Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

In this video, a variety of applications from neuron endoscopy will be presented in the first application. How to use the endoscope to score disease severity in a mouse model of colitis will be shown in the second application. The use of endoscopy to monitor mucosal wound healing in vivo will be demonstrated in the last application.

The use of fluorescence endoscopy to detect intestinal tumors and the technique to intra mucosally inject agents will be presented ultimately intestinal wound healing, inflammation, and cancer. Agenesis can be assessed based on the findings of the endoscopic analysis. The main advantage of this technique over existing methods like histological analysis of experimental colitis, is that with this technique, you can perform intra individual follow-up examinations and repetitively assess mucosal alterations.

Therefore, this method can help answer key questions in the field of experimental intestinal inflammation, such as what is the temporal establishment of colitis. The implications of this technique extend to the therapy of colorectal cancer because of the possibility to functionally characterize neoplastic lesions. Though this method can provide insight into the development of colorectal cancer, it can also be applied to other systems such as inflammatory bowel disease or infectious colitis.

Generally, individuals new to this method will struggle because of the challenge of handling the small scale endoscope Fluorescence. Endoscopy expands the potential of wide light endoscopy to molecular imaging by specific targeting of cellular structures by fluorescently labeled traces during endoscopic examinations. To induce colorectal cancer in the experimental animals, first use a one milliliter syringe equipped with a 30 gauge needle to interperitoneal inject 10 milligrams per kilogram are freshly prepared mutagenic as oxy methane into each mouse.

Challenge the experimental mouse group with repetitive cycles of 3%weight per volume, dextran sulfate sodium or DSS from day zero to 7 21 to 28, and 42 to 49 to induce the inflammatory driven colorectal cancer. Agenesis supplying the mice with autoclave water in between the challenges on the day of the colonoscopy. After confirming sedation by toe pinch, use a buttoned cannula to instill two milliliters of fluid enema into the colon of an experimental animal.

If significant fecal loading is suspected, then while waiting for the mouse to defecate, connect a small animal veterinary endoscopic workstation to the camera unit for white light endoscopy. Configure the settings of the light source to excite the traces to be injected, and then integrate an appropriate band pass filter between the telescope and the camera. Now, place the mouse on the examination table and insert the endoscope very carefully.

To avoid perforation open both valves of the sheath. One valve should be connected to the air pump. The other should be sealed with an index finger to control the air.

Then slowly and carefully inflate the colon, advancing the endoscope only as far as the right colon flexor to avoid perforation about 4.5 centimeters from the anus. Now, pull back slowly on the endoscope and examine the mucosa for inflammatory or malignant alterations, and to assess the whole circumference of the bowel. Then to retrieve a biopsy sample, introduce biopsy forceps carefully through the working channel until the tip of the forceps is visible on the monitor.

Open and close the forceps carefully to avoid perforation and move them to the site of pathology. For interra mucosal application of pharmacological agents, pre-fill a flexible injection tube completely with the agent of interest, and then push the tube through the working channel until the cannula is visible on the monitor. Then attach a fine syringe and insert the needle into the submucosa At a 15 to 30 degree angle facing the bevel in the direction of the mucosa, gently administer up to 50 microliters of the therapeutic agent.

After a successful injection, the mucosal will exhibit a characteristic lifting to perform fluorescence endoscopy, administer the agent of interest intravenously prior to examination. Check the optimal time point between injection of your fluorescently labeled tracer, and the imaging procedure, which is dependent on tracer pharmacology. Then to view the injected agent by fluorescent endoscopy, configure the settings of the band pass filter system in accordance to the excitation and emission wavelengths of the injected tracer.

Finally, image the tissue after application of the tracer to acquire a target to background ratio reading After the induction of colitis.Mice. Exhibit weight loss beginning on day three with a maximum loss of body weight of 19%occurring at day seven in accordance with the loss of body weight. The neuron endoscopic index of colitis severity score increases at day seven after DSS start indicating massive inflammatory damage of the colonic mucosa, which is ameliorated by day 13.

Also at day seven, after the DSS start, the histological damage is significantly higher in DSS treated mice compared to controls as reflected by epithelial denudation, mucosal ulcerations and increased neutrophil infiltration, all of which are significantly improved by day 13. In these representative images from a routine endoscopy, mucosal wounds were induced mechanically by miniature biopsy forceps with a one millimeter diameter wound. Healing was then monitored by daily endoscopic examination and quantified by measurement of the residual wound area with image editing software.

The individual wound area closure over time was then expressed by the quotient of actual wound area per the initial wound area. For example, a day three after wound generation, 41%of the wound area was recovered, whereas at seven days, the wound was completely healed. As usual, at the end of an experiment, wounds can be resected for histological evaluation, X vivo as in these representative images of hematin and ESN stained wound beds at day zero and five, approximately 80 days after tumor induction by as oxy methane and three cycles of DSS multiple colon tumors, as well as macroscopic signs of chronic inflammation such as granulated mucosa can be observed.

Endoscopically fluorescent endoscopy of the neuron intestine without application of a tracer, results in a lack of detection of any specific signal, and no interactions with colonic tissue or fecal autofluorescence are observed. In contrast, immediately after intravenous application of Fitz Dextran, the fluorochrome can be observed at the colonic mucosa and may be used for assessment of the increased vascularity in regions of chronic inflammation or malignant mucosa. Accordingly, quantification of the fluorescence intensity can be used to measure the significantly increased uptake of the fluorochrome within malignant tissue as compared to non-affected colonic mucosa.

Once mastered, this technique can be done in five to 10 minutes if it is performed properly. While attempting this procedure, it's important to remember to advance the rigid endoscope carefully through the marine colon. After watching this video, you should have a good understanding of how to assess epithelial wound healing, intestinal inflammation, and cancer agenesis while performing marine colonoscopy.