We thank Sonja Dufentester and Elke Weber for expert technical assistance. We thank Faekah Gohar for proofreading the manuscript and Stefan Br&#252;ckner for medical informatics support. This work was supported by an interdisciplinary grant from the Else-Kr&#246;ner-Fresenius-Stiftung (2012_A94). D. Bettenworth was supported by a research fellowship from the Faculty of Medicine, Westf&#228;lische Wilhelms-Universit&#228;t M&#252;nster. M. Br&#252;ckner was supported by a &#8220;Gerok&#8221; rotational position of the Deutsche Forschungsgemeinschaft (DFG SFB1009B8).&#160;We thank Heike Blum for illustration of the mouse cartoon.

All animal experiments were approved by the Landesamt f&#252;r Natur, Umwelt und Verbraucherschutz (LANUV) according to German Animal Protection Law.
1. Materials and Experimental Setup
<ol>
	<li>Animal care
	<ol>
		<li>Use female or male mice of any strain weighing 20 to 25 g and house them according to local animal care legislation.</li>
		<li>Feed mice with special chow for rodents and apply alfalfa-free chow at least three days prior to fluorescence examinations to minimize endoluminal auto-fluorescence.</li>
		<li>Provide autoclaved drinking water ad libitum.</li>
	</ol>
	</li>
	<li>Induction of acute DSS-induced colitis
	<ol>
		<li>Prepare a 3% (w/v) dextran sulfate sodium (DSS, molecular weight: 36,000&#8211;50,000 Da) solution by dissolving 3 g of DSS in 100 ml autoclaved water. Offer this solution as the exclusive drinking water to mice ad libitum and calculate 5 ml of DSS-solution per mouse/day. Feed control mice with autoclaved water without DSS ad libitum9.</li>
	</ol>
	</li>
	<li>Induction of colorectal cancer
	<ol>
		<li>Dissolve mutagenic azoxymethane (AOM) (CAUTION! May cause cancer and genetic damage!) in sterile isotonic saline to obtain a final concentration of 1 mg/ml. Apply a single dose of 10 mg AOM per kg bodyweight intra-peritoneally using a 1 ml syringe (30 G)10.</li>
		<li>Challenge mice (excluding control mice) with repetitive cycles of 3% (w/v) DSS from day 0 to 7, day 14 to 21, day 28 to 35 and day 42 to 49 to induce inflammatory driven colorectal cancerogenesis. Feed mice with autoclaved water only in between these challenges (see Figure 4A for a detailed time schedule). Feed control mice with autoclaved water throughout the experiment.</li>
	</ol>
	</li>
	<li>Preparation of fluorescence endoscopy (FE)
	<ol>
		<li>Use Fluorescein-Isothiocyanat (FITC) - dextran (molecular weight 70,000 Da; FITC:Glucose = 1:250) for detection of colonic adenoma by visual enhancement of dysplasia associated vascular pattern.</li>
		<li>Administer 60 mg FITC-conjugated dextran diluted in 100 &#181;l PBS intravenously 5 min before fluorescence endoscopic examination.</li>
	</ol>
	</li>
	<li>Anesthesia
	<ol>
		<li>Provide continuous isoflurane supply for anesthesia (1.5 L O2/min; 1.5&#8211;2 vol% isoflurane [2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane]). Use special veterinary anesthesia equipment with a facemask to tightly control anesthesia.</li>
	</ol>
	</li>
	<li>Preparation of enema
	<ol>
		<li>Instill 2 ml of fluid enema (contents: disodium hydrogenphosphate 1.5% (w/v) and sodium dihydrogenphosphate 11% (w/v)) into the colon if significant fecal loading is suspected that may obscure the view.</li>
	</ol>
	</li>
</ol>
2. Technical Equipment
<ol>
	<li>Use a veterinary endoscopic workstation that is developed and approved for the use of small animal endoscopy. Connect the workstation to a camera unit, a xenon light source, an air pump and to a conventional PC monitor for white light endoscopy. Then connect the camera and the miniature rigid telescope (1.9 mm outer diameter, 10 cm length; Figure 5).</li>
	<li>Use endoscope sheath with working channel (Figure 5D) for the application of biopsy forceps or injection tube. Use the sheath without working channel for diagnostic colonoscopy.</li>
	<li>Configure the settings of the light source for fluorescence endoscopy to excite used tracers (e.g., 490 nm for FITC-conjugated dextran). Additionally, integrate an appropriate bandpass filter between the telescope and the camera (e.g., 525 nm for FITC-conjugated dextran).</li>
	<li>Insert flexible biopsy forceps (3 Charr., 28 cm) through the working channel of the endoscope to obtain biopsies.</li>
	<li>Introduce flexible injection tube (0.96 mm) through the working channel for topical, intramucosal or endoluminal administration of diagnostic or therapeutic agents.</li>
	<li>Use a heatable examination table with a temperature of 42 &#176;C. This prevents mice becoming hypothermic during the examination.</li>
</ol>
3. Anesthesia of Animals
<ol>
	<li>Place mouse in a small but leakproof box and administer isoflurane (100% (v/v), 5 vol%, 3 L/min). Wait until the mouse loses consciousness.</li>
	<li>Transfer the mouse onto the examination table for endoscopy. Continue isoflurane inhalation via face mask with a dose 100% v/v, 1.5 vol%, 1.5 L/min. Always apply eye ointment to prevent eye dryness while under anesthesia.</li>
	<li>Evaluate efficacy of anesthesia by checking the reflexes. Check the &#8216;turn around reflex&#8217;: if sufficiently anaesthetized, a mouse laying on its back should not turn around. Check the &#8216;toes reflex&#8217;: when anesthesia is adequate, soft pinching in between the animal&#8217;s toes should not lead to withdrawal of leg (stage of surgical tolerance).</li>
</ol>
4. Colonoscopy
<ol>
	<li>Lay anesthetized mouse prone /on its back on the examination table.</li>
	<li>Administer 2 ml of enema via buttoned cannula into the colon if significant fecal loading is suspected that may obscure the view. Wait for mice to defecate after administering the enema. Insert the endoscope very carefully to avoid perforation.</li>
	<li>Open both valves of the sheath with one of them being connected to the air pump. Seal the other valve with your index finger to dispense air. Inflate colon with air, slowly and carefully, especially in case of biopsy or injection.</li>
	<li>Advance endoscope only as far as the right colonic flexure to avoid perforation (4&#8211;5 cm from anus).</li>
	<li>Diagnostic colonoscopy
	<ol>
		<li>Examine the mucosa for inflammatory or malignant alterations while pulling back the endoscope. Pull back slowly to assess the whole circumference of the bowel. Assess intraluminal pathologies using appropriate established scoring systems as required.</li>
		<li>To warrant identical endoscopic position for image acquisition during repetitive visualizations of wound areas, note the distance between the murine anus and the mucosal lesion. Moreover, use the tip of the biopsy forceps as a spacer to achieve identical distance between the endoscope and the wound area during image acquisition. The wound size is related to the size of the endoscope sheath, which comprises 3 mm. 
		NOTE: Place endoscope in identical position by optical comparison with photo documentation of previous examinations. Measure lesions in the same angle and distance at each follow up endoscopic examination.</li>
	</ol>
	</li>
	<li>Biopsy procedure
	<ol>
		<li>Take biopsies with the help of two investigators. Introduce the biopsy forceps carefully through the working channel until the tip of the forceps is visible on the monitor to the second investigator. Open and close forceps carefully to avoid perforation.</li>
		<li>Move forceps to the site of pathology.</li>
	</ol>
	</li>
	<li>Injection procedure
	<ol>
		<li>Perform injection procedure with the help of two investigators. Pre-fill flexible injection tube (0.96 mm) completely with the agent to be administered. Push tube through the working channel until the cannula (30 G) is visible on the monitor to the second investigator. Prepare the fine syringe and gently administer the requested amount of diagnostic or therapeutic agent. Injection volumes should be 50 &#181;l maximum.</li>
		<li>Insert the needle into the submocosa at an angle of 15&#8211;30 degrees. Face the bevel in direction of the mucosa. The mucosa shows a characteristic lifting sign after successful injection.</li>
	</ol>
	</li>
	<li>Fluorescence endoscopy (FE)
	<ol>
		<li>Administer 60 mg FITC-conjugated dextran diluted in 100 &#181;l PBS intravenously prior to fluorescence endoscopic examination.</li>
		<li>Check the optimal time point between injection of your fluorescent labeled tracer and the imaging procedure which is dependent on tracer pharmacology. Configure settings of bandpass filter system in accordance to excitation and emission wavelength of the tracer used. Perform fluorescence endoscopy for non-specific blood volume tracers (e.g., FITC) immediately after intravenous injection of the fluorescent dye to assess the vascular pattern of the mucosal surface.</li>
		<li>Consider imaging several hours after tracer application in case of targeted tracers or &#8216;smart probes&#8217; to provide a better target to background-ratio.</li>
		<li>Perform photo- and video-documentation of the results.</li>
	</ol>
	</li>
</ol>
5. Post-colonoscopy
<ol>
	<li>Separate the mouse in a vacant cage and lay it on a paper towel to protect the mouse from aspirating the litter. Warm the mouse with a redlight lamp to prevent hypothermia. Observe the mouse and do not leave unattended until it has regained sufficient consciousness to maintain sternal recumbency. Once completely conscious, place the mouse back to its respective cage.</li>
	<li>At the end of experiment, place mouse in a small but leakproof box and administer CO2 (100% (v/v), 100 vol%, 3 L/min). Wait until the mouse loses complete consciousness and stops breathing. Dispatch mouse by neck fracture. Perform abdominal laparotomy and explant the colon. Open the colon longitudinally and wash it for further histological or molecular evaluation.</li>
</ol>

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The authors have nothing to disclose.

Epithelial wound healing is an ongoing process. Continuous physiological exfoliation of surface cells within the gastrointestinal mucosa occurs requiring frequent regeneration of epithelial cells 16. Consequently, impaired wound healing has an immense impact on several diseases including gastrointestinal ulcers and 17 anastomotic leakage 18. Evaluation of molecular background as well as potential drug candidates to stimulate epithelial healing may only be incompletely performed in cell cu...

Animal models have greatly enriched our understanding of numerous intestinal pathologies. The laboratory mouse (Mus musculus) has emerged as a prime animal model in biomedical research due to its abundant genetic and genomic information and is readily available in transgenic and knockout strains. In addition to enhancing understanding disease pathogenesis, animal models are also importantly used for testing drug candidates as well as preclinical diagnostic or therapeutic interventions. However, despite the variety of mouse models mimicking human disease, many diagnostic and interventional options that are routinely used in patient care are not available for mice. Accordingly, surveillance strategies to monitor the course of murine disease or the effect of therapeutic interventions are often limited to indirect observations or post mortem analyses. While non-invasive procedures exist for monitoring mice vitality like disease activity indices, quantification of weight loss or gain, blood, urine and feces analyses, these are only indirect indicators and are biased by inter-individual variability. Additionally, post mortem analyses prevent longitudinal observations at repetitive time points. Sophisticated imaging techniques to monitor disease activity in mice have only recently been introduced 1,2. Although these imaging techniques allow for repetitive analyses, they only provide a descriptive and often imprecise view on the gut, do not enable direct mucosal visualization or allow diagnostic or therapeutic interventions such as biopsy acquisition or topical and intramucosal application of drug candidates.
Recently, high-resolution endoscopic systems for use in live mice have been developed 3,4. For the first time these endoscopic techniques allow direct visualization of endoluminal colonic disease pathologies such as wound healing or intestinal inflammation providing objective, real-time status allowing longitudinal studies in the same animal at repetitive time points. Aside from allowing repeated biopsies in an individual mouse, endoscopic systems can also be used to therapeutically influence a distinct tumor or localized inflammation by allowing direct application of a substance to the area of interest. Furthermore, as therapeutic and control substances can be delivered directly to the area of interest, this can be performed in the same mouse, excluding inter-individual variability. These systems have now been employed for the assessment of colonic inflammation, wound healing, laparoscopic liver biopsies and orthotopic induction of liver tumors 8 and tumor development using various scoring systems such as the murine endoscopic index of colitis severity (MEICS) 5-7. MEICS consists of five parameters to assess inflammation: thickening of the colon wall, changes of the vascular pattern, presence of fibrin, granularity of the mucosal surface, and stool consistence.
In this protocol we describe the use of rigid endoscopy in murine models of intestinal wound healing, inflammation and colon cancer. First, we demonstrate the endoscopic evaluation of wound healing and colonic inflammation as well as longitudinal assessment of colitis activity and the study of cancerogenesis in the murine colon. Beyond the descriptive use of murine endoscopy, we provide detailed instructions on the use of endoscopic instrumentation to obtain biopsies, and the topical and intramucosal application of different components of interest (e.g., drug candidates or tumor cells). Finally, we demonstrate the use of murine fluorescence endoscopy, which employs sophisticated molecular imaging techniques, in the setting of colorectal tumors.

<table><tbody><tr><td colspan="4">Reagents</td></tr><tr><td>Alfalfa-free diet</td><td>Harlan Laboritories, Madison, USA</td><td>2014</td><td></td></tr><tr><td>Azoxymethane (AOM)</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>A5486</td><td></td></tr><tr><td>Bepanthen eye ointment</td><td>Bayer, Leverkusen, Germany</td><td>80469764</td><td></td></tr><tr><td>Dextran sulphate sodium (DSS)</td><td>TdB Consulatancy, Uppsala, Sweden</td><td>DB001</td><td></td></tr><tr><td>Eosin</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>E 4382</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>E 9884</td><td></td></tr><tr><td>Falcon Tube 50 ml</td><td>BD Biosciences, Erembodegem, Belgium</td><td>352070</td><td></td></tr><tr><td>Florene 100 V/V</td><td>Abbott, Wiesbaden, Germany</td><td>B506</td><td></td></tr><tr><td>Haematoxylin</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>HHS32-1L</td><td></td></tr><tr><td>Isopentane (2-Methylbutane)</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>M32631-1L</td><td></td></tr><tr><td>Methylene blue</td><td>Merck, Darmstadt, Germany</td><td>1159430025</td><td></td></tr><tr><td>O.C.T. Tissue Tek compound&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Sakura, Zoeterwonde, Netherlands</td><td>4583</td><td></td></tr><tr><td>Omnican F - canula</td><td>Braun, Melsungen, Germany</td><td>9161502</td><td></td></tr><tr><td>Phosphate buffered saline, PBS</td><td>Lonza, Verviers, Belgium</td><td>4629</td><td></td></tr><tr><td>Sodium Chloride 0.9%</td><td>Braun, Melsungen, Germany</td><td>5/12211095/0411</td><td></td></tr><tr><td>Standard diet</td><td>Altromin, Lage, Germany</td><td>1320</td><td></td></tr><tr><td>Tissue-Tek Cryomold</td><td>Sakura, Leiden, Netherlands</td><td>4566</td><td></td></tr><tr><td>Vitro &amp;ndash; Clud&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>R. Langenbrinck, Teningen, Germany</td><td>04-0002&amp;nbsp;</td><td></td></tr><tr><td colspan="4">Equipment</td></tr><tr><td>AIDA Control</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>20 096020</td><td></td></tr><tr><td>Bandpass filter</td><td>Semrock, Rochester, USA</td><td>HC 716/40</td><td></td></tr><tr><td>Bandpass filter</td><td>Semrock, Rochester, USA</td><td>HC 809/81</td><td></td></tr><tr><td>Biopsy Forceps, 3 Fr., 28 cm</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>61071ZJ</td><td></td></tr><tr><td>Dell Monitor</td><td>Dell, Frankfurt am Main, Germany</td><td>U2412Mb</td><td></td></tr><tr><td>Examination Sheath, 9 Fr.</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>61029D</td><td></td></tr><tr><td>Examination Sheath, 9 Fr.</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>61029C</td><td></td></tr><tr><td>Fiber Optic Light Cable, 3.5 mm</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>69495NL</td><td></td></tr><tr><td>Fluorescein Blue Filter System</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>20100032</td><td></td></tr><tr><td>Fluorescein Barrier Filter</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>20100033</td><td></td></tr><tr><td>Foot switch</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>20010430</td><td></td></tr><tr><td>HOPKINS Telescope, 1.9 mm, Length 10 cm</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>1830231</td><td></td></tr><tr><td>SCB D-light P&amp;nbsp;</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>20 133720</td><td></td></tr><tr><td>SCB tricam SL II</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>20 2230 20</td><td></td></tr><tr><td>Tubing set instruments VETPUMP II</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>69811</td><td></td></tr><tr><td>Tricam PDD PAL</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>20221037</td><td></td></tr><tr><td>UniVet Porta</td><td>Groppler Medizintechnik, Deggendorf, Germany</td><td>BKGM 0451</td><td></td></tr><tr><td>Vetpump 2</td><td>Karl Storz - Endoskope, Tuttlingen, Germany</td><td>69321620</td><td></td></tr></tbody></table>

murine endoscopy for in vivo multimodal imaging of carcinogenesis and assessment of intestinal wound healing and inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

In vivo monitoring of intestinal wound healing 
During routine endoscopy, mucosal wounds were induced mechanically by miniature biopsy forceps with a diameter of 3 French (equals 1 mm; Figure 1A). Subsequently, wound healing was monitored by daily endoscopic examinations and quantified by measurement of the residual wound area using image editing software, e.g., ImageJ (Figure 1B). The individual wound closure over time is expresse...

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

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

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

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

murine-endoscopy-for-vivo-multimodal-imaging-carcinogenesis

Research

JoVE Journal

Medicine

18.5K Views.  University Hospital Münster. Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy. 

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

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...

Cancer Research

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

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).

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.

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Bioengineering

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

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) face and is frequently associated with a poor clinical outcome. While, for instance, GvHD manifestations of the skin are usually responsive to established immune-suppressive therapies and are, hence, not taking a fatal course, the presence and the intensity of intestinal GvHD, especially of the mid-to-lower parts of the gut, strongly influence the outcome and overall survival of patients with acute GvHD. Therapeutic options are essentially limited to the classic immune-suppressive agents yielding only moderate disease-mitigating effects. Hence, detailed knowledge about the tissue-resident immune cascade, changes in the intestinal microbiota, and the stromal response prior, upon, and after intestinal GvHD onset are urgently needed to understand the events and mechanisms underlying its pathogenesis and to develop innovative therapeutic options. Murine models of GvHD are frequently employed to identify and functionally assess molecules and pathways putatively driving intestinal GvHD. However, means to specifically monitor and evaluate intestinal inflammation over time are essentially lacking since established scores to assess and grade acute GvHD are routinely comprised of various parameters which rather reflect&#160;systemic GvHD manifestations. The detailed evaluation of intestinal GvHD has been restricted to studies using euthanized mice, thereby essentially excluding longitudinal (i.e., kinetic) analyses of the colonic compartment under a given experimental condition (e.g., antibody-mediated blockade of a proinflammatory cytokine) in live mice (i.e., in vivo). The mini-endoscopic in situ assessment of the distal colon of allo-HCT-treated mice described here allows a) a detailed macroscopic evaluation of different aspects of intestinal inflammation and b) the option to collect tissue samples for downstream analyses at various time points over the course of the observation period. Overall, the mini-endoscopic approach provides a major advance in preclinical noninvasive monitoring and assessment of intestinal GvHD.

Here, we present a protocol that describes allogeneic hematopoietic stem cell transplantation and allows repetitive mini-endoscopic evaluations of the distal colon in situ for the presence, characteristics, and severity of colonic inflammation within live mice suffering from intestinal graft-versus-host disease.

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell ...

Immunology and Infection

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount importance when studying diseases of the gastrointestinal (GI) tract. The murine colonic pinch biopsy model is a useful tool to define these processes. Additionally, the interplay between gut luminal content (e.g., microbes) and the colon can be studied. However, wound induction and the ability to track wound closure over time in a reliable manner can be challenging. Moreover, tissue preparation and orientation must be carried out in a standardized way to optimally interrogate histologic and molecular changes. Here, we present a detailed method describing biopsy-induced injury and the monitoring of wound closure through repeat colonoscopies. An approach is described that ensures consistent and reproducible measurements of wound size, the ability to collect the wound bed for molecular analyses as well as visualize the wound bed upon sectioning of tissues. The ability to successfully carry out these techniques allows for studies of the acute injury response, wound healing and luminal-host interactions within the colon.

Here, we provide a detailed description of the procedure to induce colonoscopic-guided pinch biopsies in mice and track wound closure in real time. Additionally, methods for the preparation of tissues for histological, immunohistochemical and molecular analyses of the wound bed are provided.

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount ...

Laser Ablation and Intravital Microscopy to Study Intestinal Remodeling

Investigating intestinal recovery in vivo is an exquisite technical challenge. A lack of longitudinal imaging protocols has prevented deeper insights into the cell and tissue scale dynamics that orchestrate intestinal regeneration. Here, we describe an intravital microscopy method that locally induces tissue damage at the single crypt scale and follows the regenerative response of the intestinal epithelium in living mice. Single crypts or larger intestinal fields were ablated by a high-intensity multiphoton infrared laser in a time- and space-controlled manner. Subsequent long-term repetitive intravital imaging enabled the tracking of the damaged areas over time and allowed for the monitoring of crypt dynamics during tissue recovery over a period of multiple weeks. Crypt remodeling events such as crypt fission, fusion, and disappearance were observed in the neighboring tissue upon laser-induced damage. This protocol enables the study of crypt dynamics both in homeostatic and pathophysiological settings, such as aging and tumor initiation.

Here, we present a method to obtain images of the intestine upon laser-induced wounding. By exposing the mouse intestine to a multiphoton laser, the loss of a single or multiple crypt(s) is induced locally. By repeatedly imaging the damaged area over months, the real-time dynamics of intestinal recovery is captured.

Investigating intestinal recovery in vivo is an exquisite technical challenge. A lack of longitudinal imaging protocols has prevented deeper insights into ...

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

Summary

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Chapters in this video

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

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

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

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

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes

Laser Ablation and Intravital Microscopy to Study Intestinal Remodeling