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

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 mouse 1 .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 gland 4 .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. Video LinkThe video component of this article can be found at

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

“…We recommend practicing this step by injecting trypan blue or some other sterile traceable dye inside of the duct and preparing whole mammary mounts to confirm targeting of the ductal tree. Additionally, other protocols describing intraductal injection of reagents have been published 33,34 , which may be useful for developing proper technique. Issues with viral preparation or infection of the ductal lumen can also be investigated by using a mCherry expressing adenovirus.…”

Low-Level Laser Therapy Attenuates LPS-Induced Rats Mastitis by Inhibiting Polymorphonuclear Neutrophil Adhesion

“…When pregnancy was confirmed, the female rats were divided into 4 groups of 45 animals each. Seventy-two hr after parturition, the rats of groups A and B were infused 40 µ g (shown to be the optimal dose of LPS for induction of stable mastitis in our previous studies) LPS into the inguinal mammary glands (left and right fourth glands) as described by Barham et al [ 3 ]. Groups C and D were infused an equivalent sterile pyrogen-free physiological saline.…”

“…CD62L and CD11b/CD18 are important classes of adhesion molecules expressed in bovine PMNs, which mediate the migration of activated circulating PMNs through the blood-milk barrier [ 26 ]. Intercellular adhesion molecule-1 (ICAM-1)-mediated firm adhesion of leukocytes to endothelial cells is a requisite step in PMN transmigration [ 3 , 35 ]. Platelet/endothelial cell adhesion molecule 1 (PECAM-1) expressed in the endothelial cell plasma membrane is also critically involved in the mechanism of PMN transmigration [ 23 ].…”

“…The development of noninvasive, nontoxic and non-pollutant methods for the treatment of mastitis represents a constant concern of veterinarians worldwide. Low-level laser therapy (LLLT), which fits in this category, has been promoted for its beneficial effects on tissue healing and pain relief [ 3 ]. The use of LLLT in treating inflammations has been the focus of various researchers around the world.…”

High resolution 3D MRI of mouse mammary glands with intra-ductal injection of contrast media

“…Ducts have also been imaged in genetically-engineered mice expressing fluorescent proteins using Olympus OV100 Fluorescence Imaging [9], as well as using neutral red dye and a variety of fluorescent dyes [10]. In addition, an IVIS bioluminescence system has been used to visualize inflammation in glands and ducts [11]. …”