急性视网膜模型评估血视网膜屏障违约和潜在的药物治疗
Summary
一个低成本，易于使用和强大的制度的建立，以评估可能改善组胺引起血视网膜屏障破坏的潜在治疗。血管渗漏，穆勒细胞活化和神经过程的连续性被用来评估损伤反应及其逆转与潜在的药物，脂氧素A4。
Abstract
A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of Müller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of Müller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.
Introduction
越来越多的证据支持体血视网膜屏障（BRB）1-5和其相似的血脑屏障（BBB）6,7的存在。血脑屏障的折衷已经紧密连锁因果或作为诊断标志物的慢性神经变性疾病如阿尔茨海默氏病（AD）8,9和急性病症如谵妄10。机械分析上市这些病状和发现的潜在的药物靶标一般是由脑的有限访问性和网络复杂阻碍。替代品，如在体内成像11，脑器官型培养12，原代细胞培养13,14和共培养系统15已经产生。然而，大多数这些模型需要特殊的仪器，实验长周期或多个标记来识别细胞。血脑屏障和BRB之间的功能和结构相似性，以及镝之间的相关性两个sfunctions已经争论16-19。此外，更容易获得的，定义良好的细胞类型和层状结构已经允许充分表征视网膜作为窗口到大脑。血脑屏障和BRB的结构和功能的身份留在详细地进行比较。然而，视网膜病变，尤其是BRB违约，也被紧紧与各种疾病的进展，包括糖尿病18-19和AD 21,22相关联。因此，感兴趣的是建立一个BRB功能障碍系统不仅划定机制，但也以筛选潜在药物。在这份报告中，用一个简单的急性视网膜文化协议，实现BRB功能障碍是制定并提交。
增加血脑屏障通透性和AD样病理改变已经建立了与组 胺，促炎症介质12孵育大脑器官文化。因此，在所提出的系统中，组胺是申请灭蝇灯对体外培养的视网膜诱导BRB功能障碍。从几个物种，如小家鼠和普通牛视网膜，进行了测试。由于它们的商业可用性和相似之处人体组织，新鲜的猪眼球，用来提供此报告的数据。组胺和/或其他药物孵育后，视网膜被免疫染色几种蛋白12，例如免疫球蛋白G（IgG）的，血液的主要成分之一处理进行评估;胶质纤维酸性蛋白（GFAP），神经胶质激活一个公知的标记;和微管相关蛋白2（MAP2），一个特定的神经元细胞骨架蛋白对微管组装至关重要。此外，视网膜的层状结构允许Müller细胞和神经节细胞的过程的详细分析，如在它们的宽度和连续性变化。因此几个附加参数可用来评估的后果BRB违反在早期阶段和评估的潜在治疗逆转的效果好。
血管渗漏（BVS），神经胶质细胞的活化和神经元细胞的损伤响应：在该协议中，筛选药物的潜在反转效果从三个方面进行评估。几个量化方法被利用，例如，通过免疫染色，处理并通过增强滤波器示出神经元过程的连续性的宽度测量的强度中所示的表达水平。为了更好地说明该方法，并帮助解释结果，脂氧素A4（LXA4），响应炎性损伤和衰减内皮功能障碍23内源性合成的化合物，已被选定为示范的目的。
Protocol
Representative Results
Discussion
In this report, we present a powerful ex vivo acute retinal model of BRB dysfunction using the swine retina. This model system does not require special instruments and can be easily adapted under most laboratory settings. However, to obtain a successful result, several steps require close attention. After obtaining the eyeballs from the source, they must be kept at 4 °C or on ice and processed as soon as possible. When the effect of a treatment is being analyzed, two halves of the same retina must be used -…
Declarações
The authors have nothing to disclose.
Acknowledgements
Bringhurst Meats (Berlin, NJ) is acknowledged for their genuine help in providing the swine eyeballs.
Materials
| DMEM | Life Technologies | 11965-092 | |
| HBSS | Life Technologies | 14170-112 | |
| Sucrose | J.T.Baker | 4072-05 | |
| Histamine | Sigma | H7125-1G | |
| Penicillin-Streptomycin | Invitrogen | ||
| PFA | Electron Microscopy Sciences | 15710 | |
| Freezing Media | Triangle Biomedical Sciences | TFM-5 | |
| Normal Goat Serum | Rockland | D104-00-0050 | |
| Triton X-100 | Sigma | T8787 | |
| GFAP Antibody | Millipore | AB5804 | |
| MAP2 Antibody | EMD Millipore | MAB3418 | |
| FITC conjugated Donkey anti-rabbit IgG | Jackson ImmunoResearch Laboratories, Inc. | 711-095-152 | |
| Cy3 conjugated Donkey anti-mouse IgG | Jackson ImmunoResearch Laboratories, Inc. | 715-165-150 | |
| mounting medium containing DAPI | Vector Laboratories, Inc. | H-1200 | |
| Laser Confocal Microscope | Nikon | Eclipse Ti microscope | |
| ImageJ | National Institutes of Health | 1.45s |
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