在2008年和2010（BVS），BONFOR / Gerok奖学金O型137.0015（BVS），BONFOR / Gerok奖学金O型137.0019（FT），德意志研究联合会/ DFG（BVS）STA 1135 / 2-1，中国支持吕迪格基金会资助留学基金委号2008627116（ZL），并通过Geuder AG公司，海德堡（图2）的无限制授权。 H. Skottman实验室的成员，芬兰坦佩雷大学表示衷心的感谢提供派生图2所示的RPE胚胎。

在眼科研究动物处理伦理:我们获得了来自医学院，波恩大学伦理委员会的批准，并坚持由研究协会在视觉与眼科（ARVO）规定的指导方针。此外，所有的程序是由北莱茵 - 威斯特伐利亚州监管当局的批准。动物室内举行一个专门的工厂在空调房间与18之间的温度 - 20°C，暴露在日光经常在与自由获取食物和水的标准化单独的笼子。 注意:为了确保动物手术亲和力，动物保健评分表之后，其中包括以下明确动物排除标准:20％的重量损失相比，重量入院;动物的表观发绀;动物不寒而栗，有绞痛或协调不能移动; 。共济失调/感觉异常， 如瘫痪;冷漠;极端切割汽车（皮肤伤口，断肢）。 &lt;/ P&gt; 1.器械灭菌<ol><li>将可重复使用器械超声波浴。 </li><li>加入500毫升蒸馏水和2ml仪器消毒剂。 </li><li>使用扫描功能15分钟的清洁工具。 </li><li>从超声波浴中取出仪器，用蒸馏水5分钟彻底冲洗。 </li><li>插入釜仪器和使用标准程序（器具的消毒下121°C 20分钟）。 </li></ol> 2.仪器准备<ol><li>建立和保持一个无菌区，在一个封闭的房间工作，戴着医用磨砂，口罩和发罩。消毒前，戴无菌手术手套的手。有关详细办法见28。 </li><li>将消毒器械上无菌的悬垂性。 </li><li>放置1ml注射器填充有附着到27克的针注射，将10毫升注射器余额盐溶液（BSS）40毫克曲安西龙，和5ml注射器ö悬垂˚F润滑剂。 </li><li>将3-0丝线，7-0薇乔，眼棒（停止结膜/巩膜出血），捻线机海绵纱布，伤口缝合带（注视玻切尖管），以及吊灯endoillumination纤维的悬垂线。 </li><li>解开地下25吊灯endoilluminator并连接到使用无菌技术，光机（见步骤2.1）。玻璃体切除术组，包括高速vitrector和卡带丘连接到使用无菌技术玻璃体切割机（见步骤2.1）。 </li><li>打开500毫升BSS瓶，并根据制造商的说明连接解决方​​案，文丘里录像带。 </li></ol> 3.动物的麻醉和定位的制备<ol><li>称取动物以确保准确的用药剂量。 </li><li>准备用1注射器含0.35毫克/千克氯胺酮和0.25毫克/千克美托咪定为开始27克肌肉注射针（IM）麻醉。翻转注射器混合。 </li><li>准备2注射器1/3Ť他的剂量以维持运转时的麻醉。 </li><li>制备含有20毫升5％葡萄糖溶液和18g针皮下注射静脉输液替代注射器。 </li><li>给前玻璃体切除瞳孔扩张散瞳眼的3×1滴一滴。 </li><li>封面兔毛毯麻醉注射前的平静，在后肢（臀中肌）和周围按摩注射部位注射，持续30秒。 注:IM麻醉的第一枪大约持续1 - 2小时。根据兔子的大小，药物耐受性，脂肪层，压力和体温。麻醉消失的第一个迹象是眼球震颤（必须由医生进行监测），随后的注射去年30 - 45分钟。 </li><li>确认适当的麻醉，通过验证催眠，反射降低，镇痛和动物的肌肉松弛。 </li><li>在给颈部皮肤褶皱皮下注射（PT。3.3），一旦兔子是无意识的。 </li><li>添加methylcellulose润滑剂每5 - 在操作眼10分钟，在非操作眼添加润滑剂和磁带保护盖。 </li><li>放置在与盖披手术台被覆兔如在最佳位置上的棉毡（鼻通过毛毯的模具中稍微升高，因此它是与眼表面能级）手术显微镜下。眼睛对准垂直于显微镜物镜。 </li><li>用直肠体温计（常温39±1℃）29确保适当的身体核心温度。 </li><li>用剪刀（在刀片一些药膏）剪切睫毛，以减少术后感染。 </li><li>消毒用2-眼 - 3滴为0.1g / ml的聚维酮碘的局部为1分钟，并用无菌的BSS冲洗。 </li><li>盖眼在中间的眼睛预切割开无菌的悬垂性，然后用（粘）手术切口悬垂12×17厘米盖。 </li></ol> 4.玻璃体切割术<ol><li> Proptose安全眼用INV 3-0丝线erted卡尺，进行结膜peritomy。 <ol><li>切割用剪刀vannas接近角膜缘，但远远不够从血管（约1毫米的距离）结膜。 </li><li>通过创建一个"T切割"解剖结膜。一是扩大平行剪的peritomy到了limbus然后垂直切开结膜在"T"形式约6 - 6毫米。小心地将结膜直言分开。 </li></ol></li><li>在对右眼/ OD（4点钟左眼/ OS）8时许迈向视神经小心插入刀片的尖端方向进行使用23政microvitreoretinal（MVR）刀片巩膜。慢慢缩回在同一方向的叶片，并避免扩大巩膜。 </li><li>插入，并使用缝合定制侧端口浸液套管27 7-0缝合丝线和设置眼内压（IOP）在24毫米汞柱。 </li><li>凌晨2点OD（10点进行了25微克平头套管针巩膜;时钟操作系统）与步骤4.2。 </li><li>插入地下25吊灯光线进入平头套管针，用胶带固定，并在约 30％的打开光源。 </li><li>如果需要的话，用＃20解剖刀更好眼可视除去水肿角膜上皮。 </li><li>执行一个类似在巩膜上OD 10点（凌晨2时OS），（预）处的U形各地巩膜缝线7-0不绑结，并插入玻璃体切割器顶端步骤4.2。 </li><li>玻璃体切割开始围绕30入境口岸，然后继续在视盘和切割玻璃体切成小块在最大使用高速vitrector的fibrae medullares。 2000 - 3000切/分钟，在最大吸气。 200毫米汞柱使用玻璃体切割机的所述参数设置（ 见表1） </li><li>通过保持高速vitrector在后极一个执行通过分离从视网膜玻璃体一个玻璃体后脱离（PVD）次（如果可行的话轻轻地）高级光盘，而只有在最大吸31。 200毫米汞柱无切削。 </li><li>注入约 50微升（20毫克）曲安西龙或稀释的荧光素（ 约 0.1毫克/毫升）玻璃体内到玻璃体中可视化和促进（近总）除去在后极和midperiphery浮动玻璃体。避免在镜头下穿越而过。如果气体填充需要建议压痕由（熟练）助理刮胡子周边玻璃体。 </li><li>添加20单位/毫升肝素和0.5毫克肾上腺素0.001毫克/毫升终浓度到BSS输注溶液中平行或步骤4.10之后。 注:由于肝素/肾上腺素不注射人工它们的影响取决于输液流速推迟。 </li></ol> 5.装载射手注:本文所描述的工作不赫尔辛基宣言的原则下回落;它不涉及人类患者。在这里，站ndard RPE细胞从胎儿人眼，培养和分化的未涂覆10微米厚的聚酯（PET），根据我们的先前公布的协议14插入隔离。从波恩大学的伦理委员会，获得与人类胎儿的物质工作的权限。可替代地，HES-RPE是从Skottman实验室（手稿中制备），在那里他们被根据由Vaajasaari 等人所述的技术中培养运32。用于这些细胞的一个权限已从R.科赫研究所，柏林，德国获得。 <ol><li>准备与眼科的BSS植入的3倍之前，冲洗细胞培养。 </li><li>填用10ml眼科级BSS标准细胞培养皿（100×20毫米）。 </li><li>添加细胞培养插管进入BSS和在光学显微镜下居中菜。 </li><li>切出一个2.4×1.1毫米植入物用钝，椭圆形，定做针获得平，豆形底具有两个长边和两个圆边。 </li><li>通过与BSS第二端口轻轻淹没针冲洗出植入到BSS填充定制装载站（ 图1）。 </li><li>任选切割植入物（&lt;0.5mm）的一个圆形端部，只是以获得第三边缘。 </li><li>确保植入物是在通过确保单层是细胞载体上上行的正确方向。要改变定位小心使用两个手术刀。 </li><li>轻轻地完全推植入利用持针器，直到所有的植入物的固定尖端的内部射手仪器。柱塞应保持缩回。 </li><li>请BSS下装载站的"装"射击枪，直到植入的时刻。 </li></ol> 6.植入<ol><li>引桥连接到气密注射器可扩展41克视网膜下注射针神经视网膜（确保所有气泡已被紧急疏散从管！）。 </li><li>注入BSS（钙和镁/ CM）视网膜下，从而创造大约2的疱视网膜脱离（BRD） - 3-盘直径（DD）。每眼两个BRD可以安全地得到提升。 </li><li>放大性视网膜至1.5毫米，垂直23政VR-剪刀。视网膜下空间是现在用于植入或进一步操纵访问。 </li><li>扩展巩膜（精确）用1.4毫米的切口刀至20g的方法。 </li><li>尝试使用20G的虚拟射手通过巩膜传球，因为需要确保加载射手平稳，但舒适的过渡放大。 </li><li>通过巩膜与加载的射手27最好通过在24毫米汞柱。 </li><li>办法性视网膜边缘并从视网膜前位置视网膜下弹出植入物。 </li><li>调整种植与半封闭的23政剪刀，镊子或41克的针，以确保它是retina-下定位以及从性视网膜合理了。 </li></ol> 7。结束操作<ol><li>卸下地下25吊灯和输液插管。 </li><li>缝合巩膜所有。 </li><li>由8点巩膜（前最后缝合巩膜）注入25微升（10毫克），曲安西龙。 </li><li>检查/触诊调节眼压，并通过为30G针头/注射器注入BSS，如果需要的话。 </li><li>缝合结膜与7-0薇乔。 </li><li>删除proptosing 3-0丝线吊带慢（避免眼眶深部静脉丛！）。 </li><li>下盖添加地塞米松/抗生素软膏。 </li><li> 1小时的位置覆盖有与操作眼朝上（W / O型气体），或下（空气/气体）毯。 </li><li>直到它重新获得足够的意识，以保持胸骨靠着不要让动物无人值守。 </li><li>麻醉消失完全前不要运兔，这可以通过注射美托咪定倒车代理等于量所给出的美托咪啶可加快。 </li></ol> 8.术后动物护理<ol><li>保持兔适当的条件（温度，光照，食物，水，空间等 ），并在一个专门的机构密切监测之下。 </li><li>确保动物是很好的休息， 即 ，食物或水匮乏没有长时间。 </li><li>寻找任何伤口或受伤，尤其是在注射部位。 </li><li>保持伤口干燥，防止感染。给予抗生素怀疑感染时:地塞米松1毫克/克，硫酸新霉素3500 IU /克，多粘菌素B 6000国际单位/克软膏进行术后1周，每天两次涂在眼球表面。 </li><li>每日两次添加未来7天的术后地塞米松/抗生素软膏更好的眼表面再生，减少术后疼痛。 </li><li>给全身镇痛药（Carprofene 4毫克/千克，每日两次），初始48小时。 </li><li>无人看管，直到它已经恢复了足够的意识，以保持胸骨靠着不要让动物。 </li><li>不要返回一个神兽经历了手术给公司其他动物，直到完全康复。 </li><li>不要暴露动物不必要的困扰。 </li></ol> 9. SLO / SD-OCT指导<ol><li>准备和使用注射针筒1含0.175毫克/千克氯胺酮和0.125毫克/千克美托咪定为开始27克肌肉注射针（IM）麻醉。翻转注射器混合。 </li><li>添加润滑剂至少每5分钟滋润眼睛并保持清晰的SD-OCT成像，可选可以使用自定义的隐形眼镜。 </li><li>附加一个钢平台到头枕稳定动物在所需位置。 </li><li>放置在钢平台的兔子，把它的眼睛垂直探针。 </li><li>从下腮骨（下颌骨）避免气管握住兔子的头。 </li><li>倾斜动物的头由大约45度朝向的SD-OCT探针，以优化在植入物上的观看角度。 </li><li>使用30度镜头及选择以下参数进制OCT成像[HS]:单行30度设置，设置为100（平均）ART模式和值设置为15 ART模式扫描体积20×20度的设置进行扫描;不要求高分辨率模式。 </li><li>用SLO红外线反射成像找到植入物（ 图2A）的焦平面;最佳的对焦当达到（全部）种植体边缘锋利14。 </li><li>完成后，加入地塞米松1毫克/克，硫酸新霉素3500 IU /克，多粘菌素B 6000国际单位/克软膏下盖。 </li></ol>

RB，BVS，ZL，NE和Geuder公司已经在射手提交的欧洲专利申请。 NB是Geuder公司的员工。此视频文章出版费用由Geuder AG支付。

使用兔模型，一个安全的和可重复的方法，提出了对移动运营商的培养RPE的transvitreal交付与定制设计的仪表射手视网膜下腔。所描述的方法提供了一个短/优化的手术技术，便于学习，因为它涉及到玻璃体标准技术与视网膜演习。成果是一个干净的玻璃体视网膜界面，以及眼内注射，避免流体的湍流在植入部位，在低眼压引起大疱性视网膜脱离（BRD），防止视网膜并通过干燥巩膜的损伤，和兔子的适当位置极大地方便了。 然而我们提醒，如几帧内手术并发症，可能会发生的任何时间，阻碍了植入的成功，例如眼内出血，麻醉过程中的重要步骤，例如在植入衰落断，由于仪器操作或低眼压BRD的塌陷， - [Rabbit死亡由于长期运行过程中过度剂量麻醉，血压偏低引起缺氧性脑损伤，或热疗。然而，这些并发症随时间而减少，因为它们迅速地解决和通过增加外科小组经验的解决。 一些并发症可以通过以下几个简单的，但关键步骤减少。润滑剂应添加每5 - 10分钟，以防止在操作过程中的角膜，巩膜和结膜损伤，并保持清晰的眼内媒体，如干燥/变黑巩膜可以是切口裂开的原因，这又导致低眼压和/或从巩膜术泄漏。应添加肝素，以防止纤维蛋白膜，使得特别是视网膜下植入有挑战性和同时加入肾上腺素减少下肝素16出血的形成。应避免过长肝素/肾上腺素曝光时间（&gt; 1小时），以防止角膜埃德由内皮失代偿33，高血压危象或术中死亡毫安。细致玻璃体切除应仪（入口）端口来执行，以避免视网膜和/或脉络膜分队。眼内器械应指着后极部，以避免镜头触摸（医源性引起白内障的形成）或（入门网站）视网膜损伤。眼内侧端口输注插管，应使用的，因为它衰减周围的植入区域中的喷流，从而防止性视网膜不受控制撕裂和BRD的崩溃。应避免BRD感应在中线（从视神经纵轴）或附近的光学骨髓纤维，以防止大量的医源性视网膜脱离。最后，最后但并非最不重要BRD应在低眼压被诱导，以避免视网膜下的BSS注射使用过度流速可能导致视网膜损害（ 例如 ，通过拉伸）。 许多研究变量，如细胞嘉莉ř变体，胎儿，成人或干细胞衍生的RPE细胞来源，免疫抑制剂的选择等 ，可以探讨14,26,27,34。进一步改善，如无血清培养RPE方法，在视网膜下腔xenoRPE的表征，拆除主机RPE层14或战略种植体支抗的都在进步当前的工作。 迄今为止所描述的技术已在5个不同的兔子株，包括南美栗鼠混蛋，混蛋龙猫/ KBL混合动力车，新西兰白/红十字会，新西兰白色（白化病）和荷兰束带使用。雄性和雌性兔手术，兔子在至少1.5千克或2个月的年龄（取决于物种）。大多数手术都对有色兔（Chinchilla的私生子或栗鼠混蛋混合动力）与权重之间的2.5 - 3公斤。 所有兔子的菌株，我们有机会一起工作似乎有一定的特殊性。鉴于exclus在2009-13德国灰鼠混蛋应变的青紫蓝兔的香港专业教育学院的可用性，我们已经收集与这些动物最丰富的经验。不幸的是不再可用，因为繁殖已经停产，但相比非常好新西兰白/红十字会除了在后者更有利厚巩膜和更大的眼睛卷。龙猫混蛋杂种显著术中纤维蛋白形成，并要求肝素/肾上腺素使用如上文所述，以确保成功视网膜演习。该协议也已经在非色素白兔（新西兰白色）进行的，但是尤其BRD创建和视网膜下注入更有挑战性给出对比度降低升值。诱导玻璃体后脱离的可行性似乎并不兔子应变依赖在我们手中。 Transvitreal视网膜下递送是可能的选择的未来外科策略给予它最通讯<html>路线时下临床访问视网膜。因此，许多其他团体已经提出这样的技术为培养的RPE在载体在动物工作11,15,23,35支持。 Aramant 等 36有一个仪器，哪个地方，而不是推自己的水凝胶包裹的软植入视网膜下其目标网站。图曼等人的设计中。利用中空刮板，它通过浮动它关闭通过流体注射19释放载体负载接枝。前两种策略需要仪器，相比epiretinally appositioned仪器时，这在我们看来是比较容易出现并发症，视网膜下插入。蒙特苏马等 22所描述的视网膜下插入仪器视网膜下植入芯片的猪交付，但因为对我们所知，没有进一步的工作，已经出版。我们已经能够进行一些修改，以猪延长所描述的技术。 jove_content"&gt;我们优选的细胞的载体是10微米厚的聚对苯二甲酸酯（PET）膜。从外科的角度来看，这种材料具有良好的刚度和弹性参数，除了其广泛的通用性在细胞培养实验，我们发现，具有扩展四氟乙烯类似的经历当乳酸-共-乙醇酸（PLGA），以及复合纳米纤维（PLGA或PET）和超薄的PET 26 - （膨体聚四氟乙烯）由PET，聚乳酸/ capronolactic酸（PLCL）或聚静电纺丝37或纳米纤维膜。的PET膜与我们的金属射手仪器使用的，它们有一个偶尔倾向表现出静电荷，从而从27。超薄聚酰亚胺膜可以在我们手中不可与上面概述的协议视网膜下腔植入射手挑战其喷射（在准备稿）。 MARMOR 等 。系统地研究自发RESO医源性局部视网膜脱离38-41视网膜下液rption。即使按照视网膜下腔操作，这些被发现被术后第4天在平静的手术吸收。不进行激光光凝以固定视网膜切开的边缘。虽然相比于人手术直觉相反时，则不需要空气/气体填塞。除非细致除去周边玻璃体的可以实现的，尤其是在上级象限，这可能实际上导致从视网膜切开站点始发巨大视网膜撕裂。建议只执行与后续的20％SF6气体填充流体空气交换打捞术医源性视网膜脱离或万一某一特定植入位置需要保护。 虽然神经视网膜的机械诱发消融可引起兔42,43 RPE和光感受器的损害，其程度差别很大（甚至定期BSS）德待处理的因素例如所用眼压，注射器类型，注射量与由此引起的视网膜拉伸， 等等 。我们还测试了通常建议钙/镁的BSS促进支队42-44，却发现它会导致术中晶状体混浊（特别是随着温度的升高），并显著延迟，甚至损害视网膜再附着27。 20慢视网膜下注射 - 因此用100微升注射器定期BSS的30微升体积建议;注射针运动应尽量少让周围的密封性视网膜并防止Bruch膜损伤。一些医源性损害可能由RPE伤口愈合解决，复位后外核层厚度的观测相对保存，表明RPE /感光复杂的可以承受这种障碍，也是由人45描述。 基于细胞的疗法或视网膜修复需要临床前阿尼玛升测试之前，监管部门的批准，并开始人体安全性研究。前者有所不同国家的国家。此处所描述的兔模型可作为用于建立或者甚至进行监管部门的所有要求的成本效益和较少有挑战性的平台。此外，它可能会成为后来在最终的多中心临床试验或沿途的技术的进一步改进外科医生的培训。

年龄相关性黄斑变性（AMD）是视力障碍，在工业发达国家50岁以上老年人最常见的原因，因为它会导致中心视力丧失。约15％的这些患者中从"湿"疾病的形式，其中新血管形成从脉络膜起源和破坏视网膜功能1受损。该变型可通过用的抗血管生成药物2重复帧内玻璃体注射高度有效的疗法来治疗。然而，绝大多数患者（〜85％）从干燥的形式，其特征是视网膜色素上皮细胞（RPE）根据细胞外沉积（ 例如 ，玻璃疣）受到损害。这些沉积物导致RPE功能障碍导致在黄斑视网膜萎缩。由于缺乏任何疗效的治疗方案，AMD演变成一个密集发展的研究领域，有许多不同的疗效的治疗方法正在测试中。手术RPE更换一个有吸引力的未来可能性，以战胜这一令人衰弱的疾病3。 自体视网膜色素上皮移植替换黄斑功能失调或丢失的RPE，并具有恢复其生理功能4-9的潜力。此手术技术具有与视网膜色素上皮分化的协议从人胚胎干细胞（hESC细胞）和诱导多能干细胞（IPSC）发展的突破口，使科学家RPE无限细胞源移植10。 RPE移植是目前公认的干细胞衍生的治疗有吸引力首次在人类应用。眼睛提供体内监测工具11-13优秀的外科手术进入和复杂的。 移植的视网膜色素上皮，一种方法是用一种微创递送的细胞悬浮液，可选地，为了更好地保持RPE特征和移植功能，阿尔季音响官方载体SUBSTR为RPE更换茨（支架）正在考虑4,14,15。大动物模型所需的临床前验证，但对动物的处理和手术技术缺失迄今为止16-23详细技术信息。 我们和其他人11,24尽管一些反证25，建议使用刚性但弹性载体衬底的，因为它提供了更安全的处理，保留单层完整性和功能性。随着时间的推移，我们已经测试了几个定制设计的仪器及配套技术小区载波支持RPE移植的植入视网膜下腔（SRS）。我们利用术录像， 体内扫描激光眼底镜与谱域光学相干断层扫描（SLO / SD-OCT）和组织学结合以评价植入成功14,26,27。在这里，我们为我们目前推荐用于兔视网膜下植入RPE，这是在5种不同品系兔，7细胞载体材料和4 RPE细胞来源遍及150多个程序进行测试。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>s30 ultrasonic cleaning unit</td>
			<td>Elmasonic</td>
			<td>100 4631</td>
			<td>2.75L</td>
		</tr>
		<tr>
			<td>DE-23&#160; autoclave</td>
			<td>Systec</td>
			<td>C 2209</td>
			<td>23L</td>
		</tr>
		<tr>
			<td>Syringe&#160;</td>
			<td>BD&#160;</td>
			<td>300013 
			300995&#160; 
			301285 
			300294 
			300330&#160;</td>
			<td>1ml x3 
			2ml x3 
			5ml x1 
			10ml x1 
			20ml x1</td>
		</tr>
		<tr>
			<td>Needle&#160;</td>
			<td>BD</td>
			<td>305196 
			305136</td>
			<td>18G x 1&#160; 
			27G x5</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Feather</td>
			<td>2975#20</td>
			<td>blade#20 x3</td>
		</tr>
		<tr>
			<td>Surgical drape</td>
			<td>HARTMANN 
			LOHMANN &#38; RAUSCHER</td>
			<td>277 502 
			25 440</td>
			<td>60x40 cm x2 
			12x17 cm</td>
		</tr>
		<tr>
			<td>Ocular sticks</td>
			<td>LOHMANN &#38; RAUSCHER</td>
			<td>16 516</td>
			<td>66x5 mm</td>
		</tr>
		<tr>
			<td>Twister gauze sponges</td>
			<td>HARTMANN</td>
			<td>481 274</td>
			<td>x2</td>
		</tr>
		<tr>
			<td>Closure strips</td>
			<td>HARTMANN</td>
			<td>540 686</td>
			<td>x4</td>
		</tr>
		<tr>
			<td>Opmi Visu CS Microscope</td>
			<td>Zeiss</td>
			<td>N/a</td>
			<td>incl. fundus imaging system BIOM II</td>
		</tr>
		<tr>
			<td>Chandelier endoillumination</td>
			<td>Geuder</td>
			<td>G-S03503 + 
			G-S03504</td>
			<td>25G incl. trocar</td>
		</tr>
		<tr>
			<td>Light machine</td>
			<td>Geuder</td>
			<td>G-26033</td>
			<td>Xenotron III</td>
		</tr>
		<tr>
			<td>Vitrectomy machine</td>
			<td>Geuder</td>
			<td>G-60000</td>
			<td>MegaTRON S4 S4/ HPS</td>
		</tr>
		<tr>
			<td>Vitrector</td>
			<td>Geuder</td>
			<td>G-46301</td>
			<td>MACH2 vitreous cutter 23G</td>
		</tr>
		<tr>
			<td>Venturi cassette&#160;</td>
			<td>Geuder</td>
			<td>G-60700</td>
			<td></td>
		</tr>
		<tr>
			<td>Sideport-infusion cannula</td>
			<td>Geuder</td>
			<td>custom&#160;</td>
			<td>1x23G</td>
		</tr>
		<tr>
			<td>3-0 silk suture</td>
			<td>ETHICON</td>
			<td>V546G</td>
			<td>x1</td>
		</tr>
		<tr>
			<td>Caliper</td>
			<td>Geuder</td>
			<td>G-19135</td>
			<td>x2</td>
		</tr>
		<tr>
			<td>Vannas scissors</td>
			<td>Geuder</td>
			<td>G-19777</td>
			<td>x1</td>
		</tr>
		<tr>
			<td>Sclerotomie blade</td>
			<td>Ziemer</td>
			<td>21-2301</td>
			<td>1x23G 
			1x20G</td>
		</tr>
		<tr>
			<td>7-0 silk suture</td>
			<td>ETHICON</td>
			<td>EH6162H</td>
			<td>x1</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Geuder</td>
			<td>G-32320</td>
			<td>x2</td>
		</tr>
		<tr>
			<td>Iris forceps</td>
			<td>Geuder</td>
			<td>G-18910</td>
			<td>x1</td>
		</tr>
		<tr>
			<td>Colibri forceps</td>
			<td>Geuder</td>
			<td>G-18950</td>
			<td>x1</td>
		</tr>
		<tr>
			<td>Extendible subretinal injection needle</td>
			<td>DORC</td>
			<td>1270.EXT</td>
			<td>41G</td>
		</tr>
		<tr>
			<td>VR scissor</td>
			<td>Geuder</td>
			<td>G-36542</td>
			<td>25G</td>
		</tr>
		<tr>
			<td>Grieshaber forceps holder</td>
			<td>Alcon</td>
			<td>712.00.41</td>
			<td>23G</td>
		</tr>
		<tr>
			<td>Curved scissor forceps tips</td>
			<td>Alcon</td>
			<td>723.52</td>
			<td>23G</td>
		</tr>
		<tr>
			<td>Implant loading station</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>SYLGARD 184 Silicone Elastomer Kit</td>
		</tr>
		<tr>
			<td>blunt oval implant trephine&#160;</td>
			<td>Geuder</td>
			<td>custom-made&#160;</td>
			<td>2.4 x 1.1 mm&#160;</td>
		</tr>
		<tr>
			<td>Shooter dummy</td>
			<td>Geuder</td>
			<td>G-32227</td>
			<td>x1</td>
		</tr>
		<tr>
			<td>Shooter</td>
			<td>Geuder</td>
			<td>G-S03443</td>
			<td>x1</td>
		</tr>
		<tr>
			<td>Flute needle</td>
			<td>DORC</td>
			<td>1281.SD</td>
			<td>20G (Vacuum)</td>
		</tr>
		<tr>
			<td>Manual microliter syringe</td>
			<td>Hamilton</td>
			<td>24535</td>
			<td>100&#181;l</td>
		</tr>
		<tr>
			<td>Tissue culture plates</td>
			<td>Greiner bio-one</td>
			<td>&#160;664160</td>
			<td>100 x 20 mm</td>
		</tr>
		<tr>
			<td>Spectralis Multi-Modality 
			Imaging System</td>
			<td>Heidelberg 
			Engineering</td>
			<td>N/a</td>
			<td>Spectralis HRA 
			&#160;+ OCT</td>
		</tr>
		<tr>
			<td colspan="4">Drugs and solutions</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Active agent</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Mucadont-IS</td>
			<td>Merz Hygiene</td>
			<td>virucidal instrument disinfectant&#160;</td>
			<td>2L</td>
		</tr>
		<tr>
			<td>Mucocit&#160;T</td>
			<td>Merz Hygiene</td>
			<td>Aldehyde-free instrument disinfectant</td>
			<td>2L</td>
		</tr>
		<tr>
			<td>Ketamin 10%</td>
			<td>WDT</td>
			<td>Ketamine&#160;</td>
			<td>10ml (100mg/ml)</td>
		</tr>
		<tr>
			<td>Domitor</td>
			<td>Orion Pharma</td>
			<td>Medetomidine hydrochloride</td>
			<td>10ml (1mg/ml)</td>
		</tr>
		<tr>
			<td>Antisedan</td>
			<td>Orion Pharma</td>
			<td>Atipamezole hydrochloride</td>
			<td>10ml (5mg/ml)</td>
		</tr>
		<tr>
			<td>Neosynephrin POS 10%</td>
			<td>URSAPHARM</td>
			<td>Phenylephrine HCl</td>
			<td>10ml</td>
		</tr>
		<tr>
			<td>Mydriacyl</td>
			<td>Alcon</td>
			<td>Tropicamid</td>
			<td>10ml (5mg/ml)</td>
		</tr>
		<tr>
			<td>Methocel 2%</td>
			<td>Omni Vision</td>
			<td>hydroxypropyl methylcellulose</td>
			<td>10g</td>
		</tr>
		<tr>
			<td>PURI CLEAR</td>
			<td>ZEISS</td>
			<td>Balance salt solution (BSS)&#160;</td>
			<td>500ml</td>
		</tr>
		<tr>
			<td>Glucose 5%&#160;&#160;</td>
			<td>B.Braun</td>
			<td>Glucose 5%&#160; solution</td>
			<td>100ml</td>
		</tr>
		<tr>
			<td>Heparin-Natrium-25 000</td>
			<td>Ratiopharm</td>
			<td>Heparin</td>
			<td>5ml (2500 unit/ml)</td>
		</tr>
		<tr>
			<td>Suprarenin</td>
			<td>SANOFI</td>
			<td>Epinephrine</td>
			<td>1ml (1mg/ml)</td>
		</tr>
		<tr>
			<td>Triamcinolone</td>
			<td>University of Bonn pharmacy</td>
			<td>preservative-free Triamcinolone&#160;</td>
			<td>1ml (40mg/ml)&#160;</td>
		</tr>
		<tr>
			<td>Isoptomax eye ointment</td>
			<td>Alcon</td>
			<td>dexamethasone 1 mg/g 
			neomycin sulfate 3,500 IU/g 
			polymyxin B sulfate 6,000 IU/g</td>
			<td>10ml</td>
		</tr>
		<tr>
			<td>Betaisodona</td>
			<td>Mundipharma</td>
			<td>Povidon-Iod</td>
			<td>30ml (1g/10ml)</td>
		</tr>
		<tr>
			<td>Optive</td>
			<td>ALLERGAN</td>
			<td>sodium carboxymethylcellulose glycerol</td>
			<td>10ml</td>
		</tr>
	</tbody>
</table>

a step by step protocol for subretinal surgery in rabbits

年龄相关性黄斑变性（AMD），视网膜色素变性，与其他的RPE相关疾病是在成人中工业发达国家视力的不可逆损失的最常见的原因。 RPE移植似乎是一个有希望的疗法，因为它可以代替功能失调的RPE，恢复其功能，并由此视力。 在这里，我们描述了一个支架移植培养的RPE单层家兔的视网膜下空间（SRS）的方法。后玻璃体异种移植是用由具有一个聚四氟乙烯（PTFE）涂覆的柱塞20号金属喷嘴的定制射手输送到SRS。目前的技术发展在全球150兔手术超过6年。可以用在体内成像的非侵入性和重复如随后灌注固定组织学谱域光学相干断层扫描（SD-OCT）获得术后随访。 钍E法具有易于学习和高成功率定义良好的步骤。兔被认为是在临床转化临床前研究用一只大眼睛的动物模型。在这种情况下兔子是具有成本效益的，也许方便替代其他大眼的动物模型。

从视网膜下注入所描述的方法，结果列于表2中。植活所示下视网膜有长约的成功率61％时，进行一个芯玻璃体切割术及上升到76％，当玻璃体后脱离诱导。这些数字包括了长约动物谁死要么术或在第3天，术后21％。这种技术可以用于同时植入在不同的视网膜区域2的支架的一只眼睛。 兔子进行术后体内后续使用的SD-OCT和组织学处理如史丹泽等人所述。27（ 图2）的图。一个简单的移植后2A示出在聚酯膜（PET）的植入培养的视网膜色素上皮的扫描激光检眼镜红外反射图像。该种植体周围晕对应于感光萎缩。 图。图2B示出了相应的SD-OCT中，通知视网膜，主要外核层（ONL）变薄，在SD-OCT上述植入超反射带，而相邻于植入物的神经视网膜显示接近正常的反射频带。这些结果表明无创伤输送的图。图2C示出了其示出围绕性视网膜站点可能为医源性操作的结果视网膜下疤痕和ONL萎缩植入物的苏木素/曙红（H / E）染色，过的PET的连续但不规则着色层。布鲁赫的植入膜下也出现了连续并脉络膜包含一些零散的红细胞。这些形态的结果是可比的SD-OCT和加强一个无创伤分娩的论文。 <img alt="图1" src="/files/ftp_upload/53927/53927fig1.jpg" /> 图<html> URE 1: 装入射击与人iPSC的RPE培养在PET细胞载体。 a）显示冲出使用定制的针。B）豆形植入物从细胞培养削减植入物。C）植入物的定位之前加载。D）与植入射手的加载。 <a href="https://www.jove.com/files/ftp_upload/53927/53927fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53927/53927fig2.jpg" /> 图2: 培养人HES-RPE对PET细胞载体4周兔视网膜下腔。 a）示出的SLO红外反射图像，绿线划定在图中所示的横截面。 2B，B）对应的SD-OCT。C）H / E染色，详见文字或26,27。ove.com/files/ftp_upload/53927/53927fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <table fo:keep-together.within-page="1"><colgroup><col /><col /></colgroup><tbody><tr height="21"><td height="21" style="height:21px;width:97px;"> 参数 </td><td style="width:131px;"> 使用的设置 </td></tr><tr height="21"><td height="21" style="height:21px;">玻璃体切割术</td><td> 6000切/分钟</td></tr><tr height="21"><td height="21" style="height:21px;">真空</td><td> 200毫米汞柱</td></tr><tr height="21"><td height="21" style="height:21px;">上升时间</td><td> 1秒</td></tr><tr height="21"><td height="21" style="height:21px;">空气</td><td> 24毫米汞柱</td></tr><tr height="21"><td height="21" style="height:21px;">灌溉</td><td> 24 水柱 </td></tr><tr height="21"><td height="21" style="height:21px;"> DIATHermy </td><td> 30％ </td></tr></tbody></table> 表 1: 玻璃体切割机参数设置。 <table fo:keep-together.within-page="1"><tbody><tr><td> 玻璃体切割术 </td><td> 注入 </td><td> 兔操作 </td><td> 成功的植入 </td><td> 植入失败 </td><td> 死亡 </td><td> 成功率％ </td></tr><tr><td>核心玻璃体切割术</td><td>宠物</td><td>三十</td><td> 19 </td><td> 4 </td><td> 7 </td><td> 63.33 </td></tr><tr><td></td><td> PET + RPE </td><td> 70 </td><td> 42 </td><td> 12 </td><td> 16 </td><td> 60 </td></tr><tr><td> PVD，±纤溶酶瓦特/ PPV </td><td>宠物</td><td> 28 </td><td> 21 </td><td> 2 </td><td>五</td><td> 75 </td></tr><tr><td></td><td> PET + RPË </td><td> 22 </td><td> 17 </td><td> 2 </td><td> 3 </td><td> 77.27 </td></tr></tbody></table> 表2: 最后的150业务，包括方法和植入式的总结。

Watch this Scientific Journal Video about A Step by Step Protocol for Subretinal Surgery in Rabbits at JoVE.com

A Step by Step Protocol for Subretinal Surgery in Rabbits

视网膜色素上皮细胞（RPE）替换策略和基于基因的疗法被认为对于几种视网膜退化性疾病。对于临床转化，大眼睛的动物模型需要研究适用于患者的手术技术。在这里，我们提出了视网膜手术对视网膜色素上皮移植，这是通用的，具有成本效益的面向兔模型。

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 ...

a-step-by-step-protocol-for-subretinal-surgery-in-rabbits

Research

JoVE Journal

Medicine

15.3K 次观看.  University of Bonn. 视网膜色素上皮细胞（RPE）替换策略和基于基因的疗法被认为对于几种视网膜退化性疾病。对于临床转化，大眼睛的动物模型需要研究适用于患者的手术技术。在这里，我们提出了视网膜手术对视网膜色素上皮移植，这是通用的，具有成本效益的面向兔模型。 

视频: A Step by Step Protocol for Subretinal Surgery in Rabbits

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

采用TaqMan从儿童凳诺如病毒的检测和Genogrouping一步法RT-PCR技术

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

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 ...

Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2. In HIV infection the syndrome occurs at a younger age.
HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal.
The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.

Frailty syndrome is commonly seen in the aged and reflects multi-system physiological change. However, with reduced functional reserve and resilience frailty is also known to be common in the HIV infected population. This study outlined an easily administered screening test to identify HIV patients with frailty. When significant components of frailty are identified, clinicians will be able to focus on amelioration of the problem and promote reversion to the pre-frail state.

在感染艾滋病毒的个体的测量弱者。体弱患者的识别是第一步改良与弱者拨回

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Step By Step: Microsurgical training method combining two nonliving animal models

The learning of microsurgical techniques and the maintenance of microsurgical skills have been traditionally based on the use of living animals, mainly laboratory rats. This method although extremely valuable can be economically demanding both for the surgeon and the sponsoring institution; it also requires special training facilities that may not always be available or accessible. Furthermore ethical concerns can limit the use of living animals for training purposes. Alternative training methods, such as inert tubes and gloves have not gained popularity among surgeons since they do not offer an experience similar to that of a clinical situation. Non-living animal models include the use of chicken thighs and wings; they offer a practice experience that resembles a clinical situation to a considerable extent. This type of training is relatively cheap and easily available. The microscope and instruments required can be acquired over the internet, and the chicken pieces can be bought at the local supermarket.
This approach allows a motivated trainee to rehearse different types of surgical techniques several times at a reasonable expense, helping to develop or maintain his surgical expertise if more complex facilities are not available. On the current manuscript we describe how to setup a small practice station, how to dissect the specimens, and how to practice both with the chicken thighs and with the chicken wings in a progressive fashion. This approach takes advantage on the versatility of the chicken thigh model and the small size of the chicken wing Brachial artery.

Here we describe how to set up a small microsurgical practice station and a simple and inexpensive method for the training of microsurgery with non-living animal models.

一步一步:显微外科培训方法相结合的两个无生命的动物模型

The learning of microsurgical techniques and the maintenance of microsurgical skills have been traditionally based on the use of living animals, mainly ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

异位自体肾移植的猪模型:有步骤，分步协议

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.

This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery.

采取下一步:神经整形正交后肢移植模型，以最大限度地提高大鼠的功能恢复

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

使用化学活检进行移植质量评估

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

Step-by-Step Stapedotomy through Transcanal Exclusive Endoscopic Approach

In recent years there has been an increasing trend in the use of the endoscope to treat a variety of middle ear pathologies, including otosclerosis. Several studies comparing traditional microscopic and endoscopic stapes surgery have reported similar hearing results and an overall low rate of complications. The endoscope has unraveled its full potential in demanding settings of stapes surgery, such as unfavorable anatomy of the oval window niche or revision cases. Reduced manipulation of the chorda tympani and low rate of post-operative dysgeusia are further benefits to mention for endoscopic stapes surgery.
Being a one-handed technique, management of bleeding, positioning, and crimping of the prosthesis may be challenging for novice endoscopic surgeons, so some training in endoscopic ear surgery is recommended before performing endoscopic stapedotomy. The problem of sharing the surgical field between the endoscope and the operating instruments could be easily overcome if proper instruments positioning is understood. One-handed bleeding control in the narrow space of the ear canal may represent an issue during the elevation of the tympano-meatal flap, possibly discouraging the surgeon since the preliminary steps of surgery. Following appropriate technique to raise the flap and the collaboration with the anesthesiology team in keeping the blood pressure low guarantee an adequate bleeding control in most cases.
The aim of this article is to describe the entire surgical procedure of a transcanal exclusive endoscopic stapedotomy, from operating room set up and patient positioning to post-operative care. A step-by-step description of the surgical maneuvers with technical hints is reported, to guide the surgeon across the procedure and allow any ear surgeon to perform stapes surgery endoscopically.

The aim of this article is to provide a step-by-step method for endoscopic stapes surgery from operating room setting and patient positioning to post-operative care. This work would represent a guide for any otologic surgeon who is willing to treat otosclerosis with endoscopic transcanal technique.

通过经卡全线内窥镜方法进行逐步镫骨切除术

In recent years there has been an increasing trend in the use of the endoscope to treat a variety of middle ear pathologies, including otosclerosis. ...

A Two-Step Method for Percutaneous Transhepatic Choledochoscopic Lithotomy

Intrahepatic and extrahepatic choledocholithiasis is a challenge in the field of biliary surgery. We present our experience using a two-step percutaneous transhepatic choledochoscopic lithotomy (PTCSL) procedure to treat challenging biliary stones. We retrospectively reviewed 81 patients with intrahepatic and extrahepatic choledocholithiasis treated using this two-step PTCSL from January 2013 to January 2020, including 40 males and 41 females, with an average age of 66 years. In contrast to traditional percutaneous transhepatic cholangioscopy (PTCS), a channel was established directly through a 16F Amplatz sheath, and the stone in the channel was removed with the aid of a nephroscope. The clinical efficacy and complications of all patients were analyzed. Eighty-one patients (81/81, 100%) had their biliary stones successfully removed; 62/81 patients (76.5%) had biliary stones completely removed after the first operation; 17/81 patients (21%) underwent a second operation; 2/81 patients (2.5%) needed a third operation to completely remove the stones. The incidence of severe bleeding during the operation was 0%, and there were no deaths. The use of the two-step PTCSL method is safe and efficacious, and contributes to a better prognosis of intrahepatic and extrahepatic choledocholithiasis.

This "two-step method" significantly improved the success rate of percutaneous transhepatic choledochoscopy and achieved a better prognosis of intrahepatic and extrahepatic choledocholithiasis.

经皮经肝胆胆镜截石术的两步法

Intrahepatic and extrahepatic choledocholithiasis is a challenge in the field of biliary surgery. We present our experience using a two-step percutaneous ...