An in vitro model of the Blood Brain Barrier (BBB)
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1
Imperial College London, Parkinson's Disease Research Group, United Kingdom
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2
Hammersmith Hospital Campus, Neuroscience Centre, Burlington Danes Building, United Kingdom
The blood brain barrier (BBB) is a dynamic interface between the body and the brain which is actively engaged in the regulatory functions of the central nervous system and is the most important barrier involved in regulating the transport of molecules between the blood-stream and the brain. The BBB also prevents entry of harmful substances into the brain while regulating the flux of ions, nutrients and metabolites. Therefore BBB is vital to homeostasis at the tissue, organ and organism level. It is composed of brain microvascular endothelial cells which are underlined by a continuous basement membrane lined by astrocyte and pericyte cells which produce growth factors and react against toxins and damage to the endothelial cells. Normally, tight junctions between adjacent lateral endothelial membranes seal the BBB, limiting entrance of compounds into the central nervous system. Transport of certain molecules, e.g. essential amino acids, neurotransmitter precursors, into the brain is facilitated by active transport mechanisms. Such active transport systems are rarely carriers for drugs, which have to rely on their lipophylicity to cross the BBB. However, there are a whole host of potential drugs for CNS disorders which cannot be utilized due their inability to cross the BBB.
Targeted drug delivery across the BBB to the central nervous system is a large challenge for the treatment of neurological disorders. Additionally the development of contrast agents for imaging applications that cross the BBB without disrupting it would also have many applications e.g. diagnostics for brain cancer and other neurodegenerative diseases such as Alzheimer’s disease. For this reason there is a pressing need for the development of efficient delivery systems with the ability to enhance cellular uptake of existing drugs. To facilitate such studies we need accurate in vitro models of the BBB.
Cell culture model: A number of in vitro BBB models have been utilized however the model which replicates the BBB most closely and allows transport studies to be carried out is a transwell co-culture involving endothelial (ECV304) cells and astrocytes (C6). These cells have been chosen as other endothelial cells such as PMB cells, detach from the membrane after 3 days which limits their use. It is critical to use a co-culture system since astrocytic factors facilitate tight endothelial cell connections and the presence of typical transport mechanisms thus mimicking the human BBB.
Transport studies: Transport mechanisms across the BBB can be broadly divided into three types, namely, passive, carrier-mediated, and vesicular transport (i) Lipid-soluble, nonpolar substances can cross the BBB by passive diffusion across the BBB as the large surface area of the lipid membranes of the endothelium offers an effective diffusive route for these compounds. (ii) Polar substances and small peptides can be transported across the endothelium by carrier-mediated influx, while (iii) certain proteins, such as insulin and transferrin can be taken up by receptor-mediated transcytosis.
After formation of the cell culture model, test drugs are introduced at increasing concentrations into the upper chamber (circulatory side of the model) and incubated for different time periods at 37°C. After incubation for pre-fixed times, the upper and lower culture media will be collected and analysed for the clearance rates with time of the drug across the BBB from the basolateral to the apical side of the barrier.
It is essential to assess the integrity of the BBB prior to conduction studies and to see whether the drugs in question have a direct effect on the integrity of the BBB. The integrity can be assessed by examining the paracellular permeability - i.e. leakiness along with its ability to transport molecules. Assessment of the leakiness can be assessed using fluorescent (APTS)-labelled dextran of varying molecular weights whilst the activity of the transport systems can be assessed by examining the transport of diazepam across the BBB with subsequent HPLC detection. Positive controls will also be run by incubating the BBB with a drug that will increase its permeability e.g. methamphetamine.
Mechanisms of uptake: To determine whether uptake of a drug is though the lipid bilayer is active or passive (e.g. by diffusion through the lipid bilayer or ion channels across the concentration gradient) vesicular transport across the membrane from the to the basolateral side of the membrane can be blocked utilizing active transport inhibitors such as cyclosporine A, indomethacin or MK571.
Integrity of the BBB: For a number of studies it is essential to ascertain whether the drug in question is actually toxic to the BBB itself. Cell viability can be measured utilising a number of assay: (1) the Neutral Red (NR) assay (3- amino-7-dimethylamino-2-methylphenazine hydrochloride), which measures the lysosomal accumulation of NR dye in viable cells; (2) the MTT assay, which measures the intracellular enzymatic conversion of MTT [3-(4,5-dimethylthiazol- 2-yl)- 2,5-diphenyltetrazolium bromide] to formazan by dehydrogenase enzymes in viable cells; (3) the lactate dehydrogenase (LDH) assay, which is a measure of membrane integrity; and finally (4) a Live-Dead assay by confocal microscopy, which also measures membrane integrity and detect apoptosis in a live mode. It is essential to use a range of cell viability assays as each method tests a specific cell function and will thus avoid false positive results when testing toxicity.
Conference:
Pharmacology and Toxicology of the Blood-Brain Barrier: State of the Art, Needs for Future Research and Expected Benefits for the EU, Brussels, Belgium, 11 Feb - 12 Feb, 2010.
Presentation Type:
Oral Presentation
Topic:
Presentations
Citation:
Dexter
DT
(2010). An in vitro model of the Blood Brain Barrier (BBB).
Front. Pharmacol.
Conference Abstract:
Pharmacology and Toxicology of the Blood-Brain Barrier: State of the Art, Needs for Future Research and Expected Benefits for the EU.
doi: 10.3389/conf.fphar.2010.02.00021
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Received:
11 Mar 2010;
Published Online:
12 Mar 2010.
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Correspondence:
David T Dexter, Imperial College London, Parkinson's Disease Research Group, London, United Kingdom, d.dexter@imperial.ac.uk