Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

Summary

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

Abstract

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using "intestinal sacs" are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (P_app) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Introduction

The intestinal epithelial barrier of the gastrointestinal tract is a mucosal surface area estimated at 400 m² in the human adult. Consequently, it is constantly exposed to challenge from microbes, ingested drugs, nutrients and bacterial toxins. The host must not only distinguish between tolerable commensal bacteria and potential pathogens, but must prevent these species and their secreted molecules from crossing the epithelial barrier, while at the same time allowing absorption of nutrients. Thus, the role of the intestinal epithelium is to act as a selective barrier to the luminal contents ¹. This is achieved, in part, by the innate epithelial defense system at the mucosa, which acts through a responsive biological system consisting of constitutive and inducible mechanisms ².

Loss of epithelial barrier function is a pathology that is characteristic of a number of gastrointestinal diseases. In vivo examination of epithelial barrier function may be assessed through oral gavage of a tracer molecule and subsequent serum analysis ³. However, this technique offers no indication as to the site of barrier dysfunction. In vitro and ex vivo assessment of transepithelial resistance using Transwell systems ³ and Ussing chambers ^4,5 respectively, are commonly employed as surrogate markers of epithelial barrier function, but lack the contributing disease physiology of animal models ⁶. In this protocol we describe an ex vivo tissue preparation model that allows direct and localized assessment of intestinal integrity and which may be used to assess mucosal barrier function at a number of levels. Importantly, this technique may be applied to animal models of disease, or may be pharmacologically manipulated to allow in depth interrogation of mucosal barrier dysfunction.

Protocol

All animal work in this protocol is performed with strict adherence to University of Newcastle Animal Ethics Committee approved procedures.

1. Preparation of Instruments, Culture Media and Dishes

1. Pre-warm Media 199 (TC199) or Dulbecco's Modified Eagle Medium (DMEM) media to 37 °C. Pre-oxygenate the medium by bubbling with 95% O₂/5% CO₂. Check that the medium has a final pH of 7.3.
2. Prepare suture by cutting two 5 cm sections for each sac. Loop the sutures into an unclosed knot.

2. Dissection and Preparation of the Gastrointestinal Tract

1. Withdraw solid food 12 hr before euthanasia. If desired, place animals on nutrient gel supplements during this time.
2. Euthanize mice by sodium pentobarbitone overdose ([200 mg/Kg], intraperitoneal injection) followed by cervical dislocation in accordance with institutional ethics protocols and spray 70% ethanol onto the abdomen and thorax.
3. Using a scissors, make a horizontal incision in the middle of the abdomen and expose the peritoneum.
4. Proceed to separate and the remove the gastrointestinal tract by cutting the upper small intestine from the stomach at the pyloric sphincter and cutting the large intestine at the anal verge. Use a forceps to gently remove the mesentery. Place the intestinal tract in pre-warmed, oxygenated medium.
5. Identify the section of intestine to be assessed for permeability (Figure 1) and cut this section free from the rest of the intestinal tract.
   1. In order to maintain consistency between animals, measure sections of duodenum and jejunum relative to the stomach, and measure sections of colon and ileum relative to the cecum.
   2. When selecting tissue segments, note the presence of mucosa-associated lymphoid tissues such as Peyer's patches. These can be identified as small nodules on the serosal side of the lumen.
   3. Using a 1 ml syringe, gently flush the luminal contents of the intestinal segment into a petri dish with pre-heated PBS (37 °C). These faecal contents may be discarded or stored at -80 °C for future analysis as desired.

3. Preparation of Intestinal Sacs

1. Prepare a 1 ml syringe with a 300 µl volume of the test compound or molecule. For mucosal integrity, a 1 mg/ml solution of FITC-Dextran M.Wt. 4,400 may be used. Probes ranging from 4,400-70,000 Da in size may be used for increased sensitivity. Securely fit a small animal vascular catheter onto the syringe.
2. Measure 5 cm from the opening of the intestinal segment and tie the segment securely closed with a suture-loop at this point. Gently place a pre-tied suture-loop around the opening of the intestine and insert the blunted catheter. Pull the noose closed so as to secure the intestinal segment and release the 300 µl volume from the syringe into the intestine, ensuring that all the solution is injected.
3. Gently remove the catheter while simultaneously pulling the suture noose to secure closure of the intestinal sac. Cut the intestinal sac loose from the intestine and place into a 50 ml conical tube filled with 20 ml of oxygenated medium, preheated to to 37 °C.

4. Measurement of Permeability

1. Place conical tubes containing the intestinal sacs in a heated water bath set to 37 °C. At 0, 30, 60, 90 and 120 min time points, take a 100 µl sample from the conical tube and transfer to a 96 well plate, replacing the volume with 100 µl of fresh media in each instance.
2. After the final sample is taken, cut open sacs at the point of suture and down the length of the segment, exposing the mucosal surface.
3. Measure the length and width of each intestinal segment. If desired, snap freeze the segments and store at -80 °C for protein or biochemical analysis, or alternatively, store in RNA stabilization solution for molecular assays.
4. Construct a standard curve of log dilutions for FITC-tagged molecules ranging from 1 to 1 x 10^-6.
5. Measure samples and standards for FITC on a fluorescent plate reader, FITC excitation/emission: 495 nm/519 nm.

5. Calculation of Apparent Permeability for Each Individual Intestinal Sac

1. Convert time units to sec.
2. For each time point, calculate the cumulative concentration, Q
   
   Q_t = (C_t*V_r) + (Q_{t sum}* V_s), Where:
   
   Q_t = Cumulative concentration at time t
   C_t = Concentration at time t
   V_r = Volume at receiver side
   Q_{t sum} = Sum of all previous Q_t
   V_s = Volume sampled
    
3. Plot Q versus time (T) and calculate the slope: δQ/δt
4. Calculate the apparent permeability (P_app)
   
   P_app = (δQ/δt)/(A*Co), Where:
   
   A = Area of tissue
   C₀ = Initial concentration

Results

This protocol may be used to examine regional changes in intestinal barrier function in animal models of gastrointestinal disease. By measuring the flux of a paracellular probe across the mucosal surface at varying areas of the gastrointestinal tract⁷, the integrity of the epithelial tight junctions can be assessed. In addition, by varying the nature of the paracellular probe by size (Figure 2) or hydrophobicity (Figure 3), the degree of epithe...

Discussion

Here, we have detailed the isolation and preparation of intestinal sacs to assess mucosal barrier function ex vivo. Intestinal sac preparations have primarily been utilized in pharmaceutical research, examining the absorption of candidate drugs across the intestine. However, this assay is equally well suited for the study of intestinal disease. Intestinal permeability may vary greatly by region and site specific assessment of permeability allows a better understanding of the regional importance of mucosal integr...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dekantel  Non-absorbable Silk suture	Braintree Scientific	SUT-S 116
Media 199 (TC199)	Life Technologies	11043-023	No phenol red as this interferes with fluorescence
Dulbecco's Modified Eagle Medium (DMEM)	Life Technologies	21063-045	No phenol red as this interferes with fluorescence
N-acetylcysteine	Sigma Aldrich		Use at 10 mM in media
Small animal vascular cathether: Physiocath	Data Sciences International	277-1-002
FITC-Dextran 4,400 MW	Sigma Aldrich	FD-4
FITC-Dextran 20,000 MW	Sigma Aldrich	FD-20
FITC-Dextran 70,000 MW	Sigma Aldrich	FD-70