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Özet

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

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

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 controversial^4,5,6. 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 microbiotas^{6,7,8,9,10,11,12}.

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 mortality^14,19. 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 human^21,22, 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 therapuetics^23,24,25. 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 appendix^26,27,28. 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 cancer^37,38. 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 procedures^39,40. 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.

Protocol

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.

Representative Results

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 abdomen incision using the palpation method is nearly 70% (Supplemental Information, Table S2). The appendectomy procedures mainly comprise of six important steps (Figure 3). During the appendectomy surgery, a midline incision as small as 0.5–1.0 cm in length is critical to minimize surgical trauma (Figure 3A). In the case of intraperitoneal leakage, ligating mesangial vessels and closing the stump of the caecum are necessary, using running sutures instead of simple ligation (Figure 3B,C,D,E).

Evaluation of IgA as a biomarker in appendectomy mice

To confirm the appendectomy, the large intestine of mice and fecal content were harvested post-surgery to determine the levels of IgA specific plasma cells and secretory IgA (sIgA) concentrations. The appendectomy mice exhibited a significant reduction in the percentage of B220-IgA+ specific plasma cells compared to the sham group (Figure 4A,B). Moreover, compared to their own initial state prior to surgery, sham mice maintained the fecal sIgA concentration over 14 days post-surgery, whereas appendectomy group showed a significant slgA reduction on day 14 (Figure 4C).

Validation of colitis-associated colorectal cancer in appendectomy mice

Compared to 3% DSS, at which most mice in the appendectomy group showed severe complications and significant body weight loss (Figure 5A), 2% DSS in combination with AOM provided 80% of the survival rate in both sham and appendectomy groups, with reasonable body weight changes (Figure 5E). At the end of three cycles of treatment, the full-length large bowel was harvested for pathological assessment. Visual inspection and H&E staining showed the colonic tumors with colon inflammation and different degrees of adenocarcinomas (well-, moderate-, and poorly differentiated adenocarcinomas) in mice treated with 2% AOM/DSS (Figure 6).

Figure 1: Schematic illustration of establishing murine appendectomy model of chronic colitis-associated colorectal cancer.
(A) A flow chart of the main surgical steps (first three are critical) of appendectomy procedure. (B) Induction of colorectal cancer. Seven days after the appendectomy, AOM and DSS were administered simultaneously. Mice weights were recorded at various times (red triangle). At day 70 post-surgery, mice were sacrificed, and intestinal tissues were harvested for further assessments. Please click here to view a larger version of this figure.

Figure 2: Pre-determination of the caecal patch location in mice prior to abdominal incision.
(A) Summary of the likelihood of finding caecum position. Anatomical images of three most common positions of the caecum at the (B) low right, (C) middle, and (D) low left abdomen of the mouse. Please click here to view a larger version of this figure.

Figure 3: Critical steps of murine appendectomy.
(A) Small incision arranged 0.5–1.0 cm; (B) Pulling out the exact portion of the caecal patch after pre-position of the caecum. (C) Ligation of mesenteric vessels accompanying the caecum. (D) Marking the cutting region of the caecum with the 4-0 suture. (E) Closing the stump with running sutures using the 8-0 suture. (F) Closing the muscular layer with interrupted sutures using the 8-0 suture. Please click here to view a larger version of this figure.

Figure 4: Quantification of IgA specific plasmas cells and fecal sIgA concentration after appendectomy.
(A) Representative dot plots for cells stained with anti-IgA and B220 in the whole large intestine of sham and appendectomy mice 1 day and 14 days post-surgery. Lymphocytes in lamina propria were freshly prepared and stained with FITC anti-mouse IgA, PE anti-mouse CD45R/B220 for flow cytometry. Numbers within plots indicate percentages of cells in respective areas; (B) Quantitative determination of IgA specific plasma cells in sham and appendectomy groups. The data represents mean ± SD; n = 5–8 mice; *p < 0.05, **p < 0.01, and ***p < 0.001. (C) Fecal sIgA concentrations before and after the surgery. Fecal contents were collected at designated timepoints, and levels of sIgA were tested by ELISA. Each color dot represents the same mouse over a period of 14 days. Statistical analysis was performed using an unpaired t-test, with p < 0.05 considered statistically significant. Please click here to view a larger version of this figure.

Figure 5: Animal survival status of chronic colitis-associated colorectal cancer murine model induced by AOM/DSS combination after appendectomy.
(A) The survival curve obtained using 3% DSS for the colitis induction in sham and appendectomy groups. Severe complications of anal prolapse (B) and mural abscess indicated the endpoint of the animal (C). (D) The survival curve obtained using 2% DSS for the colitis induction in sham and appendectomy groups; (E) Average body weight change of mice over 70 days. The data represents mean ± SD, n = 6 mice. Please click here to view a larger version of this figure.

Figure 6: Images of chronic colitis-associated colorectal tumors induced by combination treatment of 2% DSS and 10 mg/kg AOM.
Photographs of the dissected colon with tumors under (A) visual inspection and (B) animal operation microscope. H&E staining images of colonic tissues showing (C) colonic inflammation, (D) well-differentiated adenocarcinoma, (E) moderately differentiated adenocarcinoma, and (F) highly differentiated adenocarcinoma. Black arrow indicates the mucosal ulcer, black arrowhead indicates adenocarcinoma, white arrow indicates neutrophil infiltration between glands. Please click here to view a larger version of this figure.

Supplementary Table 1: Literature summary of surgical methods used for appendicitis induction in commonly used mice strains. Please click here to download this file.

Supplementary Table 2: Locations of the caecum in mice after laparotomy. Exposed organ after incision indicates the organ found directly at the pre-located position after laparotomy. The position of the caecum shows the caecum location relative to the abdominal region/the caecum position relative to the intestine. Exposed indicates that the caecum was below the muscular layer of the abdomen and above other parts of the intestine. Embedded indicates that the caecum was covered by the intestine. Please click here to download this file.

Discussion

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 as guidance to increase the precision for pre-localization of the cecum (Figure 2). The exploitation of anatomical features to remove the caecal patch minimized disruption of unintended parts of the bowel, thereby decreasing the rate of potential complications of appendectomy and infection of the abdominal cavity.

Three critical steps are involved in this model. First, visual examination and palpation prior to laparotomy can be helpful to determine the general location of the caecum. Despite the fact that the caecum is most likely present at the left iliac region of the abdomen, the caecal patch protruding from the apex of the caecum is often dissociative (i.e., move around or embedded) and is more likely to be found beneath the middle peritoneum (Figure 2). Second, a small incision at the middle line of the abdomen and accurate exposure of caecal patch is preferred. This is because there is a thin spot at the middle line of the abdomen, at which cutting into this position may decrease potential injury of the abdominal musculature layer. In addition, to avoid the volvulus upon appendectomy, which is detrimental to mice, accurate exposure of the caecal patch minimizes the unintended disturbance of intestine, reducing the risk of volvulus occurrence. Moreover, minimal exposure of the abdominal cavity can ensure that the intestine remains uncontaminated, warm, and moist; otherwise, excessive exposure may cause significant heat loss and drying of tissues⁴¹.

Third, disinfection and closure of the caecal stump with running sutures instead of simple ligation are necessary to prevent potential bleeding and disinfection of caecal contents of the stump. The level of IgA-specific plasma cells and fecal sIgA in the large bowel of mice are significantly reduced by an appendectomy, suggesting that the caecal patch is the main site for the production of IgA at least in the initial period³³. In the large bowel, sIgA function as a mucosal health defender by stabilizing the normal colonization of commensal flora and counteracting pathogenic microbes⁴². Removal of the appendix reduces the production of IgA and may disrupt the mucosal homeostasis.

The high mortality rate and clinical signs of excessive colitis induction (i.e., severe bloody stool, anal prolapse, and abdominal abscess^43,44) were observed in mice treated with 3% DSS (Figure 5). However, 2% DSS induction after three cycles caused none of these complications, and the survival rate was maintained above 80%. This indicates that the combined use of DSS and AOM should be validated, since the lack of integrity of GALTs may, to some extent, reduce the tolerance of mice to the AOM and DSS combination. The observational studies from clinical-based evidence suggest that the appendix is strongly associated with the development of IBD and colorectal cancer^14,45,46. However, the biological functions and underlying mechanisms of the appendix in the pathogenesis of chronic gut diseases is still unclear. It appears that the appendix acts as an important part of GALTs and as a reservoir for gut commensal flora to regulate health or diseased states^17,18,31. Establishing this repeatable and cost-effective murine appendectomy model can be used for studying the roles of the appendix in IBD and colorectal cancer in the context of microbiota homeostasis, cancer prevention, and immunotherapy.

Materials

Azoxymethane（AOM	Sigma-Aldrich,Inc.	A5486
Dextran Sulfate Sodium Salt（DSS	MP Biomedicals,Inc.	160110
Entoiodine	Shanghai likon high technology disinfection co. LTD	310102
digital caliper	Ningbo yuanneng trading co. LTD	4859263
4-0 Silk Sutures	Yuanlikang co. LTD	20172650032
8-0 Prolene Sutures	Yuanlikang co. LTD	20172650032
Electric coagulation pen	Chuang mei medical equipment co. LTD	28221777292
disposable syringe 1ml	Shengguang medical products co. LTD	3262-2014
disposable syringe 10ml	Shengguang medical products co. LTD	3262-2014
75% Medicinal alcohol	Shandong anjie high-tech disinfection technology co. LTD	371402AAJ008
Pentobarbital sodium salt	Sigma-Aldrich,Inc.	57-33-0
Physiological Saline	Shandong qidu pharmaceutical co. LTD	H37020766
Absorbent Cotton Swab	Henan ruike medical co., LTD	RK051
Surgical Instruments-Ophthalmic	Jinzhong Shanghai co.LTD	WA3050