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="Figure 1" class="xfigimg" src="/files/ftp_upload/64710/64710fig01.jpg" /> 
Figure 1: Schematic of the FITC-dextran assay. T&#8722;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 &#181;L of 80 mg&#183;mL&#8722;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 &#181;L 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. 
	&#8203;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 &#34;OK&#34; 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&#39;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 &#181;L 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 &#181;L 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&#183;mL&#8722;1 FITC dextran, dilute by a factor of 1:1 serially 7-10 times. 
	NOTE: The concentrations, thus, must read 20 mg&#183;mL&#8722;1, 10 mg&#183;mL&#8722;1, 5 mg&#183;mL&#8722;1, 2.5 mg&#183;mL&#8722;1, 1.25 mg&#183;mL&#8722;1, 0.625 mg&#183;mL&#8722;1, 0.3125 mg&#183;mL&#8722;1, etc.</li>
	<li>Plate 100 &#181;L 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>

<ol>
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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIGHT+signals+directly+to+intestinal+epithelia+to+cause+barrier+dysfunction+via+cytoskeletal+and+endocytic+mechanisms.">LIGHT signals directly to intestinal epithelia to cause barrier dysfunction via cytoskeletal and endocytic mechanisms.</a> Gastroenterology. 132 (7), 2383-2394 (2007).</li><li>Schmitz, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+tight+junction+structure+contributes+to+the+impaired+epithelial+barrier+function+in+ulcerative+colitis.">Altered tight junction structure contributes to the impaired epithelial barrier function in ulcerative colitis.</a> Gastroenterology. 116 (2), 301-309 (1999).</li><li>Fouts, D. E., Torralba, M., Nelson, K. E., Brenner, D. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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)&#160;</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="Figure 2" class="xfigimg" src="/files/ftp_upload/64710/64710fig02.jpg" /> 
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 &#177; SEM. Each dot represents one mouse. Unpaired Student&#39;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>

Watch this Scientific Journal Video about Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran at JoVE.com

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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5.9K Views.  Centre de recherche du Centre hospitalier de l’Université de Montréal (CRCHUM). 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.

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

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of Crohn&rsquo;s Disease - Crohn&rsquo;s Colitis. Acute colitis may present with abdominal pain and distention, malabsorption, diarrhea, hematochezia and mucus in the stool. We are beginning to understand the complex interactions between the environment, genetics, and epithelial barrier dysfunction in Inflammatory Bowel Disease and animal models of colitis have been essential in advancing our understanding of this disease. One popular model involves supplementing the drinking water of mice with low-molecular weight Dextran Sodium Sulfate (DSS), resulting in epithelial damage and a robust inflammatory response in the colon lasting several days 1.Variations of this approach can be used to model acute injury, acute injury followed by repair, and repeated cycles of DSS interspersed with recovery modeling chronic inflammatory diseases 2. After a single four-day treatment of 3% DSS in drinking water, mice show signs of acute colitis including weight loss, bloody stools, and diarrhea. Mice are euthanized at the conclusion of the treatment course and at necropsy dissected colons are processed and can be 'Swiss rolled" 3 to allow microscopic analysis of the entire colon or infused with formalin as "sausages" to allow macroscopic analysis. Tissue is then embedded in paraffin, sectioned, and stained for histologic review.

Murine Colitis Modeling using Dextran Sulfate Sodium DSS

Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of tissues.

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and pharmacological similarities to that of the mammals. GI intolerance during clinical testing of drug candidates is common and may pose a serious threat to human health. Testing for GI toxicity in preclinical mammalian models can be expensive in terms of time, test compound, and labor. The high-throughput method presented here may be used to predict GI safety issues. Compared to mammalian models, this method allows for more expedient assessment of test compound effects on GI transit while using low quantities of compound. In this method, larval zebrafish (7 days post fertilization) are fed food containing a fluorescent label. After feeding, each larval fish is placed into a well of a 96-conical-bottom-well plate and dosed with test compound (dissolved in water) or the vehicle. As gut transit occurs, fecal matter accumulates on the bottom of the wells, and the rate at which this happens is monitored by measuring fluorescence from the bottom of the well repeatedly over time using a plate spectrophotometer. The fluorescence from larvae in a given treatment group are averaged and these values are graphed along with standard error, for each measurement time, yielding a curve representing average transit of food over time. Effects on gut transit time are identified by comparing the area under the curve for each treatment group to that of the vehicle-treated group. This method detected changes in zebrafish GI transit time induced by drugs with known clinical GI effects; it can be employed to interrogate dozens of treatments for GI effects per day. As such, safer compounds can be quickly prioritized for mammalian testing, which expedites discovery and proffers 3Rs advancement.

The goal of this protocol is to measure the transit time of fluorescently labeled food through the gut of larval zebrafish in a high throughput fashion.

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and ...

Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for generation of transgenics for in vivo imaging. These unique capabilities make the zebrafish an ideal platform to study ocular lens development and physiology. Our recent findings that an Aquaporin-0, Aqp0a, is required for stability of the anterior lens suture, as well as for the shift of the lens nucleus to the lens center with age led us to develop tools especially suited to analyzing the properties of zebrafish lenses. Here we outline detailed methods for lens dissection that can be applied to both larval and adult lenses, to prepare them for histological analysis, immunohistochemistry and imaging. We focus on analysis of lens suture integrity and cortical cell morphology and compare data generated from dissected lenses with data obtained from in vivo imaging of lens morphology made possible by a novel transgenic zebrafish line with a genetically encoded fluorescent marker. Analysis of dissected lenses perpendicular to their optical axis allows quantification of the relative position of the lens nucleus along the anterior-posterior axis. Movement of the lens nucleus from an initial anterior position to the center is required for normal lens optics in adult zebrafish. Thus, a quantitative measure of lens nuclear position directly correlates with its optical properties.

These protocols were developed to analyze cortical lens morphology, structural integrity of the zebrafish lens sutures in fixed and live lenses and to measure the position of the zebrafish lens nucleus along the anterior-posterior axis.

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for ...

Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice

Intermittent fasting (IF), a dietary intervention involving periodic energy restriction, has been considered to provide numerous benefits and counteract metabolic abnormalities. So far, different types of IF models with varying durations of fasting and feeding periods have been documented. However, interpreting the outcomes is challenging, as many of these models involve multifactorial contributions from both time- and calorie-restriction strategies. For example, the alternate day fasting model, often used as a rodent IF regimen, can result in underfeeding, suggesting that health benefits from this intervention are likely mediated via both caloric restriction and fasting-refeeding cycles. Recently, it has been successfully demonstrated that 2:1 IF, comprising 1 day of fasting followed by 2 days of feeding, can provide protection against diet-induced obesity and metabolic improvements without a reduction in overall caloric intake. Presented here is a protocol of this isocaloric 2:1 IF intervention in mice. Also described is a pair-feeding (PF) protocol required to examine a mouse model with altered eating behaviors, such as hyperphagia. Using the 2:1 IF regimen, it is demonstrated that isocaloric IF leads to reduced body weight gain, improved glucose homeostasis, and elevated energy expenditure. Thus, this regimen may be useful to investigate the health impacts of IF on various disease conditions.

The current article describes a detailed protocol for isocaloric 2:1 intermittent fasting to protect and treat against obesity and impaired glucose metabolism in wild-type and ob/ob mice.

Intermittent fasting (IF), a dietary intervention involving periodic energy restriction, has been considered to provide numerous benefits and counteract ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog skin. Since then, this technique has been applied to many different tissues to study the physiological parameters of transport across membranes. The Ussing chamber method is preferable to other methods because native tissue can be used, making it more applicable to what is happening in vivo. However, because native tissue is used, throughput is low, time is limited, and tissue preparation requires skill and training. These chambers have been used to study specific transporter proteins in various tissues, understand disease pathophysiology such as in Cystic Fibrosis, study drug transport and uptake, and especially contributed to the understanding of nutrient transport in the intestine. Given the whole epithelial transport process of a tissue, not only transepithelial pathways, but also paracellular pathways are important. Tight junctions are a key determinant of tissue specific paracellular permeability across the intestine. In this article, the Ussing chamber technique will be used to assess paracellular permselectivity of ions by measuring transepithelial conductance and dilution potentials.

Intestinal epithelium confers not only nutrient absorption but protection against noxious substances. The apical-most epithelial intercellular junction, i.e., the tight junction, regulates paracellular solute and ion permeability. Here, a protocol for the preparation of mucosal sheets and assessment of the ion selectivity of tight junctions using Ussing chamber technique is described.

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog ...

Monitoring Gut Acidification in the Adult Drosophila Intestine

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the proper functioning of the gut. One such region, the copper cell region (CCR), is localized to the middle midgut and consists, in part, of a group of cells known as copper cells. Copper cells are involved in gastric acid secretion, an evolutionarily conserved process whose precise role is poorly understood. This paper describes improvements in the current protocol used to assay for acidification of the adult Drosophila melanogaster gut and demonstrates that it can be used on other species of flies. In particular, this paper demonstrates that gut acidification is dependent on the fly's nutritional status and presents a protocol based on this new finding. Overall, this protocol demonstrates the potential usefulness of studying Drosophila copper cells to uncover general principles underlying the mechanisms of gut acidification.

Monitoring Gut Acidification in the Adult Drosophila Intestine

Here, we present a standardized protocol for monitoring gut acidification in Drosophila melanogaster with optimal output. We first use this protocol for gut acidification monitoring in Drosophila melanogaster and then demonstrate its use in non-model Drosophila species.

The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the ...