Central retinal vascular trunk deviation in unilateral normal-tension glaucoma.

PURPOSE: To investigate whether the position of the central retinal vascular trunk (CRVT), as a surrogate of lamina cribrosa (LC) offset, was associated with the presence of glaucoma in normal-tension glaucoma (NTG) patients. METHODS: The position of the CRVT was measured as the deviation from the center of the Bruch's membrane opening (BMO), as delineated by spectral-domain optical coherence tomography imaging. The offset index was calculated as the distance of the CRVT from the BMO center relative to that of the BMO margin. The angular deviation of CRVT was measured with the horizontal nasal midline as 0° and the superior location as a positive value. The offset index and angular deviation were compared between glaucoma and fellow control eyes within individuals. RESULTS: NTG eyes had higher baseline intraocular pressure (P = 0.001), a larger ß-zone parapapillary atrophy area (P = 0.013), and a larger offset index (P<0.001). In a generalized linear mixed-effects model, larger offset index was the only risk factor of NTG diagnosis (OR = 31.625, P<0.001). A generalized estimating equation regression model revealed that the offset index was larger in the NTG eyes than in the control eyes for all ranges of axial length, while it was the smallest for the axial length of 23.4 mm (all P<0.001). CONCLUSIONS: The offset index was larger in the unilateral NTG eyes, which fact is suggestive of the potential role of LC/BMO offset as a loco-regional susceptibility factor.

Biomechanical Properties of Bruch's Membrane-Choroid Complex and Their Influence on Optic Nerve Head Biomechanics.

Purpose: The purpose of this study was to measure the rupture pressure and the biomechanical properties of porcine Bruch's membrane (BM)-choroid complex (BMCC) and the influences of BM on optic nerve head (ONH) tissues. Methods: The biomechanical properties of BMCC were extracted through uniaxial tensile tests of 10 BMCC specimens from 10 porcine eyes; the rupture pressures of BMCC were measured through burst tests of 20 porcine eyes; and the influence of BM on IOP-induced ONH deformations were investigated using finite element (FE) analysis. Results: Uniaxial experimental results showed that the average elastic (tangent) moduli of BMCC samples at 0% and 5% strain were 1.60 ± 0.81 and 2.44 ± 1.02 MPa, respectively. Burst tests showed that, on average, BMCC could sustain an IOP of 82 mm Hg before rupture. FE simulation results predicted that, under elevated IOP, prelamina tissue strains increased with increasing BM stiffness. On the contrary, lamina cribrosa strains showed an opposite trend but the effects were small. Conclusions: BMCC stiffness is comparable or higher than those of other ocular tissues and can sustain a relatively high pressure before rupture. Additionally, BM may have a nonnegligible influence on IOP-induced ONH deformations.

Photoreceptor Layer Thickness Changes During Dark Adaptation Observed With Ultrahigh-Resolution Optical Coherence Tomography.

Purpose: To examine outer retinal band changes after flash stimulus and subsequent dark adaptation with ultrahigh-resolution optical coherence tomography (UHR-OCT). Methods: Five dark-adapted left eyes of five normal subjects were imaged with 3-µm axial-resolution UHR-OCT during 30 minutes of dark adaptation following 96%, 54%, 23%, and 0% full-field and 54% half-field rhodopsin bleach. We identified the ellipsoid zone inner segment/outer segment (EZ[IS/OS]), cone interdigitation zone (CIZ), rod interdigitation zone (RIZ), retinal pigment epithelium (RPE), and Bruch's membrane (BM) axial positions and generated two-dimensional thickness maps of the EZ(IS/OS) to the four bands. The average thickness over an area of the thickness map was compared against that of the dark-adapted baselines. The time-dependent thickness changes (photoresponses) were statistically compared against 0% bleach. Dark adaptometry was performed with the same bleaching protocol. Results: The EZ(IS/OS)-CIZ photoresponse was significantly different at 96% (P < 0.0001) and 54% (P = 0.006) bleach. At all three bleaching levels, the EZ(IS/OS)-RIZ, -RPE, and -BM responses were significantly different (P < 0.0001). The EZ(IS/OS)-CIZ and EZ(IS/OS)-RIZ time courses were similar to the recovery of rod- and cone-mediated sensitivity, respectively, measured with dark adaptometry. The maximal EZ(IS/OS)-CIZ and EZ(IS/OS)-RIZ response magnitudes doubled from 54% to 96% bleach. Both EZ(IS/OS)-RPE and EZ(IS/OS)-BM responses resembled dampened oscillations that were graded in amplitude and duration with bleaching intensity. Half-field photoresponses were localized to the stimulated retina. Conclusions: With noninvasive, near-infrared UHR-OCT, we characterized three distinct, spatially localized photoresponses in the outer retinal bands. These photoresponses have potential value as physical correlates of photoreceptor function.

In Vivo Choroidal Vascular Lesions in Diabetes on Swept-Source Optical Coherence Tomography.

Diabetes induces microvascular diseases including diabetic retinopathy and choroidopathy which reciprocally promote the pathogenesis, although optical coherence tomography images of diabetic choroidopathy remains to be documented. Here we evaluated the qualitative characteristics of choroidal vascular lesions in patients with diabetes and their association with diabetic retinopathy on swept-source optical coherence tomography (SS-OCT) images. We retrospectively reviewed 110 consecutive eyes of 110 patients with diabetes and 35 eyes of 35 healthy subjects for whom SS-OCT images (6x6-mm scans centered on the fovea) of sufficient quality were acquired. The curve of chorioretinal sections was flattened using Bruch's membrane as a reference surface, followed by generation of en-face images. We characterized choroidal vascular lesions and evaluated their association with the logarithm of the minimum angle of resolution visual acuity (logMAR VA), retinal and choroidal thicknesses, and diabetic retinopathy severity. En-face SS-OCT images showed unvisualized vessels in Sattler's layer in 33 eyes (30.0%). Focal narrowing was seen in choroidal vessels in Haller's layer in 56 eyes (50.9%). The choroidal vessels ended in the superficial or middle portion of Haller's layer, referred to as vascular stumps, in 20 eyes (18.2%). Diabetic eyes had these findings more frequently than nondiabetic eyes. The subfoveal choroid was thicker in eyes with focal vascular narrowing and vascular stumps than in eyes without such lesions. Vascular stumps in Haller's layer were significantly related to diabetic retinopathy severity, logMAR VA, and central retinal and choroidal thicknesses. These novel findings on SS-OCT images would promote the better understanding of complicated pathogenesis in diabetic retinopathy and choroidopathy.

Albumen Transport to Bruch's Membrane and RPE by Choriocapillaris Caveolae.

PURPOSE: The choriocapillaris (CC), the capillary network of the choroid, is positioned adjacent to Bruch's membrane (BM) and the RPE. The aim of this study was to clarify the mechanism(s) for transport of serum albumen from CC lumen to RPE. METHODS: Alexa647 conjugated to BSA (BSA-A647) or PBS was administrated via the femoral vein to young and aged wild-type (WT; C57BL/6J) mice and Caveolin-1 knockout mice (Cav1(-/-)). Mice were perfused with PBS and killed at 30 minutes, 1 hour, and 4 hours after injection. Eyecups were cryopreserved, and cryosections were analyzed on a Zeiss 710 confocal microscope. Bovine serum albumin conjugated to gold nanoparticles (BSA-GNP) was administrated through the left common carotid artery. Mice were perfused with PBS and killed at 30 minutes after injection. Eyecups were embedded after fixation, and 70-nm-thick sections were analyzed on a Hitachi H7600 transmission electron microscope. RESULTS: In eyes of WT young mice, BSA-A647 was transported to the RPE at 30 minutes and diffused to the photoreceptor layer by 1 hour. In contrast, most BSA-A647 was found in the CC in Cav1(-/-) eyes. The majority of BSA-GNP found in the CC of young WT mice was on the luminal side in caveolae at 30 minutes after injection. In aged WT mice, BSA-GNPs were found in defective tight junctions between endothelial cells and appeared trapped at the diaphragm of fenestrations. CONCLUSIONS: Normally, CC carefully regulates transport system of BSA from lumen to BM by caveolae-mediated transcytosis; however, endothelium cells of aged control WT mice have leaky tight junctions and lacked regulated BSA transport.

Retinal Biochemistry, Physiology and Cell Biology.

The vitreous, the vasculature of the retina, macular pigments, phototransduction, retinal pigment epithelium, Bruch's membrane and the extracellular matrix, all play an important role in the normal function of the retina as well as in diseases. Understanding the pathophysiology allows us to target treatment. As ocular angiogenesis, immunity and inflammation are covered elsewhere, those subjects will not be discussed in this chapter.

Influence of translaminar pressure dynamics on the position of the anterior lamina cribrosa surface.

PURPOSE: To determine how the translaminar pressure difference (TLPD) and gradient (TLPG) influence the position of anterior lamina cribrosa (LC) surface. METHODS: Twenty-six eyes of 26 healthy subjects were subjected to enhanced-depth imaging volume scanning of the optic nerve using spectral-domain optical coherence tomography (SD-OCT). The anterior LC surface depth (LCD) relative to the Bruch's membrane (BM) opening was measured at 11 equidistant planes, and the LC thickness (LCT) was measured at three locations (superior midperipheral, midhorizontal, and inferior midperipheral). Intraocular pressure and lumbar cerebrospinal fluid pressure (CSFP) were measured on the same day as the SD-OCT examination. The TLPD was defined as the difference between IOP and CSFP (i.e., IOP-CSFP), and the TLPG as the TLPD divided by LCT (i.e., TLPD/LCT). RESULTS: Subjects were aged 63.4 ± 8.0 years and comprised 12 males and 14 females. Regression analyses revealed a significant association between a larger mean LCD and male sex (P = 0.002), and between a larger central LCD and male sex (P ≤ 0.012), larger TLPD (P = 0.048), and higher TLPG (P = 0.029). There was no significant association between IOP, CSFP, and LCT, and either the mean LCD (P = 0.438, 0.368, and 0.416, respectively) or central LCD (P = 0.284, 0.085, and 0.144, respectively). CONCLUSIONS: A larger central LCD was associated with larger TLPD and higher TLPG in healthy eyes, which indicates that the translaminar pressure dynamics may play a role in the position of the anterior LC surface relative to BM opening in healthy human eyes.

Biochemical restoration of aged human Bruch's membrane: experimental studies to improve retinal pigment epithelium transplant survival and differentiation.

Suspensions of human embryonic stem cell-derived retinal pigment epithelium (hES-RPE) and human fetal RPE resurface aged and age-related macular degeneration (AMD) Bruch's membrane to a limited degree at day 21 in organ culture. Survival and differentiation of hES-RPE and human fetal RPE on aged or AMD Bruch's membrane are enhanced greatly (200%) if a biologically synthesized extracellular matrix (bovine corneal endothelial cell extracellular matrix) is laid down on Bruch's membrane prior to transplantation. Transplanted RPE survival is enhanced even more (400-1,000%) if Bruch's membrane is treated with bovine corneal endothelial cell-conditioned medium during organ culture of hES-RPE or fetal RPE on aged or AMD Bruch's membrane. Future efforts are focused on identifying the bioactive components of bovine corneal endothelial cell-conditioned medium, so that this material can be reconstituted for clinical use as an adjunct to improve RPE transplant survival and differentiation in AMD eyes.

A method to enhance cell survival on Bruch's membrane in eyes affected by age and age-related macular degeneration.

PURPOSE: To determine whether conditioned medium (CM) derived from bovine corneal endothelial cells (BCECs) can support transplanted cells on aged and age-related macular degeneration (AMD) Bruch's membrane (BM). METHODS: Retinal pigment epithelium (RPE) cells derived from human embryonic stem cells (hES-RPE) and cultured fetal and aged adult RPE were seeded onto the inner collagenous layer of submacular BM-choroid-sclera explants generated from aged and AMD human donor eyes. Paired explants were cultured in BCEC-CM or CM vehicle. To assess cell behavior after attachment to BM was established, explants were harvested after 21 days in culture. To assess whether sustained exposure to BCEC-CM was necessary for improved cell survival on BM, short exposure to BCEC-CM (3, 7, 14 days) was compared with 21-day exposure. Explants were harvested and evaluated by scanning electron and light microscopy. Extracellular matrix (ECM) deposition after exposure to BCEC-CM was evaluated following RPE cell removal after day 21 on tissue culture dishes or on BM. RESULTS: BCEC-CM significantly enhanced hES-RPE, fetal RPE, and aged adult RPE survival on BM, regardless of submacular pathology. Although shorter BCEC-CM exposure times showed significant improvement in cell survival compared with culture in CM vehicle, longer BCEC-CM exposure times were more effective. BCEC-CM increased RPE ECM deposition on tissue culture plastic and on BM. CONCLUSIONS: The results of this study indicate that RPE survival is possible on AMD BM and offer a method that could be developed for enhancing transplanted cell survival on AMD BM. Increased ECM deposition may account for improved cell survival after culture in BCEC-CM.

Atypical retardation patterns in scanning laser polarimetry are associated with low peripapillary choroidal thickness.

PURPOSE: Scanning laser polarimetry (SLP) results can be affected by an atypical retardation pattern (ARP). One reason for an ARP is the birefringence of the sclera. The purpose of this study was to investigate the influence of the peripapillary choroidal thickness (pChTh) on the occurrence of ARP. METHODS: One hundred ten healthy subjects were investigated with SLP and spectral domain OCT. pChTh was measured in B-scan images at 768 positions using semiautomatic software. Values were averaged to 32 sectors and the total peripapillary mean. Subjects were divided into four groups according to the typical scan score (TSS) provided by the GDxVCC: group 1 TSS, 100; group 2 TSS, 90-99; group 3 TSS, 80-89; group 4 TSS, <80. RESULTS: Mean pChTh (± SD) in 110 healthy subjects was 141 µm (±49 µm). There was a significant correlation between pChTh and TSS (r = 0.608; P < 0.001). In TSS groups 1 to 4, mean pChTh was 168 µm (±38 µm), 148 µm (± 48 µm), 119 µm (±35 µm), and 92 (±42 µm). Mean pChTh of TSS groups 3 and 4 was significantly lower than that of TSS group 1 (P < 0.001). CONCLUSIONS: Low values of TSS resulting from the appearance of ARP in SLP are associated with low peripapillary choroidal thickness. Reduced choroidal thickness may result in an increased amount of confounding light getting to the SLP light detectors.

Cell-deposited matrix improves retinal pigment epithelium survival on aged submacular human Bruch's membrane.

PURPOSE: To determine whether resurfacing submacular human Bruch's membrane with a cell-deposited extracellular matrix (ECM) improves retinal pigment epithelial (RPE) survival. METHODS: Bovine corneal endothelial (BCE) cells were seeded onto the inner collagenous layer of submacular Bruch's membrane explants of human donor eyes to allow ECM deposition. Control explants from fellow eyes were cultured in medium only. The deposited ECM was exposed by removing BCE. Fetal RPE cells were then cultured on these explants for 1, 14, or 21 days. The explants were analyzed quantitatively by light microscopy and scanning electron microscopy. Surviving RPE cells from explants cultured for 21 days were harvested to compare bestrophin and RPE65 mRNA expression. Mass spectroscopy was performed on BCE-ECM to examine the protein composition. RESULTS: The BCE-treated explants showed significantly higher RPE nuclear density than did the control explants at all time points. RPE expressed more differentiated features on BCE-treated explants than on untreated explants, but expressed very little mRNA for bestrophin or RPE65. The untreated young (<50 years) and African American submacular Bruch's membrane explants supported significantly higher RPE nuclear densities (NDs) than did the Caucasian explants. These differences were reduced or nonexistent in the BCE-ECM-treated explants. Proteins identified in the BCE-ECM included ECM proteins, ECM-associated proteins, cell membrane proteins, and intracellular proteins. CONCLUSIONS: Increased RPE survival can be achieved on aged submacular human Bruch's membrane by resurfacing the latter with a cell-deposited ECM. Caucasian eyes seem to benefit the most, as cell survival is the worst on submacular Bruch's membrane in these eyes.

Sources of retinal pigment epithelium (RPE) for replacement therapy.

Stem cells, with their capacity to regenerate and replace diseased tissues, have recently been proposed as having great potential in the treatment of age-related macular degeneration (AMD). A stem cell therapeutic approach could operate to replace either the retinal pigment epithelium (RPE), the neurosensory retina or a combination of both. From the scientific perspective, RPE replacement alone is likely to be far more straightforward than rebuilding the complex circuitry of the neurosensory retina. Furthermore, recent advances with induced pluripotent stem cells have raised the real possibility of transplanting healthy 'young' autologous RPE into patients with early signs of AMD. At this stage, however, it is useful to reconsider some of the earlier clinical studies that used suspensions of autologous RPE cells harvested from the peripheral retina. These showed that isolated RPE cell suspensions had little capacity to recreate a monolayer on the diseased Bruch's membrane of AMD. To counter this problem, researchers from Southampton in the UK report the use of a synthetic polymer alternative to Bruch's membrane, which could provide a scaffold for future RPE derived from stem cells or possibly reopen opportunities for autologous RPE cells harvested from the peripheral retina.

The dynamic nature of Bruch's membrane.

Bruch's membrane (BM) is a unique pentalaminar structure, which is strategically located between the retinal pigment epithelium (RPE) and the fenestrated choroidal capillaries of the eye. BM is an elastin- and collagen-rich extracellular matrix that acts as a molecular sieve. BM partly regulates the reciprocal exchange of biomolecules, nutrients, oxygen, fluids and metabolic waste products between the retina and the general circulation. Accumulating evidence suggests that the molecular, structural and functional properties of BM are dependent on age, genetic constitution, environmental factors, retinal location and disease state. As a result, part of the properties of BM are unique to each human individual at a given age, and therefore uniquely affect the development of normal vision and ocular disease. The changes occurring in BM with age include increased calcification of elastic fibres, increased cross-linkage of collagen fibres and increased turnover of glycosaminoglycans. In addition, advanced glycation end products (AGEs) and fat accumulate in BM. These age-related changes may not only influence the normal age-related health of photoreceptor cells, but also the onset and progression of diseases like retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Undoubtedly, BM is the site of drusen development. Confluent drusen and uncontrolled activation of the complement cascade are most likely the first signs of AMD. Furthermore, the nature of adhesive interactions between the RPE and BM are instrumental in the development of retinal detachments and proliferative retinal disease. Finally, BM is passively or actively involved in a range of other retinal disorders such as Pseudoxanthoma elasticum (PXE), Sorsby's Fundus Dystrophy and Malattia Leventinese. Here, we review the dynamic nature of Bruch's membrane, from molecule to man, during development, aging and disease. We propose a simple and straightforward nomenclature for BM deposits. Finally, we attempt to correlate recently published mRNA expression profiles of the RPE and choroid with molecular, structural and functional properties of BM. Our review may shed light on the complex involvement of BM in retinal pathology, notably age-related macular degeneration.

Biomechanical properties of native basement membranes.

Basement membranes are sheets of extracellular matrix that separate epithelia from connective tissues and outline muscle fibers and the endothelial lining of blood vessels. A major function of basement membranes is to establish and maintain stable tissue borders, exemplified by frequent vascular breaks and a disrupted pial and retinal surface in mice with mutations or deletions of basement membrane proteins. To directly measure the biomechanical properties of basement membranes, chick and mouse inner limiting membranes were examined by atomic force microscopy. The inner limiting membrane is located at the retinal-vitreal junction and its weakening due to basement membrane protein mutations leads to inner limiting membrane rupture and the invasion of retinal cells into the vitreous. Transmission electron microscopy and western blotting has shown that the inner limiting membrane has an ultrastructure and a protein composition typical for most other basement membranes and, thus, provides a suitable model for determining their biophysical properties. Atomic force microscopy measurements of native chick basement membranes revealed an increase in thickness from 137 nm at embryonic day 4 to 402 nm at embryonic day 9, several times thicker that previously determined by transmission electron microscopy. The change in basement membrane thickness was accompanied by a large increase in apparent Young's modulus from 0.95 MPa to 3.30 MPa. The apparent Young's modulus of the neonatal and adult mouse retinal basement membranes was in a similar range, with 3.81 MPa versus 4.07 MPa, respectively. These results revealed that native basement membranes are much thicker than previously determined. Their high mechanical strength explains why basement membranes are essential in stabilizing blood vessels, muscle fibers and the pial border of the central nervous system.

An experimental study of the elastic properties of the human Bruch's membrane-choroid complex: relevance to ageing.

AIM: To investigate the mechanical properties (stress-strain relation, elasticity, hysteresis, response to stress spikes and drops) of isolated human Bruch's membrane-choroid, as well as the effect of ageing and aged related macular degeneration (AMD). METHODS: 13 Bruch's membrane-choroid complexes were obtained from human donors (21-97 years). Two samples (aged 85 and 95) showed signs of AMD including large, soft drusen, choroidal neovascularisation, and/or disciform scars. Various hydrostatic pressures (stress) were applied to the choroidal surface of mid-peripheral samples mounted in a modified open Ussing chamber. Linear scans of the tissue were recorded by optical coherence tomography (OCT) and the pressure induced deformation (strain), elasticity, hysteresis, and response to pressure spikes and drops measured. RESULTS: The elasticity of human Bruch's membrane-choroid complex decreased linearly with ageing (p<0.001) after the age of 21 with an approximate reduction of 1% per year. The decrease was not exaggerated in AMD. The recoil capacity of Bruch's membrane-choroid was not affected by ageing. The response to pressure spikes/drops was similar in age matched normal and AMD eyes. The results suggest that although the aged induced decrease in Bruch's membrane elasticity may contribute to breaks in this membrane in AMD leading to neovascularisation this is not sufficient. The presence of other factors is required for its development. CONCLUSION: The elasticity of Bruch's membrane-choroid complex decreases with age while recoil capacity does not. The decrease was not exaggerated in AMD.

Hematopoietic stem cells provide repair functions after laser-induced Bruch's membrane rupture model of choroidal neovascularization.

Vascular repair by adult hematopoietic stem cells (HSCs) is well-appreciated because these cells are known for their plasticity. We have shown that adult HSCs differentiate into endothelial cells and participate in both retinal and choroidal neovascularization. We asked whether HSCs participated in the wounding response by forming astrocytes, retinal pigment epithelia (RPE), macrophages, and pericytes. Lethally irradiated C57BL6/J mice were reconstituted with HSCs from mice homozygous for green fluorescent protein (GFP) and then subjected to laser-induced rupture of Bruch's membrane. After immunohistochemical examination of ocular tissue, GFP(+) astrocytes were observed concentrated along the edge of the laser wound, where they and mural cells closely ensheathed the neovasculature. GFP(+) vascular endothelial cells and macrophages/microglia were also evident. Large irregularly shaped GFP(+) RPE cells constituted approximately 93% of RPE cells adjacent to the edge of the denuded RPE area. In regions farther away from the wound, GFP(+) RPE cells were integrated among the GFP(-) host RPE. Thus, postnatal HSCs can differentiate into cells expressing markers specific to astrocytes, macrophages/microglia, mural cells, or RPE. These studies suggest that HSCs could serve as a therapeutic source for long-term regeneration of injured retina and choroid in diseases such as age-related macular degeneration and retinitis pigmentosa.

Iris pigment epithelium attachment to aged submacular human Bruch's membrane.

PURPOSE: To determine whether iris pigment epithelium (IPE) cells can attach to aged submacular human Bruch's membrane and to assess whether IPE cells express the integrin subunits that may be necessary to bind to the known extracellular matrix ligands present in Bruch's membrane. METHODS: IPE cells were seeded onto the RPE basement membrane (RPEbm) or inner collagenous layer (ICL) of aged submacular Bruch's membrane as microaggregates or were expanded in culture until enough cells could be obtained for seeding. Cell morphology and the percentage of cell coverage were determined 1 or 7 days after seeding. Messenger RNA was extracted from cultured and uncultured IPE cells and analyzed by RT-PCR. The expression of integrin subunits alpha1 to alpha6 and beta1 mRNA was examined. RESULTS: Coverage by uncultured IPE was low on both surfaces at day-1 (RPEbm, 7.9% +/- 4.8%; ICL, 5.0% +/- 2.5%) with few intact cells present. Culturing IPE improved attachment with similar coverage on both surfaces and no significant difference between day-1 (RPEbm, 89.9% +/- 9.1%; ICL, 63.4% +/- 26.5%) and day-7 (RPEbm, 97.8% +/- 2.3%; ICL, 94.7% +/- 6.6%). By day-7, cell morphology and coverage on both surfaces was variable, ranging from few intact cells to a high degree of coverage by flattened cells. All integrin subunits studied were expressed in cultured cells, whereas alpha2, alpha3, and alpha4 showed less or no expression in uncultured cells. CONCLUSIONS: Upregulation of integrin mRNA expression may be one explanation for the difference in coverage by cultured versus uncultured IPE cells. The presence of dead, dying, or flattened cells at day 7 indicates that IPE may not survive or differentiate on aged submacular Bruch's membrane.

Reengineering of aged Bruch's membrane to enhance retinal pigment epithelium repopulation.

PURPOSE: An earlier study showed that age-related changes in the inner collagen layer (ICL) inhibit RPE cell repopulation of human Bruch's membrane. The present study was undertaken to determine the effect of cleaning and/or an extracellular matrix (ECM) protein coating on the reattachment, apoptosis, proliferation, and final surface coverage of the transplanted RPE cells. METHODS: Explants of aged Bruch's membrane with ICL exposed were prepared from five human cadaveric eyes (donor ages, 69-84 years) and treated with Triton X-100 and/or coated with a mixture of laminin (330 microg/mL), fibronectin (250 microg/mL), and vitronectin (33 microg/mL). Viable human fetal and ARPE-19 cells (n = 15,000) were plated onto the surface and the RPE reattachment, apoptosis, and proliferation ratios were determined on the modified surfaces. Cells were cultured up to 17 days to determine the surface coverage. Ultrastructure of the modified Bruch's membrane and RPE morphology were studied with transmission and scanning electron microscopy. RESULTS: Reattachment ratios of fetal human RPE and ARPE-19 cells were similar on aged ICL (41.5% +/- 1.7% and 42.9% +/- 2.7%, P > 0.05). The reattachment ratio increased with ECM protein coating and decreased with detergent treatment. Combined cleaning and coating restored the reattachment ratio of the fetal RPE cells, but failed to increase the reattachment ratio of ARPE-19 cells. The highest apoptosis was observed on untreated ICL. Cleaning and the combined procedure of cleaning and ECM protein coating decreased fetal RPE cell apoptosis. Only RPE cells plated on cleaned or cleaned and ECM-coated ICL demonstrated proliferation that led to substantial surface coverage at day 17. CONCLUSIONS: Age-related changes that impair RPE repopulation of Bruch's membrane can be significantly reversed by combined cleaning and ECM protein coating of the ICL. Development of biologically tolerant techniques for modifying the ICL in vivo may enhance reattachment of the RPE and its repopulation of aged ICL.