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

The Walter and Eliza Hall Institute of Medical Research animal ethics committee approved each of the procedures described in these protocols.

1. Preparation of Experimental Mice

1. Compose experimental cohorts of at least 4 gender matched 6-8 week old C57BL/6 mice (M. musculus) that are bred and housed in the same specific pathogen free (SPF) animal facility/room and provided with autoclaved food and water. The use of female mice will permit co-housing of mice from different lines, limit box numbers, and reduce cage-to-cage variation.
2. Ensure that mice are at least 6 weeks of age to enable endoscopy procedures using the 3.0 mm diameter endoscopy sheath.
3. Use mice of the same genetic background, and where possible, from cages stored on the same rack to reduce variations associated with the intestinal microflora. Variations in microflora between animal facilities should be taken into consideration when determining the appropriate control mice and genotypes for each experiment²².
4. Mark mice by ear/toe clippings, tattoos, or similar to allow for easy identification.
5. Weigh mice on Day 0, to determine baseline experimental weights.
6. Perform endoscopy (see Section 5) of mice on Day 0 to record baseline colonic phenotypes.

2. Pre-clinical Trial in an Epithelial Damage and Acute Colitis Model

1. Prepare a solution of the therapeutic agent to be tested. For therapeutics delivered by oral gavage (p.o.), prepare solutions to a maximum volume of 100 µl. For therapeutics delivered by intraperitoneal (i.p.) injection, prepare solutions to a maximum volume of 200 µl.
   NOTE: Repeated i.p. or p.o. drug administration over prolonged periods of time can result in altered mouse behavior. Alternate the i.p. injection site to avoid prolonged discomfort. For p.o. administration, provide the mice with autoclaved sunflower seeds as a ‘treat’ to minimize the adverse association with the gavage procedure.
2. On Day 1, administer the therapeutic of interest (prepared in step 2.1) and appropriate vehicle controls. In the example provided (Figure 6A), 5 µg of recombinant human (rh) iInterleukin (IL)-11 protein was dissolved in 200 µl phosphate buffered saline (PBS) and administered i.p.
3. Determine the timing and frequency of the administrations, which will be dependent on the pharmacokinetic profile established for the therapeutic reagent that is being tested. Figure 1 outlines prophylactic treatment using twice daily i.p. injections over the course of the experiment.
4. Monitor, including stool consistency and the presence of blood, and weigh each mouse daily. On the days of treatment, weighing should coincide with administration of the therapeutic drug to minimize stress to the mice caused by repeated handling.
5. On Day 3, prepare 2.5% (2.5 g/100 ml) DSS solution in the drinking water routinely provided to the mice by the animal facility. Approximately 5 ml/mouse/day of DSS solution is required for the experiment. The consumption of the DSS water may change if the ambient temperature of the animal facility fluctuates.
   NOTE: DSS is an irritant and should be handled in accordance with MSDS instructions.
6. Provide fresh DSS to the mice ad libitum in clean water bottles for 5 continuous days. On the evening of Day 5, provide normal drinking water in addition to mashed food pellets and a protein supplement provided in small petri dishes (100 g food pellets/10 g protein shake/10 ml water). This prevents dehydration and assists with minimizing severe weight-loss.
7. Euthanize mice by CO₂ intoxication and collect tissues for subsequent histological analysis on the morning of Day 8, or when the mice experience ≥15% weight-loss for more than three consecutive days, whichever occurs first.
   NOTE: Adjust the DSS dose between 1-4% (w/v) depending on the microflora of the animal facility and the batch of DSS. Carry out, routine batch testing to establish the appropriate DSS dosage and circumvent batch-to-batch variation. The appropriate dosage should be based on weight-loss (not greater than 15% of original weight) and confirmed colitis histopathology.

3. Pre-clinical Trial in a Chronic Colitis or Colitis-associated Cancer Model

1. For the chronic colitis model begin from step 3.7.
2. For the colitis-associated cancer (CAC) model, on Day 1, inject each mouse with 10 mg/kg (w/w) Azoxymethane (AOM, 250 µl, i.p.).
   NOTE: AOM is a carcinogen and should be handled in accordance with MSDS instructions.
3. Stock solutions of AOM are prepared as 10 mg/ml aliquots and stored at -20 °C. On the day of injection, the AOM stock solution is thawed and diluted to 1 mg/ml in PBS. Repeated freezing and thawing of the AOM stock solution should be avoided.
4. The AOM dose may be adjusted between 8-12 mg/kg. Routine batch testing is required to establish the appropriate AOM dosage in order to circumvent toxicity to mice due to batch-to-batch variation.
5. During Days 1-7 following AOM administration weights will be stable and for this reason weight monitoring can be omitted during Days 1-7 to avoid handling animals that will excrete cytotoxic metabolites in their feces.
6. On Day 7 all bedding needs to be changed following cytotoxic procedures. Once the bedding change is complete, the animals no longer require cytotoxic handling procedures, as they will no longer be excreting cytotoxic metabolites.
7. On Day 8, prepare 2.5% (2.5 g/100 ml) DSS solution (as described in step 2.5) and provide to the mice ad libitum.
8. Weigh mice daily throughout the experiment and monitor for signs of illness, including ruffled fur, hunching, bloody stools and reduced movement.
9. On Day 13, remove the DSS solution and provide the mice with normal drinking water until Day 28. Days 8-28 constitute one ‘cycle’ of the chronic colitis/CAC protocol.
10. On Days 13-28, when the mice are given normal drinking water, provide the mice with mashed food pellets and a protein supplement provided in small petri dishes (100 g food pellets/10 g protein shake/10 ml water). This prevents dehydration and assists with minimizing severe weight-loss.
    NOTE: The protein supplements in mash should not be provided when the mice are receiving DSS-containing drinking water in order to ensure consistency in DSS consumption.
11. On Day 20 perform the second endoscopic examination to monitor individual mouse health (see Section 5).
12. On Day 29 repeat the DSS cycle (Cycle 2: 2.5% (w/v) DSS provided during Days 29-33 and normal drinking water during Days 34-50).
13. On Day 40 perform the third endoscopic examination to monitor individual mouse health and determine tumor burden (see Section 5).
14. If no tumors are visible by endoscopy on Day 40, begin a third DSS cycle on Days 50-72 (Cycle 3: 2.5% (w/v) DSS provided during Days 50-55 and normal drinking water during Days 56-72). Generally, tumors are visible by Day 40 and therefore the third cycle of DSS can be omitted.
15. Once tumors become visible by endoscopic examination, assign each mouse an endoscopic tumor score (see Section 5) and assign individual mice to cohorts with similar baseline tumor burdens.
16. On Day 42 (or when tumors are visible), administer the therapeutic compound or relevant vehicle control (See Section 2.2). The timing of the doses will be dependent on the pharmacokinetic profile of the therapeutic. An example of an intervention treatment injected i.p. three times weekly in mice with established tumors is provided (Figure 2).
17. Perform endoscopic exams once per week over the duration of the therapeutic treatment to monitor tumor burden. Generally, 4 weeks of a therapeutic treatment is sufficient to observe an objective treatment response. Following cessation of the treatment, a cohort of mice can also be monitored by endoscopy for tumor relapse.
18. Euthanize mice by CO₂ intoxication and collect tissues for biochemical and/or histological analysis on the morning of Day 72 or when mice experience ≥15% weight-loss for more than three consecutive days, whichever occurs first.

4. Pre-clinical Trial in a Sporadic Colorectal Cancer Model

1. On Day 1, inject each mouse i.p. with 10 mg/kg AOM (see step 3.2). The AOM dose may be adjusted between 8-12 mg/kg. Routine batch testing is required to establish the appropriate AOM dosage in order to circumvent toxicity to mice due to batch-to-batch variation.
2. Repeat the injection on the first day of each week for the following 5 weeks (Figure 3). The mice should be handled according to cytotoxic safety procedures for the entire 6 weeks.
3. Weigh each mouse on the day of AOM injection to minimize distress to the mice caused by repeated handling.
   NOTE: AOM is a carcinogen and should be handled in accordance with MSDS instructions.
4. All animals should be handled according to cytotoxic safety procedures for the 6 weeks of AOM i.p. injections required for this protocol.
5. During week 8 perform endoscopic examination to monitor for emerging tumors (see Section 5). Endoscopic examination should be performed every two weeks until tumors become visible. Generally, wild-type C57Bl/6 animals begin to develop tumors by approximately 40 weeks after the initial administration of AOM.
6. Once tumors become visible by endoscopic examination, assign each mouse an endoscopic tumor score (see Section 5) and assign individual mice to a cohort with similar tumor burdens prior to the commencement of therapeutic treatment.
7. During week 40 (or when tumors are first visible), administer the therapeutic compound or the relevant vehicle control.
8. The timing of the doses will be dependent on the pharmacokinetic profile of the therapeutic (see step 2.1). An example of an intervention treatment injected i.p. 3 times weekly in mice with established tumors is provided (Figure 3).
9. Perform endoscopic examination weekly for the duration of the therapeutic treatment periods to monitor tumor burden. Generally, 4 weeks of a therapeutic treatment is sufficient for an objective treatment response. Following treatment cessation, a cohort of mice can also be monitored by endoscopy for tumor relapse.
10. Euthanize mice by CO₂ intoxication and collect tissues for biochemical and/or histological analysis of the colon and tumors during week 45, or following 4 weeks of therapy.

5. Endoscopic Examinations

1. The endoscopy equipment should be assembled according to standard procedures¹⁶.
2. Sterilize and clean the endoscope probe with 70% ethanol or an anti-bacterial lubricant.
3. Videos can be recorded using a laptop or desktop computer and standard media software, such as iMovie. A standard computer monitor, rather than a medical grade monitor, is sufficient to visualize the colon during the endoscopy procedure (Figure 4).
4. Anesthetize groups of 5-6 mice simultaneously in a chamber with 3% isoflurane in 100% O₂ at a rate of 0.2-0.4 L/min. Once the mice are anesthetized, which is confirmed by toe pinch; the isoflurane levels should be altered to 0.5-2% for maintenance.
5. Remove an individual mouse from the chamber, and place it ventral side up with its head secured in a nose-cone. Full anesthesia should be monitored and the hind legs should be adjusted so that they are stretched out behind the mouse.
6. Place a vet ointment on the eyes of the mice to prevent dryness.
7. Hold the tail of the mouse where it meets the lower spine to clearly reveal the anus. Insert the rigid endoscope sheath into a beaker of water and adjust the airflow to allow the release of one small bubble at a time.
8. The airflow will permit the colon to inflate. If the airflow is too strong, air will be pumped into the stomach of the mouse. No additional lubrication is required.
9. Carefully insert the rigid endoscope sheath into the rectum. Generally, the endoscope can be inserted up to 3 cm, at which point the colon in mice curves and is not accessible to the rigid endoscope.
10. A common difficulty during endoscopy procedures is blockage of access to the lumen of the colon due to fecal matter. Avoid this by gently massaging of the belly of mice prior to the procedure to encourage defecation, or carefully maneuvering the endoscope around the fecal matter during the procedure. Peristalsis can also occur, during which the endoscope should be held in position until the colon musculature relaxes.
11. Initiate video recording at any stage of the endoscopy procedure. Disease scoring can be recorded by an experienced assistant during the procedure, or at a later time point from the video files.
12. Following the endoscopic exam, return the mice to their cage and monitor them during recovery from anesthesia. Animals are generally awake and mobile within 2 min following removal from the isoflurane administering nose cone.
13. Ensure that these procedures are performed by an experienced scientist. Each endoscopy procedure will take approximately 2 min per mouse.

6. Disease Scoring

1. To monitor colitis, score videos for the Murine Endoscopic Index of Colitis Severity (MEICS)¹⁶, which documents changes in (i) the thickness of the colon wall indicated by transparency, (ii) stool consistency, (iii) blood vessel integrity and presence, (iv) ulcers and areas of regeneration that present in a granular fashion, (v) bleeding indicated by fibrin deposits (Figure 5a).
2. To monitor tumor burden, score videos for tumor incidence and size. Tumor size is determined by the diameter of the colonic lumen occupied by the tumor¹⁶ (Figure 5b). It is common to use both colitis and tumor scoring parameters for a single mouse. Using endoscopy, individual tumors and the health of the colonic mucosa can be monitored over time, providing a read-out for the success of new therapeutics in a single animal.
3. (Optional) Still images can be extracted from the videos for generation of representative figures (Figure 6).