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.
