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。然后，一边拿着截面积直钳，用剪刀剪沿边取出角膜的角膜和巩膜。使用细的曲线和直线镊子，小心剥离巩膜和脉络膜相差的对视神经视网膜。这一步需要耐心快速释放的视网膜，可导致流泪。取下镜头和任何多余的虹膜和视网膜16的碎片。 </li></ol> 2。水冲洗<ol><li>在24孔盘和盖水（约500微升，与至少一个45微米的过滤器过滤）将视网膜。 </li><li>每30分钟1小时换一次水，至少4-5次。 </li><li>发布在水中过夜，在室温下轻轻摇动。 </li></ol> 注意: 洗涤步骤是非常重要的，因为它们有助于从血管神经视网膜层分离。 3。胰蛋白酶文摘<ol><li>除去水，并培育的视网膜，在3％的胰蛋白酶（1:250 Difco公司）在0.1M三羟甲基氨基甲烷缓冲液（pH为7.8）的溶液中在37℃下温和无摇晃。胰蛋白酶消化完毕，当组织开始解体的迹象（约1.5小时）。 注意: 避免快速晃动因为这可能会损坏血管。 </li><li>小心地用移液器出胰蛋白酶到一个单独的使用显微镜避免接触视网膜。这多余的胰蛋白酶后，可用于操纵船舶时，在显微镜下，在第4.3步。 </li></ol> 4。分离血管<ol><li>过滤后的水加入到视网膜。然后，尝试将与钳内界膜剥离，如有必要，用剪刀。摇匀，用温和搅拌5分钟。小心，移液管的水，在显微镜下，以避免损坏视网膜。 </li><li>重复水洗（各5分钟），以帮助网络的船只从附着的视网膜组织。时，很少有没有剩余碎片在水中洗净后，水冲洗完成。 </li><li>在解剖显微镜下小心的操作，现在需要到进一步释放船只的网络。下面是一些有用的技术，它可用于在序列或尾学生职业建构基于技术的偏好，以达到期望的结果: <ol class="lalpha"><li>用200μl移液器，小心吸取水和上下相邻的船只，吹水在血管，引起温柔的搅拌。 </li><li>使用200μL吸管，吸液管的胰蛋白酶和帮助大衣墙壁吸管。然后，小心吸取整个血管网和一次有助于打破无血管组织。 注意: 上面的视神经血管网络的边缘而不是中心的移液管尖，也能防止血管粘到顶端。 </li><li>用细镊子轻轻提起血管离开了水的，撞出非血管成分。 </li><li>如果上述步骤不起作用，加胰蛋白酶对视网膜，帮助分解组织，并在37°C孵育几分钟。然后，消除胰蛋白酶和重复步骤4.1-4.3。 </li></ol></li></ol> &lt;&gt;注意:请一定要会接触到的所有工具，胰蛋白酶（如移液器吸头，镊子）船舶涂层。这将有助于避免血管粘连设备。 5。可视化的血管<ol><li>将一个干净的玻片上一滴水。用200μl吸管或镊子，转移消化的船只入水。确保血管平面式展现水的表面张力，有助于开展现在透明的血管网络，如果组织不展开，添加额外的水，轻轻摇晃滑动，以方便展开。 </li><li>非常缓慢吹打或使用纸巾去除多余的水分。 </li><li>允许的幻灯片完全晾干。 </li><li>通过相应的协议与PAS /苏木或H＆E染色的染色幻灯片。 注意:PAS /苏木是有用的吞吐量血管壁和细胞的细胞核，H＆E染色是很有用的d执行示范以及红细胞。 </li><li>脱水和装入（安装介质Permount）。 </li></ol>

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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. 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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. 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作者宣称，他们有没有竞争经济利益。

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 ...

trypsin-digest-protocol-to-analyze-retinal-vasculature-mouse

Universidad de Cartagena UDEC

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JoVE Journal

Neuroscience

18.3K 次观看.  Northwestern University Feinberg School of Medicine. 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。

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

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

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

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

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in 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

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

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

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

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

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

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

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

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

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

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, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

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

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

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

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

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

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and hypothalamus that supplies blood to the brain and surrounding structures. It has been implicated in several cerebrovascular disorders, including cerebral amyloid angiopathy (CAA)-associated vasculopathies, intracranial atherosclerosis and intracranial aneurysms. Studies of the molecular mechanisms underlying these diseases for the identification of novel drug targets for their prevention require animal models. Some of these models may be transgenic, whereas others will involve isolation of the cerebro-vasculature, including the CoW.The method described here is suitable for CoW isolation in any mouse lineage and has considerable potential for screening (expression of genes, protein production, posttranslational protein modifications, secretome analysis, etc.) studies on the large vessels of the mouse cerebro-vasculature. It can also be used for ex vivo studies, by adapting the organ bath system developed for isolated mouse olfactory arteries.

We describe here a reproducible 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 ...