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.

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.
<img alt="FITC-dextran oral gavage in vivo fluorescence measurement; experimental flowchart; mouse model." class="xfigimg" src="/files/ftp_upload/64710/64710fig01.jpg" data-altupdatedat="1747911167000" data-alt="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. <a href="https://www.jove.com/files/ftp_upload/64710/64710fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Administration of FITC-dextran
<ol>
	<li>Prior to administering FITC-dextran, fast the mice for 4 h while maintaining ad libitum access to water. 
	NOTE: The fasting must preferably be initiated at the beginning of the light cycle (in the morning). The mice may be transferred to a new cage without bedding during fasting to limit coprophagy.</li>
	<li>Prepare 200 µL of 80 mg·mL−1 4 kDa FITC-dextran (see Table of Materials) diluted in sterile 1x phosphate-buffered saline (PBS) (per mouse). Freshly prepare the samples immediately prior to administration, and keep them protected from light.</li>
	<li>Administer 200 µL of the FITC-dextran suspension via oral gavage to each mouse using a 38 mm 22 G sterilized, curved gavage needle with a ball- or pear-shaped tip (see Table of Materials). Start a timer after the first gavage, and wait for 5-10 min before gavaging the next mouse to allow for in vivo measurements (step 2), always maintaining 1 h post gavage. Keep the remaining FITC-dextran suspension for the standard curve. 
	​NOTE: Immediately following the gavage, food may be replaced to ensure the formation of feces.</li>
</ol>
2. In vivo fluorescence measurement
<ol>
	<li>Anesthetize the mice 1 h after the gavage using 2.5% isoflurane or an alternative anesthetic. Confirm that the animal is appropriately anesthetized by pinching the toe or the tail and ensuring that the animal does not react.
	<ol>
		<li>Remove the fur from the abdominal area using an electric shaver, and generously apply ophthalmic lubricating ointment to the eyes to prevent drying. Then, place the mice individually in the imaging chamber lying dorsally. 
		NOTE: One control mouse must be included that receives PBS or saline instead of FITC-dextran to account for background during in vivo imaging.</li>
	</ol>
	</li>
	<li>Image the mice using a fluorescence imaging chamber (see Table of Materials). Acquire images of the abdominal region, setting the laser length at 470 nm and the resolution at 2.0 mm.
	<ol>
		<li>Start the machine and software by clicking and holding the start button. Allow the system to warm up. 
		NOTE: The system may need 20 min or more to warm up, so the machine must be started early so as not to interfere with imaging the mice at 1 h after gavage.</li>
		<li>Click on Device status and ensure all the configured devices show "OK" before proceeding.</li>
		<li>If needed, warm up the appropriate laser by clicking on Laser control and then clicking on the Laser name button of the desired laser.</li>
		<li>Start a new study by clicking on New study. Save under the appropriate file with the desired name.</li>
		<li>Click on Study options, then enter the Specimen ID, and choose the correct laser and experiment.</li>
		<li>Open the imaging chamber and place the animal dorsally on the scanning plate. Secure the limbs and tail with tape, and ensure that the nose and mouth fit snugly in the anesthesia tube, maintaining 2.5% isoflurane.</li>
		<li>Adjust the height of the scanning plate so that the scanning region is slightly ventral from the animal's midline. Adjust the plate by turning the adjustment knob inside the imaging chamber. Close and lock the imaging chamber door.</li>
		<li>Select the area to be scanned by using the Draw tool. Include the whole width of the abdomen from just above the liver to the rectum. Click on the Modify tool to adjust the area once drawn.</li>
		<li>Set the scan resolution to 2.0 mm, and then click on Next. Once power automation is completed, ensure that the settings are correct, and make any adjustments needed. Click on Start to begin the scan.</li>
		<li>Once the scan is complete, remove the animal from the imaging chamber, and place it in an incubator to maintain body temperature while recovering from anesthesia.</li>
		<li>Click on Continue study to maintain the settings, and then repeat steps 2.2.5-2.2.10 until all the mice have been scanned.</li>
		<li>Click on the power button and hold it for 3 s to power down the imaging chamber.</li>
	</ol>
	</li>
	<li>Evaluate the fluorescence by comparing the abdominal fluorescence of each animal and the control mouse on uniformly scaled images using a software associated with the imaging system used (see Table of Materials).
	<ol>
		<li>Open the image files by finding them under the chosen file name. Open all the files that have synchronized settings at the same time.</li>
		<li>Using the image settings toolbar, use the Sync image and Sync scale buttons to sync the settings for the images, allowing for an accurate comparison.</li>
		<li>Save the images with their adjusted scales.</li>
	</ol>
	</li>
</ol>
3. Fluorescence measurement in fecal samples and plasma
<ol>
	<li>Collect a fecal pellet from each mouse in a sterile tube 4 h after gavage. Keep the tubes in the dark on ice.</li>
	<li>Anesthetize the mice via intraperitoneal injection of 240 mg/mL pentobarbital sodium (dilution, 1:100; see Table of Materials). Administer at a dose of 0.03 mL/g body weight.
	<ol>
		<li>Collect blood samples of at least 700 µL in a tube made for plasma collection containing heparin or EDTA to prevent clotting (see Table of Materials) by inserting a glass capillary tube into the retro-orbital plexus14. 
		NOTE: Alternative methods for blood collection include cardiac puncture or withdrawal from the tail vein. As this is a terminal procedure, the mice need to be euthanized via cervical dislocation or an alternative humane method. Follow local animal ethics committee recommendations for euthanasia.</li>
	</ol>
	</li>
	<li>Centrifuge the blood samples at 9,390 x g for 10 min at room temperature. Transfer the plasma to a new sterile tube, and keep it in the dark on ice.</li>
	<li>Dilute 50 mg of fecal samples in 200 µL of 1x PBS and dilute the plasma 1:2 with 1x PBS. The dilution ratio can be modified based on the intensity of the fluorescence signal.</li>
	<li>Generate a standard curve using serial dilutions of FITC-dextran in 1x PBS. Starting with the highest concentration of 20 mg·mL−1 FITC dextran, dilute by a factor of 1:1 serially 7-10 times. 
	NOTE: The concentrations, thus, must read 20 mg·mL−1, 10 mg·mL−1, 5 mg·mL−1, 2.5 mg·mL−1, 1.25 mg·mL−1, 0.625 mg·mL−1, 0.3125 mg·mL−1, etc.</li>
	<li>Plate 100 µL samples and standards in an opaque black 96-well plate. Include a PBS blank. Read the fluorescence on a fluorescent plate reader (see Table of Materials) with the absorption at 530 nm and the excitation at 485 nm. 
	NOTE: The samples and standards can be plated in duplicate or triplicate, and then their fluorescence values are averaged.</li>
	<li>Determine the concentration of FITC-dextran per sample by comparing the fluorescence to the known concentrations of the standard curve. In the samples, multiply the concentration by the dilution factor (step 3.5).</li>
</ol>

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The authors declare that they have no disclosures.

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 &#181;L of 80 mg&#183;mL&#8722;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&#183;kg&#8722;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&#39;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 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&#39;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.

<table><tbody><tr><td>50 ppm Fe Diet (10% Inulin)</td><td>Envigo Teklad</td><td>TD.190651</td><td>Representative Results</td></tr><tr><td>50 ppm Fe Diet (FeSO4)</td><td>Envigo Teklad</td><td>TD.190723</td><td>Representative Results</td></tr><tr><td>BALB/c Mice 49-55 Days, Female</td><td>Charles River&amp;nbsp;</td><td>028BALB/C</td><td>Representative Results</td></tr><tr><td>BD 1 mL Syringe Tuberculin Slip Tip</td><td>Becton, Dickinson and Company</td><td>309659</td><td>For gavage</td></tr><tr><td>BD Microtainer Tubes - With LH (Lithium Heparin)</td><td>Becton, Dickinson and Company</td><td>365965</td><td>For plasma collection</td></tr><tr><td>Centrifuge 5420</td><td>Eppendorf</td><td>S420KN605698</td><td></td></tr><tr><td>Curved Gavage Needle (Gavage Cannula) 7.7.0 38 mm x 22 G</td><td>Harvard Apparatus Canada</td><td>34-024</td><td>No longer available - A potential alternative is available at Instech Labs (FTP-22-38)&amp;nbsp;</td></tr><tr><td>Euthanyl (Pentobarbital Sodium) 240 mg/mL</td><td>Bimeda-MTC Animal Health Inc.</td><td>141704</td><td>1/100 dilution; Administered via intraperitoneal injection at 0.03 mL/g body weight</td></tr><tr><td>FITC-dextran 4</td><td>TdB Labs</td><td>20550</td><td></td></tr><tr><td>Heparinized Capillary Tubes</td><td>Kimble Chase Life Science and Research</td><td>2501</td><td>For retro-orbital blood collection</td></tr><tr><td>Microplate, PS, 96-well, Flat-bottom (Chimney Well), Black, Flutrac, Med. Binding</td><td>Greiner Bio-one</td><td>655076</td><td></td></tr><tr><td>MiniARCO Clipper Kit</td><td>Kent Scientific</td><td>CL8787-KIT</td><td>For hair removal</td></tr><tr><td>Optix MX2 and Optix Optiview</td><td>Advanced Research Technologies</td><td>2.02.00.6</td><td>Fluorescence imaging machine and software</td></tr><tr><td>Phosphate Buffered Saline 1x (PBS)</td><td>Wisent Inc</td><td>311-010-LL</td><td></td></tr><tr><td>Puralube Vet Ointment</td><td>Dechra</td><td>12920060</td><td>Ophthalmic ointement to prevent eye damage during anesthesia</td></tr><tr><td>Spark Multiplate Reader</td><td>Tecan</td><td>30086376</td><td></td></tr></tbody></table>

assessment of gut barrier integrity in mice using fluorescein-isothiocyanate-labeled dextran

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 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.
<img alt="In vivo fluorescence imaging, FITC-dextran graphs; plasma, feces analysis; control vs. inulin diet." class="xfigimg" src="/files/ftp_upload/64710/64710fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
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. <a href="https://www.jove.com/files/ftp_upload/64710/64710fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

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 ...

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Research

JoVE Journal

Biology

7.9K Views.  Centre de recherche du Centre hospitalier de l’Université de Montréal (CRCHUM). Deze methode helpt bij het beantwoorden van vragen over de vraag of een aandoening de integriteit van de darmbarrière verandert of vragen of een mogelijke behandeling de integriteit van de darmbarrière kan behouden. Het grote voordeel van deze techniek is dat het een niet-invasieve, low-burden techniek biedt om de darmpermeabiliteit in vivo te evalueren en te kwantificeren. Om te beginnen, versnel de muizen gedurende vier uur en dien vervolgens 200 microliter van de FITC-dextran suspensie t...

Video: Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

Medicine

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

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 &#34;intestinal sacs&#34; 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 (Papp) 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.

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

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.

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Neuroscience

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

Immunology and Infection

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.

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 ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

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.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

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 ...

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...

FITC-Dextran Feeding: A Method to Quantify Intestinal Permeability in C. elegans

Source: Le, T. A. N., et al. <a href="https://www.jove.com/video/60419/measuring-effects-bacteria-chemicals-on-intestinal-permeability">Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of&#160;Caenorhabditis elegans.</a>&#160;J. Vis. Exp. (2019).
This video introduces a method of to visualize and measure the integrity of the C. elegans intestine lumen by feeding worms FITC-labeled dextran.&#160; The example protocol shows the assay done with 3,3&#8217;-diindolylmethane (DIM)-treated and pathogen-fed worms.

Source: Le, T. A. N., et al. Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of&#160;Caenorhabditis elegans.&#160;J. Vis. ...

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

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