OUR BLOOD-BRAIN BARRIER MODEL (2/2)

2- Transport experiments using the in vitro blood-brain barrier.

On the day of the experiments, Ringer-HEPES (NaCl 150mM, KCl 5.2mM, CaCl2 2.2mM, MgCl2 6H2O 0.2mM, NaHCO3 6mM, HEPES 5mM, glucose 2.8mM) is added to the lower compartment (abluminal side) of a six-well plate (2.5 ml per well). One filter containing a confluent monolayer of endothelial cells is transferred in the first well of the six-well plate containing Ringer-HEPES.

A 1.5 ml Ringer-HEPES volume containing labelled drugs is placed in the upper compartment (luminal side). At 10, 15, 20, 30, 45 and 60 min after addition of the test compound, the insert is transferred to another well of the six-well to minimize the possible passage of substances from the lower to the upper compartment.

Figure 3 : Schema of the permeability studies.

Triplicate monolayers and triplicate filters coated with collagen only are assayed for each labelled drug.
Incubations are performed on a rocking platform at 37°C.
At the end of each incubation period, two aliquots (500 µl ) of abluminal and luminal liquid were performed.

4-Analytical procedures.

a) Radioactive analysis.

Radioactivity is determined in representative samples from each lower compartiment (3 filters) and from the initial solution containing [14C] or[3H] -labelled drug.

b) HPLC analysis

Because most of the molecules are not radiolabelled, the concentrations were performed by HPLC.

5- Data analysis: Endothelial permeability coefficient.

To obtain a concentration-dependant transport parameter, the clearance principle is used. For each time, the increment in cleared volume between successive sampling events is calculated by dividing the amount of transported solute by the donor chamber concentration. The total volume cleared at each point is calculated by summing the incremental cleared volumes up to the given time point :

                       [C]A X VA
Clearance ( l) = -----------------
                          [C]L

where [C]L is the initial luminal tracer concentration, [C]A the abluminal tracer concentration, and VA the volume of the abluminal chamber.
During the 60-min experiment, the clearance volume increases linearly with time. The average volume cleared is plotted versus time, and the slope is estimated by linear regression analysis to give the mean and the SE to the estimate. The slope of the clearance curves for the coculture is denoted PSt, where PS is the permeability x surface area product (in microliters per minute). The slope of the clearance curve for the control filter is denoted PSf.

The PS value for the endothelial monolayer (PSe) was calculated from:

 1               1               1
--------- = ------------ - ------------
 Pse            PSt            PSf

The PSe values were divided by the surface area of the Millicell-PC (4.2 cm2) to generate the endothelial permeability coefficient (Pe, in centimeters per minute).

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