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. Overall design: 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.