调控硬膜淋巴系统减轻蛛网膜下腔出血后早期脑损伤的实验研究

An experimental study on modulating the dural lymphatic system to alleviate early brain injury after subarachnoid hemorrhage

目的通过调控硬膜淋巴系统(DL)的功能改善小鼠蛛网膜下腔出血(SAH)后早期脑损伤,探索硬膜淋巴管作为SAH后治疗靶点的可能性。方法实验采用C57BL/6J小鼠,通过颈内动脉刺破法诱导小鼠SAH模型。设立假手术组、SAH模型组(SAH组)以及血管内皮生长因子(VEGF)C干预组(采用重组VEGF-C胶体干预后,再进行SAH模型的诱导)。采用免疫荧光染色方法观察重组VEGF-C胶体干预10 d后DL的形态学变化,并与胶体组(仅应用胶体,不加重组VEGF-C)对比,通过伊文思蓝引流实验验证DL的功能变化情况。比较3组小鼠SAH后的皮质脑血流灌注量、脑含水量,以及神经功能损伤情况(以Garcia神经功能评分标准评估),并通过蛋白质免疫印迹法检测各组凋亡蛋白Caspase-3的表达情况。结果与胶体组比较,VEGF-C干预组小鼠的DL生成了更多的淋巴管分支,管腔直径也显著增大[胶体组为(24.8±4.8)μm,VEGF-C干预组为(31.2±3.9)μm,P<0.01],同时引流至VEGF-C干预组颈深淋巴结中的伊文思蓝显色显著深于胶体组。与SAH组相比,VEGF-C干预组在SAH后早期脑损伤期的皮质血流灌注恢复更为显著,脑含水量更低[分别为(76.7±1.6)%和(81.2±1.1)%,P<0.01],神经功能受损更轻[Garcia评分分别为(10.7±1.3)分和(14.0±0.9)分,P<0.01];同时脑组织内凋亡蛋白caspase-3的表达水平也更低(分别为0.559±0.034和0.712±0.036,P<0.01)。结论通过调控DL的功能能够有效减轻小鼠SAH后的早期脑损伤。

Objective To alleviate the early brain injury in mice after subarachnoid hemorrhage(SAH)by regulating the function of dural lymphatics(DL),and to explore the possibility of DL as a therapeutic target after SAH.Methods SAH model of C57BL/6J mice was induced by puncture of internal carotid artery.The sham operation group,SAH model group(SAH group)and vascular endothelial growth factor(VEGF)C intervention group(recombinant VEGF-C colloid intervention was followed by SAH model induction)were established.The morphological changes of DL after 10 days of recombinant VEGF-C colloid intervention were observed by immunofluorescence staining and compared with the colloid group(without recombinant VEGF-C).We evaluated the functional changes of DL by Evans blue drainage assay,and then cerebral blood perfusion,brain water content and neurological function injury(based on the Garcia score)of the three groups of mice after SAH were compared.Finally the expression of apoptotic protein caspase-3 in each group was determined by using Western blot method.Results Compared with the colloid group,the DL in the VEGF-C intervention group generated more lymphatic branches and the mean lumen diameter of lymphatic vessels was significantly increased(24.8±4.8μm vs.31.2±3.9μm,P<0.01).The Evans blue color in the deep cervical lymph nodes in the VEGF-C group was significantly darker than that in the colloid group.Compared with the SAH group,the recovery of cortical blood perfusion in the VEGF-C intervention group was more significant,the brain water content was lower[(76.7±1.6)%vs.(81.2±1.1)%,P<0.01],and the neurological function was less impaired[Garcia score:10.7±1.3 points vs.14.0±0.9 points,P<0.01).Meanwhile,the expression level of apoptosis protein caspase-3 in brain tissue was lower(0.559±0.034 vs.0.712±0.036,P<0.01).Conclusion Regulating the DL function can effectively reduce the early brain injury after SAH in mice.