优化视网膜静脉遮挡小鼠模型以限制变异性
1Department of Pathology & Cell Biology; Vagelos College of Physicians and Surgeons,Columbia University, 2Department of Molecular Pharmacology and Therapeutics; Vagelos College of Physicians and Surgeons,Columbia University, 3Department of Neurology; Vagelos College of Physicians and Surgeons,Columbia University, 4The Taub Institute for Research on Alzheimer’s Disease and the Aging Brain; Vagelos College of Physicians and Surgeons,Columbia University
概要
在这里，我们描述了使用孟加拉玫瑰和激光引导视网膜成像显微镜系统进行视网膜静脉阻塞的优化方案，并建议最大限度地提高其在转基因菌株中的可重复性。
Abstract
视网膜静脉阻塞（RVO）的小鼠模型通常用于眼科研究神经视网膜中的缺氧缺血损伤。在本报告中，提供了一种详细的方法，指出了关键步骤，并提供了优化建议，以在不同转基因小鼠品系中实现一致的成功闭塞率。RVO小鼠模型主要包括静脉内施用光敏剂染料，然后使用连接到眼科引导激光的视网膜成像显微镜进行激光光凝。三个变量被确定为遮挡一致性的决定因素。通过调整孟加拉玫瑰给药后的等待时间并平衡基线和实验激光输出，可以限制实验之间的变异性，并获得更高的遮挡成功率。该方法可用于研究以视网膜水肿和缺氧缺血性损伤为特征的视网膜疾病。此外，由于该模型诱导血管损伤，它也可以应用于研究神经脉管系统、神经元死亡和炎症。
Introduction
视网膜静脉阻塞 （RVO） 是一种常见的视网膜血管疾病，2015 年影响了全球约 2800 万人1。RVO导致工作的老年人和老年人视力下降和丧失，这是一种持续的视力威胁疾病，估计在近十年内会增加。RVO 的一些不同病变包括缺氧缺血性损伤、视网膜水肿、炎症和神经元丢失2。目前，这种疾病的一线治疗是通过给予血管内皮生长因子（VEGF）抑制剂。虽然抗VEGF治疗有助于改善视网膜水肿，但许多患者仍面临视力下降3。为了进一步了解这种疾病的病理生理学并测试潜在的新治疗线，需要为不同的小鼠品系构建功能性和详细的RVO小鼠模型方案。
已经开发了小鼠模型，实现了与人类患者相同的激光设备，并与缩放到鼠标正确尺寸的成像系统配对。这种RVO小鼠模型于2007年首次报道4，并由Ebneter等人4，5进一步建立。最终，Fuma等人对该模型进行了优化，以复制RVO的关键临床表现，例如视网膜水肿6。自该模型首次报道以来，许多研究已经使用它，使用光敏染料，然后用激光对视网膜主要静脉进行光凝。然而，在使用这种方法的研究中，施用染料的数量和类型、激光功率和暴露时间差异很大。这些差异通常会导致模型的可变性增加，使其难以复制。迄今为止，还没有已发表的研究详细说明其优化的潜在途径。
本报告介绍了 C57BL/6J 菌株中的 RVO 小鼠模型的详细方法和他莫昔芬诱导的内皮半胱天冬酶 9 敲除 （iEC Casp9KO） 菌株，具有 C57BL/6J 背景，与 RVO 病理学相关，作为转基因小鼠的参考菌株。先前的一项研究表明，内皮半胱天冬酶-9的非凋亡激活会引起视网膜水肿并促进神经元死亡8。使用该菌株的经验有助于确定并提供对潜在修改的见解，以定制RVO小鼠模型，该模型可适用于其他转基因菌株。
Protocol
该协议遵循视觉和眼科研究协会（ARVO）关于在眼科和视力研究中使用动物的声明。啮齿动物实验由哥伦比亚大学机构动物护理和使用委员会（IACUC）批准和监测。 注意：所有实验都使用重约20g的两个月大的雄性小鼠。 1.他莫昔芬用于絮状基因诱导遗传消融的制备和给药 注意：视网膜血管直径可能受动物体重的影响。确保用于实验的所?…
Representative Results
RVO小鼠模型旨在成功实现视网膜静脉闭塞，导致缺氧缺血损伤，血液视网膜屏障破裂，神经元死亡和视网膜水肿8。 图1 显示了确保可重复性的步骤时间表，实验设计的示意图，并概述了可以根据实验问题进一步优化的步骤。可以修改的三个主要步骤是孟加拉玫瑰给药后的等待时间、基线激光功率和实验激光输出。在本报告中，C57BL / 6J小鼠以及来自诱导性?…
Discussion
小鼠RVO模型为进一步了解RVO病理学和测试潜在治疗方法提供了途径。虽然鼠标RVO模型在该领域被广泛使用，但需要模型的当前详细协议来解决其可变性并描述模型的优化。在这里，我们提供了一个指南，其中包含经验示例，说明可以改变哪些内容，以便在一组实验动物中获得最一致的结果，并提供可靠的数据。
RVO小鼠模型的两个最重要的元素是光敏染料的激光输出和成功静?…
開示
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国家科学基金会研究生研究奖学金计划（NSF-GRFP）DGE – 1644869（致CCO），国家眼科研究所（NEI）5T32EY013933（致AMP）和国家老龄化研究所（NIA）R21AG063012（致CMT）的支持。
Materials
| Carprofen | Rimadyl | NADA #141-199 | keep at 4 °C |
| Corn Oil | Sigma-Aldrich | C8267 | |
| Fiber Patch Cable | Thor Labs | M14L02 | |
| GenTeal | Alcon | 00658 06401 | |
| Ketamine Hydrochloride | Henry Schein | NDC: 11695-0702-1 | |
| Lasercheck | Coherent | 1098293 | |
| Phenylephrine | Akorn | NDCL174478-201-15 | |
| Phoneix Micron IV with Meridian, StreamPix, and OCT modules | Phoenix Technology Group | ||
| Proparacaine Hydrochloride | Akorn | NDC: 17478-263-12 | keep at 4 °C |
| Refresh | Allergan | 94170 | |
| Rose Bengal | Sigma-Aldrich | 330000-5G | |
| Tamoxifen | Sigma-Aldrich | T5648-5G | light-sensitive |
| Tropicamide | Akorn | NDC: 174478-102-12 | |
| Xylazine | Akorn | NDCL 59399-110-20 |
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