Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-κB Reporter Transgenic

Summary

Described here is a technique in which lipopolysaccharide is injected into the lactating mouse mammary gland via the nipple to simulate mastitis, a condition commonly caused by bacterial infection. Lipopolysaccharide injection results in increased nuclear factor kappa B (NF-κB) signaling, visualized through bioluminescent imaging of an NF-κB luciferase reporter mouse.

Abstract

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as pre-clinical models to test interventions. In these methods, we describe a technique in which lipopolysaccharide (LPS) is injected into the lactating mouse mammary gland via the nipple, effectively modeling mastitis, or inflammation, of the gland. This simulated infection results in increased nuclear factor kappa B (NF-κB) signaling, as visualized through bioluminescent imaging of an NF-κB luciferase reporter mouse¹.

Our ultimate goal in developing these methods was to study the inflammation associated with mastitis in the lactating gland, which often includes redness, swelling, and immune cell infiltration^2,3. Therefore, we were keenly aware that incision or any type of wounding of the skin, the nipple, or the gland in order to introduce the LPS could not be utilized in our methods since the approach would likely confound the read-out of inflammation. We also desired a straight-forward method that did not require specially made hand-drawn pipettes or the use of micromanipulators to hold these specialized tools in place. Thus, we determined to use a commercially available insulin syringe and to inject the agent into the mammary duct of an intact nipple. This method was successful and allowed us to study the inflammation associated with LPS injection without any additional effects overlaid by the process of injection. In addition, this method also utilized an NF-κB luciferase reporter transgenic mouse and bioluminescent imaging technology to visually and quantitatively show increased NF-κB signaling within the LPS-injected gland⁴.

These methods are of interest to researchers of many disciplines who wish to model disease within the lactating mammary gland, as ultimately, the technique described here could be utilized for injection of a number of substances, and is not limited to only LPS.

Protocol

All animal experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee.

1. Preparing Transgenic Mice

1. Female mice, positive for the NGL (NF-κB-GFP-Luciferase) reporter transgene (described previously¹) are used for this procedure. When females are at least 6 weeks of age, mate them with an FVB wild type male. Harem breeding may be used (up to 4 females in a cage with 1 male). (Please note: FVB strain mice were used for the current study. Darker strain mice such as C57Bl6 may also be used, but these mice must be shaved prior to ima....

Discussion

Here we utilize a transgenic NF-κB reporter mouse and an optimized injection technique to model mastitis, inflammation of the breast tissue most commonly caused by bacterial infection. The most critical step in this procedure is the intraductal injection itself.

In practice, it is usually apparent whether the intraductal injection has been successful. The injection of the agents (LPS or PBS) should feel fluid and there should be little to no resistance as the syringe is emptied. The .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lipopolysaccharide from Escherichia coli (LPS)	Sigma	L 2880	serotype 055:B5
PBS	Mediatech, Inc.	46-013-CM
31 Gauge insulin syringe	Becton Dickinson	328438	Short needle
Forceps	World Precision Instruments	15908
Dissecting scope	Leica	M3Z
Terrell Isoflurane, USP	MINRAD INC. for Rx Elite	NDC 66794-011-25
IVIS 200 imaging system	Caliper (formerly Xenogen)	N/A
Syringe for luciferin injection	Becton Dickinson	309597	1cc; 26G5/8
D-Luciferin Firefly, sodium salt monohydrate (Luciferin substrate)	Biosynth Chemistry and Biology	L-8240	Dilute to 10 mg/ml