Molecular characterization of perivascular drainage pathways in the murine brain

Abstract

Perivascular compartments surrounding central nervous system (CNS) vessels have been proposed to serve key roles in facilitating cerebrospinal fluid flow into the brain, CNS waste transfer, and immune cell trafficking. Traditionally, these compartments were identified by electron microscopy with limited molecular characterization. Using cellular markers and knowledge on cellular sources of basement membrane laminins, we here describe molecularly distinct compartments surrounding different vessel types and provide a comprehensive characterization of the arachnoid and pial compartments and their connection to CNS vessels and perivascular pathways. We show that differential expression of plectin, E-cadherin and laminins α1, α2, and α5 distinguishes pial and arachnoid layers at the brain surface, while endothelial and smooth muscle laminins α4 and α5 and smooth muscle actin differentiate between arterioles and venules. Tracer studies reveal that interconnected perivascular compartments exist from arterioles through to veins, potentially providing a route for fluid flow as well as the transport of large and small molecules.

The role of brain barriers in fluid movement in the CNS: is there a ‘glymphatic’ system?

“…It is becoming increasingly apparent that under normal conditions, such flows likely do not exist in the neuropil of the gray matter. Rather than a 'glymphatic' system as originally proposed [91,124,125], the weight of the evidence now suggests the existence of a perivascular fluid system for the CNS, with convective flow or dispersion along the perivascular spaces of larger vessels and then diffusion predominantly regulating CSF/ISF exchange at the level of the neurovascular unit associated with CNS microvessels, as now proposed by several groups [6,74,80,96,137,161,162,192]. It remains possible that pericapillary convection occurs in the basal lamina in addition to diffusion to formally link up the arteriolar and venular perivascular fluid compartments and allow a fully convective circulation pathway [74,137]; regardless, CSF/ISF exchange may at least partly occur through the neuropil of the gray matter at the capillary level by diffusion, as neurons and all the other constituents of the NVU are not located further than about 10-20 µm from the pericapillary spaces, a distance that has likely been optimized for the effective diffusion of glucose, oxygen, and countless other substances from the circulation [3,192].…”

“…The perivascular spaces (PVS) 1 of cerebral blood vessels have in recent years been the subject of increasing research focus as pathways for CSF/ISF exchange [1,74,89,91,95,112,137,138,162], but controversy exists over their precise role [58,77,96,161,162]. Indeed, the glial components (astrocyte foot processes) that provide the outer boundary of the PVS within the parenchyma have been proposed to serve a special function for CNS clearance and waste turnover, forming the basis for a so-called 'glymphatic' circulation [91,124] that may potentially allow a more complete exchange of CSF and ISF at both superficial and deep sites spanning the entire neural axis.…”

“…1a) may be identified on all larger cerebral blood vessels in both the subarachnoid spaces (leptomeningeal blood vessels) and within the parenchyma. Filled with a fluid in potential communication with both CSF and ISF but also containing connective tissue, scattered cells, and basement membranes (BM), the PVS is a real compartment within the vascular connective tissue space of the adventitia [63,97,108,137,158,194,200] and possibly also the tunica media BM of large vessels [24,137,189] as well as a potential compartment within the basal lamina of capillaries [74,91,137,148]. Potential routes of entry from the CSF into the PVS include specialized pores, termed stomata, recently demonstrated on the adventitial lining cells of leptomeningeal vessels in the SAS of the rat by scanning electron microscopy [137] (Fig.…”

“…Similarly, it is proposed that the astrocytic layer forms an effective 'second barrier' surrounding the endothelial tube, with connexin 43 gap junctions linking adjacent astrocyte endfeet [64], and the whole complex being sufficiently tightly knit to restrict ready entry of leukocytes and mast cells to the brain parenchyma except in pathological conditions [57]. It is also possible that matrix components of the basal lamina (e.g., specific laminin composition [158]) may play a more important role than has been appreciated [74], e.g., in restricting the entry of cells into the parenchyma after their migration across brain endothelial cells or the transfer of very large macromolecules (e.g., ~ 460 kDa ferritin [20]) from the ECS to the perivascular space for clearance.…”

“…Interactions of endothelial cells (and astrocytes) with components of the extracellular matrix (ECM) and basal lamina are facilitated by integrins, αβ heterodimeric receptors that bind extracellularly to laminins and fibronectin to link the ECM to the cytoskeleton, and dystroglycan, a complex of α and β subunits that binds to laminins, agrin, and perlecan to provide additional links of the ECM to the cytoskeleton. Numerous additional linkages within the ECM and basal lamina occur between laminins, proteoglycans, fibronectin, and type IV collagen (arrows), among others (Adapted from [11,86,168,201] with additional information incorporated from [3, 62, 117, 182]) parenchyma [19,20,74]; indeed, recent experimental work has demonstrated that intracisternally infused small single-domain antibodies (17 kDa; 4.5 nm apparent hydrodynamic diameter) more fully access perivascular basement membranes than similarly infused large full length immunoglobulin G antibodies (150 kDa; 10 nm) [137]. A recent molecular characterization of the PVS with respect to laminin composition and other markers by Sorokin and colleagues provides new, critical information for future work in this area [74].…”