Blood–brain barrier dysfunction in l-ornithine induced acute pancreatitis in rats and the direct effect of l-ornithine on cultured brain endothelial cells
Authors: Walter Fruzsina R. , Harazin András , Tóth Andrea E. , Veszelka Szilvia , Santa-Maria Ana R. , Barna Lilla , Kincses András , Biczó György , Balla Zsolt , Kui Balázs , Maléth József , Cervenak László , Tubak Vilmos , Kittel Ágnes , Rakonczay Zoltán , Deli Mária A. 
Published: February 2022

Abstract
Background
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.

Methods
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.

Results
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.

Conclusion
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.