纵<em>在体内</em>的Cerebrovasculature影像：相关性疾病CNS
Summary
该原稿描述的过程使用纵向双光子显微镜体内淀粉样蛋白斑积累期间跟踪cerebrovasculature的重塑。减薄头骨制备使荧光染料的可视化，以评估在阿尔茨海默氏病的小鼠模型中脑血管损伤的进展。
Abstract
大脑脉管系统的重塑是脑病理学的一个共同特征。 体内成像技术是检测脑可塑性或损害的发生加班费和相对于神经元活性或血流的基础。 体内双光子显微镜允许在客厅大脑细胞大单位结构和功能可塑性的研究。特别是，减薄-颅骨窗制剂允许的感兴趣区域（ROI）的皮质区域的可视化而不诱导显著脑炎症。皮质的投资回报率的重复成像会议是可行的，许多中枢神经系统疾病的进展过程中提供疾病特点随时间变化的表征。这种技术在访问内250μm的大脑的软脑膜结构依赖于检测通过基因细胞标记和/或活体染料编码的荧光探针。后者（ 例如，荧光葡聚糖）被用于映射LUMIN脑结构的人舱。锗本文所述的协议是使用淀粉样蛋白沉积物的体内标记物，甲氧基O4，以评估阿尔茨海默氏病（AD）进展。我们还描述了用于跟踪血管变化和淀粉样沉积的采集后图像处理。虽然目前专注于AD模型，描述的协议是有关在那里发生的病理变化脑血管病其他中枢神经系统疾病。
Introduction
脑血管是一个多细胞结构，这是在解剖学和功能上耦合到神经元。船只的动态重构发生在整个大脑发育和中枢神经系统（CNS）1,2的病状的进展期间。它已被广泛接受脑损伤是几种中枢神经系统疾病，包括癫痫，阿耳茨海默氏病（AD），创伤性脑损伤的一个标志和脑炎3,4。因此， 在体内追踪脑血管变化建模中枢神经系统疾病时，从发病进入慢性期变得显著。由于脑血管修改常与神经元损伤或可塑性伴随发生时，神经血管成像是一个关键切入点破译CNS疾病的病理生理机制。
这个协议描述了一个纵向的双光子基于程序来跟踪cerebrovasculature的重构的小鼠模型AD，由于淀粉样蛋白斑块沉积5-7的大，小口径血管脑血管缺陷标志着一个渐进的病理。此过程允许淀粉样蛋白沉积和其位置和生长的跟踪的可视化对于神经血管重塑整个病程。重要的荧光染料的cerebrovasculature和淀粉样蛋白斑在转基因小鼠公元8可视化每个成像会议召开前被注入。通过减薄头骨颅窗口ROI的重复成像会议是非侵入性的和选择的方法在活老鼠的大脑2,5,9,10评估神经血管重塑。
下面的步骤概括了手术方案，图像采集和处理。淀粉样脑血管病（CAA）主要在软脑膜大与穿透性小动脉的早期发展的特点。
Protocol
Representative Results
Discussion
体内双光子显微镜开放式颅骨技术提供大量成像领域13,14无限成像会议的优势。然而，该技术也是在感兴趣区域14产生炎症，经常不相容或影响神经血管读数15。相反，减薄头骨颅技术不会导致神经炎症，使脑血管结构的可靠的成像和菌斑积累10,14。通过这种技术提出的第二个优点是稀疏的颅骨窗口的成功率可达80％〜90％，对于有经验的操纵者。成功的重大?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
笔者想承认法甲法兰西驳L'épilepsie（MA到-L），国家研究所德拉桑特等德拉RECHERCHE MEDICALE格兰特AVENIR R12087FS（FJ到），从蒙彼利埃大学教育资助（为FJ）和格兰特距离联邦倒拉RECHERCHE河畔乐Cerveau（以NM）。我们承认Chrystel的乐峰在IPAM在蒙彼利埃的体内成像核心平台设施的技术援助。我们也感谢玛丽Vernov（威尔康乃尔医学院）校对稿件。
Materials
| methoxy-X04 | tocris | 4920 | use 10 mg/Kg |
| FITC-Dextran 70Kda | sigma | 46945 | use 100 mg/Kg |
| gelfoam/Bloxang | Bausch and Lomb | ||
| micorsurgical blade | surgistar | 6900 | must be sharp and not dented |
| povidone-iodine | betadine | antisceptic solution | |
| binocular stereomicroscope | olympus | SX10 | optimal image contrast is crucial for this procedure |
| 2-photon microscope | zeiss | Zeiss LSM 710mp | |
| fine scissors-toughcut | Fine science tools | 14058-09 | this scissors are optimized for cutting skin and soft tissue |
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