作者要感谢:亚历克西娅·桑德斯女士和米歇尔女士McPhedran的实验援助;冯海宁女士电极制作竹篙艾伦博士和乔纳森博士医生的手术援助;员工的皇家维多利亚眼耳医院的生物动物保健研究中心教授罗布牧羊犬一般指导各地和葫芦科蔓草Nayagam博士和梁定邦先生的批评意见，对草案的手稿形式。 这项工作是在仿生学研究所和墨尔本圣文森特医院。经费是由伊恩·波特基金会，约翰T·里德慈善信托基金，以及通过其特别研究计划在澳大利亚研究理事会仿生视觉科学和技术授予澳大利亚仿生视觉（BVA）。仿生学研究所承认它接收来自维多利亚州政府通过其运营的基础设施支援计划的支持。

1。眼球摘除后固定这个协议假定，这个话题又被单方面与在脉络膜上电极阵列植入。 <ol><li>准备后缀解决方案玻璃肖特瓶。 <ol class="lalpha"><li>戴维森的固定液，2个100毫升<ul><li> 2毫升福尔马林（37％甲醛） </li><li> 10毫升冰醋酸（99.7％） </li><li> 35毫升乙醇（98％） </li><li> 53毫升蒸馏水水</li></ul></li><li> 50％乙醇，2个100毫升</li><li> 70％乙醇，2个100毫升</li></ol></li><li> Transcardially灌注须用温水（37°C）肝素生理盐水，然后由冷（4°C）中性缓冲福尔马林（10％）。 </li><li>配合缝线双眼优越缘（或位置，是远程从脉络膜上阵列） - 一个直肌，作为一个具有里程碑意义。 </li><li>去核的眼睛，维持任何外部导线植入眼。 </li><li>将EAC戴维森的固定液在100毫升ħ眼离开修复后在室温下。 </li><li>戴维森的固定液中的18 - 36小时后，转移到50％乙醇（室温）6 - 8小时，最后70％的乙醇（室温）。 </li><li>样品冷藏在4℃和存储，直到清扫。 </li></ol>完整和加压眼金球奖已经以这种方式存储长达3个月所得到的组织学无不利影响。 2。鉴定和组织映射上述固定协议（第1步），使巩膜半透明与阵列现在是可见的-包括单个电极位点（ 图3）。 <ol><li>取出植入全球乙醇和仔细地修剪掉多余的组织，包括结膜和Tenon囊。修剪视神经2 - 3毫米的长度（ 图3A）。 </li><li>使用组织学染料MARKIN预定义的颜色代码标签电极，克计划，照顾不弄脏染料。 </li></ol><ul><li>我们选择市售一套专门的组织学染料（参见附录）。仔细涂抹少量的染料与细笔尖的油漆刷（Roymac大小00）的组织，并在空气中干燥5分钟。 </li><li>对于我们的目的，我们选择了:绿色活性电极（）（）最大限度地刺激，阈上水平，研究期间，红色等活性电极;黄色直径较大的返回电极;蓝色额外的指南和/或解剖距离标志（ 图3B和3C）。 </li><li>可能需要一些试验和错误，发现是稳定的染料，在整个随后的步骤。例如，在图4中可以看出，比红色染料，绿色染料更有弹性。在70％乙醇稀释该染料有助于改善脂质penet口粮和组织涂层。 </li><li>它是有用的绘制或拍摄的染色地球以供将来参考。 </li></ul><ol start="3"><li>返回到70％的乙醇脱水的染料。 </li><li>重复步骤2.1未植入控制全球。 </li><li>步骤1.3，使用的缝线对齐控制眼植入眼镜像对。 </li><li>位于植入物植入眼（NB透光性巩膜和来自步骤2.2的染料标记允许准备植入数组中的位置的可视化的镜像位置上的控制大致（作为电极阵列具有相同的尺寸），将有机硅模板）。 </li><li>执行步骤2.2和2.3的对照眼，植入电极使每个站点有一个控制对解剖学上具有可比性（ 即镜匹配当量）位置。 </li></ol> 3。解剖和嵌入<ol><li>从眼睛中取出植入物阵列和删除前面的眼睛（包括角膜，虹膜和镜头）。取出玻璃体液从剩余的眼杯（后室）。 </li><li>解剖植入眼成多个代表带（〜2毫米厚），每个含有染料标记区域（ 图3D）的一个子集。需要注意的是，应选择的条带的方向，以协助评估的组织反应的植入物7上的各个方面。 </li></ol>它是有用的，以供将来参考，做一个记录染料地区各条和它们的相对位置和颜色上都存在。 <ol start="3"><li>将在其一侧的条带成浅水池液化琼脂（4％，80 - 90℃），与侧被切断的第一朝下（ 图3E）。 </li><li>琼脂后，周围的样本，并放入一个纸巾盒，泡沫填充物（ 图3F）支持削减。 </li><li>重复步骤3.1-3.3控制眼 - 德ING护理解剖镜匹配的条状。 </li><li> 通过标准（自动化过夜）石蜡加工技术处理所有的录音带。 </li><li>同方首先被削减朝下嵌入石蜡组织处理。 </li></ol> 4。切割和染色<ol><li>定期音符从步骤2和3到5微米的部分剪切石蜡块。 </li><li>收集从每个染色区域的部分和安装在幻灯片。 </li></ol>每个染色斑点巩膜紧邻该区域现在可以进行评估，它是在阵列中的相应的电极（ 图4）的最接近的信心。 <ol start="3"><li>染色进行免疫随意。例如染色和免疫组化， 如图5所示:ħE条款; Luxol蓝快（LFB）;甲酚紫; Masson染色蓝色;碘酸希夫（PAS），皮尔斯&#39;普鲁士蓝站;抗-谷氨酰胺合成酶（GS），抗神经丝蛋白（NF）;抗胶质原纤维酸性蛋白（GFAP）和4&#39;，6 - 二脒-2 - 苯基吲哚（DAPI）。 </li><li>标准和特殊污渍进行详细: <ol><li> H＆E染色徕卡multistainer浴的的ST5020阵列（徕卡生物系统公司）提供的协议进行。 </li><li> LFB和甲酚紫（11日修改）: <ol><li>脱蜡在二甲苯中的变化（3×2分钟）和2乙醇（100％，100％）（2×1分钟），并在自来水中冲洗（45秒）。 </li><li>水合物的部分，以95％的乙醇。 </li><li>地方在LFB解决方案11在37 - 42°C（过夜）。 </li><li>冲洗掉多余染色用70％乙醇。 </li><li>在蒸馏水中冲洗。 </li><li>在稀碳酸锂的地方（8％）（几秒钟）。 </li><li>在70％乙醇（几秒钟）的区分。 </li><li>在蒸馏水中冲洗。 </li><li>重复步骤4.4.2.6至4.4.2.8，如果有必要，直到进程B无色ackground。 </li><li>甲酚紫醋酸溶液在37℃（10分钟）的地方。 </li><li>不要用水清洗。 </li><li>脱水3无水酒精（1×30秒20秒，2）和3二甲苯中的变化（1×1分30秒，2×1分钟）。 </li><li>安装盖玻片DPX。 </li></ol></li><li> Masson染色蓝（11日修改）: <ol><li>脱蜡为4.4.2.1。 </li><li>使部分水（2分钟）。 </li><li>细胞核染色使用韦格特铁苏木（2分钟）。 </li><li>自来水中清洗，在蒸馏水中漂洗。 </li><li>染色布里希大红酸性品红溶液（5分钟）。 </li><li>在蒸馏水中冲洗。 </li><li>分化磷磷钨酸，直到胶原蛋白是淡粉色（6分钟）。 </li><li>在蒸馏水中冲洗。 </li><li>苯胺蓝染液（1分）。 </li><li>在1％乙酸（1分钟），洗井。 </li><li>脱水，安装和盖玻片为pER 4.4.2.12和4.4.2.13。 </li></ol></li><li>首席助理秘书长（11日修改）: <ol><li>脱蜡为4.4.2.1。 </li><li> 1％（15分钟）高碘酸氧化。 </li><li>自来水再用蒸馏水清洗。 </li><li>希夫氏试剂（15分钟）中的染色。 </li><li>水洗涤（5分钟）。 </li><li>染液与Harris苏木精（1分钟）。 </li><li>简要清洗自来水。 </li><li>分化在0.5％，酸醇（1 DIP）。 </li><li>以及自来水中清洗。 </li><li>蓝斯科特的自来水（1分）。 </li><li>水洗净。 </li><li>脱水，安装和盖玻片每4.4.2.12和4.4.2.13。 </li></ol></li><li>皮尔斯&#39;普鲁士蓝染色中所描述的11。 </li></ol></li><li>免疫荧光染色进行详细: <ol><li>脱蜡为4.4.2.1。 </li><li>在蒸馏水中冲洗（2×5分钟）。 </li><li>进行抗原修复使用0.01％柠檬酸缓冲液，pH值6在75 - 80°C（20分钟）ð允许在0.01％的柠檬酸盐缓冲液中（20分钟）冷却。 </li><li>洗部分在磷酸盐缓冲盐水（PBS）（2×5分钟）。 </li><li>细胞膜的通透在洗涤缓冲液中（10分钟）。 </li><li>孵育血清中的块溶液（2小时）。 </li><li>孵育部分在一个单一的抗体稀释液稀释（正常山羊serum/0.1％的Triton X-100/PBS的10％）的主要抗体（在冰箱中过夜）。每个初级抗体滴度示于表1。 </li><li>过夜温育后，洗涤在洗涤缓冲液（5×3分钟）的部分。从这点上，保持在黑暗的部分。 </li><li>孵育部分相应的二次抗体滴度为1:500一个特定的时间（ 见表1）。 </li><li>洗在洗涤缓冲液（3×5分钟）的部分。 </li><li>孵育DAPI（1:25,000 / PBS）（30分钟），部分。 </li><li>洗在洗涤缓冲液（3×5分钟）的部分。 </li><li>安装和盖玻片使用荧光NT安装媒体。 </li></ol></li></ol>准备两两比较试验样品和对照样品。

<ol>
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Sci. 47, 3514-3522 (2006).</li><li>Ray, A., Colodetti, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+analysis+of+retinal+neurons+under+electrical+stimulation.">Immunocytochemical analysis of retinal neurons under electrical stimulation.</a> Brain Res. 1255, 89-97 (2009).</li><li>Nakauchi, K., Fujikado, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threshold+suprachoroidal-transretinal+stimulation+current+resulting+in+retinal+damage+in+rabbits.">Threshold suprachoroidal-transretinal stimulation current resulting in retinal damage in rabbits.</a> J. Neural. Eng. 4, S50-S57 (2007).</li><li>Shepherd, R. K., Murray, M. T., Houghton, M. E., Clark, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+electron+microscopy+of+chronically+stimulated+platinum+intracochlear+electrodes.">Scanning electron microscopy of chronically stimulated platinum intracochlear electrodes.</a> Biomaterials. 6, 237-242 (1985).</li><li>Villalobos Villa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Safety of a Wide-Field Suprachoroidal Retinal Prosthesis. , (2012).</li><li>Nayagam, D. A. X., Allen, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pre-Clinical+Model+for+Chronic+Electrical+Stimulation+of+the+Retina+via+Suprachoroidal+Electrodes.">A Pre-Clinical Model for Chronic Electrical Stimulation of the Retina via Suprachoroidal Electrodes.</a> , (2012).</li><li>Nayagam, D. A. X., Villalobos, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Suprachoroidal+Retinal+Prosthesis+is+Safe+in+a+Chronic+Implantation+Model.">A Suprachoroidal Retinal Prosthesis is Safe in a Chronic Implantation Model.</a> in Vision and Ophthalmology Annual Meeting., Vol. 4928/A327. , (2011).</li><li>Cicione, R., Shivdasani, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+cortex+responses+to+suprachoroidal+electrical+stimulation+of+the+retina:+effects+of+electrode+return+configuration.">Visual cortex responses to suprachoroidal electrical stimulation of the retina: effects of electrode return configuration.</a> J. Neural Eng. 9, 036009 (2012).</li><li>Schuettler, M., Stiess, S., King, B. V., Suaning, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+implantable+microelectrode+arrays+by+laser+cutting+of+silicone+rubber+and+platinum+foil.">Fabrication of implantable microelectrode arrays by laser cutting of silicone rubber and platinum foil.</a> J. Neural Eng. 2, S121-S128 (2005).</li><li>Morimoto, T., Kamei, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+implantation+of+newly+developed+suprachoroidal-transretinal+stimulation+prosthesis+in+dogs.">Chronic implantation of newly developed suprachoroidal-transretinal stimulation prosthesis in dogs.</a> Invest. Ophthalmol. Vis. Sci. 52, 6785-6792 (2011).</li><li>Bron, A. J., Tripathi, R. C., Tripathi, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Wolff's anatomy of the eye and orbit. , (2001).</li><li>Stein, J. J., Johnson, S. A., Berson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+coverage+of+beta+cells+in+the+cat+retina.">Distribution and coverage of beta cells in the cat retina.</a> J. Comp. Neurol. 372, 597-617 (1996).</li><li>Dagnelie, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychophysical+evaluation+for+visual+prosthesis.">Psychophysical evaluation for visual prosthesis.</a> Annu. Rev. Biomed Eng. 10, 339-368 (2008).</li></ol>

作者什么都没有透露。

临床前研究，评估植入物的安全作为首要考虑因素。之前视网膜假体可植入人体内，以验证它不会造成任何伤害，它是必不可少的。这需要植入物的生物相容性17-23以及任何潜在的损害，可能是由于慢性电刺激24-26的评估。类似的神经假体，如人工耳蜗植入的经验，提供了洞察广泛的刺激和物理参数，不会造成组织损伤27。然而，在视网膜其他的植入部位具有不同的特征，因此，精确的安全限制（例如最大电荷密度）从以往的研究在其它系统中，不能假定。电荷密度是最高的活性电极网站，与此相对应的最大的潜在损害。因此直接相邻的组织本地化的方法个人主动电极会大大增加，这些研究的实用性。也会以高亮显示特定区域的植入物相邻的组织仔细检查。这种对象的方法允许少的部分，以进行分析，检查"最坏的情况"，同时保留了信心。 巩膜染料本地化这里介绍的方法已经得到了广泛的测试与手形和成型硅胶/ Pt电极阵列28-30。它已被用于与薄膜聚酰胺阵列的7,31和薄膜有机硅/铂阵列32。一些视网膜假体的研究采用"子弹头"形电极与巩膜内植入33。本发明的技术应该是有效的，用于评估这种类型的电极阵列。猫的眼睛巩膜薄（后极部的厚度在90 - 200微米）允许在一个数组中的单独电极的可视化。然而，即使从个人电德不可见，使用有机硅模板阵列的轮廓时，可以看出（在步骤2.6中所用的相同），它们的位置可以很容易地推断出。 染料的映射限制了需要取得非相关的组织切片，与染料的礼物，因为只有部分得到。能够准确地推断出电极位置不仅可以让相关的组织病理学分析和统计力量更大，但通过减少以及劳动力成本的幻灯片和刀片的数量，时间和成本管理上具有重大影响。粘附，并能承受组织处理，同时也未染色的组织切片中可见染料的使用是一个重要的初始代价。知识的解剖组织的染料和方向的位置，并在嵌入过程中的辅助切削过程。这包括正确地取向的块，实现了部分未斜切的，并给出了一个完整的representati上的组织。因此重要的是，执行切割的人存在于染料的应用程序时，剥离和嵌入。 总体协议是强大的轻微改动，特别是与固定时序。然而，眼睛解剖和染料标记步骤微妙的程序，受益于良好的手巧，以避免损坏试样。虽然戴维森的固定液已经证明是有效减少人造的植入物附近的视网膜脱离，但它不能阻止直接在解剖过程中的机械损伤。戴维森的固定液不容易与一些特殊的组织学和免疫程序。因此，有必要修改/优化这些步骤，上面详细。染色时间和浓度的增量变化，直到取得最佳结果 - 更多详细信息，请参阅补充材料。 量化视网膜组织学仍然是个问题。视网膜非均质的厚度和细胞密度的变化，随着距离的增加从中央区34,35。因此，植入眼内的邻近组织，不能用于比较和控制眼代替。成对匹配的对照眼的每个部分为我们提供了一个更强大的统计比较的病理变化进行比较时，主题36对侧眼视网膜假肢的疗效和安全性的成果更好地进行评估。必须采取特别护理，以尽量减少斜向切割，因为这会影响量化。 总之，上面描述的方法显着改善了我们的组织病理学分析，这反过来又导致了一个更有效的临床前的工作流程。使用这些方法的临床前研究的结果直接导致了3个RP患者的临床试验中，已安全植入suprachoroiDAL视网膜假肢。这些方法有关的视网膜刺激和​​其他眼部植入物需要进行评估的病理反应长期植入。这种方法提供了有价值的优势保​​持细胞结构和登记的病理植入利息区域。虽然没有具体测试，可以使用这些程序的修改可能在其他物种和解剖位置，特别是当一个阵列被植入表面。

视网膜假体，可能很快就会成为有用的临床干预治疗各种形式的严重的视力丧失。某些情况下，如视网膜色素变性（RP），结果是在广泛的眼睛中的光感受器变性，这些是负责神经信号转导的光线进入细胞。然而，一些在其他层的视网膜细胞依然存在，并且是电刺激视网膜假体1的潜在目标。从人体临床试验的早期结果已经有前途的电极阵列植入在视网膜前，视网膜4,5 2,3，巩膜内6个地点的。这些设备都是由不同的材料具有不同的形状，但它们都使用电脉冲激活的其它可行的视网膜神经元，以创建视觉知觉。 我们更广泛的群体（澳大利亚仿生视觉）已经开发出具有渐进的视网膜植入sed的临床试验研究的3 RP患者植入脉络膜上的电极阵列（临床试验号:NCT01603576）。 图1A显示这个脉络膜视网膜假体的原型。 在临床研究中患者的安全是最重要的，因此，在开始人体试验之前，我们进行了广泛的急性和慢性试验在临床前模型（ 图1B）。组织病理学评估缩小外科手术，遍历电极设计，最终建立的植入物的安全性是必不可少的。为了保持高效的工作流程与标准的临床病理实践相吻合，石蜡包埋技术。也希望采用与临床标准，如苏木精和伊红（H＆E），这是很容易解释由病理学家的染色程序。免疫组织化学分析的技术，能够适应，我们也希望为了进一步促进细胞组织的评价。 的生物相容性的植入材料，和慢性电刺激的安全，需要仔细评估。重要的是要能个别刺激电极阵列内的相邻的眼组织的组织病理学检查。然而，阵列的需要删除的前处理的样品进行测试，这样他们可以，因为它们的金属部件不能容易地剖。因此，我们建立的组织编制并不允许组织的定位与地图就植入电极7。除了 ​​缺乏组织定位方法，是标准的基于甲醛的固定和加工技术，导致广泛的工件和眼睛的各个层中的收缩率差异（ 图2），由于视网膜脱离。由于非均匀性吨他的视网膜上，这是很难与对照组织进行有效的比较，特别是对于定量测量。这些组合限制复杂准确的评估所造成的潜在损害我们的植入阵列。 在这里，我们提出新的技术，为克服这些局限性。我们已经修改了专门眼球固定和加工技术8-10组织学石蜡基保持兼容。我们已经修改了标准的组织学和免疫组化染色，根据临床病理学家反馈给比较的结果。巩膜组织半透明渲染后，基于染料的颜色代码马克眼组织相邻的每个单独的电极，除去从脉络膜上腔阵列之前。通过标记的电极阵列和单个电极站点，样品可以准确地收集从电极相邻的区域。匹配的样品可以收集日E控制眼，为了方便两两比较。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>The Davidson marking system</td>
			<td>Bradley Products</td>
			<td>1163-4 - red 1163-5 - blue 1163-2 - yellow 1163-1- green</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Polyclonal Anti-Glial Fibrillary Acidic Protein Antibody</td>
			<td>Millipore</td>
			<td>AB5804</td>
			<td>1:1500, overnight incubation</td>
		</tr>
		<tr>
			<td>Mouse Monoclonal Anti-Glutamine Synthetase Antibody</td>
			<td>Millipore</td>
			<td>MAB302</td>
			<td>1:100 overnight incubation</td>
		</tr>
		<tr>
			<td>Monoclonal Mouse Anti-Neurofilament 200 Antibody</td>
			<td>Sigma-Aldrich</td>
			<td>N0142</td>
			<td>1:100 overnight incubation</td>
		</tr>
		<tr>
			<td>4&#39;,6-diamindino-2-phenylindole, dilactate (dilactate)</td>
			<td>Life Technologies</td>
			<td>D3571</td>
			<td>1:25,000 30 min incubation</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse IgG</td>
			<td>Life Technologies</td>
			<td>A11029</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Superfrost 90&#176; yellow</td>
			<td>Grale Scientific</td>
			<td>SF41299</td>
			<td></td>
		</tr>
		<tr>
			<td>FLEX IHC Microscope Slides</td>
			<td>DAKO</td>
			<td>K802021-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 goat anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>A11037</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Life Technologies</td>
			<td>PCN5000</td>
			<td>10% serum/0.1% Triton X-100/PBS</td>
		</tr>
		<tr>
 			<td></td>
			<td></td>
			<td></td>
			<td>
			Non-Standard Solution Preparation for Special Stains
			1% Aqueous Cresyl Violet Stock Solution
			<ul>
				<li>Cresyl violet 1 g</li>
				<li>Distilled water 100 ml</li>
			</ul>
			Cresyl Violet Acetate Working Solution
			<ul>
				<li>1% aqueous cresyl violet stock solution 9 ml</li>
				<li>Distilled water 51 ml</li>
				<li>10% acetic acid 0.5 ml</li>
			</ul>
			Biebrich Scarlet-Acid Fuchsin Solution
			<ul>
				<li>1% Beihrich scarlet 90 ml</li>
				<li>1% acid fuchsin 10 ml</li>
				<li>Glacial acetic acid 1 ml</li>
			</ul>
			Phosphomolybdic-Phosphtungstic Acid Solution
			<ul>
				<li>Phosphomolybdic acid 2.5 g</li>
				<li>Phosphotungstic acid 2.5 g</li>
				<li>Distilled water 100 ml</li>
			</ul>
			Aniline Blue Solution
			<ul>
				<li>Aniline blue 2.5 g</li>
				<li>Glacial acetic acid 2 g</li>
				<li>Distilled water 100 ml</li>
			</ul>
			Solutions for Immunohistochemistry
			Wash Buffer Solution
			<ul>
				<li>0.1% Triton X-100 in Phosphate buffered saline (PBS)</li>
			</ul>
			Serum Block Solution
			<ul>
				<li>10% normal goat serum/0.1% Triton X-100 in PBS</li>
			</ul>
			SUPPLEMENTARY MATERIAL
			Luxol Fast Blue
			The purpose of performing a Luxol Fast Blue (LFB) stain was to identify the ganglion cells in the ganglion cell layer through the counterstain of cresyl violet. While LFB stains myelin, the cresyl violet stain binds to the Nissl substances in neurons, composed of rough endoplasmic reticulum and ribosomes. Numerous cells in the retina contain Nissl substance including amacrine cells. These cells usually are located in the inner boundary layer of the inner nuclear layer, but displaced amacrine cells can be found in the ganglion cell layer. The staining of Nissl substance is helpful in differentiating large, heavily stained ganglion cells from smaller displaced amacrine cells. To aid visualisation of these cells, we modified the cresyl violet working solution protocol by increasing the volume of the cresyl violet stock solution in the working solution. We increased the volume in 1.0 ml increments from 6.0 ml to 9.0 ml, in turn reducing the volume of distilled water. Assessing the ease of visualising and identifying the ganglion cells, optimal staining was achieved with 9 ml of additional cresyl violet stock solution and 51 ml of distilled water. Cresyl violet is a basic dye, though to dye the Nissl substance, it is required to be used in an acidic solution, and therefore the volume of acetic acid remained unchanged at 0.5 ml.
			Periodic Acid Schiff
			The Periodic Acid Schiff (PAS) stain was performed to highlight polysaccharides especially glycogen, found in basement membranes and fungal cell walls, indicating a fungal infection. The process of the PAS stain is to oxidise the hydroxyl groups in polysaccharides using periodic acid, producing aldehyde groups. The application of the Schiff reagent, binds to the aldehyde groups producing a magenta color with haematoxylin counterstaining producing purple cell nuclei. Our slides showed weak staining at the inner limiting membrane. This may be due to the polysaccharides present, as not all polysaccharides can be oxidised with a standard time frame, especially those containing anionic polysaccharides. We therefore increased the duration of the oxidation step from 10 min to 12, 15, and 20 min. We found that a 15 min oxidation step was most effective in PAS staining.
			Masson&#8217;s Trichrome Blue
			The principle of the Masson&#39;s trichrome blue stain is to stain collagen and muscle, which in our retinal slides can be used to indicate an increase in collagen tissue which may indicate fibrosis due to injury. This stain is sensitive to fixation techniques, so alterations were needed to obtain optimal staining. The process of this stain is to initially stain cytoplasm and muscle fibres red using the acidic Biebrich scarlet-acid fuchsin red dye. Differentiation with phosphomolybdic/phosphotungstic acid competes with the red dye in the collagen fibres. In the sclera, the large phosphomolybdic/phosphotungstic acid molecules are able to penetrate the loose connective tissue, as opposed to the dense muscle fibres which are penetrable to small molecules only. Aniline blue dye is then applied to replace the acid in the collagen fibres producing blue dyed sclera. To optimise this stain in retinal sections, the duration of staining with Biebrich scarlet-acid fuchsin dye was reduced to create a balance between the blue and the red dye. The use of Davidson&#39;s fixative also required an extended differentiation time to remove the red dye from the collagen. We trialled differentiation at 5, 6, 8, and 10 min, with optimal results occurring at 6 min. Differentiation also varies on the thickness and cutting of smooth sections, with greater than 6 min differentiation possibly being required.
			Glial Fibrillary Acidic Protein (GFAP) Antibody
			Polyclonal, host species is rabbit, has a molecular weight of 50 kDa. GFAP antigen is an intermediate filament found in the cytoplasm (the cytoplasm of astrocytes and M&#252;ller cells in the retina), the antigen is made form 428 amino acids and has a molecular weight of 49512 Da.
			Neurofilament-200 antibody
			Monoclonal, host species is mouse, clone N52, isotype IgG1, it recognises phosphorylated and non-phosphorylated forms of the heavy neurofilaments which have a molecular weight of 200 kDa. The antibody will bind to an epitope in the tail domain of the neurofilament. The antibody will stain neurofilaments in the neuronal perikarya, dendrites and axons.
			Glutamine Synthetase (GS) antibody
			Monoclonal, clone GS-6, host species is mouse, has a molecular weight of 45 kDa and antibody isotype IgG2a. The GS antigen is found in the cell cytoplasm (cytoplasm of M&#252;ller cells in the retina), the antigen has 373 amino acids and a molecular weight of 42,064 Da.
			</td>
		</tr>
	</tbody>
</table>

techniques for processing eyes implanted with a retinal prosthesis for localized histopathological analysis

视网膜假体的最新发展，重要的是要评估这些设备的安全性在临床前研究，开发可靠的技术。然而，标准固定，准备，组织学和自动化程序，是不理想的。这里，我们描述用于评估植入物直接相邻的视网膜的健康的新的程序。视网膜假体设有电极阵列与眼组织接触。以前的方法一直没能在空间上本地化阵列内的相邻的单独电极的眼组织。此外，标准的组织处理结果往往是人造的毛脱离评估时植入眼睛视网膜层。因此，一直难以评估的局部破坏，如果存在，植入的电极阵列植入和刺激引起的。因此，我们开发出了一种用于识别和定位植入ê相邻的眼组织lectrodes使用（彩色编码）染料标记的方案，我们修改了一个的眼球固定技术，以尽量减少人造的视网膜脱离。这种方法也使巩膜半透明，使个别电极和植入物的特定部分的本地化。最后，我们使用了一个匹配的控制，提高功率的组织病理学评估。总之，这种方法可植入眼睛的视网膜细胞结构可靠，高效的歧视和评估。

图4示出了图3中描述的样本从切割得到的代表性的部分。根据上述协议，在厚度为5微米，与H＆E染色部分被切断。保留样品带的总形状差异最小组织文物（ 图4A）。这两种染料标记，巩膜上可见，虽然绿色染料更弹性比红（ 图4B）。视网膜层artifactually脱落，视网膜的形态被保留（ 图4C）。视网膜组织相邻的染料标记，这对应于在阵列中的电极的位置，可以很容易地确定。 图5示出了根据当前协议制备的有代表性的特殊染色组织切片和免疫组织化学。请参阅图5 caption和补充材料的更详细的具体污渍和修改它们的用法。修改后，这些污渍和免疫组化相当于既定标准 - 作为病理学家核实。 <table border="1"><tbody><tr><td>不同的一次抗体和滴度（括号内） </td><td> GFAP（1:1500） </td><td> NF200（1:100） </td><td> GS（1:100） </td></tr><tr><td>不同的二次抗体滴度（括号内） </td><td>的Alexa Fluor 594 </td><td>的Alexa Fluor 488 </td><td>的Alexa Fluor 488 </td></tr><tr><td>孵化二次抗体的持续时间</td><td> 1小时</td><td> 2小时</td><td> 45分钟</td></tr></tbody></table> 表1中。抗体类型，效价和贴标期限。 <img alt="图1" src="/files/ftp_upload/50411/50411fig1.jpg" /> 图1。 Suprachoro新娘原型电极阵列。 A）微距摄影的临床级模压阵列B）手工制作的潜伏期阵列的显微照片。 <img alt="图2" src="/files/ftp_upload/50411/50411fig2.jpg" /> 图2。前视网膜组织学的标准组织学方法被用来准备这些，H＆E染色，眼脉络膜上电极阵列植入的部分。）正交部分通过电极阵列腔（描绘星）。视网膜脱离的外眼组织。下方的注入区域（箭头所示），这一点尤其明显。这是不可能的，以确定视网膜的部分相邻的阵列中每个单独的电极。比例尺= 1毫米。B）的盒装地区面板更高的放大倍率。在视网膜Laye的有几个，定期间隔，文物RS（箭头），以及主要脱离绒毡层（反射层的猫科动物的眼睛）。比例尺= 100微米。C）更高的放大倍率在面板B的盒装地区。箭头指示的光感受器的外段，以及色素上皮细胞，保持不变，表明脱离是一个人造的副作用的处理，而不是在体内的创伤或病理引起的。比例尺= 20微米。 <img alt="图3" src="/files/ftp_upload/50411/50411fig3.jpg" /> 图3。本地化和夹层的电极邻近组织。AD）高动态范围的宏观照片去核的眼睛，与脉络膜上电极阵列原位。固定与戴维森的固定液，A）发表阵列去除，前巩膜半透明使可视化单个阵列中的电极（单箭头指示的例子）B）电极与预定义的染料颜色标记。）染料标记，定期距解剖标志使用保持一致性，整个实验，D）去除后阵列，眼睛解剖由手入样品条。彩色箭头表示两个相邻电极网站巩膜上。黄色虚线表示的切片平面E）内嵌入的琼脂块。F的样品条的宏观照片）从面板E琼脂插入一个样品条，切出由泡沫活检垫支持在包埋盒中的下宏观照片在处理过程中，以尽量减少的部分的运动。 <img alt="图4" src="/files/ftp_upload/50411/50411fig4.jpg" /> 图4。新的视网膜组织学。 </s<html>阮富仲&gt;的上述样品带钢（ 图3D），含有电极口袋的，是石蜡嵌入，切片在5微米和染色ħ＆E. A）绿色和红色染色区域（由箭，同图3D表示）是可见的截取的截面图3D中的黄色虚线上面的水平。比例尺= 1000微米。的视网膜是不沾边，既不阵列下方的口袋，也没有远程B）盒装地区可见红色和绿色染料从Panel A的箭头表示，整个完整的视网膜。比例尺= 500微米C）盒装区B组，完整的，附加，视网膜相邻的绿色染料。比例尺= 50微米。知道的染料指示的位置的电极的位置，我们可以自信地评估相邻的视网膜组织的损坏，如果有的话，通过电刺激引起的。 ve_content"FO:保持together.within"页面="总是"&gt; <img alt="图5" src="/files/ftp_upload/50411/50411fig5.jpg" /> 图5。实施例的修改视网膜cytohistochemistry的使用特殊的着色剂和免疫荧光。Davidson的固定剂的使用，而不是标准的福尔马林固定技术，需要改变通常的染色协议中所概述的主要协议文本。病理学家已经证实，这些修改导致在同等染色。Å）H E条款;苏木渍细胞核紫伊红染色细胞的细胞质中，胶原蛋白和支持组织的各种色调的粉红色B）LFB髓鞘蓝色污渍，同时甲酚紫染液可以用来识别神经节细胞内的核周质蓝尼氏体染色和突出的卫星细胞周围的细胞C）Masson染色蓝色;布里希猩红酸品红溶液污渍肌肉FI红色的BER，而苯胺蓝染色的胶原蛋白，在这种情况下，巩膜，蓝色D）PAS;糖蛋白成分的基底膜和结缔组织成分被染成紫色，而苏木counterstains细胞核紫色E）皮尔斯&#39;普鲁士蓝;于出血的部位，形成含铁血黄素从退化的红血细胞和铁配合物的释放产生紫色的颜色而加入中性红染色的颜色溶酶体红色F）GS，这神经递质的降解酶，发现在Müller细胞13中 （绿色），可以看到在两个方向延伸Müller细胞机构内的核层和的Müller细胞endfeet形成内界膜，G）NF-200在这个沉重的链骨架蛋白（绿色）发现在胞体和流程，与其他神经纤维的交联，以保持结构的神经元14,15。神经节可以看出，在细胞和其轴突神经节细胞层，神经纤维层，同时还强调了猫科动物的视网膜的内核层中的位于外边界（boarder），水平细胞的轴突。用DAPI复染（蓝色），允许可视化的视网膜层H）GFAP在Müller细胞和星形胶质细胞增殖，这种细胞骨架蛋白（红色）在16胶质细胞增生，可以看到排队Müller细胞形成薄的扩展通过内部神经纤维层外层视网膜。用DAPI复染（蓝色​​）允许可视化的视网膜层。比例尺= 50微米的所有面板。在每幅图像的顶部示出视网膜内层;，视网膜外层是在每个图像的底部所示，不含面板E，显示subscleral反应。

Watch this Scientific Journal Video about Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis at JoVE.com

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

直接相邻的各个电极的视网膜刺激视网膜细胞结构可视化的技术。

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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

19.7K 次观看.  Bionics Institute. 直接相邻的各个电极的视网膜刺激视网膜细胞结构可视化的技术。

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

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

表演和加工为淀粉样蛋白前脂肪垫活检

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

科研

医学

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

如何从MR图像测量皮质折叠:一个循序渐进的教程，以计算本地Gyrification指数

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-) and nitrate (NO3-) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

为含量测定一氧化氮生物活性分析技术

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

处理人减少乳房整形和乳房切除术的组织细胞培养

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

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.

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 ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

定量自体荧光眼底视网膜疾病的评价

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

一种用于兔视网膜下手术分步协议

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

痰液诱导和实验室处理方法

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

通过鱼眼研究糖尿病: 成人 tg (fli: EGFP) 斑马鱼视网膜血管的显微解剖、可视化和分析

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...