This is a protocol that studies how the small intestine handles materials of different physical properties and how it distributes those materials in space. It's a complementary tool in our study of gut mechanosensitivity. The main advantages of this protocol are that it can be used to study materials or mixtures with different physical properties and that the resolution allows us to resolve regional transits over short intestinal segments.
This technique adds to our understanding of gut mechanosensitivity. It helps us understand how the small intestine makes transit decisions based on the mechanical cues it receives from the intraluminal contents. To begin, prepare the gavage by attaching a feeding tube of 18-gauge thickness and 50-millimeters length to a 1-milliliter syringe and drawing 200 microliters of RITC solution or microsphere solution.
Next, manually restrain the fasted animal using the one-handed restraint technique, then gently insert the feeding tube through the mouth and esophagus of the mouse until it enters the stomach. Once inserted, slowly expel the syringe contents into the stomach and carefully remove the tube from the mouse. Following the gavage, return the mouse to the cage.
Before beginning the dissections, turn on the in vivo imaging instrument so it reaches the desired temperature. After euthanasia, place the mouse in the supine position on a dissection stage and pin its four appendages on the stage to access the abdomen. Next, wet the surface of the abdomen with 70%ethanol.
Next, use micro-dissection forceps to pull the skin and make a transverse incision using sharp surgical scissors 1 centimeter above the anus. To expose the intraperitoneal cavity, continue the incision vertically up the abdomen until the rib cage. Gently handle the bowels and appreciate their orientation in the abdominal cavity before manipulating them.
Cut the proximal to the gastroesophageal junction, thus separating the stomach from the esophagus and gently unravel the colon, cecum, and small intestine by pulling slowly in the opposite direction. Use micro-dissection scissors to separate the attachments of the mesentery to the bowels. Later, using the forceps, transfer the dissected bowels to the measurement sheet.
Place the stomach at 0 millimeters and arrange the bowels along the ruler until 200 millimeters, then cut the bowel at 200 millimeters with micro-dissection scissors and repeat this procedure of bowel alignment. Once the tissue is arranged on the ruler, arrange the cecum to parallel with the tissue, but not in direct contact with it. Place the measurement sheet with the dissected tissue in a dark area so the fluorescence can be preserved until it is time to image.
To start ex vivo imaging, open the imaging software. Log in and initialize the imaging instrument to be ready for image acquisition. For RITC gavage, set Excitation at 535 nanometers and Emissions, 600 nanometers.
While for green microsphere beads, set Excitation at 465 nanometers and Emission at 520 nanometers. Now, set the exposure to Auto and select the field of view. After ensuring that the intestines have not shifted during transport, place the measurement sheet into the instrument within the field of view.
Securely shut the door to the instrument and select Snapshot to photograph the field of view. Save collected pictures to a flash drive for analysis. Next, save individual captures of the fluorescent and photograph images.
An overlay of the photograph and fluorescence indicates the location of fluorescent materials in the gastrointestinal tract. Open the fluorescent and photograph image files in picture editing software for analysis. Adjust the pixel size of both images to have the exact dimensions and close the photograph file.
For the fluorescent image, use an eraser tool to remove the background and make it transparent. Create a new layer. Choose a black fill to the layer and drag the layer to lie below the layer with the fluorescent image and make an entirely black background.
Save the new fluorescent image containing only the fluorescent signal on a black background as a new TIF file. Open the new fluorescent and photograph images in ImageJ. Turn each image into a 30-bit image by choosing Image, followed by Type and 30-bit.
Create a merged image of both by choosing Image, followed by Color and Merge Channels. In the dialogue box that opens, select the photograph file for the gray channel and the fluorescent file Under any colored channels. Turn off the scale of the merged image by selecting Analyze, followed by Set Scale and Click to Remove Scale.
Select the Rectangle tool in ImageJ. Draw a rectangle around a section of the small bowel while paying close attention to the width of this region of interest, as it should remain constant between all regions of interest. Duplicate the region of interest by selecting Image, followed by Duplicate and select only the channel corresponding to the colored channel.
Again, use the Rectangle tool to draw a region of interest over the whole new image and retrieve a fluorescence profile by selecting Analyze, followed by Plot Profile. Open the list of values and copy them to spreadsheet software. Repeat steps from drawing of the region of interest to retrieving a fluorescence profile for each section of the small bowel as described previously on a different ruler row.
In the spreadsheet software, multiply each average intensity value by the region of interest rectangle's constant width created in the previous step. This will yield the real intensity value along the small bowel at each point. Shown here are the average fluorescence traces along the length of the small bowel.
The distribution of fluorescent material varies according to the material properties of the intraluminal contents. Increasing the number of bins used to sort the same raw data sets reveals granular trace features unresolvable with fewer bins. Smaller bins decrease measurement uncertainty, increase spatial resolution, and better reflect the distributive component of small bowel motility.
The geometric center fails to completely characterize the spatial distribution of intraluminal contents. This limitation of the geometric center measurement is evident in the comparison between liquids and larger beads. The fluorescent trace of the larger beads is more distributed, but it averages out to a similar point as that of the liquid, regardless of binning granularity, which highlights the limitation of solely focusing on the geometric center.
To account for the distributive nature of small bowel contractions, a power spectral analysis was incorporated in the present study. Plotting the power spectrum demonstrates that as bin sizes shrink, we can observe significantly dominant frequencies in the larger beads spectrum, but not in that of the small beads. Some, but not all, of those additional dominant frequencies are present in the liquid spectrum.
The most delicate steps of this protocol are the gavage and the dissection. These should be standardized by the experimenters prior to performing this technique for experimental purposes. This is a terminal experiment, as the animal needs to be sacrificed.
When possible, maximize the use of your animal model by performing other non-terminal experiments before this one. Our group has recently used this technique to support our finding of gut touch sets where the gut uses epithelial mechanoreceptors to sense finer physical properties, much like our fingers do.
