This work was funded by NIH grant CA113734 awarded to F Yull and the Department of Veterans Affairs (TS Blackwell).

All animal experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee.
1. Preparing Transgenic Mice
<ol>
 <li>Female mice, positive for the NGL (NF-&kappa;B-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). (...

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Here we utilize a transgenic NF-&kappa;B 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 ...

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

intraductal injection of lps as a mouse model of mastitis: signaling visualized via an nf-&kappa;b reporter transgenic

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-&kappa;B) signaling, as visualized through bioluminescent imaging of an NF-&kappa;B 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 infiltration2,3. 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-&kappa;B luciferase reporter transgenic mouse and bioluminescent imaging technology to visually and quantitatively show increased NF-&kappa;B 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.

Watch this Scientific Journal Video about Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-ÎºB Reporter Transgenic at JoVE.com

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-&amp;kappa;B Reporter Transgenic

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-&kappa;B) signaling, visualized through bioluminescent imaging of an NF-&kappa;B luciferase reporter mouse.

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-&kappa;B Reporter Transgenic

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as ...

intraductal-injection-lps-as-mouse-model-mastitis-signaling

Research

JoVE Journal

Medicine

19.1K visualizações.  Vanderbilt University Medical Center. Aqui descrito é uma técnica na qual lipopolissacárido é injectado na glândula mamaria de rato lactante através do bocal para simular a mastite, uma condição geralmente causada por infecção bacteriana. Resultados lipopolissacarídeos injeção em maior fator nuclear kappa B (NF-kB) de sinalização, visualizado através de imagem de bioluminescência de um rato repórter NF-kB luciferase.