Structural Validation of the Intermediate Leptomeningeal Layer in the Human Central Nervous System

Traditionally, the human central nervous system (CNS) is described as having three meningeal layers, from outer to inner: dura mater, arachnoid mater, and pia mater. The arachnoid and pia mater are called the leptomeninges, and the space between them is filled with cerebrospinal fluid (CSF). Using gross dissection, light microscopy, and ultrastructural methods on fresh postmortem and cadaveric fetal to adult age CNS specimens (N=61), we demonstrate the presence of a fibro-cellular intermediate leptomeningeal layer (ILL) in the human CNS along the entire neural axis, from the cortex to the caudal end of the spinal cord. ILL divides the subarachnoid space (SAS) into two distinct structural compartments, traversed by vessels and nerves. The ILL shows unique structural features, such as dips into the sulci and fissures of the brain as a double-fold membrane carrying the vessels, bears intra-layer trabeculae, and creates the perivascular sheath. In the spinal cord, ILL shows the presence of distinct meningothelial cells with macrophage-like properties. Moreover, throughout the neural axis, it appears to be a non-sieved barrier, characterized by the presence of tight and adherens junctions. Significance An intermediate leptomeningeal layer encloses the central nervous system in humans Departing from the traditional understanding, the integrated analysis of our macroscopic, microscopic, and ultrastructural study provides robust support for an intermediate leptomeningeal layer (ILL) between arachnoid and pia in the subarachnoid space (SAS) of the human central nervous system (CNS) along the entire neural axis. The ILL is a fibro-cellular macroscopic structure, with restricted permeability, that divides the cerebrospinal fluid (CSF)-filled SAS into two distinct structural compartments. In the spinal cord, ILL revealed the presence of distinct meningothelial cells with macrophage-like properties, suggesting a possible role in immune surveillance. The ILL may redefine the established concept of protective coverings of CNS, CSF circulation dynamics, and the role of leptomeninges in health and disease, including drug delivery.