Name: optimizing the setup and conditions for ex vivo electroretinogram to study retina function in small and large eyes
Uploaded: 2022-06-27T13:00:00
Description: Watch this Scientific Journal Video about Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes at JoVE.com

这项工作得到了国家眼科研究所对Vinberg博士的资助EY02665和EY031706以及国际视网膜研究基金会的支持，美国国立卫生研究院核心资助（EY014800），以及纽约州纽约州纽约州预防失明研究对犹他大学眼科与视觉科学系的无限制资助。Frans Vinberg博士还是预防失明研究/H. James博士和Carole Free职业发展奖的获得者，以及ARVO EyeFind资助的Silke Becker博士。我们感谢斯克里普斯研究所的Anne Hanneken博士提供用于图2E所示记录的供体眼睛。

所有使用小鼠的实验均按照NIH实验动物护理和使用指南进行，并得到犹他大学机构动物研究委员会的批准。用于演示此视频的猪眼是从屠宰场（约翰逊维尔可持续猪资源）获得的。在脑或心脏死亡后，通过犹他州狮子会眼库、圣地亚哥眼库或生命共享从人类捐赠者那里获得眼睛，并同意用于研究用途，这些眼睛已获得 FDA、器官采购组织协会 （AOPO） 和美国眼库协会的完全认可。人类捐赠者眼睛的...

<ol>
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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+sodium+ion+concentration+on+the+electroretinogram+of+the+isolated+retina+of+the+frog.">The effect of sodium ion concentration on the electroretinogram of the isolated retina of the frog.</a> Journal of Physiology. 167 (1), 156-168 (1963).</li><li>Granit, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+components+of+the+retinal+action+potential+in+mammals+and+their+relation+to+the+discharge+in+the+optic+nerve.">The components of the retinal action potential in mammals and their relation to the discharge in the optic nerve.</a> Journal of Physiology. 77 (3), 207-239 (1933).</li><li>Donner, K., Hemila, S., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-dependence+of+rod+photoresponses+from+the+aspartate-treated+retina+of+the+frog+(Rana+temporaria).">Temperature-dependence of rod photoresponses from the aspartate-treated retina of the frog (Rana temporaria).</a> Acta Physiologica Scandinavica. 134 (4), 535-541 (1988).</li><li>Green, D. 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最初由Holmgren于1865年开发，用于测量两栖动物视网膜10的视网膜光响应，技术限制最初阻止了ERG的广泛使用。尽管如此，Ragnar Granit和其他人的开创性研究确定了ERG的细胞起源，并测量了体外光感受器和ON双极细胞反应11，12，13。从那时起，改进的方法允许更广泛地使用离体ERG记录14?...

视网膜电图测量视网膜功能对光的反应1.它是研究视网膜生理学和病理生理学以及衡量视网膜疾病治疗成功与否不可或缺的一部分。体内ERG被广泛用于评估完整生物体的视网膜功能，但它具有显着的局限性2，3。其中，体内ERG中单个视网膜细胞类型的定量分析受到阻碍，因为它记录了从所有视网膜细胞到光刺激的潜在变化的总和，因此重叠了反应4。此外，它不容易允许向视网膜添加药物，容易受到全身影响，并且信噪比相对较低。这些缺点在研究分离视网膜功能的离体ERG中被消除2，3，5，6。离体ERG允许通过添加药理学抑制剂和易于评估治疗剂来记录特定视网膜细胞类型的大而稳定的反应，治疗剂可以添加到超级熔体中。同时，它消除了全身效应的影响，消除了生理噪音（例如，心跳或呼吸）。
在 离体 ERG中，视网膜或视网膜样品被分离并安装在标本支架3，5的圆顶上。标本支架被组装好，连接到灌注系统，该系统为视网膜提供加热的含氧介质，并放置在显微镜的载物台上，显微镜载物台经过修改以提供计算机控制的光刺激。为了记录光引起的响应，将样品支架连接到放大器、数字化仪和记录系统（图 1）。该技术允许通过改变光刺激的参数并添加药理学试剂来分离杆和视锥光感受器、ON双极细胞和Müller胶质细胞的反应。
现有的膜片钳或多电极阵列（MEA）设置可以转换为 记录离 体ERG，可以与市售的 离体 ERG适配器或定制的聚碳酸酯计算机数控（CNC）加工的标本支架结合使用，以测量小动物模型（如小鼠）视网膜中的光响应。这种修改增加了 离体 ERG的可及性，同时最大限度地减少了对专用设备的需求。与先前报道的经视网膜 离体 ERG方法7相比，样品支架的设计简化了安装技术并集成了电极，消除了对微电极的操作。标本支架内的灌注速率和温度是影响光感受器和ON双极细胞反应特性的重要因素。通过调整这些条件，可以在较长时间内可靠地从分离的小鼠视网膜记录离 体 ERG。优化的实验条件允许从较大的视网膜（包括大型动物眼睛和人类供体眼睛）的视网膜中离 体 ERG记录8。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2 mm socket</td>
			<td>WPI</td>
			<td>2026-10</td>
			<td>materials to prepare electrode</td>
		</tr>
		<tr>
			<td>Ag/AgCl Electrode</td>
			<td>World Precision Instruments</td>
			<td>EP1</td>
			<td>materials to prepare electrode</td>
		</tr>
		<tr>
			<td>Ames&#39; medium</td>
			<td>Sigma Aldrich</td>
			<td>A1420</td>
			<td>perfusion media</td>
		</tr>
		<tr>
			<td>barium chloride</td>
			<td>Sigma Aldrich</td>
			<td>B0750</td>
			<td>potassium channel blocker</td>
		</tr>
		<tr>
			<td>DL-AP4</td>
			<td>Tocris</td>
			<td>0101</td>
			<td>broad spectrum glutamatergic antagonist</td>
		</tr>
		<tr>
			<td>OcuScience Ex Vivo ERG Adapter</td>
			<td>OcuScience</td>
			<td>n/a</td>
			<td>ex vivo ERG specimen holder</td>
		</tr>
		<tr>
			<td>Threaded luer connector</td>
			<td>McMaster-Carr</td>
			<td>51525K222 or 51525K223</td>
			<td>materials to prepare electrode</td>
		</tr>
	</tbody>
</table>

optimizing the setup and conditions for ex vivo electroretinogram to study retina function in small and large eyes

视网膜神经元光反应的测量对于研究健康视网膜的生理学、确定视网膜疾病的病理变化和测试治疗干预至关重要。 离体 视网膜电图（ERG）允许通过添加特定的药理学试剂和独立于全身影响评估组织内在变化来量化孤立视网膜中单个细胞类型的贡献。视网膜光响应可以使用专门的 离体 ERG标本支架和记录装置进行测量，该装置由现有的膜片钳或微电极阵列设备修改而成。特别是，ON双极细胞以及光感受器的研究受到离 体 ERG中光反应的缓慢但逐渐下降的阻碍。提高灌注速度和调节灌注液温度可改善 离体 视网膜功能，并最大限度地提高响应幅度和稳定性。 离体 ERG独特地允许研究单个视网膜神经元细胞类型。此外，最大化响应幅度和稳定性的改进允许研究大型动物以及人类供体眼睛的视网膜样本中的光响应，使 离体 ERG成为用于研究视网膜功能的技术库的宝贵补充。

离体ERG能够记录可重复和稳定的光感受器和ON双极细胞光反应，例如，来自小鼠视网膜（图2A-C）。记录来自人类供体视网膜的光感受器反应是可能的，死后剜除延迟长达5小时（图2D）和ON-bipolar细胞反应具有<20分钟剜除延迟（图2E）。获得大反应的重要参数包括仔细的解剖技术、高灌注速率和接近生...

Watch this Scientific Journal Video about Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes at JoVE.com

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

对现有多电极阵列或膜片钳设备的修改使 离体 视网膜电图更广泛地使用。记录和维持离体光反应的改进方法有助于研究健康视网膜、眼病动物模型和人类供 体 视网膜中的感光器和ON双极细胞功能。

优化离 体 视网膜电图的设置和条件，以研究小眼睛和大眼睛的视网膜功能

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in ...

该协议展示了在离体ERG中维持视网膜功能所需的条件，特别是由ON双极细胞产生的极其敏感的B波。使用离体ERG，我们可以以高信噪比测量不同类型的视网膜神经元的功能，同时允许轻松引入药物。该方法特别适合量化药物对视网膜功能和疾病的疗效。首先，通过将离体ERG样品支架通过头级连接到差分放大器来转换多电极阵列设置。放大器必须插入多电极阵列系统接口板的模拟输入。使用多电极阵列的记录软件记录和存储来自离体ERG的输入数据。将差分放大器的增益设置为100，并根据数字化仪规格增加额外的10倍电压放大。将低通滤波器设置为 100 赫兹。或者，要转换膜片钳设置，可通过上级将离体ERG样品支架连接到差分放大器，差分放大器必须连接到膜片钳放大器的头级。使用膜片钳系统软件和数字化仪记录和存储来自离体ERG的输入数据。设置参数，如前所示。将具有适当波长的LED连接到显微镜。要控制光刺激，请使用由数字化仪的模拟输出控制的LED驱动器。使用记录软件控制LED，该软件将触发光刺激。然后，使用光电二极管在标本支架中视网膜的位置校准LED的光输出。如有必要，插入中性密度滤光片以调暗光路中的光强度。要准备电极，请将氯化银颗粒电极插入螺纹诱饵连接器中。用热胶填充诱饵连接器的内部，然后从无螺纹侧将一个两毫米的插座插入热胶中。将 EP-1 电极的银线焊接到两毫米插座上。用螺纹上的O形圈将完成的电极拧入离体ERG样品支架的电极端口中。对于大眼睛，包括人类供体眼睛，清洁眼球中剩余的结缔组织并去除眼前段和晶状体。使用手术刀从角膜缘切开约三毫米。将弯曲的解剖剪刀插入切口并沿着角膜缘切割，用晶状体去除眼睛的前部。获取视网膜标本进行离体视网膜电图检查，并用三到六毫米的一次性活检打孔。要将组织安装在标本支架上，请将标本支架的下半部分放入一个大的培养皿中，并用含氧的 Ames'培养基填充它，使标本支架的圆顶刚刚被淹没。用细镊子小心地抓住视网膜的边缘，并将视网膜转移到离体标本支架的圆顶上，感光器面朝上。从艾姆斯溶液中取出标本支架，注意视网膜保持在原位。完全干燥试样支架的板，以最大程度地减少噪声、电串扰和信号分流。然后用四个螺钉组装试样支架的两半并连接灌注管路。驱动样品支架下半部分的电极，并将阳极电缆连接到视网膜内侧，将阴极电缆连接到感光器侧。每分钟至少用一毫升含氧的 Ames'培养基灌注标本支架 10 到 20 分钟，以使轻微反应稳定下来。离体ERG能够记录来自小鼠视网膜的感光器和ON双极细胞光反应。记录来自人类供体视网膜的光感受器反应是可能的，死后眼球摘除延迟长达5小时，ON双极细胞反应延迟不到20分钟。在理想条件下，两种细胞类型的反应幅度和动力学随着时间的推移相对稳定，但在将视网膜安装在标本支架上40至45分钟后显示出缓慢下降。样品支架中的温度降低大大降低了光感受器和ON双极细胞的动力学。然而，它降低了B波的振幅，但没有降低A波的振幅。相反，减慢灌注速率降低了光受体和ON双极细胞反应的振幅，但不影响A波或B波的隐含时间。停止灌注10分钟，然后再灌注导致ON双极细胞功能完全丧失，光感受器反应保留。足够的灌注速度和生理温度对于获得稳定的反应至关重要，特别是从离体ERG中的ON双极细胞。该协议允许深入探索人类黄斑和外围光感受器功能的差异，回答以前只能在非人类灵长类动物中进行的问题。

Research

JoVE Journal

Neuroscience

Optimizing the Setup and Conditions for Ex Vivo Electroretinogram to Study Retina Function in Small and Large Eyes

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

科研

神经科学

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

鼠标后脑<em&gt;前体内</em&gt;企业文化来研究面部Branchiomotor神经元迁移

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

使用电生理评估功能，在啮齿动物的视网膜和远程肢体缺血预适应的保护作用

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Rabies Necropsy Techniques in Large and Small Animals

大、小动物狂犬病的解剖技术

The New York State Department of Health (NYSDOH) Rabies Laboratory receives between 6,000 to 9,000 specimens annually and performs rabies testing for the ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

体内 大型动物视网膜神经节细胞及视神经功能及结构的评估方法

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

优化离 体 视网膜电图的设置和条件，以研究小眼睛和大眼睛的视网膜功能

优化离体视网膜电图的设置和条件，以研究小眼睛和大眼睛的视网膜功能