In Vitro Blood-Brain Barrier Models Using Brain Capillary Endothelial Cells Isolated from Neonatal and Adult Rats Retain Age-Related Barrier Properties

Fuyuko Takata,Takashi Machida,Atsushi Yamauchi,Junichi Matsumoto,Shinya Dohgu,Keisuke Shibata,Yasufumi Kataoka,Schuichi Koizumi,Kayoko Fujishita,Youichi Shinozaki,Kaoru Sato,Stefan Liebner

doi:10.1371/journal.pone.0055166

Abstract

The blood–brain barrier (BBB) restricts the entry of circulating drugs and xenobiotics into the brain, and thus its permeability to substances is a critical factor that determines their central effects. The infant brain is vulnerable to neurotoxic substances partly due to the immature BBB. The employment of in vitro BBB models to evaluate permeability of compounds provides higher throughput than that of in vivo animal experiments. However, existing in vitro BBB models have not been able to simulate the intrinsic neonatal BBB. To establish a neonatal BBB model that mimics age-related BBB properties, the neonatal and adult in vitro BBB models were constructed with brain endothelial cells isolated from 2- and 8-week-old rats, respectively. To evaluate BBB functions, transendothelial electrical resistance, permeability of sodium fluorescein and Evans blue-albumin, and transport of rhodamine123 were measured. Radiolabelled drugs were used for BBB permeability studies in the neonatal and adult BBB models (in vitro) and in age-matched rats (in vivo). The neonatal BBB model showed lower barrier and p-glycoprotein (P-gp) functions than the adult BBB model; these were well associated with lower expressions of the barrier-related proteins and P-gp, and a different distribution pattern of immunostained barrier-related proteins. Verapamil (a P-gp inhibitor) significantly increased the influx of rhodamine 123, supporting functional P-gp expression in the neonatal BBB model. Valproic acid, but not nicotine, showed higher BBB permeability in the neonatal BBB model, which was well in accordance with the in vivo BBB property. We established a neonatal BBB model in vitro. This could allow us to assess the age-dependent BBB permeability of drugs.

Highlights

The developing brain is known to be vulnerable to neurotoxic substances including therapeutic drugs
TEER values increased, reached a peak during a period from 3 to 4 days after co-culture and decreased to the levels seen at the start of co-culture in the adult blood-brain barrier (BBB) model
This time course of TEER values was observed in the neonatal BBB model even though peak TEER values were extremely low compared with those of the adult model

Summary

Introduction

The developing brain is known to be vulnerable to neurotoxic substances including therapeutic drugs. The BBB is comprised of brain microvessel endothelial cells (BMECs) and perivascular cells (pericytes and astrocytes) This barrier functions as an interface between the brain parenchyma and circulating blood. There are reports showing that the BBB in newborn animals allows compounds with low BBB permeability, in the adult brain, to penetrate into the brain parenchyma [6,7]. This suggests that ontogenic differences in endothelial barrier properties at the BBB may account for different responses to drugs in the CNS between infant and adult humans. Determining the permeability of individual drugs through the infant BBB is required to predict and/or evaluate the potential neurotoxicity

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Conclusion