用于访问视网膜下腔的另类和验证注射法<i>通过</i>一经巩膜后路
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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，?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Hamilton Model 62 RN SYR | Hamilton | 87942 | Syringe x 1 |
| Hamilton Needle 33G, 1.0", 20 DEG, point 3 (304 stainless steel) | Hamilton | 7803-05 | Needles x 6 |
| Vannas Curved Scissors | Ted Pella, INC. | 1347 | 5mm Blade |
| 22.5 Degree Microsurgery Knife | Wilson Ophthalmic Corp. | 91204 | |
| Ketaject | Phoenix | NDC 57319-609-02 | Ketamine |
| Anased | Lloyd Laboratories | NDC 61311-482-10 | Xylazine |
| Fluorescein 10% AK-Fluor | Akorn | NDC 17478-253-10 | 100mg/ml |
| 0.9% Saline USP | Hospira | NDC 0409-4888-50 | 0.9% NaCl |
| Antibiotic Ointment | Akorn | NDC 17478-235-35 | Ophthalmic |
| Water Circulating Pump | Gaymar | TP-500 T/Pump | P/N 07999-000 |
| sd-OCT | Bioptigen | R-Series | Commercial |
| Fundus Camera | Phoenix Research Laboratories | MICRON III | |
| Tweezers Type 3 | Ted Pella, INC. | 5385-3SU | |
| 2.5% Phenylephrine | Paragon BioTeck | NDC 42702-102-15 | Ophthalmic |
| IMARIS8 | Bitplane | Version 8.1.2 | |
| ImageJ | NIH | V1.8.0_77 | |
| Hypromellose 2.5% | Goniovisc | AX0401 | Methylcellulose |
| Eye Drops (Rinse) | Bausch & Lomb | Saline Solution | |
| Microscope | Zeiss | Stemi 2000 | Microscope |
| Light source | Fostec | P/N 20520 | Light source |
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