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). (...

<ol>
	<li>Everhart, M. B., Han, W., Sherrill, T. P., Arutiunov, M., Polosukhin, V. V., Burke, J. R., Sadikot, R. T., Christman, J. W., Yull, F. E., Blackwell, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duration+and+intensity+of+NF-&#954;B+activity+determine+the+severity+of+endotoxin-induced+acute+lung+injury.">Duration and intensity of NF-&#954;B activity determine the severity of endotoxin-induced acute lung injury.</a> The Journal of Immunology. 176, 4995-5005 (2006).</li><li>Notebaert, S., Carlsen, H., Janssen, D., Vandenabeele, P., Blomhoff, R., Meyer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+NF-&#206;&#186;B+activity+during+Escherichia+coli-induced+mammary+gland+infection.">In vivo imaging of NF-&#206;&#186;B activity during Escherichia coli-induced mammary gland infection.</a> Cellular Microbiology. 10, 1249-1258 (2008).</li><li>Elazar, S., Gonen, E., Livneh-Kol, A., Rosenshine, I., Shpigel, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+recruitment+in+endotoxin-induced+murine+mastitis+is+strictly+dependent+on+mammary+alveolar+macrophages.">Neutrophil recruitment in endotoxin-induced murine mastitis is strictly dependent on mammary alveolar macrophages.</a> Veterinary Research. 41, 10 (2010).</li><li>Connelly, L., Barham, W., Pigg, R., Sait-Jean, L., Sherrill, T., Cheng, D. S., Chodosh, L. A., Blackwell, T. S., Yull, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+nuclear+factor+kappaB+in+mammary+epithelium+promotes+milk+loss+during+mammary+development+and+infection.">Activation of nuclear factor kappaB in mammary epithelium promotes milk loss during mammary development and infection.</a> J. Cell. Physiol. 222, 73-81 (2010).</li><li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injections+in+mice.">Retro-orbital injections in mice.</a> Lab Animal. 40, 155-160 (2011).</li><li>Plante, I., Stewart, M. K., Laird, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+mammary+gland+development+and+function+in+mouse+models.">Evaluation of mammary gland development and function in mouse models.</a> J. Vis. Exp. (21), e2828 (2011).</li><li>Behbod, F., Kittrell, F. S., LaMarca, H., Edwards, D., Kerbawy, S., Heestand, J. C., Young, E., Mukhopadhyay, P., Yeh, H., Allred, D. C., Hu, M., Polyak, K., Rosen, J. M., Medina, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intraductal+human-in-mouse+transplantation+model+mimics+the+subtypes+of+ductal+carcinoma+in+situ.">An intraductal human-in-mouse transplantation model mimics the subtypes of ductal carcinoma in situ.</a> Breast Cancer Research. 11, R66 (2009).</li><li>Zheng, J., Watson, A. D., Kerr, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+expression+analysis+of+lipopolysaccharide-induced+mastitis+in+a+mouse+model.">Genome-wide expression analysis of lipopolysaccharide-induced mastitis in a mouse model.</a> Infection and Immunity. 74, 1907-1915 (2006).</li><li>Nguyen, D., Beeman, N., Lewis, M., Schaack, J., Neville, M. C., Ip, M. M., Asch, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Intraductal Injection into the Mouse Mammary Gland. Methods in Mammary Gland Biology and Breast Cancer Research. , 259-270 (2000).</li><li>Lee, J. W., Paape, M. J., Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+bovine+soluble+CD14+reduces+severity+of+experimental+Escherichia+coli+mastitis+in+mice.">Recombinant bovine soluble CD14 reduces severity of experimental Escherichia coli mastitis in mice.</a> Veterinary Research. 34, 307-316 (2003).</li><li>Elazar, S., Gonen, E., Livneh-Kol, A., Rosenshine, I., Shpigel, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+neutrophils+but+not+mammary+alveolar+macrophages+in+a+murine+model+of+acute+Escherichia+coli+mastitis.">Essential role of neutrophils but not mammary alveolar macrophages in a murine model of acute Escherichia coli mastitis.</a> Veterinary Research. 41, 53 (2010).</li><li>Bannerman, D. D., Paape, M. J., Hare, W. R., Sohn, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+levels+of+LPS-binding+protein+in+bovine+blood+and+milk+following+bacterial+lipopolysaccharide+challenge.">Increased levels of LPS-binding protein in bovine blood and milk following bacterial lipopolysaccharide challenge.</a> Journal of Dairy Science. 86, 3128-3137 (2003).</li><li>Gonen, E., Vallon-Eberhard, A., Elazar, S., Harmelin, A., Brenner, O., Rosenshine, I., Jung, S., Shpigel, N. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+4+is+needed+to+restrict+the+invasion+of+Escherichia+coli+P4+into+mammary+gland+epithelial+cells+in+a+murine+model+of+acute+mastitis.">Toll-like receptor 4 is needed to restrict the invasion of Escherichia coli P4 into mammary gland epithelial cells in a murine model of acute mastitis.</a> Cellular Microbiology. 9, 2826-2838 (2007).</li><li>Notebaert, S., Demon, D., Vanden Berghe, ., Vandenabeele, T., P, ., Meyer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+mediators+in+Escherichia+coli-induced+mastitis+in+mice.">Inflammatory mediators in Escherichia coli-induced mastitis in mice.</a> Comparative Immunology, Microbiology, and Infectious Diseases. 31, 551-565 (2008).</li><li>Osterman, K. L., Rahm, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactation+mastitis:+bacterial+cultivation+of+breast+milk+symptoms,+treatment,+and+outcome.">Lactation mastitis: bacterial cultivation of breast milk symptoms, treatment, and outcome.</a> Journal of Human Lactation. 16, 297-302 (2000).</li><li>Sordillo, L. M., Streicher, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+Gland+Immunity+and+Mastitis+Susceptibility.">Mammary Gland Immunity and Mastitis Susceptibility.</a> Journal of Mammary Gland Biology and Neoplasia. 7, 135-146 (2002).</li><li>Blackwell, T. S., Yull, F. E., Chen, C. L., Venkatakrishnan, A., Blackwell, T. R., Hicks, D. J., Lancaster, L. H., Christman, J. W., Kerr, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+genetically+altered+mice+to+investigate+the+role+of+nuclear+factor-kappa+B+activation+and+cytokine+gene+expression+in+sepsis-induced+ARDS.">Use of genetically altered mice to investigate the role of nuclear factor-kappa B activation and cytokine gene expression in sepsis-induced ARDS.</a> Chest. 116, 73S-74S (1999).</li><li>Stathopoulos, G. T., Sherrill, T. P., Cheng, D. S., Scoggins, R. M., Han, W., Polosukhin, V. V., Connelly, L., Yull, F. E., Fingleton, B., Blackwell, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+NF-kappaB+activation+promotes+urethane-induced+lung+carcinogenesis.">Epithelial NF-kappaB activation promotes urethane-induced lung carcinogenesis.</a> Proceedings of the National Academy of Sciences. 104, 18514-18519 (2007).</li><li>Blackwell, T. S., Hipps, A. N., Yamamoto, Y., Han, W., Barham, W. J., Ostrowski, M. C., Yull, F. E., Prince, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-&#954;B+signaling+in+fetal+lung+macrophages+disrupts+airway+morphogenesis.">NF-&#954;B signaling in fetal lung macrophages disrupts airway morphogenesis.</a> The Journal of Immunology. 187, 2740-2747 (2011).</li><li>Chiang, S. H., Bazuine, M., Lumeng, C. N., Geletka, L. M., Mowers, J., White, N. M., Ma, J. T., Zhou, J., Qi, N., Westscott, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+kinase+IKKepsilon+regulates+energy+balance+in+obese+mice.">The protein kinase IKKepsilon regulates energy balance in obese mice.</a> Cell. 138, 961-975 (2009).</li><li>Singh, M. V., Kapoun, A., Higgins, L., Kutschke, W., Thurman, J. M., Zhang, R., Singh, M., Yang, J., Guan, X., Lowe, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2+/calmodulin-dependent+kinase+II+triggers+cell+membrane+injury+by+inducing+complement+factor+B+gene+expression+in+the+mouse+heart.">Ca2+/calmodulin-dependent kinase II triggers cell membrane injury by inducing complement factor B gene expression in the mouse heart.</a> The Journal of Clinical Investigation. 119, 986-996 (2009).</li></ol>

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 次观看.  Vanderbilt University Medical Center. 这里描述的是一种技术，其中脂多糖注入泌乳小鼠乳腺通过乳头模拟乳腺炎，通常由细菌感染引起的一个条件。脂多糖（LPS）的结果中增加核因子kappa B（NF-κB）信号，通过NF-κB荧光素酶报告基因小鼠的生物发光成像的可视化。