Passage of low-density lipoproteins through Bruch's membrane and choroid

Abstract

Plasma lipoproteins are thought to transport cholesterol, vitamins and carotenoids to the retinal pigment epithelium (RPE) for ultimate use by the photoreceptors. However, to reach the RPE, these lipoprotein particles must cross Bruch's membrane. We examined the reflection coefficient of Bruch's membrane (BrM) to low-density lipoprotein (LDL). Bruch's membrane and choroid were removed from 47 bovine eyes. Specimens were placed in a Ussing chamber and perfused with phosphate-buffered saline (PBS) with (31 specimens) or without (16 specimens) fluorescent low-density lipoproteins (DiI-LDL). The hydraulic conductivity of the tissue was determined for both calf and cow eyes. In the perfusions with DiI-LDL, the fluorescence intensity emitted by DiI-LDL in the efflux was measured and the reflection coefficient of BrM/choroid preparations to DiI-LDL determined. Leakage tests were done to confirm tissue integrity. Several specimens were examined using scanning electron microscopy (SEM) to examine tissue integrity before and after perfusion. Leak testing confirmed that BrM was intact both before and after perfusion. The average hydraulic conductivity of BrM/choroid perfusion of calf eyes with PBS alone was 1.42 ± 0.55 × 10(-9) m/s/Pa (mean ± SD, n = 11). The average hydraulic conductivity of the cow eyes was 4.94 ± 1.48 × 10(-10) m/s/Pa (n = 5), nearly a 3-fold decrease with age. While the flow rate remained constant during the PBS perfusions, it decreased as a function of time during perfusion with DiI-LDLs. Our major finding was of fluorescence in the effluent collected in all perfusions with DiI-LDLs, demonstrating passage of LDL through the tissue. The average reflection coefficient of calf BrM/choroid preparations to DiI-LDL was 0.58 ± 0.25 (n = 23); a similar distribution of reflection coefficients was seen in tissue from cow eyes (0.51 ± 0.33, n = 8). Our data suggested that the DiI-LDL was modestly hindered and/or captured by the tissue. This might explain the progressive decrease of hydraulic conductivity with continued perfusion of DiI-LDL.

Figure 1

The perfusion system used in the current study.

Figure 2

(A) SEM image of calf BrM before perfusion; (B) SEM of calf BrM after perfusion.

Figure 3

The flow rate and the pressure drop as a function of perfusion time in (A) PBS perfusion of calf tissue and (B) DiI-LDL perfusion of calf tissue.

Figure 4

The initial tissue hydraulic conductivity (L_pi) of the calf BrM preparations as a function of the DiI-LDL reflection coefficient (σ). (A) BrM facing upstream. (B) Choroid facing upstream. The dashed line in 5A is a linear regression to the data with p < 0.02 and a correlation coefficient of 0.73.