帕金森病扩大的血管周围间隙增加及类淋巴系统功能障碍的机制研究

The enlarged perivascular space increased and the mechanism of dysfunction of glymphatic system in Parkinson disease

目的 评估帕金森病(Parkinson disease,PD)患者扩大血管周围间腔数量,并通过动物实验研究其可能的机制。方法 临床研究部分:纳入30例帕金森病患者和35名健康对照参与研究,通过MRI对扩大的血管周围间隙(enlarged perivascular spaces,EPVS)进行显像并计数。动物实验部分:将90只C57BL/6J小鼠随机分成对照组、模型组与实验组(每组n=30),模型组与实验组纹状体注射α-突触核蛋白预制纤维(α-synuclein preformed fibrils,α-syn PFFs)构建PD模型,实验组用舒尼替尼灌胃抑制脑膜淋巴管增生,采用免疫荧光检测脑膜中淋巴管内皮透明质酸受体-1(lymphatic vessel endothelial receptor-1,LYVE-1)、大脑中水通道蛋白4(aquaporin 4,AQP4)表达,量化伊文思蓝(Evans blue,EB)在脑实质的分布评估类淋巴功能,免疫组织化学染色检测脑内磷酸化α-syn(phosphorylated α-syn,pα-syn)表达。结果 临床研究中,PD患者脑内基底节区[10(7,12)vs. 7(5,9),P<0.001]及半卵圆中心[2(1,3)vs. 1(0,2),P<0.001] EPVS数量均多于健康对照。动物研究中:与对照组相比,造模4个月后模型组小鼠脑膜LYVE-1阳性区域面积减少[(8.36%±0.74%)vs.(11.12%±1.32%),P<0.01],类淋巴系统引流[(10.78%±0.02%)vs.(14.25%±0.01%),P<0.001]降低,清除功能降低[(8.02%±0.01%)vs.(5.46%±0.01%),P<0.001],伴随pα-syn的聚集及AQP4极性下降[(0.44±0.04)vs.(0.51±0.04),P<0.01];实验组小鼠脑膜LYVE-1阳性面积[(2.50%±0.54%)vs.(11.12%±1.32%),P<0.001]、类淋巴引流[(3.55%±0.01%)vs.(14.25%±0.01%),P<0.001]、清除功能[(15.31%±0.01%)vs.(5.46%±0.01%),P<0.001]进一步受损,AQP4极性明显降低[(0.28±0.02)vs.(0.51±0.04),P<0.001],皮层[(8.12%±0.38%) vs.(2.85%±0.43%),P<0.001]、纹状体[(7.84%±0.32%) vs.(3.62%±0.31%),P<0.001]及黑质[(8.71%±0.34%)vs.(2.38%±0.15%),P<0.001] α-syn表达量均较模型组增加,差异均具有统计学意义。结论 PD患者EPVS数目增多提示类淋巴系统功能障碍,这可能是脑膜淋巴管受损通过介导病理性α-syn沉积破坏AQP4极性引起的。

Objective This study aimed at assessing the number of enlarged perivascular spaces(EPVS)and explore the potential mechanism of dysfunction of the glymphatic system in Parkinson disease(PD).Methods Clinical study:Magnetic resonance imaging(MRI)was used to show EPVS and quantify the number of EPVS in 30 PD patients and35 normal controls(NC).Animal research:Ninety C57 BL/6J mice randomly were divided into three groups:control,model and experimental groups.Experimental group mice were treated with Sunitinib(60 ml/kg)via gavage daily for 2 weeks.Then model and experimental groups were injected with 2.5μlα-synuclein preformed fibrils(α-syn PFFs)into bilateral striatum respectively to establish PD model.Then experimental group was treated with Sunitinib(60 ml/kg)every five days to restrain the development of meningeal lymphatic vessels(mLVs).The expression of Lymphatic Vessel Endothelial Receptor-1(LYVE-1)in meninges and aquaporin 4(AQP4)polarity in brain were detected by immunofluorescence.The function of glymphatic system was assessed by quantifying the amount of Evans blue(EB)in brain parenchyma.Theα-syn positive inclusions in brain were detected by immunohistochemistry.Results The number of EPSV increased in PD patients compared to NC in basal ganglia[10(7,12)vs.7(5,9),P<0.001]and centrum semiovale[2(1,3)vs.1(0,2),P<0.001].After four months of being injected withα-syn PFFs,LYVE-1 positive area in meninges[(8.36%±0.74%)vs.(11.12%±1.32%),P<0.001],the drainage[(10.78%±0.02%)vs.(14.25%±0.01%),P<0.001]and clearance function[(8.02%±0:01%)vs.(5.46%±0.01%),P<0.001]of glymphatic system decreased in model group compared with control group together with aggregation ofα-syn and impaired polarization of AQP4[(0.44±0.04)vs.(0.51±0.04),P<0.01]in brain.For the experimental group,LYVE-1 positive area significantly reduced[(2.50%±0.54%)vs.(11.12%±1.32%),P<0.001],the drainage[(3.55%±0.01%)vs.(14.25%±0.01%),P<0.001]and clearance[(15.31%±0.01%)vs.(5.46%±0.01%),P<0.0001]function of glymphatic system and AQP4 polarity[(0.28±0.02)vs.(0.51±0.04),P<0.0001]further worsened,accompanied by more severe accumulation of pα-syn in cortex[(8.12%±0.38%)vs.(2.85%±0.43%),P<0.001],striatum[(7.84%±0.32%)vs.(3.62%±0.31%),P<0.001]and substantia nigra[(8.71%±0.34%)vs.(2.380±0.15%),P<0.001]than model group.Conclusion There an increase in number of EPSVs,an indicator of the glymphatic dysfunction,in PD patients,that may attribute to the impairment of AQP4 polarization secondary to the damaged mLVs cause accumulation of pathologicalα-syn.