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Aleksandra Rutkowska
Medical University of Gdańsk, Poland
Abstract: The in vitro blood-brain barrier (BBB) models were initially built on a membrane in a transwell system with a single cell type, usually the brain vascular pericytes or endothelial cells (ECs). Recently, several advances have been made to build more sophisticated and physiologically relevant in vitro models of the BBB. Here, we present a tri-cell BBB model comprised of conditionally immortalised human brain microvascular ECs, pericytes and astrocytes. The cells are seeded in the classical transwell system or can be grown into a 3D spheroid. In a transwell or spheroid form, the model can be applied to study drug BBB permeability, disruption or breakdown of the BBB, transmigration of the immune cells, and many others. We use these models to study the pathophysiology of the BBB in multiple sclerosis and, in particular, the mechanisms involved in the disruption of the BBB, its protection and recovery. Both, the transwell and spheroid models, can also be used to measure the transmigration of immune cells. We measure the chemotaxis of CD4+ cells isolated from multiple sclerosis patients and modulate their function in the search for novel therapy. Here, we present some of our preliminary results obtained with these BBB models and discuss the shortcomings and challenges ahead.
Fionä Caratis1, Tomomi Furihata2, Bartosz Karaszewski3, Aleksandra Rutkowska1
1Department of Anatomy and Neurobiology, Medical University of Gdańsk, Gdańsk, Poland
2Laboratory of Clinical Pharmacy & Experimental Therapeutics, School of Pharmacy,
Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
3Department of Adult Neurology, Medical University of Gdańsk & University Clinical Center, Gdańsk, Poland
Abstract: Multiple sclerosis (MS) is a chronic, progressive, neuroinflammatory and neurodegenerative disease characterised by the entry of peripheral immune cells into the central nervous system via a damaged blood-brain barrier (BBB). The infiltrating immune cells propagate the inflammatory signalling and attack the myelin sheaths surrounding the neuronal axons, leading to their neurodegeneration and death. Oxysterol 7α,25OHC is a natural ligand for the Epstein-Barr virus-induced receptor 2 (EBI2, GPR183) which, among other functions, regulates immune cell migration. Using a human in vitro BBB spheroid model, comprised of endothelial cells, pericytes and astrocytes, we characterised the expression of EBI2 and EBI2-related enzymes after inflammatory stimuli in the cells forming the BBB. We also looked at the integrity of the BBB after stimulation with human cerebrospinal fluid and serum collected from healthy and MS patients. Finally, we investigated the migration of immune cells via opened BBB.
Jakub Jurczyk1,Weronika Krzyżanowska1, Lucyna Pomierny-Chamioło2, Bartosz Pomierny1
1Department of Toxicological Biochemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Krakow, Poland
2Department of Toxicology, Faculty of Pharmacy, Jagiellonian University Medical College, Poland
Abstract: Ischemic stroke is one of the leading cause of death and disability worldwide. Some evidence highlights the beneficial properties of hydrogen sulfide (H2S) donors in animal models of brain ischemia, but these data are inconclusive. This study was undertaken to verify the neuroprotective effects of AP39 - a novel molecule that releases H2S in the mitochondrial space of neural cells. For this purpose, a model of 90-minutes middle cerebral artery occlusion (MCAO) in a rat was performed along with the techniques of neurological deficit and infarct volume assessment. AP39 (100 nmol/kg, i.v.) was administered 10 minutes after reperfusion. Using the Seahorse XF analyzer, the functional parameters of the isolated mitochondria (efficiency of the oxidative phosphorylation and glycolysis) from frontal cortex and dorsal striatum were determined. Next, the integrity of the blood-brain barrier (BBB) was assessed. AP39 resulted in a reduction in the infarct volume and neurological deficit. The modulating effect of AP39 on the oxygen consumption rate and the level of glycolysis has been demonstrated. AP39 showed its protective potential on the BBB integrity. Results indicate that AP39 may have a beneficial effect in ischemic diseases, however, further work on the molecular mechanism of its action is necessary.
Ewelina Czuba-Pakuła1, Sebastian Głowiński2, Sławomir Wójcik1, Grażyna Lietzau1, Przemysław Kowiański1,2
1Division of Anatomy and Neurobiology, Department of Anatomy, Medical University of Gdansk, Gdańsk, Poland
2Institute of Health Sciences, Pomeranian University in Slupsk, Słupsk, Poland
Abstract: The most important consequence of hypercholesterolemia is increased BBB permeability, resulting from changes in the level of protein synthesis and distribution in the basal membrane of the cerebral vessel wall. This leads to a disturbance in the flow of metabolites of the cholesterol synthesis pathway through the wall of the cerebral vessels, which may intensify inflammatory and neurodegenerative processes. Among many vascular wall proteins and associated enzymes, agrin (Agr), matrix metalloproteinase-9 (MMP9), and aquaporin-4 (AQP4) may be of importance. We analyzed the level of vascular wall proteins Agr, MMP9, and AQP4 by performing immunohistochemical staining and BBB permeability changes using labeled fluorescein isothiocyanate–dextran. The study included the prefrontal cortex, motor cortex, hippocampus, and striatum in 3-month-old and 12-month-old mice in the C57/BL6 control and the LDLR-/-/Apo E-/- „knock-out” group. The present study showed that both the development of hypercholesterolemia, the age of the mice studied, and the location of the lesions in the brain had an impact on BBB function. Differences in the content of proteins responsible for maintaining the integrity of functional barrier systems have a significant impact on BBB permeability, especially in younger mice. The development of the vascular system that occurs with age may limit the effects of these disorders and seal the BBB.
