In the present study, fluorescein-isothiocyanate-labeled (FITC) dextran is administered to mice via oral gavage to evaluate intestinal permeability both in vivo and in plasma and fecal samples. As gut barrier function is affected in many disease processes, this direct and quantitative assay can be used in diverse research areas.
Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of plasma inflammatory markers and bacterial translocation to the spleen and lymph nodes, the gold standard directly quantifies the ability of selected molecules to traverse the gut mucosal layer toward systemic circulation. This article uses a non-invasive, cost-effective, and low-burden technique to quantify and follow in real time the intestinal permeability in mice using fluorescein-isothiocyanate-labeled dextran (FITC-dextran). Prior to oral supplementation with FITC-dextran, the mice are fasted. They are then gavaged with FITC-dextran diluted in phosphate-buffered saline (PBS). One hour after the gavage, the mice are subjected to general anesthesia using isoflurane, and the in vivo fluorescence is visualized in an imaging chamber. This technique aims to assess residual fluorescence in the abdominal cavity and the hepatic uptake, which is suggestive of portal migration of the fluorescent probe. Blood and stool samples are collected 4 h after oral gavage, and the mice are sacrificed. Plasma and fecal samples diluted in PBS are then plated, and the fluorescence is recorded. The concentration of FITC-dextran is then calculated using a standard curve. In previous research, in vivo imaging has shown that fluorescence rapidly spreads to the liver in mice with a weaker gut barrier induced by a low-fiber diet, while in mice supplemented with fiber to strengthen the gut barrier, the fluorescent signal is retained mostly in the gastrointestinal tract. In addition, in this study, control mice had elevated plasma fluorescence and reduced fluorescence in the stool, while inversely, inulin-supplemented mice had higher levels of fluorescence signals in the gut and low levels in the plasma. In summary, this protocol provides qualitative and quantitative measurements of intestinal permeability as a marker for gut health.
The gut barrier plays an important role in both health and disease. It requires a complex balance between allowing the required nutrients to permeate into the circulation from the intestinal lumen while simultaneously preventing the penetration of pro-inflammatory molecules, such as pathogens or antigens1. Increased permeability can be caused by many gastrointestinal disorders, such as liver disease or inflammatory bowel diseases (IBDs)2,3. For example, in ulcerative colitis (UC), an IBD, chronic inflammation leads to the breakdown of tight junctions, the subsequent disruption of the gut barrier, and the translocation of bacteria, potentially perpetuating mucosal and systemic inflammation4.
Gut barrier integrity is, therefore, an important marker of gut health. However, current methods for the measurement of intestinal permeability have many limitations. For instance, methods measuring plasma inflammatory markers or bacterial translocation to the spleen and lymph nodes are indirect5,6. Other methods can be invasive and time-intensive. This article describes a non-invasive and cost-effective assay that directly and quantitatively measures intestinal permeability. This assay uses fluorescein-isothiocyanate-labeled dextran (FITC-dextran) to follow intestinal permeability in real time by measuring fluorescence in vivo. In addition, measuring FITC-dextran levels in the plasma and feces quantifies the intestinal permeability (Figure 1).
The FITC-dextran permeability assay has been used previously in many different contexts, including in animal models of Parkinson's disease7, sepsis8, ischemic stroke9, and burn injury10. Additionally, this assay has been used recently to aid in understanding how the gut microbiome may be implicated in different disease processes and how it may be targeted or manipulated as a potential therapeutic. For example, it has been used to study the microbiome and microbiome-based therapeutics in aging11, IBDs12, colorectal cancer13, and autism spectrum disorder11. As gut barrier function is implicated in numerous aspects of health and disease, this assay has been used widely. Its relative simplicity and low time burden make it ideal for testing the in vivo conditions suspected to alter gut barrier integrity. Its quantitative results are useful for determining the effectiveness of a potential treatment.
In this study, the effect of diet on gut barrier function was evaluated using the FITC-dextran assay. The intestinal permeability of mice receiving a control diet and the intestinal permeability of mice receiving an inulin-supplemented diet were compared. Inulin is a beneficial oligosaccharide that has been shown to improve gut barrier function12,13. For in vivo fluorescence measurements (background), one additional untreated mouse was used as a negative control and received PBS instead of FITC-dextran. This experiment demonstrates that the FITC-dextran assay is a valuable tool for evaluating intestinal permeability.
All procedures were performed following the Canadian Council on Animal Care guidelines after approval by the Institutional Animal Care Committee of the CRCHUM. Eight-week-old female BALB/c mice, obtained from a commercial source (see Table of Materials), were used for the present study. The animals received dietary supplementation with 10% inulin wt/wt for 2 weeks. A control group received a similar diet lacking the inulin supplement. Mice had ad libitum access to the diet. An overview of the assay is shown in Figure 1.
Figure 1: Schematic of the FITC-dextran assay. T−4– 4 h prior to gavage, food access was removed. T0– FITC-dextran was administered via oral gavage. T1 – 1 h post gavage, the in vivo fluorescence was evaluated. T4– 4 h post gavage, the fecal and plasma samples were collected, and the fluorescence was measured. Please click here to view a larger version of this figure.
1. Administration of FITC-dextran
2. In vivo fluorescence measurement
3. Fluorescence measurement in fecal samples and plasma
The analysis of the in vivo fluorescence showed that mice who received only the control diet had a higher hepatic intake of FITC-dextran and higher levels of residual fluorescence in the abdominal cavity compared to the mice who received the inulin-supplemented diet (Figure 2A). Some fluorescence was visible in the caecum of the mice that received the inulin diet, but there was little to no hepatic intake, indicating that these diets protected against increased intestinal permeability.
The fluorescence levels in plasma and fecal samples work to reinforce and quantify their in vivo counterparts. The mice that received the inulin-supplemented diet had significantly lower levels of FITC-dextran in their plasma compared to the mice that received only the control diet (Figure 2B). This indicates that they had improved gut barrier function because less FITC-dextran could permeate the intestinal barrier into the circulation. Concordantly, the mice that received the inulin diet had significantly higher levels of FITC-dextran in their feces than the mice that received only the control diet (Figure 2C). This reinforces that they had intact gut barrier function as the FITC-dextran remained in the colon until excretion, as is considered normal. The lower levels of FITC-dextran in the feces of the control mice indicate that it permeated through the intestinal barrier into the circulation rather than being appropriately excreted. The high levels of FITC-dextran in the plasma reinforce this finding.
Figure 2: Dietary supplementation with inulin decreases the translocation of FITC-dextran through the gut barrier. (A) The residual fluorescence and hepatic uptake of FITC-dextran. Red = highest intensity; dark purple = lowest intensity. Maximum fluorescence of 2.15 x 103, minimum fluorescence of 0.378. (B) The plasmatic concentration of FITC-dextran. P = 0.010. (C) The fecal concentration of FITC-dextran. P = 0.00003. N = 4 per group. Data are represented as mean ± SEM. Each dot represents one mouse. Unpaired Student's t-test. Please click here to view a larger version of this figure.
The gut barrier function is an integral part of many different disease processes. Thus, assessing intestinal permeability in a non-invasive, cost-effective, and quantifiable way is essential for accurately representing these diseases in animal models. The FITC-dextran assay provides the possibility for this representation. However, this protocol involves several critical steps that must be completed accurately to obtain reliable results. Firstly, ensuring the use of appropriately sized FITC-dextran is essential. For examining in vivo permeability, 4 kDa FITC-dextran is the optimal molecular weight, and as the molecular weight increases, the permeability decreases15. Thus, using FITC-dextran of a different molecular weight may provide confusing or unreliable results. Additionally, it is important to note the time of each gavage and to adjust the time points for in vivo data collection and the collection of plasma and feces accordingly. For example, if two mice are gavaged 10 min apart, the in vivo fluorescence readings and the collection of feces and plasma must also occur 10 min apart. Comparing the fluorescence at the same time points allows for a more accurate representation of the differences in permeability. Furthermore, the order in which the animals from different groups are tested should be alternated to prevent a clustering effect due to timing. Instead of testing all the animals in Group A first, then all the animals in Group B second (AAABBB), it is recommended for the group to be switched after each animal (ABABAB).
This assay can be modified to include only the evaluation of plasma and fecal samples if there is a lack of access to an imaging machine. Though direct fluorescence imaging in vivo allows for the visualization of hepatic intake and residual abdominal fluorescence, evaluating fluorescence in the plasma and fecal samples still provides a quantitative measurement of intestinal permeability. Furthermore, as demonstrated by the described experiment, the fluorescence levels in the plasma and feces correlate well with the in vivo imaging. Additionally, this assay can be modified to include only the in vivo imaging. This allows the animals to be kept alive to continue testing other parameters or monitor how intestinal permeability changes over time. The ability to modify this assay, therefore, makes it accessible, yet still quantitative. Finally, the dosage of 200 µL of 80 mg·mL−1 FITC-dextran given to each mouse has been used previously and was shown to be effective in mice with small differences in body weight16. Furthermore, it is important to note that all the mice used in the representative results section weighed approximately 20 g, allowing the same dosage to be used for each mouse. To account for differences in body weight, however, FITC-dextran can be administered at a dosage of 0.6-0.8 mg/g body weight, for example17. Crucially, regardless of the dosage used, it is important to limit the amount gavaged to each mouse to less than 10 mL·kg−1 to prevent complications or discomfort18.
Though the FITC-dextran assay provides an effective method for evaluating gut barrier function, it still has some limitations. One limitation of this model is that it requires fasting the mice for several hours, meaning it is unreliable to compare these results to those from mice that have not been fasted. Additionally, fasting may affect the outcomes in certain models that require strict feeding schedules, such as when measuring blood glucose in animal models for diabetes.
Despite these limitations, the FITC-dextran assay remains an effective method for analyzing intestinal permeability as it is quantitative, versatile, cost-effective, and less invasive than many classical methods. For example, common probes used for measuring intestinal permeability are small saccharide probes or Cr-EDTA, which have some advantages19. However, some saccharide probes have only region-specific permeability. As they are hydrolyzed in the distal portion of the small intestine, they provide no insight into colonic permeability19. On the other hand, Cr-EDTA can provide information about colonic permeability but requires measurements for 24 h, making the time burden of this method much higher than that of the FITC-dextran assay20. Furthermore, neither of these methods provides the direct in vivo imaging of this assay. Therefore, the FITC-dextran assay provides a relatively simple, direct, and effective option as compared to alternative methods for measuring intestinal permeability.
Finally, in disease processes such as IBDs4, Alzheimer's disease21, and liver disease2, intestinal permeability is an important parameter that could be measured using the FITC-dextran assay to improve studies. For example, in developing novel treatments, such as immunotherapies for IBDs, this assay can be used to test the efficacy of the therapeutic for maintaining gut barrier integrity. Considering that impaired gut barrier function may be implicated in perpetuating the chronic inflammation in UC, for example, examining how well a therapeutic protects against increased permeability is important4. This is only one example, but the FITC-dextran assay is an accessible and quantifiable way to measure intestinal permeability in many different areas and aspects of research.
The authors have nothing to disclose.
This work was funded by a grant from the Natural Sciences and Engineering Research Council of Canada (grant RGPIN-2018-06442 to MMS). We thank the animal facility at the CRCHUM and Dr. Junzheng Peng from the Cardiovascular Phenotyping Platform.
50 ppm Fe Diet (10% Inulin) | Envigo Teklad | TD.190651 | Representative Results |
50 ppm Fe Diet (FeSO4) | Envigo Teklad | TD.190723 | Representative Results |
BALB/c Mice 49-55 Days, Female | Charles River | 028BALB/C | Representative Results |
BD 1 mL Syringe Tuberculin Slip Tip | Becton, Dickinson and Company | 309659 | For gavage |
BD Microtainer Tubes – With LH (Lithium Heparin) | Becton, Dickinson and Company | 365965 | For plasma collection |
Centrifuge 5420 | Eppendorf | S420KN605698 | |
Curved Gavage Needle (Gavage Cannula) 7.7.0 38 mm x 22 G | Harvard Apparatus Canada | 34-024 | No longer available – A potential alternative is available at Instech Labs (FTP-22-38) |
Euthanyl (Pentobarbital Sodium) 240 mg/mL | Bimeda-MTC Animal Health Inc. | 141704 | 1/100 dilution; Administered via intraperitoneal injection at 0.03 mL/g body weight |
FITC-dextran 4 | TdB Labs | 20550 | |
Heparinized Capillary Tubes | Kimble Chase Life Science and Research | 2501 | For retro-orbital blood collection |
Microplate, PS, 96-well, Flat-bottom (Chimney Well), Black, Flutrac, Med. Binding | Greiner Bio-one | 655076 | |
MiniARCO Clipper Kit | Kent Scientific | CL8787-KIT | For hair removal |
Optix MX2 and Optix Optiview | Advanced Research Technologies | 2.02.00.6 | Fluorescence imaging machine and software |
Phosphate Buffered Saline 1x (PBS) | Wisent Inc | 311-010-LL | |
Puralube Vet Ointment | Dechra | 12920060 | Ophthalmic ointement to prevent eye damage during anesthesia |
Spark Multiplate Reader | Tecan | 30086376 |