Here we have described a protocol to guide others in investigating the mechanisms of dysbiosis in disease, from collecting fecal samples to using the Swiss roll method to study changes in the gut. To begin, spray the chest and sides of the euthanized mouse with 70%ethanol, and carefully open the skin and peritoneal cavity to expose the gastrointestinal tract. Isolate the cecum, and use sterile surgical scissors to cut it in half.
Briefly expose the cecum, and cut 0.5 centimeters proximally from the ileum and 0.5 centimeters distally at its junction with the colon. Transfer the isolated cecum onto a sterile Petri dish. Use a sterile spatula to transfer the cecal content into sterile tubes, and store the aliquots in a 80 degrees Celsius freezer.
Re-suspend fresh or previously frozen fecal pellets in sterile saline in a 1 to 20 proportion, and vortex until homogenized. Pass the homogenate through a 30-micrometer-pore nylon filter to remove large particulate matter. Centrifuge at 79G for five minutes, and collect the supernatant to use for transplantation.
Oral gavage 100 microliters of the slurry per germ-free recipient mouse for three consecutive days, followed by gavage every three days for two weeks. Gently place the mouse in restraints on the preheated mouse holder, and leave the tail outside. Carefully tape down the top without pinching it so as not to stress the mouse.
Allow the mouse to rest in the holder, and place it on the tail cuff machine platform for three to five minutes, covered by a sheet to acclimate. Collect at least three rounds of systolic pressure measurements using tail cuff plethysmography, and average the measurements from all the rounds for an average systolic pressure for each animal. Aseptically collect cecal contents from the euthanized mouse.
To harvest the intestines and other tissues, locate the tissue in the mouse, and excise them with scissors to examine the role of the gut microbiota in cardiometabolic health. On day one, dissect the mouse gut from the anal side to the stomach side. And place the entirety of the isolated gastrointestinal tract in a Petri dish containing PBS.
Gently pull the proximal end from the stomach end, and remove the surrounding fat and connective tissue by hand. Isolate the small intestine, and make a Z-type zigzag with each length. Then, cut to obtain the duodenum, jejunum, and ileum.
Isolate the colon by cutting the section of the intestine below the cecum. Cut the duodenum, jejunum, ileum, and colon. Use a syringe and a needle with a ball tip to carefully flush and wash the gut inside with PBS without tearing the intestine.
Place the gut on filter paper, and label the paper with the name of the section and then P at the top left corner for proximal or D for distal at the bottom left corner. Cut the gut longitudinally with ball-tip scissors. Open the gut on the filter paper, and wash with more PBS as needed.
Sandwich the gut between two filter papers, and staple the filter papers at four points or corners near the gut. Soak in 10%formalin neutral buffer solution, and shake using a platform rocker at five rpm overnight at room temperature. On day two, heat the prepared 2%agarose in distilled water with a stir bar in a beaker covered with aluminum foil.
To retrieve the tissues, strip the upper filter paper, and roll the gut from the proximal side so that the proximal side goes inside first, and roll inward so that the lumen is inside on the slide as well. Pin with a 30-gauge needle or two as needed. Aspirate one milliliter of agarose using disposable graduate transfer pipettes, and pour the agarose on a rolled gut section on a flat surface while avoiding air bubbles in the tissues.
After the agarose cools down and solidifies, use a razor blade to trim the extra agarose around the tissue section. Finally, place the gut sections in tissue processing or embedding cassettes, and soak them in 70%ethanol at four degrees Celsius. The estimation of species biodiversity in cecal content obtained from mice on normal salt diets and high-salt diets is shown here.
Changes in the gut microbiota in response to the high salt included a decrease in bacterial biodiversity. Non-metric multidimensional scaling shows that the bacteria from normal salt and high-salt-diet mice form separate clusters. High salt is associated with an increased Firmicutes to Bacteroidetes ratio.
Fecal microbiota transplantation from high-salt-fed mice predisposes germ-free mice to angiotensin II-induced hypertension. Transferring high-salt-induced dysbiotic gut microbiota was associated with significantly increased systolic pressure in germ-free mice compared to mice that received normal salt gut microbiota. The representative images show apolipoprotein A-I stain in ilia obtained from mice that had hyperlipidemia without or with proteinuria.
This protocol aligns steps to achieve successful FMT in key experimental considerations for both human and rodent donors.
