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Materials

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Immunology and Infection

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

Published: February 11th, 2021

DOI:

10.3791/62093

1Department of Pathology, University of Michigan, Ann Arbor

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD).

Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.

We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease.

The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

The intestinal mucosa encompasses a single layer of columnar intestinal epithelial cells (IECs), underlying lamina propria immune cells and the muscularis mucosae. Besides its role in the absorption of nutrients, the intestinal epithelium is a physical barrier that protects the body interior from luminal commensal bacteria, pathogens, and dietary antigens. In addition, IECs and lamina propria immune cells coordinate the immune response inducing either tolerance or response depending on the context and stimuli. It has been reported that the disruption of the epithelial barrier can precede the onset of pathologic mucosal inflammation and contribute to inflammatory bowel....

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All animal experiments were conducted in accordance with the guidelines and policies of the National Institutes of Health and approved by the Institutional Animal Care & Use Committee at the University of Michigan.

1. Preoperative preparation

NOTE: This method was generated employing adult mice from C57BL/6 genetic background, aged 8 - 12 weeks. All mice were kept under strict specific pathogen free conditions with ad libitum access to normal chow and water. Resul.......

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A schematic representation of the ileal loop and pcLoop models is depicted in Figure 1 and Figure 2, respectively. The anatomical pictures display the critical steps of the procedure including exteriorization of the intestinal segment (Figure 1B and Figure 2B), identification of an appropriate location for ligations that allows minimal disturbance of blood supply (

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The mechanisms responsible for dysregulation of intestinal barrier function and immune cell recruitment under pathologic conditions such as IBD are incompletely understood. Here, we detail a robust in vivo murine model that employs a well-vascularized exteriorized intestinal segment of either ileum or proximal colon and allows for assessment of intestinal permeability, neutrophil migration studies as well as other applications.

The iLoop is a non-recovery surgery that is performed on live anim.......

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The authors thank Dr. Sven Flemming of the University of Wuerzburg for his contributions to the establishment of the proximal colon loop model, Sean Watson for the management of the mouse colonies and Chithra K. Muraleedharan for helping with the acquisition of the pictures of the iLoop model. This work was supported by the German Research Foundation/DFG (BO 5776/2-1) to KB, R01DK079392, R01DK072564, and R01DK061379 to C.A.P.

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Name Company Catalog Number Comments
Equipment and Material
BD Alcohol Swabs BD 326895
BD PrecisionGlide Needle, 25G X 5/8" BD 305122
BD PrecisionGlide Needle, 30G X 1/2" BD 305106
BD 1ml Tuberculin Syringe Without Needle BD 309659
15ml Centrifuge Tube Corning 14-959-53A
Corning 96-Well Solid Black Polystyrene Microplate FisherScientific 07-200-592
Corning Non-treated Culture Dish, 10cm MilliporeSigma CLS430588
Cotton Tip Applicator (cotton swab), 6", sterile FisherScientific 25806 2WC
Dynarex Cotton Filled Gauze Sponges, Non-Sterile, 2" x 2" Medex 3249-1
EZ-7000 anesthesia vaporizer (Classic System, including heating units) E-Z Systems EZ-7000
Falcon Centrifuge Tube 50ml  VWR 21008-940
Fisherbrand Colored Labeling Tape FisherScientific 15-901-10R
Halsey Needle Holder (needle holder)  FST 12001-13
Kimwipes, small (tissue wipe) FisherScientific 06-666
1.7ml Microcentrifuge Tubes  Thomas Scientific  c2170
Micro Tube 1.3ml Z (serum clot activator tube) Sarstedt  41.1501.105
Moria Fine Scissors FST 14370-22
5ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap (35 µm nylon mesh) Falcon 352235
Puralube Vet Ointment, Sterile Ocular Lubricant Dechra 12920060
Ring Forceps (blunt tissue forceps) FST 11103-09
Roboz Surgical 4-0 Silk Black Braided, 100 YD FisherScientific NC9452680
Semken Forceps (anatomical forceps) FST 1108-13
Sofsilk Nonabsorbable Coated Black Suture Braided Silk Size 3-0, 18", Needle 19mm length 3/8 circle reverse cutting  HenrySchein SS694
Student Fine Forceps, Angled FST 91110-10
10ml Syringe PP/PE without needle Millipore Sigma  Z248029
96 Well Cell Culture Plate Corning 3799
Yellow Feeding Tubes for Rodents 20G x 30 mm Instech FTP-20-30
Solutions and Buffers
Accugene 0.5M EDTA Lonza 51201
Ammonium-Chloride-Potassium (ACK) Lysing Buffer BioWhittaker 10-548E
Hanks' Balanced Salt Solution Corning 21-023-CV
Phosphate-Buffered Saline without Calcium and Magnesium Corning 21-040-CV
Reagents
Alexa Fluor 647 Anti-Mouse Ly-6G Antibody (1A8) BioLegend 127610
CD11b Monoclonal Antibody, PE, eBioscience (M1/70) ThermoFisher 12-0112-81
CountBright Absolute Counting Beads Invitrogen C36950
Dithiotreitol FisherScientific BP172-5
Fetal Bovine Serum, heat inactivated R&D Systems 511550
Fluorescein Isothiocyanate-Dextran, average molecular weight 4.000 Sigma 60842-46-8
Isoflurane Halocarbon 12164-002-25
Leukotriene B4 Millipore Sigma 71160-24-2
PerCP Rat Anti-Mouse CD45 (30-F11) BD Pharmingen 557235
Purified Rat Anti-Mouse CD16/CD32 (Mouse BD FC Block) BD Bioscience 553142
Recombinant Murine IFN-γ Peprotech 315-05
Recombinant Murine TNF-α Peprotech 315-01A

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