1，建立无菌区的<ol><li>唐合适的服装，包括帽子和口罩。戴防护眼镜，在处理室时。 </li><li>准备处理区域。当使用一个加工室，维持风扇的连续操作，以创建在室内的正压。 </li><li>收集所有用品。检查到期日期，包装完整性，灭菌指标。 </li><li>洗手，不要非无菌手套。 </li><li>打开无菌包，以建立一个无菌区。当使用一个加工室，放在桌上的包和无菌条件下打开包裹。不利用外部1.5英寸的卷体的无菌区的一部分，因为这部分被认为是未消毒。 </li><li>使用无菌技术，开放的拖放无菌物品到无菌区，除非预先做好的包已经组成部分。 </li><li>移动显微镜，使其上面的无菌区域，打开显微镜的光源，并调整目镜是必要的。 </li><li> OBTAIN角膜组织被剥离（在保存介质），并置于旁边的制备无菌处理区域。松开观察室的盖子，让它停留在观察室的基础，没有搞线程的顶部。丢弃非无菌手套。 </li><li>打开一对无菌手套和无菌袍毛巾。 </li><li>进行手术擦洗。穿上无菌衣，再用无菌手套。 </li><li>打开仪器箱，让所有用品都可以访问。打开水不渗透的悬垂性，以创建工作区。 </li><li>附针5毫升注射器。使用止血钳，通过从斜角放置一个弯曲1毫米制备地下​​30针，并从基体的另一个弯曲1毫米（以形成一"S"形状）。 </li><li>通过将柱塞插入注射器和手指拧紧柱塞组装玻璃注射器的台盼蓝染色。连接套管。 </li><li>连接套管的平衡盐溶液（BSS）的瓶子。 </li><li>从真空块分开的环钻打孔并预留。视显微镜的视野内居中真空块。 </li><li>通过调整显微镜的设置带来的真空块成为关注焦点。 </li></ol> 2，程序供体组织准备<ol><li>观察室使用方形铝箔作为阻隔的掀盖。用另一只手使用镊子，轻轻抓住了角膜巩膜轮辋去除角膜。更换观察室的盖子。 </li><li>完全踩下吸入注射器中，然后将角膜上皮侧朝下的真空块上，并确保它的中心。 （ 图1） </li><li>在使用导柱真空块轻轻下休息环。慢慢松开吸入注射器的活塞从事真空。拆下座椅环，备用。 （ 图2）。 </li><li>执行部分环钻术，以高分通过后弹力膜。 </li><li>调整显微镜变焦和聚焦，使该组织及可视化。 </li><li&gt;倾斜真空块，并用无菌棉签矛放置在角膜缘以除去多余的介质。 （ 图3） </li><li>将足够的染色在角膜缘染色的进球后弹力膜的边缘。允许污渍保留在血管内皮细胞为60-90秒。 （ 图4） </li><li>移除污点: <ol><li>倾斜真空块倒污垢角膜和上纱布垫。 </li><li>用手术长矛从角膜缘区域去除多余的污渍，注意不要接触内皮细胞。 （ 图5） </li></ol></li><li>用几滴BSS的冲洗染色液角膜，无菌棉签长矛，去除多余的BSS。 （ 图6） </li><li>放置在巩膜缘1-2滴BSS以防止组织干燥。根据需要重复整个过程。 </li><li>执行后弹力膜部分剥离: <ol><li>开始当管路连接s到真空块，使用开放镊子，将一个尖的得分标记的边缘，并轻轻分开后弹力膜和内皮远离基质。 （ 图7）。 </li><li>分开后弹力膜和内皮细胞不超过1毫米的得分边缘和继续剥离360°左右。 （ 图8）。 </li><li>注意任何微小的泪水位置放置点与皮肤标记真空块。 注意:如果该环钻没有通过后弹力膜完全渗透，使用所制备的地下30针的斜面边缘，完成通过后弹力膜在有需要时，随后染色的第二应用在需要的分数。如果后弹力膜的粘附性是阻碍局部解剖，根据需要取出trephinated禁区外的内皮细胞。 </li></ol></li><li>使用镊子去除重叠的得分标记任何内皮标记。 </li> &lt;LI&gt;考虑了先前标记的微撕裂的位置和旋转真空块，使最大的是位于6点钟位置。这将成为护翼的铰链。 </li><li>使用镊子，抓住后弹力膜在12点。轻轻剥离朝铰链分开后弹力膜的基质。通过停止从得分标记分离2毫米创建的铰链。 注:瓣的周缘开始分离过程中进行滚动。 （ 图9）。 </li><li>轻轻地将瓣放回原处的基质。用BSS和拭子长矛unscroll的内皮细胞，并将其返回到其原始位置。 （ 图10A-C） </li><li>从后弹力膜和基质之间用无菌棉签长矛删除这样的襟翼保持在适当的位置残留的液体。 （ 图11） </li><li>用消毒棉签长矛来吸收多余的水分从靠近转轴处的巩膜。使用皮肤标记来绘制Ñ​​慈姑上巩膜缘指向铰合部的中心，用无菌拭子矛除去任何过量的墨水。 （ 图12） </li><li>通过按压连接到真空块状注射器的活塞释放真空。而真空被切断，使用镊子从真空块除去角膜。 </li><li>观察室使用方形铝箔作为阻隔的掀盖。放置在角膜的观察室，内皮面朝上，并取代观察室的盖子。 </li><li>重复上面的操作步骤与队友角膜（如适用）。用纱布处理所述第二组织前清洁真空块。 </li><li>拧紧的观看室盖（次），并从处理区域除去组织。 </li><li>通过根据生物危害废物管理计划处置项目分解无菌区。脱去污染的手套和礼服。洗手，不要非无菌手套。 </li><li>进行后切裂隙灯评价和角膜的镜面反射镜（次）并记录结果。 </li></ol>

<ol>
	<li>Fernandez, M., Afshari, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Keratoplasty:+From+DLEK+to+DMEK.">Endothelial Keratoplasty: From DLEK to DMEK.</a> Middle East African Journal of Ophthalmology. 17 (1), 5-8 (2010).</li><li>Kruse, F., 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+Stepwise+Approach+to+Donor+Preparation+and+Insertion+Increases+Safety+and+Outcome+of+Descemet+Membrane+Endothelial+Keratoplasty.">A Stepwise Approach to Donor Preparation and Insertion Increases Safety and Outcome of Descemet Membrane Endothelial Keratoplasty.</a> Cornea. 30 (5), 580-587 (2011).</li><li>Dapena, I., Moutsouris, K., Droutsas, K., Ham, L., van Dijk, K., Melles, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+'No-Touch'+Technique+for+Descemet+Membrane+Endothelial+Keratoplasty.">Standardized 'No-Touch' Technique for Descemet Membrane Endothelial Keratoplasty.</a> Archives of Ophthalmology. 129, 88-94 (2011).</li><li>Tourtas, T., Laaser, K., Bachmann, B., Cursiefen, C., Kruse, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet+Membrane+Endothelial+Keratoplasty+versus+Descemet+Stripping+Automated+Endothelial+Keratoplasty.">Descemet Membrane Endothelial Keratoplasty versus Descemet Stripping Automated Endothelial Keratoplasty.</a> American Journal of Ophthalmology. 153 (6), 1082-1090 (2012).</li><li>Guerra, F., Anshu, A., Price, M., Price, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Keratoplasty:+fellow+eyes+comparison+of+Descemet+stripping+automated+endothelial+keratoplasty+and+Descemet+membrane+endothelial+keratoplasty.">Endothelial Keratoplasty: fellow eyes comparison of Descemet stripping automated endothelial keratoplasty and Descemet membrane endothelial keratoplasty.</a> Cornea. 30 (12), 1382-1386 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2012 Eye Banking Statistical Report: Surgical Use and Indications for Corneal Transplant Statistical Report Analysis. , (2012).</li><li>Price, M., Price, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet's+stripping+endothelial+keratoplasty.">Descemet's stripping endothelial keratoplasty.</a> Current Opinion Ophthalmology. 18 (4), 290-294 (2007).</li><li>Woodward, M., Titus, M., Mavin, K., Shtein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Donor+Tissue+Preparation+for+Endothelial+Keratoplasty.">Corneal Donor Tissue Preparation for Endothelial Keratoplasty.</a> Journal of Visualized Experiments. 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T., Birbal, R., Ham, L., van der Wees, J., Melles, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+tissue+preparation+for+Descemet+membrane+endothelial+keratoplasty.">Donor tissue preparation for Descemet membrane endothelial keratoplasty.</a> Journal of Cataract Refractive Surgery. 34 (9), 1578-1583 (2008).</li><li>Kruse, F. E., Laaser, K., Cursiefen, C., Heindl, L. M., Schlotzer-Schrehardt, U., Riss, S., Bachmann, B. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stepwise+approach+to+donor+preparation+and+insertion+increases+safety+and+outcome+of+Descemet+membrane+endothelial+keratoplasty.">A stepwise approach to donor preparation and insertion increases safety and outcome of Descemet membrane endothelial keratoplasty.</a> Cornea. 30 (5), 580-587 (2011).</li><li>Rudolph, M., Kaaser, L., Bachmann, B., Cursiefen, C., Epstein, D., Kruse, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+higher-order+aberrations+after+Descemet&#8217;s+membrane+endothelial+keratoplasty.">Corneal higher-order aberrations after Descemet&#8217;s membrane endothelial keratoplasty.</a> Ophthalmology. 119 (3), 528-535 (2012).</li><li>Price, F., Price, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+endothelial+keratoplasty.">Evolution of endothelial keratoplasty.</a> Cornea. 32, 28-32 (2013).</li><li>Dapena, I., Dapena, L., Dirisamer, M., Ham, L., Melles, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+acuity+and+endothelial+cell+density+following+Descemet+membrane+endothelial+keratoplasty+(DMEK).">Visual acuity and endothelial cell density following Descemet membrane endothelial keratoplasty (DMEK).</a> Arch Soc Esp Oftalmol. 86 (12), 395-401 (2011).</li></ol>

哈桑Tausif和Navasuja Chandrasekaran博士什么都没有透露。博士。勉，Shtein和伍德沃德是医疗董事密歇根州眼库。 Mavin先生，提多先生和Johnson女士是中西部眼库的员工。

角膜内皮细胞层起着通过脱水维持角膜透明度的关键作用。内皮细胞的疾病都可以导致显著视力障碍。 DMEK旨在与供体移植物，包括后弹力层和内皮细胞层取代病变血管内皮细胞。供体组织编制了一个完整的DM和足够的内皮细胞计数是移植组织的生存至关重要。糖尿病供体组织筹备过程中撕裂可导致组织损失和捐助组织的浪费。 DMEK的广泛应用已经在准备移植的组织和掌握复杂的手术技术所面临的挑战限制。凭借改进的仪器和眼睛准备银行预切组织的可用性，此过程有可能成为标准治疗角膜内皮疾病的潜力。 目前的技术供体组织编制包括使用空气气泡的到DM从基质中分离，以促进从供体角膜10的切除移植物组织。 DMEK接枝制备的SCUBA（浸没角膜使用背景远）技术涉及收获Optisol或平衡盐溶液（BSS）下的供体组织，减少了表面张力和撕裂11的机会。然而，在DMEK接枝制备的另一种技术是离开基质的外周缘产生的接枝是更容易的手柄和刀片12。供体组织，也可以用自定义的环钻准备离开完整的DM铰链同时抬起移植与BSS 13。 相比于此前公布的技术到76％，这可能反映了这些名头50组织正在准备两个眼库技术员14,15的总体成功率与我们的技术DMEK移植的准备是有限的。然而，结果却提高在组织前的第二半缩减。此外，我们的技术不需要专门的仪器，使DMEK方便和更具成本效益。视力恢复可以更快，更好地DMEK由于缺乏间质界面5。研究也表明在角膜上比DSEK或PKP 16的后表面上较少的高阶像差。移植物排斥的发生率是显著下与DMEK比其他角膜移植术的技术17。内皮细胞密度下DMEK媲美角膜后弹力层剥除自动内皮角膜移植术（DSAEK）18。 DMEK提供了一个微创手术保留眼球的结构完整性，减少术后散光，伤口渗漏，术后青光眼与长期局部使用类固醇的风险。 总之，DMEK允许替换患病内皮而不除去接受者角膜基质或附加组织。手术方法是在部分具有挑战性，由于供体组织编制的可重复性。眼库筹备DMEK可以允许DMEK类似于以前角膜内皮移植术的技术改进适应。

角膜内皮移植术（EK）已经迅速取代于2012年6穿透性角膜移植术（PKP）作为首选治疗内皮疾病占美国移植手术的50％以上的步骤。PKP是由长期的视觉康复高度散光的限制和屈光不稳定，全球诚信和缝线相关并发症的损失。初代EK程序，包括深板层内皮角膜（DLEK）参与角膜基质偏厚供体组织移植的人工清扫。这些程序成功地移植供体血管内皮组织，同时保持一个完整的角膜表面，但在技术上具有挑战性，由于不规则角膜板层接口，1导致有限的最佳矫正视力。选择目前EK程序后弹力层剥除内皮角膜移植术（DSEK），提供角膜CLAR性与更快的恢复视力，减少术后屈光度，散光，并降低外伤伤口裂开的风险，同时保持角膜表面7。此过程涉及去除病变后弹力膜和内皮细胞和置换部分的厚度供体角膜组织，包括一个新的内皮细胞层。供体角膜的解剖与角膜板层刀来准备后组织被移植8。由眼银行的供体组织的制备一直在快速增长，可以说，这个程序的成功。 2005年至2012年的近16倍的DSEK程序在美国对应目前正在编写的眼睛6银行组织的近一半。 虽然DSEK可以提高视力，以20/40以上，多数患者没有达到正常视力，由于间质混浊的界面和增强的光从另外捐助基质组织7的散落一地。该EK手术技术已经发展到后弹力膜内皮角膜移植术（DMEK）涉及的只给予体后弹力膜（DM）和血管内皮细胞的移植。本程序有谁达到正常视力，视力恢复快，而血管内皮抑制9的风险较低的患者比例较高。然而，准备供体组织是更具挑战性的是由于移植物更薄的尺寸。

<table><tbody><tr><td>VisionBlue</td><td>D.O.R.C., Zuiland, The Netherlands</td><td>NA</td><td></td></tr><tr><td>Barron vacuum punch</td><td>Katena, Denville, New Jersey</td><td>K20-2112</td><td></td></tr></tbody></table>

corneal donor tissue preparation for descemet's membrane endothelial keratoplasty

后弹力膜内皮角膜移植术（DMEK）是角膜移植，其中只有一个单细胞层，角膜内皮细胞，连同其基底膜（Descemet膜）被引入到接收方的后基质3的形式。与角膜后弹力层剥除自动内皮角膜移植术（DSAEK），其中更多的捐助基质的介绍，不自然基质对基质的界面创建。其结果，角膜的天然解剖学保留尽可能允许改善恢复时间和视力4。角膜内皮移植术（EK）是首选的治疗内皮功能障碍的过程。 EK的优点包括快速恢复视力，保护眼部的完整性和最小的屈光变化，由于采用了小型，外围切口1。 DSAEK利用制备偏厚的基质和内皮细胞供体组织。快速SUCCESS和利用这个程序可以归因于眼睛银行可用性制备预切割的组织。眼库准备供体组织的好处包括:消除需要专门的设备备份供体组织的情况下，组织穿孔制备过程中的手术室和可用性。此外，高体积制备由眼库的技术人员供体组织可以提供供体组织的改进的质量。 DSAEK可能限制最佳矫正视力，由于创作的供体和受体角膜之间的间质界面。消除这个接口仅为捐助后弹力膜和内皮DMEK移植可以改善视觉效果和EK 5后减少并发症的发生。类似DSAEK，长期成功和验收DMEK的是依赖于易于预切割的可用性，眼库制备供体组织。在这里，我们提出了一个阶梯式的供体组织编写这可能会降低一些巴里ERS眼银行面临提供DMEK移植。

从基质适当分离，DM和内皮正在准备预切组织的DMEK必不可少的。五十捐献角膜为65±6岁（范围55-75），死亡2,616±321个/ mm 2至5.9±2.3天（范围1-10）和内皮细胞密度的处理时间（ECD）（平均年龄范围2,049-3,247）被用于由两个眼库的技术人员来评估手术制备技术的可行性和可重复性。成功率为72％，前25例，上升至80％，下半年76％的成功率。点二列相关（RPB）来确定，术前组织因子的相关性。捐赠者的年龄（RPB = 0.18），死亡的处理时间（RPB = 0.07）和幼儿（RPB = -0.11），并没有相关的成功筹备组织。后制备的2676±284（范围2,041-3,205）ECD组（n = 37）为不显著不同于预先制备ECD（p值= 0.72）。不成功FUL结果导致从DM泪（10％）和重度细胞损失（14％）。这些案件也难以重新安装组织和DM边缘不完整的染色有关。 患者的治疗效果的眼部银行评估为临床成功和DMEK相关的不良事件必要的数据，可能有助于提高手术技术的适应练习和利用眼库准备组织。九预减DMEK组织提供了五个医生为9例。一个案例中有一个错位1天，1周，而不需要rebubbling。视力在第1天不等20/400光感和1周提高到一百〇三分之二十零（范围20/30〜20/200）。 <img alt="图1" src="/files/ftp_upload/51919/51919fig1highres.jpg" /> 图1:组织安装。角膜组织安装并居中的环钻块。 SS ="jove_content"FO:保together.within页="总是"&gt; <img alt="图2" src="/files/ftp_upload/51919/51919fig2highres.jpg" /> 图2:组织环钻术。真空环安装和角膜组织部分trephined包括后弹力膜。 <img alt="图3" src="/files/ftp_upload/51919/51919fig3highres.jpg" /> 图3:存储介质删除。染色申请前多余的存储介质被删除。 <img alt="图4" src="/files/ftp_upload/51919/51919fig4highres.jpg" /> 图4台盼蓝的应用。应用台盼蓝染色的后弹力膜表面带馅井的60-90秒。 <html> _upload / 51919 / 51919fig5highres.jpg"/&gt; 图5污渍清除，冲洗台盼蓝与BSS。 <img alt="图6" src="/files/ftp_upload/51919/51919fig6highres.jpg" /> 图6污渍清除。完全去除污渍。 <img alt="图7" src="/files/ftp_upload/51919/51919fig7highres.jpg" /> 图7后弹力膜剥离。绑钳用于执行360°局部解剖。 <img alt="图8" src="/files/ftp_upload/51919/51919fig8highres.jpg" /> 图8:后弹力膜剥离，开始剥离后弹力膜。 <img alt="&lt;/ HTML"><html> "图9"SRC ="/文件/ ftp_upload / 51919 / 51919fig9highres.jpg"/&gt; 图9。后弹力膜剥离，继续剥离后弹力膜。 <img alt="图10" src="/files/ftp_upload/51919/51919fig10highres.jpg" /> 图10重新定位后弹力膜。 （一）返回后弹力膜基质床（B）BSS应用后弹力膜扬起帮助和后弹力膜（三）完善扬起到原来的位置。 <img alt="图11" src="/files/ftp_upload/51919/51919fig11highres.jpg" /> 图11。平衡盐溶液去除。从挡板下面去除多余的BSS的。 2"SRC ="/文件/ ftp_upload / 51919 / 51919fig12highres.jpg"/&gt; 图12铰大关。铰链与皮肤标记鉴定。

Watch this Scientific Journal Video about Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty at JoVE.com

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

角膜内皮移植术是一种外科技术不断发展的进步导致的移植物薄，每个继承1移植。在这里，我们提出了一个技术控制的手动组织剥离，使血管内皮和后弹力捐助角巩膜按键膜移植2的安全和可重复的分离。

角膜供体组织准备后弹力层内皮角膜移植术

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, ...

corneal-donor-tissue-preparation-for-descemet-s-membrane-endothelial

Universidad San Sebastian

Research

JoVE Journal

Medicine

15.2K 次观看.  University of Michigan. 角膜内皮移植术是一种外科技术不断发展的进步导致的移植物薄，每个继承1移植。在这里，我们提出了一个技术控制的手动组织剥离，使血管内皮和后弹力捐助角巩膜按键膜移植2的安全和可重复的分离。 

视频: Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

腺病毒基因治疗对糖尿病性角膜病变:对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

科研

发育生物学

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly.
The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision4,5,6. The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina5,6. The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve5,6.
The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.

简化技术<em&gt;原位</em&gt;切除角膜和视网膜组织剜人眼地球

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

医学

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

供体角膜内皮角膜移植术的组织准备

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2.
However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types.

角膜组织工程: 人角膜间质神经相互作用的体外模型

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Microstereolit​​hography和静电结合，以产生膜配备有利基的角膜再生

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

生物工程

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

小鼠角膜移植模型，研究实体器官移植的最常见形式

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

An &#8220;All-laser&#8221; Endothelial Transplant

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an intended depth, and a near infrared diode laser to weld the corneal tissue. The proposed technique enables to reach the three main goals in endothelial keratoplasty: a precise control in the thickness of the donor tissue; its easy insertion in the recipient bed and a reduced risk of donor lenticule dislocation. The donor cornea thickness is measured in the surgery room with optical coherence tomography (OCT), in order to correctly design the donor tissue dimensions. A femtosecond laser is used to cut the donor cornea. The recipient eye is prepared by manual stripping of the descemetic membrane. The donor endothelium is inserted into a Busin-injector, the peripheral inner side is stained with a proper chromophore (a water solution of Indocyanine Green) and then it is pulled in the anterior chamber. The transplanted tissue is placed in the final and correct location and then diode laser welding is induced from outside the eyeball. The procedure has been performed on more than 15 patients evidencing an improvement in surgery performances, with a good recovery of visual acuity and a reduced donor lenticule dislocation event.

An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

"全激光"内皮移植

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

生物材料膜上人与羊角膜内皮细胞层的生长

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

We describe use of full-field optical coherence microscopy as a method for high quality assessment of corneal donor stroma. This protocol can be used to identify features indicative of health or disease, and is aimed at improving the screening and selection of donor tissues, and hence the results of keratoplasty.

全场光学相干显微镜，用于角膜移植物基质特征的组织学样分析

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting ...

免疫与感染

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

角膜供体组织准备后弹力层内皮角膜移植术

摘要

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此视频中的章节

腺病毒基因治疗对糖尿病性角膜病变:对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

简化技术

供体角膜内皮角膜移植术的组织准备

角膜组织工程: 人角膜间质神经相互作用的体外模型

Microstereolithography和静电结合，以产生膜配备有利基的角膜再生

小鼠角膜移植模型，研究实体器官移植的最常见形式

"全激光"内皮移植

生物材料膜上人与羊角膜内皮细胞层的生长

全场光学相干显微镜，用于角膜移植物基质特征的组织学样分析

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

角膜供体组织准备后弹力层内皮角膜移植术

摘要

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腺病毒基因治疗对糖尿病性角膜病变:对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

简化技术

供体角膜内皮角膜移植术的组织准备

角膜组织工程: 人角膜间质神经相互作用的体外模型

Microstereolit​​hography和静电结合，以产生膜配备有利基的角膜再生

小鼠角膜移植模型，研究实体器官移植的最常见形式

"全激光"内皮移植

生物材料膜上人与羊角膜内皮细胞层的生长

全场光学相干显微镜，用于角膜移植物基质特征的组织学样分析

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

Microstereolithography和静电结合，以产生膜配备有利基的角膜再生