Abstract：
Abnormal alpha-synuclein (αSyn) and iron accumulation in the brain play an important role in Parkinson\'s disease (PD). Herein, we aim to visualize αSyn inclusions and iron deposition in the brains of M83 (A53T) mouse models of PD in vivo. The fluorescent pyrimidoindole derivative THK-565 probe was characterized by means of recombinant fibrils and brains from 10- to 11-month-old M83 mice. Concurrent wide-field fluorescence and volumetric multispectral optoacoustic tomography (vMSOT) imaging were subsequently performed in vivo. Structural and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) at 9.4 T as well as scanning transmission x-ray microscopy (STXM) were performed to characterize the iron deposits in the perfused brains. Immunofluorescence and Prussian blue staining were further performed on brain slices to validate the detection of αSyn inclusions and iron deposition. THK-565 showed increased fluorescence upon binding to recombinant αSyn fibrils and αSyn inclusions in post-mortem brain slices from patients with PD and M83 mice. Administration of THK-565 in M83 mice showed higher cerebral retention at 20 and 40 min post-intravenous injection by wide-field fluorescence compared to nontransgenic littermate mice, in congruence with the vMSOT findings. SWI/phase images and Prussian blue indicated the accumulation of iron deposits in the brains of M83 mice, presumably in the Fe3+ form, as evinced by the STXM results. In conclusion, we demonstrated in vivo mapping of αSyn by means of noninvasive epifluorescence and vMSOT imaging and validated the results by targeting the THK-565 label and SWI/STXM identification of iron deposits in M83 mouse brains ex vivo.
摘要：
异常α-突触核蛋白(αSyn)和脑内铁积累在帕金森病(PD)中起重要作用。在这里，我们旨在观察体内PD的M83（A53T）小鼠模型的大脑中αSyn内含物和铁沉积。荧光嘧啶吲哚衍生物THK-565探针通过10至11个月大的M83小鼠的重组原纤维和大脑进行表征。随后在体内同时进行宽场荧光和体积多光谱光声断层扫描（vMSOT）成像。进行9.4T的结构和磁敏感加权成像（SWI）磁共振成像（MRI）以及扫描透射X射线显微镜（STXM）以表征灌注大脑中的铁沉积物。在脑切片上进一步进行免疫荧光和普鲁士蓝染色以验证αSyn内含物和铁沉积的检测。THK-565在与PD和M83小鼠患者的死后脑切片中的重组αSyn原纤维和αSyn内含物结合后显示出增加的荧光。与非转基因同窝小鼠相比，在M83小鼠中施用THK-565在静脉注射后20和40分钟通过宽视野荧光显示出更高的脑retention留，与vMSOT的发现一致。SWI/相位图像和普鲁士蓝表明M83小鼠大脑中铁沉积物的积累，大概是Fe3+的形式，如STXM结果所示。总之,我们通过非侵入性落射荧光和vMSOT成像证明了αSyn的体内定位，并通过靶向THK-565标记和SWI/STXM鉴定M83小鼠大脑中的铁沉积物来验证结果。
