小鼠模型的激光诱导脉络膜新生血管
Summary
Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.
Abstract
The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch’s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD.
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects.
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.
Introduction
年龄相关性黄斑变性（AMD）是失明的个体50 1-3岁以上的主要原因之一。 AMD可分为两种形式：萎缩性（“干”）AMD和新生血管（“湿”），AMD。前者的特征在于视网膜色素上皮细胞（RPE），脉络膜，和光感受器地理萎缩，而后者的特征在于侵入从脉络膜异常血管的插入外视网膜层造成渗漏，出血和纤维化，最终导致失明1,2。这两种形式中，新生血管性AMD占绝大多数视力减退1。幸运的是，这种形式有许多有效的药物管理选项，而它的萎缩对应目前还没有成熟的医学治疗3。此外，由于新生血管形态已经很容易地重新投降的动物模型，它已经被越来越广泛地接触到基本的AMD研究探索潜在的病理机制，以便开发新型疗法4。
由Ryan等人开发的实验性脉络膜新生血管形成（CNV）的第一动物模型。在非人灵长类5。布鲁赫膜通过激光光凝该模型诱发破裂，这引起导致血管生成类似于见于新生血管性AMD的局部炎症反应。血管生成后的激光诱导的组织病理学进展，发现以模仿新生血管性AMD，这证实了模型的有效性6。非人灵长类提供最相似解剖人类，但不幸的是，维护费用昂贵，不能轻易遗传操作，并且有疾病进展7的慢时间过程。与此相反，啮齿动物模型是更经济有效的维护，可以遗传操作相对容易，并具有更快的凑疾病进展的RSE（实验可以在周的时间尺度来进行与个月）。这些实验应该只在有色啮齿类动物中进行，因为它是非常困难的白化动物来可视化。
鼠标激光诱导CNV模型，首先由坎波基亚罗集团在90年代末10开发，已经成长为在大多数最近的研究11-16的占主导地位的动物模型。由于CNV的复杂，目前还不清楚发病机理，激光模式已在湿性AMD的研究，从学习驾驶血管生成来评估新的治疗模式为未来的人类使用的分子机制各个方面的应用。例如，Sakurai等人和埃斯皮诺萨，Heidmann 等 。使用的激光模型，探讨巨噬细胞对CNV的使用转基因小鼠和药理耗尽处理15，16中的发展的影响。吉亚尼等 。和Hoerster 等。用的光学相干断层扫描（OCT），以图像的激光诱导的CNV在努力表征CNV的进展和比较组织病理学结果以看到的OCT成像12,17的调查结果。最后，涉及玻璃体内注射的抗血管生成剂的研究已被用作先决条件人体试验并分别在显影的第一代中新生血管性AMD今天10,18,19的管理中使用的抗VEGF剂是至关重要的。
替代模式的实验CNV利用手术的方法，诱导CNV。此过程涉及注射促血管生成物质 （如重组病毒载体过量表达血管内皮生长因子，视网膜下注射RPE细胞和/或聚苯乙烯珠），以模仿见于新生血管性AMD的增加的VEGF表达，以引起血管生成8,20的目标。然而，这种方法产生新血管形成的显着较低的发生率;这些研究显示，在CNVC57 / BL6小鼠中发生注射抗小鼠8,14的相同应变出现在激光光凝法的〜70％的成功率的31％。由于这些原因，并给予使用啮齿类动物对非人类灵长类动物的优势，激光诱导CNV的小鼠模型已成为CNV的标准动物模型对于大多数新生血管性AMD的研究实验8。
鼠标的眼睛是微乎其微的，细腻的组织一起工作。眼可视化的视网膜机动是困难的，需要大量的练习，直到掌握为止。这个任务是由一个事实，即它必须与显性和非优势手学习复杂。此外，以显现视网膜所需的精细动作已经被学习之后，双手和脚踏板操作激光之间的协调是重要的。在本文中，我们试图以蒸馏学习所有参与的激光诱导的CNV进程内的物理操纵的挑战edure成一个指南，将帮助运营商实现快速成功，这种模式。
Protocol
Representative Results
Discussion
有成功的激光诱导后会影响激光传输和由此产生的CNV病变的发展多因素。这些因素应控制为与标准化，以便能有最可靠的结果。最相关的这些因素中的鼠标选择（基因型，年龄和性别），麻醉剂的选择，以及激光设置。
使用的特定小鼠模型可以对CNV的发展过程中一个显著效果。最广泛使用的基因型是C57BL / 6小鼠。从该动物得到的供应商可以影响所得CNV大小。普尔等人。?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge Jonathan Chou, MD for his assistance on preparation and editing of the final manuscript and Wenzhong Liu for the OCT data. We would also like to acknowledge support from the Macula Society Research Grant (AAF), support from an unrestricted grant to Northwestern University from Research to Prevent Blindness, Inc., New York, NY, USA, and support from NIH-EY019951.
Materials
| 532 nm (green) argon ophthalmic laser | IRIDEX | GLx | any ophthalmic 532 nm (green) argon laser can be used |
| slit lamp | Carl Zeiss | 30SL-M | any slit lamp can be used as long as it is compatible with the laser |
| tribromoethanol | Sigma | T48402-25G | used to make anesthetic |
| tert-amyl alcohol | Sigma | 152463-1L | used to make anesthetic |
| amber glass vials + septa | Wheaton | WH-223696 | tribromoethanol storage |
| tissue wipes | VWR | 82003-820 | miscellaneous |
| 1% tropicamide | Falcon Pharmaceuticals | RXD2974251 | pupillary dilation |
| 0.5% tetracaine hydrochloride | Alcon | 0065-0741-12 | topical anesthesia |
| artificial tears | Alcon | 58768-788-25 | hydration |
| heat therapy pump (for animal warming) | Kent Scientific | HTP-1500 | used to maintain animal body temp |
| warming pad | Kent Scientific | TPZ-0510EA | maintains animal body temperature |
| 30 gauge insulin needles | BD | 328418 | IP anesthesia injection |
| scale | American Weigh Scale | AWS-1KG-BLK | mouse weighing |
| cover slip (25 mm x 25 mm) | VWR | 48366089 | flatten cornea to visualize mouse retina |
| xylazine | obtained from institution | obtained from institution | anesthesia |
| ketamine | obtained from institution | obtained from institution | anesthesia |
| Volocity | PerkinElmer | used for volumetric re-construction | |
| ImageJ | National Institutes of Health | used for image analysis |
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