Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

Summary

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

Abstract

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Introduction

Murine endoscopy has been performed as a useful research tool for over a decade^1-3. To date, most studies employing murine endoscopy have used rigid endoscopes, although some have also used flexible sigmoidoscopy. Murine endoscopy provides immediate results and a more objective estimate of the extent of intestinal normalcy, severity of inflammation, and tumor progression compared to indirect measures, such as body weight loss, diarrhea, and histology (which is suboptimal when lesions are patchy), while offering insights on the overall health of the colon. Most notably, endoscopy allows for the repeated assessment of laboratory animal models over time, as opposed to traditional histological examination that requires the animal to be euthanized and colons to be harvested for analysis¹. Despite these advantages, the use of murine endoscopic technology has not been widespread and is limited by the lack of standardized protocols for implementation and scoring of pathological findings. Properly implemented, murine endoscopy holds great promise to further facilitate our understanding and characterization of animal models of multiple chronic gastrointestinal disease states, including inflammatory bowel diseases, colitis, and colorectal tumors.

The utility of murine endoscopic technology hinges upon its reproducibility and objectivity, which requires the existence of standardized examination procedures and a consistent, non-redundant, and reliable scoring system for intestinal pathologies. At least three descriptive scoring systems for endoscopic assessment of colitis^4-6, as well as for colonic tumors^4,7,8 in mice have been published. However, these reported approaches and scoring systems are not readily comparable across studies. In many cases, there is no clear definition of the criteria used for classification of lesions, and when criteria are given, they vary widely. Moreover, there are no reported scoring systems that integrate the assessment of both colitis and tumors, two of the most common colonic pathologies that can occur simultaneously and have interactive effects on outcome, into a single measurement tool. Finally, the endoscopic scoring systems that do exist for colitis often have inflammatory categories with limited discrimination (i.e. narrow scoring options over short integer scales, often 1-4) to properly represent disease progression scenarios and enable the use of parametric statistical analysis.

In this paper, we describe the use of a flexible endoscope to assess the severity of murine colitis and colonic tumors and describe a standardized protocol for employing this technology in the anatomical assessment of the perianal region, rectum, and distal colon. We illustrate effective troubleshooting during endoscopy to minimize trauma and image artifacts, and we describe a reproducible scoring system based on validated published clinical parameters. The scoring system integrates evaluation of both intestinal inflammation and tumors using a high level of discrimination with 12 possible grades of inflammation, tumor mapping and plotting options, and a novel decimal unit system (i.e., decimal identifiers) to highlight findings with specific diagnostic value (i.e. tumor development, complications during endoscopy, etc.). The use of decimal identifiers allows for rapid tracking of relevant data sets for further downstream analysis. Finally, we perform reliability and validity testing of the scoring system in multiple murine models of colitis and colitis-associated cancer (dextran sulfate sodium (DSS) induced-colitis, Clostridium difficile infection, and azoxymethane/DSS-induced colonic cancer).

Protocol

1. Setting up the Endoscopy System

1. Set up the endoscopy system before anesthetizing the mice, following the manufacturer's instructions. Use a video ureteroscope designed for humans for this protocol (Figure 1). The outer diameter of the insertion tube is 9.9 Fr (3.3 mm), its distal end is 8.5 Fr (2.8 mm), and the inner diameter of the instrumentation channel is 6 Fr (1.2 mm).
2. Although the use of endoscopes have not been linked to disease transmission [9], sterilize the endoscope and lens with 70% ethanol¹⁰ or with appropriate techniques when working with spore-forming microorganisms. Prepare bacteriostatic sterile lubricant and non-scratching wipe tissues to saturate the endoscope tip.
3. Attach a 3 ml syringe full of air (or a suitable CO₂ gas mixture) to the top of the instrumentation channel.
4. Turn on the power, and set up the white balance in normal white light, as well as narrow band imaging (NBI) mode, when applicable. Since 2001, NBI has provided diagnostic imaging benefits as it emphasizes fine blood vessel structures on mucous membranes, including ulcerative and tumorous lesions, which have characteristic abnormal vascularization¹¹. NBI-filter mode restricts the wavelength of captured light to 390-445 nm and 530-550 nm, which highlights structures with hemoglobin.
5. Print the assessment scoring form (Figure 2) to record your findings. Become familiar with expected normal and NBI images (Figure 3A) and the definition and grades of each criterion before the exam (see Section 6.1).
6. Prepare a colorant-based solution on phosphate-buffered saline (PBS) for chromoendoscopy, if desired. In chromoendoscopy, the exam is performed using a dye as contrast medium to highlight mucosal changes. Use methylene blue (0.1-1%) and indigo carmine (0.1%) solutions (Figure 3B). Note that methylene blue is an oxidation-reduction agent that can produce oxidizing radicals, indirect cross-linking of amino acid residues, and DNA degradation¹², which could interfere with sample analysis.

2. General Anesthesia of the Mouse

1. Choose the anesthetic agent and administration route. Consult a veterinarian from an animal resource facility or Institutional Animal Care and Use Committee (IACUC) regarding potential interference of medications with the immune system and research goals. Some medications may influence cytokine profile expression in animals, including rodents¹³. Use injectable tribromoethanol for terminal procedures or isoflurane for survival endoscopies, along with appropriate controls.
2. For induction, administer 4-5% of isoflurane in 100% oxygen at a rate of 0.2-0.5 L/min; use 0.5-2% for maintenance. This protocol is not appropriate for all animals. Consult your facility veterinarian for proper instructions and training. Abide by relevant IACUC recommendations to achieve the highest standards for animal welfare. Make sure that the mouse if fully anesthetized.
3. ‘Strap’ the anesthetized mouse to a supporting surface table in a dorsal or ventral position using laboratory adhesive tape. Beware of the anatomical orientation on your endoscope screen. Secure its lower extremities and tail. A band of tape can also be placed over the chest to prevent movement away from the respirator, but avoid restricting thoracic ventilatory movements.

3. Detailed Endoscopic Examination

1. Look for perianal lesions before inserting the endoscope into the rectum, then advance the scope through the anus.
2. Coat the endoscope with sterile surgical lubricant. Be sure not to coat the surface of the CCD (charge coupled device) sensor.
3. Insert the endoscope and start recording. Recorded videos are useful for meticulous evaluation of endoscopy if needed after the examination. Try to perform the endoscopy in the least possible length of time to minimize intestinal/mucosal irritation.
4. Apply air by using a syringe to slowly set apart the intestinal walls. Depending on the size of the mouse, use 1-2 ml of air. Avoid excessive air insufflation; it may cause respiratory distress and death of the anesthetized mouse.
5. Advance the endoscope forward as you clear the way with air or gentle rotation of the scope. Do not advance the endoscope if you do not see the intestinal lumen. Perforations or rectal/colonic tears can occur at this stage, especially in young mice (<8-10 weeks old). Take pictures as necessary.
6. As the scope advances, assess if there is spontaneous bleeding (defined as naturally occurring mucosal hemorrhaging not associated with traumatic endoscopy). Spontaneous bleeding can be observed as the endoscope enters the rectum and colon.
7. Assess the transparency of the colonic wall. Transparency is defined as the ability to visualize the intramural blood vessels in the colon and that of the other surrounding viscera (Figure 4). Gentle motion of the scope (back-forward, clockwise, counterclockwise) will help determining if blood vessels can be seen in the context of other viscera. Mesenteric adipose tissue can also be seen. Note if the rectum inside the pelvic cavity appears less transparent than the colonic wall in the abdominal cavity.
8. Inspect to determine the type and number of focal lesions (edematous areas, erosions, ulcers, tumorous masses). This will be recorded in the scoring form using the colonic inflammation score and the decimal identifiers that have been developed.
9. Once the area where the colon flexes caudally into the proximal colon is reached, withdraw the endoscope slowly and look for the presence of contact bleeding (which occurs due to mucosal friability after the endoscope is advanced or removed). It is not necessary to scrape the colon surface intentionally. Healthy colorectal mucosa does not bleed after proper endoscopic examination.

4. Biopsy

1. When biopsies are needed, insert the biopsy forceps through the endoscope channel dedicated for instrumentation. Monitor the advancement of the forceps on the endoscope screen to prevent intestinal perforation.
2. Open the biopsy cups of the forceps to about a 60-80° cup-to-cup angle, and direct them to the area of interest. Excessively opened forceps or pressure may grasp extra layers of intestinal tissue, which results in a biopsy-induced perforation of healthy transparent intestinal walls. If perforation occurs, euthanize the animal during anesthesia following your IACUC recommendations. Perforations result in endotoxemia and peritonitis.
3. Close the biopsy cups, and pull them out while keeping the forceps closed.
4. Remove the specimen from the forceps by flushing the tissue with a solution that is gentle to the tissue and the forceps. RNA later and PBS are common working solutions; if needed, use the needle to dislodge the sample. Avoid eroding the forcep cups or cutting edges.

5. Recovery from Anesthesia

1. Upon completion of the examination, provide a warm environment to the mouse until it recovers. Use either an electric light, or a warm pad set at 37 °C. Avoid overheating; peripheral vasodilation compromises animal recovery. Monitor the mouse closely until full recovery.

6. Integrated Endoscopic Scoring System of Colorectal Health with Decimal Identifiers

1. Use the scoring form (Figure 2) and the parameter definitions below (Endoscopic Assessment of Murine Colorectal Inflammation and Tumors Using a Decimal-weighted Scoring System: Parameter Definitions, and Protocol) for examples and the optional 'Distal-Proximal Endoscopic Colitis scoring system' (D-PECS) as guidelines for nonbiased assessment and detailed record keeping.
2. Score the four parameters of colorectal inflammation described on the scoring system form (Figure 2). Meticulously evaluate the perianal region, intestinal transparency, type of intestinal bleeding (if present), and presence and quantity of focal lesions (erosive, ulcerative, and/or tumorous).
3. When assessing tumors for research purposes, examine the quantity, location, and size, and describe them using the colonic map and bar graph on the scoring form. The numerical sum of the scores obtained and number of lesions (when applicable) will determine the background colonic inflammation, the most predominant type of lesion, and the number of lesions.

Results

The protocol and images presented in this study were prepared with an Olympus URF-V flexible endoscope¹⁴ and NBI^11,15. Video images were saved in MPEG-2 format and pictures in JPG, TIF, PNG, or DICOM format with maximum 1,280 x 1,024 pixels, transferable to standard USB flash portable memories. NBI enhances the visual contrast of hemoglobin-containing tissues. Normal light and NBI imaging are key features to illustrate the normal anatomy of the colorectal mucosa and the differences between...

Discussion

There are several considerations regarding modification and troubleshooting of the endoscopic technique in mice. Critical aspects of the procedure that need to be mastered during the use of flexible endoscopy include the adjustment of the air volume needed to prevent abdominal distension and respiratory compromise of the mouse, and minor technical aspects of view control with coordination of the angulation and torque controls. Torque control is important to minimize the risk of intestinal perforation. When handled proper...

Materials

Name	Company	Catalog Number	Comments
Isoflurane, USP	Webster Veterinary
Surgical lubricant	Savage laboratories	surgilube, 0281-0205-45
Phosphate Buffered Saline	Thermo Scientific	SH30256
RNAlater	Ambion	AM7021
Methylene Blue 1% Aqueous Solution	Fisher Science Education	S96393
Flexible digital ureteroscope	Olympus America	URF-V
Video system center	Olympus America	VISERA Pro OTV-S7 Pro
Xenon light source	Olympus America	VISERA Pro CLV-S40 Pro
Video recorder	MediCapture, Inc.	MediCap USB200
Flexible biopsy cup forceps	Olympus America	FBC-3115
Anesthesia machine	Euthanex Corporation	EZ-7000 Classic System