摘要
目的探究脊髓损伤(SCI)导致脑膜淋巴管(MLVs)引流功能障碍是否诱发远隔大脑区域的继发性神经炎症及神经元损伤。方法选取52只雌性C57BL/6小鼠,按随机数字表法分为假手术组、SCI组、阴性对照腺相关病毒组(阴性对照组)及过表达血管内皮生长因子-C(VEGF-C)腺相关病毒组(VEGF-C组),每组13只。假手术组仅切除椎板,但不损伤脊髓。SCI组、阴性对照组及VEGF-C组采用改良Allen法打击装置损伤T9节段脊髓,建立SCI模型。阴性对照组和VEGF-C组于SCI建模前4周经枕大池注射3μl阴性对照腺相关病毒和过表达VEGF-C腺相关病毒。伤后56 d,通过枕大池注射Alexa Fluor™647卵清蛋白(OVA-647)示踪剂,2 h后评估各组颈深淋巴结(dCLN)中OVA-647占比;采用免疫荧光检测各组MLVs标志物淋巴管内皮透明质酸受体-1(LYVE-1)占比及大脑皮层、海马、中脑、丘脑中小胶质细胞标记物离子钙结合适配器分子1(Iba1)的表达量;通过ELISA法检测各组上述各脑区肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)水平;采用尼氏染色法检测各组上述各脑区的神经元数量。结果伤后56 d,假手术组dCLN中OVA-647占比高于SCI组和阴性对照组(P<0.01),而SCI组和阴性对照组dCLN中OVA-647占比低于VEGF-C组(P<0.05)。伤后56 d,假手术组硬脑膜LYVE-1占比高于SCI组和阴性对照组(P<0.01),而SCI组和阴性对照组硬脑膜LYVE-1占比低于VEGF-C组(P<0.05)。伤后56 d,与假手术组相比,SCI组和阴性对照组大脑皮层、海马、中脑、丘脑Iba1阳性小胶质细胞数量均增加(P<0.01);与SCI组和阴性对照组相比,VEGF-C组上述各脑区Iba1阳性小胶质细胞数量均减少(P<0.01)。伤后56 d,与假手术组相比,SCI组和阴性对照组上述各脑区TNF-α及IL-1β水平均升高(P<0.05);与SCI组和阴性对照组相比,VEGF-C组上述各脑区TNF-α及IL-1β水平均降低(P<0.05)。伤后56 d,与假手术组相比,SCI组和阴性对照组上述各脑区神经元存活数量均减少(P<0.05);与SCI组和阴性对照组相比,VEGF-C组上述各脑区神经元存活数量均增加(P<0.05)。结论SCI可导致MLVs引流功能障碍,进而引起远隔大脑区域的继发性神经炎症和神经元损伤。
Objective To determine whether spinal cord injury(SCI)triggers secondary neuroinflammation and neuronal injury in remote brain regions by impairing the drainage function of the meningeal lymphatic vessels(MLVs).Methods Fifty-two female C57BL/6 mice were assigned with the random number table into four groups(n=13 per group):sham group,SCI group,adeno-associated virus negative control group(negative control group),and adeno-associated virus overexpressing VEGF-C group(VEGF-C group).The sham group underwent laminectomy without spinal cord injury.In the SCI group,negative control group and VEGF-C group,T9 contusion was made to establish the SCI models using a modified Allen's impactor.At 4 weeks before SCI modeling,the negative control group and VEGF-C group were injected via the cisterna magna with 3μl adeno-associated virus for negative control or adeno-associated virus for VEGF-C overexpression.At 56 days after injury,Alexa Fluor™647 ovalbumin conjugate(OVA-647)was injected via the cisterna magna as a tracer.Two hours later,the proportion of OVA-647 in the deep cervical lymph nodes(dCLN)was detected.Immunofluorescence was performed to assess the proportion of MLVs marker lymphatic vessel endothelial hyaluronan receptor-1(LYVE-1)and expression levels of microglial marker ionized calcium-binding adaptor molecule 1(Iba1)in the cerebral cortex,hippocampus,midbrain,and thalamus across the experimental groups.ELISA was employed to quantify the levels of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)and Nissl staining was used to assess neuronal counts in these regions.Results At 56 days after injury,the OVA-647 proportion in the dCLN was higher in the sham group than that in the SCI group and negative control group(P<0.01),whereas the SCI group and negative control group showed a lower OVA-647 proportion in the dCLN than the VEGF-C group(P<0.05).At 56 days after injury,the dural LYVE-1 proportion was higher in the sham group than that in the SCI group and negative control group(P<0.01),whereas it was lower in the SCI group and negative control group than that in the VEGF-C group(P<0.05).At 56 days after injury,the count of Iba1-positive microglia across all the above-mentioned regions was increased in the SCI group and negative control group(P<0.01),compared with that in the sham group,whereas it was reduced in these regions in the VEGF-C group,compared with that in the SCI group and negative control group(P<0.01).At 56 days after injury,TNF-αand IL-1βlevels in these regions were both elevated in the SCI group and negative control group when compared with those in the sham group(P<0.05),whereas they were reduced in the VEGF-C group,compared with those in the SCI group and negative control group(P<0.05).At 56 days after injury,neuronal survival in the regions was decreased in the SCI group and negative control group,compared with that in the sham group(P<0.05),whereas it was increased in the VEGF-C group,compared with that in the SCI group and negative control group(P<0.05).Conclusion SCI can induce secondary neuroinflammation and neuronal damage in remote brain regions by impairing the drainage function of MLVs.
作者
陈义
唐渝
闵令霞
谭明亮
丛勃雅
侯景明
冯州
Chen Yi;Tang Yu;Min Lingxia;Tan Mingliang;Cong Boya;Hou Jingming;Feng Zhou(Department of Rehabilitation Medicine,First Affiliated Hospital of Army Medical University,Chongqing 400038,China)
出处
《中华创伤杂志》
北大核心
2025年第11期1103-1111,共9页
Chinese Journal of Trauma
基金
国家自然科学基金(82301569,82472607)
重庆市中青年医学高端人才(YXGD202460)
重庆市医学青年拔尖人才(YXQN202417)。
关键词
脊髓损伤
大脑
炎症
神经元
脑膜淋巴管
Spinal cord injuries
Cerebrum
Inflammation
Neurons
Meningeal lymphatic vessels
