Evaluating the Effects of Fecal Microbiota Transplantation in an Interleukin-10 Deficient Mouse

Overview

This video demonstrates a procedure of fecal microbiota transplantation in an interleukin-10 deficient mouse with colon inflammation. Colonized fecal microbes in the gut release secondary metabolites, eliciting anti-inflammatory cytokine release and reducing colon inflammation.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Collection of fresh fecal pellets

1. Prepare sterile paper towels, blunt-end forceps, and 50 mL conical tubes.
   1. Place some paper towels and forceps in separate autoclave bags and autoclave them at 180 °C in dry heat for 30 min. Use sterile conical tubes as well. Weigh the conical tubes and write down their weight on the tubes.
2. Turn on the biosafety cabinet in the animal room.
3. Take an autoclaved clean mouse cage without any bedding and place it in the biosafety cabinet. Remove the cover and the food rack and place them inside the cabinet.
4. Place some sterile paper towels on the bottom of the cage and place the metal rack back on top of the cage.
5. Identify age-matched fecal donors and place the mouse cage in the biosafety cabinet. Open the cage and gently grab a donor mouse (C57BL/6J) by the tail and place it on the metal rack on top of the clean cage.
6. Place the cage cover on top of the rack and wait for the animal(s) to defecate.
   NOTE: Put a few mouse littermates on the rack simultaneously.
7. Collect the fecal pellets and put them in a sterile 50 mL conical tube. Pool the pellets by sex. Do not mix fecal pellets collected from males and females.
8. Weight the tube again and calculate the weight of the fecal pellets.

2. Preparation of fecal suspension

1. Prepare a sterile solution (10% glycerol in normal saline).
2. Add 10 mL of 10% glycerol/normal saline to the conical tube for each gram of fecal pellets.
   NOTE: Increase the solution volume to 20 mL if necessary. This study used 1 mL of solution for each pellet (5-10 mg) as well.
3. Homogenize the mixture at a low speed with a benchtop homogenizer or a blender inside a fume hood to resuspend the feces (3 X 30 s).
4. Filter the fecal suspension through 2 layers of sterile cotton gauze (10.2 cm x 10.2 cm). Store the filtrate temporarily in a refrigerator for up to 6 h or package it into sterile cryogenic vials and store it in a -80 °C freezer.
5. Thoroughly clean the homogenizer or blender following a standard procedure.

3. Administration of fecal suspension by oral gavage

1. Thaw the frozen fecal suspension on ice if using frozen samples. Mix the thawed fecal suspension by vortexing.
2. Transfer the fresh or thawed fecal suspension to 1 mL syringes.
   NOTE: Each mouse will receive a total of 200 µL of fecal suspension, and each IL-10^-/- mouse in the control group will get 200 µL of 10% glycerol/normal saline.
3. Weigh the mice and choose the right gavage needle size and maximum dosage volume.
   NOTE: For mice with a bodyweight between 20-25 grams, use a 20 G 3.81 cm curved gavage needle with a 2.25 mm ball. Please check gavageneedle.com for more information.
4. Test the gavage needle by measuring the length from the tip of the mouse's nose to the xiphoid process (bottom of the sternum). Fill the syringe with 10% glycerol/saline or fecal suspension and remove air bubbles inside the syringe and the needle.    
   NOTE: If the needle is longer than the length, put a mark on the needle shaft/tubing at the level of the nose. Do not pass the needle/tubing through the animal past that point to prevent gastric perforation.
5. Place one mouse cage in the biosafety cabinet, remove the plastic cage cover, and leave the metal rack in place.
6. Grab one mouse by the tail and put it on the metal rack. Hold the mouse by the tail with one hand and use the thumb and middle fingers of another hand to restrain the animal by grasping the skin over the shoulders. This way, the forelegs are stretched out to the side, which will prevent the front feet from pushing the needle out. Gently extend the animal's head backward and hold the head in place with one hand.       
   NOTE: Practice mouse handling until the experimenter has full confidence before proceeding to the experiment.
7. Place the gavage needle on top of the tongue inside the mouth. Gently advance along the upper palate until the needle reaches the esophagus. Pass the needle smoothly in one motion. Do not force the needle if any resistance is felt. Take the needle out and try again.
8. Once the needle is properly placed and verified, slowly administer the material by pushing the syringe attached to the needle. Do not rotate the needle or push the needle forward, which may rupture the esophagus. After dosing, gently pull the needle out.
9. Return the mouse to its home cage. Monitor the animal for 5-10 min by looking for signs of labored breathing or distress. Monitor the mice again between 12-24 h after the FMT.

Materials

Name	Company	Catalog Number	Comments
BD Syringe, 1 mL	Fisher Scientific	14-829-10F
Blunt end forceps	Knipex	926443
C57BL/6J mice	Jackson Lab	664
Centrifuge	Eppendorf	5415R
Conical tubes	ThermoFisher	339650
Curved feeding Needles	Kent Scientific	FNC-20-1.5-2
Glycerol	MilliporeSigma	G5516
IL-10 knockout mice	Jackson Lab	4366
USP normal saline	Grainger	6280
Vaporizer	Euthanex Corp.	EZ-108SA