The Brain Cognition and Brain Disease Institute (BCBDI), NMPA Key Laboratory for Research and Evaluation of Viral Vector Technology in Cell and Gene Therapy Medicinal Products, Shenzhen Key Laboratory of Viral Vectors for Biomedicine Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen, 518055, China.
Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences-Wuhan National Laboratory for Optoelectronics, 430071, Wuhan, PR of China.
Mol Psychiatry. 2024 Mar;29(3):545-552. doi: 10.1038/s41380-022-01580-0. Epub 2022 Apr 28.
Astrocytes constitute a major part of the central nervous system and the delineation of their activity patterns is conducive to a better understanding of brain network dynamics. This study aimed to develop a magnetic resonance imaging (MRI)-based method in order to monitor the brain-wide or region-specific astrocytes in live animals. Adeno-associated virus (AAVs) vectors carrying the human glial fibrillary acidic protein (GFAP) promoter driving the EGFP-AQP1 (Aquaporin-1, an MRI reporter) fusion gene were employed. The following steps were included: constructing recombinant AAV vectors for astrocyte-specific expression, detecting MRI reporters in cell culture, brain regions, or whole brain following cell transduction, stereotactic injection, or tail vein injection. The astrocytes were detected by both fluorescent imaging and Diffusion-weighted MRI. The novel AAV mutation (Site-directed mutagenesis of surface-exposed tyrosine (Y) residues on the AAV5 capsid) significantly increased fluorescence intensity (p < 0.01) compared with the AAV5 wild type. Transduction of the rAAV2/5 carrying AQP1 induced the titer-dependent changes in MRI contrast in cell cultures (p < 0.05) and caudate-putamen (CPu) in the brain (p < 0.05). Furthermore, the MRI revealed a good brain-wide alignment between AQP1 levels and ADC signals, which increased over time in most of the transduced brain regions. In addition, the rAAV2/PHP.eB serotype efficiently introduced AOP1 expression in the whole brain via tail vein injection. This study provides an MRI-based approach to detect dynamic changes in astrocytes in live animals. The novel in vivo tool could help us to understand the complexity of neuronal and glial networks in different pathophysiological conditions.
星形胶质细胞构成了中枢神经系统的主要部分，描绘其活动模式有助于更好地理解大脑网络动力学。本研究旨在开发一种基于磁共振成像（MRI）的方法，以便在活体动物中监测全脑或特定区域的星形胶质细胞。携带人类胶质纤维酸性蛋白（GFAP）启动子驱动 EGFP-AQP1（水通道蛋白 1，一种 MRI 报告基因）融合基因的腺相关病毒（AAV）载体被用于该研究。具体步骤包括：构建用于星形胶质细胞特异性表达的重组 AAV 载体，在细胞培养、脑区或全脑中转导后检测 MRI 报告基因，立体定向注射或尾静脉注射。通过荧光成像和弥散加权 MRI 检测星形胶质细胞。与 AAV5 野生型相比，新型 AAV 突变（AAV5 衣壳表面暴露的酪氨酸（Y）残基的定点突变）显著增加了荧光强度（p<0.01）。携带 AQP1 的 rAAV2/5 的转导导致细胞培养物中 MRI 对比的滴度依赖性变化（p<0.05）和脑内尾状核-壳核（CPu）的变化（p<0.05）。此外，MRI 显示 AQP1 水平与 ADC 信号之间具有良好的全脑一致性，在大多数转导的脑区中，这些信号随时间增加。此外，rAAV2/PHP.eB 血清型通过尾静脉注射可有效地在全脑中引入 AQP1 表达。本研究提供了一种基于 MRI 的方法来检测活体动物中星形胶质细胞的动态变化。这种新型的体内工具可以帮助我们理解不同病理生理条件下神经元和神经胶质网络的复杂性。
