Department of Synthetic Neurotechnology, Pirogov Russian National Research Medical University, Moscow 117997, Russia.
Faculty of Medicine, Sechenov First Medical University, Moscow 119991, Russia.
Int J Mol Sci. 2023 Aug 11;24(16):12688. doi: 10.3390/ijms241612688.
The development of new neurotherapeutics depends on appropriate animal models being chosen in preclinical studies. The cuprizone model is an effective tool for studying demyelination and remyelination processes in the brain, but blood-brain barrier (BBB) integrity in the cuprizone model is still a topic for debate. Several publications claim that the BBB remains intact during cuprizone-induced demyelination; others demonstrate results that could explain the increased BBB permeability. In this study, we aim to analyze the permeability of the BBB for different macromolecules, particularly antibody conjugates, in a cuprizone-induced model of demyelination. We compared the traditional approach using Evans blue injection with subsequent dye extraction and detection of antibody conjugates using magnetic resonance imaging (MRI) and confocal microscopy to analyze BBB permeability in the cuprizone model. First, we validated our model of demyelination by performing T2-weighted MRI, diffusion tensor imaging, quantitative rt-PCR to detect changes in mRNA expression of myelin basic protein and proteolipid protein, and Luxol fast blue histological staining of myelin. Intraperitoneal injection of Evans blue did not result in any differences between the fluorescent signal in the brain of healthy and cuprizone-treated mice (IVIS analysis with subsequent dye extraction). In contrast, intravenous injection of antibody conjugates (anti-GFAP or non-specific IgG) after 4 weeks of a cuprizone diet demonstrated accumulation in the corpus callosum of cuprizone-treated mice both by contrast-enhanced MRI (for gadolinium-labeled antibodies) and by fluorescence microscopy (for Alexa488-labeled antibodies). Our results suggest that the methods with better sensitivity could detect the accumulation of macromolecules (such as fluorescent-labeled or gadolinium-labeled antibody conjugates) in the brain, suggesting a local BBB disruption in the demyelinating area. These findings support previous investigations that questioned BBB integrity in the cuprizone model and demonstrate the possibility of delivering antibody conjugates to the corpus callosum of cuprizone-treated mice.
新神经治疗药物的开发依赖于临床前研究中选择合适的动物模型。铜诱导模型是研究大脑脱髓鞘和髓鞘再生过程的有效工具，但铜诱导模型中的血脑屏障 (BBB) 完整性仍然存在争议。一些出版物声称，在铜诱导的脱髓鞘过程中，BBB 保持完整；而其他研究结果则表明 BBB 通透性增加。在这项研究中，我们旨在分析不同大分子，特别是抗体偶联物，在铜诱导的脱髓鞘模型中的 BBB 通透性。我们比较了传统的方法，使用 Evans 蓝注射，随后进行染料提取，并使用磁共振成像 (MRI) 和共聚焦显微镜检测抗体偶联物，以分析铜诱导模型中的 BBB 通透性。首先，我们通过进行 T2 加权 MRI、扩散张量成像、定量 RT-PCR 检测髓鞘碱性蛋白和蛋白脂质蛋白 mRNA 表达的变化以及髓鞘卢索快速蓝组织染色来验证我们的脱髓鞘模型。腹腔内注射 Evans 蓝在健康和铜处理小鼠的脑内荧光信号之间没有差异 (IVIS 分析，随后进行染料提取)。相比之下，在铜饮食 4 周后静脉注射抗体偶联物 (抗 GFAP 或非特异性 IgG)，通过对比增强 MRI（用于钆标记抗体）和荧光显微镜（用于 Alexa488 标记抗体），均在铜处理小鼠的胼胝体中观察到偶联物的聚集。我们的结果表明，具有更高灵敏度的方法可以检测大分子（如荧光标记或钆标记的抗体偶联物）在大脑中的积累，这表明脱髓鞘区域的局部 BBB 破坏。这些发现支持了先前的研究，这些研究对铜诱导模型中的 BBB 完整性提出了质疑，并证明了将抗体偶联物递送到铜处理小鼠胼胝体的可能性。
