作者要感谢迈克尔·戈德堡为制备稳定siRNA溶液和希瑟·托宾她与动物的援助。这项研究是由美国国防部创新奖W81XWH-08-1-0659为台达电子。 SK的博士后训练是由苏珊科曼摹基金会（KG101329）的支持。作者要感谢克里斯汀约翰逊准备原理图。

1。注射液的制备<ol><li>在磷酸盐缓冲盐水（PBS）或其他感兴趣的水溶液制备的0.2％Evans蓝染料。这一重要的染料是用于评估导管内注射的成功和有用的技术开发的专业知识时，建议作为可视化助剂。 </li></ol>所需要的量将取决于待注射的腺体的数量和位置。雌性小鼠的所有10个乳腺可以被注入，但由于较小的腺大小，腺对1和5通常注射10微升的溶液。所有其他的乳腺对注射用20μl。这些卷都足以填满整个导管树。 2。术前准备<ol><li>记录体重每只小鼠。如同所有的临床前研究，动物权重应定期监测（每周两次或以上），以评估潜在的毒性。 </li><li>麻醉米乌斯使用异氟醚室和敷眼部润滑剂。在实验过程中的小鼠会继续经由鼻锥使用异氟烷在氧气吸入2-4％的浓度进行麻醉。仔细监视鼠标的变化呼吸频率，相应地调节异氟醚的水平。 </li><li>美洛昔康注射（5-10毫克/千克）的程序为皮下镇痛前。 </li><li>申请过的非处方脱毛膏乳头区。等待5分钟，然后轻轻取出松散的头发使用圆周运动棉花尖端涂药。使用湿用温水湿纸巾除去霜。剃须不会由于损坏的乳头的风险推荐。 </li><li>轻轻向下录音四肢固定鼠标的立体镜之下。 </li><li>清洁注射部位用酒精棉签。 </li></ol> 3。导管内注入<ol><li>找到合适的乳头立体镜下被注入。用细微解剖故作多情zers，以消除任何死皮覆盖乳头开口。 </li><li>负载10-20微升注射液加入到50微升注射器上贴上33 G，金属轮毂针。有时候，注射后，少量（1微升或更少）可以在拔出针头漏出的乳头。因此，建议注入11或21微升分别占潜在损失。 </li><li>轻轻握住乳头与细镊子和其轻轻抬起来定位它注射。这是没有必要削减乳头。 </li><li>注射液缓慢，尽量减少所造成的乳腺导管管腔内快速移动的流体的潜在损害。喷射率应保持在约40微升/分钟。 </li></ol> 4。术后护理<ol><li>观察注射部位。应该有创伤的乳头区域或周围组织没有任何迹象。肿胀的乳头周围的区域可能表明一个乳腺脂肪垫注射过早地r除一个成功的导管内注入。 </li><li>从鼻锥取出的动物，并移动到恢复一个单独的笼子里。放置在热灯下笼，防止低温和协助恢复。小鼠单独圈养，并会密切监察，直到他们恢复意识和流动性。 </li></ol> 5。乳腺组织分析<ol><li>经研究完成后，将小鼠颈椎脱臼在一个隔室下面的CO 2的压缩气体安乐死。乳腺切下并可以用于整个装载制备11，组织学，或者RNA ​​和蛋白质分离。 </li></ol>

<ol>
	<li>Dooley, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ductal+lavage+for+detection+of+cellular+atypia+in+women+at+high+risk+for+breast+cancer.">Ductal lavage for detection of cellular atypia in women at high risk for breast cancer.</a> J Natl Cancer Inst. 93, 1624-1632 (2001).</li><li>Jacobs, L., Sukumar, S., Stearns, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraductal+therapy+for+the+prevention+of+breast+cancer.">Intraductal therapy for the prevention of breast cancer.</a> Curr Opin Investig Drugs. 11, 646-652 (2011).</li><li>King, B. L., Love, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intraductal+approach+to+the+breast:+raison+d'etre.">The intraductal approach to the breast: raison d'etre.</a> Breast Cancer Res. 8, 206 (2006).</li><li>Murata, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ductal+access+for+prevention+and+therapy+of+mammary+tumors.">Ductal access for prevention and therapy of mammary tumors.</a> Cancer Res. 66, 638-645 (2006).</li><li>Stearns, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+and+clinical+evaluation+of+intraductally+administered+agents+in+early+breast+cancer.">Preclinical and clinical evaluation of intraductally administered agents in early breast cancer.</a> Sci Transl Med. 3, 106ra108 (2011).</li><li>Flanagan, M., Love, S., Hwang, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Status+of+Intraductal+Therapy+for+Ductal+Carcinoma+in+Situ.">Status of Intraductal Therapy for Ductal Carcinoma in Situ.</a> Curr Breast Cancer Rep. 2, 75-82 (2010).</li><li>Nguyen, D. -. A., 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> Methods in mammary gland biology and breast cancer research. , 259-270 (2000).</li><li>Behbod, F., et al. <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 Res. 11, R66 (2009).</li><li>Bu, W., Xin, L., Toneff, M., Li, L., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+vectors+for+stably+introducing+genes+into+mammary+epithelial+cells+in+vivo.">Lentivirus vectors for stably introducing genes into mammary epithelial cells in vivo.</a> J Mammary Gland Biol Neoplasia. 14, 401-404 (2009).</li><li>Reddy, J. P., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RCAS-TVA+system+for+introduction+of+oncogenes+into+selected+somatic+mammary+epithelial+cells+in+vivo.">The RCAS-TVA system for introduction of oncogenes into selected somatic mammary epithelial cells in vivo.</a> J Mammary Gland Biol Neoplasia. 14, 405-409 (2009).</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. , e2828 (2011).</li></ol>

作者并无任何财务披露。

转基因和基因敲除小鼠是研究各个基因在乳腺癌体内的作用非常宝贵的工具。然而，这是昂贵和费时的，以产生这些动物和基因敲除通常是无处不在的，有时会令我们无法认识到基因对乳腺的具体效果。因此，靶向敲低会减轻在其他器官中的非特异性的副作用和毒性问题。 导管内注射的siRNA这里提出使在小鼠乳腺的局部基因敲除。它是一个快速的方法靶向和局部药物递送到乳腺不限于siRNA递送。这样的乳腺特异性药物递送避免了非特异性的副作用和毒性的其他器官，并允许基因变异的研究在乳腺发育和乳腺肿瘤的形成过程中的特定时间点。没有证据显示硅核糖核酸颗粒在非注射腺，肝脏或其他器官提供了进一步的证据表明，这种方法提出了在乳腺局部的基因敲除的优选方式。此外，胰管内注入可以每周或每两周重复在几个月内以允许长期监测的治疗剂。 这种技术开辟了可能性用于测定的导管内输送各种试剂对小鼠乳腺导管。在充分表征的小鼠模型中局部递送进展加快的非侵入性的应用中，有针对性的在人类中的治疗策略。

小鼠乳腺是由一个复杂的导管肺泡树收敛到在乳头中央管。在小鼠以及人类中，大多数乳腺肿瘤起源于上皮细胞衬乳管。目前可用的治疗包括静脉化疗，放疗和手术治疗。局部导管的治疗已经探索1-3，并提供在减少的毒性和副作用方面显著优点。然而，在这些可以更广泛地应用于一些挑战依然存在。在小鼠乳腺执行程序相媲美的能力，有利于导管内干预的表征。这里，我们提出的递送试剂的直接进入小鼠乳腺导管的非侵入性方法。 导管内输送的两种乳腺癌患者和小鼠模型的化疗剂已被证明是没有证据的高效全身毒性或长期的病理改变3-6。此外，肿瘤细胞注射及癌基因的慢病毒载体传递已经显示出以前通过导管注射7-10是可行的。 除了药物释放，这个过程是乳腺基因通过导管内注射的siRNA沉默本地有用。注射少量的siRNA的解决方案是在不改变乳腺的解剖学和生理学有效。在我们的实验室，我们最近表明在转基因小鼠的关键调解人的基因，自发发展造成预防肿瘤进展（Brock 等人 ，未发表）的乳腺肿瘤成功的沉默。我们已经能够以没有明显的组织损伤或毒性重复每两周注射到相同的乳头最多7次在2周的时间间隔。 导管内注射可在成年小鼠，以解决发育定时孕期，哺乳期和退化或肿瘤的发生和发展过程中的目标后期阶段有关乳腺发育opmentally特定的事件。处女女年仅8周龄时已注入非侵入性在我们的实验室。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>isoflurane</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>2-4%</td>
		</tr>
		<tr>
			<td>meloxicam</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>5-10 mg/ml</td>
		</tr>
		<tr>
			<td>Evans blue dye</td>
			<td>Sigma-Aldrich</td>
			<td>E2129-10G</td>
			<td>0.2% in PBS</td>
		</tr>
		<tr>
			<td>hair removing cream</td>
			<td>Veet</td>
			<td>&nbsp;</td>
			<td>Choose sensitive option</td>
		</tr>
		<tr>
			<td>stereoscope</td>
			<td>Zeiss</td>
			<td>Stemi DV4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>micro-dissecting tweezers</td>
			<td>Roboz</td>
			<td>rs-4976</td>
			<td>With a 0.10 x 0.06 mm tip made of dumostar</td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>7637-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>33-gauge needle</td>
			<td>Hamilton</td>
			<td>7803-05</td>
			<td>point style 4, 12 degree bevel (standard)</td>
		</tr>
		<tr>
			<td>siGLO cyclophilin B control siRNA</td>
			<td>Dharmacon</td>
			<td>D-001610-01-05</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

intraductal injection for localized drug delivery to the mouse mammary gland

在此我们描述了一个协议，提供各种试剂通过导管注射小鼠乳腺。局部给药和敲低内的乳腺上皮细胞的基因已经难以实现由于缺乏适当的靶向分子，是独立的，如怀孕和哺乳期的发育阶段的。本文中，我们描述了用于试剂的局部递送到乳腺在通过导管注射到小鼠的乳头在成年期的任何阶段的技术。注射剂可以在活体小鼠中进行，在麻醉状态下，并允许非侵入性和局部药物输送到乳腺。此外，注射剂可以重复在几个月而不损坏乳头。重要的染料，如伊文思蓝是来学习的技术非常有帮助。经导管内注射蓝色染料，整个导管树变得肉眼可见的。此外，荧光标记试剂也允许在乳腺内的可视化和分布。这种技术可适于多种化合物，包括的siRNA，化疗剂，和小分子。

乳头可以使用立体显微镜一旦头发已经在其周围（ 图1A）的区域被去除容易地本地化。掌握注射技术，建议注射伊文思蓝染料和监测腺体的完整性（ 图1B和C）。这种方法也允许适当体积被注入到各压盖作为一个可以在视觉上评估该染料是否达到整个导管系统（ 图1C和图2A）的测定。注射Evans蓝的图像的图像然后可以相对于整体式乳腺，其中整个导管树沾有胭脂红明矾染料（ 图2B）。 请注意，该喷射方法的鲁棒性是高度依赖于操作者。例如，穿孔的导管将导致乳腺脂肪垫注射和注射该溶液过快，可能会损坏导管上皮大公ls和引发炎症反应。成功的导管内注射使局部药物输送到乳腺，并减少非特异性的副作用常常观察到，否则。 一个腺体的一例图象注射荧光标记的siRNA靶向亲环素（一种非必需基因）示于图3。 <img alt="图1" src="/files/ftp_upload/50692/50692fig1.jpg" /> 图1。通过与文思蓝染料乳头腹股沟乳腺注入。 A）注射液与20μl伊文思蓝染料后乳头注入和 B之前外观）。没有肿胀或组织损伤是观察。C）一旦打开皮肤，染料允许整个乳腺导管树的可视化。 <img alt="图2" src="/files/ftp_upload/50692/50692fig2.jpg" /> 图2。整个安装埃文斯蓝的分析遭离弃的腺体。 A）一种解剖腺从动物切除和脂肪后的同一腺与胭脂红染料注射B）整装染色后立即铺展在载玻片上的代表性图像已被清除。以A）的比较证实了注射液填补了乳腺导管树和腺体在解剖学上是完整的。 <img alt="图3" src="/files/ftp_upload/50692/50692fig3.jpg" /> 图3。新鲜的组织样本导管内交付荧光的siRNA。代表形象，切除48小时postinjection。

Watch this Scientific Journal Video about Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland at JoVE.com

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

一种协议，用于非侵入性导管内输送水性试剂的小鼠乳腺进行说明。该方法利用局部注射到专门针对乳腺导管乳腺的乳头。这种技术可适于多种化合物，包括的siRNA，化疗剂和小分子。

导管内注射局部给药的小鼠乳腺

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

intraductal-injection-for-localized-drug-delivery-to-mouse-mammary

Research

JoVE Journal

Biology

34.8K 次观看.  Boston Children's Hospital and Harvard Medical School. 一种协议，用于非侵入性导管内输送水性试剂的小鼠乳腺进行说明。该方法利用局部注射到专门针对乳腺导管乳腺的乳头。这种技术可适于多种化合物，包括的siRNA，化疗剂和小分子。 

视频: Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

小鼠乳腺上皮细胞形成过程中Lactogenic分化的微球体

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

科研

生物学

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

变化在大鼠乳腺形态和患乳腺癌的风险

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

乳腺的发育和功能在小鼠模型的评价

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

啮齿动物附睾精子磷酸分析

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

对小鼠乳腺冰冻切片间接免疫荧光

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

针对鼠标内耳给药方法的比较研究：Bullostomy<em&gt;对战</em&gt;鼓室注射

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

完整、全小鼠乳腺的分离及细胞外基质表达及腺体形态学分析

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

小鼠部分胆管结扎术: 局部梗阻性胆汁淤积症的对照模型

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.