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Gupta, Gaurav (2025) Establishment of horse blood-brain barrier model for the study of drug delivery to the brain. PhD thesis, University of Nottingham.

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Abstract

The lack of a benchmark standard in vitro model of blood-brain barrier (BBB) is the principal reason behind high failure rates of central nervous system drugs in clinical trials during their development phase. In the last few decades, in vitro models of BBB have been developed using brain endothelial cells from various species like rat, mice, pig, and cattle However, no in vitro BBB model till date has been developed using horse brain endothelial cells. Hence, the main aim of this study was to establish an in vitro primary horse brain endothelial cell (HBEC) transwell model of BBB for determining the how much drugs are transported and handled by the BBB by measuring various pharmacokinetic parameters.

Phylogenetic analysis of multi-drug efflux molecules, evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model. Species having only 1:1 orthologue genes to these multi-drug efflux molecules were included. This study revealed that these molecules are phylogenetically closest to similar molecules of donkey and there the horse was divergent from other species.

The next step established and standardised a method to isolate HBECs from horse brains. Different conditions were optimised to ensure that near-pure population of HBECs were being cultured. For this, addition of 4µg/ml puromycin for first 3 days of culture yielded the highest purity. These HBECs were then characterised to confirm retention of BBB phenotype by using PCR, immunofluorescence and Western blot for various cell type biomarkers and key protein. It was determined that these HBECs had similar key features to the BBB. In addition, the application of scanning electron microscopy brought to light, for the first time, the detailed structure of cell-cell junctions in HBECs. Further characterisation revealed that the presence of astrocyte conditioned medium (ACM) and puromycin affected the expression of multi-drug efflux transporter protein, P-Glycoprotein (P-gp). Furthermore, presence of both ACM and puromycin significantly enhanced P-gp expression.

Transwell model of horse BBB was standardised for drug transport studies by culturing HBECs in presence of transport medium (in essence control) or ACM or astrocyte co-culture or in a combination of transport medium and ACM. The transwell model with ACM performed most optimally in terms of highest transendothelial electrical resistance assay (TEER) and FITC-dextran permeability assay. Drug transport studies were undertaken on this transwell model of the horse BBB. Eight drugs were selected which were substrate for three important multi-drug efflux molecules. The results of drug studies using the HBEC Transwell model did reveal that paracellular movement of drugs was likely to the main transport process taking place for several drug. This could be suggestive of leaky cell-cell junctions. This finding is also corroborated by low-to-moderate TEER values observed in these studies.

Therefore, the project has established and standardised a method for isolation and culture of primary HBECs. Also, for the first time this project has established methodology of novel transwell model of Horse BBB. Nevertheless, additional improvements will be required to establish this model as a candidate for use in drug development for central nervous system.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Paine, Stuart Robinson, Bob Rauch, Cyril
Keywords:	Blood-brain barrier, In vitro models, Brain endothelial cells, Drug transportation, Transwell model
Subjects:	Q Science > QP Physiology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Veterinary Medicine and Science
Item ID:	80798
Depositing User:	Gupta, Gaurav
Date Deposited:	24 Jul 2025 04:40
Last Modified:	24 Jul 2025 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/80798

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