Blood-brain barrier dysfunction in L-ornithine induced acute pancreatitis in rats and the direct effect of L-ornithine on cultured brain endothelial cells

In severe acute pancreatitis (AP) the CNS is affected manifesting in neurological symptoms. Earlier research from our laboratory showed blood-brain barrier (BBB) permeability elevation in a taurocholate-induced AP model. Here we aimed to further explore BBB changes in AP using a different, non-invasive in vivo model induced by L-ornithine. Our goal was also to identify whether L-ornithine, a cationic amino acid, has a direct effect on brain endothelial cells in vitro contributing to the observed BBB changes.

We demonstrated BBB damage in the L-ornithine-induced rat AP model suggesting a general, AP model independent effect. L-ornithine induced oxidative stress, decreased barrier integrity and altered BBB morphology in a culture BBB model. These data suggest a direct effect of the cationic L-ornithine on brain endothelium. Endothelial surface glycocalyx injury was revealed both in vivo and in vitro, as an additional novel component of the BBB-related pathological changes in AP.

AP was induced in rats by the intraperitoneal administration of L-ornithine-HCl. Vessel permeability and the gene expression of the primary transporter of L-ornithine, cationic amino acid transporter-1 (Cat-1) in the brain cortex, pancreas, liver and lung were determined. Ultrastructural changes were followed by transmission electron microscopy. The direct effect of L-ornithine was tested on primary rat brain endothelial cells and a triple co-culture model of the BBB. Viability and barrier integrity, including permeability and TEER, nitrogen monoxide (NO) and reactive oxygen species (ROS) production and NF-κB translocation were measured. Fluorescent staining for claudin-5, occludin, ZO-1, β-catenin, cell adhesion molecules Icam-1 and Vcam-1 and mitochondria was performed. Cell surface charge was measured by laser Doppler velocimetry.

In the L-ornithine-induced AP model vessel permeability for fluorescein and Cat-1 expression levels were elevated in the brain cortex and pancreas. On the ultrastructural level surface glycocalyx and mitochondrial damage, tight junction and basal membrane alterations, and glial edema were observed. L-ornithine decreased cell impedance and elevated the BBB model permeability in vitro. Discontinuity in the surface glycocalyx labeling and immunostaining of junctional proteins, cytoplasmic redistribution of ZO-1 and β-catenin, and elevation of Vcam-1 expression were measured. ROS production was increased and mitochondrial network was damaged without NF-κB, NO production or mitochondrial membrane potential alterations. Similar ultrastructural changes were seen in L-ornithine treated brain endothelial cells as in vivo. The basal negative zeta potential of brain endothelial cells became more positive after L-ornithine treatment.