这项工作是支持的部分通过Regione威尼托（疗养Finalizzata n.292/2008找实习和找实习疗养Finalizzata 2009）的赠款。作者感谢利用人眼地球上的2009/2010年度的汇总数据医生三Griffoni。

1。 在原位角膜切除眼金球奖<ol><li>约15分钟后方可使用的空气层流柜的切换。清洁层流罩，使用70％异丙醇。戴上手术帽和口罩等个人防护服。擦洗手和前臂，用无菌毛巾擦干。戴无菌手套和礼服或袖子使用无菌技术。 </li><li>设立无菌区，放置无菌无菌仪器托盘。验证该仪器是无菌的。打开无菌工具包和翻转到无菌避免任何污染领域。 </li><li>随着瓶（眼罐），其中包含眼睛地球仪，使它们相邻的易用性不育领域的边缘定位所有不育材料和工具（ 见表2）。 </li><li>标签的瓶子，用事先准备好的保存/存储介质，如表3所示，并将随着我PVP的空气层流罩（碘聚乙烯吡咯烷酮），硫代硫酸钠和无菌生理盐水溶液表面上M PBS（磷酸盐缓冲液）。 </li><li>因为它们在冰箱中保存在4°C，使眼内组织和解决方案，以达到正常室温避免反复加热/冷却循环。保持溶液的盖和内侧的眼睛罐子朝上，下到各自的罐子。 </li><li>从眼睛罐子使用无菌镊子取出纱布和海绵。沉浸在无菌的I-PVP 0.5％，为2分钟，以净化眼地球全球。无菌纸盒的盖子放置在两钳，无菌领域之外。硫代硫酸钠0.1％，为1分钟使用另一无菌镊子全球转移。使用相同的镊子，瓶无菌生理盐水溶液（PBS）的全球转移，离开它，直到它运行。其他眼部全球从T重复同样的程序他同样的捐助。 </li><li>工作下方的空气层流罩，包裹地球，用无菌纱布（绷带）离开角膜和巩膜约5毫米，从发现角膜。在手保持足够的压力，眼球，可以简单地举行。整个手术过程中，组织应保持湿润。 </li><li>用剪刀沿钳去除所有结膜最终遗骸。保持上述操作与其他文书分开使用的工具。使用手术刀刀片执行从缘地区3-4毫米的巩膜切口，然后由360°延长切口，避免穿孔底层葡萄膜组织或造成任何角膜曲率变形。切四个大切口，留下四个避免玻璃体分泌小的差距。所有四个差距被切断，确保没有任何其他组织的去除。 </li><li>小巩膜斑块的存在可能妨碍切割，所以确保到排版lete巩膜切除术与显微剪刀。应执行此操作，而不损害脉络膜，视网膜和玻璃体。 </li><li>检查切口，以确保它是完整的。如果切口已执行正确的corneo的巩膜轮辋坚持只有在对应点与角膜睫状肌机构。 </li><li>无菌区的中心附近设置包裹眼全球。完成使用镊子，以保持固定巩膜缘切除手用来拉睫状体，脉络膜，巩膜的corneo按钮向下，远离角膜去除。 </li><li>轻轻分开从corneo巩膜按钮任何剩余的粘连。无论是拉的方式，可能会导致跨角膜紧张，也不会允许它回落下来到前房巩膜corneo轮辋。 </li><li>被选中的角膜内皮细胞密度，使用低渗的解决方案和检查细胞死亡率，使用1 TRYP蓝染色1分钟左右，细胞，然后计算在光学显微镜下测量细胞/毫米2。前房应该面对的盖子时后要面对的培养皿的底部。手动使用100X倍率网格的目标下，内皮细胞密度可算。接受移植的细胞计数在意大利最低是2200内皮细胞/毫米2。 </li><li>先前准备的角膜保存在RT（室温），如表3所示的存储介质使用角膜爪转移corneo巩膜轮辋。 </li><li>检查一个自然的晶状体后房型。如果巩膜准备，从全球的葡萄膜的所有残留物和玻璃体。小心拆开包装后的剩余部分返回其各自的眼睛罐子，使用无菌技术。重复使用新工具的其他全球的过程。修复角膜机智Ĥ角膜爪下的存储介质（机关文化）保持在31°C。切除的角膜， 如图1a所示，然后可以用于角膜移植或研究。 </li><li>到适当的垃圾箱不必要的材料和生物危险废物处置。一旦工作完成后，用70％异丙醇喷雾清洁工作区域。 </li><li> 28天之后，如果角膜保持所有的评价指标[如形态，细胞计数，透明度，厚度和微生物学测试 - 细菌和真菌的使用BACTEC 9240仪器（碧迪，米兰，意大利），它可以被运送到医院使用的传输介质，如表4所示。角膜应该被删除，并放置在储存，运输介质下空气层流柜。 </li><li>作为，在存储过程中，角膜的厚度比它通常用于移植所需大小，眼库使用的溶胀剂，以减少厚度。我们使用6％右旋糖酐T500的角膜回到其正常大小的传输介质。虽然可以使用4-8％的葡聚糖T500的6％，已对我们最好的。这使它容易对角膜移植外科医生，只要他们收到。 </li></ol> 2。剜眼地球后摘除眼角膜，视网膜<ol><li>切除术，角膜移植或研究选择其形态和角膜内皮细胞密度的基础上，并放置在存储或保存介质在31°C。眼全球剩余然后研究为进一步利用手术的目的，如巩膜隔离处理，视网膜等。 </li><li>视网膜切除术之前，确保眼全球，是完整的，只有角膜与巩膜的一部分被删除。如果凝胶状玻璃体分泌出它变得难以去除的视网膜组织。 </li><li>如果捐赠者是65岁以下，巩膜保存在无菌PBS手术后血清学检测目的。在这种情况下，巩膜不切断，只有玻璃体被删除，并保留整个巩膜。 </li><li>视网膜切除，开始有一小截的移动使用无菌镊子和剪刀的神经对视神经的巩膜角膜切口。持有的部分巩膜使用镊子和切直奔避免损害玻璃体切割部分的视神经神经剪刀。 </li><li>从巩膜脉络膜斑应分开，因为它们粘在一起。削减整个巩膜暴露脉络膜组织。巩膜一旦被完全切断，分离与剩余的玻璃体切割附近的视神经巩膜。 图2a所示，整个球会是一个与脉络膜覆盖的玻璃体可见。 </li><li>使用两个无菌镊子取出脉络膜层，轻轻采摘一双钳和与其他删除。另外，脉络膜层可以用剪刀去除，但最好应避免，因为它可能造成视网膜的损害。脉络膜层的部分删除，并削减它方便搬迁。 </li><li>一层透明的视网膜脉络膜层一旦被删除，将清楚地看到，在图2b所示。脉络膜层有一个室内布鲁赫的膜，这也可以被删除，但它是用肉眼很难辨别。 </li><li>同样，用两个镊子取出视网膜层， 如图2c所示。这是很容易隔离高的金额，如果切除视神经附近视网膜。所有的眼睛层现在可以看到图2d所示。 </li><li>一旦视网膜组织已被删除，它可以用于不同的目的，因此在随后的步骤和使用条件为保存相应可能会有所不同。例如，可放置在视网膜组织含有的RNA稳定试剂（如RNAlater）或裂解液（RNA或DNA提取），Dispase酶/胰蛋白酶（细胞的分离），固定的解决方案（前嵌入组织在石蜡或华侨城的试管免疫组化）。 </li><li>清除所有的工作区和清洁区，用70％异丙醇喷雾。 </li></ol> 3。代表结果在图1a所示的corneo巩膜轮辋是正确切除的角膜与巩膜RIM和内皮细胞的形态，这一般用于穿透性角膜移植角膜所需的金额。如果切除角膜前基质混浊的存在损坏，可以用于角膜内皮角膜移植术/后板层角膜移植术（血管内皮细胞间质的一部分）。反之，如果内皮细胞层被破坏，或如果角膜内皮细胞密度&lt;2200细胞/毫米2（阿莲标准），可用于角膜前板层角膜前基质的一部分上皮。如果内皮细胞计数小于所需的，可以用于角膜前板层角膜移植术或研究，而如果角膜完全损坏，它可以用于研究显示在图1B，3B和3C或丢弃。的损害可能是一个组织处理不当的原因，之前白内障手术，导致基质或后弹力层内皮疤痕，在病人的生命等人身伤害的发展，因此，它成为必要的检查每一个组织在光学显微镜下裂隙灯然后罚款疤痕/折叠/分队。角膜器官培养（约31°）通常用于在欧洲国家或低温条件下（约4°），通常在美国使用。如两个不同的条件下保存我们用机关文化，因为它有助于再生大坝中年，上皮细胞重新的振兴具有很高的验尸时内皮细胞检查，4周的贮存期，使规划的手术足够的窗口，提供足够的时间为微生物（细菌和真菌）和血清学检测等角膜可以在7天的传输介质保存完好。 巩膜可确保所有脉络膜斑块的温柔去除部分削减的一部分。玻璃体液体排出时，视神经，脉络膜或视网膜被大幅削减，如在图4所示。 RNA分析，如果切除的视网膜应保存在RNAlater尽快组织切除。一般1X1厘米的脉络膜和视网膜层切除后24小时保持在RNAlater在4°C下保存在-80°新鲜Eppendorf管（安全锁管，2毫升）C，直到使用或随干冰。视网膜组织应切除视神经附近的两钳距离虹膜或睫状肌的保证金，以避免与其他色素结构的污染，应作为RNA立即转移到维护容器变得不稳定，而检索的视网膜。 <img src="/files/ftp_upload/3765/3765fig1.jpg" alt="图1" /> 图1一个好的和质量差的角膜之间的比较：一）正确切除的角膜。通常是这样的角膜器官培养下保存4周。微生物（使用仪器BACTEC 9240）和形态（台盼蓝染色测定细胞死亡率与100X放大倍率的光学显微镜下观察细胞的细胞和）检查执行运送前到医院（ 图3a）; b）在弯曲的角膜，最终导致移植排斥反应，由于高弹力的褶皱和高内皮细胞损伤裂隙灯显微镜检查使用。这种角膜通常用于水库[地球或将被丢弃。 <img src="/files/ftp_upload/3765/3765fig2.jpg" alt="图2" /> 图2。使用，以确保准确的视网膜组织切除的方法。 一）解剖巩膜眼全球使用95毫米弯曲或钝端无菌剪刀和100毫米，0.70毫米的牙齿无菌镊子统治11X224毫米刀片。巩膜应一方持有切割钳对视神经直接用剪刀剪。脉络膜层现在可以轻松地看到，b）使用两个100毫米0.70毫米的牙齿无菌镊子统治11X2，脉络膜被删除它除了撕裂，从而揭示了底层透明的视网膜层; C）透明的视网膜层切除非常仔细地附近没有使用相同的镊子玻璃体液体排泄的视神经。镊子都应该保持每一侧的视网膜和组织应轻轻地被删除挑动Ğ视神经附近的移动，对角膜，视网膜组织获得良好的数额。可以切成不同大小的组织和分析实验所用; D）不同层次的眼睛角膜切除后，可以在这个数字来看。首先遵循不损害眼睛的其他部分由视网膜脉络膜的身体被删除。 <img src="/files/ftp_upload/3765/3765fig3.jpg" alt="图3" /> 图3 a）良好的内皮细胞密度（&gt; 2200个细胞/ mm 2）和形态（少多态性，变性低，没有营养不良和0％。100X放大倍率的光学显微镜下看细胞密度和形态的不同类型的比较。死亡率）在角膜，这是适合用于移植; 二）贫困内皮细胞密度（约1200 - 1400细胞/ mm 2）和形态（非常高的多态性和德劲台盼蓝染色后观察细胞一起约30-40％的总死亡率eration）; c）在血管内皮细胞部分损坏而从眼部全球检索角膜内皮表支队。这也证实了在该地区的染色观察细胞的死亡率，台盼蓝; D）弹力的褶皱，形成弯曲，营造跨角膜紧张，激烈等在切除时使用的仪器，由于角膜不当。该图显示了厚厚的发达国家之间的角膜光学区和外围发现的医源性褶皱。 <img src="/files/ftp_upload/3765/3765fig4.jpg" alt="图4" /> 图4。脉络膜和视网膜组织的损害，同时切除角膜最终导致玻璃液的排泄。难以消费质量好的组织无线玻璃体视网膜被牵连大地的污染。 <img src="/files/ftp_upload/3765/3765table1.jpg" alt="表1" /> 表1使用人眼地球在2009年和2010年的Fondazione Banca银行执行的活动范围。Degli Occhi（威尼斯，意大利）。组织约60％被用于移植，从而为科学和医学研究的眼地球的高数（40％左右）。 <img src="/files/ftp_upload/3765/3765fig1sup.jpg" alt="补充图1" /> 补充图1。解剖人类的眼睛5。人的眼睛的不同部位都在这个数字表示，按照组织的确切位置。

<ol>
	<li>Fay, A., Goldman, L., Ausiello, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diseases+of+the+visual+system.">Diseases of the visual system.</a> Cecil Medicine. , (2007).</li><li>Parekh, M., Megaw, R., Ray-Chaudhuri, A., Ahmad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patents+in+Limbal+Stem+Cell+Biology.">Patents in Limbal Stem Cell Biology.</a> Recent patents on Regenerative Medicine. 1, 207-212 (2011).</li><li>Pharm, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Eye:+The+physiology+of+human+perception.">The Eye: The physiology of human perception.</a> Britannica educational publishing. 36, 19-27 (2011).</li><li>Lang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ophthalmology: A pocket textbook atlas. , 115-117 .</li><li>Liesbeth, P. e. l. s. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+Culture:+The+method+of+choice+for+preservation+of+human+donor+corneas.">Organ Culture: The method of choice for preservation of human donor corneas.</a> Br. J. Ophthalmol. 81, 523-525 (1997).</li><li>Meyer, J. S., Howden, S. E., Wallace, K. A., Verhoeven, A. D., Wright, L. S., Capowski, E. E., Pinilla, I., Martin, J. M., Tian, S., Stewart, R., Pattnaik, B., Thomson, J., Gamm, 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=Optic+vesicle-like+structures+derived+from+human+pluripotent+stem+cells+facilitate+a+customized+approach+to+retinal+disease+treatment.">Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment.</a> Stem Cells. , (2011).</li><li>Trifunovic, D., Karali, M., Camposampiero, D., Ponzin, D., Banfi, S., Marigo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-resolution+RNA+expression+atlas+of+retinitis+pigmentosa+genes+in+human+and+mouse+retinas.">A high-resolution RNA expression atlas of retinitis pigmentosa genes in human and mouse retinas.</a> Invest. Ophthalmol. Vis. Sci. 49, 2330-2336 (2008).</li><li>Mora, P., Montanini, L., Ferrari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Retina. 30, 1555 (2010).</li><li>Claybon, A., Bishop, A. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+a+Mouse+Eye+for+a+Whole+Mount+of+the+Retinal+Pigment+Epithelium.">Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium.</a> J. Vis. Exp. (48), e2563 (2011).</li><li>Skeie, J. M., Tsang, S. H., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evisceration+of+Mouse+Vitreous+and+Retina+for+Proteomic+Analyses.">Evisceration of Mouse Vitreous and Retina for Proteomic Analyses.</a> J. Vis. Exp. (50), e2795 (2011).</li></ol>

作者没有根本的利害冲突。

内皮损伤或高弹力的褶皱数，如轻微的缺陷可能导致，而温度改变角膜移植失败或处理不当会损害视网膜组织的完整性至关重要，正确的切除和妥善保存角膜和视网膜组织。本文的目的是显示角膜和视网膜组织如何可以分离出最佳诱导移植或研究的目的，可能会危及其使用的损害或改建。所需的最小细胞密度（&gt; 2200个细胞/ mm 2），良好的形态，如细 ​​胞大小，结构间的边界，并具有?...

角膜和视网膜切除的材料。 <table border="1"><tbody><tr><td> 产品描述 </td><td> 尺寸 </td><td> 公司/机构 </td></tr><tr><td>守卫一次性手术刀</td><td>刀片尺寸15 </td><td>斯旺 - 莫顿</td></tr><tr><td>无菌绷带</td><td> 5厘米×5厘米，8个分层，5个</td><td> artsana </td></tr><tr><td>一次性无菌医疗毛巾</td><td> 35×50厘米</td><td> U.Jet </td></tr><tr><td>无菌剪刀</td><td>叶片24毫米/总长度。 95毫米弯曲，钝</td><td> e.janach </td></tr><tr><td>无菌镊子</td><td>不锈钢-100毫米11×2 0.70毫米的牙齿排除</td><td> e.janach </td></tr><tr><td>角膜爪 - 一次性医疗器械</td><td></td><td> NIIOS（Hippocratech） </td></tr&gt; <tr><td></td><td> 筹备工作 </td><td></td></tr><tr><td> PBS </td><td> [10倍] D / W（900毫升蒸馏水100毫升PBS） </td><td> Sigma-Aldrich公司</td></tr><tr><td>钠，硫代</td><td> 1克钠硫代1的PBS [1X]升</td><td> Sigma-Aldrich公司</td></tr><tr><td>我的PVP </td><td> 5克我PVP在1升D / W </td><td> Sigma-Aldrich公司</td></tr></tbody></table> 表2。表描述用于切除角膜和视网膜的公司，他们都收到的材料。 存储介质材料（2000毫升）。 <table border="1"><tbody><tr><td> 组件 </td><td> 供应商 </td><td> 目录编号 </td><td> 浓度 </td><td> 数量 </td></tr><tr><td>的MEM（1X），液体</td><td> Invitrogen公司</td><td> 32360-034 </td><td></td><td> 1900年毫升</td></tr><tr><td>丙酮酸钠</td><td> Invitrogen公司</td><td> 11360-039 </td><td> 1MM（10毫升/升） </td><td> 20毫升</td></tr><tr><td> L-谷氨酰胺</td><td> Invitrogen公司</td><td> 25030-032 </td><td> 2MM（10毫升/升） </td><td> 20毫升</td></tr><tr><td>抗生素/抗霉菌</td><td> Sigma-Aldrich公司</td><td> A5955-20ML </td><td> 10毫升/ L </td><td> 20毫升</td></tr><tr><td>新生牛血清</td><td> Invitrogen公司</td><td> 26010-74 </td><td> 2％（20毫升/升） </td><td> 40毫升</td></tr></tbody></table> 表3。存储介质材料。 存储介质的制备 添加的所有成分，使用如上在一个罐子里给出具体的浓度，并拌匀。过滤网使用蠕动泵的帮助下，孔径0.2微米的过滤器（Millipore公司，意大利米兰）。在瓶在室温下保存的介质。 传输介质材料（2000毫升）。 <table border="1"><tbody><tr><td> 组件 </td><td> 供应商 </td><td> 目录编号 </td><td> 浓度 </td><td> 数量 </td></tr><tr><td>的MEM（1X），液体</td><td> Invitrogen公司</td><td> 32360-034 </td><td></td><td> 1800毫升</td></tr><tr><td>丙酮酸钠</td><td> Invitrogen公司</td><td> 11360-039 </td><td> 1MM（10毫升/升） </td><td> 20毫升</td></tr><tr><td> L-谷氨酰胺</td><td> Invitrogen公司</td><td> 25030-032 </td><td> 2MM（10毫升/升） </td><td> 20毫升</td></tr><tr><td>新生牛血清</td><td> Invitrogen公司</td><td> 26010-74 </td><td> 2％（20毫升/升） </tD&gt; <td> 40毫升</td></tr><tr><td>葡聚糖T500 </td><td> pharmacosmos </td><td> 551005004007 </td><td> 6％（60克/升） </td><td> 120克/升</td></tr></tbody></table> 表4。传输介质材料。 传输介质的制备 添加葡聚糖6％〜1.5升的MEM，一夜之间离开它。加入其余成分在媒体和过滤0.2微米的过滤器（Millipore公司，意大利米兰），使用真空泵。在瓶在室温下保存的介质。

a simplified technique for in situ excision of cornea and evisceration of retinal tissue from human ocular globe

眼球摘除是从捐助者的尸体离开地球的休息不受干扰检索眼全球的过程。切除是指眼组织，尤其是角膜的检索，通过削减从眼部全球分开。剜是去除称为视网膜这里的内部器官的过程。眼全球包括角膜，巩膜，玻璃体，晶状体，虹膜，视网膜，脉络膜，肌肉等（增刊图1）。当病人患有角膜损伤，角膜需要删除与一个健康的，必须由keratoplastic手术移植。视网膜功能的遗传性疾病或缺陷，可能危及视力。可用于各种外科手术，如眼银行，人的角膜或巩膜移植和眼组织的研究人眼地球。然而，有对人体的角膜和视网膜切除提供的资料很少，可能是由于到Limited无障碍人体组织。大多数的研究，描述类似的程序进行的动物模型。研究的科学家们依靠正常解剖和保守的眼组织的可用性，以延长人类眼睛的发育，稳态和功能上的知识。我们收到眼地球的高金额，其中约40％（ 见表1），其中用于研究目的，我们是能够执行这些组织的大量实验，确定技术，消费和定期维护。 角膜是股骨头缺血性的组织，使在视网膜上的光传输，为此，要始终保持良好的透明度程度。在角膜缘地区，这是一个水库的干细胞，有利于上皮细胞的重建和限制结膜增生，维持角膜的​​透明度和清晰度。大小和日<html>角膜ickness是明确的目标的关键，无论是在他们的变化可能导致心烦意乱，视力不清。角膜由5层;一）上皮细胞，B）基质层，鲍曼的C），D）后弹力膜和e）血管内皮细胞。所有层应正常运作，以确保清晰的视野。4,5,6。脉络膜巩膜和视网膜之间的中间中山装，布鲁赫膜界内部，并负责在眼内的血流量。脉络膜也有助于调节温度和供应营养视网膜外层5,6。视网膜的神经组织，包括眼全球（增刊图1）的背面，由两部分组成层：photoreceptive的一部分，和非接受的一部分。视网膜接收的光线从角膜和晶状体，它转换成化学能最终传送到大脑视神经5,6帮助。 ove_content“&gt;本文的目的是提供解剖从人类眼部地球的角膜和视网膜组织的协议。避免交叉污染与癌旁组织和保持RNA的完整性等组织旨在为研究目的是不可或缺的基本重要性于（i）特征的眼组织的转录，（二）隔离干细胞再生医学工程，（三）评价组织从正常/受影响的学科之间的组织学差异，在本文中，我们描述的技术，我们目前使用的删除角膜，眼全球脉络膜和视网膜组织。在这里，我们为人类眼全球，角膜和视网膜组织的切除剥离了详细的协议。影随行，将有助于研究人员了解一个适当的检索技术珍贵的人体组织，这是很难找到定期。

Watch this Scientific Journal Video about A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe at JoVE.com

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

本文介绍了一个消费角膜简化技术和剔骨从全球人类尸体捐赠者的眼部视网膜组织。这里描述的技术将有助于消费的质量好，可用于移植，手术或研究目的，不损害其他组织的眼部全球的组织。

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 ...

a-simplified-technique-for-situ-excision-cornea-evisceration-retinal

Research

JoVE Journal

Medicine

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

20.7K 次观看.  Fondazione Banca Degli Occhi del Veneto O.N.L.U.S.. 本文介绍了一个消费角膜简化技术和剔骨从全球人类尸体捐赠者的眼部视网膜组织。这里描述的技术将有助于消费的质量好，可用于移植，手术或研究目的，不损害其他组织的眼部全球的组织。

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

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

技术收集人舌菌状（味道）乳头

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

科研

医学

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

一个生产缺血性损伤模型的简化技术

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

人类癫痫术后皮层组织的多电极阵列录音

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

隔离和小鼠主动脉切除;心血管疾病研究的一个多功能的技术

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

从整体组织和活检建立人类上皮Enteroids和Colonoids的

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

人工撕裂制剂对暴露于干燥后人类角膜上皮细胞代谢活动的影响

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

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 ...

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

视网膜器官诱导系统，用于从人类多能干细胞中提取3D视网膜组织

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...

Human Subcutaneous Adipose Tissue Sampling Using a Mini-Liposuction Technique

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate training and strict adherence to aseptic technique, subcutaneous adipose samples can be safely and efficiently obtained in a non-clinical setting by researchers. Following the administration of local anesthetic lateral to the umbilicus, a 14 G needle attached to a 5 or 10 mL syringe is inserted through the skin into the subcutaneous tissue. Under suction, the syringe is moved in a reciprocating, slicing motion to isolate fragments of adipose tissue. Withdrawing the plunger is enough to ensure that adipose tissue fragments are aspirated through the needle into the syringe. A single biopsy can collect about 200 mg of tissue. This biopsy technique is very safe for both participants and research staff. Following the biopsy, participants can resume most everyday activities, although they should avoid swimming and overly strenuous activities for 48 h to avoid excessive bleeding. Participants can safely undergo 2 biopsies within a single day, meaning that the technique can be applied in before-after acute intervention studies.

The manuscript and associated video demonstrate a percutaneous biopsy technique to obtain samples of subcutaneous adipose tissue from areas surrounding the umbilicus. This method is a low-risk and efficient way to investigate a range of parameters (e.g., gene or protein expression, enzyme activity, lipid content) within adipose tissue.

使用微型吸脂技术进行人体皮下脂肪组织取样

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate ...