The goal of our study was to measure differences in flux and permeability of macromolecules across BM/Ch as related to RS, and to do so using the smallest piece of tissue possible. The latter will facilitate future studies using small animals such as mice and to evaluate regional variations in diffusion across BM/Ch in human eyes. To accomplish this we applied a simple and inexpensive modification to a commercially available Ussing chamber to permit the use of small pieces of BM/Ch. As shown in , this modification involves the use of an insert produced from two pieces of exposed x-ray film. A hole of any size can be drilled in the film and the tissue held in place between the two pieces by a thin film of silicon grease.
In our modified Ussing setup the ability to follow diffusion across BM/Ch requires that BM be intact for the duration of the experiment. To determine whether all five layers of BM are intact, we compared BM from eyes fixed immediately after enucleation and with the RPE intact (A) with BM/Ch preparations (B) by TEM. demonstrates that all five layers of BM remain intact after the procedure, with the only noticeable difference being the washout of plasma protein. Elastic and collagen fibers as well as the endothelial and RPE basement membranes remained intact. It should be noted that in all experiments it is the RPE basement membrane that is directly exposed to the fluid in the chamber.
Figure 2. Transmission electron microscopy of BM/Ch preparations. (A) BM from an eye immediately fixed after enucleation and with RPE intact. (B) BM from a BM/Ch preparation. Scale bars, 500 nm. bm, basement membrane; ic, inner collagenous zone; elastic, elastic (more ...)
Since the integrity of BM could be compromised by poor dissection of the tissue, failure to remove RPE cells from BM, or the disintegration of the tissue during mounting, or during the time course of the experiment, the integrity of each BM/Ch preparation was evaluated at the end of each experiment using SEM. shows that RPE cells were successfully and completely removed from the surface of BM (compare A1 and A2 with C) without collateral damage to BM (A1, A2). BM appeared intact because there was no evidence of exposed collagen fibers, as would be predicted due to tearing of the basal lamina. Examination of the opposing surface revealed a web of connective tissue fibers resembling the suprachoiodal tissue that connects the Ch and sclera (B). We observed no evidence that the tissue had deteriorated during our experiments. Thickness (L) of BM/Ch explants was measured by SEM of explants in cross-section and was 58.1 ± 1.51 μm (mean ± SD, n = 3).
Figure 3. Evaluation of tissue integrity via SEM. At the end of each experiment, tissues were fixed and prepared for SEM. (A1) The surface of BM, corresponding to the RPE-BL is relatively smooth, with no apparent gaps or tears. (A2) Low-magnification view of a (more ...)
To determine how flux across BM/Ch is influenced by RS, we established a concentration gradient of all four tracer molecules across BM/Ch and applied Fick's first law to calculate the flux and permeability coefficient of each molecule (see the Materials and Methods section). Although others have used fluorescent tracers to measure flux across BM/Ch preparations, we found that this did not suit the design of our experiments. The reason for this was that we could not achieve a high enough specific activity of labeling of the reporter molecules to perform experiments with 1.8-mm2 tissue samples in a 24- to 48-hour experiment, and we wanted to be able to compare multiple tracers in the same experiment without introducing the need for each reporter to be differentially labeled. Furthermore, labeling of proteins with fluorescent tracers is typically accomplished by modification of the Ε amino acid of a lysine residue, which significantly alters charge characteristics and potentially affects RS. To circumvent these limitations we chose to use gel exclusion chromatography to quantify all four tracers, without structural modification, at each time point simultaneously. Examples of chromatograms and standard curves indicating the limits of detection for each standard are shown in .
Experiments were performed to examine the diffusion of all four tracers in both the BM to Ch () and Ch to BM () directions. We were able to detect cytosine at the first time point of 4 hours regardless of the direction of diffusion (, ). RNase A was quantifiable at 4 hours in the BM to Ch direction () but was not quantifiable until 8 hours in the Ch to BM direction (). Similarly, albumin was first observed in the BM to Ch direction at 4 hours () but the peak was too small to quantify; however, by 8 hours a significant amount of albumin was observed to have diffused. In the Ch to BM direction albumin was first detected at 16 hours (). Ferritin was first detected at 24 hours in the BM to Ch experiments (), but was reliably observed in the Ch to BM experiments only at the 36-hour time point (). Importantly, even at 36 hours, ferritin was not detected in every experiment in either direction. To ensure that our data were not skewed by absorption of the tracers to the explants, we quantified the amount of each tracer in both compartments at each time point. Essentially 100% of each tracer was recovered at each time point.
Figure 5. (A) Representative chromatograms obtained for tracers in the BM to Ch direction. Elution profiles at different time points are overlayed as indicated. (B) Note that peaks can be examined using data acquisition software independently so that scaling can (more ...)
Figure 6. (A) Representative chromatograms obtained for tracers in the Ch to BM direction. Elution profiles at different time points are overlayed as indicated. (B) Note that peaks can be examined using data acquisition software independently so that scaling can (more ...)
The flux of each tracer remained relatively constant throughout the time course of each experiment (A), although the diffusion of tracers did not have a significant impact on the concentration gradient through the time course of the experiment (). Flux was greatest for the smallest tracer, cytosine, and diminished with increasing RS (B). Ferritin had the slowest average flux (0.1 ± 0.08 nmol cm−2 h−1 from Ch to BM and 0.09 ± 0.14 nmol cm−2 h−1 from BM to Ch) and was detected only at the 36-hour time point in the Ch to BM direction and at the 24- and 36-hour time points in the BM to Ch direction. For all four tracers flux appeared greater in the BM to Ch direction compared with that in the Ch to BM direction (A). For cytosine and RNase A this difference was statistically significant (P < 0.05).
Figure 7. Total diffusion flux and relationship to RS. (A) The flux of proteins through the tissue remained constant throughout the time course of the experiment. Runs in the Ch to BM direction are indicated by solid symbols and runs in the BM to Ch direction are (more ...)
Change in concentration gradient of tracers as a function of time. Protein gradients in experiments performed from BM to Ch or Ch to BM as indicated. Lines are the regression fit through the data which are given as mean ± SD.
The permeability coefficient (P
) for each tracer was calculated using Eq. (2)
(see the Materials and Methods section) at each time point at which the tracer was detected. Like flux, P
remained constant with time (A) and decreased with increasing RS
for cytosine, RNase A, and albumin (A, B). In contrast to the difference in flux, however, P
was similar for albumin and ferritin. Thus, we conclude that the size exclusion limit of BM exceeds the RS
of ferritin, but that the permeability of BM to molecules with RS
similar to or greater than that of albumin is limited, suggesting that albumin and ferritin are near the physical size exclusion limit of BM.
Permeability coefficients (P) of tracers. (A) P remained relatively constant throughout the duration of the experiment. (B) P varied exponentially with RS but appeared to be saturated for albumin and ferritin.