Pl, V.; Bitsika, S.; Giannetto, M.; Ladron-de-Guevara, A.; Gahn Martinez, D.; Mori, Y.; Nedergaard, M.; Mollgard, K.

Traditionally, the meninges are described as 3 distinct layers, dura, arachnoid and pia. Yet, the classification of the connective meningeal membranes surrounding the brain is based on the postmortem macroscopic examination. Ultrastructural and single cell transcriptome analyses have documented that the 3 meningeal layers can be subdivided into several distinct layers based on cellular characteristics. We here re-examined the existence of a 4th meningeal membrane, Subarachnoid Lymphatic-like Membrane or SLYM in Prox1-eGFP reporter mice. Imaging of freshly resected whole brains showed that SLYM covers the entire brain and brain stem and forms a roof shielding the subarachnoid cerebrospinal fluid (CSF)-filled cisterns and the pial vasculature. Thus, SLYM is strategically positioned to facilitate periarterial influx of freshly produced CSF and thereby support unidirectional glymphatic CSF transport. Histological analysis showed that SLYM in spinal cord and parts of dorsal cortex fused with the arachnoid barrier layer, while in the basal brain stem formed a 1-3 layered membrane subdividing the subarachnoid space into two compartments. However, great care should be taken when interpretating the organization of the delicate leptomeningeal membranes in tissue sections. We show that hyperosmotic fixatives dehydrate the tissue with the risk of shrinkage and dislocation of fragile membranes in postmortem preparations.