Brigham and Women's Hospital, Center for Neurologic Diseases, Partner Multiple Sclerosis Center, Harvard Medical School, Department of Neurology, Boston, Massachusetts 02115, USA.
J Biomed Opt. 2011 Feb;16(2):021109. doi: 10.1117/1.3533312.
Myelin loss and axonal degeneration predominate in many neurological disorders; however, methods to visualize them simultaneously in live tissue are unavailable. We describe a new imaging strategy combining video rate reflectance and fluorescence confocal imaging with coherent anti-Stokes Raman scattering (CARS) microscopy tuned to CH(2) vibration of myelin lipids, applied in live tissue of animals with chronic experimental autoimmune encephalomyelitis (EAE). Our method allows monitoring over time of demyelination and neurodegeneration in brain slices with high spatial resolution and signal-to-noise ratio. Local areas of severe loss of lipid signal indicative of demyelination and loss of the reflectance signal from axons were seen in the corpus callosum and spinal cord of EAE animals. Even in myelinated areas of EAE mice, the intensity of myelin lipid signals is significantly reduced. Using heterozygous knock-in mice in which green fluorescent protein replaces the CX(3)CR1 coding sequence that labels central nervous system microglia, we find areas of activated microglia colocalized with areas of altered reflectance and CARS signals reflecting axonal injury and demyelination. Our data demonstrate the use of multimodal CARS microscopy for characterization of demyelinating and neurodegenerative pathology in a mouse model of multiple sclerosis, and further confirm the critical role of microglia in chronic inflammatory neurodegeneration.
髓鞘损失和轴突退化在许多神经疾病中占主导地位；然而，在活体组织中同时可视化它们的方法尚不可用。我们描述了一种新的成像策略，将视频速率反射和荧光共焦成像与相干反斯托克斯拉曼散射（CARS）显微镜结合，该显微镜调谐到髓鞘脂质的 CH(2)振动，应用于患有慢性实验性自身免疫性脑脊髓炎（EAE）的动物的活体组织中。我们的方法允许以高空间分辨率和信噪比实时监测脑切片中的脱髓鞘和神经退行性变。在 EAE 动物的胼胝体和脊髓中，可以看到脱髓鞘和轴突反射信号丢失的脂质信号严重丢失的局部区域。即使在 EAE 小鼠的髓鞘区域，髓鞘脂质信号的强度也显著降低。使用杂合敲入小鼠，其中绿色荧光蛋白取代标记中枢神经系统小胶质细胞的 CX(3)CR1 编码序列，我们发现激活的小胶质细胞区域与反射和 CARS 信号改变的区域共定位，反映轴突损伤和脱髓鞘。我们的数据证明了多模态 CARS 显微镜在多发性硬化症小鼠模型中用于表征脱髓鞘和神经退行性病变的用途，并进一步证实了小胶质细胞在慢性炎症性神经退行性变中的关键作用。
