Arango-Lievano Margarita, Giannoni Patrizia, Claeysen Sylvie, Marchi Nicola, Jeanneteau Freddy
Inserm, U1191, Institute of Functional Genomics; CNRS, UMR-5203; Université de Montpellier;
Inserm, U1191, Institute of Functional Genomics; CNRS, UMR-5203; Université de Montpellier.
J Vis Exp. 2016 Dec 6(118):54796. doi: 10.3791/54796.
Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 μm of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer's disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.
脑脉管系统重塑是脑部病变的一个常见特征。体内成像技术对于检测随时间推移以及与神经元活动或血流相关的脑血管可塑性或损伤至关重要。体内双光子显微镜可用于研究活体大脑中大型细胞单元的结构和功能可塑性。特别是，薄颅骨窗口制备可使感兴趣的皮质区域（ROI）可视化，而不会引发明显的脑部炎症。对皮质ROI进行重复成像 sessions 是可行的，这为众多中枢神经系统疾病进展过程中疾病特征随时间的变化提供了表征。这种能够观察大脑250μm范围内软脑膜结构的技术依赖于对由遗传细胞标记物和/或活性染料编码的荧光探针的检测。后者（例如荧光葡聚糖）用于绘制脑血管结构的管腔部分。与本文所述方案相关的是使用淀粉样蛋白沉积物的体内标记物甲氧基 - O4来评估阿尔茨海默病（AD）的进展。我们还描述了用于跟踪血管变化和淀粉样蛋白沉积的采集后图像处理。虽然目前专注于AD模型，但所描述的方案与发生病理性脑血管变化的其他中枢神经系统疾病相关。
