Abstract
BackgroundExcitotoxicity is a central pathological pathway in many neurological diseases with blood–brain barrier (BBB) dysfunction. Kainate, an exogenous excitotoxin, induces epilepsy and BBB damage in animal models, but the direct effect of kainate on brain endothelial cells has not been studied in detail. Our aim was to examine the direct effects of kainate on cultured cells of the BBB and to test three anti-inflammatory and antioxidant drugs used in clinical practice, simvastatin, edaravone and dexamethasone, to protect against kainate-induced changes.MethodsPrimary rat brain endothelial cell, pericyte and astroglia cultures were used to study cell viability by impedance measurement. BBB permeability was measured on a model made from the co-culture of the three cell types. The production of nitrogen monoxide and reactive oxygen species was followed by fluorescent probes. The mRNA expression of kainate receptors and nitric oxide synthases were studied by PCR.ResultsKainate damaged brain endothelial cells and made the immunostaining of junctional proteins claudin-5 and zonula occludens-1 discontinuous at the cell border indicating the opening of the barrier. The permeability of the BBB model for marker molecules fluorescein and albumin and the production of nitric oxide in brain endothelial cells were increased by kainate. Simvastatin, edaravone and dexamethasone protected against the reduced cell viability, increased permeability and the morphological changes in cellular junctions caused by kainate. Dexamethasone attenuated the elevated nitric oxide production and decreased the inducible nitric oxide synthase (NOS2/iNOS) mRNA expression increased by kainate treatment.ConclusionKainate directly damaged cultured brain endothelial cells. Simvastatin, edaravone and dexamethasone protected the BBB model against kainate-induced changes. Our results confirmed the potential clinical usefulness of these drugs to attenuate BBB damage.
Highlights
Excitotoxicity has a pivotal role in many neurological diseases, including stroke, traumatic brain injury, epilepsy and neurodegenerative disorders like multiple sclerosis, Alzheimer’s, Huntington’s and Parkinson’s diseases [1,2,3]
Effect of kainate on cell viability of rat brain endothelial cells, astrocytes and pericytes Viability and integrity of brain endothelial, astrocyte and pericyte cell layers were monitored with real-time impedance measurement for 24 h after kainate treatment (10 and 100 μM) (Fig. 1a)
In conclusion, we proved for the first time that the excitotoxin kainate directly damaged cultured brain endothelial cells
Summary
Excitotoxicity has a pivotal role in many neurological diseases, including stroke, traumatic brain injury, epilepsy and neurodegenerative disorders like multiple sclerosis, Alzheimer’s, Huntington’s and Parkinson’s diseases [1,2,3]. Barna et al Fluids Barriers CNS (2020) 17:5 endogenous or exogenous excitotoxins, like N-methyld-aspartate (NMDA), α-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid (AMPA) and kainate, act on specific receptor families. Excitatory neurotransmitters are fundamental for physiological processes, but the excessive stimulation of these receptors causes excitotoxicity, the damage or death of the nerve cells [4]. In research kainate is used to induce epilepsy in animal experiments in which excitotoxicity and neuronal damage and blood–brain barrier (BBB) leakage and neurovascular changes are observed [5]. An exogenous excitotoxin, induces epilepsy and BBB damage in animal models, but the direct effect of kainate on brain endothelial cells has not been studied in detail. Our aim was to examine the direct effects of kainate on cultured cells of the BBB and to test three anti-inflammatory and antioxidant drugs used in clini‐ cal practice, simvastatin, edaravone and dexamethasone, to protect against kainate-induced changes
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