Nicole Vella (Macquarie University) for providing reagents; Alexia Saunder (Bionics Institute; BI), Michelle McPhedran (BI), Chris Williams (BI) for experimental support; the Royal Victorian Eye and Ear Hospital (RVEEH) Biological Research Centre staff for animal care; Sue Pierce (RVEEH) for veterinary advice; Anthony Burkitt (Bionic Vision Australia; BVA), Tamara Brawn (BVA) and the BVA staff for administrative support. 
This research was supported by the Australian Research Council (ARC) through its Special Research Initiative (SRI) in Bionic Vision Science and Technology grant to Bionic Vision Australia (BVA). The Bionics Institute receives Operational Infrastructure Support from the Victorian Government and also acknowledges support from the Bertalli Family Trust and the J T Reid Charitable Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The Bionic Vision Australia Consortia authors for this manuscript are (a-z): 
Penelope J. Allen, Owen Burns, Kate E. Fox, Kumaravelu Ganesan, David J. Garret, Hamish Meffin, Joel Villalobos, and Jonathan Yeoh.

注:所有程序批准了皇家的维多利亚眼耳医院动物研究与伦理委员会（AEC RVEEH;＃10-199AB）。受试者根据国家卫生及（2013年）和"防止虐待动物法"医学研究理事会的"实践的护理和使用动物用于科学目的澳洲代码"处理（1986;和修正）。所有手术，临床评估和电程序进行麻醉下，并尽一切努力，以减少痛苦。 1.眼球摘除术和内固定注:按照详细的同伴手稿16所描述的眼球摘除和固定的程序，以围绕设备的电缆或玻璃体切割口格外小心，如果存在的话。简单地说，这涉及到: <ol><li> Transcardially灌注用温生理盐水后冷中性buffe主题红色的福尔马林。 </li><li>配合缝合眼球，作为地标。 </li><li>剜除眼球16，保持设备电缆及附件补丁/标签。 </li><li>后期修复眼18戴维森的解决方案 - 36小时。 </li><li>转移到50％乙醇为6 - 8小时。 </li><li>转移到70％乙醇和冷藏（4℃），直到解剖。 </li></ol> 2.电极拆卸和解剖。 注意:不是所有视网膜植入物将具有相同的外形尺寸，但一般会有一个电极阵列和某些形式的柔性和适形的载体材料。被临时固定到视网膜器件具有其中金属钉穿透阵列和眼球后部，保持这些共同一个钉孔。 <ol><li> Visualise植入物与在那里被固定到视网膜上的点。 <ol><li>用15度的刀片进行圆周跨角膜切口，取出角膜瓣为expOSE底层光圈和镜头。 </li><li>使用15度的刀片，disinsert透镜的悬韧带的纤维和全盘取下镜片，露出后房与视网膜植入物和金属钉原位 。 </li></ol></li><li>根据正在执行的种植和研究，在进一步解剖删除多余的组件。 注意:在本实施例中，视网膜前阵被评估由容纳在保形硅载体中的原型密封金刚石电极和电子封装（在内部产生的;参见20）。 <ol><li>在这里，仔细解剖精用手术刀提取金刚石电极包。离开植入物的有机硅主体随着视网膜粘性和铂布线的残余（ 图2）。如果嵌入式阵列/外壳不被调查的设备设计的一部分，则省略该步骤（2.2）。 </li></ol></li><li>小心解剖一个样品，它包括在所期望的方向的粘性和周围的组织。用细清扫剪刀剪下全部厚度条从眼球后部，包括巩膜，脉络膜和视网膜。取一个样品，这给钉的纵向横截面，示出所有相邻的粘性的视网膜层（ 图2） 注:供试品的所需取向可以有所不同，取决于所希望的具体结果。例如，如果一个人希望检查的粘性损坏视盘然后视盘应包含在样品内的接近。 </li></ol> 3.脱水，嵌入，安装，打磨，染色和成像<ol><li>脱水样品在渐进中乙醇阶段三天<ol><li>脱水在70％乙醇的样品2小时两次。 </li><li>脱水在80％的乙醇的样品2小时两次，然后O / N。 </li><li>脱水在90％的乙醇的样品2小时TWIC即</li><li>脱水在100％乙醇的样品2小时两次，然后O / N。 </li></ol></li><li>脱水在100％丙酮2小时的样品的两倍。 <ol><li>从丙酮中取出样品，并观察其光镜，因为它晾晒在室温下。样品转移到环氧树脂（参考步骤3.4）在开始之前卷曲/折叠。 注:从软组织造成倒塌细胞收缩去除液体。当组织卷曲/折叠发生的升值开发的经验。 </li></ol></li><li>根据制造商的指示准备的医疗级环氧树脂。以固化树脂，使用55℃，1小时或24小时，在RT。放置在真空室中进行约5分钟〜50毫巴以除去气泡。手动调节真空作为一个过度的真空会导致环氧混合煮沸，将不能有效地发生脱气 注:同时用3.2步执行步骤3.3。 </li><li>嵌入SAMP乐清晰的环氧树脂。浸入眼组织中脱气的环氧树脂在适当密封的容器中，并离开O / N在室温固化。 </li><li>照顾通过调整大小和重新嵌入固化的环氧块嵌入到所需的取向（ 图2）的样品。再嵌入含眼组织，使得粘性的长轴被定向为平行于所述模具的底部的调整块（黄色杯- 图3A）。 </li><li>安装树脂研磨试样座和研磨样品-使用碳化硅纸（230 250转与水有关，手工研磨）（从800网格; 图3C和3E）。 </li><li>对于染色，浸在甲苯胺蓝染色地面3 - 5分钟，或直到污渍发展（ 图3F）。 </li><li>用自来水冲洗（ 图3G）。 </li><li>图像具有高功率清扫范围检体的接地表面以可视化的蜂窝层视网膜（ 图3H）。适用于上述检体中的环氧树脂的顶部表面上的一滴蒸馏水以平滑衍射在空气 - 环氧接口。使用的光纤"鹅颈"光源照亮样本。 </li><li>重复步骤3.6 - 3.9，每次研磨掉样品的预先设定的厚度（试样保持器的最小精确和可重复的磨削幅度为20微米）。 </li></ol>

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Authors have nothing to disclose. Irfan Durmo is an employee of Cochlear Ltd.

标准组织学技术目前无法处理原位硬金属植入物由于在切割这些对象与金属，玻璃或甚至金刚石刀片的限制。在我们的配套文件16，我们发现，使用修改的全眼球固定技术可以减少人为的视网膜分层。在目前的手稿，一个既定的研磨和抛光技术可视化人工耳蜗植入原位 21-23修改视网膜假体。的钛粘性，用来固定电极阵列到视网膜，epiretinally，包埋在环氧树脂沿与周围的眼组织。这种树脂块，然后导向适当且逐步地/抛光以揭示所述组织形态学紧邻金属粘性。该块在不同深度的抛光表面的图像用一个功能强大的解剖显微镜。这种技术是有用的:可视化和evaluat荷兰国际集团邻近视网膜植入物的组织反应;评估与植入物的植入相关的外科手术创伤;以确定生物反应到硬金属部件;并测量植入物和视网膜的表面之间的距离。 这种技术将在未来的安全性研究可用于在邻近于在眼睛视网膜的粘性或其他硬（ 如，金属）物体区域的原位可视化。这在评估假肢上涨到视网膜epiretinally的临床前安全性直接应用。它也可以是用于评估组织损伤的视网膜的区域与位于子视网膜位置的植入物接触是有用的。 有几种方法，以验证该技术已被正确地执行。在每个阶段，视网膜应保持附着在眼球的外层。如果没有人为的视网膜脱离总值（GDP），这可能会印度语吃与固定的问题。当样品被嵌入和重新定向在最终树脂阻断视网膜应接近正交与磨削面块;这将最大限度地减少斜向切割。检查增量研磨步骤来遍历一个对象（如视网膜粘性）所需（已知步长）的数目与对象的尺寸相应地关联是有用的。 该技术可以被优化以几种方式。与研磨过程相关联的环氧块的表面上的划痕可减小逐渐变细级抛光。对于本研究中，我们使用的800，1000，1200，2400和4000级碳化硅纸。钻石膏也可以用来改善表面光洁度。更精细的表面光洁度给出更高质量的图像，但在附加的抛光时间的成本。另一个重要的考虑因素对于提高该技术的结果是OPTI的选择和质量CS和照明用于图像捕获。其他基本的组织学染色 - 特别是尼氏染色，可代替甲苯胺蓝的可以使用，但可能需要进一步优化。一些污渍会弄脏树脂以及该组织（ 例如 ，曙红），因此一个浅抛光，可能需要在染色后除去背景变色。专门的污渍，荧光染料和免疫组化染色没有尝试，但除非一个非常特定的结果是期望的，在每个磨削级别执行这些污渍所需的时间可能是令人望而却步。然而，有可能的嵌入步骤（步骤3.4）24之前以染色组织作为一个整体。 该技术的主要限制是，一旦所感兴趣的区域已被磨掉，它不能被检索，因此，为谨慎起见，在各种放大倍数在研磨和抛光的每个阶段，捕捉许多（可能冗余）的图像。这是还重要的是使用较小的增量为每个磨削深度调整。这种技术的另一个限制是该光学放大倍率和分辨率的组织固定在玻璃载片上，并与一个标准（传输）光学显微镜观察比较的那个。进行原型设计和评估一个新颖的植入装置的安全性的目的，病理评估总值为主要兴趣。该技术提供了用于观察与视网膜粘性相关的临床相关损害的有效方法。通过实践，收集研磨，抛光和拍摄一个给定的样本（一旦嵌入）所需的总时间是不相上下的时候，将采取部分中的蜡块或冰冻切片。 还有要延伸到视网膜植入物的范围之外的应用对本技术的潜力。这种技术适合于评估所述组织邻近硬植入物，其中植入物的提取不feasibLE或会损坏接口。例如，该技术可以扩展到评价从金属（ 例如 ，铂，镍钛诺等 ），不能用常规的组织学技术切割，制成的植入物，如某些脑深部或外周神经的电极，插管用于药物递送，血管支架或矫形假肢。

色素性视网膜炎（RP）是一种遗传性疾病，导致感光体，它们是细胞中负责转导光视网膜的最外层的普遍丧失，在光子的形式，成神经活性。重要的是，患者的RP通常具有剩余的神经元在其视网膜的其它层，它们仍然起作用。视网膜假体能够通过针对这些存活的神经元电刺激来激活他们的视觉通路1,2恢复一些有限的视野，这些患者中。从临床试验的感性效果已经显示出可喜的早期结果和最近一些设备已被批准用于商业用途。目前，存在其中临床视网膜假肢已被定位的三个主要解剖学位置:epiretinally 3,4，视网膜下5,6和suprachoroidally 7,8。不同的设备利用不同的材料和它们的形式被定制到它们被植入的位置。然而，它们都构成视觉知觉通过激活视网膜的剩余神经元的电脉冲。 存在用于任何医疗假体损伤周围组织因初始放置的机械作用或随后持续力的潜力。在可植入刺激器，如视网膜假肢的情况下，存在的电气参数应该是在安全范围内的额外的考虑因素。病人的安全是最重要的，所以设备必须在临床前研究推进到临床设置9-15前的严格测试。在我们的同伴文章中，我们描述了用于评估周围定位在脉络膜上腔16的植入物的眼睛的局部组织病理学的方法。在本手稿中，我们描述用于可视眼组织周围的电极阵列上涨到视网膜epiretinally，在临床前的技术（FE线）模型（ 图1）。 在视网膜前的位置是定位视觉假体是最常用的位置。这里位于电极阵列通常固定到与该穿透眼睛17-20的所有层的金属粘着性视网膜。之前在本手稿中描述的技术，这是难以准确评估视网膜和直接围绕一个粘性其他组织。用中性缓冲的福尔马林标准眼球固定造成人为的视网膜损伤，由于视网膜和巩膜对粘性的固定点的差速运动。因此所造成的粘性和视网膜前阵的任何实际损害不能精确观察。另外，切片的眼组织不能与原位视网膜粘性进行作为金属物体不能容易地切断与传统组织学装置;去除粘性前组织处理也并不可取，因为这也导致人为的视网膜损伤。 本研究的目的是双重的:1），以减少视网膜脱离伪影，使得所引起的粘性和视网膜植入阵列的任何损坏能够可靠地评估;和2）以可视化相邻的粘性视网膜结构而不删除。为了达到目的，图1中，新的固定技术被利用，从而降低了人为的视网膜剥离（如在同伴文章16描述）。为了达到目的，2，我们修改的嵌入，研磨，和抛光技术中，最初在原位观察耳蜗植入电极21-23的开发。在这个手稿中描述的方法允许周围视网膜的可视化和邻近粘着在原位 ，同时尽量减少人为的视网膜损伤，因此允许所造成的粘性和视网膜前阵的任何潜在损害精确的评估。

<table><tbody><tr><td>Acetone</td><td>Chem-Supply</td><td>AA008</td><td>Propanone BHD Medical grade</td></tr><tr><td>Epo-Tek 301 Epoxy</td><td>Epoxy Technology</td><td>Part A 1675-54-3 Part B 9046-10-0</td><td></td></tr><tr><td>Ethanol 70-75% v/v</td><td>Merck PTY LTD</td><td>4.10261</td><td>Alcohol</td></tr><tr><td>Ethanol</td><td>Merck PTY LTD</td><td>90143</td><td>Alcohol</td></tr><tr><td>Toluidine blue O</td><td>Sigma-Aldrich</td><td>T3260</td><td></td></tr><tr><td>Ethylenediamine Tetraacetic Acid</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>TegraPol grinding/polishing machine</td><td>Struers</td><td>TegraPol-25</td><td></td></tr><tr><td>AccuStop specimen holder</td><td>Struers</td><td>Accustop</td><td></td></tr><tr><td>Light microscope</td><td>Leica</td><td>MZ16</td><td></td></tr><tr><td>Objective lens</td><td>Leica</td><td>2.0x Planapo Objective</td><td></td></tr><tr><td>Digital Microscope Camera</td><td>Leica</td><td>DFC-420C</td><td></td></tr><tr><td>Microscope Software</td><td>Leica&amp;nbsp;</td><td>Application Suite v4.1.0</td><td></td></tr></tbody></table>

techniques for processing eyes implanted with a retinal prosthesis for localized histopathological analysis: part 2 epiretinal implants with retinal tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices currently entering the market. In order to evaluate the safety of these devices, in preclinical studies, reliable techniques are needed. However, the hard metal components utilised in some retinal implants are not compatible with traditional histological processes, particularly in consideration for the delicate nature of the surrounding tissue. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinally with a metal tack.
Retinal prostheses feature electrode arrays in contact with eye tissue. The most commonly used location for implantation is the epiretinal location (posterior chamber of the eye), where the implant is secured to the retina with a metal tack that penetrates all the layers of the eye. Previous methods have not been able to assess the proximal ocular tissue with the tack in situ, due to the inability of traditional histological techniques to cut metal objects. Consequently, it has been difficult to assess localized damage, if present, caused by tack insertion.
Therefore, we developed a technique for visualizing the tissue around a retinal tack and implant. We have modified an established technique, used for processing and visualizing hard bony tissue around a cochlear implant, for the soft delicate tissues of the eye. We orientated and embedded the fixed eye tissue, including the implant and retinal tack, in epoxy resin, to stabilise and protect the structure of the sample. Embedded samples were then ground, polished, stained, and imaged under various magnifications at incremental depths through the sample. This technique allowed the reliable assessment of eye tissue integrity and cytoarchitecture adjacent to the metal tack.

定影协议大幅减少人为的脱离和视网膜16的分层。环氧块内的试样的方向是使用中所述的两步嵌入处理取得一致。增量研磨过程所需手巧的中等水平，以达到最佳的效果，但得益于其提供的精确控制增量分辨率可调样品架。在所有情况下（N = 5）的粘性位于和地面/抛光与可取的和一致的结果。毗邻鞋钉的视网膜是解析并适当染色。抛光用等级P＃800的碳化硅纸环氧树脂块的表面就足以图像嵌入组织的细胞的宏观结构。更高等级的纸张，或金刚石浆料可以用来进一步抛光表面在任何给定深度如果需要的话。解剖显微镜和光学纤维"鹅颈"二极管灯吨源被认为是合适的成像地面块和嵌入的组织样本的表面上。光源的位置是变化的通过试验和错误来找到位置和角度，给了通过显微镜的最佳照明和对比度。加入一滴蒸馏水到块的表面上，在样品上方，是有用的，以减少在环氧空中接口的光路的衍射和/或光滑的失真。 图4示出了可视化紧邻钛视网膜的视网膜组织的例子的图像钉使用这种技术。非人为的视网膜脱离和折页上可以看到硅氧烷（ 图4A）的任一侧。粘性轴是可见的嵌入硅;粘性的头已经渗透视网膜和巩膜。有在未染色视网膜硅氧烷（ 图4C）的任一侧的非人为的视网膜脱离。该技术已经表明，在这种情况下，疗法e是相邻的视网膜中的一侧上的粘着性和压缩性视网膜解体由于斜插入角度。注意，呈现的图像仅仅是的技术中，不能代表不粘损伤组织病理学在一般的成功的插图。 <img alt="图1" src="/files/ftp_upload/52348/52348fig1highres.jpg" /> 图1.放置在视网膜的电极阵列。（A）眼显示后极部巩膜，脉络膜和视网膜退化（缺乏光感受器）的放大截面示意图。电极阵列中描绘了蓝色，贴epiretinally。一个视网膜前电极阵列（B）的计算机辅助绘图。一，集成电路（"芯片"）和电极包; B，钛视网膜粘性; C，医用级硅胶住房; D，铅出口点。 A组从原来的illustrati修改所提供的礼貌仿生视觉澳大利亚，版权贝丝十字。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52348/52348fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52348/52348fig2highres.jpg" /> 图2.去除电极和眼睛的解剖。一个去核的猫科动物的眼睛原位视网膜粘性高动态范围照片，微距（A）固定后与戴维森的固定液维持视网膜结构16中，去核的眼睛解剖。电极阵列包被从硅酮载体去除（虚线正方形轮廓指示电极阵列的原始位置）与剩余的植入物的粘着性（箭头）和有机硅主体。（B）的眼解剖与相邻的纵截面钉（虚线）。的粘着性保持嵌入在眼罩（箭头）的后壁，通过巩膜主要稳定。物为树脂嵌入和研磨（右段）制备含有粘性的组织切片，而制备用于标准组织学处理16（左段）含有被删除的电极阵列下方的视网膜上的一节。 0.5 mm的增量标尺显示在每个面板的底部边缘。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52348/52348fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52348/52348fig3highres.jpg" /> 图3.环氧树脂灌封和研磨的粘性和视网膜组织。嵌入式环氧树脂的照片，并对准组织样本，研磨，染色和粘性和视网膜组织的成像。（A）的交<html> K个样本包埋在环氧树脂块。使用的模具，将样品这样取向的纵向平面是平行于模具的底部。（B）中的环氧块中，安装在研磨试样保持器的固化后的环氧嵌段含有粘性样品（C）中 ，准备用于研磨（D）的样品定向的基本组织，使成像部分包含视网膜细胞层和粘着性的纵向轴线（E）的使用碳化硅纸在旋转磨床将样品地面（F）的接地表面块被甲苯胺蓝染色，以确定视网膜层。（G）中的块用自来水漂洗以除去过量的污点。（H），蓝染色视网膜层和粘着使用大功率清扫范围进行成像的甲苯胺。 广告/ 52348 / 52348fig4highres.jpg"/&gt; 图4.高和低功率的图像的粘着性和视网膜。在各点在研磨过程中图像用解剖范围，显示了粘性（星号）的纵向部分通过眼睛和巩膜（"S"穿透出射），相邻的硅（散列）和甲苯胺蓝染色和未染色的视网膜（"R"）。注意这些例子的图像仅用于说明本可视化技术，并不能代表全部视网膜植入物或粘着性插入组织学的结果。铂接线地面残存可见的面板AD（&#39;W&#39;）。（A）低功耗，大头钉穿透视网膜和巩膜的未染色的图像。（B）的视网膜脱离的高功率形象和折叠相邻未染色的视网膜到在粘性轴的中心的硅氧烷载体中图像A（C）的低放大率图像。 <str翁&gt;（D）的图像C.（E）的粘着性。在一个单独的样品的中心的高放大率图像，无硅酮载体的粘着性剑柄下方甲苯胺蓝染色的视网膜上的高功率图像显示，之前立即磨粘性轴（F）正常甲苯胺蓝染色的视网膜结构（GCL:视网膜神经节细胞层，INL:内核层，ONL:外核层; PR:感光; T:猫绒毡层灵芝）使用相同的研磨技术，可视化。比例尺在每块面板是:A和C = 2毫米; B和D = 500微米; E = 200微米; F = 100微米。

Watch this Scientific Journal Video about Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks at 

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

为处理眼植入视网膜假体的局部病理组织学分析技术:第2部分视网膜前植入视网膜大头钉

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

techniques-for-processing-eyes-implanted-with-retinal-prosthesis-for

Universidad San Sebastian

Research

JoVE Journal

Medicine

11.5K 次观看.  Bionics Institute. Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis. 

视频: Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

视网膜色素上皮的生理与病理生理学研究的实验模型

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

科研

神经科学

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

老鼠的眼睛剜除术的远程高通量的表型

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

医学

Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.

Techniques for visualizing retinal cytoarchitecture directly adjacent to individual electrodes within a retinal stimulator.

技术处理眼睛的视网膜假体植入局部组织病理学分析

With  the recent development of retinal prostheses, it is important to develop  reliable techniques for assessing the safety of these devices in ...

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

评估和量化年龄相关性黄斑变性小鼠模型中视网膜色素上皮病理的协议

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

遗传学

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

视网膜平片准备成像和免疫组化优化方案

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

生物学

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of&#160;unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole ...

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 ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

新型人类组织特征视网膜培养技术的特征

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

为处理眼植入视网膜假体的局部病理组织学分析技术:第2部分视网膜前植入视网膜大头钉

摘要

探索更多视频

此视频中的章节

视网膜色素上皮的生理与病理生理学研究的实验模型

老鼠的眼睛剜除术的远程高通量的表型

技术处理眼睛的视网膜假体植入局部组织病理学分析

评估和量化年龄相关性黄斑变性小鼠模型中视网膜色素上皮病理的协议

视网膜平片准备成像和免疫组化优化方案

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

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

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

新型人类组织特征视网膜培养技术的特征

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

为处理眼植入视网膜假体的局部病理组织学分析技术:第2部分视网膜前植入视网膜大头钉

摘要

探索更多视频

此视频中的章节

视网膜色素上皮的生理与病理生理学研究的实验模型

老鼠的眼睛剜除术的远程高通量的表型

技术处理眼睛的视网膜假体植入局部组织病理学分析

评估和量化年龄相关性黄斑变性小鼠模型中视网膜色素上皮病理的协议

视网膜平片准备成像和免疫组化优化方案

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

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

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

新型人类组织特征视网膜培养技术的特征

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

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