作者感谢露丝Zelkha，MS，她在成像慷慨援助。这项研究是由临床科学家发展计划奖K12EY021475到ME支持，授予R01 EY024349-01A1到ARD，从国家眼科研究所，美国国立卫生研究院，并从研究的无限制资助防盲核心补助EY01792。 ARD是一个职业发展奖从研究到防止失明的收件人。

在动物身上进行的所有程序符合视觉与眼科声明在视觉研究使用动物研究协会。十四四到六个月大的男/女C57BL / 6J野生型小鼠用于:对损伤模型十只小鼠和四个对照动物（未受损角膜）。 1.动物的制备<ol><li>麻醉小鼠用100毫克/千克氯胺酮腹腔注射5毫克/公斤甲苯噻嗪混合物。麻醉生效后，捏脚趾确认麻醉深度;动物不应该反应。 </li><li>在进行实验来验证没有任何先前的角膜损伤的前检查裂隙灯下眼。 </li><li>灌输1％盐酸丁卡因的到眼睛上的下降。经过60 - 90秒，测试角膜反射，确认没有感觉。盐酸丙美卡因0.5％，可替代使用。 </li><li>当角膜感觉的损失一定，轻轻地放在5％碘伏对眼睛和眼睛周围的头发的下降。擦去降1分钟后，或它涂在眼部周围的头发，以防止头发从与刀尖干扰。 </li></ol> 2.打磨角膜上皮<ol><li>戴上外科手套和隔离衣。准备所需的工具:无菌旋转毛刺和一对无菌镊子。为了更好的控制，使用弯曲的镊子。放置在过程中的无菌片的工具。 </li><li>使用弯曲镊子，通过从鼻侧抓住盖子，轻轻proptosing眼稳定的眼睛。避免用太多的压力。 </li><li>在手术显微镜，刮胡子360°左右的角膜缘区使用无菌旋转毛刺的两倍。周围的角膜缘去5的速度 - 每回合持续6秒。 注:毛刺有一个固定的速度。然而，它在既定的速度转动，在"端螺母"应被完全收紧。 </li><li>从角膜缘删除整个角膜上皮与旋转毛刺了limbus。为更好的效果，移动以循环方式的工具和保持它稍微平行于角膜表面到其尖端的侧面工作。小心不要弄伤基质没有重新涂刷，其中上皮细胞已被移除的区域。不要使用对眼睛的压力。根据需要清洁画笔笔尖。 </li><li>灌输无菌磷酸盐缓冲盐水的对角膜的下降和擦去用软清洁组织的任何已分离的上皮片。 </li><li>刮剩下的上皮细胞，如果有的话。 </li><li>在手术过程中需要注意的是鼠标尾部。 注:如果发生任何动静，麻醉深度已经消退，需要进一步注入。三分之一到主注入量的三分之二应该足够了。不要过量使用麻醉药物的动物。 </li><li>使用它们的另一只眼睛前消毒所有的工具。 </li></ol> 3.确认完全去除上皮<ol><li>申请为1mg / ml荧光素钠一滴在角膜上。清洁角膜30秒后，并检查其与钴蓝色滤色器，在显微镜下，以验证完全去除上皮。 注:具有上皮缺损的角膜区用绿色显示。 </li></ol> 4.手术后护理<ol><li>放在一个加热毯动物并将其保存在一个安全的地方（ 例如，在笼子里），而下恢复。不要让一个无意识的动物在该公司有意识的人的。 </li><li>应用1％红霉素眼药膏。这也将防止眼睛干涩。继续申请了一个星期抗生素的日常。 </li><li>注入0.1毫克/手术后公斤丁丙诺啡皮下和后再次12小时。继续注射每天两次两天。 </li><li>观察动物30 - 45分钟，直到从麻醉中完全恢复。 注意:腹面对呼吸肌轻度运动表明动物的健康，而下恢复。 </li></ol>

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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+limbal+mesenchymal+cells+support+the+growth+of+human+corneal+epithelial+stem/progenitor+cells.">Human limbal mesenchymal cells support the growth of human corneal epithelial stem/progenitor cells.</a> Invest. Ophthalmol. Vis. Sci. 55 (10), 6953-6959 (2014).</li><li>Pajoohesh-Ganji, A., Pal-Ghosh, S., Tadvalkar, G., Stepp, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+goblet+cells+and+their+niche:+implications+for+corneal+stem+cell+deficiency.">Corneal goblet cells and their niche: implications for corneal stem cell deficiency.</a> Stem Cells. 30 (9), 2032-2043 (2012).</li><li>McCloy, R. A., Rogers, S., Caldon, C. 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The authors declare that they have nothing to disclose.

本文介绍了一种可重复的和相对简单的技术来创建LSCD的小鼠模型。但是也有一些值得一提的这个模型的几个重要方面。第一，使用的化学物质11，12不同的机型，损伤主要涉及表面上皮（和基底膜），与底层角膜基质或其它人工结构的损害最小。因此，仅存在有限炎症和疤痕，这两者都可以变得复杂的程序，并使其更难以评估旨在角膜缘干细胞的任何干预的结果。一个有趣的最近研究由李等人。 10，其比较使用与手术和化学处理的不同组合的旋转毛刺，还发现毛刺更安全和兔创建LSCD更有效。其次，模式似乎至少长达三个月1是稳定的。创建这个模型包括使用旋转毛刺，仪器是熟悉的人谁与角膜的工作，以及为那些谁做鼠标伤人研究临床医生。它比使用钝锅铲优点是损伤更均匀和可重复性1。 为了更好地破坏角膜缘干细胞，角膜缘区域进行处理两次。基于对老鼠的眼睛的工作经验，待遇超过两回合的经常擦伤角膜缘血管，引起出血。万一该技术在其他动物中使用，这个步骤可能依赖于角膜上皮厚度进行修改。在这项研究中，用0.5毫米的毛刺尖端的机动工具是利用;在其它形状和尺寸毛刺提示也可与可用于代替，这取决于角膜的规格和在眼睛尺寸。 Li 等人 。 10报道了2.5毫米毛刺以适于一致地除去兔角膜和角膜缘上皮细胞。 为了获得最佳结果，角膜缘和角膜上皮细胞中以循环方式被剃光并使用了毛刺的侧面。以防止动物毛发从与刀尖干扰时，聚维酮碘滴散布在眼睛周围的头发，而不是被擦掉。该工具不应该在一个地方经营静态，因为这可能会导致穿孔。在一个非常缓慢的步伐可能会导致出血治疗角膜缘;每一轮近6秒的速度是适当的C57BL / 6J小鼠的眼睛。关键是要避免重新涂刷裸露的基质，因为这将导致更多的炎症和间质混浊。而研磨上皮压力不应施加到眼球。在手术后，角膜用荧光素染料染色，以确保完全的上皮去除。它防止动物恢复期间眼球干燥是很重要的。限制是，这种技术无法去除干细胞的100％。这也是有些运营商依赖和需要实践给予合作nsistent结果。 当研究角膜上皮表型，整装染色，以评估该模型的结果的最佳方法。这是因为它允许整个角膜上皮被一次评估，而横截面只会给每个特定部分的信息。 这种控制技术可以在其他动物诱发LSCD小鼠，而且很可能也是如此。它提供了LSCD一个合适的模型来研究新的治疗方法，并发现本病的病理生理的更广泛的方面。与一些改变，也可以是在学习角膜伤口愈合，血管发生和淋巴管生成以及有用。

角膜上皮是必需的，以保持角膜的清晰度和完整性。它是不断在整个寿命通过在角膜和结膜的交界处驻留在角膜缘-一个狭窄区域的上皮干细胞更新。这些自我更新角膜缘干细胞在再生角膜上皮至关重要的作用，这两个正常和损伤后。这些干细胞的部分或完全耗尽将导致复发性角膜糜烂，疼痛，角膜瘢痕和新血管形成，杯状细胞的外观，并且如果不进行治疗，角膜失明。这种情况被称为角膜缘干细胞缺陷（LSCD），并且可以是特发性;遗传;或获得由于化学或热损伤，长期佩戴隐形眼镜，和慢性炎症1-6。 研究LSCD需要一个合适的动物模型，不仅模仿人类的疾病，但是可重复的和可持续的，与至少一个伤害到其他角膜和眼结构的坐骑。这种模式是必要的，以评估治疗和澄清在分子和细胞水平的疾病机制。如1如前所述，通过使用一个旋转毛刺，人们可以很容易地开发LSCD的小鼠模型，具有上述优点并持续至少三个月。这项研究的目的是提出LSCD简单，重复性好，可持续的小鼠模型。 旋转毛刺是一个方便的工具，均匀地去除上皮细胞不损伤下面的基质1。它已被用于诱导伤口愈合研究中央角膜糜烂7,8。兹-呈现技术在小鼠创建LSCD以前尚未报道。此前推出了一个不太均匀伤病特别是在角膜缘和一个更多变型1,9用钝刮刀刮结果上皮细胞，与更多的修复邻方法F普通角膜上皮1。不同于钝刮，旋转毛刺去除上皮基底膜以及7，8，10。其他报道的方法以切断干细胞涉及使用化学物质，如氢氧化钠，正 - 庚醇，和苯扎氯铵，不仅可以诱导不想要的损伤底层眼构造，但也可能导致显著炎症和随后的角膜混浊或上皮鳞状上皮化生11-15。旋转毛刺不会与这些严重并发症。手术去除角膜缘上皮细胞，单独或与化学品的使用10，11，16，更难以进行，而不是在动物细眼和薄角膜缘上皮细胞（如小鼠）的最好的选择。此外，奇怪的是，limbectomy仍可能留下一些落后10角膜缘上皮。 将导致LSCD与新生血管形成的下述技术ð结膜模仿患者完全LSCD的介绍，并持续至少三个月1。它适用于那些谁旨在研究小鼠LSCD或伤口愈合病理生理学，免疫学，和潜在的治疗方法。可能的话，有一些修改，这个程序可以在大鼠或家兔10执行。作为旋转毛刺有效地除去上皮细胞，它可以在有关角膜上皮磨损/伤人及在任何相关的治疗或分子生物学研究中的任何研究中使用。

<table border="0" cellpadding="0" cellspacing="0" width="1091">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Rotating burr: AlgerBrush II rust ring remover&#160;</td>
			<td style="width:277px;">Rumex International Co, Clearwater, FL</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 16-141</td>
			<td style="width:351px;">0.5 mm burr. Also available from other companies.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Surgical microscope</td>
			<td style="width:277px;">Wild Heerbrugg, Switzerland</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160; Wild M691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Digital camera</td>
			<td style="width:277px;">Nikon, Thailand</td>
			<td style="width:144px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Nikon FS-2 slit lamp</td>
			<td style="width:277px;">Nikon, Japan</td>
			<td style="width:144px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Polyclonal anti-CK12 antibody</td>
			<td style="width:277px;">Santa Cruz Blotechnology, CA,</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; SC-17101</td>
			<td>1:100 concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Monoclonal anti-CK8 antibody</td>
			<td style="width:277px;">TROMA-I-s, Iowa City, IA</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; AB_531826</td>
			<td>1:50 concentration</td>
		</tr>
	</tbody>
</table>

a simple mechanical procedure to create limbal stem cell deficiency in mouse

角膜缘干细胞缺陷（LSCD）是故障或角膜缘上皮干细胞，然后将角膜上皮被替换结膜损失的状态。患有复发性角膜上皮缺损，疼痛，炎症，视力遭受损失。 先前，LSCD的鼠模型进行了说明，并相对于其他两个模型。的目标是产生LSCD的一致小鼠模型，这两个模仿人类的表型和持续足够长以使得可以研究疾病的病理生理学和评估新的治疗方法。这里，该技术进行更详细的说明。 用旋转毛刺的电动工具被设计为从角膜表面除去锈环或平滑翼状胬肉床的患者。它是一个合适的装置以产生所需的LSCD模型。这是一个现成的，易于使用的工具，用细尖，使得它适合于小眼睛的工作，如小鼠。它的应用可以防止不必要的眼创伤，它不会导致不必要的伤害，由于经常与化学损伤模型的情况。相对于钝刮，它消除了与基底膜上皮。在这个协议中，角膜缘区域被研磨两次，然后整个角膜上皮被剃光从角膜缘到角膜缘。为了避免基质伤害，呵护被带到曾经的上皮细胞已经删除不要刷角膜表面。

在执行此技术后一个月，角膜的100％开发浅表血管新生（ 图1A。照片拍摄的同时，在明场和钴蓝色的过滤器连接到裂隙灯摄像机）。在眼中的18/20（90％），新生血管介入整个角膜表面（ 图1A）。在2/20（10％），在三四个角膜象限观察新血管形成，但是它仍然达到角膜中心。 为了验证LSCD的发展，整个装载免疫进行1。这个步骤不是该协议的一部分，并且可以根据目标和利益而改变。三个变化被认为是LSCD指标:无细胞角蛋白（CK）12，具体到正常角膜上皮细胞17，18中间丝; CK8的外观，简单的上皮细胞标记<s向上&gt; 20，其不存在于正常的角膜;和角膜表面上杯状细胞的存在。为了评估角膜上皮表型，对于CK12和CK8双重免疫染色上整装样品损伤三个月后进行，如先前1,9所述。杯状细胞存在被MUC5AC-评估杯状细胞特异性粘蛋白免疫染色1，15，1945天以下上皮去除。杯状细胞的出现时间减少。对于最好的结果，它在受伤后进行染色不到两个月是重要的。 正常，未受损伤的角膜明示CK12-一个成熟的角膜上皮细胞17 18 -throughout角膜表面的特异性标记物，但它们是没有任何CK8的或杯状的整装样品的角膜部分细胞（MUC5AC）（ 图1B） 。在比较正常的眼睛，CK12表达上万人受伤几乎不存在<html> Ÿ模型角膜，而他们显著显示CK8阳性和杯状细胞（ 图1B）。这些观察表明LSCD的发展。 如果有兴趣的定量免疫荧光结果，使用图像-J对通过在显微镜下相同的设置拍摄的原始图像不变量化分别免疫染色20密度。简言之，选择整装样品的角膜部分，测量该区域的集成密度和面积，并计算经校正的密度为背景20。 CK12的至CK8密度比以前提出了关于样品1的更大的数目。相比于对照的减少率（正常角膜）和杯状细胞的角膜表面上的外观表明LSCD的发展。 658 / 54658fig1.jpg"/&gt; 图1: 角膜表型小鼠创建LSCD后。裂隙灯检查表明，所有角膜个月（A，比例尺为0.5mm）产生某种程度的新生血管。正常角膜表达CK12遍布角膜，但没有任何CK8或杯状细胞（MUC5AC）的。 CK12是在LSCD模型不存在，而CK8和杯状细胞上出现的角膜（B，比例尺:200微米）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54658/54658fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse at JoVE.com

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

下面的文章提供了一个简单，可重复的方法来有效地创建角膜缘干细胞缺乏（LSCD）的一个可持续发展的小鼠模型。该动物模型是在测试和比较的用于角膜缘干细胞的疾病的治疗的效力是有用的。

一个简单的机械过程来创建在小鼠角膜缘干细胞缺乏症

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

a-simple-mechanical-procedure-to-create-limbal-stem-cell-deficiency

Nanyang Technological University

Research

JoVE Journal

Medicine

8.3K 次观看.  University of Illinois at Chicago. 下面的文章提供了一个简单，可重复的方法来有效地创建角膜缘干细胞缺乏（LSCD）的一个可持续发展的小鼠模型。该动物模型是在测试和比较的用于角膜缘干细胞的疾病的治疗的效力是有用的。 

视频: A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

科研

医学

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

离体骨骼肌肌的力学分析

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

一个简单的方法小鼠肺插管

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Parabiosis in Mice: A Detailed Protocol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 18641, the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival2. In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

联体小鼠:一个详细的协议

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

外科手术切除为鼠标肋骨:模型进行大规模的长骨修复

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

一个简单的设备，迅速准备小鼠肠的全坐骑

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

静脉介入型号:研究旁路移植通畅一个合适的模型

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

在小鼠模型中将胶质母细胞瘤治疗干细胞的鼻内递送

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...