The molecular and biophysical characterization of the human blood-nerve barrier: current concepts

Abstract

The internal microenvironment in peripheral nerves is highly regulated in order to maintain normal axonal impulse transmission to or from the central nervous system. In humans, this regulation is facilitated by specialized tight junction (TJ)-forming endoneurial microvascular endothelial cells and perineurial myofibroblasts that form multiple concentric layers around nerve fascicles. The endoneurial endothelial cells come in direct contact with circulating blood and, thus, can be considered the blood-nerve barrier (BNB). Studies on the molecular and biophysical properties of the human BNB in vivo or in situ are limited. Owing to the recent isolation of primary human endoneurial endothelial cells and the development of simian virus 40 large T-antigen immortalized cell lines, data are emerging on the structural and functional characteristics of these cells. These data aim to increase our understanding of how solutes, macromolecules, nutrients and hematogenous leukocytes gain access into or are restricted from the endoneurium of peripheral nerves. These concepts have clinical relevance in understanding normal peripheral nerve homeostasis, the response of peripheral nerves to external insult and stresses such as drugs and toxins and the pathogenesis of peripheral neuropathies. This review discusses current knowledge in this nascent and exciting field of microvascular biology.

Figure 1. Peripheral nerve anatomy and characteristics of primary endoneurial endothelial cells

A digital photomicrograph of an axial cryostat section of human sciatic nerve stained with fluoresceinated UEA-1 demonstrates the multilayered anatomical organization of peripheral nerves, with blood vessels (BV) within the epineurium and endoneurium indicated (A). A digital phase contrast photomicrograph demonstrates confluent, spindle-shaped pHEndECs six days after being cultured on rat tail collagen-coated CellBIND^® tissue culture plates at the onset of a flow-dependent leukocyte trafficking assay (B). Proliferating pHEndECs strongly bind UEA-1, as expected for human vascular endothelial cells (C) with more intense perinuclear staining seen at higher magnification (D), as shown by these direct immunocytochemistry digital photomicrographs of fixed cells cultured on rat-tail collagen coated glass coverslips. Intracellular vWF expression, another marker of vascular endothelial cells, is demonstrated on fixed, permeabilized proliferating pHEndEC cultures grown on rat-tail collagen coated glass coverslips (E), with intracellular perinuclear expression verified at higher magnification (F), based on these digital indirect immunocytochemistry photomicrographs. Scale bars 100 µm for 1A–C and 1E, 5 µm for 1D and 1F.

Figure 2. Human BNB intercellular junctional complex

This figure illustrates the known AJ and TJ proteins expressed by the human BNB in vitro, and their accessory or regulatory proteins and their membrane localization guided by knowledge of other restrictive barrier forming endothelial cells. There is immunocytochemistry evidence for ZO-1, ZO-2, claudin-5, occludin and VE-Cadherin expression at intercellular contact sites, without direct evidence for claudin-12 and claudin-19. pHEndEC β-catenin expression has been demonstrated by PCR and western blot. GFRα1, the receptor for GDNF, complexed with RET, is expressed by pHEndECs, and mediates recovery of restrictive barrier characteristics at the human BNB in vitro following serum withdrawal, dependent on RET-tyrosine kinase mediated signaling. There is preliminary evidence that the Ras-MAPK signaling pathway acts downstream of RET, with the net effect being cytoskeletal reorganization of F-actin filaments, with relocation towards the cytoplasmic membrane and intercellular contact sites, providing the scaffold for more continuous AJ and TJ and fewer intercellular clefts. Cyclic adenosine monophosphate (cAMP), acting via protein kinase A (PKA) has been shown to play a minor role in this process, independent of GDNF, with a mild additive effect observed when multiple mitogens are administered to induce pHEndEC recovery from diffuse endothelial injury. Schwann cells and pericytes are potential cellular sources for GDNF in peripheral nerves.

Figure 3. Human BNB transporters

This figure depicts the known transporters and their putative cellular localization and actions at the BNB based on PCR, western blot, flow cytometry and immunocytochemical studies performed with primary and immortalized human endoneurial endothelial cells in vitro. The influx transporters are shown on the luminal side, while efflux transporters are shown on the abluminal side. It is hypothesized that MCT-1 may a dual role as an influx and efflux transporter dependent on endoneurial energy requirements and the presence of toxic metabolites. Adherens and tight junctions should prevent paracellular transport of small polar molecules from the bloodstream into the endoneurium. Weibel-Palade bodies (WPB), known to contain vWF and P-selectin, are also shown.

Figure 4. Hunan endoneurial endothelial cell adhesion molecules and effect of proinflammatory cytokines

The constitutive expression of CAMs by the human BNB in vitro is depicted in the upper image, with a time-dependent increase in cytoplasmic protein expression observed following exogenous treatment of confluent cultures with 10 U/mL TNF-α and 20 U/mL IFN-γ over 48 hours. CD31 and CD34 expression have been described, but the effect of cytokine treatment on their expression has not been evaluated. Cytoplasmic JAM-A expression by pHEndECs has been described in vitro, but its expression was not regulated by proinflammatory cytokine treatment (not shown in figure). Using a flow-dependent in vitro BNB-leukocyte trafficking model, ICAM-1 significantly contributed to pathogenic peripheral blood mononuclear cell adhesion and transmigration, dependent on interactions with α_Mβ₂ integrin, rather than its more ubiquitously expressed counterligand, α_Lβ₂ integrin. Video microscopy shows that these mononuclear cells firmly arrest and congregate at contact sites between confluent endothelial cells, with a subset of leukocytes slowly migrating across the BNB via the paracellular route without significant retraction of neighboring endothelial cells from each other during the course of a 30-minute assay with estimated in vivo capillary flow rates based on red blood cell velocities (refer to published supplementary data for reference [61] for videos). Further studies are needed to determine whether transcellular trafficking occurs at the human BNB in vitro and in vivo, as observed in other microvascular transmigration model systems.