This study outlines a method for inducing localized inflammation in mice by creating an air pouch and administering lipopolysaccharide (LPS). The subsequent analysis of inflammatory exudate allows for the quantification of neutrophil infiltration.
Place an anesthetized mouse on the sterile surgical pad attached to a breathing unit with a continuous flow of oxygen and an anesthetic gas.
Using a filter, load the syringe with sterile air. Attach a needle to the syringe and inject sterilized air subcutaneously into the back of the mouse to create an air pouch within the tissue layers. Monitor the mouse until recovery.
Re-anesthetize the mouse and administer lipopolysaccharide, LPS, into the air pouch. LPS binds to the toll-like receptor 4, TLR-4 on the tissue-resident macrophages and dendritic cells, activating them.
The activated cells release various inflammatory mediators like cytokines and chemokines, that attract neutrophils and other immune cells. These mediators also increase vascular permeability of the nearby blood vessels, enabling immune cells and soluble factors to enter the air pouch from the bloodstream.
As fluid accumulates within the pouch, it forms an exudate comprising inflammatory cells and various bioactive molecules.
Euthanize the mouse and inject a suitable wash buffer into the air pouch. Collect the inflammatory exudate into a microcentrifuge tube and centrifuge. Discard the supernatant and resuspend the cells in the wash buffer.
Analyze the inflammatory exudate to quantify the neutrophil ratio and determine the extent of their infiltration.
After anesthetization of the mice on day zero, use a 0.22-micron filter attached to a five-milliliter syringe to obtain a three-milliliter volume of sterilized air. Lift the back skin of the anesthetized mouse with tweezers and subcutaneously use a 26-gauge by 3/8th-inch needle to inject three milliliters of the sterilized air.
After treatment, remove the mice from the breathing unit and place them into a well-set cage. Monitor the mice to ensure they are alive until they start to move around. On day three, inject an additional three milliliters of sterilized air into the previously-established air pocket to sustain the air pouch.
On day six, inject different treatments into the air pouch. Inject one milliliter of PBS as a negative control, and inject one milliliter of one microgram per milliliter LPS as the positive control to induce local inflammation. After six hours, sacrifice the mice.
For each air pouch, inject one milliliter of wash buffer to wash the air pouch and collect the inflammatory exudate in a 15-milliliter centrifuge tube. Then, wash the air pouch with two milliliters of wash buffer twice, and collect the inflammatory exudate in the same centrifuge tube.
Centrifuge at 100 x g for 10 minutes at room temperature. Discard the supernatant and resuspend the cells in one milliliter of wash buffer. Count the cells to quantify the neutrophil ratio, using the automatic hematology analyzer.
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