Establishment of a method for evaluating endothelial cell injury by TNF-α in vitro for clarifying the pathophysiology of virus-associated acute encephalopathy.
BACKGROUND
Virus-associated acute encephalopathy (VAE) is a severe central nervous system complication caused by common viral infections in children. The pathophysiology of VAE is thought to be endothelial injury. This study was designed to establish an in vitro VAE model for evaluating endothelial injury caused by the proinflammatory cytokine TNF-α.


METHODS
Transwell-grown human umbilical vein endothelial cells (HUVECs) monolayers were incubated with serially diluted TNF-α. Transendothelial electrical resistance (TER) was measured using impedance spectroscopy. Permeability changes of HUVECs after TNF-α treatment were determined by fluorescein isothiocyanate (FITC)-conjugated dextran. Moreover, TNF-α-induced morphological changes in claudin-5 and apoptosis were observed by immunofluorescent staining.


RESULTS
The decrease in TER, time of TER recovery to baseline, and increase in permeability were all dependent on TNF-α concentration. Immunofluorescent staining showed that claudin-5 was delocalized after TNF-α treatment in a dose-dependent manner. In addition, some apoptotic cells were observed at high TNF-α concentrations.


CONCLUSIONS
TER measurement combined with a permeability assay could be useful for evaluating vascular endothelial cell permeability in an in vitro model. These evaluation methods will contribute to both the development of specific treatments focusing on vascular permeability, and the search for a novel therapeutic strategy in VAE treatment.
Department of Pediatrics, Fukushima Medical University, Fukushima, Japan.
Kyohei
K
Miyazaki
Department of Pediatrics, Fukushima Medical University, Fukushima, Japan.
Koichi
K
Hashimoto
Department of Pediatrics, Fukushima Medical University, Fukushima, Japan.
Masatoki
M
Sato
Department of Pediatrics, Fukushima Medical University, Fukushima, Japan.
Masahiro
M
Watanabe
Department of Basic Pathology, Fukushima Medical University, Fukushima, Japan.
Naoki
N
Tomikawa
Department of Pediatrics, Fukushima Medical University, Fukushima, Japan.
Shuto
S
Kanno
Department of Pediatrics, Fukushima Medical University, Fukushima, Japan.
Yukihiko
Y
Kawasaki
Department of Pediatrics, Fukushima Medical University, Fukushima, Japan.
Nobuo
N
Momoi
Department of Pediatrics, Fukushima Medical University, Fukushima, Japan.
Mitsuaki
M
Hosoya
D017209:Apoptosis / Q000187:drug effects; D001927:Brain Diseases / Q000821:virology*; D002648:Child; D062507:Claudin-5 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D066298:In Vitro Techniques; D010539:Permeability; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity*
C107466:CLDN5 protein, human; D062507:Claudin-5; D014409:Tumor Necrosis Factor-alpha