这项工作是由美国国立卫生研究院资助CA113734到F Yull和退伍军人事务部（TS布莱克韦尔）获得。

所有的动物实验，范德比尔特大学实验动物护理和使用委员会的批准。 1。准备转基因小鼠<ol><li> NGL（N F-κB-G FP-L uciferase）记者转基因（前面描述1）阳性的雌性小鼠，用于此过程。当女性的年龄至少6周，交配与FVB野生型男性。后宫养殖可以使用（在一个笼子里，男1例，女4例）。 （请注意：FVB应变小鼠用于目前的研究中，也可以使用较深的菌株如C57BL6小鼠，但必须被剃光这些小鼠成像前）。 </li><li>交配后15天开始，定期检查的女性，怀孕的征兆（大突出的周长）。每一个女性一旦怀孕，必须分开，从男性的繁殖笼，一旦女性怀孕后。 </li><li>观看分居女性d注意的出生日期时，会出现一个垃圾箱。一胎至少6个幼崽，需要有足够的哺乳期。八天出生后的垃圾，进行协议（的时间安排示意图参见图1）。 </li></ol> 2。注射剂<ol><li>溶解E.大肠杆菌 LPS在PBS中至终浓度为0.1微克/微升。 50μl的该溶液中，或共5微克，将被注入到乳腺。要用于控制注射的PBS单独准备的第二管。 </li><li>建立了一个的解剖范围和异氟醚麻醉设备。鼻锥装置应固定的解剖范围，将鼠标放在舞台。一个很好的光源，是否也应该准备的范围本身或从额外的聚光灯。 </li><li>一旦设置完成后，启动异氟醚机和空气流通。 </li></ol> 3。导管内注射LPS <ol><li>用50μl的LPS溶液装入短针注射器。设置这一边。 </li><li>首先将女性的异氟醚填充框，然后一次呼吸减慢麻醉的小鼠被注入，将她搬上舞台，并把她的鼻子，鼻锥体内。用脚趾的捏确认鼠标是完全麻醉。在整个过程中，监视鼠标的呼吸，并相应地调整异氟醚的水平。笼中的一窝幼崽，女性已经从应小心放在一边，直到大坝手术后可以返回。 </li><li>使用一小滴70％乙醇作的被毛平躺着，并帮助可视化鼠标的小腹连接到正确的腹股沟乳腺乳头。 </li><li>重点解剖的范围，以获得最佳的乳头。小心地用细镊子，以进一步绘制出乳头的皮肤一组，但不要刺穿或以任何方式伤害乳头。 “钳子应该永远不会被紧紧地夹住操纵组织在一起的时候，但使用的光保持需要的最小量的应力对乳头。 </li><li>使用镊子在左手轻轻握住乳头的地方。慢慢带来到视图使用右手的字段中的预装注射器。应保持的注射器，以便没有重新定位有发生，为了注入的内容。 </li><li>一旦注射器是在视图中，瞄准仔细的乳头的中心内的小导管开口，和插入注射器。 </li><li>不重新定位的手的情况下，注入的注射器的内容和取下针头从乳头。 </li><li>装入的第二注射器用50微升PBS控制喷射。重新定位鼠标在舞台上，以达到最佳效果＃4左腹股沟乳腺乳头的另一端鼠标的小腹。重复步骤3.4至3.7的PBS装注射器。 </li><li>一旦这两个注射的并发症ETE，停止麻醉，将鼠标移动到恢复笼。监控的女性，以确保在5分钟内，她成为移动。允许鼠标保持在1小时的恢复笼。 1小时后复苏，将女性的背部与她关进笼子一窝幼崽。应定期监测小鼠注射后6小时，然后再进行成像。根据我们的经验，无不良后果，无论是在女性的幼崽指出，注射和成像之间的期间。但是，如果遇险的迹象中观察到的女性（ 如驼背的姿势，咬或防范的注射部位，一般嗜睡）或幼崽（没有动静，皮肤的色调偏蓝）实验应停止使用。 </li></ol> 4。生物光学成像NF-κB活动的可视化<ol><li>一旦小鼠已被运送到的摄像设备，开始成像过程，通过打开的软件和机器初始化。打开的是oflurane机和空气流量，确保麻醉流到两个盒和成像室。 </li><li>在清澈的麻醉中的异氟醚麻醉下，将鼠标。一旦有所放缓呼吸，用脚趾的捏确认鼠标是完全麻醉。然后，眼窝注射用无菌注射器注入100μL荧光素板鼠标。 </li><li>立即将鼠标的成像室和设在那里的异氟醚鼻锥体放在她的鼻子下。鼠标应该是仰卧。每一个脚固定在舞台上，用胶带。 </li><li>输入适当的设置的IVIS软件采取白光摄影图像和图像的生物发光活性。曝光时间是10秒。采集图像。 </li><li>一旦已经获得的图像，让鼠标恢复，然后将她的背部与她的小狗在笼子里，并定期监测。 </li><li>照相和发光图像被自动重叠并显示在屏幕上，如在图2中。分析所获取的图像，通过将感兴趣区域的或“ROI”圈的大小相等的鼠标的下腹部（1以上的PBS注入腺和一个在LPS注射腺）的每一侧。设置的读出的光子。数字显示每个区域内检测到的发光（ 见图2B）。使用该号码进行统计分析。 </li><li>为了显示最佳的图像，进一步调整阈值的信号显示。这不会改变总的发光读出，但将允许你去除背景信号和可视化的控制和LPS处理腺之间的差异。减少的发光量，直到PBS注射腺背景信号是免费的。这应该留下经LPS处理的腺仍然明显，在图像中，如在图2中看到的信号的增加。 </li><li>的其他与乳腺组织从女性的验尸报告收集，分析部分，包括蛋白质和RNA的检测，就可以完成。 </li></ol> 5。代表性的成果要获得这些方法的解释结果，LPS（PBS）必须被注入到乳腺导管，使相邻的组织不受干扰。注射到皮下组织乳头周围将导致在炎症由于损伤而不是模拟感染。在PBS中稀释的注射液的台盼蓝染料学习的技术时，是有帮助的， 图3A示出一个成功的注射腺注入用100μl的在PBS中的台盼蓝（trypan blue）。如图所示，只在导管的树应该充满注入的溶液，并在注射部位不应该有大丸剂。在这种情况下A“丸”是一个建立的注射液在乳头或乳头周围组织，不输液导管树。这是sh自己在图3B中 。请注意，在图3所示的做法注射完成了21天的哺乳期女性。在泌乳的高度期间（8-10天，在哪个阶段注入LPS是使用目前的协议），牛奶是丰富的，它是很难以可视化的管道和确定注射，如果已正确完成。因此，在哺乳期的稍后的时间点是更好的做法注射。 成功的注射LPS到乳腺导管导致增加腺内的NF-κB活性。 IVIS成像记者鼠标时，这应该是显而易见的，并在LPS注射腺应具有相当高的荧光素酶活性（由光子计数测量），如在图2中所示的（平均比PBS注射腺，脂多糖腺读取200％的的PBS控制信号）。发光在LPS处理腺体排版的一个统计上的显着增加虽然完成一组4只4 ARED向对侧控制实现。另外一个研究中还利用导管乳腺注射或E.大肠杆菌或的LPS到NF-κB记者的转基因。我们自己的工作，笔者看到一个记者活动6小时后显着增加，E.大肠杆菌注射液2。 请注意，由于在大脑和其他腹腔器官的构NF-κB活性，本报记者小鼠的头部和腹部一直显示信号成像时，即使在基线之前的任何操作。因此，该信号应该被忽略关于这个实验。一个明显的信号应保持在LPS注射的乳腺内读出的图像进行调整，以尽量减少在PBS腺背景发光后， 图2示出的图像的背景降低。 不成功的腺体注射最有可能导致损伤的乳头。这将导致指出，皮肤的水平炎症，会是明显的，在成像。这种类型的伤人信号将是非常强大的，局限于乳头的地方发生的损害，而不是整个腺体产生，作为成功后，LPS注射入管道。 <img alt="图1" src="/files/ftp_upload/4030/4030fig1.jpg" /> 图1。过程示意图。 <img alt="图2" src="/files/ftp_upload/4030/4030fig2.jpg" /> 图2。代表结果由IVIS生物发光成像。（A）获取的图像与背景的减少，在LPS注射腺中揭示荧光素酶的活性增加。 （B）图像从一个地区的利益量化补充。 <a href="https://www.jove.com/files/ftp_upload/4030/4030fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alT =“图3”SRC =“/ files/ftp_upload/4030/4030fig3.jpg”/&gt; 图3。练习注射：注射染料通过乳腺导管内注射。交付通过乳头要测试的精确度，在第21天的小鼠的哺乳期用100μl的在PBS中稀释的台盼蓝染料注入。牺牲后，腺体被曝光的可视化。 （A）成功注入和（B）在乳头丸染料。

<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>

没有利益冲突的声明。

在这里，我们利用转基因NF-κB记者的鼠标和优化注射技术模型乳腺炎，乳房组织的炎症最常见的原因是细菌感染。在此过程中最关键的一步是导管内注射本身。 在实践中，这通常也是显而易见注射导管内是否已经成功。注射剂（LPS或PBS）应该感到流体，应该有很少或几乎没有阻力的注射器被清空。最常见的陷阱是导致阻塞通道进入腺体，以乳头的损伤。这可以是可视化的寻?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 大肠杆菌脂多糖 （LPS） </td><td>西格玛</td><td> ł2880 </td><td>血清型055：B5 </td></tr><tr><td> PBS </td><td>敏达公司</td><td> 46-013-CM </td><td></td></tr><tr><td> 31计胰岛素注射器</td><td> Becton Dickinson公司</td><td> 328438 </td><td>短针</td></tr><tr><td>镊子</td><td>世界精密仪器</td><td> 15908 </td><td></td></tr><tr><td>解剖范围</td><td>徕卡</td><td> M3Z </td><td></td></tr><tr><td>特雷尔异氟醚，USP </td><td>接收精英MINRAD INC。 </td><td> NDC 66794-011-25 </td><td></td></tr><tr><td> IVIS 200成像系统&lt;/ TD&gt; <td>显卡尺（原精诺真） </td><td> N / A </td><td></td></tr><tr><td>萤光素注射的注射器</td><td> Becton Dickinson公司</td><td> 309597 </td><td> 1CC 26G5 / 8; </td></tr><tr><td>萤火虫，D-荧光素钠盐一水合物 （荧光素基底） </td><td> Biosynth化学和生物</td><td> L-8240 </td><td>稀释至10毫克/毫升</td></tr></tbody></table>

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

疾病进展和相关的机制，严格学习阶段，最终的临床前模型测试干预是必要的，人类疾病的动物模型。在这些方法中，我们描述了一种技术，其中脂多糖（LPS）被注入到通过乳头，有效建模乳腺炎，或炎症，腺体泌乳小鼠乳腺。增加核因子kappa B（NF-κB）信号，这个模拟感染的结果，显示通过生物 ​​发光成像的NF-κB荧光素酶报告基因小鼠1。 我们的最终目标是研究开发这些方法有关的炎症，乳腺炎在哺乳期腺，通常包括发红，肿胀，免疫细胞浸润2,3。因此，我们敏锐地意识到，切口或任何类型的受伤的皮肤，乳头，或腺体介绍LPS可以在我们的方法中不能使用，因为该方法可能会混淆的读出的炎症。我们也想要一个直接的方法，并不需要特制的手绘移液器或利用显微操作器来保存这些专门的工具。因此，我们确定了一个完整的乳头进入乳腺导管，使用市售的胰岛素注射器和注射剂。此方法是成功的，并允许我们研究与LPS注射相关的炎症，由注射的过程中没有任何额外的效果重叠。此外，该方法还利用NF-κB的荧光素酶报告基因的转基因小鼠和生物发光成像技术的视觉和定量显示增加的NF-κB信号在LPS注射压盖4。 谁愿意在哺乳期乳腺疾病模型，因为归根结底，这项技术的许多学科的研究人员感兴趣的是这些方法这里描述的可用于注射的物质的数量，并不限定于仅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

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。

导管内可视化通过NF-κB记者的转基因小鼠模型的乳腺炎：信号注射LPS

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

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

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。

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

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

鼠标膀胱壁注射液

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

科研

医学

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

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

乳腺癌的一种新的转基因小鼠模型：转移性乳腺癌通过瞄准导管上皮与腺病毒Cre重组酶的启动

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

通过重组腺相关病毒载体注射立体的α-突触核蛋白基于大鼠模型为帕金森氏病的发展

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains. At postnatal day 1 (P1), we used 4-hydroxytamoxifen (4HT) to activate Cre recombinase activity from the Cdh5-CreErt2 transgene to cleave the floxed allele of Ccm2. CCM lesions in mouse brains were analyzed at P8. For micro-CT, iodine based Lugol&#39;s solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 &#181;m, which lead to a minimum feature size of approximately 25 &#181;m. This resolution is sufficient to measure CCM lesion volume and number globally and accurately, and provide high-quality 3-D mapping of CCM lesions in mouse brains. This method enhances the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

小鼠脑海绵体畸形的诱导及显微 CT 成像

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

穿刺诱导的虹膜新生血管虹膜虹膜小鼠模型的建立

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

油酸注射液在猪急性呼吸窘迫综合征模型中的研究

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

毛利精密切肝片作为肝脏生物学的Ex Vivo模型

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

通过胆管 TNBS 输液治疗慢性胰腺炎的鼠标模型

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...