Name: a simple mechanical procedure to create limbal stem cell deficiency in mouse
Uploaded: 2016-11-17T13:00:00
Description: Watch this Scientific Journal Video about A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse at JoVE.com

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...

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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...

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?...

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 ...

Research

JoVE Journal

Medicine

A Mouse Model of in Utero Transplantation

A Mouse Model of in Utero Transplantation

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

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

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  ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for 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

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

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

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 ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

球囊损伤诱发小鼠腹主动脉 Myointimal 增生的研究

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

Protocol to Create Chronic Wounds in Diabetic Mice

在糖尿病小鼠中创建慢性伤口的协议

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

研究小鼠心肌梗死的低温损伤模型

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...