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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		<tr>
			<td>Entoiodine</td>
			<td>Shanghai likon high technology disinfection co. LTD</td>
			<td>310102</td>
			<td></td>
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			<td>digital caliper</td>
			<td>Ningbo yuanneng trading co. LTD</td>
			<td>4859263</td>
			<td></td>
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			<td>4-0 Silk Sutures</td>
			<td>Yuanlikang co. LTD</td>
			<td>20172650032</td>
			<td></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>
			<td></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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		<tr>
			<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>
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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 ...

The overall protocol describes easily performed surgical procedures of removing the appendix known as caecal patch in mice. The murine appendectomy model can be used to study the biological roles of appendix in pathogenesis of the human gastrointestinal disease. The human appendix is a disputable organ.Recent studies have suggested that the appendix plays an important role in maintaining the gut health. To study the biological function of appendix, generating a disease-associated appendectomy animal model is critical. In this protocol, we will describe a simple and a safe surgical steps of removing appendix, known as caecal patch in mice.To ensure the success of this model, there are three key steps as you will see in the following video. First, locate the caecum of the mice before the surgery. Second, make a small incision in the abdomen.Third, disinfect and close the caecum stump. The first part, mice appendectomy. Prepare sterile surgical instruments and cleaning solutions before the surgery.Anesthetize and secure the mouse on a platform in a supine position by placing four strips of medical adhesive tape across the limbs to prevent postural movement during the surgery. Gently touch the whole abdomen of the mouse to find the feeling of bump. Cover the drape and disinfect the shaved area of mouse abdomen as wide as possible using two times of entoiodine.Make a longitudinal incision ranging from 0.5 centimeter to one centimeter at the midline of abdomen. According to the predetermined location of the caecum, seek the caecum and gently exteriorize it for approximately one centimeter long to identify the caecal patch. Use a pre-warmed and sterile physiological saline to hydrate the intestine.To prevent potential complications of post-operative bleeding and infection, ligate the mesenteric blood vessels of caecum using the 8-0 suture. Hydrate the caecum with the saline. Mark the resection region using the 4-0 suture with open loop near the apex of the caecum at the proximal colon.Cut off the caecal patch below the marked resection using micro-scissors and wipe out the residual caecal content with medical cotton swabs. Disinfect the stump of caecum with entoiodine. Close the stump with running suture using the 8-0 suture.Carefully remove the 4-0 thread loop, previously used for marking the resection at the stump of caecum. Sterilize the sutured position using entoiodine. Rehydrate the cutting area with the saline.Close the musculature layer with running suture using 8-0 suture thread. Close the skin layers using interrupted sutures with 4-0 suture thread. Disinfect the surgical cut twice with entoiodine.Then, remove iodine using 75%medical alcohol. Gently flip the mouse back. Subcutaneously inject 0.1 milligram per kilogram body weight of buprenorphine, and 0.4 milliliters of physiological saline.And rest the mouse on the heat pad, until it returns to consciousness. Put the mouse back to the sterile cage, and closely monitor the mouse for the signs of pain for three days post the surgery to ensure its recovery. The second part:Induction of Colitis-Associated Colorectal Cancer with Azoxymethane, AOM, and Dextran Sodium Sulfate, DSS, seven days post-appendectomy.Thaw one aliquot of AOM and dilute it to the 1 milligram per milliliter using 0.9%normal saline. The entire preparation process is carried out in the fume hood. Dissolve four grams DSS powder in 200 milliliters autoclaved water solution to prepare 2%DSS.Each cage contains five mice. Make an intraperitoneal injection of AOM with 0.01 milliliter per gram per mouse basing on its weight. Replace the autoclaved water with 2%DSS for five days.The mice will be fed with autoclaved water for additional two cycles. The third part:Results. Flow and ELISA results indicate the successful removal of the caecal patch.This procedure can contribute to significant reduction of IgA-specific plasma cells, an obvious change of secretory IgA in large bowel, compared to 3%DSS, which has high mortality rate and significant weight change because of severe bloody stool, anus prolapse, and abdominal abcess. The 2%DSS is suitable for the whole process for its high survival rate to nearly 80%and reasonable weight change in both sham and appendectomy groups. As shown in these images, we have successfully induced the colitis-associated colorectal tumors.In H&E staining images, black arrow indicates mucosal ulcer, arrowhead indicates adenocarcinoma, and a white arrow indicates neutrophil infiltration between glands. Different stages of adenocarcinoma can be seen in both sham and appendectomy groups. This cost-effective Murine Appendectomy Model can be used for answering some important questions such as:what's the role of appendix in the regulation of gut bacteria and how it participates in the immune response in the gut-associated lymphoid tissue.Remember, to avoid the damage in adhesion of intestine, the pre-location of caecum and small skin incision are important. In addition, we used the 2%DSS to induce colitis. But a higher concentration of DSS may be used for other mice strains.

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