Autocrine TNF-α Increases Penetration of Myelin-Associated Glycoprotein Antibodies Across the Blood-Nerve Barrier in Anti-MAG Neuropathy.

BACKGROUND AND OBJECTIVES: Deposition of myelin-associated glycoprotein (MAG) immunoglobulin M (IgM) antibodies in the sural nerve is a key feature in anti-MAG neuropathy. Whether the blood-nerve barrier (BNB) is disrupted in anti-MAG neuropathy remains elusive.We aimed to evaluate the effect of sera from anti-MAG neuropathy at the molecular level using our in vitro human BNB model and observe the change of BNB endothelial cells in the sural nerve of anti-MAG neuropathy. METHODS: Diluted sera from patients with anti-MAG neuropathy (n = 16), monoclonal gammopathies of undetermined significance (MGUS) neuropathy (n = 7), amyotrophic lateral sclerosis (ALS, n = 10), and healthy controls (HCs, n = 10) incubated with human BNB endothelial cells to identify the key molecule of BNB activation using RNA-seq and a high-content imaging system, and exposed with a BNB coculture model to evaluate small molecule/IgG/IgM/anti-MAG antibody permeability. RESULTS: RNA-seq and the high-content imaging system showed the significant upregulation of tumor necrosis factor (TNF-α) and nuclear factor-kappa B (NF-κB) in BNB endothelial cells after exposure to sera from patients with anti-MAG neuropathy, whereas the serum TNF-α concentration was not changed among the MAG/MGUS/ALS/HC groups. Sera from patients with anti-MAG neuropathy did not increase 10-kDa dextran or IgG permeability but enhanced IgM and anti-MAG antibody permeability. Sural nerve biopsy specimens from patients with anti-MAG neuropathy showed higher TNF-α expression levels in BNB endothelial cells and preservation of the structural integrity of the tight junctions and the presence of more vesicles in BNB endothelial cells. Neutralization of TNF-α reduces IgM/anti-MAG antibody permeability. DISCUSSION: Sera from individuals with anti-MAG neuropathy increased transcellular IgM/anti-MAG antibody permeability via autocrine TNF-α secretion and NF-κB signaling in the BNB.

Characterization of the structure and control of the blood-nerve barrier identifies avenues for therapeutic delivery.

The blood barriers of the nervous system protect neural environments but can hinder therapeutic accessibility. The blood-brain barrier (BBB) is well characterized, consisting of endothelial cells with specialized tight junctions and low levels of transcytosis, properties conferred by contacting pericytes and astrocytes. In contrast, the blood-nerve barrier (BNB) of the peripheral nervous system is poorly defined. Here, we characterize the structure of the mammalian BNB, identify the processes that confer barrier function, and demonstrate how the barrier can be opened in response to injury. The homeostatic BNB is leakier than the BBB, which we show is due to higher levels of transcytosis. However, the barrier is reinforced by macrophages that specifically engulf leaked materials, identifying a role for resident macrophages as an important component of the BNB. Finally, we demonstrate the exploitation of these processes to effectively deliver RNA-targeting therapeutics to peripheral nerves, indicating new treatment approaches for nervous system pathologies.

Utilization of focused ultrasound for opening of the blood-nerve barrier.

Objective. Focused ultrasound (FUS) use with and without microbubbles (MB) for investigation of the blood-nerve barrier (BNB) within the peripheral nervous system (PNS) has been performed in this study. We evaluate the feasibility of BNB opening in a rodent sciatic nerve model by direct vision FUS treatment and provide preliminary results of magnetic resonance guided FUS (MRgFUS).Approach. Twenty rodent bilateral sciatic nerves were investigated. Rodents were treated using a benchtop FUS system to directly visualize nerve FUS studies. Definity MB, Evans blue dye (EB) and latex micro beads were injected during studies. Selected animals underwent further compound muscle action potential (CMAP) studies. Sonication peak pressure (MPa), width, duty-cycle and duration as well as MB concentration were varied to investigate effective pressure threshold. Further preliminary MRgFUS studies were performed on selected animals. Immunohistochemistry and histological analysis under florescent microscopy were performed at termination of experiments to verify treatment outcomes.Main results. Three ultrasound pressures and three microbubble concentrations at a single sonication frequency (476.5 kHz) were performed under direct open targeting. Histological analysis demonstrated nerve internal architecture disruption at 1.2 MPa with 166.7µl kg-1while 0.3 MPa, with 40µl kg-1MB concentration was the lower threshold for consistently observed disruption of the BNB without anatomical microarchitecture disruption. EB leakage was confirmed at the target region in histological evaluation of nerve following MB injection and FUS sonication. Supra-harmonic emissions were detected during FUS exposures following MB injection but not at baseline reference, indicating effective MB response and stable cavitation. CMAP amplitudes showed delayed onset latency and lower amplitudes in sonicated nerves compared to control nerves without evidence of complete conduction block, suggesting a transient BNB disruption, while at lower limit pressure subtle conduction changes were observed. In MRgFUS, targeted nerves demonstrated further contrast agent leak as well as supra-harmonic frequency detection.Significance. Opening of the BNB in the PNS was achieved using FUS and MB in a rodent model. Ongoing work aims to refine FUS parameters for drug delivery into the nerve after experimental transient BNB disruption.

MicroRNA-21-5p functions via RECK/MMP9 as a proalgesic regulator of the blood nerve barrier in nerve injury.

Both nerve injury and complex regional pain syndrome (CRPS) can result in chronic pain. In traumatic neuropathy, the blood nerve barrier (BNB) shielding the nerve is impaired-partly due to dysregulated microRNAs (miRNAs). Upregulation of microRNA-21-5p (miR-21) has previously been documented in neuropathic pain, predominantly due to its proinflammatory features. However, little is known about other functions. Here, we characterized miR-21 in neuropathic pain and its impact on the BNB in a human-murine back translational approach. MiR-21 expression was elevated in plasma of patients with CRPS as well as in nerves of mice after transient and persistent nerve injury. Mice presented with BNB leakage, as well as loss of claudin-1 in both injured and spared nerves. Moreover, the putative miR-21 target RECK was decreased and downstream Mmp9 upregulated, as was Tgfb. In vitro experiments in human epithelial cells confirmed a downregulation of CLDN1 by miR-21 mimics via inhibition of the RECK/MMP9 pathway but not TGFB. Perineurial miR-21 mimic application in mice elicited mechanical hypersensitivity, while local inhibition of miR-21 after nerve injury reversed it. In summary, the data support a novel role for miR-21, independent of prior inflammation, in elicitation of pain and impairment of the BNB via RECK/MMP9.

Selective blood-nerve barrier leakiness with claudin-1 and vessel-associated macrophage loss in diabetic polyneuropathy.

Diabetic polyneuropathy (DPN) is the most common complication in diabetes and can be painful in up to 26% of all diabetic patients. Peripheral nerves are shielded by the blood-nerve barrier (BNB) consisting of the perineurium and endoneurial vessels. So far, there are conflicting results regarding the role and function of the BNB in the pathophysiology of DPN. In this study, we analyzed the spatiotemporal tight junction protein profile, barrier permeability, and vessel-associated macrophages in Wistar rats with streptozotocin-induced DPN. In these rats, mechanical hypersensitivity developed after 2 weeks and loss of motor function after 8 weeks, while the BNB and the blood-DRG barrier were leakier for small, but not for large molecules after 8 weeks only. The blood-spinal cord barrier remained sealed throughout the observation period. No gross changes in tight junction protein or cytokine expression were observed in all barriers to blood. However, expression of Cldn1 mRNA in perineurium was specifically downregulated in conjunction with weaker vessel-associated macrophage shielding of the BNB. Our results underline the role of specific tight junction proteins and BNB breakdown in DPN maintenance and differentiate DPN from traumatic nerve injury. Targeting claudins and sealing the BNB could stabilize pain and prevent further nerve damage. KEY MESSAGES: • In diabetic painful neuropathy in rats: • Blood nerve barrier and blood DRG barrier are leaky for micromolecules. • Perineurial Cldn1 sealing the blood nerve barrier is specifically downregulated. • Endoneurial vessel-associated macrophages are also decreased. • These changes occur after onset of hyperalgesia thereby maintaining rather than inducing pain.

Oligoclonal IgG bands in chronic inflammatory polyradiculoneuropathies.

BACKGROUND: Cerebrospinal fluid (CSF) albumincytologic dissociation represents a supportive diagnostic criterion of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).Few studies have investigated possible systemic or intrathecal humoral immune response activation in CIDP.Aim of our study was to investigate whether the search of oligoclonal IgG bands (OCBs) might provide additional data helpful in CIDP diagnostic work-up. METHODS: Forty-eight consecutive patients with CIDP (34 men, mean age 59.4, range 16-83) were recruited. CSF analysis included nephelometric measurement of albumin and IgG concentrations, calculation of QALB, QAlbLIM and intrathecal IgG synthesis, and OCBs detection with isoelectric focusing. Data were compared with those from CSF and serum of 32 patients with Guillain-Barré syndrome (GBS), 18 patients with anti-myelin associated glycoprotein (MAG) antibody neuropathy, 4 patients with multifocal motor neuropathy and 32 patients with non-inflammatory neuropathies (NINPs). RESULTS: Patients with CIDP and anti-MAG antibody neuropathy had significantly higher CSF albumin concentrations and QALB values than NINPs (p=0.0003 and p=0.0095, respectively). A total of 9 (19%) patients with CIDP presented identical serum and CSF OCBs ('mirror pattern') versus 3 patients (16.6%) with anti-MAG antibody neuropathy, 13 patients (40.6%) with GBS and 12.5% patients with NINPs. Only one patient with CIDP showed unique-to-CSF OCBs. First-line therapy was effective in 80.4% of patients with CIDP, irrespective of CSF findings. CONCLUSIONS: Compared with NINP, CIDP, GBS and anti-MAG antibody neuropathies had a significantly increased CSF protein and blood-spinal nerve root barrier damage. Intrathecal humoral immune response is rare in our patients with CIDP. Systemic oligoclonal activation is more frequent, but not significantly different from what was detected in the control groups.

Netrin-1 as a Multitarget Barrier Stabilizer in the Peripheral Nerve after Injury.

The blood-nerve barrier and myelin barrier normally shield peripheral nerves from potentially harmful insults. They are broken down during nerve injury, which contributes to neuronal damage. Netrin-1 is a neuronal guidance protein with various established functions in the peripheral and central nervous systems; however, its role in regulating barrier integrity and pain processing after nerve injury is poorly understood. Here, we show that chronic constriction injury (CCI) in Wistar rats reduced netrin-1 protein and the netrin-1 receptor neogenin-1 (Neo1) in the sciatic nerve. Replacement of netrin-1 via systemic or local administration of the recombinant protein rescued injury-induced nociceptive hypersensitivity. This was prevented by siRNA-mediated knockdown of Neo1 in the sciatic nerve. Mechanistically, netrin-1 restored endothelial and myelin, but not perineural, barrier function as measured by fluorescent dye or fibrinogen penetration. Netrin-1 also reversed the decline in the tight junction proteins claudin-5 and claudin-19 in the sciatic nerve caused by CCI. Our findings emphasize the role of the endothelial and myelin barriers in pain processing after nerve damage and reveal that exogenous netrin-1 restores their function to mitigate CCI-induced hypersensitivity via Neo1. The netrin-1-neogenin-1 signaling pathway may thus represent a multi-target barrier protector for the treatment of neuropathic pain.

Impairment and restoration of the blood-nerve barrier and its correlation with pain following gradual nerve elongation of the rat sciatic nerve.

Purpose: This study aimed to evaluate the time course of impairment and restoration of the blood-nerve barrier (BNB) following gradual elongation of the sciatic nerve and to clarify its association with nociception.Materials and Methods: The right femur was lengthened at a rate of 1.5 mm/day for 10 days. Von Frey tests were performed until 50 days after lengthening. Compound muscle action potentials (CMAPs) were measured to assess gross dysfunction of the elongated nerve. Evans blue-albumin tracing and immunohistochemistry for endothelial barrier antigen (EBA), rat endothelial cell antigen-1 (RECA-1), and CD68 for qualitative and quantitative analysis of the BNB and macrophage infiltration were performed for up to 50 days after cessation of lengthening in three segments of the sciatic nerves.Results: Paw-withdrawal threshold was significantly decreased at 7 days from initiation and began to recover from day 25 after lengthening. CMAPs showed delayed latency and attenuated amplitude but recovered at day 30 after cessation. On days 10 and 30 after cessation, spotted leakage of Evans blue-albumin in the endoneurium was observed, and the ratio of EBA/RECA-1-positive microvessels was significantly decreased, which subsequently recovered simultaneously in all segments on day 50 after cessation. Macrophages did not infiltrate the BNB at any time point.Conclusion: The restoration of BNB function following gradual nerve elongation was associated with the resolution of mechanical allodynia. Our findings provide insight into the association between nerve stretch injury and chronic nociception in adult male rats, which are potentially relevant to human orthopedic procedures and chronic neuropathic pain.

Blood-Nerve Barrier (BNB) Pathology in Diabetic Peripheral Neuropathy and In Vitro Human BNB Model.

In diabetic peripheral neuropathy (DPN), metabolic disorder by hyperglycemia progresses in peripheral nerves. In addition to the direct damage to peripheral neural axons, the homeostatic mechanism of peripheral nerves is disrupted by dysfunction of the blood-nerve barrier (BNB) and Schwann cells. The disruption of the BNB, which is a crucial factor in DPN development and exacerbation, causes axonal degeneration via various pathways. Although many reports revealed that hyperglycemia and other important factors, such as dyslipidemia-induced dysfunction of Schwann cells, contributed to DPN, the molecular mechanisms underlying BNB disruption have not been sufficiently elucidated, mainly because of the lack of in vitro studies owing to difficulties in establishing human cell lines from vascular endothelial cells and pericytes that form the BNB. We have developed, for the first time, temperature-sensitive immortalized cell lines of vascular endothelial cells and pericytes originating from the BNB of human sciatic nerves, and we have elucidated the disruption to the BNB mainly in response to advanced glycation end products in DPN. Recently, we succeeded in developing an in vitro BNB model to reflect the anatomical characteristics of the BNB using cell sheet engineering, and we established immortalized cell lines originating from the human BNB. In this article, we review the pathologic evidence of the pathology of DPN in terms of BNB disruption, and we introduce the current in vitro BNB models.

Targeting the blood-nerve barrier for the management of immune-mediated peripheral neuropathies.

Healthy peripheral nerves encounter, with increased frequency, numerous chemical, biological, and biomechanical forces. Over time and with increasing age, these forces collectively contribute to the pathophysiology of a spectrum of traumatic, metabolic, and/or immune-mediated peripheral nerve disorders. The blood-nerve barrier (BNB) serves as a critical first-line defense against chemical and biologic insults while biomechanical forces are continuously buffered by a dense array of longitudinally orientated epineural collagen fibers exhibiting high-tensile strength. As emphasized throughout this Experimental Neurology Special Issue, the BNB is best characterized as a functionally dynamic multicellular vascular unit comprised of not only highly specialized endoneurial endothelial cells, but also associated perineurial cells, pericytes, Schwann cells, basement membrane, and invested axons. The composition of the BNB, while anatomically distinct, is not functionally dissimilar to that of the well characterized neurovascular unit of the central nervous system. While the BNB lacks a glial limitans and an astrocytic endfoot layer, the primary function of both vascular units is to establish, maintain, and protect an optimal endoneurial (PNS) or interstitial (CNS) fluid microenvironment that is vital for proper neuronal function. Altered endoneurial homeostasis as a secondary consequence of BNB dysregulation is considered an early pathological event in the course of a variety of traumatic, immune-mediated, or metabolically acquired peripheral neuropathies. In this review, emerging experimental advancements targeting the endoneurial microvasculature for the therapeutic management of immune-mediated inflammatory peripheral neuropathies, including the AIDP variant of Guillain-Barré syndrome, are discussed.

Biology of the human blood-nerve barrier in health and disease.

A highly regulated endoneurial microenvironment is required for normal axonal function in peripheral nerves and nerve roots, which structurally consist of an outer collagenous epineurium, inner perineurium consisting of multiple concentric layers of specialized epithelioid myofibroblasts that surround the innermost endoneurium, which consists of myelinated and unmyelinated axons embedded in a looser mesh of collagen fibers. Endoneurial homeostasis is achieved by tight junction-forming endoneurial microvessels that control ion, solute, water, nutrient, macromolecule and leukocyte influx and efflux between the bloodstream and endoneurium, and the innermost layers of the perineurium that control interstitial fluid component flux between the freely permeable epineurium and endoneurium. Strictly speaking, endoneurial microvascular endothelium should be considered the blood-nerve barrier (BNB) due to direct communication with circulating blood. The mammalian BNB is considered the second most restrictive vascular system after the blood-brain barrier (BBB) based on classic in situ permeability studies. Structural alterations in endoneurial microvessels or interactions with hematogenous leukocytes have been described in several human peripheral neuropathies; however major advances in BNB biology in health and disease have been limited over the past 50 years. Guided by transcriptome and proteome studies of normal and pathologic human peripheral nerves, purified primary and immortalized human endoneurial endothelial cells that form the BNB and leukocytes from patients with well-characterized peripheral neuropathies, validated by in situ or ex vivo protein expression studies, data are emerging on the molecular and functional characteristics of the human BNB in health and in specific peripheral neuropathies, as well as chronic neuropathic pain. These early advancements have the potential to not only increase our understanding of how the BNB works and adapts or fails to adapt to varying insult, but provide insights relevant to pathogenic leukocyte trafficking, with translational potential and specific therapeutic application for chronic peripheral neuropathies and neuropathic pain.

Characteristics of the nerve barrier and the blood dorsal root ganglion barrier in health and disease.

A variety of barriers ensures the protection of the peripheral nervous system from noxious blood-borne or surrounding stimuli. In this review, anatomy and functioning of the blood nerve barrier (BNB) and the blood DRG barrier (BDB) will be presented and key tight junction proteins described: ZO-1, claudin-1, -3, -5, -11, -12, -19, occludin, and tricellulin. Different diseases can lead to or be accompanied by nerve barrier disruption; impairment of nerve barriers in turn worsens pathology. Peripheral nerve injury, diabetic neuropathy and inflammatory polyneuropathy cause an increased permeability of BNB and BDB. Knowledge and understanding of these mechanisms might ultimately lead to the invention of drugs to control barrier function and help ameliorating neurological diseases.

The Role of Endothelial Dysfunction in Peripheral Blood Nerve Barrier: Molecular Mechanisms and Pathophysiological Implications.

The exchange of solutes between the blood and the nerve tissue is mediated by specific and high selective barriers in order to ensure the integrity of the different compartments of the nervous system. At peripheral level, this function is maintained by the Blood Nerve Barrier (BNB) that, in the presence, of specific stressor stimuli can be damaged causing the onset of neurodegenerative processes. An essential component of BNB is represented by the endothelial cells surrounding the sub-structures of peripheral nerves and increasing evidence suggests that endothelial dysfunction can be considered a leading cause of the nerve degeneration. The purpose of this review is to highlight the main mechanisms involved in the impairment of endothelial cells in specific diseases associated with peripheral nerve damage, such as diabetic neuropathy, erectile dysfunction and inflammation of the sciatic nerve.

In situ molecular characterization of endoneurial microvessels that form the blood-nerve barrier in normal human adult peripheral nerves.

The blood-nerve barrier (BNB) formed by tight junction-forming endoneurial microvessels located in the innermost compartment of peripheral nerves, and the perineurium serve to maintain the internal microenvironment required for normal signal transduction. The specific molecular components that define the normal adult human BNB are not fully known. Guided by data derived from the adult human BNB transcriptome, we evaluated the in situ expression of 25 junctional complex, transporter, cell membrane, and cytoskeletal proteins in four histologically normal adult sural nerves by indirect fluorescent immunohistochemistry to determine proteins specifically expressed by restrictive endoneurial microvascular endothelium. Using Ulex Europaeus Agglutinin-1 expression to detect endothelial cells, we ascertained that the selected proteins were uniformly expressed in ≥90% of endoneurial microvessels. P-glycoprotein (also known as adenosine triphosphate-binding cassette subfamily B member 1) and solute carrier family 1 member 1 demonstrated restricted expression by endoneurial endothelium only, with classic tight junction protein claudin-5 also expressed on fenestrated epineurial macrovessels, and vascular-specific adherens junction protein cadherin-5 also expressed by the perineurium. The expression profiles of the selected proteins provide significant insight into the molecular composition of normal adult peripheral nerves. Further work is required to elucidate the human adult BNB molecular signature in order to better understand its development and devise strategies to restore function in peripheral neuropathies.

CD8 T cell-derived perforin aggravates secondary spinal cord injury through destroying the blood-spinal cord barrier.

Perforin plays an important role in autoimmune and infectious diseases, but its function in immune inflammatory responses after spinal cord injury (SCI) has received insufficient attention. The goal of this study is to determine the influence of perforin after spinal cord injury (SCI) on secondary inflammation. Compared recovery from SCI in perforin knockout (Prf1-/-) and wild-type(WT)mice, WT mice had significantly lower the Basso mouse score (BMS), CatWalk XT, and motor-evoked potentials (MEPs) than Prf1-/- mice. Spinal cord lesions were also more obvious through glial fibrillary acidic protein (GFAP), Nissl, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. Furthermore, the blood-spinal cord barrier (BSCB) disruption was more severe and inflammatory cytokine levels were higher. Flow cytometry indicated that perforin mainly originated from CD8 T cells. With flow cytometry and enzyme-linked immunosorbent assay (ELISA), human cerebrospinal fluid (CSF) yielded similar results. Together, this study firstly demonstrated that CD8 T cell-derived perforin is detrimental to SCI recovery in the mouse model. Mechanistically, this effect occurs because perforin increases BSCB permeability, causing inflammatory cells and related cytokines to infiltrate and disrupt the nervous system.

Tissue plasminogen activator and neuropathy open the blood-nerve barrier with upregulation of microRNA-155-5p in male rats.

The blood-nerve barrier (BNB) consisting of the perineurium and endoneurial vessels is sealed by tight junction proteins. BNB alterations are a crucial factor in the pathogenesis of peripheral neuropathies. However, barrier opening, e.g. by tissue plasminogen activator (tPA), can also facilitate topical application of analgesics. Here, we examined tPA both in the pathophysiology of neuropathy-induced BNB opening or via exogenous application and its effect on the cytoplasmatic tight junction protein anchoring protein, zona occludens-1 (ZO-1), the adherens molecule JAM-C and microRNA(miR)-155-5p. Specifically, we investigated whether tPA alone and barrier opening lead to pain behavioral changes, i.e. hyperalgesia, or whether these effects require further factors. Male Wistar rats underwent chronic constriction injury (CCI) or were treated by a single perisciatic application of recombinant (r)tPA. CCI elicited mechanical allodynia, tPA mRNA upregulation, macrophage invasion, BNB leakage for large molecule tracers, downregulation of ZO-1 and JAM-C mRNA/protein, and a loss of immunoreactivity of both in perineurium and endoneurial cells. Similarly, after perisciatic rtPA injection, ZO-1 and JAM-C mRNA as well as cytosolic/membrane protein and ZO-1 immunoreactivity were downregulated, and the BNB was opened. Neither mechanical hypersensitivity nor macrophage infiltration was observed after rtPA in contrast to CCI. Mechanistically, miR-155-5p, which is known to destabilize barriers and tight junction proteins like claudin-1 and ZO-1, was increased in CCI and to lesser extent after rtPA application. In summary, tPA transiently opens the BNB possibly via miR-155-5p. However, tPA does not provoke allodynia in the absence of a neuropathic stimulus like a ligation or inflammation.

Increased IP-10 production by blood-nerve barrier in multifocal acquired demyelinating sensory and motor neuropathy and multifocal motor neuropathy.

OBJECTIVE: Dysfunction of the blood-nerve barrier (BNB) plays important roles in chronic inflammatory demyelinating polyneuropathy (CIDP) and multifocal motor neuropathy (MMN). The aim of the present study was to identify the candidate cytokines/chemokines that cause the breakdown of the BNB using sera from patients with CIDP and MMN. METHODS: We determined the levels of 27 cytokines and chemokines in human peripheral nerve microvascular endothelial cells (PnMECs) after exposure to sera obtained from patients with CIDP variants (typical CIDP and multifocal acquired demyelinating sensory and motor neuropathy [MADSAM]), MMN and amyotrophic lateral sclerosis (ALS), and healthy controls (HC), using a multiplexed fluorescent bead-based immunoassay system. RESULTS: The induced protein (IP)10 level in the cells in both the MADSAM and MMN groups was markedly increased in comparison with the typical CIDP, ALS and HC groups. The other cytokines, including granulocyte colony-stimulating factor,vascular endothelial growth factor (VEGF) and interleukin-7, were also significantly upregulated in the MADSAM group. The increase of IP-10 produced by PnMECs was correlated with the presence of conduction block in both the MADSAM and MMN groups. CONCLUSION: The autocrine secretion of IP-10 induced by patient sera in PnMECs was markedly upregulated in both the MADSAM and MMN groups. The overproduction of IP-10 by PnMECs leads to the focal breakdown of the BNB and may help to mediate the transfer of pathogenic T cells across the BNB, thereby resulting in the appearance of conduction block in electrophysiological studies of patients with MADSAM and MMN.

GDNF enhances human blood-nerve barrier function in vitro via MAPK signaling pathways.

The human blood-nerve barrier (BNB) formed by endoneurial microvascular endothelial cells, serves to maintain the internal microenvironment in peripheral nerves required for normal axonal signal transduction to and from the central nervous system. The mechanisms of human BNB formation in health and disease are not fully elucidated. Prior work established a sufficient role for glial-derived neurotrophic factor (GDNF) in enhancing human BNB biophysical properties following serum withdrawal in vitro via RET-tyrosine kinase-dependent cytoskeletal remodeling. The objective of the study was to ascertain the downstream signaling pathway involved in this process and more comprehensively determine the molecular changes that may occur at human BNB intercellular junctions under the influence of GDNF. Proteomic studies suggested expression of several mitogen-activated protein kinases (MAPKs) in confluent GDNF-treated endoneurial endothelial cells following serum withdrawal. Using electric cell-substrate impedance sensing to continuously measure transendothelial electrical resistance and static transwell solute permeability assays with fluoresceinated small and large molecules to evaluate BNB biophysical function, we determined MAPK signaling was essential for GDNF-mediated BNB TEER increase following serum withdrawal downstream of RET-tyrosine kinase signaling that persisted for up to 48 hours in vitro. This increase was associated with reduced solute permeability to fluoresceinated sodium and high molecular weight dextran. Specific GDNF-mediated alterations were detected in cytoskeletal and intercellular junctional complex molecular transcripts and proteins relative to basal conditions without exogenous GDNF. This work provides novel insights into the molecular determinants and mechanisms responsible for specialized restrictive human BNB formation in health and disease.