Name: trypsin digest protocol to analyze the retinal vasculature of a mouse model
Uploaded: 2013-06-13T13:00:00
Description: Watch this Scientific Journal Video about Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model at JoVE.com

部分支持这项工作是由NIH RO1 EY019951（AAF），伊利诺伊州防盲协会（JCC，AAF），无限制的资金从研究到眼科部防盲（RPB），纽约和RPB医学学生团契格兰特（JCC）。

1。视网膜准备<ol><li> Enucleate老鼠的眼睛。用一只手，打开眼睑，使眼可见。用另一只手，用弯钳（弯朝上），适用于上方和下方的压力方面的轨道直到截止突出。轻轻关闭钳在连续运动enucleate眼睛眼睛后方面解除。 </li><li>修复眼用10％中性福尔马林缓冲液中至少24小时。 </li><li>将眼PBS溶液在小培养皿。 </li><li>在显微镜下仔细解剖视网膜，照顾，以避免引起大的眼泪。清扫剪刀在角膜上切。然?...

<ol>
	<li>Kuwabara, T., Cogan, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+retinal+vascular+patterns.+I.+Normal+architecture.">Studies of retinal vascular patterns. I. Normal architecture.</a> Arch. Ophthalmol. 64, 904-911 (1960).</li><li>Kuwabara, T., Cogan, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+vascular+patterns.+VII.+Acellular+change.">Retinal vascular patterns. VII. Acellular change.</a> Invest. Ophthalmol. 4, 1049-1064 (1965).</li><li>Bell, W. R., Green, W. R., Goldberg, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathologic+and+trypsin+digestion+studies+of+the+retina+in+incontinentia+pigmenti.">Histopathologic and trypsin digestion studies of the retina in incontinentia pigmenti.</a> Ophthalmology. 115, 893-897 (2008).</li><li>Danis, R. P., Wallow, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HRP/trypsin+technique+for+studies+of+the+retinal+vasculature.">HRP/trypsin technique for studies of the retinal vasculature.</a> Invest. Ophthalmol. Vis. Sci. 27, 434-437 (1986).</li><li>Kim, S. H., Chu, Y. K., Kwon, O. W., McCune, S. A., Davidorf, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+studies+of+the+retina+in+a+new+diabetic+model;+SHR/N:Mcc-cp+rat.">Morphologic studies of the retina in a new diabetic model; SHR/N:Mcc-cp rat.</a> Yonsei Med. J. 39, 453-462 (1998).</li><li>Cuthbertson, R. A., Mandel, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+mouse+retina.+Endothelial+cell-pericyte+ratio+and+capillary+distribution.">Anatomy of the mouse retina. Endothelial cell-pericyte ratio and capillary distribution.</a> Invest. Ophthalmol. Vis. Sci. 27, 1659-1664 (1986).</li><li>Laver, N. M., Robison, W. G., Pfeffer, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+procedures+for+isolating+intact+retinal+vascular+beds+from+diabetic+humans+and+animal+models.">Novel procedures for isolating intact retinal vascular beds from diabetic humans and animal models.</a> Invest. Ophthalmol. Vis. Sci. 34, 2097-2104 (1993).</li><li>Midena, E., 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=Studies+on+the+retina+of+the+diabetic+db/db+mouse.+I.+Endothelial+cell-pericyte+ratio.">Studies on the retina of the diabetic db/db mouse. I. Endothelial cell-pericyte ratio.</a> Ophthalmic Res. 21, 106-111 (1989).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+30.">Chapter 30.</a> Diabetic Retinopathy. , (2005).</li><li>Rosenblatt, B., Benson, W. E., Yannoff, M., Duker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+6.19+Diabetic+Retinopathy.">Chapter 6.19 Diabetic Retinopathy.</a> Ophthalmology. , (2008).</li><li>Friedenwald, 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=A+new+approach+to+some+problems+of+retinal+vascular+disease.">A new approach to some problems of retinal vascular disease.</a> Am. J. Ophthalmol. 32, 487-498 (1949).</li><li>Podesta, 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=Bax+is+increased+in+the+retina+of+diabetic+subjects+and+is+associated+with+pericyte+apoptosis+in+vivo+and+in+vitro.">Bax is increased in the retina of diabetic subjects and is associated with pericyte apoptosis in vivo and in vitro.</a> Am. J. Pathol. 156 (10), 1025-1032 (2000).</li><li>Dagher, Z., 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=Studies+of+rat+and+human+retinas+predict+a+role+for+the+polyol+pathway+in+human+diabetic+retinopathy.">Studies of rat and human retinas predict a role for the polyol pathway in human diabetic retinopathy.</a> Diabetes. 53, 2404-2411 (2004).</li><li>Puro, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinovascular+physiology+and+pathophysiology:+new+experimental+approach/new+insights.">Retinovascular physiology and pathophysiology: new experimental approach/new insights.</a> Prog. Retin. Eye Res. 31, 258-270 (2012).</li><li>Roy, S., Nasser, S., Yee, M., Graves, D. T., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+long-term+siRNA+strategy+regulates+fibronectin+overexpression+and+improves+vascular+lesions+in+retinas+of+diabetic+rats.">A long-term siRNA strategy regulates fibronectin overexpression and improves vascular lesions in retinas of diabetic rats.</a> Mol. Vis. 17, 3166-3174 (2011).</li><li>Li, Q., 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=Diabetic+eNOS-knockout+mice+develop+accelerated+retinopathy.">Diabetic eNOS-knockout mice develop accelerated retinopathy.</a> Invest. Ophthalmol. Vis. Sci. 51, 5240-5246 (2010).</li><li>Connor, K. M., 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=Quantification+of+oxygen-induced+retinopathy+in+the+mouse:+a+model+of+vessel+loss,+vessel+regrowth+and+pathological+angiogenesis.">Quantification of oxygen-induced retinopathy in the mouse: a model of vessel loss, vessel regrowth and pathological angiogenesis.</a> Nature Protocols. 4, 1565-1573 (2009).</li></ol>

胰蛋白酶的消化是一个标准的方法，以评估视网膜的血管。不幸的是，它在技术上具有挑战性，如果没有正确执行，并可能导致在标本亏损率很高。此外，在小鼠体内的过程是特别困难的，可以限制这一技术的应用在常用眼疾基因动物模型。本文提供了关于如何执行程序的有效和一致的小鼠眼睛的指导。 在协议中，有几个关键步骤。首先，它是至关重要的，视网膜仔细解剖?...

可视化的视网膜血管解剖各种眼科疾病，如糖尿病性视网膜病变的机制，是一个非常重要的方法。它允许一个评估最早的血管异常，包括微血管瘤，毛细血管变性，和周细胞损失8,9。到今天为止，已经有几个技术分析视网膜血管。已用于各种染料灌注突出的船只，但是都共享了类似的限制。注射很少强调除非整个视网膜血管在高压下，破裂的风险和损坏的船只1。血管内皮细胞的免疫染色荧光标记ĝ凝集素B4（共轭的Alexa Fluor 594; I21413; Invitrogen公司; 1:100稀释）和视网膜平面坐骑可以突出整体建筑的船只，但没有详细的可视化毛细血管基底膜和周。有人指出由Friedenwald的船只可以突出碘酸-希夫（PAS）或苏木精伊红（H＆E）染色10平坐骑染色的视网膜。然而，染色呈非特异性的船只，以及突出的无血管组织，使其难以区分的船只。在20世纪60年代，科根和桑原开发的胰蛋白酶消化技术，使得它更容易显现视网膜血管消化的非血管成分的视网膜1。自那时以来，胰蛋白酶消化已成为金标法分析血管视网膜2-5。然而，重要的是要注意到，已经描述了其他替代技术的隔离的血管。渗透裂解的使用已被用于隔离的血管，并允许生化研究的组织11,12，但没有被使用作为一个主要的解剖学研究方法的程序。组织印刷方法一直用来隔离微血管和大段，允许学习能力的脉管13日电紧张的架构。从理论上讲，这种技术也可以用来研究解剖变​​化，作为船舶的质量是高的。但是，它是仅能够分离出整个血管网络段。虽然这些方法不能代替胰蛋白酶消化，重要的是要注意，它们具有不同的优点和缺点，在这方面是互补的。 胰蛋白酶消化法在技术上具有挑战性，并很难执行一致6,7。此外，人们已经注意到，胰蛋白酶消化的小鼠模型上是特别困难的，尤其是当一个人渴望保护视网膜6,7整体的血管结构。挑战包括：（1）在消化的视网膜，造成损失的脉管系统，以及无血管组织，（2）在消化，需要广泛的机械清扫继而可能导致损伤血管床，（3）分离度不佳导致非特异性染色血管的无血管组织。几个手稿都强调这些挑战，但都没有提供详细和一致的协议来克服他们14,15。这份手稿将引进一步一步的方法，详细介绍了具体处理特别困难的步骤提示进行胰蛋白酶消化小鼠和大鼠视网膜技术。 图1中所示的示意性概述。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>10% neutral buffered formalin</td>
			<td>Fischer Scientific</td>
			<td>SF100-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin (1:250)</td>
			<td>Amresco</td>
			<td>0458-50G</td>
			<td>Make 3% trypsin in 0.1 M Tris</td>
		</tr>
		<tr>
			<td>TRIZMA base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td>Create 0.1 M Tris Buffer (pH 7.8 @ 37 &deg;C)</td>
		</tr>
		<tr>
			<td>TRIZMA</td>
			<td>Sigma</td>
			<td>T3253-500G</td>
		</tr>
		<tr>
			<td>Steritop sterile vacuum bottle</td>
			<td>Millipore</td>
			<td>SCGPS05RE</td>
			<td>Create filtered water</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Any microscope that allows good visualization of the retina is adequate. (10x is sufficient with working distance of 24 mm)</td>
		</tr>
		<tr>
			<td>Straight scissors</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td>Cutting edge: 8 mm; Tip diameter: 0.2 mm; 10 cm</td>
		</tr>
		<tr>
			<td>Straight Forceps (Inox)</td>
			<td>Dumont #5</td>
			<td>11252-20</td>
			<td>Biologie tip; 0.05 x 0.02 mm; 11 cm</td>
		</tr>
		<tr>
			<td>Curved Forcepts (Dumostar)</td>
			<td>Dumont #7</td>
			<td>11297-10</td>
			<td>Biologie tip; 0.07 x 0.04 mm; 11.5 cm</td>
		</tr>
		<tr>
			<td>Dubnoff Metabolic Shaker Incubator</td>
			<td>Precision Scientific</td>
			<td>BDG59442</td>
			<td>Shaker optional</td>
		</tr>
		<tr>
			<td>24-well dish</td>
			<td>Falcon</td>
			<td>353047</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>VWR</td>
			<td>82027-132</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

trypsin digest protocol to analyze the retinal vasculature of a mouse model

胰蛋白酶消化的金标准方法来分析视网膜血管1-5。它允许对整个网络的可视化的复杂的三维视网膜血管和通过创建一个二维的平面安装的血管通道互连消化后的非血管成分的视网膜毛细血管。这使得一个研究各​​种病理血管的变化，如微血管瘤，毛细血管变性，内皮细胞异常周细胞比率。然而，该方法在技术上具有挑战性的，特别是在老鼠，这已成为最广泛使用的动物模型来研究视网膜因为遗传操作便于6,7。在小鼠眼，这是特别困难的，以完全除去非血管的组件，同时保持视网膜血管的整体结构。到今天为止，我有一个文学缺乏详细描述胰蛋白酶消化技术n的鼠标。这份手稿提供了详细的一步一步的方法在小鼠视网膜中的胰蛋白酶消化，同时也提供了如何解决困难的步骤。

一个成功的程序的最终产物是一台安装了整个网络的小鼠视网膜血管，维持的体系结构，与PAS /苏木素或H＆E染色， 图2-4中所示。 如图3所示，可以看出，内皮细胞和周细胞的明显分化。在视网膜，内皮细胞的细胞核中是椭圆形或细长的，并完全位于血管壁内的。周细胞的细胞核小，呈球形，密集的染色，一般有一个突起的位置沿毛细血管壁。 胰蛋白...

Watch this Scientific Journal Video about Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model at JoVE.com

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

胰蛋白酶的消化是一个最常用的方法来分析视网膜血管。这份手稿中详细介绍的方法，包括关键的改动，优化技术，并删除无血管组织，同时保持整体架构的船只。

胰蛋白酶精华协议分析视网膜血管小鼠模型

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

Research

JoVE Journal

Neuroscience

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

科研

神经科学

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

一个视神经挤压伤的小鼠模型，研究视网膜神经节细胞生存

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Morphometric Analyses of Retinal Sections

视网膜节的形态分析

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

从大鼠脑的解剖脑表面血管及相关脑膜和脉络丛的可视描述，

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

角膜微囊含量：血管生成在小鼠眼模型

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

在小鼠大脑的各个区域端粒酶活性：非放射性端粒酶重复序列扩增法（TRAP）分析

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

视频成像和时空地图来分析胃肠动力小鼠

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

Protocol for Isolating the Mouse Circle of Willis

协议隔离威利斯的鼠标圈

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and ...