This method helps answer questions about whether a condition alters gut barrier integrity or questions of whether a potential treatment can maintain gut barrier integrity. The main advantage of this technique is that it offers a non-invasive, low-burden technique to evaluate and quantify intestinal permeability in vivo. To begin, fast the mice for four hours, then administer 200 microliters of the FITC-dextran suspension via oral gavage to each mouse using a 38 millimeter 22 gauge sterilized curved gavage needle.
Start a timer after the first gavage and wait five to 10 minutes between each gavage to allow in vivo measurements, maintaining one hour post gavage. Keep the remaining FITC-dextran suspension for the standard curve. Start the machine and software by clicking and holding the start button and allow the system to warm up.
Click on Device status and ensure all the configured devices show OK before proceeding. Then click on New study and save the file with the desired name. Next, click on Study options.
Enter the specimen ID and choose the correct laser and experiment. Then remove the fur from the abdominal area using an electric shaver, and apply veterinary ointment generously to the eyes to prevent drying. Open the imaging chamber and place the animal dorsally on the scanning plate.
Then secure the limbs and tail with tape. Next, select the area to be scanned by using the Draw tool. Ensure to include the whole width of the abdomen from just above the liver to the rectum.
Then click on the Modify tool to adjust the area once drawn. Set the scan resolution to 2.0 millimeters and click on Start to begin the scan. Next, open the image files by finding them under the chosen file name and simultaneously open all the files with synchronized settings.
Using the image settings toolbar, use the Sync image and Sync scale buttons to sync the image settings for an accurate comparison. Then save the images with the adjusted scales. Compare the abdominal fluorescence of each animal and the control mouse on uniformly scaled images using software associated with the imaging system.
Collect a fecal pellet from each mouse in a sterile tube four hours post gavage. Then keep the tubes on ice and place them in the dark. Next, centrifuge the blood samples at 9, 390 x g for 10 minutes at room temperature.
Then transfer the plasma to a new sterile tube and keep it on ice in the dark. Dilute 50 milligrams of fecal samples in 200 microliters of PBS, and dilute the plasma with PBS in a one to two ratio. Plate 100 microliter samples and standards in a opaque black 96-well plate.
Read the fluorescence on a fluorescent plate reader with excitation at 485 nanometers and absorption at 530 nanometers. Finally, determine the concentration of FITC-dextran per sample by comparing the fluorescence to the known concentrations of the standard curve. 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.
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. 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. The lower levels of FITC-dextran in the feces of the controlled 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 reinforced this finding. Timing is essential when attempting this procedure. Fluorescence reading and sample collection should occur as closely as possible to one hour and four hours post gavage for each individual mouse.
