This work was supported by grants from Crohn&#8217;s and Colitis Foundation (D.C.M) and New York Crohn&#8217;s Foundation (D.C.M. and A.J.D.).&#160;The authors thank Ms. Carmen Ferrara for assistance with creating the video accompaniment to this article.

All procedures described here were approved by the Institutional Animal Care and Use Committee of Weill Cornell Medicine.&#8221; To: &#8220;All procedures described here were approved by the Institutional Animal Care and Use Committees of Weill Cornell Medicine and Stony Brook University.
1. Colonoscopy and wound induction
<ol>
	<li>Pre-assemble the components of the endoscope by first inserting the 1.9 mm rigid bore endoscope into the sheath (Figure 1A-...

<ol>
	<li>Boal Carvalho, P., Cotter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+Healing+in+Ulcerative+Colitis:+A+Comprehensive+Review.">Mucosal Healing in Ulcerative Colitis: A Comprehensive Review.</a> Drugs. 77 (2), 159-173 (2017).</li><li>Chen, M. L., Sundrud, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+Networks+and+T-Cell+Subsets+in+Inflammatory+Bowel+Diseases.">Cytokine Networks and T-Cell Subsets in Inflammatory Bowel Diseases.</a> Inflammatory Bowel Diseases. 22 (5), 1157-1167 (2016).</li><li>Habtezion, A., Nguyen, L. P., Hadeiba, H., Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+Trafficking+to+the+Small+Intestine+and+Colon.">Leukocyte Trafficking to the Small Intestine and Colon.</a> Gastroenterology. 150 (2), 340-354 (2016).</li><li>Halfvarson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+human+gut+microbiome+in+inflammatory+bowel+disease.">Dynamics of the human gut microbiome in inflammatory bowel disease.</a> Nature Microbiology. 2, 17004 (2017).</li><li>Johansson, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+penetrate+the+normally+impenetrable+inner+colon+mucus+layer+in+both+murine+colitis+models+and+patients+with+ulcerative+colitis.">Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis.</a> Gut. 63 (2), 281-291 (2014).</li><li>Luissint, A. C., Parkos, C. A., Nusrat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+the+Intestinal+Barrier:+Leukocyte-Epithelial+Cell+Interactions,+Cell+Junction+Remodeling,+and+Mucosal+Repair.">Inflammation and the Intestinal Barrier: Leukocyte-Epithelial Cell Interactions, Cell Junction Remodeling, and Mucosal Repair.</a> Gastroenterology. 151 (4), 616-632 (2016).</li><li>Pineton de Chambrun, G., Blanc, G., Peyrin-Biroulet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+evidence+supporting+mucosal+healing+and+deep+remission+as+important+treatment+goals+for+inflammatory+bowel+disease.">Current evidence supporting mucosal healing and deep remission as important treatment goals for inflammatory bowel disease.</a> Expert Review of Gastroenterology &amp; Hepatology. 10 (8), 915-927 (2016).</li><li>Fung, K. Y., Putoczki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Models+of+Inflammatory+Bowel+Disease+and+Colitis-Associated+Cancer.">In Vivo Models of Inflammatory Bowel Disease and Colitis-Associated Cancer.</a> Methods in Molecular Biology. 1725, 3-13 (2018).</li><li>Jurjus, A. R., Khoury, N. N., Reimund, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+inflammatory+bowel+disease.">Animal models of inflammatory bowel disease.</a> Journal of Pharmacological and Toxicological Methods. 50 (2), 81-92 (2004).</li><li>Mizoguchi, A., Takeuchi, T., Himuro, H., Okada, T., Mizoguchi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+engineered+mouse+models+for+studying+inflammatory+bowel+disease.">Genetically engineered mouse models for studying inflammatory bowel disease.</a> Journal of Pathology. 238 (2), 205-219 (2016).</li><li>Seno, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+colonic+mucosal+wound+repair+requires+Trem2+signaling.">Efficient colonic mucosal wound repair requires Trem2 signaling.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (1), 256-261 (2009).</li><li>Alam, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microenvironment+of+injured+murine+gut+elicits+a+local+pro-restitutive+microbiota.">The microenvironment of injured murine gut elicits a local pro-restitutive microbiota.</a> Nature Microbiology. 1, 15021 (2016).</li><li>Alam, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+signaling+regulates+commensal-mediated+mucosal+homeostasis+and+restitution+and+requires+formyl+peptide+receptor+1.">Redox signaling regulates commensal-mediated mucosal homeostasis and restitution and requires formyl peptide receptor 1.</a> Mucosal Immunology. 7 (3), 645-655 (2014).</li><li>Kuhn, K. A., Manieri, N. A., Liu, T. C., Stappenbeck, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-6+stimulates+intestinal+epithelial+proliferation+and+repair+after+injury.">IL-6 stimulates intestinal epithelial proliferation and repair after injury.</a> PLoS One. 9 (12), 114195 (2014).</li><li>Leoni, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+A1,+formyl+peptide+receptor,+and+NOX1+orchestrate+epithelial+repair.">Annexin A1, formyl peptide receptor, and NOX1 orchestrate epithelial repair.</a> Journal of Clinical Investigation. 123 (1), 443-454 (2013).</li><li>Manieri, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosally+transplanted+mesenchymal+stem+cells+stimulate+intestinal+healing+by+promoting+angiogenesis.">Mucosally transplanted mesenchymal stem cells stimulate intestinal healing by promoting angiogenesis.</a> Journal of Clinical Investigation. 125 (9), 3606-3618 (2015).</li><li>Miyoshi, H., Ajima, R., Luo, C. T., Yamaguchi, T. P., Stappenbeck, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt5a+potentiates+TGF-beta+signaling+to+promote+colonic+crypt+regeneration+after+tissue+injury.">Wnt5a potentiates TGF-beta signaling to promote colonic crypt regeneration after tissue injury.</a> Science. 338 (6103), 108-113 (2012).</li><li>Neurath, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+tumor+development+and+wound+healing+using+endoscopic+techniques+in+mice.">Assessment of tumor development and wound healing using endoscopic techniques in mice.</a> Gastroenterology. 139 (6), 1837-1843 (2010).</li><li>Montrose, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonoscopic-Guided+Pinch+Biopsies+in+Mice+as+a+Useful+Model+for+Evaluating+the+Roles+of+Host+and+Luminal+Factors+in+Colonic+Inflammation.">Colonoscopic-Guided Pinch Biopsies in Mice as a Useful Model for Evaluating the Roles of Host and Luminal Factors in Colonic Inflammation.</a> American Journal of Pathology. 188 (12), 2811-2825 (2018).</li><li>Bruckner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+endoscopy+for+in+vivo+multimodal+imaging+of+carcinogenesis+and+assessment+of+intestinal+wound+healing+and+inflammation.">Murine endoscopy for in vivo multimodal imaging of carcinogenesis and assessment of intestinal wound healing and inflammation.</a> Journal of Visualized Experiments. (90), (2014).</li></ol>

The authors have nothing to disclose.

Ensuring consistent and accurate biopsies as well as measurements of wound size are of paramount importance when attempting to effectively evaluate the rate of wound closure in this model. Therefore, several measures should be taken to be confident that the procedures are being correctly carried out. First, the depth of the biopsy must not be too shallow or deep. If too shallow, there will not be a sufficient window to evaluate wound closure. Figure 2 demonstrates an optimal biopsy depth and...

The gastrointestinal (GI) tract is a complex organ system&#160;given its multiple functions, host cell types (e.g. epithelial, immune, stromal, etc.) as well as trillions of microbes. In light of this complexity, diseases of the GI tract often involve the interplay of all of these factors. For example, inflammatory bowel diseases (IBD) are associated with cycles of inflammation and remission in the GI tract, involving the activation of inflammatory cells, dysbiosis, and epithelial repair1,2,3,4,5,6,7. Having appropriate model systems to study IBD and other inflammatory conditions of the GI tract is critical for elucidating the pathogenesis of disease. Several models exist to study IBD pathogenesis including genetically engineered mice and the use of chemicals such as dextran sodium sulfate (DSS) in rodents8,9,10. Limitations of these models include an inability to precisely control the induction of inflammation as well as difficulties in evaluating wound healing. Alternative methods to mimic aspects of IBD pathogenesis could prove useful for developing therapies.
Colonoscopic-guided pinch biopsies in mice offer a useful model system to study the pathogenesis of the inflammatory response, wound healing, as well as host-microbe interactions in the colon. This approach was first used as an experimental tool in 2009, which demonstrated its utility for studying the acute inflammatory response and wound healing in the gut11. Subsequent studies utilized this technique to evaluate the roles of different signaling pathways as well as the gut microbiota, in colonic wound healing11,12,13,14,15,16,17,18. More recently, our group used this model to investigate the importance of sphingosine-1-phosphate signaling and bacteria in the acute response to colonic injury19. Although useful, carrying out colonoscopic-guided pinch biopsies in mice and evaluating subsequent tissue changes can be technically challenging. For instance, perforation of the bowel can occur upon induction of injury and ensuring consistent measurements of the wound bed through serial colonoscopies can be difficult. Additionally, orienting the colonic tissue properly to visualize the wound bed for histological or immunohistochemical analyses can be challenging. Although some information does exist regarding these methods18,20, a precise step-wise description of these techniques along will visual aids promises to enhance the reliability and broader utility of this model. Here, we present a detailed method to carry out colonoscopic-guided pinch biopsies in mice, track wound closure over time and prepare the tissue to enable histologic and molecular analyses of the wound bed. Creating a standard method to carry out these techniques can expand the use of this model to study previously uninvestigated mediators that are potentially important for GI inflammation and wound repair.

<table>
	<tbody>
		<tr>
			<td>Biopsy forceps, 3 Fr</td>
			<td>Karl Storz</td>
			<td>61071ZJ</td>
			<td></td>
		</tr>
		<tr>
			<td>Coloview Tower system</td>
			<td>Karl Storz</td>
			<td>contact company</td>
			<td></td>
		</tr>
		<tr>
			<td>Examination sheath, 9 Fr, Kit</td>
			<td>Karl Storz</td>
			<td>61029DK</td>
			<td></td>
		</tr>
		<tr>
			<td>Hopkins telescope, 0&#39;, 1.9 mm x 10 cm</td>
			<td>Karl Storz</td>
			<td>64301AA</td>
			<td></td>
		</tr>
		<tr>
			<td>isofluorane</td>
			<td>Covetrus</td>
			<td>2905</td>
			<td></td>
		</tr>
		<tr>
			<td>methylene blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140</td>
			<td></td>
		</tr>
		<tr>
			<td>micro iris scissors</td>
			<td>Integra</td>
			<td>18-1619</td>
			<td></td>
		</tr>
		<tr>
			<td>NIH ImageJ</td>
			<td>NIH</td>
			<td>N/A</td>
			<td>software available for free download from: https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Pawfly MA-60 aquarium pump</td>
			<td>Amazon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpal with #10 blade</td>
			<td>Hill-Rom</td>
			<td>372610</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The small items (lens, sheath, biopsy forceps) needed to carry out biopsies are shown in Figure 1 along with indicators of proper assembly of these components. Figure 2 shows representative images of acceptable views of the wound bed in order to accurately quantify the size of the wound bed and closure rate of the wound. An example of an ex vivo view of the wound bed is shown in Figure 3A inclusive of indicators of the perimeter of ...

Watch this Scientific Journal Video about Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes at JoVE.com

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

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 ...

This method provides an ideal approach for studying colonic wound healing and the molecular and histologic changes that occur following injury. This method can also shed light on IBD pathogenesis. The main advantage of this technique is to precisely control the location of injury and the timing of the wound healing process.Individuals performing this method for the first time may have difficulty inserting the colonoscope into the mouse's colon and creating consistent wounds. Repetitively practicing these techniques should overcome these issues. Given the multiple coordinated steps and nuance involved with this procedure, visual demonstration of this method is critical.Before beginning the procedure, insert a 1.9 millimeter rigid bore endoscope into an endoscope sheath. And attach the assembled endoscope to the light source and video imaging device per the manufacturer's instructions. Use the provided tubing to attach the air pump to the gas valve on the left side of the sheath next to the working channel.Ensure that the working channel is in the open position and insert three French biopsy forceps through the working channel. Advance the forceps to the end of the sheath without protruding out of the sheath. Next, place the anesthetized mouse onto an endoscopic staging platform on its ventral side and confirm the appropriate level of sedation by lack of response to pedal reflex.After applying eye ointment, fill a three milliliter syringe with an attached rat gavage needle with room temperature PBS. And insert the needle approximately one centimeter into the mouse's anus. Gently infuse with PBS until the fecal material has been cleared.Several fecal pellets should exit the mouse along with the PBS that was infused. Insert the assembled endoscope 0.5 centimeters into the anus and advance the biopsy forceps into the cleared lumen of the rectum until the full jaws of the forceps are beyond the end of the sheath. Turn the forceps 90 degrees so that the jaws open in an east-west orientation and open the tips and advance the forceps approximately one centimeter, closing and retracting the forceps in one smooth, quick motion to harvest the biopsy.To avoid fully insufflating the colon while performing the biopsy, leave the right side of the gas valve open. Immediately after the biopsy, depress the foot pedal attached to the colonoscope recording device to initiate the video recording. And firmly press an index finger against the right side of the gas valve to completely cover the opening, forcing air into the endoscope and thus into the colon.Retract the forceps back out of the sheath and into the rectal lumen while in the closed position. And place the forceps against the rectal wall immediately above the wound until the base of the jaws is aligned with the top edge of the viewing field. Continue fully insufflating the colon until a clear view of the wound can be observed.To obtain the best measurements of the wound bed, be patient then place the colonoscope at a precise angle so that you get your best image of the wound bed. Open the video in an appropriate software program and advance the video to a frame showing a point in time at which the wound bed can be easily visualized. And at which the closed forceps are above the wound bed and against the rectal wall, and the wall is taut.At each time point, obtain a snapshot of this frame and code the file name to ensure that the measurements of the wound beds are carried out in a blinded manner. To quantify the size of the wound bed, open the images in ImageJ and use the Free Hand selections tool to draw a perimeter around the wound. Under Analyze, select Measure.And the value of that measurement will automatically populate in the Results window. When all of the measurements have been acquired, calculate the size of the wound on subsequent days relative to the size on day zero in a spreadsheet. At the appropriate experimental endpoint, open the skin and abdominal muscle layers to expose the body cavity of the experimental animal and place closed forceps under the colon.Gently lift the colon to release it from the underlying mesentery and cut the tissue at its midpoint and at the anus to collect it from the mouse. Use a 20 milliliter syringe filled with ice-cold PBS and equipped with a rat gavage needle to flush out the fecal contents. And place the cleared colon onto a piece of filter paper.Open the colon longitudinally, taking care that the mesenteric side is face down against the filter paper. And use a Pasteur pipette to cover the mucosa with 0.2%methylene blue. After a few seconds, drain off the excess stain and view the colon under a dissecting microscope to locate the wound bed.Use four-inch Micro Iris Scissors to cut around the edge of the bed, being careful not to cut into the muscle layer. And use fine point tweezers to transfer the dissected tissue into a tube for snap freezing and or storage. At the appropriate experimental endpoint, open the harvested colon longitudinally on a piece of filter paper, mesenteric side down.Gently cover the tissue with your fixative of choice. Cover the tissue with parafilm in a sealed container for 4 to 6 hours before storage in 70%ethanol. On the day of the processing, remove the parafilm and add 0.2%methylene blue to the tissue, as demonstrated.After draining off the excess stain, place the tissue under a dissecting microscope to locate the wound bed. Using a scalpel with a number 10 blade, cut directly through the center of the wound bed and continue cutting through the remainder of the colon in a straight line, such that the colon is cut in half lengthwise. For cryosectioning, embed the colon pieces such that the side that was cut by the scalpel is face down in a base mold half-filled with tissue freezing medium.Secure the tissue in place with fine tweezers and place the base mold onto a metal plate or thick aluminum foil on top of dry ice to harden the freezing medium. Once the bottom portion of the medium is frozen, fill the remaining volume of the base mold with freezing medium at room temperature. And return the mold to the dry ice until the entire tissue block is frozen.Then, transfer the mold to a 80 degrees Celsius freezer until sectioning. Here, representative images of acceptable views of the wound bed for an accurate quantification of the size of the wound bed and closure rate of the wound are shown. In this ex-vivo view of a wound bed, indicators of the perimeter of the wound bed and where to cut the tissue to enable visualization of the wound bed upon sectioning can be observed.In this representative image, an H and E stain section of a wound bed can be clearly observed. It is important to acquire instant images of the wound bed at all time points to ensure an accurate assessment of the wound healing rate. Following the pinch biopsy, different agents of interest can be injected into the wound bed to test their effects on the wound healing rate.

performing-colonoscopic-guided-pinch-biopsies-mice-evaluating

Research

JoVE Journal

Immunology and Infection

4.2K Views.  Stony Brook University, Stony Brook, NY. This method provides an ideal approach for studying colonic wound healing and the molecular and histologic changes that occur following injury. This method can also shed light on IBD pathogenesis. The main advantage of this technique is to precisely control the location of injury and the timing of the wound healing process.Individuals performing this method for the first time may have difficulty inserting the colonoscope into the mouse's colon and creating consistent wounds. Repetitive...