of a proinflammatory cytokine) in live mice (i.e., in vivo). The mini-endoscopic in situ assessment of the distal colon of allo-HCT-treated mice described here allows a) a detailed macroscopic evaluation of different aspects of intestinal inflammation and b) the option to collect tissue samples for downstream analyses at various time points over the course of the observation period. Overall, the mini-endoscopic approach provides a major advance in preclinical noninvasive monitoring and assessment of intestinal GvHD.

Here, we present a protocol that describes allogeneic hematopoietic stem cell transplantation and allows repetitive mini-endoscopic evaluations of the distal colon in situ for the presence, characteristics, and severity of colonic inflammation within live mice suffering from intestinal graft-versus-host disease.

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell ...

Immunology and Infection

Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount importance when studying diseases of the gastrointestinal (GI) tract. The murine colonic pinch biopsy model is a useful tool to define these processes. Additionally, the interplay between gut luminal content (e.g., microbes) and the colon can be studied. However, wound induction and the ability to track wound closure over time in a reliable manner can be challenging. Moreover, tissue preparation and orientation must be carried out in a standardized way to optimally interrogate histologic and molecular changes. Here, we present a detailed method describing biopsy-induced injury and the monitoring of wound closure through repeat colonoscopies. An approach is described that ensures consistent and reproducible measurements of wound size, the ability to collect the wound bed for molecular analyses as well as visualize the wound bed upon sectioning of tissues. The ability to successfully carry out these techniques allows for studies of the acute injury response, wound healing and luminal-host interactions within the colon.

Here, we provide a detailed description of the procedure to induce colonoscopic-guided pinch biopsies in mice and track wound closure in real time. Additionally, methods for the preparation of tissues for histological, immunohistochemical and molecular analyses of the wound bed are provided.

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Murine Model for Studying Postoperative Ileus through Intestinal Manipulation

Postoperative Ileus Murine Model

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI is a consequence of mechanical damage during surgery, resulting in disruption of motility in the gastrointestinal tract. The mechanisms of POI are related to aberrant neuronal sensitivity, impaired epithelial barrier function, and increased local inflammation. However, the details remain enigmatic. Therefore, experimental murine models are crucial for elucidating the pathophysiology and mechanism of POI injury and for the development of novel therapies.
Here, we introduce a murine model of POI generated via intestinal manipulation (IM) that is similar to clinical surgery; this is achieved by mechanical damage to the small intestine by massaging the abdomen 1-3 times with a cotton swab. IM delayed gastrointestinal transit 24 h after surgery, as assessed by FITC-dextran gavage and fluorescence detection of the segmental digestive tract. Moreover, tissue swelling of the submucosa and immune cell infiltration were investigated by hematoxylin and eosin staining and flow cytometry. Proper pressure of the IM and a hyperemic effect on the intestine are critical for the procedure. This murine model of POI can be utilized to study the mechanisms of intestinal damage and recovery after abdominal surgery.

We describe a murine model of postoperative ileus generated via intestinal manipulation. Gastrointestinal transit function, pathologic changes, and immune cell activation were assessed 24 h after surgery.

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI ...

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

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