使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍
Summary
To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.
Abstract
Müller细胞是视网膜的主要神经胶质细胞。其最终的脚形成的视网膜在外侧和内界膜（ILM）的范围内，并在与星形胶质细胞，周细胞和内皮细胞结合它们建立血 – 视网膜屏障（BRB）。 BRB限制了血液和视网膜之间的物质运输而在ILM充当基底膜限定组织学视网膜和玻璃体腔之间的边界。标记Müller细胞是特别相关，研究视网膜屏障的物理状态，因为这些细胞是BRB和ILM的一个组成部分。既BRB和ILM经常改变视网膜病和负责疾病症状。
有几种公认的方法来研究的BRB的完整性，如Evans蓝测定法或荧光血管造影。然而，这些方法不提供关于BRB渗透性吨的范围内的信息Ø较大的分子，在纳米范围内。而且，它们不提供对其他视网膜屏障如在ILM的状态信息。研究BRB渗透性沿着视网膜ILM，我们使用的AAV基础的方法，可提供对渗透性BRB对较大分子虽然表示在疾病状态在ILM和胞外基质蛋白的状态的信息。两个变种AAV对于这样的研究有用：AAV5和ShH10。 AAV5具有天然趋光感受器，但它不能得到跨越到外视网膜给药时进入玻璃体时在ILM是完整的（ 即，在野生型视网膜）。 ShH10具有朝向胶质细胞强烈的取向和在健康和患病的视网膜将选择性标记米勒胶质细胞。 ShH10提供视网膜更高效的基因传递，其中ILM受到损害。再加上免疫和血液DNA分析这些病毒工具揭示到的视网膜疾病障碍的状态。
Introduction
Müller细胞是视网膜的主要神经胶质成分。形态，它们跨越视网膜径向和它们endfeet，与玻璃体接触，面对在ILM和后者的秘密分量。在ILM是一种基底膜约十个不同的细胞外基质蛋白（层粘连蛋白，聚集蛋白，串珠素，巢蛋白，胶原蛋白和一些硫酸肝素蛋白聚糖）组成。在开发过程中，它的存在是不可或缺的视网膜组织发生神经节细胞1-3，视神经轴突的导航和生存。但是，ILM是非本质的成人视网膜，可以在某些病理手术切除，而不会造成视网膜损伤4。在基因治疗中，此膜变成为使用自动增值服务的视网膜高效转导通过玻璃体内注射5的物理屏障。
通过他们的流程的广泛树枝状，Müller细胞提供营养和监管suppoRT既视网膜神经细胞和血管细胞。 Müller细胞也参与视网膜动态平衡的调节，在BRB 6的形成和维持。紧密连接视网膜毛细血管内皮细胞之间，Müller细胞，星形胶质细胞和周形成BRB。 BRB防止某些物质进入retina.In许多疾病，如糖 尿病性视网膜病变，视网膜静脉阻塞和呼吸系统疾病，视网膜缺氧会导致通过BRB 7-9泄漏。这种断裂是与增加血管通透性，导致血管性水肿，视网膜脱离和视网膜损伤相关联。
Müller细胞被紧密地与血管和基底膜相关联，打在两BRB和ILM完整性中起重要作用。因此，标记缪勒胶质细胞是特别相关的这些视网膜屏障的物理状态的研究。
经典同盟，BRB渗透性是用伊文思蓝测定法，包括全身性注射Evans蓝染料，其结合非共价地与血浆白蛋白的测定。此法测量从血管进入视网膜（见协议第5条）10白蛋白漏出（蛋白质中等大小，〜66 kDa的）。另外，血管渗漏可以通过荧光血管造影视网膜的证明荧光素渗漏进行可视化（小分子〜359达;见协议第6条）11。然而，这两种方法允许BRB渗透性小分子和蛋白质的评估，但它们不提供关于在ILM的完整性信息。
因此，为了研究BRB渗透性，我们使用的AAV基础的方法，让上BRB渗透性至较大的分子（ 例如，AAV颗粒，25纳米的直径）的信息。事实上，我们的方法可以检测出的AAV转基因的存在于血液中，这将表明，〜25 nm的直径的颗粒会能够渗透到血液中。这种方法还提供了在病理条件下在ILM和细胞外基质蛋白质的结构信息。两个变种AAV对于这样的研究有用：AAV5和ShH10。视网膜下注射，AAV5具有天然向性为感光细胞和视网膜色素上皮细胞12，但是它不能获得跨越到外视网膜给药时进入玻璃体在野生型视网膜中有完整的ILM 5,13。 ShH10是已工程化以特异性靶向神经胶质细胞在神经元14,15的AAV变种。 ShH10选择性标记在健康和患病的视网膜与视网膜增加的效率受损障碍16Müller细胞。再加immuhistochemistry和血液DNA分析这些病毒的工具提供的视网膜屏障的状态和它们在疾病的参与（ 图1）的信息。
Protocol
Representative Results
Discussion
该BRB调节血液和视网膜之间的分子交换。其击穿与各种疾病，如糖尿病性视网膜病或年龄相关性黄斑变性（AMD）相关联。我们最近发现，在一个肌营养不良蛋白敲除小鼠，其中显示可渗透BRB，视网膜变得更宽容的由腺相关病毒载体（AAV）介导的基因传递。然而，尽管BRB渗透性的AAV颗粒喷入眼内保持局限于此模型中的眼隔室。我们的研究结果表明，基因治疗中显示渗透BRB并不代表全身副作用附加险?…
Declarações
The authors have nothing to disclose.
Acknowledgements
We thank the imaging platform of the Institut de la Vision. We acknowledge the French Muscular Dystrophy Association (AFM) for a PhD fellowship to O.V. and Allergan INC. This work performed in the frame of the LABEX LIFESENSES [reference ANR-10-LABX-65] was supported by French state funds managed by the ANR. We thank Peggy Barbe, and Mélissa Desrosiers for technical assistance with AAV preparations. We are grateful to Stéphane Fouquet for excellent technical assistance in confocal microscopy and his expert input with the interpretation of the results.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| C57BL6J mice strain | JANVIER LABS | mice | |
| Ketamine 500 | Virbac France | anesthetic | |
| Xylazine Rompun 2% | Bayer Healthcare | anesthetic | |
| Neosynephrine 5% Faure | Europhta | dilatant | |
| Mydriaticum 0,5% | Thea | dilatant | |
| Sterdex | Novartis | anti-inflammatory | |
| Cryomatrix embedding resin | Thermo Scientific | 6769006 | |
| Superfrost Plus Adhesion Slides | Thermo Scientific | 10143352 | slides |
| anti-laminin | Sigma | L9393 | antibody |
| anti-rhodopsin clone 4D2 | Millipore | MABN15 | antibody |
| anti-glutamine synthetase clone GS-6 | Millipore | MAB302 | antibody |
| Anti-Glial Fibrillary Acidic Protein | Dako | 334 | antibody |
| PNA Lectin | Invitrogen | L32459 | probe |
| Alexa fluor conjugated secondary antibodies | Invitrogen | antibody | |
| Fluorsave reagent | Calbiochem | 345789 | mounting medium |
| QIAmp DNA Micro Kit | QIAGEN | 56304 | |
| GoTaq DNA polymerase | Promega | M3001 | |
| Evans Blue dye | Sigma | E2129 | dye |
| 5 µm filter | Millipore | ||
| Sodium Citrate | Sigma | S1804 | |
| Citric acid | Sigma | C1909-2.5KG | |
| Formamide spectrophotometric | Sigma | 295876-2L | |
| Fluorescein | Sigma | F2456 | dye |
| Micron III | Phoenix Research Labs | Microscopy system based on 3-CCD color camera, frame grabber, and off-the-shelf software enables researchers to image mouse retinas. | |
| Insulin Syringes | Terumo | SS30M3109 | |
| Syringe 10 µl Hamilton | Dutscher | 74487 | Seringue 1701 |
| Needle RN G33, 25 mm, PST 2 | Fisher Scientific | 11530332 | Intravitreal Injection |
| UltraMicroPump UMP3 | World Precision Instruments | UMP3 | Versatile injector uses microsyringes to deliver picoliter volumes |
| UltraMicroPump (UMP3) (one) with SYS-Micro4 Controller | UMP3-1 | Digital controller | |
| Binocular magnifier SZ76 | ADVILAB | ADV-76B2 | Zoom 0.66 x 5 x LEDs with stand epi and dia / Retinas dissection |
| Spring scissors straight – 8,5cm | Bionic France S.a.r.l | 15003-08 | Retinas dissection |
| Micro-ciseaux de Vannas courbe | 15004-08 | ||
| Pince Dumont 5 | 11254-20 | ||
| Veriti 96-Well Thermal Cycler | Life technologies | 4375786 | Thermocycler |
| Ultrasonic cleaner | Laboratory Supplies | G1125P1T | |
| Nanosep 30k omega tubes | VWR | ||
| Speedvac | Fisher Scientific | SC 110 A | |
| Spectrofluorometer | TECAN | infinite M1000 |
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