作者感谢沙龙Spana夫人的鼠害监测，处理和试验的帮助。博士的资金支持已经由悉尼大学和澳大利亚研究中心卓越视觉提供的。

该协议遵循悉尼大学的动物护理准则。 1.制作电极<ol><li>构造正极（一个其将接触角膜）从铂丝直径1-2毫米的短（5厘米）的长度。时尚它变成一个循环的几个毫米的直径。这个环路连接到一个常规的引线，足够长的时间，以达到你放大器的输入级（参见图4）。 </li><li>构造负极使用银/氯化银沉淀1-2毫米的直径，也连接到一个会议引线（这将在动物的嘴去）（ 见图4）。 </li><li>作为参比电极（将进入动物的臀部），用干净的皮下注射针头（23 G）的，也连接到一个适当长度的导线（ 见图4）。 </li><li>理想情况下，使用由仪器制造商提供的三引脚电缆，连接三个电极（正极û94;角膜，负→口，参考→臀部）的放大器。 </li></ol> 2.连接光刺激和ERG建立和校准<ol><li>创建（或定位）的小记录实验室，可制成暗。装备有一个以上的非长凳光发红色或红色头灯的任一个或两个。 </li><li>用照度计，以确认红色光的照度在安装过程中到达老鼠的眼睛不会超过1勒克斯。 注意:中性密度滤镜可用于降低灯的亮度和灯光的来源必须明确发出红光。如果光源发出低（可见光）波长暗适应将受到损害。 </li><li>封锁进入录音实验室所有杂散光（这往往需要恒心与不透明的胶带），并准备一个中性密度滤镜（可在张购买）足够大，以适应过来，等昏暗，任何计算机屏幕，您将在实验室。 注:杂散光和屏幕的光足以妨害暗适应鼠眼。 </li><li>连接放大器，以数据采集硬件。连接阳性，阴性和参考引线到放大器。确保计算机和LED Ganzfeld电源单元牢固地连接到一地源。 注:一些实验室有专门的接地点，连接到建筑物地面;水管是一种有效的替代。 </li><li>校准与科研质量的辐射计的LED光源。修复仪的传感器在该动物的眼睛将一个实验过程中所处位置。 </li><li>方案Ganzfeld LED的运行与逐步增加闪光能量，闪光持续时间，闪光灯重复和时间闪烁之间的全场ERG协议，称为interstimuls间隔（ISI），设置。举一个例子全场协议见表1。 注:全视野ERG从闪烁朦胧重复闪烁提高到b右闪在步骤明智的方式。双闪存程序遵循从满场的协议，使杆状和锥状响应隔离。 </li></ol> 3.前一天ERG实验<ol><li>录制黑暗中前适应SD大鼠12小时。是很方便的做到这一点，在记录实验室，一旦杂散光已被淘汰。 </li></ol> 4日ERG实验<ol><li>安排动物轻轻加热的同时录制。我们使用内置使动物的头可以放心在正确的点在入口到Ganzfeld轻金属平台。该平台具有内置管，通过它我们泵送水预热到40℃的水浴中。 注意:经验表明，这保持了动物的核心温度在37℃。 </li><li>权衡黑暗条件下的老鼠。记录体重和化妆正确的氯胺酮（60毫克/公斤）和甲苯噻嗪（5毫克/千克）剂量。抑制大鼠根TLY和腹腔注射麻醉剂。 </li><li>注意注射时间。一旦动物是无意识的（通常在5分钟），轻轻捏一只脚垫，看是否有反射反应存在检查麻醉深度。最好是等到这种反射不存在或弱，继续前。 </li><li>应用阿托品一滴proxmethacaine的，另一个角膜。 </li><li>切黑线的有10厘米长。使用一个简单的结的环路和滑环在眼睛的赤道。稍微拧紧;效果是吸引眼球微微前倾，以最小的压力。这样可以使角膜从眼皮清楚。 </li><li>应用卡波姆滴眼液在角膜表面。确保卡波姆保持在角膜表面，不会波及到眼皮或脸部。 </li><li>放置在加热平台的顶吸收床上用品。 </li><li>位置在老鼠的床上用品，用头在Ganzfeld开幕推荐的地方。 </li><li>插入INTernal温度探头插入直肠。用胶带探头线到尾位置的安全温度探头。 </li><li>插入基准电极（23政针）皮下后支腿，并连接到放大器。 </li><li>放置负电极（银/氯化银沉淀）牢固地固定在口中。为了防止这种情况滑出嘴角，加盖连接导线，以稳定的表面。 </li><li>定位正极在角膜的中心。采用显微，确保电极接触角膜平缓。 </li><li>查体温在37.0 - 37.5℃。 </li><li>一旦动物被适当地定位和电极的地方，悬垂整个安装（Ganzfeld和动物）用不透明材料（以保留暗适应）。我们用黑色的软布。 </li><li>在采集软件设置为2 kHz的采样速率为100-1000毫秒，5毫秒预收款采样的集合时间。设置带通滤波器，以1-1,000赫兹并确保采样触发品尝〜250毫秒以下闪光的时期。 </li><li>检查记录基线。它应该是免费的外来噪音，但表现出一定的放大器的噪声和振动的呼吸。 </li><li>如果基线显示多余的噪音，开始排除故障。大多数问题都与在电极位置，或接地滑脱。使用一个法拉第笼，以确保录音都是免费的外来噪音。 </li><li>运行测试闪存，0.4日志税赋cd.sm -2。一个ERG波形类似于图2A应该出现。在我们的0.4日志苏格兰人cd.sm -2闪光灯实验室典型的回答是:（a波:-474±39μV和b波:1,512±160μV，N = 11）。 </li><li>允许动物到暗重新适应10分钟。方便的是使用这些10分钟，以重新检查基线。 </li><li>稳定的信号的确认之后开始录制。 </li><li>在记录会话结束时，检查身体Temperat .. [温度茜保持。取出电极。重新卡波姆聚合物眼角膜。让动物恢复在散热垫，直到其完全的移动和积极的，返回到动物的住房之前。 </li></ol> 5.远程缺血<ol><li>在任何醒着还是anesthetised啮齿类动物进行远程缺血。 </li><li>如果将动物麻醉，把它在加热平台（上）和滑血压计袖带在后肢，明确膝关节的上部。 </li><li>如果动物被用于被处理，就可以不用麻醉执行此过程;这需要两个人。白岩松抑制动物轻轻第二应用血压计袖带和操作血压计。 </li><li>对于清醒的动物，用一块毛巾约15厘米×30〜50厘米轻轻包裹的动物，一个后肢免费。躺在动物在其上（说）左前臂背面，与持有人的手臂和躯干，以及地方之间的藏头袖带刚刚描述。 </li><li>袖带放气，并保证空气压力阀关闭。泵的袖口到160毫米汞柱的anesthetised动物，以及180毫米汞柱清醒的动物。这超出了收缩压（通常140毫米汞柱和160毫米汞柱分别）。 </li><li>保持这些压力的要求，使用手持式泵。 </li><li>之后在计划时间为缺血（我们使用的5分钟再灌注分离5分钟2期），松开气压阀放气袖带压力。 </li><li>确认远程缺血与附连到足垫皮肤温度探测器的作用。皮肤温度通常瀑布从32-30℃，超过5分钟，恢复再灌注。 </li></ol> 6.光损伤<ol><li>确保大鼠在暗适应过夜，之前的光损伤的过程。 </li><li>在肢体缺血（在我们的实验中无延迟）在适当的时候，每个动物被置于单独成有机玻璃盒，无线日水和食物地板为主的容器。 注意:光诱导损伤，只能在白化动物进行。 </li><li>开关上的预校准1000勒克斯的白光以标准时间（通常上午09点），并保持该条件24小时。 </li></ol> 7. ERG数据提取和分析<ol><li>获得ERG的平均波形。如果需要的话，校正非零基线，由减法。 </li><li>测量一个波（在中间呈现给高刺激强度）的振幅，作为基准，并在第一间的电压差（&lt;30毫秒延迟）槽（ 图1）。 </li><li>测量的b波振幅为a波的峰值和正下面波之间的电压差，通常是在80-100毫秒（ 图1）一个延迟发生。 </li><li>用傅立叶隔离振荡电位变换从60-235赫兹筛选数据，以90赫兹的过渡带<sup&gt; 7。如果需要分离的振荡电位的信号然后可从未经滤波波形中减去，以确认一个波槽的身份。 </li><li> a-和b-波峰的隐式时间（等待时间）也可以是一个有用的度量（ 图1）。使用双闪烁以分离杆响应。减去从混合反应（闪光1）隔离杆响应（ 图2）的视锥细胞反应（闪光2）。 </li><li>正常化个别光强度的波和b波振幅（后处理/后处理基线）或平均为治疗组。强度响应曲线绘制组幅度和对错误闪光能量。 </li></ol>

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<li>Sugawara, T., Sieving, P. A., Bush, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+relationship+of+the+scotopic+and+photopic+ERG+to+photoreceptor+cell+loss+in+light+damaged+rats%5BTitle%5D)+AND+%22Experimental+Eye+Research%22%5BJournal%5D)">Quantitative relationship of the scotopic and photopic ERG to photoreceptor cell loss in light damaged rats.</a> Experimental Eye Research. 70 (5), 693-705 (2000).</li>
<li>Machida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((P23H+rhodopsin+transgenic+rat%3A+Correlation+of+retinal+function+with+histopathology%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">P23H rhodopsin transgenic rat: Correlation of retinal function with histopathology.</a> Investigative Ophthalmology & Visual Science. 41 (10), 3200-3209 (2000).</li>
<li>Brandli, A., Stone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Remote+Ischemia+Influences+the+Responsiveness+of+the+Retina%5BTitle%5D)+AND+%22Observations+in+the+Rat.+Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Remote Ischemia Influences the Responsiveness of the Retina.</a> Observations in the Rat. Investigative Ophthalmology & Visual Science. 55 (4), 2088-2096 (2014).</li>
<li>Maccarone, R., Di Marco, S., Bisti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Saffron+supplement+maintains+morphology+and+function+after+exposure+to+damaging+light+in+mammalian+retina%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Saffron supplement maintains morphology and function after exposure to damaging light in mammalian retina.</a> Investigative Ophthalmology & Visual Science. 49 (3), 1254-1261 (2008).</li>
<li>Hood, D. C., Birch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+abnormal+rod+photoreceptor+activity+with+the+a-wave+of+the+electroretinogram%3A+Applications+and+methods%5BTitle%5D)+AND+%22Documenta+Ophthalmologica%22%5BJournal%5D)">Assessing abnormal rod photoreceptor activity with the a-wave of the electroretinogram: Applications and methods.</a> Documenta Ophthalmologica. 92 (4), 253-267 (1996).</li>
<li>Robson, J. G., Frishman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+rod-driven+a-wave+of+the+dark-adapted+mammalian+electroretinogram%5BTitle%5D)+AND+%22Progress+in+Retinal+and+Eye+Research%22%5BJournal%5D)">The rod-driven a-wave of the dark-adapted mammalian electroretinogram.</a> Progress in Retinal and Eye Research. 39, 1-22 (2014).</li>
<li>Hood, D. C., Birch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+COMPUTATIONAL+MODEL+OF+THE+AMPLITUDE+AND+IMPLICIT+TIME+OF+THE+B-WAVE+OF+THE+HUMAN+ERG%5BTitle%5D)+AND+%22Visual+Neuroscience%22%5BJournal%5D)">A COMPUTATIONAL MODEL OF THE AMPLITUDE AND IMPLICIT TIME OF THE B-WAVE OF THE HUMAN ERG.</a> Visual Neuroscience. 8 (2), 107-126 (1992).</li>
<li>Wachtmeister, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oscillatory+potentials+in+the+retina%3A+what+do+they+reveal%5BTitle%5D)+AND+%22Progress+in+Retinal+and+Eye+Research%22%5BJournal%5D)">Oscillatory potentials in the retina: what do they reveal.</a> Progress in Retinal and Eye Research. 17 (4), 485-521 (1998).</li>
</ol>

乔纳森·斯通是CSCM私人有限公司的董事

上述的暗适应闪光ERG方法是评估在大鼠视网膜功能的可靠方法。两者的a波和b波被光伤害减少。远程缺血预处理减轻光损伤诱导的减少在一个波和B波。这种防护视网膜功能的建议，远程缺血性预处理已引起神经保护作用，类似其他形式的防护预处理，如缺氧，缺血和运动8-10。记录设置中，光刺激的参数，和动物的状态 - 记录ERG的信号是由三组因素来决定。 录像设置<...

ERG的是由视网膜响应于光产生的，并且从眼睛的角膜表面记录的电势。当记录条件精心管理，ERG的可以以各种不同的方式来评估视网膜功能被使用。在这里，我们介绍了如何记录"闪光ERG"，时产生的视网膜暴露在一个简短的，明亮的闪光在Ganzfeld背景呈现的潜力。该Ganzfeld分散的均匀光与光的闪光灯到达视网膜整体大致均匀。如果视网膜记录前暗适应和暗适应被保持为动物准备用于记录，ERG的获得是通过两个杆和视锥感光细胞生成的。 暗适应闪光ERG的具有特征的波形，这已在两个方面进行了分析。首先，ERG的波形的早期和晚期成分被区分开来，并与神经元的序人活化在视网膜上。最早的组件是一个短潜伏期负向电位的一个波（ 图1）。这之后是一个正向电位，称为b波。 b波的上升阶段表示振荡，这被认为是一个单独的部件（振荡电位或有机磷）。在一个波被认为是由光感受器产生，b波由内核层细胞和有机磷由无长突细胞1。 根据刺激强度，反应非常昏暗闪烁称为暗门槛响应是可能的。暗视阈响应被理解为从视网膜神经节细胞2-4生成的。第二，闪光ERG可以通过光适应来分离，或通过两个闪光协议如下所述，成棒状和锥驱动的组件。明视下的条件下，一个波是在大鼠未检测到，因为锥体人口低，但有机磷和b波是明确5。在灵长类动物，其视网膜具有较高的人口锥，既杆状和锥途径产生可探测的一个波6。 常从闪速ERG提取两个有用措施是a-和b-波的振幅，测量为在图1中，与图2中所示的典型闪光的反应。当感光体人口被减少，例如通过暴露于破坏力亮光，ERG的所有成分减少。神经保护措施，如远程缺血预处理（RIP），可以由a-和b-波（ 图3）的振幅的保存进行验证。综上所述，ERG的分析使得健康，光损伤和neuroprotected视网膜之间的比较。

<table><tbody><tr><td>PC computer</td><td></td><td></td><td></td></tr><tr><td>Powerlab, 4 channel acquistion hardware</td><td>AD Instruments</td><td>PL 35044</td><td>Acquistion of ERG</td></tr><tr><td>Animal Bio Amp</td><td>AD Instruments</td><td>FE 136</td><td>Amplifier for ERG</td></tr><tr><td>Lab chart</td><td>AD Instruments</td><td></td><td>Signal collection software</td></tr><tr><td>Ganzfield</td><td>Photometric solutions</td><td>FS-250A</td><td>Light stimulus</td></tr><tr><td>Ganzfield operating system</td><td>Photometric solutions</td><td></td><td></td></tr><tr><td>Research Radiometer</td><td>International light technologies</td><td>ILT-1700</td><td>calibrate light series</td></tr><tr><td>Lux meter</td><td></td><td>LX-1010B</td><td>check red light illumanation</td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td></td></tr><tr><td>Lead wires</td><td>AD Instruments</td><td></td><td>Connect postive, negative ground electrodes to amplifier</td></tr><tr><td>Lead wires - alligator</td><td>AD Instruments</td><td></td><td>ground ganzfield and acquistion hardware to computer</td></tr><tr><td>Platinum wire 95%</td><td>A&amp;amp;E metals</td><td></td><td>postive electrode</td></tr><tr><td>Mouth electrode Ag/AgCl Pellet</td><td>SDR</td><td>E205</td><td>negative electode</td></tr><tr><td>26 G needle</td><td>BD</td><td></td><td>ground electode</td></tr><tr><td>Water pump</td><td></td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td></td></tr><tr><td>Tubing</td><td></td><td></td><td></td></tr><tr><td>Homeothermic blanket system with flexible probe</td><td>Harvard Appartus</td><td>507222F</td><td></td></tr><tr><td>Atropine 1% w/v</td><td>Bausch &amp;amp; Lomb</td><td></td><td>topical mydriasis</td></tr><tr><td>Proxmethycaine 0.5% w/v</td><td>Bausch &amp;amp; Lomb</td><td></td><td>topical anaesthetic</td></tr><tr><td>Visco tears eye drops</td><td>Novartis</td><td></td><td>carbomer polymer</td></tr><tr><td>Thread</td><td></td><td></td><td>retract eye lid</td></tr><tr><td>Tweezers</td><td></td><td></td><td></td></tr><tr><td>Reusable adhesive</td><td>Blu tac</td><td></td><td>Dim red headlamp. Affix electrodes</td></tr><tr><td>Absorbent bedding</td><td></td><td></td><td></td></tr><tr><td>Ketamil - ketamine 100 mg/ml - 50 ml</td><td>Troy Laboratories Pty Ltd</td><td></td><td>dissociative</td></tr><tr><td>Xylium - Xylazine 100 mg/ml - 50 ml</td><td>Troy Laboratories Pty Ltd</td><td></td><td>muscle relaxant</td></tr><tr><td>Scale</td><td></td><td></td><td></td></tr></tbody></table>

using the electroretinogram to assess function in the rodent retina and the protective effects of remote limb ischemic preconditioning

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 retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

该协议可以用于测量啮齿动物视网膜的体内视觉功能。在一个波，光感受器功能的量度，和b波，内视网膜功能的量度，被注释在图1中。 随光刺激的棒为主的ERG的信号增加时， 如图2A所示 。在〜0.4日志苏格兰人cd.sm -2和的一个波增加，直到饱和为2.5日志苏格兰人cd.sm振幅-2（未示出）的一个波变得很明显。双子闪光范式已经用于...

Watch this Scientific Journal Video about Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning at JoVE.com

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

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote 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 ...

using-electroretinogram-to-assess-function-rodent-retina-protective

Research

JoVE Journal

Neuroscience

16.2K 次观看.  University of Sydney. The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

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

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both laboratory and clinical settings. Currently, preclinical ERG and VEP measurements are performed with anesthesia to ensure stable electrode placements. However, the very presence of anesthesia has been shown to contaminate normal physiological responses. To overcome these anesthesia confounds, we develop a novel platform to assay ERG and VEP in conscious rats. Electrodes are surgically implanted sub-conjunctivally on the eye to assay the ERG and epidurally over the visual cortex to measure the VEP. A range of amplitude and sensitivity/timing parameters are assayed for both the ERG and VEP at increasing luminous energies. The ERG and VEP signals are shown to be stable and repeatable for at least 4 weeks post surgical implantation. This ability to record ERG and VEP signals without anesthesia confounds in the preclinical setting should provide superior translation to clinical data.

We show surgical implantation and Recording procedures to measure visual electrophysiological signals from the eye (electroretinogram) and brain (visual evoked potential) in conscious rats, which is more analogous to the human condition where recordings are conducted without anesthesia confounds.

植入和无线电图记录和视觉诱发电位清醒大鼠

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both ...

科研

行为学

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

血管再生在中枢神经系统中使用的小鼠视网膜评估

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

神经科学

Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain the overall health and structural integrity of the photoreceptors. The RPE is polarized, exhibiting apically and basally located receptors or channels, and performs vectoral transport of water, ions, metabolites, and secretes several cytokines.
In vivo noninvasive measurements of RPE function can be made using direct-coupled ERGs (DC-ERGs). The methodology behind the DC-ERG was pioneered by Marmorstein, Peachey, and colleagues using a custom-built stimulation recording system and later demonstrated using a commercially available system. The DC-ERG technique uses glass capillaries filled with Hank&#8217;s buffered salt solution (HBSS) to measure the slower electrical responses of the RPE elicited from light-evoked concentration changes in the subretinal space due to photoreceptor activity. The prolonged light stimulus and length of the DC-ERG recording make it vulnerable to drift and noise resulting in a low yield of useable recordings. Here, we present a fast, reliable method for improving the stability of the recordings while reducing noise by using vacuum pressure to reduce/eliminate bubbles that result from outgassing of the HBSS and electrode holder. Additionally, power line artifacts are attenuated using a voltage regulator/power conditioner. We include the necessary light stimulation protocols for a commercially available ERG system as well as scripts for analysis of the DC-ERG components: c-wave, fast oscillation, light peak, and off response. Due to the improved ease of recordings and rapid analysis workflow, this simplified protocol is particularly useful in measuring age-related changes in RPE function, disease progression, and in the assessment of pharmacological intervention.

Direct-Coupled Electroretinogram DC-ERG for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium

Here, we present a method for recording light-evoked electrical responses of the retinal pigment epithelium (RPE) in mice using a technique known as DC-ERGs first described by Marmorstein, Peachey, and colleagues in the early 2000s.

直接耦合电雷电图（DC-ERG），用于记录小鼠视网膜颜料皮皮的光唤起电响应

The retinal pigment epithelium (RPE) is a specialized monolayer of cells strategically located between the retina and the choriocapillaris that maintain ...

化学

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb &#8211; specifically the hind limb of a rat &#8211; ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

远程肢体缺血预适应:在啮齿动物的保护技术

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

医学

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.
Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2) or glutamate stress (250 &#181;m glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.
For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p &#60;0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p &#60;0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 &#181;m glutamate, an initial 7.8% reduction of a-wave amplitudes (p &#62;0.05) followed by a reduction of 1.9% (p &#62;0.05). A reduction of 83.7% of b-wave amplitudes (p &#60;0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

With this study, we introduce a standardized stress model for the isolated superfused bovine retina for future preclinical therapeutic testing. The effect of either hypoxia (pure N2) or glutamate stress (250 &#181;M glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

谷氨酸和缺氧作为离体脊椎动物视网膜应力模型

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

电图同时记录和麻醉大鼠视觉诱发电位

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

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

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment. Particularly, the study of ON-bipolar cells, but also of photoreceptors, has been hampered by the slow but progressive decline of light responses in the ex vivo ERG over time. Increased perfusion speed and adjustment of the perfusate temperature improve ex vivo retinal function and maximize response amplitude and stability. The ex vivo ERG uniquely allows the study of individual retinal neuronal cell types. In addition, improvements to maximize response amplitudes and stability allow the investigation of light responses in retina samples from large animals, as well as human donor eyes, making the ex vivo ERG a valuable addition to the repertoire of techniques used to investigate retinal function.

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

Modification of existing multielectrode array or patch clamp equipment makes the ex vivo electroretinogram more widely accessible. Improved methods to record and maintain ex vivo light responses facilitate the study of photoreceptor and ON-bipolar cell function in the healthy retina, animal models of eye diseases, and human donor retinas.

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 ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

由瞬态双边常见胡萝卜动脉闭塞引起的缺血性视网膜损伤的 Murine 模型

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

<meta charset="utf8"></head><body>用于研究视网膜病变的视网膜缺血再灌注损伤小鼠模型

Induction of Retinal Ischemia-Reperfusion Injury in a Mouse Eye Model

Ischemia-reperfusion injuries are known to cause a range of retinal pathologies, including diabetic retinopathy, glaucoma, retinal vascular occlusions, and other vaso-occlusive conditions. This manuscript presents a method for inducing ischemia-reperfusion injury in a mouse model. The method utilized anterior chamber cannulation attached to a saline reservoir, generating hydrostatic pressure to raise the intraocular pressure to 90-100 mmHg. This method effectively caused constriction of retinal capillaries to induce retinal ischemia. At the end of the ischemic period (60 min), the intraocular pressure was normalized (&#8804;20 mmHg) before removing the cannula from the anterior chamber to initiate reperfusion. Days after the ischemia/reperfusion procedure, the eyes were collected and sectioned for histological staining. The histopathology of the retinal sections was scored by evaluating eight parameters of retinal injury: folds, hemorrhage, deformation, cell loss in the ganglion cell, inner nuclear, outer nuclear, and photoreceptor layers, and damage to retinal pigment epithelial cells. This method provided a reproducible model to study the mechanisms and pathology of retinal ischemia/reperfusion injury. In addition, this model can facilitate the discovery of potential therapeutic targets to treat retinal ischemia/reperfusion injury, advancing the study of retinal pathologies and improving patient outcomes.

<meta charset="utf8"></head><body>小鼠眼模型中视网膜缺血再灌注损伤的诱导

This protocol models retinal ischemia-reperfusion injury in a mouse eye by inducing retinal ischemia via anterior chamber cannulation and intraocular pressure elevation, followed by intraocular pressure normalization to initiate the reperfusion.

小鼠眼模型中视网膜缺血再灌注损伤的诱导

Ischemia-reperfusion injuries are known to cause a range of retinal pathologies, including diabetic retinopathy, glaucoma, retinal vascular occlusions, ...

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

摘要

探索更多视频

植入和无线电图记录和视觉诱发电位清醒大鼠

血管再生在中枢神经系统中使用的小鼠视网膜评估

直接耦合电雷电图（DC-ERG），用于记录小鼠视网膜颜料皮皮的光唤起电响应

远程肢体缺血预适应:在啮齿动物的保护技术

谷氨酸和缺氧作为离体脊椎动物视网膜应力模型

电图同时记录和麻醉大鼠视觉诱发电位

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

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

由瞬态双边常见胡萝卜动脉闭塞引起的缺血性视网膜损伤的 Murine 模型

小鼠眼模型中视网膜缺血再灌注损伤的诱导

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

摘要

探索更多视频

植入和无线电图记录和视觉诱发电位清醒大鼠

血管再生在中枢神经系统中使用的小鼠视网膜评估

直接耦合电雷电图（DC-ERG），用于记录小鼠视网膜颜料皮皮的光唤起电响应

远程肢体缺血预适应:在啮齿动物的保护技术

谷氨酸和缺氧作为离体脊椎动物视网膜应力模型

电图同时记录和麻醉大鼠视觉诱发电位

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

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

由瞬态双边常见胡萝卜动脉闭塞引起的缺血性视网膜损伤的 Murine 模型

小鼠眼模型中视网膜缺血再灌注损伤的诱导

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