Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.
The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.
Intestinal epithelial cells are not only responsible for the absorption of nutrients, but also form an important barrier to defend against pathogenic microorganisms and microbial toxins. This intestinal barrier function is regulated by tight junction permeability and epithelial cell integrity1,2,3, and dysfunction of the epithelial barrier function is associated with inflammatory bowel disease (IBD). The perijunctional actomyosin ring (PAMR) lies within the cell that is closely contiguous to the tight junctions. The contraction of the PAMR, which is regulated by the myosin light chain (MLC), is crucial for the regulation of tight junction permeability4,5,6,7,8,9,10. Tumor necrosis factor (TNF) is central to intestinal barrier loss by upregulating intestinal epithelial MLC kinase (MLCK) expression and inducing occludin internalization11,12,13.
Ions such as Na+ and Cl– can cross the paracellular space by either the pore or leak pathway14. In a "leaky" epithelium, changes in TER primarily reflect altered tight junction permeability. TER measurement is a commonly used electrophysiological approach to quantify tight junction permeability, primarily to Na+ and Cl–, based on the impedance of cell monolayers. Diverse cell types, including intestinal epithelial cells, pulmonary epithelial cells, and vascular endothelial cells, have been reported for TER measurements. Advantages of this method are that TER measurements are non-invasive and can be used to monitor live cells in real-time. In addition, the TER measurement technique is useful for drug toxicity studies15.
Caco-2BBe cells are human epithelial colorectal adenocarcinoma cells with a structure and function similar to the differentiated small intestinal epithelial cells: for example, these cells have microvilli and enzymes associated with small intestinal brush border. Therefore, cultured Caco-2BBe monolayers are utilized as an in vitro model for testing barrier function.
In mice, one way to study intestinal paracellular permeability is by measuring the ability of FITC-dextran to cross from the lumen into the blood. Thus, the intestinal permeability can be assessed by gavaging FITC-dextran directly into mice and measuring the fluorescence within the blood. The following protocol describes two simple methods to assess intestinal epithelium permeability both in vitro and in vivo.
This study was approved by the Animal Care and Use Protocol of Cambridge-Suda Genomic Resource Center (CAM-SU), Soochow University.
1. Plating and Maintenance of Caco-2bbe on Porous Polycarbonate Membranes
2. Use of Epithelial Meter (Volt/Ohm) for Measuring TER
NOTE: After about 3 weeks of culture on polycarbonate membranes, Caco-2BBe cells are ready for TER measurement.
3. Murine Model of Dextran Sulphate Sodium (DSS)-induced Colitis
4. Measuring the Epithelial Barrier Permeability in DSS-induced Colitis Mice
In culture, Caco-2BBe cells grow as a monolayer and slowly differentiate into mature absorptive enterocytes that have brush borders. In this protocol, Caco-2BBe cells were plated with a high density on polycarbonate membranes, and cells reached 100% confluency one day after seeding. However, cells are undifferentiated at this stage: To fully differentiate the cells, the media is changed every 2-3 days for 3 weeks. Cells were stained with nuclei and F-actin stains to show the differences between undifferentiated and differentiated cells. Stress fibers are clearly seen in undifferentiated cells. Compared with undifferentiated cells, differentiated cells have a smaller volume, larger nuclei, and fewer stress fibers (Figure 1).
TNF is central to intestinal barrier loss through MLCK-dependent tight junction regulation. TER of cells without or with TNF treatment at different time points was analyzed. As shown in Figure 2, TNF significantly decreases the TER of the Caco-2BBe monolayer in a dose-dependent manner, suggesting that TNF increases epithelial permeability.
Administration of DSS induces acute colitis in mice. Mice treated with DSS lose weight significantly compared to their initial body weight (Figure 3A). The severity of colitis is scored by rectal prolapse, stool consistency, bleeding, and activity (Figure 3B). Cross sections of colonic tissues stained with hematoxylin & eosin show colonic mucosal damage in DSS treated mice (Figure 3C). The length of colon crypt is decreased in DSS treated mice (Figure 3D). The colon length is shortened in DSS treated mice (Figure 3E). As seen in Figure 3F, there is approximately a 2-fold increase in FITC-dextran levels in DSS treated mice compared to control mice.
Figure 1: Caco-2BBe cells cultured on polycarbonate membranes. Cells cultured 1 day (undifferentiated) and 3 weeks (fully-differentiated) after plating are stained with Hoechst 33342 (blue) for the nucleus and Alexa Fluor 594 Phalloidin for F-actin (red). Scale bar = 20 µm. Please click here to view a larger version of this figure.
Figure 2: TNF reduces the TER of the Caco-2BBe monolayer. Cells are primed with 10 ng/mL of IFNγ overnight. On the next day, the culture medium is replaced with HBSS containing 2.5 or 7.5 ng/mL of TNF. TER is measured by an epithelial meter (Volt/Ohm) at the indicated time points. Values are mean ± SEM, n = 4. Significant differences are indicated by * (p <0.05) and ** (p <0.01), as compared to TER values of cells without TNF treatment, by two-tailed t-test. Please click here to view a larger version of this figure.
Figure 3: DSS-induced colitis in mice. C57BL/6 male mice are given 3.5% DSS in drinking water for 7 days. Body weight (A) and clinical scores (B) are assessed daily for each group (mean ± SEM, n = 4 for each group). Control mice received water without DSS. (C) Histological analysis of colonic tissue sections collected on day 7 post–DSS treatment. (D) Colon crypt lengths are measured on day 7 post–DSS treatment. (E) Colon lengths are measured on day 7 post–DSS treatment. (F) Permeability of colon epithelium is measured on day 7 post-DSS treatment by FITC-Dextran gavage. Values are mean ± SEM, n = 4. Significant differences are indicated by * (p <0.05 by two-tailed t-test). Please click here to view a larger version of this figure.
Score | Rectal prolapse | Stool consistency | Bleeding | Activity |
0 | None | Normal | None | Active |
1 | Sign of prolapse | Soft | Red | Decreased activity |
2 | Extensive prolapse | Diarrhea | Gross bleeding | Lethargic |
Table 1. Disease Clinical Score
There are several critical steps in the protocol. Caco-2BBe (brush border-expressing) cells are always used for TER measurement, selected from the Caco-2 cell line for expression of brush-border proteins. Caco-2BBe cells have a villus absorptive phenotype when fully-differentiated (after about 3 weeks of culture post-confluence)17. It is necessary to avoid contamination during the measurement, and to sterilize the electrode. Because the procedure is non-sterile, measurements can only be performed for up to 8 h in most cases. During the measurement, we determined the resistance value of the two points inside and outside the insert. The resistance measurements at various sites can sometimes greatly differ because of differences in the cell state; This is a major limitation of the method. However, this variation can be significantly reduced by statistical methods and other methods. The TER method can be applied to other cells, such as determining the barrier integrity of pulmonary epithelial cells and vascular endothelial cells. In addition, TER measurements can be used in drug toxicity studies.
Administration of indigestible macromolecules, which enter the intestinal epithelium through diffusion, can be used to evaluate the intestinal permeability. The advantage of this method is that the barrier function of the intestine can be examined in vivo. There are several types of markers that can be used, including radioisotopes, polyethylene glycols, and sugars18. However, several factors should be considered for precise measurement of the permeability: (1) the position of gut barrier abnormality cannot always be precisely determined; and (2) the mucosal blood flow and gastrointestinal motility might affect the absorption and the excretion of the macromolecules.
In this study, 4 kDa FITC-dextran was administered intragastrically in mice and the amount of FITC-dextran in the mouse serum was measured. This method has numerous advantages. Oral gavage reduces possible risks that are associated with using enemas. For instance, unintentional impairment to the colonic epithelium may be caused by rectal instillation, leading to a false-positive result. Additional effects caused by stress should also be eliminated. Permeability is known to increase when the mice are stressed. It is recommended to conduct a preliminary experiment at least one week prior to the actual experiment. Performing a practice experiment will help minimize experimental noise for the real experiment. A wild-type control (no treatment) should always be included.
FITC-dextran travels through the small intestine and begins to enter the colon 3 h after gavaging. Thus, 3 h is an ideal time point for measuring small intestinal permeability. For measurement of colonic permeability, the time can be extended, but this then becomes a balance between the leakage of FITC-dextran into the lumen and clearance of these molecules from the blood stream. 4 h is generally accepted as a good time point for colonic measurements.
In conclusion, two separate methods were described to determine intestinal epithelial cell permeability both in vitro and in vivo. Whereas TER measurement and FITC-dextran gavage methods both measure paracellular permeability, they each provide independent and distinct properties of the paracellular permeability.
The authors have nothing to disclose.
We thank Dr. Jerrold R. Turner, from Brigham and Women's Hospital, Harvard Medical School, for his generous help in completing this study. This work is supported by the National Natural Science Foundation of China (grant number 81470804, 31401229, and 81200620), the Natural Science Foundation of Jiangsu Province (grant number BK20180838, and BK20140319), The Research Innovation Program for College Graduates of Jiangsu Province (grant number KYLX16-0116), Advanced Research Projects of Soochow University (grant number SDY2015_06), and Crohn's & Colitis Foundation Research Fellowship Award (grant number 310801).
22G gavage needle | VWR | 20068-608 | |
4 kDa FITC-dextran | Sigma | 46944 | |
Avertin | Sigma | T48402 | |
Black 96-well plates for fluorescence | Fisher | 14-245-197A | |
C57/B6 mice | Nanjing Biomedical Research Institute of Nanjing University | ||
Caco-2BBe cells | ATCC | CRL-2102 | |
Dextran sulphate sodium | MP Biomedicals | 2160110 | |
DMED with high glucose and sodium pyruvate | Hyclone | SH30243.01B | |
Epithelial (Volt/Ohm) Meter | Millicell-ERS | MERS00002 | |
Ethanol | Sinopharm ChemicalReagent | 10009218 | |
Falcon tube (15 mL) | Corning | 430791 | |
FBS | Gibco | 10437-028 | |
Fluorescence microscope | Olympus | FV1000 | |
Fluorometer | Biotek | Synergy 2 | |
HBSS | 138 mM NaCl, 0.3 mM Na2HPO4, 0.4 mM MgSO4, 0.5 mM MgCl2, 5.0 mM KCl, 0.3 mM KH2PO4, 15.0 mM HEPES, 1.3 mM CaCl2, 25 mM glucose | ||
IFNg | PeproTech | 315-05-20 | |
Modular Tissue Embedding Center | Leica | EG1150H | |
Serum collection tubes | Sarstedt | 41.1378.005 | |
T75 flask | corning | 430641 | |
TNF | PeproTech | 315-01A | |
Parraffin | Sigma | A6330-1CS | |
Polycarbonate membranes (Transwell) | Costar | 3413 | |
Pressure pump | AUTOSCIENCE | AP-9925 | |
Rotary Microtomy | Leica | RM2235 | |
Trypsin-EDTA | Gibco | 25200-056 | |
Xylene | Sinopharm ChemicalReagent | 10023418 |