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.

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 &#38; 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...

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...

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 disease (IBD) encompassing both ulcerative colitis and Crohn&#39;s disease1,2,3,4,5,6,7. Individuals with ulcerative colitis present excessive transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) forming crypt abscesses, a finding that has been associated with severity of disease8,9. Although compromised epithelial barrier function and excessive immune responses are hallmarks of IBD, there is a lack of experimental in vivo assays to perform quantitative assessments of intestinal permeability and immune cell recruitment into the intestinal mucosa.
The most common methods used to study intestinal epithelial permeability and PMN TEpM employ ex vivo chamber-based approaches using IEC monolayers cultured on semi-permeable porous membrane inserts10,11,12. The epithelial barrier integrity is monitored either by measurements of transepithelial electrical resistance (TEER) or the paracellular flux of the Fluorescein isothiocyanate (FITC)-labeled dextran from apical to basal compartment13,14,15. Similarly, PMN TEpM is typically studied in response to a chemoattractant that is added in the lower chamber16. PMN are placed in the upper chamber and after an incubation period, PMN that have migrated into the basal compartment are collected and quantified. While these methods are useful, easy to perform and very reproducible, they are obviously reductionist approaches and do not necessarily represent an accurate reflection of in vivo conditions.
In mice, a common assay to study intestinal paracellular permeability is by oral gavage of FITC-dextran and subsequent measurement of FITC-dextran appearance in the blood serum13,17. The disadvantage of this assay is that it represents an assessment of overall barrier integrity of the gastrointestinal tract rather than that of regional intestinal contributions. In addition, Evans blue is commonly used to evaluate vascular leakage in vivo18 and has also been employed to evaluate intestinal mucosal permeability in mouse and rat19,20,21. The quantification of Evans blue in the intestinal mucosa requires extraction from tissue employing incubation in formamide overnight. Therefore, the same tissue cannot be used to study intestinal epithelial permeability and neutrophil infiltration.
Here we highlight a simple protocol that reduces the number of animals needed to collect reproducible data on colonic mucosal permeability and leukocyte transepithelial migration in vivo. We, therefore, recommend the use of FITC-dextrans that are easily detectable in blood serum without compromising the integrity of intestinal loops which can be harvested for further analysis. Of note, the intestinal ligated loops have been used in various species (including mouse, rat, rabbit, calf) to study bacterial infection (such as Salmonella, Listeria monocytogenes and Escherichia coli)22,23,24,25 as well as intestinal permeability26; however, to the best of our knowledge there are no studies investigating mechanisms of PMN TEpM in specific regions in the intestine such as ileum or colon that are commonly involved in IBD.
Here we describe the mouse intestinal loop (iLoop) model that is a robust and reliable microsurgical in vivo method that employs a well-vascularized and exteriorized intestinal segment of either the ileum or proximal colon. The iLoop model is physiologically relevant and allows the assessment of intestinal barrier integrity and PMN TEpM on living mice under anesthesia. We demonstrate two applications: 1) quantification of serum levels of 4 kDa FITC-dextran after intraluminal administration in the iLoop 2) quantification of transmigrated PMN in the iLoop lumen after intraluminal injection of&#160;the potent chemottractant Leukotriene B4 (LTB4)27. Moreover, utilizing the iLoop model with Jam-a-null mice or mice harboring selective loss of JAM-A on IECs (Villin-cre;Jam-a fl/fl) compared to control mice, we are able to corroborate previous studies that have reported a major contribution for tight junction-associated protein JAM-A to intestinal permeability and neutrophil transmigration15,28,29,30,31.
The iLoop model is a highly functional and physiological method that can be used to corroborate in vitro assays. Furthermore, this is a versatile experimental model that allows the study of various reagents that can be injected into the loop lumen, including chemokines, cytokines, bacterial pathogens, toxins, antibodies and therapeutics.

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			<td>Equipment and Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Alcohol Swabs</td>
			<td>BD</td>
			<td>326895</td>
			<td></td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle, 25G X 5/8&#34;</td>
			<td>BD</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>BD PrecisionGlide Needle, 30G X 1/2&#34;</td>
			<td>BD</td>
			<td>305106</td>
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			<td>BD 1ml Tuberculin Syringe Without Needle</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
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		<tr>
			<td>15ml Centrifuge Tube</td>
			<td>Corning</td>
			<td>14-959-53A</td>
			<td></td>
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		<tr>
			<td>Corning 96-Well Solid Black Polystyrene Microplate</td>
			<td>FisherScientific</td>
			<td>07-200-592</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Non-treated Culture Dish, 10cm</td>
			<td>MilliporeSigma</td>
			<td>CLS430588</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Tip Applicator (cotton swab), 6&#34;, sterile</td>
			<td>FisherScientific</td>
			<td>25806 2WC</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynarex Cotton Filled Gauze Sponges, Non-Sterile, 2&#34; x 2&#34;</td>
			<td>Medex</td>
			<td>3249-1</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-7000 anesthesia vaporizer (Classic System, including heating units)</td>
			<td>E-Z Systems</td>
			<td>EZ-7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Centrifuge Tube 50ml&#160;</td>
			<td>VWR</td>
			<td>21008-940</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Colored Labeling Tape</td>
			<td>FisherScientific</td>
			<td>15-901-10R</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey Needle Holder (needle holder)&#160;</td>
			<td>FST</td>
			<td>12001-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes, small (tissue wipe)</td>
			<td>FisherScientific</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7ml Microcentrifuge Tubes&#160;</td>
			<td>Thomas Scientific&#160;</td>
			<td>c2170</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Tube 1.3ml Z (serum clot activator tube)</td>
			<td>Sarstedt&#160;</td>
			<td>41.1501.105</td>
			<td></td>
		</tr>
		<tr>
			<td>Moria Fine Scissors</td>
			<td>FST</td>
			<td>14370-22</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap (35 &#181;m nylon mesh)</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Puralube Vet Ointment, Sterile Ocular Lubricant</td>
			<td>Dechra</td>
			<td>12920060</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring Forceps (blunt tissue forceps)</td>
			<td>FST</td>
			<td>11103-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Roboz Surgical 4-0 Silk Black Braided, 100 YD</td>
			<td>FisherScientific</td>
			<td>NC9452680</td>
			<td></td>
		</tr>
		<tr>
			<td>Semken Forceps (anatomical forceps)</td>
			<td>FST</td>
			<td>1108-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Sofsilk Nonabsorbable Coated Black Suture Braided Silk Size 3-0, 18&#34;, Needle 19mm length 3/8 circle reverse cutting&#160;</td>
			<td>HenrySchein</td>
			<td>SS694</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Fine Forceps, Angled</td>
			<td>FST</td>
			<td>91110-10</td>
			<td></td>
		</tr>
		<tr>
			<td>10ml Syringe PP/PE without needle</td>
			<td>Millipore Sigma&#160;</td>
			<td>Z248029</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well Cell Culture Plate</td>
			<td>Corning</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>Yellow Feeding Tubes for Rodents 20G x 30 mm</td>
			<td>Instech</td>
			<td>FTP-20-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Solutions and Buffers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accugene 0.5M EDTA</td>
			<td>Lonza</td>
			<td>51201</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium-Chloride-Potassium (ACK) Lysing Buffer</td>
			<td>BioWhittaker</td>
			<td>10-548E</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution</td>
			<td>Corning</td>
			<td>21-023-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline without Calcium and Magnesium</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Anti-Mouse Ly-6G Antibody (1A8)</td>
			<td>BioLegend</td>
			<td>127610</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b Monoclonal Antibody, PE, eBioscience (M1/70)</td>
			<td>ThermoFisher</td>
			<td>12-0112-81</td>
			<td></td>
		</tr>
		<tr>
			<td>CountBright Absolute Counting Beads</td>
			<td>Invitrogen</td>
			<td>C36950</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiotreitol</td>
			<td>FisherScientific</td>
			<td>BP172-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, heat inactivated</td>
			<td>R&#38;D Systems</td>
			<td>511550</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein Isothiocyanate-Dextran, average molecular weight 4.000</td>
			<td>Sigma</td>
			<td>60842-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Halocarbon</td>
			<td>12164-002-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Leukotriene B4</td>
			<td>Millipore Sigma</td>
			<td>71160-24-2</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP Rat Anti-Mouse CD45 (30-F11)</td>
			<td>BD Pharmingen</td>
			<td>557235</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD FC Block)</td>
			<td>BD Bioscience</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine IFN-&#947;</td>
			<td>Peprotech</td>
			<td>315-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine TNF-&#945;</td>
			<td>Peprotech</td>
			<td>315-01A</td>
			<td></td>
		</tr>
	</tbody>
</table>

functional assessment of intestinal permeability and neutrophil transepithelial migration in mice using a standardized intestinal loop model

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.

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 (<strong class=...

Watch this Scientific Journal Video about Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model at JoVE.com

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

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 ...

The intestinal model is a widely adaptable and physiologic way to measure how leaky the gut is and how leukocytes migrate in the intestine in mice, in vivo. This method helps to better understand mechanisms underlying increased intestinal permeability and pathologic intestinal inflammation. The technique can be employed to aid in our understanding mechanisms underlying gut barrier function, and pathological inflammation, as seen in ulcerative colitis and Crohn's disease.This model employs a well vascularized, exteriorized bowel segment, either of the ileum or proximal colon, for the examination of the intestinal barrier function or immune cell recruitment in mouse. With this broad application, the intestinal loop model can provide insight in diseases resulting either from a leaky gut barrier as well as the inflammatory response. Struggles can be overcome by practicing the surgical steps.It is critically important to exteriorize a fully vascularized intestinal segment and select appropriate locations for the ligations to avoid bleeding. The intestinal loop model is a microsurgical technique, thus, as with other surgical procedures, it benefits greatly from visual demonstration and a step-by-step presentation of the method. After achieving a surgical plane of anesthesia, begin by scrubbing the fur of the abdominal midline with alcohol swabs or a gauze sponge soaked with 70%ethanol.Do not wet a wide area of fur with alcohol to prevent hypothermia. Using scissors, perform a midline laparotomy. Make a horizontal incision in the middle of the abdomen, and expose the peritoneum, taking care to not injure intra-abdominal organs.Place precut wet cotton gauze over the exposed intra-abdominal cavity. Use wet cotton swabs to mobilize and exteriorize the cecum and carefully place it on wet cotton gauze. Then, mobilize and gently exteriorize the ileum.Deploy at least six centimeters of terminal ileum on the wet cotton gauze without disrupting the mesenteric vessels and the blood supply. Keep the exposed tissues moist at all times with warm HBSS. Identify the major artery supplying the ileum in the mesentery, close to the cecum.Then, locate two ligation sites in the mesentery that are free of critical blood vessels. Firmly grab the terminal ileum with blunt tissue forceps and use fine tip forceps to fenestrate the mesentery, avoiding blood vessels. Place silk suture across the perforation, and tie a surgical knot to create the first ligation.Use the ruler to measure four centimeters away from the first ligature, and create the second ligature. Carefully cut next to each ligation with fine scissors to isolate the four centimeter ileal loop, keeping the blood supply and mesenteric membrane intact. Gently flush the content of the ileal loop segment with warm HBSS using a flexible yellow feeding tube attached to a 10 milliliter syringe.Make sure to flush the luminal contents out of the abdominal cavity to keep the surgical site clean. Ligate the two cut ends of the flushed ileal loop with silk suture. Use a one milliliter syringe with a 30 gauge needle to slowly inject 250 microliters of reagent, such as FITC-dextrans or chemokine, into the intestinal lumen.The ileal loop will inflate, causing a moderate distension of the mucosa. Close the abdominal wall using a needle holder, anatomical forceps, and 3.0 non-absorbable silk sutures with a reverse cutting needle. After drying the animal to prevent hypothermia, place it in a temperature regulated anesthesia chamber for the incubation period.Prepare the mouse for surgery and exteriorize the cecum as previously demonstrated. Using wet cotton swabs, exteriorize the entire ileum and place it on top of a wet cotton gauze. Identify the proximal colon and the blood supply located in the mesocolon.Mobilize the proximal colon and create the first ligature in an area free of vessels in the mesocolon at about 0.5 centimeters distal from the cecum. Measure two centimeters from the first ligature and create a second ligature at an area free of blood supply in the mesocolon. Using fine scissors, carefully cut next to each ligation to isolate a two centimeter long pcLoop.Gently flush the pcLoop with warm HBSS to remove feces using a flexible yellow feeding tube attached to a 10 milliliter syringe. Make sure to flush the luminal contents out of the abdominal cavity to keep the surgical site clean. Then, ligate the two cut ends of the flushed pcLoop using silk suture.Use a one milliliter syringe with a 30 gauge needle to slowly inject 200 microliters of reagent, such as FITC-dextran or chemokine, into the intestinal lumen. The pcLoop will inflate, causing a moderate distension of the mucosa. Use wet cotton swabs to gently put back the ligated pcLoop, ileum, and cecum.Close the abdominal wall using a needle holder, anatomical forceps, and 3.0 non-absorbable silk sutures with a reverse cutting needle. After the incubation period, euthanize the anesthetized mouse and collect tissues for analysis. In order to verify the accuracy of the I loop model for the assessment of intestinal permeability, an FITC-dextran pcLoop assay was performed to evaluate the role of tight junction associated protein Jam-a in the regulation of intestinal barrier function in vivo.With the pcLoop model, a 2.5 fold increase in FITC-dextran serum levels was quantified in Jam-a null mice compared to controls. Similar results were obtained with mice harboring selective loss of Jam-a on intestinal epithelial cells. The pcLoop model was used to study polymorphonuclear neutrophil, or PMN, recruitment into the intestinal mucosa and subsequent transepithelial migration in vivo.The number of PMN in the luminal content of the pcLoop was quantified by flow cytometry analysis. The number of PMN present in the segment of the proximal colon was low under physiological conditions. Pretreatment with pro-inflammatory cytokines, TNF alpha and IFN gamma, prior to surgery, resulted in augmented numbers of PMN recruited in the pcLoop lumen.Administration of the PMN chemo attractant LTB4 led to a dramatic increase in PMN counts. Immunohistochemical staining of PMN in the colonic mucosa corroborate the elevated recruitment of PMN following stimulation with cytokines and LTB4. The contribution of Jam-a to PMN transepithelial migration was studied using the pcLoop model on mice harboring selective loss of Jam-a on intestinal epithelial cells.Loss of epithelial Jam-a led to a reduced number of transmigrated PMN in the colonic lumen compared to littermate controls. A critical aspect for this technique is to remember to preserve blood supply to the exteriorized intestinal segment when performing the surgery. Following this protocol, other methods such as an intestinal permeability assay and a neutrophil transepithelial migration assay can be performed to investigate loss of barrier integrity and intestinal inflammation in vivo.

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

Abstract