Development and Characterization of a Novel Human In Vitro Blood-Nerve Barrier Model Using Primary Endoneurial Endothelial Cells

Abstract

There are phenotypic and functional differences between vascular endothelium from different tissues and between microvascular and macrovascular endothelial cells (ECs) from the same tissue. Relatively little is known about the human blood-nerve barrier (BNB). We report the development of an in vitro BNB model using primary human endoneurial ECs freshly isolated and purified from decedent sciatic nerves via endoneurial stripping, connective tissue enzymatic digestion, and density centrifugation. Primary human endoneurial ECs are spindle shaped and contact inhibited. They rapidly differentiate to form capillary-like networks and microvessels, bind Ulex Europaeus Agglutinin 1 lectin, express von Willebrand factor, and endocytose acetylated low-density lipoprotein. They also express specific transport and cellular adhesion molecules and tight junction proteins, consistent with cells that form a highly restrictive endothelial barrier similar to the blood-brain barrier. When cultured on collagen-coated transwell inserts, the primary human endoneurial ECs develop an in vitro BNB with high transendothelial electrical resistances (160 Omega x cm(2); maximal 12 days after seeding) and low solute permeability coefficient to fluoresceinated high-molecular weight (70 kDa) dextran (2.75 x 10(-3) cm/minute). This in vitro BNB model retains essential known or expected characteristics of the human BNB and has many potential applications for studies of solute, macromolecule, microbial, virus, and leukocyte interactions with this highly specialized endothelial barrier.

Establishment and Characterization of Human Peripheral Nerve Microvascular Endothelial Cell Lines: A New <i>in vitro</i> Blood-Nerve Barrier (BNB) Model

“…These molecules compose TJs and limit the paracellular permeability in order to maintain the brain microenvironment. Previous reports have indicated that PnMECs in human sural nerves express occludin, claudin-5 and ZO-1 (Kanda et al, 2004), and claudin-1 was detected in perineurium (Hirakawa et al, 2003), primary human PnMECs (Yosef et al, 2010) and human Schwann cells (Alanne et al, 2009). We also reported that rat PnMECs expressed many molecules forming tight junctions (Sano et al, 2007).…”

“…Another considerable point is the problem of purity of the cells. Yosef et al reported that primary human PnMECs have higher TEER values than FH-BNBs (Yosef et al, 2010). However, these primary cells inevitably contained a small amount of pericytes, which have been reported to promote barrier integrity of the barrier-derived endothelial cells .…”

“…Recently, primary cultured human PnMECs were isolated and extensively characterized (Yosef et al, 2010). These cells are very useful to analyze the nature of the endothelium forming the BNB; however, they have limited capacities for division because they are not immortalized, and they also contain a small percentage of non-endothelial cells including pericytes and fibroblasts.…”

Osteoblasts Have a Neural Origin in Heterotopic Ossification

“…Two cell types are common within the endoneurial compartment of peripheral nerves, specialized vascular endothelial cells, and Schwann cells necessary for myelination of axons. Claudin 5 is a marker for the specialized endoneurial endothelial cells [41,47]. Therefore, tissues were costained for osterix and claudin 5 ( Fig.…”

“…An important protein, claudin 5, has been shown to be an essential component of restrictive microvascular barriers [3]. This protein is expressed at sites of cell-to-cell contact on human endoneurial endothelial cells in vitro [47]. Mice lacking expression of claudin 5 lack a functional blood-brain barrier (BBB) and die immediately after birth [24].…”