We gratefully acknowledge support by the Harold and Pauline Price Chair in Ophthalmology and the Jules Stein Eye Institute to MBG, the NEI Core grant (EY00331-43) to SN. Research was supported in part by a generous gift from the Sakaria family to SN and MGB, and from an unrestricted grant from the Research to Prevent Blindness to the Department of Ophthalmology. We thank Charlotte Yiyi Wang at Berkeley School of Optometry for obtaining initial OCT images of subretinal injections.

动物:野生型C57BL / 6J小鼠在加州大学洛杉矶分校（UCLA）育成。所有动物为11之间 - 周17岁，并包括雄性和雌性小鼠。所有小鼠群养，保持与食品和水随意 12:12光照/黑暗周期。所有实验均按照加州大学洛杉矶分校和视觉的研究协会和眼科声明在眼科和视觉研究使用动物的机构准则进行的。 注:所有的药物和注射剂是美国药典（USP）级。 1.手术准备<ol><li>麻醉小鼠以100毫克/千克氯胺酮和8毫克/公斤赛拉嗪的盐水混合物腹膜内注射。辖麻醉的深度，使得鼠标有没有脚趾捏或角膜反射触摸。 </li><li>维持体温在37.0℃用循环水垫。 </li><li>扩张2.5％福林眼ð学生ROPS和修剪胡须，以方便可视化。晶须鼠标提供显著感觉输入，因此，晶须修整应该只删除部阻止明确前往眼睛，而不是与晶须的基极。根据我们的经验，小鼠显示该过程后正常恢复。应用甲基纤维素滴眼液，以防止干燥和减少麻醉药引起的瞬态白内障13。 </li><li>消毒手术前（ 即，聚维酮碘和乙醇或烫珠）仪器。 </li><li>制备稀释的荧光素（使用0.9％盐水0.01％）在无菌环境中（ 即 ，生物安全柜）如果可视化将被执行（见下文第3节）。 </li></ol> 2.注射整地<ol><li>准备的注射器（ 例如 ，5微升注射器）与适当的喷射量（ 例如 ，0.3〜1.0微升）。 </li><li>所以眼睛朝上，并在二清晰可见鼠标位置ssecting显微镜。 </li><li>轻轻一捏用细尖镊子时间结膜。使用弯曲Vannas剪刀大约90度的周向切口。 </li><li>重复步骤2.3与底层的眼球筋膜囊。 </li><li>而鼻旋转地球切除用细尖镊子周围的结缔组织。向注射部位大约0.5mm颞视神经工作。使用非常谨慎，以避免破坏眶窦。 </li></ol> 3.巩膜与视网膜下注射注:建议在0.9％盐水0.01％荧光素的注射可以用来协助可视化，同时学习此过程。荧光素的地形分布眼底成像（见下文第4节）进行有效记录。 <ol><li>通过用22.5度的眼用刀片轻轻搔抓眼罩使在注射部位的小巩膜切口。这个切口寿LD仅足够大以允许针的尖端穿过巩膜。 </li><li>插入斜面33克针（倾斜5 - 10°与该斜面面对并成角度平行于视网膜的巩膜注入所需量（ 例如，0.3〜1.0微升0.01％荧光素的用于学习的目的）。 注:通过每次注射前适当的溶剂和去离子水连续清洗彻底清洗维护注射器的无菌。 </li><li>压低柱塞缓慢（超过3〜秒）无需移动针，并用均匀的压力。 注意:当针在视网膜下腔，轻微的阻力将在按下柱塞可以感觉到。将没有对最小的阻力，如果针头穿刺视网膜，和高电阻，如果针不会刺入巩膜或视网膜色素上皮。 </li><li>抽出针头，以尽量减少回流前等待几秒钟。 </li><li>冲洗眼睛用无菌缓冲盐水和保证眼睛ħ作为旋转回其正常位置。 </li></ol> 4. OCT和眼底成像评估视网膜脱离<ol><li>注射来评价喷射的质量后立即和在适当的时间执行OCT成像点注射后根据需要来评估视网膜结构。 注:在类似的研究中使用OCT的实例先前已描述7,14。 <ol><li>调整并对准OCT图像定位到注射部位。注射部位应是中线为0.5mm颞视神经头。或根据需要，如果脱离超出框架不能很好地集中重复。 </li></ol></li><li>可视化视网膜脱离和染料有连接面眼底成像7,14注射区域。 注意:如果一个OCT成像系统不可用时，为实践的载体少量荧光素的注射将允许与执行荧光素血管任何眼底照相机的可视化造影使用相同的激发波长和阻塞过滤器。超荧光的局部区域将会出现在脉管系统下方，如果视网膜下空间被正确地定位在脉管将具有尖锐和明显边界。从注射水泡的边缘将由hyper-到低渗荧光过渡划定。几台仪器提供此功能的鼠标;这里使用的仪器是其他地方14条所描述。 </li></ol> 5.术后护理<ol><li>应用三联抗生素眼药霜厚厚的棉衣，以注入眼睛的角膜表面。 </li><li>将小鼠恢复干净孤笼。请勿将已经接受了手术，直到他们完全康复小鼠。 </li><li>监控麻醉恢复过程中呼吸和温度。监测动物，直到他们能保持胸骨斜卧。 </li><li>执行其他适当的术后监测和治疗，包括卡布洛芬为手术后疼痛管理一个皮下注射（5毫克/千克）。 </li></ol> 6.评估视网膜功能的电描记所（ERG） <ol><li>进行ERG分析预喷射和在适当的时间后喷射作为需要评估视网膜的功能。如果注射制成视网膜下腔，视网膜脱离应72小时内解决。 <ol><li>使用标准的ERG技术如前所述14,15前和注射后评估视网膜的功能。 </li></ol></li></ol> 7.三维重建和水泡量化卷注:OCT具有高对比度包围的视图帧内的整个脱离扫描是最佳的使用。 ImageJ的/斐济17,18和了Imaris被使用，但也可以使用其它的软件。 <ol><li>导出感兴趣的B超扫描，进口的ImageJ /斐济和作物（图像&gt;裁剪）华侨城SC的部分一个使用矩形选择工具来建模。 <ol><li>调整对比度（图像&gt;调整&gt;亮度/对比度），并用一条线连接两个部分划定任何丢失的边界。 </li><li>绘制与线工具（保持移）跨越RPE到感光层的直线。测量（分析&gt;测量）的行的长度，以获得最大分离尺寸为步骤7.8。 </li></ol></li><li>进口裁剪帧的三维重建软件（见材料表）使用"RGB到灰色"插件和MATLAB编译器运行时。 </li><li>设置使用校准参数从OCT的扫描（X，Y，Z）的体素尺寸（下图片属性）。 </li><li>执行"RGB格雷"插件（在图像属性），具有相同的权重给每个通道，创建第四个通道。删除原始红 - 绿 - 蓝通道。 </li><li>使用反转对比变化的灰色通道。店铺形象。 </li><li>点击"添加EW面"中的3D视图选项卡按钮，开始创建面的引导下4个步骤。 <ol><li>设置表面水平细节（步骤4 1）。 注意:根据我们的经验8.0至12.0是最有效的范围。 </li><li>设置最大球体大小（背景下的选择）比7.1.2测得的最大尺寸支队略少。创建面和撤消灰色通道反转（步骤4 2）。 </li><li>阈值设定为最大值，使视网膜外的负空间和脱离的表面不接触（4步骤3）来。 </li><li>设置过滤器类型的体素的数目和按大小分离在分离部位的负空间。完成表面（步骤4 4）。 注:该支队面的体积位于下的统计标签的体积。 </li></ol></li></ol>

<ol>
	<li>Garoon, R. B., Stout, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+ocular+gene+therapy+and+advances+in+treatment+of+inherited+retinal+diseases+and+exudative+macular+degeneration.">Update on ocular gene therapy and advances in treatment of inherited retinal diseases and exudative macular degeneration.</a> Curr Opin Ophthalmol. 27 (3), 268-273 (2016).</li><li>Pierce, E. A., Bennett, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Status+of+RPE65+Gene+Therapy+Trials:+Safety+and+Efficacy.">The Status of RPE65 Gene Therapy Trials: Safety and Efficacy.</a> Cold Spring Harb Perspect Med. 5 (9), a017285 (2015).</li><li>Tolmachova, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+of+Rab+escort+protein+1+following+AAV2-mediated+gene+delivery+in+the+retina+of+choroideremia+mice+and+human+cells+ex+vivo.">Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo.</a> J Mol Med (Berl). 91 (7), 825-837 (2013).</li><li>Nork, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+anatomic+consequences+of+subretinal+dosing+in+the+cynomolgus+macaque.">Functional and anatomic consequences of subretinal dosing in the cynomolgus macaque.</a> Arch Ophthalmol. 130 (1), 65-75 (2012).</li><li>Ye, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+Biodistribution+Evaluation+in+Cynomolgus+Macaques+of+rAAV2tYF-PR1.7-hCNGB3,+a+Recombinant+AAV+Vector+for+Treatment+of+Achromatopsia.">Safety and Biodistribution Evaluation in Cynomolgus Macaques of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia.</a> Hum Gene Ther Clin Dev. , (2016).</li><li>Qi, Y., 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=Trans-Corneal+Subretinal+Injection+in+Mice+and+Its+Effect+on+the+Function+and+Morphology+of+the+Retina.">Trans-Corneal Subretinal Injection in Mice and Its Effect on the Function and Morphology of the Retina.</a> PLoS One. 10 (8), e0136523 (2015).</li><li>Engelhardt, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+morphological+analysis+of+the+subretinal+injection+of+retinal+pigment+epithelium+cells.">Functional and morphological analysis of the subretinal injection of retinal pigment epithelium cells.</a> Vis Neurosci. 29 (2), 83-93 (2012).</li><li>Lambert, N. G., 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=Subretinal+AAV2.COMP-Ang1+suppresses+choroidal+neovascularization+and+vascular+endothelial+growth+factor+in+a+murine+model+of+age-related+macular+degeneration.">Subretinal AAV2.COMP-Ang1 suppresses choroidal neovascularization and vascular endothelial growth factor in a murine model of age-related macular degeneration.</a> Exp Eye Res. 145, 248-257 (2016).</li><li>Muhlfriedel, R., Michalakis, S., Garcia Garrido, M., Biel, M., Seeliger, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+technique+for+subretinal+injections+in+mice.">Optimized technique for subretinal injections in mice.</a> Methods Mol Biol. 935, 343-349 (2013).</li><li>Nusinowitz, S., 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=Cortical+visual+function+in+the+rd12+mouse+model+of+Leber+Congenital+Amarousis+(LCA)+after+gene+replacement+therapy+to+restore+retinal+function.">Cortical visual function in the rd12 mouse model of Leber Congenital Amarousis (LCA) after gene replacement therapy to restore retinal function.</a> Vision Res. 46 (22), 3926-3934 (2006).</li><li>Huang, R., 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=Functional+and+morphological+analysis+of+the+subretinal+injection+of+human+retinal+progenitor+cells+under+Cyclosporin+A+treatment.">Functional and morphological analysis of the subretinal injection of human retinal progenitor cells under Cyclosporin A treatment.</a> Mol Vis. 20, 1271-1280 (2014).</li><li>Maguire, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+efficacy+of+gene+transfer+for+Leber's+congenital+amaurosis.">Safety and efficacy of gene transfer for Leber's congenital amaurosis.</a> N Engl J Med. 358 (21), 2240-2248 (2008).</li><li>Ridder, W. . 3. r. d., Nusinowitz, S., Heckenlively, 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=Causes+of+cataract+development+in+anesthetized+mice.">Causes of cataract development in anesthetized mice.</a> Experimental Eye Research. 75 (3), 365-370 (2002).</li><li>Ridder, W. H. 3. r. d., Nusinowitz, 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+visual+evoked+potential+in+the+mouse--origins+and+response+characteristics.">The visual evoked potential in the mouse--origins and response characteristics.</a> Vision Res. 46 (6-7), 902-913 (2006).</li><li>Matynia, A., 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=Intrinsically+photosensitive+retinal+ganglion+cells+are+the+primary+but+not+exclusive+circuit+for+light+aversion.">Intrinsically photosensitive retinal ganglion cells are the primary but not exclusive circuit for light aversion.</a> Experimental Eye Research. 105, 60-69 (2012).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li></ol>

None of the authors have any commercial disclosures.

视网膜下注射是选择的方法为病毒载体的递送和干细胞衍生的疗法用于操纵光感受器和在基础研究和临床治疗视网膜色素上皮。在患者中，视网膜下注射是通常用前巩膜通过用直接可视化的针进行在玻璃体，视网膜的后部芯玻璃体切割术及渗透。如同大多数玻璃体切割程序，是很常见的，除非眼睛已经晶状体过早发生白内障形成。在小鼠中，视网膜下注射传统上与巩膜前到视网膜，经常与透镜，?...

视网膜下注射是递送细胞和病毒剂的小鼠的视网膜研究其对感光体和下面的视网膜色素上皮1,2-效果的主要手段。在小鼠中大多数视网膜下注射协议使用transcorneal或经巩膜注射部位前到赤道（ 图1）。这种方法可能会导致在包括切口和透镜的所得混浊时，神经视网膜和虹膜，视网膜出血，大量视网膜脱离和持久的视网膜下水肿3-9的玻璃体，渗透的完整性的破坏固有的附带损害。实验操作必须以评价治疗性干预3,7,10,11的作用克服这些影响。本研究提供了一种避免这些并发症的后经巩膜注射方法的详细描述和验证，减少创伤和具有定位子的高成功率视网膜空间。 注射小鼠靶向视网膜下空间往往非常难以执行并且多数研究者遇到失败的尝试，其中该载体被递送到不正确的位置或有显著视网膜损伤，例如，在一个完整的视网膜脱离6的高频。眼睛从分析中排除，因为注射的并发症的数量一般不报道在小鼠研究中，但在我们自己的经验，并与其他研究者的讨论中，失败的注射次数可以是高达50％，并且变化依赖于经验和谁正在执行注射研究者的能力。注射的成功通常通过直接眼底成像和/或光学相干断层扫描（OCT）7,9评估。在小鼠高成功率视网膜下注射容易掌握的方法可以加速实验，减少特雷的临床前研究的成本atments视网膜疾病是在美国失明的主要原因。 后部，这里描述经巩膜视网膜下注射技术是从临床和临床前协议9,12的适应。在注射的小鼠进行的非侵入性诊断评估论证温和，高度本地化的破坏和缺乏额外的抵押镜头，视网膜和视网膜色素上皮的损伤。此外，以相对较少的实践中，实验者可以实现这些结果以高成功率（80 - 90％或更好），从而降低了与这类研究相关的成本。此过程可用于递送细胞，病毒，或药理学治疗干预到光感受器和/或视网膜色素上皮中的临床前研究，并轻松地评估实验介入。

<table><tbody><tr><td>Hamilton Model 62 RN SYR</td><td>Hamilton</td><td>87942</td><td>Syringe x 1</td></tr><tr><td>Hamilton Needle 33 G, 1.0&quot;, 20 DEG, point 3 (304 stainless steel)</td><td>Hamilton</td><td>7803-05</td><td>Needles x 6</td></tr><tr><td>Vannas Curved Scissors</td><td>Ted Pella, INC.</td><td>1347</td><td>5 mm Blade</td></tr><tr><td>22.5 Degree Microsurgery Knife</td><td>Wilson Ophthalmic Corp.</td><td>91204</td><td></td></tr><tr><td>Ketaject&amp;nbsp;</td><td>Phoenix</td><td>NDC 57319-609-02</td><td>Ketamine</td></tr><tr><td>Anased</td><td>Lloyd Laboratories</td><td>NDC 61311-482-10</td><td>Xylazine</td></tr><tr><td>Fluorescein 10% AK-Fluor</td><td>Akorn</td><td>NDC 17478-253-10</td><td>100 mg/ml</td></tr><tr><td>0.9% Saline USP</td><td>Hospira</td><td>NDC 0409-4888-50</td><td>0.9% NaCl</td></tr><tr><td>Antibiotic Ointment</td><td>Akorn</td><td>NDC 17478-235-35</td><td>Ophthalmic</td></tr><tr><td>Water Circulating Pump</td><td>Gaymar</td><td>TP-500 T/Pump&amp;nbsp;</td><td>P/N 07999-000</td></tr><tr><td>sd-OCT</td><td>Bioptigen</td><td>R-Series</td><td>Commercial</td></tr><tr><td>Fundus Camera</td><td>Phoenix Research Laboratories</td><td>MICRON III</td><td></td></tr><tr><td>Tweezers Type 3</td><td>Ted Pella, INC.</td><td>5385-3SU</td><td></td></tr><tr><td>2.5% Phenylephrine</td><td>Paragon BioTeck</td><td>NDC 42702-102-15</td><td>Ophthalmic</td></tr><tr><td>IMARIS8</td><td>Bitplane</td><td></td><td>Version 8.1.2</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td>V1.8.0_77</td></tr><tr><td>Hypromellose&amp;nbsp;2.5%</td><td>Goniovisc</td><td>AX0401</td><td>Methylcellulose</td></tr><tr><td>Eye Drops (Rinse)</td><td>Bausch &amp;amp; Lomb</td><td></td><td>Saline Solution</td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Stemi 2000</td><td>Microscope</td></tr><tr><td>Light source</td><td>Fostec</td><td>P/N 20520</td><td>Light source</td></tr></tbody></table>

an alternative and validated injection method for accessing the subretinal space via a transcleral posterior approach

视网膜下注射已在人类和啮齿类动物被成功地用于递送蛋白质的治疗性干预，抗病毒剂，以及细胞到具有直接暴露于光感受器和视网膜色素上皮细胞（RPE）的interphotoreceptor /视网膜下舱。纤维蛋白溶酶原的视网膜下注射，以及最近的临床前和临床试验已经证明递送病毒载体和干细胞的个体具有先进视网膜疾病的安全性和/或功效。视网膜疾病，特别是遗传性视网膜营养不良的小鼠模型，用于测试这些治疗是必不可少的。在啮齿类动物中最常见的注射程序是用小transcorneal或经巩膜切口用前路到视网膜。通过这种方法，注射针头穿透视网膜神经上皮层破坏底层RPE和插入可以轻松尼克镜头，引起晶状体混浊及无创意马减值准备NG。通过经巩膜访问视网膜下的空间，后路避免了这些问题:针穿过巩膜从视神经约为0.5毫米，没有视网膜渗透并避免破坏玻璃体。附带损害仅限于与焦点巩膜和一过性，浆液性视网膜脱离的影响有关。该方法的简单性最大限度地减少眼外伤，确保快速视网膜复位和恢复，并具有低故障率。视网膜和RPE的损害最小允许的疗效明确评估和治疗剂本身的直接影响。该原稿描述了一种新颖的视网膜下注射技术，可用于靶向病毒载体，药剂，干细胞或诱导的多能干细胞（iPS细胞），以在具有高效力，最小的损伤，快恢复小鼠的视网膜下间隙。

后路经巩膜视网膜下注射了31健康的眼睛用0.3微升（N = 18），0.5微升（N = 8）和0.01％荧光素的1.0微升（N = 5）注射执行16个野生型小鼠。一只眼睛由于该防止结构和功能分析一个预先存在的角膜混浊排除注射。每次注射的眼睛是包括本报告。没有意外的视网膜脱离，检测也没有透镜切口，炎症反应，葡萄膜炎，或外科手术后的感染中观察到的任何眼睛的任何证据的神经视网?...

Watch this Scientific Journal Video about An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach at JoVE.com

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

用于访问视网膜下腔的另类和验证注射法<i&gt;通过</i&gt;一经巩膜后路

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

an-alternative-validated-injection-method-for-accessing-subretinal

Research

JoVE Journal

Neuroscience

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

13.1K 次观看.  University of California, Los Angeles. Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

视频: An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

科研

医学

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

神经科学

A Step by Step Protocol for Subretinal Surgery in Rabbits

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

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

一种用于兔视网膜下手术分步协议

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

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into &#39;adult&#39; rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

经瞳孔引导下视网膜下移植物的视网膜下移植物经巩膜移植视网膜变性小鼠模型

Transplantation of photoreceptor cells and retinal pigment&#160;epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. ...

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

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

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

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

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

生物学

焕活视力:干细胞疗法治疗视网膜变性

Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal diseases at advanced stages. We have shown human recombinant retina-specific laminin isoform matrix is able to support the differentiation of human embryonic stem cells (hESCs) to photoreceptor progenitors. In addition, sub-retinal injection of these cells has also shown partial restoration in the rd10 rodent and rabbit models. Sub-retinal injection is known to be an established method that has been used to deliver pharmaceutical compounds to the photoreceptor cells and retinal pigmented epithelial (RPE) layer of the eye due to its proximity to the target space. It has also been used to deliver adeno-associated viral vectors into the sub-retinal space to treat retinal diseases. The sub-retinal delivery of pharmaceutical compounds and cells in the murine model is challenging due to the constraint in the size of the murine eyeball. This protocol describes the detailed procedure for the preparation of hESC-derived photoreceptor progenitor cells for injection and the sub-retinal delivery technique of these cells in genetic retinitis pigmentosa mutant, rd10 mice. This approach allows cell therapy to the targeted area, in particular the outer nuclear layer of the retina, where diseases leading to photoreceptor degeneration occur.

作者聚焦:推进视力恢复 - 基于干细胞的视网膜疾病疗法 

We describe a detailed protocol for the preparation of post-cryopreserved hESC-derived photoreceptor progenitor cells and the sub-retinal delivery of these cells in rd10 mice.

rd10小鼠中人胚胎干细胞衍生的光感受器祖细胞的视网膜下递送

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal ...

用于访问视网膜下腔的另类和验证注射法

摘要

探索更多视频

此视频中的章节

视网膜下进行注射的鼠害提供视网膜色素上皮细胞悬浮

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

一种用于兔视网膜下手术分步协议

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

非人灵长类动物模型中视网膜色素上皮移植治疗退行性视网膜疾病

经瞳孔引导下视网膜下移植物的视网膜下移植物经巩膜移植视网膜变性小鼠模型

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

rd10小鼠中人胚胎干细胞衍生的光感受器祖细胞的视网膜下递送

rd10小鼠中人胚胎干细胞衍生的光感受器祖细胞的视网膜下递送

rd10小鼠中人胚胎干细胞衍生的光感受器祖细胞的视网膜下递送