A Method for the Isolation and Culture of Rat Peripheral Nerve Vascular Endothelial Cells

doi:10.1006/mcne.1994.1051

Molecular and Cellular Neuroscience

Volume 5, Issue 5, October 1994, Pages 413-417

https://doi.org/10.1006/mcne.1994.1051 Get rights and content

Abstract

In studying autoimmune diseases of the human peripheral nervous system (PNS), in vitro studies involving the use of cultured rat Schwann cells, neurons, and disease-inducing immune system cells have provided basic information about disease pathogenesis. For example, T-cells that induce experimental allergic neuritis have been shown in vitro to damage Schwann cells, the target cell in these diseases. However, before making contact with Schwann cells, these T-cells must first pass through the blood-nerve barrier. Despite the importance of this interaction, no studies employing PNS endothelial cells in coculture with dorsal root ganglia cells to mimic the environment of the blood-nerve barrier have been reported. This paper describes a simple method for the isolation and culture of peripheral nerve vascular endothelial cells from adult rats that should facilitate in vitro studies of the blood-nerve barrier. Endothelial cells were identified by their expression of an endothelial cell marker, Factor VIII/von Willebrand factor. Their identity was further confirmed by their inability to express Thy 1.1, a fibroblast marker, and their in vitro morphology. Purity of endothelial cell cultures was ensured by a regular program of Thy 1.1 complement depletion of fibroblasts.

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Cited by (15)

Targeting the blood-nerve barrier for the management of immune-mediated peripheral neuropathies
2020, Experimental Neurology
Citation Excerpt :
A recent molecular study characterized the transcriptome from human endoneurial microvessels as consisting of 12,881 unique transcripts expressed by endoneurial endothelial cells inclusive of an impressive array of transporters (509), chemokine and chemokine receptors, as well as over 100 junctional complex proteins (22 tight junction/JAMs; 45 adherens junction/associated; 52 cell-junction /adaptor proteins) (Palladino et al., 2017). Primary endoneurial endothelial cells isolated from different species by different laboratories (Abe et al., 2012; Argall et al., 1994; Kanda et al., 1997; Kanda et al., 2000; Kanda et al., 2003; Langert et al., 2013b; Sano et al., 2007; Ubogu, 2013; Yosef and Ubogu, 2013; Yosef et al., 2010) appear to have, however, remarkably similar cellular and molecular characteristics (Table 1). One notable distinction, however, is with observed transendothelial electrical resistance (TEER), a widely accepted quantitative measure of tight-junction integrity.
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.
Efficient Schwann cell purification by differential cell detachment using multiplex collagenase treatment
2008, Journal of Neuroscience Methods
Schwann cell purification is usually difficult due to the contamination of fibroblasts, which often become a predominant cell type in Schwann cell culture in vitro. We have developed a novel and efficient method to enrich Schwann cells by differential detachment of two types of cells. In the culture, cells were treated with a multiplex collagenase and the Schwann cells were found to detach faster than fibroblasts and thus Schwann cells could be easily isolated. Within 5 days, Schwann cell purity could reach above 99%, which was confirmed by immunostaining characterization and flow cytometric analysis. In addition, this efficient method can reach a high cell yield after two rounds of differential detachment procedures and does not require antimitotic treatment or special equipments as often needed by other reported methods.
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Regular ArticleA Method for the Isolation and Culture of Rat Peripheral Nerve Vascular Endothelial Cells

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Regular Article
A Method for the Isolation and Culture of Rat Peripheral Nerve Vascular Endothelial Cells