Method Article

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

DOI:

10.3791/59921

⸱

August 24th, 2019

 ,  ,  ,  ,  ,  ,  ,  ,  , 
1Department of General Surgery, The First Affiliated Hospital, Xi'an Jiaotong University, 2Institute of Medical Research, Northwestern Polytechnical University, 3School of Life Sciences, Northwestern Polytechnical University, 4Department of Medicine, Xi'an Jiaotong University, 5Princess Margaret Cancer Center, University of Health Network, 6Department of Pathology, Nanjing Medical University

In This Article

Summary

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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.

Abstract

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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’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 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.

Introduction

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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’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’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., 1–2 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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Protocol

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All animal procedures were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University (No. XJTULAC2019-1023).

1. Mice appendectomy

  1. House 8–10-week-old C57BL/6 male mice in a certified specific-pathogen free (SPF) environment for 1 week prior to surgery.
  2. 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 coagulation pen with needle holder, 75% medical alcohol, iodine-based scrub (e.g., entoiodine), and a package of sterile cotton swabs.
  3. Fill a 10 mL syringe with pre-warmed 0.9% physiological saline for the abdominal flush and hydration during surgery.
  4. Anesthetize a non-fasted mouse intraperitoneally (i.p.) with 1% sodium pentobarbital at a dose of 100 mg/kg. Check for the depth of anesthesia by the lack of response to pedal reflexes.
    NOTE: A single anesthetic dose is administered to ensure a full sedative effect in the mouse. Under the circumstance of short procedures, 50 mg/kg of sodium pentobarbital i.p. may be sufficient.
  5. Gently shave the abdominal hair with an electric shaver.
  6. Lay the mouse on the heating pad to prevent hypothermia.
  7. 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.
  8. Gently touch the whole abdomen to find the feeling of a bump.
    NOTE: In most cases, the feeling of a bump always indicates the exact position of the caecum. To avoid possible damage to the rest of the intestine, this pre-locating of the caecum prior to abdominal incision is important.
  9. Cover with a sterile drape and disinfect the shaved area of the abdomen by applying alternating surgical scrubs of iodine-based solution and 75% alcohol. Repeat the process 2x.
  10. Make a longitudinal incision ranging from 0.5–1.0 cm at the midline of the abdomen.
  11. According to the pre-determined location of the caecum, reach the caecum and gently exteriorize (~1 cm) to identify the caecal patch. Use a sterile, prewarmed saline solution of physiological pH to hydrate the intestine.
    NOTE: The caecal patch of a mouse is part of the caecum and characterized by the presence of white ovoid follicles on the surface.
  12. To prevent potential complications of the post-operative bleeding and infection, ligate the mesenteric blood vessels of the caecum using the 8-0 suture. Hydrate the caecum with sterile saline.
  13. Mark the resection region using the 4-0 suture with an open loop near the apex of the caecum at the proximal colon.
    NOTE: Marking the caecum with an open loop also prevents leakage of caecal content from the cut.
  14. Cut off the caecal patch below the marked resection using micro-scissors and wipe out the residual caecal content with medical cotton swabs. Then, disinfect the stump of caecum with iodine-based scrub.
  15. Close the stump with the running suture using the 8-0 suture.
  16. Carefully remove the 4-0 thread loop previously used for marking the resection at the stump of the caecum.
  17. Sterilize the sutured position with iodine-based scrub. Rehydrate the surgical site with saline again.
  18. Close the musculature layer with the running suture using 8-0 suture thread.
  19. Close the skin layers using interrupted sutures with 4-0 suture thread.
  20. Disinfect the surgical cut 2x with iodine-based scrub, then remove iodine using 75% medical alcohol.
  21. Gently flip the mouse back, subcutaneously (s.c.) inject 0.1 mg/kg body weight of buprenorphine and 0.4 mL of physiological saline and let the mouse rest on the heat pad until returning to consciousness.
  22. Put the mouse back to the sterile cage and closely monitor for signs of pain for 3 days post-surgery to ensure recovery.
    NOTE: Post-operative application of 0.05 mg/kg buprenorphine may be needed for pain relief of the individual mouse. Allow mice to recover for 7 days after surgery for further induction of colitis-associated colorectal cancer.

2. Induction of chronic colitis-associated colorectal cancer with AOM and DSS

NOTE: Perform this procedure 7 days post-appendectomy.

  1. Prepare AOM stock solution by dissolving 25 mg of AOM in 2.5 mL of 0.9% sterile saline at a concentration of 10 mg/mL.
    NOTE: AOM is light-sensitive.
    1. Aliquot 2.5 mL of prepared AOM stock solution into 5 mL glass tubes wrapped with aluminum foil and store at -20 ˚C each time upon use.
    2. Thaw one aliquot of AOM once and dilute it to a concentration of 1 mg/mL with 0.9% sterile saline (ratio 1:10).
      CAUTION: AOM is extremely carcinogenic; hence, perform the entire preparation procedure in a fume hood.
  2. Dissolve 4 g of DSS powder in 200 mL of autoclaved water to prepare 2% (w/v) DSS solution.
    NOTE: The concentration of DSS may vary depending on the mouse strain, sex, and induction cycle; 3% DSS may be used for other mice strains.
  3. Simultaneously administer the freshly prepared AOM/DSS to the mice. To do so, follow the steps below.
    1. Intraperitoneally inject 0.01 mL/g of freshly prepared AOM working solution for each mouse and replace the autoclaved water with 2% DSS solution for 5 days ad libitum.
      NOTE: Each cage contains five mice; regularly check the DSS drinking bottle to ensure no precipitate occurs during the treatment period.
    2. Weight and closely monitor each mouse every day.
      NOTE: During the administration, euthanize mice with up to 20% weight loss compared to its initial weight or signs of the huddle, squint, hypothermia, and poor activity.
  4. Provide a fresh bottle of autoclaved water on the day 6 post-AOM/DSS administration until day 21 (Figure 1B).
    NOTE: This is one complete cycle comprising of 21 days.
  5. Repeat steps 2.3–2.4 for an additional two cycles and sacrifice the mice on day 70.

3. Assessment of colonic inflammation and tumor (70 days post-AOM administration)

  1. Sacrifice the mice by CO2 inhalation at a fill rate of 10% in the euthanasia chamber followed by cervical dislocation.
  2. Harvest the entire colon from above the ileo-colic junction to the anus.
  3. Expose the lumen side by opening the colon longitudinally. Cut the whole colon into 10 cm long pieces and fix the colon tissue in 10% formalin for 72 h for hematoxylin and eosin (H&E) staining.

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Results

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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...

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Discussion

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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...

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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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’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&E staining results of colitis and colorectal tumors. Y.L. performed the surgery demonstration, 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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Azoxymethane(AOM)Sigma-Aldrich,Inc.A5486
Dextran Sulfate Sodium Salt(DSS)MP Biomedicals,Inc.160110
EntoiodineShanghai likon high technology disinfection co. LTD310102
digital caliperNingbo yuanneng trading co. LTD4859263
4-0 Silk SuturesYuanlikang co. LTD20172650032
8-0 Prolene SuturesYuanlikang co. LTD20172650032
Electric coagulation penChuang mei medical equipment co. LTD28221777292
disposable syringe 1mlShengguang medical products co. LTD3262-2014
disposable syringe 10mlShengguang medical products co. LTD3262-2014
75% Medicinal alcoholShandong anjie high-tech disinfection technology co. LTD371402AAJ008
Pentobarbital sodium saltSigma-Aldrich,Inc.57-33-0
Physiological SalineShandong qidu pharmaceutical co. LTDH37020766
Absorbent Cotton SwabHenan ruike medical co., LTDRK051
Surgical Instruments-OphthalmicJinzhong Shanghai co.LTDWA3050

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Murine Appendectomy ModelCaecal Patch RemovalColitis Associated Colorectal CancerDextran Sulfate SodiumAzoxymethane InductionIgA Specific CellsGut Microbiota HomeostasisSurgical Procedure ProtocolHistological Analysis ResultsFlow ELISA Detection

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