Using intravital microscopy, this protocol enables the real-time visualization of intestinal tissue up to a single-cell resolution and the study of highly dynamic processes such as cell shedding. In combination with the standard tracer experiments, this protocol permits the identification of both intestinal permeability disturbances in vivo and the distinction between para and transcellular permeability. This protocol could be used as the basis for the development of additional intravital microscopy approaches for visualizing other highly dynamic cellular processes of interests within various tissues.
Visual demonstration of this method is essential to understanding the surgical preparation steps and to position the living animal in a way that facilitates image acquisition on the intestinal mucosal surface. Before beginning the procedure, turn on the base and scanner box of a confocal laser scanning microscope and press start to turn on the computer. Launch the image acquisition software and select the appropriate configuration.
To set the appropriate image resolution, select configuration, hardware, resolution, bit depth, and 12. Under the acquisition menu, select XYZT for the image acquisition mode and set the objective to 20 or 40X. To set up the sequential acquisition setting, click seek and add and select between frames.
To configure sequence one, turn on the visible laser box. Set photomultiplier tube one to on and define the emission wavelength. To configure sequence two, turn on the visible laser box.
Set photomultiplier tube two to on and define the emission wavelength. Then activate the appropriate lasers. After confirming a lack of response to toe pinch, use a cotton bud to apply ointment to the eyes of the anesthetized mouse and make a one centimeter incision in the left ventricle region of the abdomen.
Use forceps to exteriorize a three to five centimeter segment of the intestine. Make two small incisions at the top and bottom of the gut segment and introduce a glass micropipette into the intestinal lumen. Then use electrocauterization to open the exteriorized intestinal segment longitudinally along the anti-mesenteric side and expose the mucosa.
Rinse the tissue briefly with saline solution to remove the fecal content. For staining of the intestinal mucosa surface, apply 100 microliters of a one milligram per milliliter of acriflavine solution in droplets onto the exposed mucosal surface and allow the stain to develop for three minutes before washing out the remaining solution with PBS. Next, apply 100 microliters of a two milligram per milliliter rhodamine dextran solution in droplets to the mucosa for three-minute incubation before washing with PBS.
Then place the anesthetized mouse supine on a cover slide mounted on the chamber rinsed with 37 degrees Celsius saline, adjusting the position if necessary, and place the preparation onto the inverted microscope stage. Immediately after placing the mouse onto the stage, turn on the light source and adjust the XY position until the illumination axis is focused on the tissue preparation. In order to select the field of vision, select the appropriate filter cube.
Open the shutter and use the macro and micro wheels to focus on the surface of the intestinal mucosa. Adjust the XY position to locate an area in which several villi can be visualized within the field of view and change the filter cube to verify that the rhodamine dextran staining is also visible in that area. When an area of interest has been identified, start the image acquisition and select sequence one to adjust the laser power, gain, and offset for sequence one.
Then select sequence two and adjust the laser power, gain, and offset for sequence two. To define the Z-stack range, open the Z-stack drop-off menu. Use the Z-access control to focus on the surface of the mucosa and click begin.
Focus on the bottom limit of where the signal is still detectable and click end. Define the numbers of Z-stacks, avoiding time-lapses longer than two minutes for two consecutive time points and open the time menu to select minimize and acquire until stop. To define the line average, select seek one, line average two, seek two, and line average two.
Then to acquire images, set the format to 1024 x 1024 pixels and the speed to 400 and click start. Intestinal epithelial cell-specific GGTase deficient conditional mice developed severe intestinal pathology as demonstrated by an increase in the histological damage score of the small intestine and increased intestinal epithelial permeability can also be detected via tracer in in-vivo experiments using orally administered FITC-dextran. Cell shedding events could be identified as cells moving out of the epithelial monolayer into the lumen, leading to temporary gaps in the ceiling of the epithelium that are finally closed by the contact between neighboring cells, a so-called zipper effect.
These gaps are observed in both control and GGTase deficient mice, although the frequency of these phenomenon is higher in the latter. Interestingly, other cells also appear to uptake dextran. These so-called permeable cell events also occur mainly in conditional knockout mice.
The immunostaining of cytoskeleton-related and tight junction proteins of interest can be performed to identify specific targets that contribute to potential epithelial permeability disruptions determined via intravital microscopy. This method can be used for the analysis of other phenomena on the surface of the intestinal mucosa, such as endothelial leakage or immune epithelial communication in the context of intestinal infection.
