An Air Pouch Mouse Model for Lipopolysaccharide-Induced Inflammatory Exudate Collection

Abstract

Source: Lu, Q., et al. Detecting Migration and Infiltration of Neutrophils in Mice. J. Vis. Exp.  (2020).

This video describes the procedure for generating an air pouch in the subcutaneous tissue of mice and collecting inflammatory exudates in response to lipopolysaccharide administration in vivo. This video also covers the analysis of the neutrophil infiltration in the air pouch.

Protocol

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

1. Air pouch assay

1. First air injection
   1. On day 0, fully anesthetize mice with 5% isoflurane for 3 min in a gas anesthesia chamber at a speed of 2 L/min, and maintain the anesthesia of each mouse in a single breathing unit with 2% isoflurane at a speed of 0.5 L/min.
      NOTE: Sufficient depth of anesthesia is ensured by pinching the toes before moving mice from the chamber to the single breathing unit. The legs should not move when the toes are pinched.
   2. Use a 0.22 µm filter attached to a 5 mL syringe to obtain a 3 mL volume of sterilized air.
   3. Lift the back skin of the anesthetized mouse with tweezers and subcutaneously inject 3 mL of sterilized air using a 26 G x 3/8" needle.
   4. After treatment, remove the mice from the breathing unit. Monitor the mice to ensure they are alive until they start to move around.
2. Second air injection
   1. On day 3, inject an additional 3 mL of sterilized air into the previously established air pocket to sustain the air pouch as described in section 1.1.
3. Treatment
   1. On day 6, 6 h before sacrifice, inject different treatments into the air pouch. Inject 1 mL of phosphate-buffered saline (PBS) as a negative control. Inject 1 mL of 1 µg/mL LPS as the positive control to induce local inflammation.
   2. Fully anesthetize mice with 5% isoflurane for 3 min in a gas anesthesia chamber at a speed of 2 L/min, and maintain the anesthesia of each mouse in a single breathing unit with 2% isoflurane at a speed of 0.5 L/min. Prepare wash buffer according to Table of Materials.
      NOTE: Sufficient depth of anesthesia is ensured by pinching the toes before moving mice from the chamber to the single breathing unit. The legs should not move when the toes are pinched.
   3. For each air pouch, wash the air pouch with 1 mL of wash buffer and collect the inflammatory exudate in a 15 mL centrifuge tube. Wash the air pouch with 2 mL of wash buffer 2x and collect the inflammatory exudate in the same centrifuge tube.
   4. Centrifuge at 100 x g for 10 min at RT. Discard the supernatant and resuspend cells in 1 mL of wash buffer. Count the cells to quantify the neutrophil ratio using the automatic hematology analyzer.
      NOTE: See representative results in Figure 1.

Results

Figure 1: Representative results of the air pouch assay. (A) Illustration of the air pouch assay. (B) Representative results of leukocyte subset infiltration in the air pouch assay. PBS: control; LPS: 1 µg/mL LPS. Data are presente...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100% Ethanol	Beijing Chemical Works
100% Methanol	Beijing Chemical Works
15 mL Conical Polypropylene Centrifuge Tube	Falcon	Falcon
23 G x 1 1/4" Needle	BD	305120
26 G x 3/8" Needle	BD	305110
30 G x 1/2" Needle	BD	305106
5 mL Syringe	BD	Z683574
50 mL Conical Polypropylene Centrifuge Tube	Falcon	14-432-22
Gas Anesthesia System	ZS Dichuang	ZS-MV-IV
Phosphate-Buffered Saline (PBS), 1×			Mix 90% ddH2O with 10% (v/v) 10×PBS, autoclaved
Phosphate-Buffered Saline (PBS), 10×			Dissolve 16 g NaCl, 0.4 g KCl, 2.88 g Na2HPO4·2H2O, 0.48 g KH2PO4 (anhydrous) in 200 mL ddH2O, adjust pH 7.4, autoclaved
Lipopolysaccharide (LPS)	Sigma	L3012
Wash Buffer in Air Pouch Assay			Dilute 0.5M EDTA to 10mM with HBSS