Three clinical experiments suggested that fecal microbiota transplantation carries the therapeutic potential for a number of human diseases. Further mechanistic studies will help bring FMT from bench to bedside. This method adopts oral gavage for the delivery route, allowing more precise volume delivery and the fast absorption.
It requires only moderate technical skills. Demonstrating the procedure will be Xioying Zhong, a senior research associate from our laboratory. Begin by preparing sterile paper towels, blunt end forceps, and 50 milliliter conical tubes.
Place some paper towels and forceps in separate autoclave bags and autoclave them at 180 degrees Celsius in dry heat for 30 minutes. Weigh the sterile conical tubes and write down their weight on the tubes. Turn on the biosafety cabinet in the animal room.
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. Place some sterile paper towels on the bottom of the cage, and place the metal rack back on top of the cage.
Identify age matched fecal donors and place the mouse cage in the biosafety cabinet. Open the cage and gently grab a donor mouse by the tail and place it on the metal rack on top of the clean cage. Place the cage cover on top of the rack and wait for the animal to defecate.
Collect the fecal pellets and put them in a sterile 50 milliliter conical tube. Pull the pellets by sex. Weigh the tube again and calculate the weight of the fecal pellets.
Prepare a sterile solution by adding 10%glycerol solution to normal saline. Add 10 milliliters of this solution to the conical tube for each gram of fecal pellets. Homogenize this mixture at a low speed three times for 30 seconds using a benchtop homogenizer inside a fume hood to resuspend the feces.
Filter the fecal suspension through two layers of sterile cotton gauze. Store the filtrate temporarily in a refrigerator for up to six hours, or package it into sterile cryogenic vials and store it in a minus 80 degree freezer. Thoroughly clean the homogenizer or blender following a standard procedure.
Thaw the frozen fecal suspension on ice if using frozen samples. Mix the thawed fecal suspension by vortexing. Transfer the fresh or thaw fecal suspension to one milliliter syringes.
Weigh the mice and choose the right gavage needle size and maximum dosage volume. Test the gavage needle by measuring the length from the tip of the mouse's nose to the bottom of the sternum. Fill the syringe with 10%glycerol in saline or fecal suspension and remove air bubbles inside the syringe and the needle.
Place one mouse cage in the biosafety cabinet. Remove the plastic cage cover and leave the metal rack in place. Open the metal rack with one hand.
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. Gently extend the animal's head backward and hold the head in place with one hand.
Place the gavage needle on top of the tongue inside the mouth. Gently advance along the upper pallet until the needle reaches the esophagus. Pass the needle smoothly in one motion.
Once the needle is properly placed and verified, slowly administer the material by pushing the syringe attached to the needle. After dosing, gently pull the needle out. Mark the mouse and return it to its home cage.
Monitor the animal for five to 10 minutes by looking for signs of labored breathing or distress. Monitor the mice again between 12 to 24 hours after the fecal microbiota transplantation. Enzyme immunoassay revealed that B type natriuretic peptide was markedly elevated in the plasma of interleukin 10 deficient mice.
A healthy donor fecal microbiota transplantation significantly mitigated the increase in B type natriuretic peptide levels. Echocardiography detected a significant decrease in the left ventricle ejection fraction in the interleukin 10 deficient mice compared to the wild type mice. The decrease was significantly abrogated by fecal microbiota transplantation.
These findings suggest that healthy donor fecal microbiota transplantation mitigated colitis induced cardiac impairment. When attempting the protocol, choosing the right gavage needle is essential. Flexible plastic feeding tubes could be a better choice for beginners.
It is also important to practice animal handling before gavage.
