Name: important endpoints and proliferative markers to assess small intestinal injury and adaptation using a mouse model of chemotherapy-induced mucositis
Uploaded: 2019-05-12T14:45:00
Description: 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

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 befo...

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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 challen...

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&#174;Vet</td>
			<td>Merck, New Jersey, USA</td>
			<td>511485</td>
			<td></td>
		</tr>
		<tr>
			<td>Rompun&#174;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&#174;, 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 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 ...

This model of chemotherapy-induced mucositis can be used to determine important endpoints and proliferated markers of small intestinal injury and compensatory hyperproliferation in mice. This method is time and cost efficient and easy to carry out. With a standardized method like this, it is easier to compare analytic endpoints with few variations.For 5-Florauracil or 5FU mucositis induction, load a 1-milliliter syringe equipped with a 27 gauge by 19 millimeter needle with the appropriate volume of 5FU and manually restrain a recipient mouse. While maintaining the mouse in a firm but gentle grip, expose the ventral side of the animal and insert the needle into the intraparitoneal cavity within one of the lower quadrants of the abdomen. Then, aspirate to ensure proper needle placement and inject the 5FU.At the appropriate experimental endpoint, 4-Bromo-Deoxyuridine or BrdU quantification, weigh a 5FU injured animal mouse and deliver 5 milligrams per kilogram of BrdU by intraperitoneal injection in a fume hood. 150 minutes after the BrdU injetion, confirm a lack of response to toe pinch, record the weight of the anesthetized mouse, and place the animal in the supine position. Perform a laparotomy to expose the abdominal cavity and make an incision of the chest cavity to introduce pneumothorax.Cut superior to the pylorus, and carefully extract retract the intestinal tissue away from the animal until the cecum is reached. Using forceps, gently clamp the proximal lumen shut and use a 10 milliliter syringe equipped with a 25 gauge needle to flush the small intestine with saline. Then place the emptied intestine onto clean blotting paper to carefully remove the excess salt solution.For tissue collection, harvest the small intestine from the injected animal as just demonstrated and record the weight of the flushed tissue. After histological processing, the tissue can be imaged under the 10x objective of a light microscope connected to a camera to obtain histological photos of the harvested tissue. To measure the area of the BrdU immunoreactive cells, open the image of interest in image J, and open the stage micrometer image.Use the straight line selection tool to draw a straight line on micrometer image to define a known distance and select set scale in the analyze menu. Enter a value in the known distance box, and define the unit of length in the unit of length box. Open an image of interest to measure the area of the BrdU immunoreactive cells per crypt, and set the color graphics to 8-bit under the image type menu.Increase the contrast of the image under process-enhanced contrast, and set the saturated pixels to 0.4%Apply a threshold to the image to segment the immuno-positivity and select image adjust. Select threshold and red, moving the threshold bar to select a threshold of around 10 to 20 percent. Then select a well-oriented crypt, and use the freehand selection tool to mark the area around it.To measure the threshold area, open the analyze tab and select analyze particles. In the analyze particle window, set the size to 20 to infinity, and click the box pixel units. Set the circularity to zero to one, and set show to outlines.Then click the display results, summarize, and include holes boxes. The measurement results will be displayed in the summary window. To measure the crypt depth, open an image in Zen Light and connect to the camera.Take snapshots in camera mode with a 20x objective, and switch to processing mode to open the snapshot. Switch to the measure view. Use the line tool to start a line at the bottom of a well-oriented crypt, finishing the line at the top of the crypt.Then export the measurements and calculate the average of the crypt depth and fill as height. The small intestine weight decreases from day 2 until day 4, suggesting a loss in the enterocyte mass. Of note, by day 5, the weight is not significantly different than the weight of day 0 on treated animals.The measured proliferation by BrdU incorporation is almost abolished at days 1 and 2, but at days 4 and 5, there are approximately four-and five-fold increases in their proliferation, respectively. This hyperproliferation is also observed when measuring the crypt depth. In the regenerative phase of mucositis, there is a strong correlation between BrdU incorporation and crypt depth that is not observed during the acute phase, suggesting that using crypt depth as an endpoint might be suitable in the acute phase of mucositis.Transgenic mice, with an insufficient L-cell secretion, demonstrate a significant loss of body weight and a decrease in small intestine weight in the recovery phase, compared to that observed in wild type 5-FU mice. Further, the transgenic mice fail to show compensatory hyperproliferation as the crypts are significantly shorter than in both wild-type mice and healthy controls. While attempting this procedure, make sure to empty the small intestine completely, and to remove the excess saline before weighing the tissue to obtain the small intestinal weight weight only.After watching this video, you should have a good understanding of how to conduct, collect, and determine important endpoints of small intestinal injury. And to effectively obtain reproducible data. Don't forget that working with chemical compounds such as 5FU and BrdU can be hazardous and that precautions, such as using a lab coat, gloves, and a fume hood should always be taken when performing these procedures.

Research

JoVE Journal

Cancer Research

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