ABC Transporter Function and Regulation at the Blood–Spinal Cord Barrier

We present here an initial characterization of ATP binding cassette (ABC) transporter function and regulation at the blood–spinal cord barrier. We isolated capillaries from rat spinal cords and studied transport function using a confocal microscopy-based assay and protein expression using western blots. These capillaries exhibited transport function and protein expression of P-glycoprotein (Abcb1), multidrug resistance protein 2 (Mrp2, Abcc2), and breast cancer-related protein (Bcrp, Abcg2). Exposing isolated capillaries to dioxin (activates aryl hydrocarbon receptor) increased transport mediated by all three transporters. Brain and spinal cord capillaries from dioxin-dosed rats exhibited increased P-glycoprotein-mediated transport and increased protein expression for all three ABC transporters. These findings indicate similar ABC transporter expression, function, and regulation at the blood–spinal cord and blood–brain barriers.

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Characterization of specific [3H]nociceptin binding in rat brain and spinal cord

The Japanese Journal of Pharmacology ◽

10.1016/s0021-5198(19)48113-4 ◽

2000 ◽

Vol 82 ◽

pp. 163

Author(s):

Toyofumi Kusaka ◽

Shizuo Yamada ◽

Ryohei Kimura

Keyword(s):

Spinal Cord ◽

Rat Brain ◽

Brain And Spinal Cord

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Isolation, culture, and downstream characterization of primary microglia and astrocytes from adult rodent brain and spinal cord

Journal of Neuroscience Methods ◽

10.1016/j.jneumeth.2020.108742 ◽

2020 ◽

Vol 340 ◽

pp. 108742 ◽

Cited By ~ 2

Author(s):

Nilesh M. Agalave ◽

Brandon T. Lane ◽

Prapti H. Mody ◽

Thomas A. Szabo-Pardi ◽

Michael D. Burton

Keyword(s):

Spinal Cord ◽

Primary Microglia ◽

Rodent Brain ◽

Brain And Spinal Cord

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Mrp1 is Essential for Sphingolipid Signaling to P-Glycoprotein in Mouse Blood–Brain and Blood–Spinal Cord Barriers

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2012.174 ◽

2012 ◽

Vol 33 (3) ◽

pp. 381-388 ◽

Cited By ~ 33

Author(s):

Tara A Cartwright ◽

Christopher R Campos ◽

Ronald E Cannon ◽

David S Miller

Keyword(s):

Spinal Cord ◽

Endothelial Cells ◽

Transport Activity ◽

Wild Type ◽

Mouse Blood ◽

P Glycoprotein ◽

Blood Brain ◽

Tnf Α ◽

Glycoprotein Transport ◽

Brain And Spinal Cord

At the blood–brain and blood–spinal cord barriers, P-glycoprotein, an ATP-driven drug efflux pump, is a major obstacle to central nervous system (CNS) pharmacotherapy. Recently, we showed that signaling through tumor necrosis factor-α (TNF-α), sphingolipids, and sphingosine-1-phosphate receptor 1 (S1PR1) rapidly and reversibly reduced basal P-glycoprotein transport activity in the rat blood–brain barrier. The present study extends those findings to the mouse blood–brain and blood–spinal cord barriers and, importantly, identifies multidrug resistance-associated protein 1 (Mrp1, Abcc1) as the transporter that mediates S1P efflux from brain and spinal cord endothelial cells. In brain and spinal cord capillaries isolated from wild-type mice, TNF-α, sphingosine, S1P, the S1PR agonist fingolimod (FTY720), and its active, phosphorylated metabolite, FTY720P, reduced P-glycoprotein transport activity; these effects were abolished by a specific S1PR1 antagonist. In brain and spinal cord capillaries isolated from Mrp1-null mice, neither TNF-α nor sphingosine nor FTY720 reduced P-glycoprotein transport activity. However, S1P and FTY720P had the same S1PR1-dependent effects on transport activity as in capillaries from wild-type mice. Thus, deletion of Mrp1 alone terminated endogenous signaling to S1PR1. These results identify Mrp1 as the transporter essential for S1P efflux from the endothelial cells and thus for inside-out S1P signaling to P-glycoprotein at the blood–brain and blood–spinal cord barriers.

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Validating Immunohistochemistry Assay Specificity in Investigative Studies: Considerations for a Weight of Evidence Approach

Veterinary Pathology ◽

10.1177/0300985820960132 ◽

2020 ◽

pp. 030098582096013

Author(s):

Joshua D. Webster ◽

Margaret Solon ◽

Katherine N. Gibson-Corley

Keyword(s):

Protein Expression ◽

Experimental Studies ◽

Negative Control ◽

Weight Of Evidence ◽

Molecular Technique ◽

Western Blots ◽

Cumulative Evidence ◽

Antibody Validation ◽

Careful Design

Immunohistochemistry (IHC) is a fundamental molecular technique that provides information on protein expression in the context of spatial localization and tissue morphology. IHC is used in all facets of pathology from identifying infectious agents or characterizing tumors in diagnostics, to characterizing cellular and molecular processes in investigative and experimental studies. Confidence in an IHC assay is primarily driven by the degree to which it is validated. There are many approaches to validate an IHC assay’s specificity including bioinformatics approaches using published protein sequences, careful design of positive and negative tissue controls, use of cell pellets with known target protein expression, corroboration of IHC findings with western blots and other analytical methods, and replacement of the primary antibody with an appropriate negative control reagent. Each approach has inherent strengths and weaknesses, and the thoughtful use of these approaches provides cumulative evidence, or a weight of evidence, to support the IHC assay’s specificity and build confidence in a study’s conclusions. Although it is difficult to be 100% confident in the specificity of any IHC assay, it is important to consider how validation approaches provide evidence to support or to question the specificity of labeling, and how that evidence affects the overall interpretation of a study’s results. In this review, we discuss different approaches for IHC antibody validation, with an emphasis on the characterization of antibody specificity in investigative studies. While this review is not prescriptive, it is hoped that it will be thought provoking when considering the interpretation of IHC results.

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