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

Begin with a restrained mouse lacking interleukin-10, an anti-inflammatory cytokine, which results in colon inflammation.

Take a syringe filled with a fecal suspension rich in diverse gut microbes, from a healthy mouse.

Next, attach an oral tube to the syringe.

Insert this tube into the mouth, guiding it downward to the stomach of the mouse.

Now, administer the fecal suspension directly into the stomach, allowing it to gradually reach the colon.

Once in the colon, the fecal microbes multiply and colonize, reestablishing the gut microbes.

The colonized microbes then release various secondary metabolites.

These metabolites help in mucin production, which repairs the colon epithelium and restores the gut integrity.

Within the colon epithelium, the fecal microbes and their metabolites interact with immune cells.

This interaction causes the immune cells to release anti-inflammatory cytokines, leading to a reduction in colon inflammation in the interleukin-10 deficient mouse.

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 thawed fecal suspension to 1-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 palate 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 5 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.