Effect of VEGF-C treatment on sorted brain microglia/macrophages. Effect of VEGF-C treatment on sorted brain microglia/macrophages

Meningeal lymphatic vessels (MLVs) promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelial growth factor-C (VEGF-C) regulates MLV development and maintenance and has therapeutic potential for treating neurological disorders. Herein, we investigated the effects of VEGF-C overexpression on brain fluid drainage and ischemic stroke outcomes in mice. Intra-cerebrospinal administration of an adeno-associated virus expressing mouse full-length VEGF-C (AAV-mVEGF-C) increased CSF drainage to the deep cervical lymph nodes (dCLNs) by enhancing lymphatic growth, and upregulated neuroprotective signaling pathways identified by single nuclei RNA sequencing of brain cells. In a mouse model of ischemic stroke, AAV-mVEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage, associated with mitigated microglia-mediated inflammation and increased BDNF signaling in brain cells. Neuroprotective effects of VEGF-C were lost upon ligation of the dCLN afferent lymphatics, and not mimicked by acute post-stroke VEGF-C injection. We conclude that VEGF-C prophylaxis promotes multiple vascular, immune, and neural responses that culminate in a protection against neurological damage in acute ischemic stroke. Overall design: For evaluating the effect of VEGFc-mediated meningeal lymphatic expansion on brain myeloid cells, we treated C57/Bl6J mice with AAV-CTRL or AAV-VEGFc for 4 weeks. Half of the samples were obtained at this time point. For the other half of samples, tMCAO was performed and the infarcted hemisphere was isolated 7 days after. Samples were processed with MACS bead-based myelin removal followed by purification of CD11b+ microglia/macrophages using MACS beads. Isolated cells were then lysed for mRNA isolation and sequencing. We then performed gene expression analysis with RNAseq data.

脑膜淋巴管（meningeal lymphatic vessels, MLVs）可促进中枢神经系统（central nervous system, CNS）的组织清除与免疫监视功能。血管内皮生长因子C（Vascular endothelial growth factor-C, VEGF-C）能够调控MLV的发育与维持，在神经系统疾病治疗中具备潜在应用价值。本研究探究了VEGF-C过表达对小鼠脑脊液引流及缺血性脑卒中预后的影响。
经脑脊髓腔给予表达小鼠全长VEGF-C的腺相关病毒（adeno-associated virus, AAV-mVEGF-C），可通过促进淋巴管生成增强脑脊液（cerebrospinal fluid, CSF）向颈深淋巴结（deep cervical lymph nodes, dCLNs）的引流，并上调经脑内单细胞核RNA测序（single nuclei RNA sequencing）鉴定得到的神经保护信号通路。
在缺血性脑卒中小鼠模型中，AAV-mVEGF-C预处理可减轻亚急性期脑梗死损伤并改善运动功能，该效应与小胶质细胞介导的炎症反应减轻及脑源性神经营养因子（brain-derived neurotrophic factor, BDNF）信号通路增强相关。
当颈深淋巴结输入淋巴管被结扎时，VEGF-C的神经保护作用会完全消失；脑卒中后急性给予VEGF-C，则无法重现上述保护效应。
综上，VEGF-C预防性给药可激活多重血管、免疫及神经应答通路，最终在急性缺血性脑卒中中发挥神经保护作用，减轻神经系统损伤。
实验整体设计：为评估VEGF-C介导的脑膜淋巴管扩增对脑内髓系细胞的影响，我们将C57/Bl6J小鼠分为两组，分别注射AAV-对照（AAV-CTRL）与AAV-VEGF-C，干预时长为4周。其中一半样本于干预结束时采集；另一半小鼠则构建短暂性大脑中动脉闭塞（transient middle cerebral artery occlusion, tMCAO）模型，并于造模后7天分离梗死侧脑组织。
样本处理采用磁珠激活细胞分选（magnetic activated cell sorting, MACS）磁珠法去除髓鞘，随后通过MACS磁珠纯化分化簇11b（cluster of differentiation 11b, CD11b）阳性小胶质细胞/巨噬细胞。分离得到的细胞经裂解后提取mRNA并进行测序，后续基于RNA测序数据开展基因表达分析。