Name: murine endoscopy for in vivo multimodal imaging of carcinogenesis and assessment of intestinal wound healing and inflammation
Uploaded: 2014-08-26T14:00:00
Description: Watch this Scientific Journal Video about Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation at JoVE.com

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 a...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="1467">
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			<td height="21" style="height:21px;width:469px;">Name</td>
			<td style="width:423px;">Company</td>
			<td style="width:320px;">Catalogue Number</td>
			<td style="width:255px;">Comment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;"></td>
			<td style="width:423px;"></td>
			<td style="width:320px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Reagents</td>
			<td style="width:423px;"></td>
			<td style="width:320px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alfalfa-free diet</td>
			<td>Harlan Laboritories, Madison, USA</td>
			<td style="width:320px;">2014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Azoxymethane (AOM)</td>
			<td style="width:423px;">Sigma - Aldrich, Deisenhofen, Germany</td>
			<td style="width:320px;">A5486</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bepanthen eye ointment</td>
			<td style="width:423px;">Bayer, Leverkusen, Germany</td>
			<td style="width:320px;">80469764</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran sulphate sodium (DSS)</td>
			<td>TdB Consulatancy, Uppsala, Sweden</td>
			<td>DB001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Eosin</td>
			<td style="width:423px;">Sigma - Aldrich, Deisenhofen, Germany</td>
			<td style="width:320px;">E 4382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Ethylenediaminetetraacetic acid (EDTA)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:423px;">Sigma - Aldrich, Deisenhofen, Germany</td>
			<td style="width:320px;">E 9884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Falcon Tube 50ml</td>
			<td>BD Biosciences, Erembodegem, Belgium</td>
			<td style="width:320px;">352070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Florene 100V/V</td>
			<td>Abbott, Wiesbaden, Germany</td>
			<td style="width:320px;">B506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescein-Isothiocyanat (FITC)&#8211;dextrane&#160;</td>
			<td style="width:423px;">Sigma - Aldrich, Deisenhofen, Germany</td>
			<td style="width:320px;">FD4-250MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Haematoxylin&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:423px;">Sigma - Aldrich, Deisenhofen, Germany</td>
			<td style="width:320px;">HHS32-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Isopentane (2- Methylbutane)</td>
			<td style="width:423px;">Sigma - Aldrich, Deisenhofen, Germany</td>
			<td style="width:320px;">M32631-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:469px;">Methylene blue</td>
			<td style="width:423px;">Merck, Darmstadt, Germany</td>
			<td style="width:320px;">1159430025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">O.C.T. Tissue Tek compound&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:423px;">Sakura, Zoeterwonde, Netherlands</td>
			<td style="width:320px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Omnican F - canula</td>
			<td style="width:423px;">Braun, Melsungen, Germany</td>
			<td style="width:320px;">9161502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Phosphate buffered saline, PBS</td>
			<td style="width:423px;">Lonza, Verviers, Belgium</td>
			<td style="width:320px;">4629</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Chloride 0,9%</td>
			<td>Braun, Melsungen, Germany</td>
			<td style="width:320px;">5/12211095/0411</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Standard diet</td>
			<td>Altromin, Lage, Germany</td>
			<td style="width:320px;">1320</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue-Tek Cryomold</td>
			<td>Sakura, Leiden, Netherlands</td>
			<td style="width:320px;">4566</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:469px;">Vitro &#8211; Clud&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:423px;">R. Langenbrinck, Teningen, Germany</td>
			<td style="width:320px;">04-0002&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td style="width:320px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td style="width:320px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td style="width:320px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AIDA Control</td>
			<td>Karl Storz - Endoskope, Tuttlingen, Germany</td>
			<td style="width:320px;">20 096020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bandpass filter</td>
			<td>Semrock, Rochester, USA</td>
			<td style="width:320px;">HC 716/40</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bandpass filter</td>
			<td>Semrock, Rochester, USA</td>
			<td style="width:320px;">HC 809/81</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biopsy Forceps, 3 Fr., 28cm</td>
			<td>Karl Storz - Endoskope, Tuttlingen, Germany</td>
			<td style="width:320px;">61071ZJ</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dell Monitor</td>
			<td>Dell, Frankfurt am Main, Germany</td>
			<td style="width:320px;">U2412Mb</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Examination Sheath, 9 Fr.</td>
			<td>Karl Storz - Endoskope, Tuttlingen, Germany</td>
			<td style="width:320px;">61029D</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Examination Sheath, 9 Fr.</td>
			<td>Karl Storz - Endoskope, Tuttlingen, Germany</td>
			<td style="width:320px;">61029C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fiber Optic Light Cable, 3.5mm</td>
			<td>Karl Storz - Endoskope, Tuttlingen, Germany</td>
			<td style="width:320px;">69495NL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescein Blue Filter System</td>
			<td>Karl Storz - Endoskope, Tuttlingen, Germany</td>
			<td style="width:320px;">20100032</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescein Barrier Filter</td>
			<td>Karl Storz - Endoskope, Tuttlingen, Germany</td>
			<td style="width:320px;">20100033</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Foot switch</td>
			<td>Karl Storz - Endoskope, Tuttlingen, Germany</td>
			<td style="width:320px;">20010430</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HOPKINS Telescope, 1.9mm, Length 10cm</td>
			<td>Karl Storz - Endoskope, Tuttlingen, Germany</td>
			<td style="width:320px;">1830231</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SCB D-light P&#160;</td>
			<td>Karl Storz - Endoskope, Tuttlingen, Germany</td>
			<td style="width:320px;">20 133720</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SCB tricam SL II</td>
			<td>Karl Storz - Endoskope, Tuttlingen, Germany</td>
			<td style="width:320px;">20 2230 20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tubing set instruments VETPUMP II</td>
			<td>Karl Storz - Endoskope, Tuttlingen, Germany</td>
			<td style="width:320px;">69811</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tricam PDD PAL</td>
			<td>Karl Storz - Endoskope, Tuttlingen, Germany</td>
			<td style="width:320px;">20221037</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UniVet Porta</td>
			<td>Groppler Medizintechnik, Deggendorf, Germany</td>
			<td style="width:320px;">BKGM 0451</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vetpump 2</td>
			<td>Karl Storz - Endoskope, Tuttlingen, Germany</td>
			<td style="width:320px;">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...

Watch this Scientific Journal Video about Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation at JoVE.com

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 ...

Research

JoVE Journal

Medicine

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

Murine Model of Wound Healing

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A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

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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 ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

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 ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...