Name: murine appendectomy model of chronic colitis associated colorectal cancer by precise localization of caecal patch
Uploaded: 2019-08-24T14:00:00
Description: Watch this Scientific Journal Video about Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch at JoVE.com

This work is partially supported by Fundamental Research Funds for the Central University (G2018KY0302), University Fundamental Research Development Fund (KT00062), National Natural Science Foundation of China (81870380), and Clinical Research Award of the First Affiliated Hospital of Xi&#8217;an Jiaotong University in China (NO.XJTU1AF-CRF-2015-029). The authors thank Dr. Chengxin Shi for his technical suggestions during the early exploration phase of the murine appendectomy model, as well as the pathologist Dr. Xi Liu for evaluation of H&#38;E staining results of colitis and colorectal tumors. Y.L. performed the surgery demonstration,&#160;did data analysis, and wrote the draft of the manuscript; J.L., G.L., Z.P., and Y.M. took part in the surgical preparation, tissue collections, and video production; M.Z. performed the flow cytometry and ELISA; Q.W. and H.X. provided the technical support of generating a clinically relevant murine model; R.X.Z. designed the study, supervised the research, and wrote and proofed the manuscript; J.S. reviewed the manuscript.

All animal procedures were approved by the Institutional Animal Care and Use Committee of Xi&#8217;an Jiaotong University (No. XJTULAC2019-1023).
1. Mice appendectomy
<ol>
	<li>House 8&#8211;10-week-old C57BL/6 male mice in a certified specific-pathogen free (SPF) environment for 1 week prior to surgery.</li>
	<li>Prepare the following sterile surgical instruments: one pair of micro-scissors, one pair of micro-forceps, two sizes (4-0 and 8-0) of sterilized non-absorbable sutures, an electric...

A murine appendectomy model of colitis-associated colorectal cancer was obtained using surgical steps with a high survival rate in mice. In most cases, since the caecum was positioned under the abdominal wall (Supplementary Table 1, Supplementary Table 2, and Figure 2), it was difficult to prejudge its location without laparotomy. In this surgical protocol, an easy step of touching the bump was introduced, and quantitative evaluation of the cecum location was also provided a...

The clinical appendectomy is a standard surgical procedure involving removal of the appendix mostly due to inflammation (e.g., appendicitis)1,2,3. However, the biological function of the vermiform human appendix remains controversial4,5,6. The appendix has been regarded as a vestigial remnant projecting from the cecum in the large bowel. Until recently, evolutionary, immunological, morphological, and microbiological studies have suggested that the appendix may possess distinct functions. These roles include the production of immunoglobins (e.g., IgA and IgG), a variety of B cells and T cells critical for adaptive immune responses within the gut-associated lymphoid tissues (GALTs), and replenishment of the large bowel with commensal microbiotas6,7,8,9,10,11,12.
Clinical epidemiological studies of patients with prior appendectomy or acute appendicitis have also revealed its potential roles in the pathogenesis of human diseases, such as inflammatory bowel disease (IBD), colorectal cancer, and non-gastrointestinal disorders (e.g., Parkinson&#8217;s disease and cardiovascular disease)13,14,15,16,17,18. For example, a large Asian population cohort study with 75,979 appendectomy patients recently showed a significant association between appendectomy and subsequent development of colorectal cancer, one of the most common malignancies with a high incidence and mortality14,19. Accordingly, establishing a suitable animal appendectomy model that resembles a human will be helpful to investigate the biological functions and molecular mechanisms of the appendix in the disease pathogenesis.
Many mammals possess an appendix or appendix-like organ, including primates, lagomorphs (e.g., rabbits), some rodents, and marsupials20. For small and commonly used laboratory animals, the rabbit possesses the vermiform appendix morphologically resembling the human21,22, but GALT in the rabbit is extremely large compared to that in humans, since the majority of lymphoid tissues are also found in Peyer&#8217;s patches located in both small and large intestines21. Additionally, the rabbit shows a different lymphoid follicular structure, T cell distribution, and immunoglobulin density from the human, which makes the studying of their appendices inappropriate21.
Mice are the most commonly used animal model to study human pathophysiology and test the various existing and novel therapuetics23,24,25. The single white large lymphoid cluster at the apex of the caecum in mice, known as the caecal patch, is thought to perform functions similar to the human appendix26,27,28. Yet, it is practically difficult to separate the caecal patch from caecum in mice. So far, the common surgical procedures for inducing appendicitis in a mouse model involve a relatively large incision (e.g., 1&#8211;2 cm) through the abdominal wall to gain access to the whole caecum (Supplemental Table 1)29,30,31,32,33,34,35,36.
Herein, to generate an appendectomy model associated with gastrointestinal disease, this report presents a facile surgical protocol for caecal patch removal in mice. This is followed by the combined administration of the genotoxic agent AOM and pro-inflammatory agent DSS for the induction of colitis-associated colorectal cancer similar to that seen in humans. IBD has been shown to be a risk factor of intestinal cancer37,38. The combination of AOM/DSS-induced chronic colitis-associated colorectal cancer has been well-established, and readers can refer to Neufert et al., and Thaker et al. for detailed procedures39,40. This reproducible and rapid murine appendectomy model can be used to study appendix-modulated bowel inflammation and colon microbiota, especially in the development and progression of IBD and colorectal cancer.

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			<td>Azoxymethane&#65288;AOM</td>
			<td>Sigma-Aldrich,Inc.</td>
			<td>A5486</td>
			<td></td>
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			<td>Dextran Sulfate Sodium Salt&#65288;DSS</td>
			<td>MP Biomedicals,Inc.</td>
			<td>160110</td>
			<td></td>
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			<td>Entoiodine</td>
			<td>Shanghai likon high technology disinfection co. LTD</td>
			<td>310102</td>
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			<td>digital caliper</td>
			<td>Ningbo yuanneng trading co. LTD</td>
			<td>4859263</td>
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			<td>4-0 Silk Sutures</td>
			<td>Yuanlikang co. LTD</td>
			<td>20172650032</td>
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			<td>8-0 Prolene Sutures</td>
			<td>Yuanlikang co. LTD</td>
			<td>20172650032</td>
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			<td>Electric coagulation pen</td>
			<td>Chuang mei medical equipment co. LTD</td>
			<td>28221777292</td>
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			<td>disposable syringe 1ml</td>
			<td>Shengguang medical products co. LTD</td>
			<td>3262-2014</td>
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			<td>disposable syringe 10ml</td>
			<td>Shengguang medical products co. LTD</td>
			<td>3262-2014</td>
			<td></td>
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			<td>75% Medicinal alcohol</td>
			<td>Shandong anjie high-tech disinfection technology co. LTD</td>
			<td>371402AAJ008</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital sodium salt</td>
			<td>Sigma-Aldrich,Inc.</td>
			<td>57-33-0</td>
			<td></td>
		</tr>
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			<td>Physiological Saline</td>
			<td>Shandong qidu pharmaceutical co. LTD</td>
			<td>H37020766</td>
			<td></td>
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			<td>Absorbent Cotton Swab</td>
			<td>Henan ruike medical co., LTD</td>
			<td>RK051</td>
			<td></td>
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			<td>Surgical Instruments-Ophthalmic</td>
			<td>Jinzhong Shanghai co.LTD</td>
			<td>WA3050</td>
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murine appendectomy model of chronic colitis associated colorectal cancer by precise localization of caecal patch

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal cancer, inflammatory bowel disease, and Parkinson&#8217;s disease. To study the function of the appendix, a gut disease-associated murine appendectomy model has been established and its step-by-step protocol is described here. This report introduces a facile protocol for caecal patch removal in mice followed by the chemical induction of chronic colitis-associated colorectal cancer using a combination of dextran sulfate sodium (DSS) and azoxymethane (AOM). IgA specific cells and IgA concentration were significantly reduced upon removal of the caecal patch in male C57BL/6 mice&#160;compared to those in the sham group. Simultaneously administering 2% DSS and AOM resulted in nearly 80% mice survival in both sham and appendectomy groups without significant body weight loss. Histological results confirmed colonic inflammation and different degrees of adenocarcinoma. This model can be used for the study of the functional role of the appendix in maintaining gut microbiota homeostasis and pathogenesis of gut colitis and malignancies, as well as for the potential development of drug targeting therapies.

Establishment of murine appendectomy model
This murine appendectomy model of chronic colitis associated colorectal cancer can be generated by following the sequential surgical and induction steps as illustrated in Figure 1. The most frequent positions of caecum are in the left and right iliac fossa followed by the middle line of the abdomen (Figure 2). The successful rate of pre-localization of caecum prior to the abd...

Watch this Scientific Journal Video about Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch at JoVE.com

Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch

The presented protocol describes a facile surgical removal of the appendix (caecal patch) in a mouse followed by the induction of inflammatory bowel disease-associated colorectal cancer. This murine appendectomy model enables investigation of the biological role of the appendix in the pathogenesis of human gastrointestinal disease.

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal ...

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