Diffusion Of Small Solutes Research Articles

Single-Molecule Displacement Mapping Indicates Unhindered Intracellular Diffusion of Small (≲1 kDa) Solutes.

While fundamentally important, the intracellular diffusion of small (≲1 kDa) solutes has been difficult to elucidate due to challenges in both labeling and measurement. Here we quantify and spatially map the translational diffusion patterns of small solutes in mammalian cells by integrating several recent advances. In particular, by executing tandem stroboscopic illumination pulses down to 400 μs separation, we extend single-molecule displacement/diffusivity mapping (SMdM), a super-resolution diffusion quantification tool, to small solutes with high diffusion coefficients D of >300 μm2/s. We thus show that for multiple water-soluble dyes and dye-tagged nucleotides, intracellular diffusion is dominated by vast regions of high diffusivity ∼60-70% of that in vitro, up to ∼250 μm2/s in the fastest cases. Meanwhile, we also visualize sub-micrometer foci of substantial slowdowns in diffusion, thus underscoring the importance of spatially resolving the local diffusion behavior. Together, these results suggest that the intracellular diffusion of small solutes is only modestly scaled down by the slightly higher viscosity of the cytosol over water but otherwise not further hindered by macromolecular crowding. We thus lift a paradoxically low speed limit for intracellular diffusion suggested by previous experiments.

Novel Combined Test for Osmotic Conductance to Glucose and Small Solute Diffusion Capacity in Peritoneal Dialysis

Novel Combined Test for Osmotic Conductance to Glucose and Small Solute Diffusion Capacity in Peritoneal Dialysis

SGLT2 inhibition does not reduce glucose absorption during experimental peritoneal dialysis.

Unwanted glucose absorption during peritoneal dialysis (PD) remains a clinical challenge, especially in diabetic patients. Recent experimental data indicated that inhibitors of the sodium and glucose co-transporter (SGLT)-2 could act to reduce glucose uptake during PD, which raises the question of whether glucose absorption may also occur via intracellular or trans-cellular pathways. We performed PD in anesthetized Sprague-Dawley rats using a fill volume of 20 mL with either 1.5% glucose fluid or 4.25% glucose fluid for 120 min dwell time to evaluate the effects of SGLT2 inhibition by empagliflozin on peritoneal water and solute transport. To assess the diffusion capacity of glucose, we developed a modified equation to measure small solute diffusion capacity, taking convective- and free water transport into account. SGLT2 inhibition markedly increased the urinary excretion of glucose and lowered plasma glucose after PD compared to sham groups. Glucose absorption for 1.5% glucose was 165 mg 95% CI (145-178) in sham animals and 157 mg 95% CI (137-172) for empagliflozin-treated animals. For 4.25% glucose, absorption of glucose was 474 mg 95% CI (425-494) and 472 mg 95% CI (420-506) for sham and empagliflozin groups, respectively. No significant changes in the transport of sodium or water across the peritoneal barrier could be detected. We could not confirm recent findings that SGLT2 inhibition reduced glucose absorption and increased osmotic water transport during experimental PD.

Curvature and bottlenecks control molecular transport in inverse bicontinuous cubic phases.

We perform a simulation study of the diffusion of small solutes in the confined domains imposed by inverse bicontinuous cubic phases for the primitive, diamond, and gyroid symmetries common to many lipid/water mesophase systems employed in experiments. For large diffusing domains, the long-time diffusion coefficient shows universal features when the size of the confining domain is renormalized by the Gaussian curvature of the triply periodic minimal surface. When bottlenecks are widely present, they become the most relevant factor for transport, regardless of the connectivity of the cubic phase.

Diffusion of Small Solute Particles in Viscous Liquids: Cage Diffusion, a Result of Decoupling of Solute-Solvent Dynamics, Leads to Amplification of Solute Diffusion.

We study the diffusion of small solute particles through solvent by keeping the solute-solvent interaction repulsive and varying the solvent properties. The study involves computer simulations, development of a new model to describe diffusion of small solutes in a solvent, and also mode coupling theory (MCT) calculations. In a viscous solvent, a small solute diffuses via coupling to the solvent hydrodynamic modes and also through the transient cages formed by the solvent. The model developed can estimate the independent contributions from these two different channels of diffusion. Although the solute diffusion in all the systems shows an amplification, the degree of it increases with solvent viscosity. The model correctly predicts that when the solvent viscosity is high, the solute primarily diffuses by exploiting the solvent cages. In such a scenario the MCT diffusion performed for a static solvent provides a correct estimation of the cage diffusion.

The human knee: A window on the microvasculature

In synovial joints, the lining cells do not have tight junctions with their neighboring cells and they have no underlying basement membrane. Therefore, the synovial fluid within the articular cavity is continuous with the interstitial fluid of the synovial intima. These features, combined with ready access to the space via arthrocentesis, permit quantitative studies of microvascular function in the knees of unanesthetized, volunteer, human subjects both with and without chronic arthritis. This brief article reviews the principal findings of such work over ∼40 years at the University of Washington. Examined variables include bidirectional fenestral diffusion of small solutes, effective blood flow, lymphatic drainage, and endothelial pore size and permeability. The latter work introduced a new method using gel filtration chromatography of paired synovial fluid (SF) and serum (S) to obtain essentially continuous SF/S ratios over a range of radii between 1 and 12 nanometers.

Altered cerebral vascular volumes and solute transport at the blood–brain barriers of two transgenic mouse models of Alzheimer's disease

We evaluated the integrity and function of the blood–brain barrier in 3xTg-AD mice aged 3–18 months and in APP/PS1 mice aged 8-months to determine the impacts of changes in amyloid and tau proteins on the brain vascular changes. The vascular volume (Vvasc) was sub-normal in 3xTg-AD mice aged from 6 to 18 months, but not in the APP/PS1 mice. The uptakes of [3H]-diazepam by the brains of 3xTg-AD, APP/PS1 and their age-matched control mice were similar at all the times studied, suggesting that the simple diffusion of small solutes is unchanged in transgenic animals. The uptake of d-glucose by the brains of 18-month old 3xTg-AD mice, but not by those of 8-month old APP/PS1 mice, was reduced compared to their age-matched controls. Accordingly, the amount of Glut-1 protein was 1.4 times lower in the brain capillaries of 18 month-old 3xTg-AD mice than in those of age-matched control mice. We conclude that the brain vascular volume is reduced early in 3xTg-AD mice, 6 months before the appearance of pathological lesions, and that this reduction persists until they are at least 18 months old. The absence of alterations in the BBB of APP/PS1 mice suggests that hyperphosphorylated tau proteins contribute to the vascular changes that occur in AD.

Actin-based confinement of calcium responses during Shigella invasion

Shigella, the agent of bacillary dysentery, invades epithelial cells by locally inducing actin reorganization. Upon cell invasion, Shigella induces calcium (Ca(2+)) signalling, but its role in invasion has remained unclear. Here we show that components involved in inositol 1, 4, 5- trisphosphate (InsP3) signalling are implicated in Shigella invasion. Although global Ca(2+) responses are dispensable for bacterial invasion, local Ca(2+) responses of unprecedented long duration are associated with invasion sites. Fluorescence recovery after photo-bleaching experiments indicate that diffusion of small solutes is hindered at Shigella-invasion sites and that diffusion hindrance is dependent on bacterially induced actin reorganization. Computational simulations and experimental challenge of the model support the notion that local accumulation of InsP3 permitted by restricted diffusion and enrichment of InsP3 receptors account for sustained local Ca(2+) increases at entry sites. Thus, cytoskeletal reorganization through diffusion hindrance shapes the duration of local Ca(2+) signals.

Mass effect on rotational diffusion of small solutes in solution

Rotational correlation times (τc) of two pairs of small solutes, CpM(CO)3 and M2(CO)10 (M=Mn, Re), are determined in various viscous alkane solutions by narrow-band IR pump broad-band IR probe spectroscopy. By choosing these pairs of molecules, which are significantly different in mass but almost identical in volume, shape and in their expected interactions with solvents, we isolate the effects of mass on τc. The effect of mass was observed clearly for these pairs of solute tracers, with heavier substitutions leading to larger τc values. In the case of the CpM(CO)3 pair, in which the moments of inertia do not change much, the effect of mass was seen in the τc vs. solvent viscosity plot as result of larger slope for heavier substitution, with no clear change in the intercept. For the M2(CO)10 pair, in which the moments of inertia change significantly, this mass effect can be observed through changes in both the slope and intercept.

Engineered Oligomerization State of OmpF Protein through Computational Design Decouples Oligomer Dissociation from Unfolding

Biogenesis of β-barrel membrane proteins is a complex, multistep, and as yet incompletely characterized process. The bacterial porin family is perhaps the best-studied protein family among β-barrel membrane proteins that allows diffusion of small solutes across the bacterial outer membrane. In this study, we have identified residues that contribute significantly to the protein–protein interaction (PPI) interface between the chains of outer membrane protein F (OmpF), a trimeric porin, using an empirical energy function in conjunction with an evolutionary analysis. By replacing these residues through site-directed mutagenesis either with energetically favorable residues or substitutions that do not occur in natural bacterial outer membrane proteins, we succeeded in engineering OmpF mutants with dimeric and monomeric oligomerization states instead of a trimeric oligomerization state. Moreover, our results suggest that the oligomerization of OmpF proceeds through a series of interactions involving two distinct regions of the extensive PPI interface: two monomers interact to form a dimer through the PPI interface near G19. This dimer then interacts with another monomer through the PPI interface near G135 to form a trimer. We have found that perturbing the PPI interface near G19 results in the formation of the monomeric OmpF only. Thermal denaturation of the designed dimeric OmpF mutant suggests that oligomer dissociation can be separated from the process of protein unfolding. Furthermore, the conserved site near G57 and G59 is important for the PPI interface and might provide the essential scaffold for PPIs.

Nisin Quantification by ELISA Allows the Modeling of Its Apparent Diffusion Coefficient in Model Cheeses

The diffusion of small solutes in cheese is of key importance for most enzymatic reactions involved in the ripening process. However, only a limited amount of data is available on salt diffusion and practically none on peptide diffusion. Nisin, a bacteriocin peptide, migrated in model cheeses made from ultrafiltered (UF) retentate. A profile concentration device and an enzyme-linked immunosorbent assay (ELISA), specifically developed for nisin quantification in cheese, were used to model the apparent diffusion coefficients for nisin according to Fick's law. This average coefficient was 49.5 μm(2)/s in UF cheese (n = 2). When 10% gelatin was added to the retentate, this value decreased to 34.4 μm(2)/s (n = 2). The two cheeses differed in their macrostructure (rheology) and microstructure (confocal microscopy). This study provides the first apparent diffusion coefficients for a peptide in cheese and supports the hypothesis that composition and structure influence the diffusion of small solutes such as peptides.

Composition of the endothelial glycocalyx and its relation to its thickness and diffusion of small solutes

The endothelial glycocalyx is well endowed with the glycosaminoglycans (GAGs) heparan sulfate, chondroitin sulfate and hyaluronan. The current studies aimed to assess the relative contributions of each of these GAGs to the thickness and permeability of the glycocalyx layer by direct enzymatic removal of each using micropipettes to infuse heparinase, chondroitinase and hyaluronidase into post-capillary venules of the intestinal mesentery of the rat. The relative losses of GAGs due to enzymatic removal were compared with stimulated shedding of glycans induced by superfusing the mesentery with 10 − 7 M fMLP. Thickness of the glycocalyx was assessed by infiltration of the glycocalyx with circulating FITC labeled 70 kDa dextran (Dx70) and measuring the distance from the dye front to the surface of the endothelium (EC), which averaged 463 nm under control conditions. Reductions in thickness were 43.3%, 34.1% and 26.1% following heparinase, chondroitinase and hyaluronidase, respectively, and 89.7% with a mixture of all three enzymes. Diffusion coefficients of FITC in the glycocalyx were determined using a 1-D diffusion model. By comparison of measured transients in radial intensity of a bolus of FITC with that of a computational model a diffusion coefficient D was obtained. Values of D were obtained corresponding to the thickness of the layer demarcated by Dx70 ( D Dx70), and a smaller sublayer 173 nm above the EC surface ( D 173), prior to and following enzyme infusion and superfusion with fMLP. The magnitude of D Dx70 was twice that of D 173 suggesting that the glycocalyx is more compact near the EC surface. Chondroitinase and hyaluronidase significantly increased both D Dx70 and D 173. However, heparinase decreased D Dx70, and did not induce any significant change for the D 173. These observations suggest that the three GAGs are not evenly distributed throughout the glycocalyx and that they each contribute to permeability of the glycocalyx to a differing extent. The fMLP-induced shedding caused a reduction in glycocalyx thickness (which may increase permeability) and as with heparinase, decreased the diffusion coefficient of solutes (which may decrease permeability). This behavior suggests that the removal of heparan sulfate may cause a collapse of the glycocalyx which counters decreases in thickness by compacting the layer to maintain a constant resistance to filtration.

Regulation of Myoendothelial Junction Formation

See related article, pages 1092–1102 In this issue of Circulation Research , Heberlein et al1 provide exciting new insight into the actions of plasminogen activator (PA) inhibitor (PAI)-1 by illuminating its role in governing the ability of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) to communicate with each other through specialized contacts defined as myoendothelial junctions (MEJs). In the resistance vasculature, MEJs are cellular extensions through the internal elastic lamina (IEL), whereby ECs make physical contact with adjacent VSMCs.2 At points of cell–cell contact, the presence of gap junction channels enables electrical coupling and intercellular diffusion of small solutes (<1 kDa) that govern vasomotor control. Cell–cell signaling through gap junction channels can be regulated acutely (eg, through phosphorylation of key serine residues on connexin subunits), and their protein complexes undergo hourly turnover in the plasma membrane. Such properties imply that the nature of cell–cell signaling through MEJs is under dynamic regulation. Despite implications that the MEJ is itself a dynamic structure, little is known about how MEJ formation and regression are regulated. New findings presented by Heberlein et al1 illustrate how dynamic MEJ formation and signaling may be enhanced or impaired via PAI-1 regulation of the PA system. Plasminogen is synthesized in the liver and released into the circulation as a zymogen of the serine protease, plasmin. The conversion of plasminogen to active plasmin is mediated by tissue (t)-PA and urokinase-type (u)-PA (ie, urokinase). Whereas t-PA is involved primarily in the dissolution of fibrin in the circulation, u-PA binds to the urokinase receptor (uPAR) (ie, CD87) on cell membranes, thereby localizing proteolytic activity to the vicinity of binding. In this manner, uPAR is integral to cell migration and adhesion through breakdown of the extracellular matrix (ECM).3,4 PAI-1, a ≈45-kDa glycoprotein, is a serine …

Gas permeation of LC films observed by smectic bubble expansion

Gas permeation through liquid crystal (LC) films was examined using hemispherical smectic bubbles. A smectic bubble, when the inside and the outside are filled with different gases, should expand or shrink toward the quasi-equilibrium state, where the influx and efflux caused by osmotic pressure are balanced. Deriving a simple formula that directly converts the quasi-equilibrated bubble radius to the gas permeation, we determined the absolute permeability coefficients of 8 simple gases through the smectic bubble. The permeability was distributed in such a wide range that carbon-dioxide had more than 20 times larger value than nitrogen, the dependence of which on the gas species was mostly dominated by their solubility into the LCs. Dividing the measured permeability by the calculated solubility, we obtained the diffusion constants as well, yet whose magnitude and the dependence on the solute size could not be explained by either conventional continuum theories or microscopic diffusion models. In order to describe the diffusion of small solutes in the liquid solvent composed of large molecules, a new theoretical framework may be necessary.

2009 ISSLS Prize Winner: What Influence Does Sustained Mechanical Load Have on Diffusion in the Human Intervertebral Disc?

An in vivo study of the effects of mechanical loading on transport of small solutes into normal human lumbar intervertebral discs (IVD) using serial postcontrast magnetic resonance imaging (MRI). To investigate the influence of a sustained mechanical load on diffusion of small solutes in and out of the normal IVD. Diffusion is an important source of disc nutrition and the in vivo effects of load on diffusion in human IVD remains unknown. Forty normal lumbar discs (on MRI) in 8 healthy volunteers were subjected to serial post contrast (Gadoteridol) 3 Tesla MRI in 2 phases. In phase 1 (control), volunteers were scanned at different time points--precontrast and 1.5, 3, 4.5, 6, and 7.5 hours postcontrast injection. In phase 2, 1 month later, the same volunteers were subjected to sustained supine loading for 4.5 hours. MRI scans were performed precontrast (preload) and postcontrast (postloading) at 1.5, 3, and 4.5 hours. Their spines were then unloaded and recovery scans performed at 6 and 7.5 hours postcontrast. In house software was used to analyze images. Repeated-measures ANOVA and pairwise comparisons at different time points in the central region of the loaded disc (LD) compared to the unloaded discs (UD) revealed significantly lower signal intensity ratios (P1.5h:P3h:P4.5h<0.001:<0.001:<0.002) indicating reduction in transport rates for the LDs. Signal intensity ratios continued to rise in LD for 3 hours into recovery phase,whereas UD at the same time point showed a decrease (mean +/- SD = 0.08 +/- 0.08 vs. -0.21 +/- 0.03). Sustained supine creep loading (50% body weight) for 4.5 hours retards transport of small solutes into the center of human IVD and it required 3 hours of accelerated diffusion in recovery state for LD to catch-up with diffusion in UD. The study supports the theory that sustained mechanical loading impairs diffusion of nutrients entering the disc and quite possibly accelerates disc degeneration.

Effect of convection on diffusion of small solutes through tissue

Transport of small solutes (MW< 500 da) through tissue is typically diffusion-dominated. In the special case of intraperitoneal therapy, hypertonic solutions are used to introduce drugs or nutrients into the body. The hyperosmolar solution induces convection from the body into the cavity, opposing solute delivery. Our hypothesis is that this convection will retard small solute transfer into peritoneal tissue, if its magnitude approaches the solute mass transfer coefficient (MTC). To test this, we measured the effect of osmotic volume flux (Fluxosm) on the mannitol MTC using plastic chambers affixed to the abdominal wall peritoneum of 12 SD rats. The chamber fixes the fluid contact area of the peritoneum and permits determination of volume and concentration versus time and the calculation of MTC. 14C-mannitol transport from the chamber to the tissue was measured during two 90 min periods of testing under isotonic or hypertonic (~500 mosm/kg) conditions. If convection from the tissue to the chamber had a significant effect, the magnitude of MTC would decrease. Our results for the isotonic case resulted in a mean ± SE (cm/min × 103) of: Fluxosm, −0.053 ± 0.027 and MTC of 3.09 ± 0.40. The hypertonic case demonstrated a markedly positive Fluxosm into the chamber of 2.33 ± 0.33 (p<0.05 vs isotonic) but no significant change in the MTC (2.80 ± 0.35), disproving our hypothesis. We conclude that this level of convection has insignificant effects on the diffusive transfer of small solutes across the peritoneum into the underlying tissue.

Physiological Properties of the Peritoneum in an Adult Peritoneal Dialysis Population over a Three-Year Period

To describe the physiological properties of the peritoneal membrane in adult patients treated with peritoneal dialysis (PD) and to analyze the effects of patient characteristics and time. Observational study. Department of Nephrology at the Sahlgrenska University Hospital. Peritoneal function was analyzed by the Personal Dialysis Capacity (PDC) test, based on the three-pore theory of capillary transport. The functional PDC variables are absorption, large-pore flow, and the area parameter (A0/deltax), which determines the diffusion of small solutes. The ultra-filtration (UF) coefficient is determined mainly by A0/deltax. All patients (n = 280) who had at least one PDC test done between September 1990 and August 1999. In 249 patients examined soon after start of PD, area was 19000 (SD 7100) cm2/cm/1.73 m2, large-pore flow 0.112 (SD 0.052) mL/min/1.73 m2, and the UF coefficient 0.071 (SD 0.032) mL/minute/mmHg/1.73 m2. Absorption was 1.54 (SD +2.64, -0.97) mL/min/1.73 m2. Large-pore flow was greater in patients with severe comorbidity than in patients with fewercomorbid conditions. Elderly patients had a lower UF coefficient than did younger patients (p < 0.05). Repeated PDC tests were performed in 208 patients during a mean observation time of 18.4 months. There was a slight increase in the slope of the area-versus-time curve of 54 cm2/cm/1.73 m2 per month (approximately 10% after 3 years, p < 0.01); all other parameters remained constant. Patient characteristics have an impact on peritoneal performance already at the start of dialysis. Peritoneal function can remain essentially stable during medium long-term PD.

MRI of articular cartilage in OA: novel pulse sequences and compositional/functional markers

Osteoarthritis (OA) is a leading cause of disability worldwide. Magnetic resonance imaging (MRI), with its unique ability to image and characterize soft tissue non-invasively, has proven valuable in assessing cartilage in OA. The development of new, fast imaging methods with high contrast show promise to improve the magnetic resonance (MR) evaluation of this disease. In addition to morphologic MRI methods, MRI contrast mechanisms under development may reveal detailed information about the physiology of cartilage. It is anticipated that these and other MRI techniques will play an increasingly important role in assessing the success or failure of therapies for OA. On December 5 and 6, 2002, OMERACT (Outcome Measures in Rheumatology Clinical Trials) and OARSI (Osteoarthritis Research Society International) held a workshop in Bethesda, MD aiming at providing a state-of-the-art review of imaging outcome measures for OA of the knee to help guide scientists and pharmaceutical companies in the use of MRI in multi-site studies of OA. Applications of MRI were initially reviewed by a multidisciplinary, international panel of expert scientists and physicians from academia, the pharmaceutical industry and regulatory agencies. The findings of the panel were then presented to a wider group of participants for open discussion. The following report summarizes the results of these discussions with respect to novel MRI pulse sequences for evaluating articular cartilage of the knee in OA and notes any additional advances that have been made since.

Molecular crowding reduces to a similar extent the diffusion of small solutes and macromolecules: measurement by fluorescence correlation spectroscopy.

Aqueous environments in living cells are crowded, with up to >50 wt% small and macromolecule-size solutes. We investigated quantitatively one important consequence of molecular crowding--reduced diffusion of biologically important solutes. Fluorescence correlation spectroscopy (FCS) was used to measure the diffusion of a series of fluorescent small solutes and macromolecules. In water, diffusion coefficients (D(o)w) were (in cm2/s x 10(-8)): rhodamine green (270), albumin (52), dextrans (75, 10 kDa; 10, 500 kDa), double-stranded DNAs (96, 20 bp; 10, 1 kb; 3.4, 4.5 kb) and polystyrene nanospheres (5.4, 20 nm diameter; 2.3, 100 nm). Aqueous-phase diffusion (Dw) in solutions crowded with Ficoll-70 (0-60 wt%) was reduced by up to 650-fold in an exponential manner: Dw = D(o)w exp (-[C]/[C]exp), where [C]exp is the concentration (in wt%) of crowding agent reducing D(o)w by 63%. FCS data for all solutes and Ficoll-70 concentrations fitted well to a model of single-component, simple (non-anomalous) diffusion. Interestingly [C]exp were nearly identical (11+/-2 wt%, SD) for diffusion of the very different types of macromolecules in Ficoll-70 solutions. However, [C]exp was dependent on the nature of the crowding agent: for example, [C]exp for diffusion of rhodamine green was 30 wt% for glycerol and 16 wt% for 500 kDa dextran. Our results indicate that molecular crowding can greatly reduce aqueous-phase diffusion of biologically important macromolecules, and demonstrate a previously unrecognized insensitivity of crowding effects on the size and characteristics of the diffusing species.

The influence of path length and matrix components on ageing characteristics of transport between the choroid and the outer retina.

To determine the relative influence of path length and matrix components on the movement of small solutes and water between the choroid and the outer retina. Human and bovine Bruch's membrane-choroid (BC) tissue samples were mounted in modified Ussing chambers, and the diffusion of taurine and hydraulic conductivity (Lp) was determined. In humans, diffusion of taurine was determined as a function of age of the donor. The relative contribution of Bruch's membrane in the BC complex to transport processes was measured after its removal by laser ablation. Similarly, the effect of choroidal path length was determined. In humans, tracking the trend of age-related thinning provided samples of various path lengths. In young bovine animals (< or =2 years old), choroidal thickness was adjusted by laser ablation. Diffusion of taurine across human BC decreased linearly from 162.7 to 105.9 nanomoles/h per 3 mm between 10 and 90 years of age (P < 0.05). Ablation of Bruch's membrane increased diffusion of taurine from 129 to 287.9 nanomoles/h per 4 mm in human (donor age 55, 74, and 82 years; P < 0.005) but caused no statistically significant change in bovine BC. Diffusion of taurine across bovine BC was greater in samples with partially ablated choroid (218 nanomoles/h per 4 mm) than in normal control samples (128.75 nanomoles/h per 4 mm). Lp was not measurable in bovine samples after complete ablation of Bruch's membrane, but did not change significantly as the choroid thinned. The data suggest that both path length and matrix components contribute to the decline of diffusion of small solutes across BC with age. The importance of matrix components was also demonstrated in restricting the movement of water while choroidal thickness played little if any role.