Purpose. To determine the effect of Stokes radius (R S) on the diffusion of molecules through Bruch's membrane (BM), and to establish a system suitable for the analysis of diffusion through small (<2 mm 2) samples of BM. Methods. Porcine BM/choroid (BM/Ch) was mounted in a modified Ussing chamber. A concentration gradient was simultaneously established for four traceR S with R S values ranging from <1.0 to 6.15 nm. Samples were collected from both chambeR S at various time points up to 36 houR S and the amount of each tracer was determined using quantitative gel exclusion chromatography. The integrity of samples was determined using scanning electron microscopy. Results. BM/Ch mounted in the chamber exhibited no obvious damage even after 36 houR S in the chamber. Flux was significantly (P < 0.05) greater in the BM to Ch direction than that in the Ch to BM direction for only two of the traceR S: cytosine and RNase A. Flux also was dependent on R S; cytosine, the smallest tracer (R S < 1 nm), exhibited the greatest flux and ferritin (R S = 6.15 nm) the least. Permeability coefficients for each tracer were determined and exhibited a power relationship with R S. Conclusions. Flux was dependent on the direction of the concentration gradient and the R S of the individual traceR S. We have successfully demonstrated that quantitative gel exclusion chromatography can be used to follow diffusion of a mixture of traceR S across BM/Ch, and that we can measure flux across BM/Ch preparations with an exposed surface area as small as 1.8 mm 2.
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience