Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Summary

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Abstract

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Introduction

In recent years, a substantial number of non-infectious colitis entities different from classical inflammatory bowel diseases (IBDs; i.e., Crohn's disease or colitis ulcerosa) have been clinically and histopathologically characterized in humans. The pathophysiological mechanisms leading to the development of these colitis forms are not completely understood partially because appropriate animal models are scarce. Diversion colitis is one of these recently described entities. Although the term was coined in 1980 by Glotzer¹, the initial description of a similar phenotype was given in 1972 by Morson². The disease develops in 50% to 91% of patients with diverting enterostomy, and its clinical intensity varies^3,4. Given the annual incidence of around 120,000 colostomy patients in the United States of America, this disease entity constitutes an important health problem.

The overall goal of developing this protocol was to provide a murine DC model that relies on a colitis trigger similar to that seen in human DC and that reproduces the primary histopathological features of the human disease. In contrast to other murine colitis models, colitis induction in our model does not require genetically modified animals (e.g., IL-7 transgenic mice, N-cadherin dominant negative mice, or TGFβ^-/- mice), the application of chemically irritating substances (e.g., dextran sulfate sodium (DSS)-induced colitis, or trinitrobenzene sulfonic acid (TNBS)-induced colitis), or the transfer of specific cell populations in immune deficient mice (as in the CD45RB^high transfer model of colitis) (for a review, see⁵). In contrast to other models, the intact immune system of our DC model allows assessment of the immunological mechanism involved in DC development. The limitation of mucosal inflammation to the excluded bowel segment allows assessment of its repercussion on mucosal immunity in other parts of the gastrointestinal tract, on the immune homeostasis in other immune compartments of the intestinal tract (e.g., Peyer's patches and mesenteriale lymph nodes), and on the immune homeostasis of the entire organism. Finally, our model constitutes an appropriate tool for investigating the mechanisms controlling local inflammatory stimuli originating from changes in the normal local environment for both the local microbiome as well as alimentary antigens.

Protocol

All methods described here have been approved by the veterinary government authority (Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern, (LALLF M-V)).

1. Preoperative Care and Preparation of the Animal

1. Upon arrival in the animal facility, divide animals (C57Bl/6) into groups of similar size, cage each group together, and keep groups constant throughout the experiments.
   NOTE: Use animals of the same sex. The results described were obtained with male mice.
   NOTE: If male animals are used, cage groups together starting when they are 7 weeks of age so that a hierarchy can be established, thus minimizing the risk of aggressive behavior during experiments.
2. At least one week before surgery, switch to a high energy (>14 MJ/kg) and high protein (>20%) feed containing all the essential trace elements and vitamins (for details, see list of materials).
   NOTE: Assure all mice weigh at least 25 g when surgery is performed.
3. Induce anaesthesia and analgesia by intraperitoneal injection of ketamine (87 mg/kg i.p.) and xylazine hydrochloride (13 mg/kg i.p.). Wait until the mouse tolerates mechanical stimulation, e.g. toe pitch, without motor response.
4. Secure the narcotized mouse with tapes in a supine position on a heat underlay positioned at the operation desk, guaranteeing stable positioning during operation and avoiding an overwhelming loss of body heat.
   NOTE: The heat underlay should have a surface temperature of 36 °C to 40 °C; the operating room temperature should be 21 °C.

2. Distal Colostomy Operation

1. Shave the abdominal hair. Before starting surgery, disinfect the operation field three times using alcohol 70% and an iodophor. Drape the operation field to guarantee for aseptic conditions.
2. Perform a 15-mm median laparotomy by incising the abdominal muscles and the peritoneum along the linea alba, thus minimizing blood loss.
3. Use two DeBakey atraumatic forceps, carefully pull the cecum, terminal ileum, and ascending and transverse colon from the peritoneal cavity.
   NOTE: Be careful to strictly limit mechanical manipulation of the intestine to prevent injury to mesenteric structures.
4. Identify the cecal pole, the ascending colon, and small intestine (Figures 1a and 1b).
   NOTE: Correct identification of the ascending colon is fundamental for correct placement of the colostomy. In cases where the anatomy of the ileocecal region is ambiguous, the presence of Peyer's patches identifies the small intestine, and the presence of formed stool characterizes the colon.
5. Use a ruler to determine the position of the future colostomy. It should be placed 20 mm distal to the ileocecal valve for a distal colostomy.
6. Make a second 3-mm incision in the abdominal wall in the upper right quadrant. Pull the previously identified colon segment through this incision to form a loop, being careful not to distort the loop.
7. Carefully pass a 22-gauge flexible i.v. cannula through the mesocolon. Take care not to damage mesenteric vascular structures.
8. Return the intestine to the peritoneal cavity.
9. Fix both ends of the flexible tube to the skin using simple stitches and a resorbable suture (e.g., polyglactin 910 or polyfil 4-0 1/2c).
10. Before closing the laparotomy, perform fluid resuscitation using an intraperitoneal injection of 0.5 mL 0.9% saline.
11. Close the peritoneum and the muscle layer with a continuous suture using a resorbable suture (e.g., polyglactin 910 or polyfil 4-0 1/2c). Close the skin with a continuous suture using a resorbable suture (e.g., polyglactin 910 or polyfil 4-0 1/2c).
12. Open the exteriorized colon loop by performing a subtotal transection using a fine scissor. Avoid all injury to the mesentery. Do not transect the colon completely.
13. Fix each colostomy opening using three single full-thickness stitches to the peritoneum and skin using a monofil, absorbable suture (e.g., polydioxanone or monofil 6-0 3/8s). The afferent loop, which is a functional end-colostomy, and the efferent loop, which is a mucous fistula, are clearly separated at this point (Figure 1c).
    NOTE: Operation time should be less than 20 minutes to limit fluid and thermal losses.
14. After finishing surgery, disinfect instruments using an aldehyde-free disinfection solution in an ultrasonic bath according to the instructions of the manufacturer.

3. Sham Operation (Colotomy)

1. Perform steps 1.1. through 2.4.
2. Use a ruler to determine the future colotomy position. The colotomy should be positioned the same distance from the ileocecal valve as the colostomy in the experimental group.
3. Open the colon at least two-thirds its circumference using fine scissors.
4. Close the colotomy with a single layer, full-thickness interrupted suture using a monofil, absorbable suture (e.g., polydioxanone, monofil 6-0 3/8s).
   NOTE: Operation time should be less than 20 minutes for an experimented surgeon, limiting fluid and thermal losses.
5. Perform steps 2.10. , 2.11. and 2.14.

4. Postoperative Care

1. Return animals to their cages. Provide a well-tempered atmosphere of 37 °C (e.g., with an infrared lamp) until mice are fully awake. Then, keep mice in a temperature- and humidity-regulated environment (21 °C; 30% ± 10% relative humidity). Allow free access to food and drinking water. To facilitate fluid uptake, provide additional water-soaked animal feed.
2. Start postoperative analgesia by injecting 0.1 mg/kg body weight buprenorphine s.c. when animals show response to mechanical stimulation. Be careful to avoid respiratory depression.
3. Supplement drinking water with 1 mg/mL tramadol for continued analgesia during the first postoperative week.
4. To compensate for decreased fluid intake due to reduced mobility, supply a Solid Drink Pad in the cage during the first postoperative week.
5. Weigh the animals and score animal behavior daily during the first week, every second day during remainder of the first month, and every third day during the second month using the disease severity score described in Kleinwort et al.⁶.

Results

Surgery is well tolerated both in the experimental (colostomy) and sham (colotomy) groups. Perioperative mortality should not exceed 10% when surgery and perioperative management is correctly performed. In the first postoperative week, significant weight loss is seen in both the experimental and sham groups. Animals of the sham group attain their weight nadir usually toward the fourth postoperative day, but it occurs one day later in the experimental group. Weight loss is more pronounced ...

Discussion

The murine model of DC presented in this protocol reliably reproduces the histopathological features of human DC (e.g., de novo development of lymphoid follicles in the submucosa of inflamed bowel segments, crypt shortening, and reductions in goblet cell numbers). Aside from this advantage, this model is induced by a very similar triggering factor and presents with a clinical course of moderate to mild severity as is the case in most affected humans.

To obtain reproducible re...

Materials

Name	Company	Catalog Number	Comments
Operation material
heat underlay, 6 watt	ThermoLux, Witte + Sutor GmbH	461265
Ethanol 70% (with methylethylketon)	Pharmacie of the University Hospital Greifswald
Wooden applicators, small cotton head onesided, wooden stick, 150 mm length	Centramed GmbH, Germany	8308370	for disinfection of operation field
Scissor	Aesculap (BRAUN)	BC064R
Atraumatic DeBakey forceps	Aesculap (BRAUN)	OC021R
Needle holder	Aesculap (BRAUN)	BM012R
Vasofix Safety i.v. cannula 22G	Braun Melsungen AG, Germany	4268091S-01
VICRYL Plus 4-0 violet braided; V-5 17 m 1/2c; 70 cm	ETHICON, Inc.	VCP994H
PDS II 6-0 monofil; V-18 13 mm 3/8c; 70 cm	ETHICON, Inc.	Z991H
BD Plastipak 1 mL (Syringes with needle with sterile interior)	BD Medical	305501
Triacid-N (N-Dodecylpropan-1,3-diamin)	ANTISEPTICA, Germany	18824-01	disinfection of surgical instruments in ultrasonic bath
Medications
ketamine 10%, 100 mg/mL (ketamine hydrochloride)	selectavet, Dr. Otto Fischer GmbH	9089.01.00	87 mg/kg i.p.
Xylasel, 20 mg/mL (xylazine hydrochloride)	selectavet, Dr. Otto Fischer GmbH	400300.00.00	13 mg/kg i.p.
NaCl 0.9%	Braun Melsungen AG, Germany	6697366.00.00
Buprenovet 0.3 mg/mL (buprenorphine)	Bayer, Germany	PZN: 01498870	0.1 mg/kg s.c.
Tramal Drops, 100 mg/mL (tramadol hydrochloride)	Grünenthal GmbH, Germany	10116838	1 mg/mL drinking water
Ceftriaxon-saar 2 g (ceftriaxone)	Cephasaar GmbH, Germany	PZN: 08844252	25 mg/kg body weight i.p.
metronidazole 5 mg/mL	Braun Melsungen AG, Germany	PZN: 05543515	12.5 mg/kg body weight i.p.
Food
ssniff M-Z Ereich	ssniff, Germany	V1184-3
Solid Drink Dehyprev Vit BIO, pouches	TripleATrading, the Netherlands	SDSHPV-75
Equipment
Nikon Eclipse Ci-L	Nikon Instruments Europe BV, Germany		light microscopy
VetScan HM 5	Abaxis, USA	770-9000	Veterinary hematology analyzer of 50 µl venous EDTA-blood
Bandelin SONOREX (Ultrasonic bath)	Bandelin electronics, Germany	RK 100 H	disinfection of surgical instruments
Software
NIS-Element BR4 software	Nikon Instruments Europe BV, Germany
GraphPad Prism Version 6	GraphPad Software, Inc.