Identification of direct connections between the dura and the brain [EAE]

The arachnoid barrier delineates the border between the central nervous system and dura mater. Although the arachnoid barrier creates a partition, communication between the central nervous system and the dura mater is crucial for waste clearance and immune surveillance. How the arachnoid barrier balances separation and communication is poorly understood. Leveraging transcriptomic data, we developed novel transgenic mice to examine specific anatomical structures that serve as routes across the arachnoid barrier. Bridging veins create discontinuities where they cross the arachnoid barrier, forming structures that we termed arachnoid cuff exit (ACE) points. The openings that ACE points create allow the exchange of fluids and molecules between the subarachnoid space and the dura, enabling the drainage of cerebrospinal fluid and limited entry of molecules from the dura to the subarachnoid space. In healthy human volunteers, MRI tracers transit along bridging veins in a similar fashion to access the subarachnoid space. Interestingly, in neuroinflammatory conditions such as experimental autoimmune encephalomyelitis, ACE points also allow cellular trafficking, representing a previously unknown route for immune cells to directly enter the subarachnoid space from the dura mater. Collectively, our results indicate that ACE points are a critical part of the anatomy of neuroimmune communication in both mice and humans that links the central nervous system with the dura and its immunological diversity and waste clearance systems. EAE was induced in mice by subcutaneous injection of MOG35–55 peptide (200 μg, CSBio) emulsified in Freund’s adjuvant (Sigma Aldrich) supplemented with 4 mg/mL of Mycobacterium tuberculosis (BD) into each flank. Pertussis toxin (200 ng, List Biologicals) was injected i.p. on day 0 and day 2 after MOG immunization. Controls were treated similarly, but MOG peptide was excluded. At peak disease (day 16), cranial leptomeninges were microdissected and placed into 50 uL nuclei isolation medium (NIM: 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl, 1 mM DTT, 1 U/mL RNase inhibitor (Takara, 2313B), protease inhibitor (1 tablet/10 mL; Thermo, A32955). Leptomeninges were chopped on ice for 10 minutes, before the volume was increased to 1 mL with NIM. Samples were then passed 10 times through a 2 mL dounce homogenizer with pestle A, then 10 times with pestle B. Nuclei were passed through a 20 um strainer, mashed with a glass pestle, and washed through two times with 0.5 mL NIM. Nuclei were spun at 1000 x g for 5 minutes at 4 oC, and resuspended in 1 % ultrapure nuclease-free BSA (Invitrogen, AM2616) in PBS with RNase inhibitor (1 U/mL) and DAPI (100 nM). Fluorescence-activated cell sorting was performed on nuclei based on forward and side scatter, and DAPI signal. Nuclei were then spun at 1500 x g for 5 minutes at 4 oC, and resuspended in 1 % ultrapure nuclease-free BSA (Invitrogen, AM2616) in PBS with RNase inhibitor (1 U/mL) to a concentration of 1500 nuclei/uL. The sorted nuclei were loaded onto a 10X Genomics Chromium platform for GEM and cDNA generation carrying cell- and transcript-specific barcodes and sequencing libraries constructed using the Chromium Single Cell 3′ Library & Gel Bead Kit v3. Libraries were sequenced on the Illumina NovaSeq6000, targeting a depth of 100,000 reads per cell.

蛛网膜屏障（arachnoid barrier）界定了中枢神经系统（central nervous system）与硬脑膜（dura mater）之间的边界。尽管蛛网膜屏障形成了物理分隔，但中枢神经系统与硬脑膜之间的通讯对于废物清除和免疫监视至关重要。目前学界对蛛网膜屏障如何平衡分隔与通讯的机制仍知之甚少。

本研究借助转录组学数据（transcriptomic data），构建了新型转基因小鼠（transgenic mice），以探究充当蛛网膜屏障跨膜通路的特定解剖结构。桥静脉（bridging veins）在穿过蛛网膜屏障时会形成不连续区域，由此构成我们命名为蛛网膜袖套出口（ACE）点的结构。ACE点所形成的开口允许蛛网膜下腔（subarachnoid space）与硬脑膜之间进行液体和分子交换，实现脑脊液（cerebrospinal fluid）的引流以及硬脑膜内分子向蛛网膜下腔的有限渗透。

在健康人体志愿者中，磁共振示踪剂（MRI tracers）可沿桥静脉以类似方式进入蛛网膜下腔。有趣的是，在实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis, EAE）这类神经炎症疾病中，ACE点同样介导细胞迁移（cellular trafficking），这代表了免疫细胞从硬脑膜直接进入蛛网膜下腔的一条此前未被发现的通路。

综上，本研究结果表明，ACE点是小鼠与人类体内神经免疫通讯解剖结构的关键组成部分，它将中枢神经系统与硬脑膜及其免疫多样性系统和废物清除系统联系起来。

研究通过向小鼠双侧腹股沟皮下注射溶于弗氏佐剂（Freund’s adjuvant, Sigma Aldrich）的MOG35–55肽（200 μg, CSBio）（佐剂中添加4 mg/mL结核分枝杆菌（Mycobacterium tuberculosis, BD）），诱导小鼠发生EAE。在MOG免疫后的第0天和第2天，经腹腔注射百日咳毒素（pertussis toxin, 200 ng, List Biologicals）。对照组采用相同处理，但不添加MOG肽。在疾病峰值期（第16天），显微解剖获取颅骨软脑膜（leptomeninges），并置于50 μL细胞核分离液（nuclei isolation medium, NIM：250 mM蔗糖、25 mM KCl、5 mM MgCl2、10 mM Tris-HCl、1 mM DTT、1 U/mL RNase抑制剂（Takara, 2313B）、蛋白酶抑制剂（1片/10 mL; Thermo, A32955））中。

将软脑膜在冰上切碎10分钟，随后用NIM将体系体积补至1 mL。随后将样本通过2 mL杜恩匀浆器（dounce homogenizer）以A杵匀浆10次，再以B杵匀浆10次。将细胞核通过20 μm滤网过滤，用玻璃杵研磨后，用0.5 mL NIM洗涤两次。将细胞核以1000×g转速在4℃下离心5分钟，重悬于含1 U/mL RNase抑制剂和100 nM DAPI的PBS配制的1%超纯无核酸酶BSA（Invitrogen, AM2616）溶液中。

基于前向散射光、侧向散射光及DAPI信号对细胞核进行荧光激活细胞分选（fluorescence-activated cell sorting）。随后将分选后的细胞核以1500×g转速在4℃下离心5分钟，重悬于含1 U/mL RNase抑制剂的PBS配制的1%超纯无核酸酶BSA溶液中，调整浓度至1500个核/μL。将分选得到的细胞核加载至10X Genomics Chromium平台，进行GEM（凝胶珠乳液）与cDNA合成，该平台搭载细胞特异性和转录特异性条形码，并使用Chromium单细胞3′文库与凝胶珠试剂盒v3构建测序文库。利用Illumina NovaSeq6000对文库进行测序，目标测序深度为每细胞100,000条reads。