Murine Fecal Isolation and Microbiota Transplantation

Vanderbilt University's Institutional Animal Care and Use Committee approved all procedures described in this manuscript. C57B1/6 male mice at 3 months of age, purchased from The Jackson Laboratory, were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals.

1. Collection, storage, and processing of human fecal samples

1. Collect a stool sample, using a sterile container if the subject is in the clinic. Refrigerate the stool samples at 4 °C within 36 h of collection until ready for processing. Alternatively, collect stool samples using a commercially available tool for easy and extended DNA stability at ambient temperature, especially for at-home use.
2. Sanitize a biosafety-level fume hood with a 10% bleach solution, or other Environmental Protection Agency-approved disinfectant.
3. Remove the stool from cool storage and bring into the fume hood; use a disposable spatula to make ~1 g aliquots and store in a -80 °C freezer until completely ready for processing.
4. Discard all disposable items in biohazard trash. Disinfect all surfaces (hood and any surfaces touched by the processor) and items being removed from the hood.

2. Aseptic collection of mouse fecal samples

NOTE: Use aseptic techniques, including sterilized instruments.

1. Euthanize the mouse by CO₂ asphyxiation. Spray the chest and sides of the mouse with 70% ethanol and carefully open the skin and peritoneal cavity to expose the gastrointestinal tract.
2. Isolate the cecum and use sterile surgical scissors to cut it in half. Briefly, expose the cecum and cut 0.5 cm proximally from the ileum and 0.5 cm distally at its junction with the colon. Transfer the isolated cecum onto a sterile Petri dish.
3. Use a sterile spatula to transfer cecal content into sterile tubes, and store aliquots in a -80 °C freezer¹⁹.
   ​NOTE: Since the majority of bacteria in the gut are anaerobes, exposure to oxygen may damage or kill the organisms during the isolation procedure in a room atmosphere. Thus, fecal samples should be isolated in anaerobic chambers to maintain the viability of the bacteria.

3. Fecal matter transplantation

1. Resuspend fresh or previously frozen fecal pellets in sterile saline in a 1:20 (w:v) proportion and vortex until homogenized.
2. Pass the homogenate through a 30 µm pore nylon filter to remove large particulate matter. Centrifuge at 79 × g for 5 min and collect the supernatant to use for transplantation.
3. Oral gavage 100 µL of the slurry per germ-free recipient mouse for 3 consecutive days, followed by gavage every 3 days for 2 weeks. Use conventional mice to study mechanisms of the gut microbiota if they have been first treated with antibiotics to eliminate the recipient's own endemic microbiota. For example, administer ceftriaxone (400 mg/kg) daily to the recipient mice for 5 consecutive days by oral gavage before gavage of the fecal slurry.
   NOTE: Studies suggest that a minimum of 2 weeks of this treatment is necessary to elicit cardiovascular changes, including blood pressure²⁰.
4. Ensure that germ-free recipient mice are single-housed in gnotobiotic film isolators and fed with sterile food and water.

4. Systolic blood pressure measurements

NOTE: Gnotobiotic mice that received FMT from conventionally housed 3-month-old C57Bl/6 mice were implanted with osmotic minipumps (Alzet, model 2002) for infusion of low-dose angiotensin II (140 ng/kg/min) for 2 weeks. Blood pressure was monitored weekly via tail cuff. The protocol for implanting osmotic minipumps has been previously reported²¹. Tail-cuff was performed as briefly summarized below. A noninvasive method of measuring blood pressure, such as tail cuff, is suitable for FMT studies in gnotobiotic mice. The detailed steps on how to perform tail cuff have been described previously²².

1. Briefly, retrieve the mice from the gnotobiotic isolators and preheat the tail-cuff machine platform and mouse holder.
2. Place the conscious mice in restraints on the heated platform and collect at least three rounds of systolic pressure measurements using tail cuff plethysmography. Perform the following steps below on 3 consecutive days prior to the proper measurement days, to train mice to being restrained in order to reduce stress.
   1. Gently place the mouse in the preheated holder and leave the tail outside. Carefully tape down the top without pinching it, so as not to stress the mouse.
   2. Allow the mouse to rest in the holder; place on the platform for 3-5 min covered by a sheet to acclimate.
3. Average the measurements from all the rounds for an average systolic pressure for each animal.

5. Assessment of FMT to cardiovascular changes

1. Following blood pressure measurement, euthanize the mice and aseptically collect cecal contents, as described in section 2 .
2. Harvest the intestines and other tissues, including the heart, aorta, liver, mesenteric arteries, and kidneys, to examine the role of the gut microbiota in cardiometabolic health. To harvest tissues, locate the tissue in the mouse and use scissors to excise them.
3. Run a metagenomic sequencing analysis on fecal samples/cecal contents collected from the donor and recipient mice to confirm engraftment of the gut microbiota after FMT²³. The first proof of successful colonization of microbiota is confirming that the donor and recipient's microbiota are similar.
4. Use the Swiss-roll technique (see section 6) on the harvested intestinal tissue, coupled with immunostaining and histology to examine morphological and cellular expression changes²⁴.

6. Making gut intestine Swiss-rolls

1. Day 1
   1. In a properly euthanized mouse sprayed with 70% ethanol, dissect the mouse gut from the anal side (fixed in retroperitoneum) to the stomach side. Place the entirety of the isolated gastrointestinal tract in a Petri dish containing phosphate-buffered saline (PBS). Gently hold the proximal end from the stomach end and remove the surrounding fat and connective tissue by hand.
   2. Isolate the small intestine (cephalad from the appendix) and make a Z-type zigzag with each length. Then, cut to obtain the duodenum, jejunum, and ileum successively, as previously described²⁵. Isolate the colon by cutting the section of the intestine below the cecum.
   3. Cut the duodenum, jejunum, ileum, and colon.
   4. Flush and wash the gut inside using PBS with a syringe and a needle with a ball tip, so as not to tear the intestine.
   5. Put the gut on filter paper. Label the paper with the name of the section (e.g., duodenum) and then 'P' at the right top corner for proximal or 'D' for distal at the right bottom corner.
   6. Cut the gut longitudinally with ball-tip scissors. Open the gut on the filter paper. Wash with more PBS as needed.
   7. Sandwich the gut between two filter papers. Staple the filter papers at four points/corners near the gut.
   8. Soak in 10% formalin neutral buffer solution (4.0 g of sodium phosphate, monobasic, 6.5 g of sodium phosphate, dibasic, 100 mL of 37% formaldehyde, 900 mL of distilled water). Shake using a platform rocker at 5 rpm at room temperature overnight.
2. Day 2
   1. Prepare 2% agarose in distilled water and heat with a stir bar in a beaker covered with aluminum foil.
   2. Retrieve the tissues; strip the upper filter paper. 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 G needle or two, as needed.
   3. Aspirate 1 mL 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.
   4. Allow the agarose to cool and solidify. Use a razor blade to trim the extra agarose around the tissue section.
   5. Put the gut sections in tissue processing/embedding cassettes (bigger than the regular ones to accommodate the increased height due to agarose). Soak in 70% ethanol at 4 °C.
   6. Prepare paraffin-embedded tissue slides and proceed to immunostaining, as outlined below.

7. Immunostaining of the gut intestinal tract

1. Deparaffinization
   1. Pass through the following baths with slides in a rack: xylene for 3 min, fresh xylene again for 3 min, xylene with 100% ethanol (1:1) for 3 min, 95% ethanol for 3 min, 70% ethanol for 3 min, and 50% ethanol for 3 min.
   2. Rinse gently with cold running tap water. Store in a bath of tap water.
2. Antigen retrieval
   1. Following deparaffinization, boil the slides in a rack in a bath of antigen retrieval buffer (0.01M trisodium citrate dihydrate at pH 6 and 0.05% Tween-20) at 100 °C for 20 min.
   2. Run under cold tap water.
3. Staining
   1. Remove the slides from the bath and place the tissue face up in a slide box with wet laboratory wipes/paper towels in the bottom. Draw an outline around the tissue with a hydrophobic marker pen.
   2. Drop Tris-buffered saline (TBS) + 0.025% Triton X-100 onto the tissue and incubate for 5 min. Repeat this step.
   3. Block with TBS + 10% fetal bovine serum (FBS) + 1% bovine serum albumin (BSA) for 2 h at room temperature. Turn the slides on their side and remove the blocking buffer on a laboratory wipe.
   4. Add primary antibody solution and incubate at 4 °C for at least 2 h or overnight. Wash gently with TBS + 0.025% Triton X-100 by gently pipetting ~200 µL over the section.
   5. Add secondary antibody solution and incubate for 1 h at room temperature. Rinse the slides 3 x 5 min with TBS by rinsing with a pipette, as in step 7.3.4.
   6. Mount with mounting medium and a coverslip.