On day zero, Myer injected with azoxymethane or a OM the following week. Their drinking water is replaced with a solution containing dextran sulfate sodium or DSS. At week three, mice are given water for the following two weeks.
This DSS water cycle is repeated two additional times. After the second DSS cycle. Mice can be assessed for tumors by mirroring endoscopy.
After week 10, mice are sacrificed and colons harvested. Colons are flushed and opened longitudinally opened. Colons are assessed for tumor size and tumor number.
A longitudinal section of the colon in thirds can be embedded and submitted for histological analysis. Using light microscopy tumors can be counted and classified. We demonstrate a protocol in which administration of the genotoxic agent a OM, followed by three cycles of the pro-inflammatory agent.
DSS rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis associated cancer individually labeled mice with tail marks or ear clips on day zero, record the baseline weight of each mouse on a table, then inject each mouse with a 10 milligram per kilogram solution of a OM exercise. Caution when handling a OM as it is a genotoxic agent on day seven, miser supplied with a 2.5%DSS solution as their drinking water for the following week. To prepare this solution, add DSS powder to distilled water and mix until dissolved.
The solution is ready after it is passed through a 0.2 micron cellulose acetate filter. To provide a continuous supply of DSS solution for seven days. Replace DSS every two to three days.
After this period, switch cages back to water for two weeks. Weigh mice two to three times per week and observe for rectal bleeding and diarrhea. Mice with large tumors may temporarily develop rectal prolapse, though this generally does not reduce survival percent.
Weight loss relative to baseline is used as a surrogate measure of colitis severity. A weight loss up to 10%along with two to three days of diarrhea and rectal bleeding can be expected in the week following a full cycle of DSS endoscopy.Following. The second or third cycle of DSS can be performed to confirm tumor growth in vivo prior to sacrifice.
As the A-O-M-D-S-S model is quite reliable in producing tumors, this step is generally not necessary. Only a few mice should be evaluated using this method as it has the potential to disrupt mouse tumors. Your working area should include a beaker of sterile phosphate, buffered saline or PBS as well as an empty beaker.
To discard used PBS draw up eight to 10 milliliters of PBS into a syringe and attach to the endoscope per use. In flushing intraluminal contents anesthetize each mouse with inhaled or injectable sedation and secure the mouse to a flat board With tape at the tail and chest mouse foot pads can be pinched. To ensure adequate sedation, use your thumb and index finger to pinch the lower abdomen of the mouse to expose the anus, advance the muren endoscope into the mouse rectum.
Gently using PBS to inflate and irrigate to improve intraluminal view and remove fecal contents. Continue to advance the endoscope slowly while observing the monitor. For the presence of gross tumors on day 70 each A-O-M-D-S-S treated mouse should harbor multiple colon tumors and be ready for assessment.
This state can be delayed weeks to months if larger tumors are desired. Prepare a PBS filled syringe attached to a gavage needle. A syringe loaded with 10%formalin basic surgical scissors, scissors with rounded tips.
A small syringe loaded with 1%Ian Blue solution and fine forceps. Also, lay out a cold tray, a away boat containing chilled PBSA new razor for sectioning and a fixation basin filled with 10%formalin euthanize each mouse by isof, fluorine and cervical dislocation or other institutionally approved method. Final weight and other measurements can be made.
At this time, lay the mouse with its ventral side exposed on a cutting board. Spray the abdomen with 70%ethanol to keep hairs free from the dissection area. Use forceps to grasp the midline of the abdomen and make a small incision through the skin to expose the peritoneum.
Use gentle traction to separate the skin from the peritoneum and expose the entire abdominal cavity. Make an incision into the peritoneum and extend this incision along the costal margin bilaterally. Use closed scissors to sweep the small intestine and colon to the side.
This will expose the mesenterical lymph nodes, which can be optionally harvested. For assessment, sweep the genital urinary tract inferiorly to expose the descending colon and rectum, excise the bladder and reproductive organs as necessary to further expose the pelvis. Being careful not to disrupt adjacent tissues.
Cut through the inferior and superior pubic rami to expose the anal rectal junction. Cut the skin around the anal rectal junction to free the colon while providing gentle upward traction. With forceps mesenteric, attachments of the colon can be excised or teased away.
It is important to retain some of the skin at the anal rectal junction as this area is often affected by dysplasia. Transect the large intestine from the small intestine just proximal to the cecum. Separate the secum from the adjacent colon using an 18 gauge gavage needle attached to a five milliliter syringe.
Flush the colon with ice cold PBS such that fluid exits in the physiologic direction using scissors with rounded edges. Open the colon along its mesentery. Paint the open colon onto the cold tray.
Ensure the tray has sufficiently thawed as to not freeze the tissue. Apply a few drops of 1%Ian Blue with a finger to better visualize tumors. Assess tumor number and size with a ruler or take photographs for digital analysis.
Small sections of tumor in adjacent normal tissue can be excised at this point for future analysis by RNA protein or immunohistochemical methods, leaving some portion of the colon for histological assessment. Apply 10%formal into the remaining colon on the cold tray via syringe or finger, allowing the tissue to fix for at least 30 seconds. In this manner, facilitates transfer to the fixation basin.
Ensure residual formin is wiped off prior to painting a new opened colon on the same tray. Transfer the colon to a basin filled with 10%formin and pinned down at edges. After three hours of fixation, discard, formin and replace with 70%ethanol.
Colons can be preserved in 70%ethanol indefinitely. Use low heat to make a solution of 2%agar and set aside a clean slide on a glass plate. Cut the fixed colon into thirds and return unused pieces to the ethanol basin.
Trim down sections to a uniform size using two razors. Embed colon sections and layers in agar on the clean slide. Repeat this process for the next colon.
Segment cutaway, excess agar, and place the embedded colon into a cassette for processing and staining with hematin and eoin. Figure one outlines the protocol for colitis associated cancer induction. Figure two shows mouse weight relative to baseline during a OM and DSS administration.
Note that in the week following each DSS cycle, mice lose five to 10%of their body weight. Weight loss in this experiment is a surrogate marker for colitis severity. Figure three shows a view of tumors in the distal colon via muren endoscopy at day 50 of A-O-M-D-S-S treatment.
Note, the multiple polypoid masses obstructing the lumen of the distal colon in panels B and C in comparison to the normal colon. In panel a figure four shows a longitudinally opened mouse colon illustrating the gross appearance of tumors. Note the higher tumor burden in the distal colon and the characteristic ated texture of the proximal colon with little tumor growth.
Figure five demonstrates tumors highlighted by application of lc and blue stain. Note how the dye emphasizes the normal texture of the colon as well as the borders of each individual tumor. Such staining can be helpful in the precise measurement of tumor areas by ruler or digital measurement.
Figure six shows the representative distribution of the average number of tumors per mouse treated with a OM and DSS. Note, the majority of tumors are located in the distal colon and are less than two millimeters in size. Figure seven shows paraffin embedded longitudinal sections of colon in a cassette and h and E stain slide.
Note that residual S and blue stain does not interfere with h and e staining. A large tumor is circled on the slide image. The designation's distal, middle and proximal result from sectioning the entire colon in thirds between the cecum and anus.
Figure eight shows the representative histology of a tumor resulting from a OM and DSS administration in the distal colon. Photo micrographs shown are stained with H and E-B-R-D-U and beta-catenin and demonstrate dysplastic changes similar to human adenocarcinomas of the colon. In this video, we have demonstrated a protocol for inflammation driven colonic tumorgenesis in mice, utilizing a single injection of a OM followed by three seven day cycles of DSS.
Over a 10 week period, this model induces tumors with histological and molecular changes closely resembling those occurring in human colitis associated cancer, and provides a highly valuable model for the study of oncogenesis and chemo prevention in this disease.
