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

The overall goal of the following experiment is to test the efficacy of new therapeutics in colitis and cancer models through inducing disease in mice to investigate therapies that inhibit the induced pathology. The therapeutic is administered prophylactically. In this video, recombinant human interleukin 11 protein is used dextran sulfate sodium or DSS is then administered in the drinking water to elicit acute mucosal damage and many of the histopathological features associated with inflammatory bowel disease.

Next, changes in disease are monitored by endoscopy in order to determine therapeutic efficacy. Ultimately, the results show reduced disease burden based on endoscopic monitoring and scoring following therapeutic treatment of mice with induced disease pathology. The main advantage of this technique is the ability for us to monitor one individual mouse over the course of the experiment compared to standard techniques for histological.

Examination of multiple cohorts of animals is required. This methodology can help us asking and answering key questions in terms of inflammation and cancer of the colon, and in particular to try to establish better manipulation of cytokine signaling may influence the progression of the disease. It's important to use these preclinical models to assess the efficacy of drugs and compounds, and this allows us really to test the same mouse at several times and to see whether these compounds have an activity.

I'll be demonstrating this procedure with Giovanni CI Ciano, an animal technician from our institute On day one, administer the therapeutic of interest and appropriate vehicle controls. In this demonstration, five micrograms of recombinant human interleukin 11 protein was dissolved in 200 microliters of phosphate buffered saline and administered intraperitoneal 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. Shown here is an outline of prophylactic treatment using twice daily intraperitoneal injections over the course of the experiment.

Monitor stool consistency and the presence of blood and weigh each mouse daily. On the days of treatment, weighing should coincide with of the therapeutic drug to minimize stress to the mice caused by repeated handling. On day three, prepare 2.5%dextran sulfate sodium or DSS solution in the drinking water routinely provided to the mice by the animal facility.

Approximately five milliliters per mouse per 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. Provide fresh DSS to the mice.

Add Liam in clean water bottles for five continuous days on the evening of day five. Provide normal drinking water in addition to mashed food pellets and a protein supplement provided in small Petri dishes. On the morning of day eight.

Proceed with mouse euthanasia as described in the text protocol to collect tissues for subsequent histological analysis. 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 and confirmed colitis histopathology.

Adjust the DSS dose between one and 4%weight per volume. Depending on the microflora of the animal facility and the batch of DSS. The endoscopy equipment should be assembled according to standard procedures.

Sterilize and clean the endoscope probe with 70%ethanol or an antibacterial lubricant. 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.

Anesthetize groups of five to six mice simultaneously in a chamber with 3%eyes of fluorine in 100%oxygen at a rate of 0.2 to 0.4 liters per minute. Once the mice are anesthetized, which is confirmed by toe pinch, the isof fluorine levels should be altered to between 0.5 and 2%For maintenance, 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.Place. Veto ointment on the eyes of the mice to prevent dryness.

Hold the tail of the mouse where it meets the lower spine to clearly reveal the anus. The airflow will permit the colon to inflate if the airflow is too strong. Air will be pumped into the stomach of the mouse.

Carefully insert the rigid endoscope sheath into the rectum. Generally, the endoscope can be inserted up to three centimeters at which point the colon in mice curves and is not accessible to the rigid endoscope. 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 the belly of mice prior to the procedure to encourage defecation. 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, ensure that these procedures are performed by an experienced scientist.

Each endoscopy procedure will take approximately two minutes per mouse following the endoscopic exam. Return the mice to their cage and monitor them during recovery from anesthesia. Animals are generally awakened mobile within two minutes following removal from the isof Lorraine administering nose cone shown here are representative results from mice exposed to DSS to model acute inflammatory bowel disease.

At day eight of the protocol untreated. Wild type mice have colitis as expected weight loss, endoscopy images and endoscopy colitis scores of control mice and those that underwent treatment with recombinant human interleukin 11 protein are shown. Weight loss is used as a standard parameter to monitor for disease associated with colitis, which is routinely used to monitor the overall health of mice as visualized through the decreased endoscopy colitis score.

Reduced disease is apparent following therapeutic treatment of mice with induced disease pathology. So once you have master this technique, it should be a, you should be able to assess the mouse in roughly two minutes. And in general, what it really allows you is to test very effectively whether or not these treatments have an effect on inflammation and cancer in a relevant preclinical model.