Detecting Migration and Infiltration of Neutrophils in Mice

This method tries to describe three frequently used methods for evaluating the migration of normal neutrophils in vivo and in vitro as well as the infiltration of pathological neutrophils in mouse arthritis model. We think this method could be useful for other researchers in this field. This protocol contains the methodological details to evaluate the migration capacity of neutrophils in the inflammation sites by using air pouch assay and adjuvant-induced arthritis.

The implications of this technique extend towards the arthritis therapy by comparing the model group with the treatment group. This method could provide insight into inflammatory disease and can be applied to inflammatory bowel disease. Demonstrating the procedure will be Qingyi Lu and Haixu Jiang, post-graduate students from our lab.

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 3/8 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 times 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. Suspend complete Freund’s adjuvant by vortexing at least five seconds, then draw 100 microliters of suspension into an insulin injector. After anesthetizing, mark the chosen paw and inject 20 microliters of complete Freund’s adjuvant from the injector into four periarticular spots on the ankle joint space.

Put the processed mice in the new chamber. Monitor the mice to ensure that they are breathing until they regain the ability to move. Every three days, use a pocket thickness gauge to measure the ankle joint diameter.

Also, assess arthritis severity by arthritis scoring criterion. Zero is normal. No evidence of erythema and swelling.

One is the mildest arthritis. Erythema and mild swelling confined to the tarsals or ankle joint. Two is the moderate arthritis.

Erythema and mild swelling extending from the ankle to the tarsals. Three is the severe arthritis. Erythema and moderate swelling extending from the ankle to metatarsal joints.

Four is the most severe arthritis. Erythema and severe swelling encompass the ankle, foot, and digits, or ankylosis of the limb. After sacrificing the mouse, remove the skin and part of the muscle from the hind leg with tweezers and scissors.

Spray the joint with 70%ethanol and remove the rest of the muscles using a paper towel. Fix the ankle joint in 4%paraformaldehyde for two days at room temperature. Then decalcify the joint in 10%EDTA for one month at room temperature and change the medium weekly.

After one month, place the tissue in a marked mold with certain volume of liquid paraffin at approximately 60 degrees Celsius to embed the tissue. Cool briefly. Set the thickness on the microtome at four micrometers and cut slices.

Then float the sections in a 43 degree Celsius water bath for a short time to expand the sections. Mount the sections onto slides and put the slides into the oven at 70 degrees Celsius for two hours. For future use, conserve the slides at minus 20 degrees Celsius.

Next, place the slides in a rack and perform washes in xylene and ethanol according to the manuscript to rehydrate at room temperature. Lastly, wash in running tap water for three minutes. Then stain in 0.1%fast green solution for five minutes.

Rinse in 1%acetic acid for 10 seconds and stain in 0.1%Safranin O staining solution for 20 minutes. After that, immerse the slides in the xylene and ethanol washing solutions according to the manuscript. Mount the tissue sections onto the object stage and observe the tissues under a microscope.

To visualize neutrophils, perform immunohistochemical staining by first baking the paraffin sections for two hours at 78 degrees Celsius. Place the slides in a rack and perform the xylene, ethanol, water, and PBS washes according to the manuscript to rehydrate at room temperature. Then add one drop of permeabilization buffer to cover the tissue.

Incubate the sections in a humidity controlled tray at 37 degrees Celsius for 10 minutes. After that, rinse the slides in PBS for three minutes three times. Avoid rinsing the tissue directly.

Next, perform heat-induced antigen epitope retrieval. Arrange the slides in a rack. Immerse the slides in the pressure boiler filled with retrieval buffer.

Put the pressure boiler in the microwave oven and set the microwave oven at 600 watts and heat the slides for 10 minutes. After boiling, keep the slides in the boiler to cool to 90 degrees Celsius. Take out the slides and rinse them in PBS for three minutes three times.

To quench endogenous peroxidase activity, immerse the slides in freshly prepared 3%hydrogen peroxide at room temperature for 15 minutes. Rinse slides in PBS for three minutes three times. Outline a large circle around the sample with a hydrophobic pen.

Avoid touching the sample. Add onto the sample 50 to 100 microliters of 3%bovine serum albumin to block and place the samples in a humidity controlled chamber at 37 degrees Celsius for 60 minutes. Then remove the blocking solution.

Add 50 microliters of PBS diluted primary antibody to each section quickly. Incubate the slides in a humidity controlled tray at four degrees Celsius overnight. On the second day, take out the tray and let it stand at room temperature for 30 minutes.

Then rinse the slides in PBS for three minutes three times. Add 50 microliters of PBS diluted secondary antibodies to the tissues. Incubate slides in a humidity controlled tray at 37 degrees Celsius for 30 minutes.

Then rinse the slides in PBS for three minutes three times. Develop in diluted DAB solution for five minutes. Avoid the development of a dark color caused by overreaction of the DAB.

Rinse the slides in distilled water. Counterstain the slides in hematoxylin for 10 seconds and rinse the slides in tap water for five minutes. Rinse in acid alcohol super fast differentiation solution for three seconds, then rinse in tap water for 10 minutes.

Immerse the slides in the ethanol and xylene washes at room temperature according to the manuscript. Fix the coverslip with mounting solution. Observe the tissue under a microscope.

In this study, air pouch experiments were performed to investigate the neutrophil recruitment stimulated by LPS in vivo. The leukocyte subsets in the air pouch exudates were much higher than in the control. Compared with the control group, the adjuvant-induced arthritis group showed significant edema in the paw.

The ankle joint diameter increased and the arthritis score rose consistently. Cartilage damage is the representative syndrome of rheumatoid arthritis. CFA challenge induced a large amount of leukocyte infiltration, significant cartilage erosion, and synovial hyperplasia.

MPO in any expression levels are representative markers of neutrophil infiltration which were significantly upregulated in the joint section. Make sure that the air is injected into the skin, but not into the muscle. We can also investigate the formation of neutrophil extracellular traps by immunohistochemical staining of the ankle joint tissue sections with anti-p84 or These methods can be used to study other inflammatory diseases such as inflammatory bowel disease.