Abstract
BackgroundSepsis and jaundice are common conditions in newborns that can lead to brain damage. Though lipopolysaccharide (LPS) is known to alter the integrity of the blood-brain barrier (BBB), little is known on the effects of unconjugated bilirubin (UCB) and even less on the joint effects of UCB and LPS on brain microvascular endothelial cells (BMEC).Methodology/Principal FindingsMonolayers of primary rat BMEC were treated with 1 µg/ml LPS and/or 50 µM UCB, in the presence of 100 µM human serum albumin, for 4 or 24 h. Co-cultures of BMEC with astroglial cells, a more complex BBB model, were used in selected experiments. LPS led to apoptosis and UCB induced both apoptotic and necrotic-like cell death. LPS and UCB led to inhibition of P-glycoprotein and activation of matrix metalloproteinases-2 and -9 in mono-cultures. Transmission electron microscopy evidenced apoptotic bodies, as well as damaged mitochondria and rough endoplasmic reticulum in BMEC by either insult. Shorter cell contacts and increased caveolae-like invaginations were noticeable in LPS-treated cells and loss of intercellular junctions was observed upon treatment with UCB. Both compounds triggered impairment of endothelial permeability and transendothelial electrical resistance both in mono- and co-cultures. The functional changes were confirmed by alterations in immunostaining for junctional proteins β-catenin, ZO-1 and claudin-5. Enlargement of intercellular spaces, and redistribution of junctional proteins were found in BMEC after exposure to LPS and UCB.ConclusionsLPS and/or UCB exert direct toxic effects on BMEC, with distinct temporal profiles and mechanisms of action. Therefore, the impairment of brain endothelial integrity upon exposure to these neurotoxins may favor their access to the brain, thus increasing the risk of injury and requiring adequate clinical management of sepsis and jaundice in the neonatal period.
Highlights
The blood-brain barrier (BBB) is a dynamic interface between blood and brain compartments that protects nerve tissue from insults
In order to evaluate the impairment of rat brain microvascular endothelial cells (BMEC) viability by exposure to LPS and unconjugated bilirubin (UCB), we assessed necrosis-like cell death by determining the release of intracellular lactate dehydrogenase (LDH) into the incubation medium
We have demonstrated that LPS treatment caused a concentration- and time-dependent decrease in transendothelial electrical resistance (TEER) and increased permeability to tracers in rat BMEC co-cultured with astrocytes [10], but, so far, such features have not been explored in cells exposed to UCB or to both neurotoxins, either in co-cultures or in BMEC mono-cultures
Summary
The blood-brain barrier (BBB) is a dynamic interface between blood and brain compartments that protects nerve tissue from insults. Brain microvascular endothelial cells (BMEC), possessing unique properties, are considered the main constituents of the barrier. They regulate the selective passage of substances through the expression of specific influx and efflux transport systems [1]. Pathological conditions affecting the integrity of intercellular junctions, BBB transporters or the basement membrane impair the barrier function of the BBB, which can lead to or further increase brain damage. Though lipopolysaccharide (LPS) is known to alter the integrity of the blood-brain barrier (BBB), little is known on the effects of unconjugated bilirubin (UCB) and even less on the joint effects of UCB and LPS on brain microvascular endothelial cells (BMEC)
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