Assessing the Permeability of Two Barriers in the Post-capillary Neurovascular Unit Part 2: Measuring R2
In Part 1 we explained that the composite barrier in our model of the post-capillary NVU is determined by RBBB = RBlB + R2 + RBrB where is R2 is the resistance of the ‘perivascular space’ in our chip. Unlike the other two, the perivascular space is not a regulated cellular barrier that we need to model with as much accuracy as possible. And our chip makes no attempt at accuracy other then by creating a space intermediate to the two actual barriers. Still, even free space has a resistance, and therefore a permeability, and the bigger and more complex the space, the larger this resistance will become. So the next order of business is to measure R2 in our device and to compare it to RBLB and RBRB. If it is larger or comparable to these, we should not try to ‘subtract’ it from a composite method. In that case we should find another way to answer the question we care about, which is how do inflammatory signals including cellular cross-talk in the NVU, impact the function of these two barriers. If it is much smaller than the other two, we can ignore it and make a composite measurement by adding dye to the blood side of a chip and measuring its appearance on the brain side in an experiment that looks like this:
Doing this same measurement, but without cells or coatings, is the method to measure R2. Pelin completed this task and actually with and without coatings and found similar answers …
| Dye added to well | Dye added to insert | ||
| Permeability (cm/min) | With Coating (n = 2) | 3.11E-4 (2.97; 3.24) | 3.23E-4 (3.77; 2.69) |
| Permeability (cm/min) | Without Coating (n = 2) | 3.58E-4 (3.54; 3.63) | 3.07E-4 (4.27; 1.86) |
| Resistance (min/cm) | With Coating | 3219 | 3094 |
| Resistance (min/cm) | Without Coating | 2790 | 3260 |
… where ‘insert’ is the astrocyte compartment.
Roughly, R2 ~ 3000 min/cm. By comparison the threshold from ‘tight’ to ‘leaky’ barriers is 6E-4 cm/min so RBlB ~ 1600 min/cm. While theoretically R2 can be subtracted from a composite measurement, this is not ideal. R2 is nearly 2X the size of the resistance of the barrier we want to measure and has a considerably uncertainty. This will make the measurement of RBlB noisy and unreliable. So it is best to avoid a method for the measurement of the blood barrier permeability that requires the measurement and subtraction of R2.
