Glymphatic系统功能损害影响toxic milk小鼠脑铜清除的机制研究

The deposition of copper in brain tissue is the main cause of neurological symptoms in patients with hepatolenticular degeneration (Wilson’s disease, WD), and its transport and clearance mechanisms are unknown. We recently found that the free copper concentrations in toxic milk(tx) mice brain tissue significantly elevated during penicillamine treatment. The pilot experiments revealed copper clearance through brain lymphatic drainage pathway, as well as the impaired glymphatic system, in tx mice. Thus, we speculate that copper can be cleared by glymphatic system, and WD copper deposition may damage glymphatic system function and affect brain copper clearance. We plan to use tx mice as WD model, combine with two-photon imaging, microdialysis and other techniques to comfirm that tx mouse glymphatic system damage leads to the brain copper removal disorder. And we plan to investigate the role of copper deposition of astrocytes in the damage brain copper clearing function of tx mouse glymphatic system. The completion of this project will help to elucidate the mechanism of brain copper clearance, and provide a new target for improving the treatment of brain WD patients.

铜在脑组织的沉积是肝豆状核变性（Wilson's disease, WD）患者出现神经症状的主要原因，其转运和清除机制不明。本课题组前期发现WD动物模型toxic milk（tx）小鼠予青霉胺驱铜后脑间质液游离铜升高，预实验发现阻断小鼠脑淋巴引流可引起脑间质液游离铜升高，并发现tx小鼠的glymphatic系统功能受损。因此，我们推测脑铜可经glymphatic系统清除，WD脑铜沉积可损害glymphatic系统功能而影响脑铜清除。我们拟以tx小鼠为WD模型，结合双光子活体成像、微透析等技术明确tx小鼠存在glymphatic系统功能损害并导致脑铜清除障碍，并探讨星型胶质细胞铜沉积在tx小鼠glymphatic系统功能损害导致脑铜清除障碍中的作用和相关机制。本项目的完成有助于进一步阐明WD脑铜清除的相关机制，并为改善脑型WD患者治疗现状提供新的治疗靶点。

肝豆状核变性（Wilson disease，WD）是因ATP7B 基因突变导致其铜转运功能减弱，铜在脑组织的沉积，是WD患者出现神经症状的主要原因，其转运和清除机制不明。本项目组通过阻断脑淋巴液经颈部淋巴管引流途径，发现小鼠纹状体组织间液游离铜浓度升高，提示提示了ISF 中铜离子可能通过glymphatic系统排出。予小鼠立体定向下脑注射小分子荧光示踪剂， 发现tx小鼠荧光示踪剂清除减弱，提示tx小鼠glymphatic系统清除能力减弱。通过小脑延髓池注射CuCl2 后，检测小鼠纹状体ISF游离铜浓度的动态变化；纹状体注射CuCl2 后，检测小鼠注射点旁铜染色观察注射点周围铜的含量和分布，发现tx小鼠脑铜清除能力下降。给予青霉胺驱铜后进一步发现tx小鼠纹状体ISF游离铜较对照鼠升高，给与3月龄tx小鼠予青霉胺长期驱铜后，发现tx小鼠和DL小鼠的纹状体组织透析液铜浓度升高程度无明显差异，青霉胺治疗tx小鼠组较非治疗tx小鼠升高幅度有所降低。提示经过驱铜后，tx小鼠 glymphatic系统清除能力改善。通过免疫荧光技术检测3月龄tx小鼠AQP4在星型胶质细胞终足的表达，发现星型胶质细胞终足AQP4表达下降，初步提示glymphatic系统清除能力下降可能与AQP4在星型胶质细胞终足的极化率降低有关。本项目基本完成了目标 1：证实通过小鼠ISF中铜可经glymphatic系统清除；2：明确tx小鼠存在glymphatic系统功能损害，并影响了脑铜的清除。

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