Retinal Pigment Epithelium Cell Density Research Articles

In-vitro safety assessment of meropenem on human retinal pigment epithelium (RPE)

PurposeEndophthalmitis is a severe infection accompanied by inflammation that affects the anterior and posterior parts of the eye. It is typically treated with a combination of antibiotics that cover various microorganisms. However, retinal pigment epithelium (RPE) cells are highly susceptible to damage from intravitreal injection therapy. This study aimed to investigate the impact of clinically relevant concentrations of meropenem (alone or in combination with vancomycin) on the viability and inflammation of RPE cells. DesignIn-vitro Study. MethodsRPE cells from passages 5–7 were treated with different concentrations of meropenem (1/4x, x, and 4x; [x = 16 mg/L]), vancomycin (30 mg/L), and meropenem (x) plus vancomycin for 24 h. The morphology assessment and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay were performed to evaluate cytotoxicity due to drug treatment. Real-time PCR was used to measure the relative expression of apoptotic genes (BCL-2 and BAX) and inflammation biomarkers (IL-1b and IL-6). ResultsMeropenem (alone or in combination with vancomycin) did not have any significant effect on RPE cell morphology, density, and viability. Gene expression analysis confirmed these results, showing no significant changes in the BCL-2/BAX ratio in drug-treated RPE cells compared to controls. Treatment with meropenem significantly induced the expression of IL-1b at all studied concentrations. Additionally, at concentrations of x and 4x, it also significantly increased the expression of IL-6, which was dose-dependent. However, this effect was not observed with vancomycin alone or in combination with meropenem. ConclusionsThe results of this study suggest that meropenem, either alone or in combination with vancomycin, does not induce RPE cytotoxicity. There was an upregulation of IL-1b and IL-6 in meropenem monotherapy, the clinical implication of which should be elucidated in future in-vivo or clinical studies.

IMI-Nonpathological Human Ocular Tissue Changes With Axial Myopia.

To describe nonpathological myopia-related characteristics of the human eye. Based on histomorphometric and clinical studies, qualitative and quantitative findings associated with myopic axial elongation are presented. In axial myopia, the eye changes from a spherical shape to a prolate ellipsoid, photoreceptor, and retinal pigment epithelium cell density and total retinal thickness decrease, most marked in the retroequatorial region, followed by the equator. The choroid and sclera are thin, most markedly at the posterior pole and least markedly at the ora serrata. The sclera undergoes alterations in fibroblast activity, changes in extracellular matrix content, and remodeling. Bruch's membrane (BM) thickness is unrelated to axial length, although the BM volume increases. In moderate myopia, the BM opening shifts, usually toward the fovea, leading to the BM overhanging into the nasal intrapapillary compartment. Subsequently, the BM is absent in the temporal region (such as parapapillary gamma zone), the optic disc takes on a vertically oval shape, the fovea-optic disc distance elongates without macular BM elongation, the angle kappa reduces, and the papillomacular retinal vessels and nerve fibers straighten and stretch. In high myopia, the BM opening and the optic disc enlarge, the lamina cribrosa, the peripapillary scleral flange (such as parapapillary delta zone) and the peripapillary choroidal border tissue lengthen and thin, and a circular gamma and delta zone develop. A thorough characterization of ocular changes in nonpathological myopia are of importance to better understand the mechanisms of myopic axial elongation, pathological structural changes, and psychophysical sequelae of myopia on visual function.

Anatomic Peculiarities Associated with Axial Elongation of the Myopic Eye.

To describe anatomical peculiarities associated with axial elongation in the human myopic eye. Reviewing the results of previous histomorphometrical investigations of enucleated human globes, as well as reviewing findings obtained in population-based studies and hospital-based clinical investigations of myopic patients and non-myopic individuals. Myopic axial elongation is associated with a change from a mostly spherical eye shape to a prolate ellipsoid form. It is combined with choroidal and scleral thinning, most pronounced at the posterior pole and less pronounced in the fundus midperiphery. In the fundus midperiphery, the retina and density of the retinal pigment epithelium (RPE) and photoreceptors decrease with a longer axial length, while in the macular region, retinal thickness, RPE cell density, and choriocapillaris thickness are not related to axial length. With axial elongation, a parapapillary gamma zone develops, leading to an enlargement of the optic disc-fovea distance and a decrease in angle kappa. Axial elongation is also correlated with an increase in the surface and volume of Bruch's membrane (BM), while BM thickness remains unchanged. Axial elongation causes moderately myopic eyes to show a shift of BM opening to the foveal direction so that the horizontal disc diameter becomes shorter (with a consequent vertical ovalization of the optic disc shape), a temporal gamma zone develops, and the optic nerve exit takes an oblique course. Features of high myopia are an enlargement of the RPE opening (myopic parapapillary beta zone) and BM opening (secondary macrodisc), elongation and thinning of the lamina cribrosa, peripapillary scleral flange (parapapillary delta zone) and peripapillary choroidal border tissue, secondary BM defects in the macular region, myopic maculoschisis, macular neovascularization, and cobblestones in the fundus periphery. These features combined may be explained by a growth in BM in the fundus midperiphery leading to axial elongation.

Myopic macular Bruch’s membrane defects

PurposeTo examine histologic characteristics of macular Bruchś membrane defects (BMD) in axially elongated eyes. DesignHistomorphometric study. MethodsUsing light microscopy, we examined enucleated human globes for BMDs. ResultsIn 247 eyes, BMDs were detected in 15 (6.1%) eyes (axial length:27.0–36.0 mm), in 10 of them in the macular region. Prevalence and size of BMDs (mean:1.93 ± 1.62 mm; range:0.22mm–6.24 mm) correlated with longer axial length (OR:1.52; 95%CI:1.19,1.94; P = 0.001) and higher prevalence of scleral staphylomas (OR:16.3; 95%CI:2.67,99.3; P < 0.001). The BMDs were smaller than corresponding gaps in the retinal pigment epithelium (RPE) (1.93 ± 1.62 mm versus 2.61 mm ± 1.73 mm; P = 0.003), and larger than corresponding gaps in the inner nuclear layer (0.43 ± 0.76 mm; P = 0.008) and inner limiting membrane bridges (0.13 ± 0.33 mm; P = 0.001). Choriocapillaris thickness, BM thickness and RPE cell density did not vary (all P > 0.05) between the BDM border and adjacent areas. In the BMD, choriocapillaris and RPE were absent. The sclera was thinner in the BDM area than in adjacent areas (0.28 ± 0.19 mm versus 0.36 ± 0.13 mm; P = 0.006). ConclusionsBMDs as hallmarks of myopic macular degeneration are characterized by longer gaps in the RPE and smaller gaps in the outer nuclear layer and inner nuclear layer, by localized scleral thinning, and by a spatial association with scleral staphylomas. Thickness of the choriocapillaris and density of the RPE cell layer, both absent within the BDMs, do not vary between the BMD border and adjacent regions. The results suggest an association between BDMs and absolute scotomas, stretching of the adjacent retinal nerve fiver layer, and an axial elongation-associated stretching effect on BM as etiology of the BDMs.

Photoreceptor density in relation to axial length and retinal location in human eyes

The purpose of the study was to examine the density of retinal photoreceptors and retinal pigment epithelium (RPE) cells in relation to myopic axial elongation in human eyes. Using light microscopy, we assessed the density of photoreceptors and RPE cells at the ora serrata, equator, and midperiphery (equator/posterior pole midpoint), and the RPE cell density additionally at the posterior pole, in enucleated human globes. The study included 78 eyes (mean age: 59.2 ± 15.6years; range: 32-85years) with a mean axial length of 27.3 ± 3.6mm (range: 21.5-37.0mm). Close to the ora serrata, at the equator and midperiphery, photoreceptor and RPE cell density was 246 ± 183, 605 ± 299 and 1089 ± 441 photoreceptors/mm and 56.1 ± 13.7, 45.2 ± 15.1, and 48.8 ± 15.6 RPE cells/mm, respectively. Densities of both cell types in all three regions were positively correlated with each other (all P < 0.001) and decreased with longer axial length (all P < 0.001) and longer distance between the ora serrata and the posterior pole (all P < 0.001), most marked at the midperiphery and least marked close to the ora serrata. The PRE cell density at the posterior pole was not significantly (P = 0.35) related to axial length. The photoreceptor density at the ora serrata (beta:- 0.33) and equator (beta: - 0.27) and RPE cell density at the ora serrata (beta: - 0.27) decreased additionally with the presence of glaucoma. The findings suggest that the axial elongation-related decrease in photoreceptor and RPE cell density is most marked at the midperiphery, followed by the equator and finally the ora serrata region. It suggests that the axial elongation-related enlargement of the eye wall predominantly takes place in the retro-equatorial region, followed by the equatorial region.

Photoreceptor density in dependence of axial length and retinal location in human eyes

AbstractPurpose: To examine the density of retinal photoreceptors and retinal pigment epithelium (RPE) cells in relation to myopic axial elongation in human eyes.Methods: Using light microscopy, we counted the density of photoreceptors and RPE cells at the ora serrata, the equator, and at the midpoint between the equator and the posterior pole (MEPP) in enucleated human globes .Results: The study included 78 eyes (mean age: 59.2 ± 15.6 years; range: 32–85 years) with a mean axial length of 27.3 ± 3.6 mm (range: 21.5–37.0 mm). Close to the ora serrata, at the equator and at the MEPP, the photoreceptor and RPE cell density was 11.8 ± 8.8, 29.0 ± 14.3 and 52.3 ± 21.1 cells/48 μm and 27.0 ± 6.6, 21.7 ± 7.2 and 23.4 ± 7.5 cells/480 μm, respectively. Densities of both cell types in all regions measured were positively correlated with each other (all p &lt; 0.001), and decreased with longer axial length (all p &lt; 0.001) and retinal length (all p &lt; 0.001), most marked at the MEPP and least marked close to the ora serrata. Close to the ora serrata, at the equator and at the MEPP, differences between both globe sides in photoreceptor and RPE cell density were 3.98 ± 3.91, 8.93 ± 8.03 and 11.8 ± 11.1 cells/48 μm and 4.67 ± 4.37, 4.27 ± 3.13 and 5.30 ± 4.65 cells/480 μm, respectively. They were not significantly (all p &gt; 0.05) related with axial length and did not differ (all P &gt; 0.05) between both globe sides.Conclusions: The axial elongation‐associated decrease in photoreceptor and RPE cell density is most marked at the MEPP, followed by the equator and finally the ora serrata region. It suggests that the axial elongation‐associated enlargement of the eye wall predominantly takes place in the retro‐equatorial region, followed by the equatorial region. Side differences in cell densities suggest different elongation rates between both sides of the globe.

Myopic macular Bruch's membrane defects

AbstractPurpose: To examine peculiarities of macular Bruch's membrane defects (BMD) in axially elongated eyes.Methods: Using light microscopy, enucleated human globes were histomorphometrically.Results: The study included 247 eyes (axial length: 20.0–37.0 mm). BMDs were detected in 15 (6.1%) eyes (axial length: 30.6 ± 2.4 mm) (1.9 ± 0.9 BMD/eye with BMDs), located in the macular region in 10 eyes. Mean BMD size was 1.93 ± 1.62 mm (range: 0.22 mm–6.24 mm). Prevalence and size of BMDs were associated with longer axial length (OR: 1.52; p = 0.001) and higher prevalence of scleral staphyloma (OR: 16.3; p &lt; 0.001). BMD width was smaller than the defect width in the overlying retinal pigment epithelium (RPE) layer (p = 0.003) and larger than the length of a defect in the overlying inner nuclear layer (0.43 ± 0.76 mm; p = 0.008) and the width of an inner limiting membrane bridge (0.13 ± 0.33 mm; p = 0.001). Choriocapillaris thickness (8.8 ± 5.0 μm vs. 7.9 ± 3.2 μm; p = 0.94), BM thickness (3.1 ± 0.6 μm versus 3.0 ± 0.5 μm; p = 0.43) and RPE cell density (31.6 ± 8.4 cells/480 μm vs. 27.4 ± 8.8 cells/480 μm; p = 0.051) did not differ significantly between the region at the BDM margin and neighbouring regions. The sclera was thinner in the BDM region than in the neighbouring regions (0.28 ± 0.19 mm vs. 0.36 ± 0.13 mm; p = 0.006).Conclusions: BMDs as hallmark of myopic macular degeneration are characterized by a larger defect in the overlying RPE layer, smaller defects in the overlying retinal outer nuclear layer and inner nuclear layer, localized scleral thinning and an association with scleral staphylomas. Thickness of the choriocapillaris and density of the RPE cell layer, both absent within the BDMs, do not differ between the BMD margin and neighbouring regions. The results suggest an association between BDMs and absolute scotomas, stretching of the overlying retinal nerve fiver layer, an axial elongation‐associated stretching effect as aetiology of the BDMs, and a biomechanical role BM may play.

Histology of neovascular myopic macular degeneration

To assess the histological correlate of neovascular or exudative myopic macular degeneration (nMMD) in highly myopic human eyes, we examined histomorphometrically histologic sections of enucleated eyes of Caucasian patients. The study included 284 eyes (age: 61.9 ± 13.7 years; range: 24–89 years; axial length: 25.5 ± 3.1 mm; range: 20–37 mm). An nMMD was detected in 5 eyes (axial length: 29.6 ± 2.6 mm; range: 26.0–31.0 mm). All these eyes showed within or close to the nMMD a macular Bruch’s membrane (BM) defect, fibrous tissue with erythrocyte-filled blood vessels, and proliferations of irregularly pigmented and irregularly piled-up retinal pigment epithelium (RPE) cells each of which was connected with a curled-up, PAS (Periodic-Acid-Shiff)-positive membrane. The nMMD lesions were covered by proliferated RPE cells. RPE cells were not detected within the retina. In binary regression analysis, a higher nMMD prevalence was associated with a higher prevalence of macular BM defects (odds ratio: > 1000; P < 0.001), while the association with axial length was not significant (P = 0.43) in that model. After adjustment for the presence of macular BM defects, the nMMD prevalence was not associated with BM thickness (measured at the posterior pole, equator-posterior pole midpoint, equator and shortly posterior to the ora serrata) (P = 0.10; P = 0.87; P = 0.40; and P = 0.36, respectively), RPE cell layer thickness (P = 0.83; P = 0.79; P = 0.31; P = 0.38, resp.), RPE cell density (P = 0.56; P = 0.91; P = 0.47; P = 0.87, resp.), choriocapillaris thickness (P = 0.47; P = 0.93; P = 0.41; P = 0.75, resp.), and choriocapillaris density (P = 0.99; P = 0.94; P = 0.17; P = 0.97, resp.). The results suggest that nMMD is characterized by a fibrous pseudo-metaplasia of the RPE and is strongly associated with macular BM defects, without other detected histomorphometric differences in thickness or density of the RPE, BM, and choriocapillaris.

Choriocapillaris Flow Deficits in Normal Chinese Imaged by Swept-Source Optical Coherence Tomographic Angiography

To evaluate the ocular and systemic determinants of the choriocapillaris flow deficits percentage (CC FD%) in normal eyes. Observational cross-sectional study. Healthy Chinese participants without ocular or systemic diseases underwent detailed ophthalmic evaluations, including swept-source optical coherence tomography angiography (SS-OCTA) with 6-×6-mm macular choriocapillaris images. The CC FD% was assessed in circular regions with diameters of 1.0 and 5.0 mm, rings with 1.0- to 2.5-mm diameters, and rings with 2.5- to 5.0-mm diameters. The study included 830 individuals (mean ± SD age 58.66 ± 8.75 years). CC FD% (mean ± SD 22.05% ± 1.13%) was the lowest in the 2.5- to 5.0-mm ring, followed by the 1.0- to 2.5-mm ring, and highest in the 1.0-mm circle. In multivariable analysis, a higher CC FD% was associated with older age (β=0.16 [95% confidence interval {CI}] 0.08-0.24, P < .001), higher intraocular pressure (β=0.34 [95% CI 0.25-0.42], P < .001), higher serum concentrations of high-density lipoprotein cholesterol (β=0.24 [95% CI 0.17-0.32], P < .001), and lower image quality score (β=-0.22 [95% CI -0.30 to -0.14], P < .001). CC FD% was also independent of axial length. In healthy adult Chinese individuals, a higher CC FD% was associated with older age, higher intraocular pressure, and higher high-density lipoprotein cholesterol serum concentration. These factors may influence clinical assessments of the choriocapillaris. The lack of an association between CC FD% and axial length is consistent with similar findings for Bruch membrane thickness, macular retinal thickness, and macular retinal pigment epithelium cell density.

Retinal Pigment Epithelium Cell Density and Bruch\u2019s Membrane Thickness in Secondary versus Primary High Myopia and Emmetropia

To assess differences between secondary high myopia (SHM) due to congenital glaucoma and primary high myopia (PHM) and non-highly myopic eyes (NHM) in the relationships between axial length and Bruch’s membrane (BM) thickness and retinal pigment epithelium (RPE) density. The histomorphometric study included human globes enucleated for reasons such as malignant uveal melanoma, end-stage painful secondary angle-closure glaucoma and congenital glaucoma. BM thickness and RPE cell density were measured upon light microscopy. The investigation included 122 eyes (mean axial length: 26.7 ± 3.7 mm; range: 20.0–37.0 mm): 7 eyes with SHM (axial length: 33.7 ± 2.1 mm; range: 31.0–37.0 mm), 56 eyes with PHM (mean axial length: 29.1 ± 2.4 mm; range: 26.0–36.0 mm) and 59 eyes in the NHM-group (axial length: 23.5 ± 1.3 mm; range: 20.0–25.5 mm). In the SHM group, longer axial length was associated with lower RPE cell density at the posterior pole (standardized regression coefficient beta: 0.92; non- standardized regression coefficient B: −2.76; 95% confidence interval (CI): −4.41, −1.10;P = 0.01), at the midpoint posterior pole/equator (beta: −0.87; B: −3.60; 95% CI: −6.48, −0.73;P = 0.03), and at the equator (beta: −0.88; B: −0.95; 95% CI: −1.68, −0.23; P = 0.02), but not at the ora serrata (P = 0.88). In the PHM-group and NHM group, RPE cell density at the posterior pole (P = 0.08) and ora serrata (P = 0.88) was statistically independent of axial length, while at the midpoint posterior pole/equator (P = 0.01) and equator (P < 0.001), RPE cell density decreased with longer axis. BM thickness in the SHM group decreased with longer axial length at the posterior pole (beta: −0.93;B: −0.29; 95% CI: −0.39, −0.14; P = 0.003), midpoint posterior pole/equator (beta: −0.79; B: −0.22; 95% CI: −0.42, −0.02; P = 0.035) and equator (beta: −0.84; B: −0.21; 95% CI: −0.37, −0.06; P = 0.017), while in the PHM-group and NHM-group, BM thickness at any ocular region was not statistically significantly correlated with axial length (all P > 0.05). In the SHM-group, but not in the PHM-group or NHM-group (P = 0.98), lower BM thickness was associated with lower RPE cell density (beta: 0.93; B: 0.09; 95% CI: 0.04, 0.14; P = 0.007), while in the eyes without congenital glaucoma the relationship was not statistically significant. In SHM in contrast to PHM, BM thickness and RPE cell density decrease in a parallel manner with longer axial length. The findings fit with the notion of BM being a primary driver in the process of axial elongation in PHM as compared to SHM.

Myopia: Anatomic Changes and Consequences for Its Etiology.

The process of emmetropization is the adjustment of the length of the optical axis to the given optical properties of the cornea and lens after the end of the second year of life. Up to the end of the second year of life, the eye grows spherically. Axial elongation in the process of emmetropization after the second year of life is associated with a thinning of the retina and a reduced density of retinal pigment epithelium (RPE) cells in the equatorial and retroequatorial region, and a thinning of the choroid and sclera, starting at the equator and being most marked at the posterior pole. In contrast, retinal thickness and RPE density in the macular region and thickness of Bruch membrane (BM) in any region are independent of axial length. It led to the hypothesis that axial elongation occurs by the production of additional BM in the equatorial and retroequatorial region leading to a decreased RPE density and retinal thinning in that region and a more tube-like than spherical enlargement of the globe, without compromise in the density of the macular RPE cells and in macular retinal thickness. The increased disc-fovea distance in axially myopic eyes is caused by the development and enlargement of parapapillary, BM-free, gamma zone, whereas the length of macular BM, and indirectly macular RPE cell density, and macular retinal thickness, remain constant.

The Construction of Retinal Pigment Epithelium Sheets with Enhanced Characteristics and Cilium Assembly Using iPS Conditioned Medium and Small Incision Lenticule Extraction Derived Lenticules

In vitro generation of a functional retinal pigment epithelium (RPE) monolayer sheet is useful and promising for RPE cell therapy. Here, for the first time, we used induced pluripotent stem (iPS) supernatant as the conditioned medium (iPS-CM) and femtosecond laser intrastromal lenticule (FLI-lenticule) as a scaffold to construct an engineered RPE sheet. There are significant enhancements in RPE cell density, transepithelial electrical resistance (TER) and inhibitions of ultraviolet C (UVC)-irradiated apoptosis when RPE cells are cultured in iPS supernatant/Dulbecco’s modified Eagle’s medium (DMEM)-F12 of 1/2 (iPS-CM) compared with those in normal medium (NM, DMEM-F12). Using the assay of a panel of cytokines, combined with transcriptome and protein analyses, we discover that iPS-CM contains high levels of platelet-derived growth factor AA (PDGF-AA), insulin-like growth factor binding protein (IGFBP)-2, transforming growth factor (TGF)-α and IGFBP-6, which are responsible for the upregulation of gene and protein markers with RPE phenotypes and downregulation of gene and protein markers with epithelial-mesenchymal transition (EMT) phenotypes for RPE cells in iPS-CM when compared to those in NM. Moreover, compared to cultures on tissue culture plates (TCP), RPE cells on FLI-lenticule display more microvilli and cilium in accordance with the results in terms of RNA-Seq data, quantitative polymerase chain reaction (qPCR) expression, immunofluorescence staining, and western blot assays. Furthermore, acellular FLI-lenticule exhibits biocompatibility after rabbit subretinal implantation by 30 days through electroretinography and histological examination. Thus, we determined that engineered RPE sheets treated by iPS-CM in conjunction with FLI-lenticule scaffold aid in enhanced RPE characteristics and cilium assembly. Such a strategy to construct RPE sheets is a promising avenue for developing RPE cell therapy, disease models and drug screening tools. Statement of SignificanceIn vitro generation of a functional RPE monolayer sheet is useful and promising for RPE cell therapy. Here, we constructed engineered RPE sheets treated by iPS-CM in conjunction with FLI-lenticule scaffolds to help in enhanced RPE characteristics and cilium assembly. Such a strategy to generate RPE sheets is a promising avenue for developing RPE cell therapy, disease models and drug screening tools.

Bruch\u2019s Membrane Thickness and Retinal Pigment Epithelium Cell Density in Experimental Axial Elongation

To assess anatomical changes in eyes with progressive myopia, we morphometrically examined the eyes of guinea pigs with lens-induced axial elongation. Starting at an age of 3–4 weeks, guinea pigs in the experimental group (n = 20 animals) developed unilateral lens-induced axial elongation by wearing goggles for 5 weeks compared to a control group of 20 animals without intervention (axial length:8.91 ± 0.08 mm versus 8.74 ± 0.07 mm; P < 0.001). Five weeks after baseline, the animals were sacrificed, and the eyes enucleated. As measured histomorphometrically, Bruch’s membrane thickness was not significantly correlated with axial length in either group at the ora serrata (P = 0.41), equator (P = 0.41), midpoint between equator and posterior pole (MBEPP) (P = 0.13) or posterior pole (P = 0.89). Retinal pigment epithelium (RPE) cell density decreased with longer axial length at the MBEPP (P = 0.04; regression coefficient beta = −0.33) and posterior pole (P = 0.01; beta = −0.40). Additionally, the thickness of the retina and sclera decreased with longer axial length at the MBEPP (P = 0.01; beta = −0.42 and P < 0.001; beta = −0.64, respectively) and posterior pole (P < 0.001; beta = −0.51 and P < 0.001; beta = −0.45, respectively). Choroidal thickness decreased at the posterior pole (P < 0.001; beta = −0.51). Experimental axial elongation was associated with a thinning of the retina, choroid and sclera and a decrease in RPE cell density, most markedly at the posterior pole. Bruch’s membrane thickness was not related to axial elongation.

In situ regeneration of retinal pigment epithelium by gene transfer of E2F2: a potential strategy for treatment of macular degenerations.

The retinal pigment epithelium (RPE) interacts closely with photoreceptors to maintain visual function. In degenerative diseases such as Stargardt disease and age-related macular degeneration, the leading cause of blindness in the developed world, RPE cell loss is followed by photoreceptor cell death. RPE cells can proliferate under certain conditions, suggesting an intrinsic regenerative potential, but so far this has not been utilised therapeutically. Here, we used E2F2 to induce RPE cell replication and thereby regeneration. In both young and old (2 and 18 month) wildtype mice, subretinal injection of non-integrating lentiviral vector expressing E2F2 resulted in 47% of examined RPE cells becoming BrdU positive. E2F2 induced an increase in RPE cell density of 17% compared with control vector-treated and 14% compared with untreated eyes. We also tested this approach in an inducible transgenic mouse model of RPE loss, generated through activation of diphtheria toxin-A gene. E2F2 expression resulted in a 10-fold increase in BrdU uptake and a 34% increase in central RPE cell density. Although in mice this localised rescue is insufficiently large to be demonstrable by electroretinography, a measure of massed retinal function, these results provide proof-of-concept for a strategy to induce in situ regeneration of RPE for the treatment of RPE degeneration.

Bruch´s membrane thickness in relationship to axial length.

PurposeTo assess a potential role of Bruch´s membrane (BM) in the biomechanics of the eye, we measured its thickness and the density of retinal pigment epithelium (RPE) cells in various ocular regions in eyes of varying axial length.MethodsHuman globes, enucleated because of an ocular tumor or end-stage glaucoma were prepared for histological examination. Using light microscopy, the histological slides were histomorphometrically examined applying a digitized image analysis system.ResultsThe study included 104 eyes with a mean axial length of 27.9±3.2 mm (range:22.6mm-36.5mm). In eyes without congenital glaucoma, BM was significantly thickest (P<0.001) at the ora serrata, followed by the posterior pole, the midpoint between equator and posterior pole (MBEPP), and finally the equator. BM thickness was not significantly correlated with axial length (ora serrata: P = 0.93; equator:P = 0.31; MBEPP:P = 0.15; posterior pole:P = 0.35). RPE cell density in the pre-equatorial region (P = 0.02; regression coefficient r = -0.24) and in the retro-equatorial region (P = 0.03; r = -0.22) decreased with longer axial length, while RPE cell density at the ora serrata (P = 0.35), the MBEPP (P = 0.06; r = -0.19) and the posterior pole (P = 0.38) was not significantly correlated with axial length. Highly myopic eyes with congenital glaucoma showed a tendency towards lower BM thickness and lower RPE cell density at all locations.ConclusionsBM thickness, in contrast to scleral and choroidal thickness, was independent of axial length in eyes without congenital glaucoma. In association with an axial elongation associated decrease in the RPE cell density in the midperiphery, the findings support the notion of a biomechanical role BM may play in the process of emmetropization/myopization.

Gene transfer of E2F2 induces in situ regeneration of retinal pigment epithelium

SummaryThe retinal pigment epithelium (RPE) interacts closely with photoreceptors and is important for maintaining visual function. In degenerative diseases such as age‐related macular degeneration (AMD), the leading cause of blindness in the developed world, RPE cell loss is followed by photoreceptor cell death. RPE cells can proliferate under certain conditions, suggesting an intrinsic regenerative potential, but so far this has not been utilised therapeutically. Here we use E2F2, a potent transcriptional regulator of cell proliferation, to induce RPE regeneration in vitro and in vivo. Gene transfer of E2F2 induced upregulation of Ki67 and uptake of BrdU in growth arrested ARP19 cells in vitro. In both, young and old C57Bl/6 mice, subretinal lentiviral delivery of E2F2 to the RPE caused a 40‐fold ±27.2 increase in E2F2 positive RPE cells that correlated with a 10‐fold ±4.7 increase in BrdU positive cells and a mean increase of RPE cell density. E2F2 also induced BrdU uptake and increased cell density in the central RPE of RPECreER/DTA mice, where pathology, induced by the activation of diphtheria toxin‐A, was strongest. These results provide proof of concept for a strategy to treat progressive RPE cell loss by in situ regeneration.

Retinal pigment epithelium cell density in relationship to axial length in human eyes.

To assess associations between axial length and density of retinal pigment epithelium (RPE) cells in various ocular regions. The histomorphometric investigation included histological sections of enucleated eyes of Caucasian patients. Using a light microscope, we counted the number of RPE cells on Bruch's membrane at the ora serrata, in the pre-equatorial region, the equatorial and retro-equatorial region, at the midpoint equator/posterior pole, and at the posterior pole. The study included 65 globes with a mean axial length 25.9±3.5mm (range: 21.0-34.0mm). Retinal pigment epithelium (RPE) cell count in the equatorial to retro-equatorial region (p<0.001; correlation coefficient r2 : 0.44), in the pre-equatorial region (p<0.001; r2 : 0.39) and at the midpoint equator/posterior pole (p=0.03; r2 : 0.12) decreased with increasing axial length. Retinal pigment epithelium (RPE) cell count at the ora serrata (p=0.49) and posterior pole (p=0.44) was not significantly correlated with axial length. As a corollary, mean RPE cell density was higher (p<0.001) at the posterior pole than at the midpoint equator/posterior pole or at the ora serrata region, where it was higher than in pre-equatorial region (p<0.001) and in the equatorial to retro-equatorial region (p<0.001). The decrease in the RPE cell density mainly in the equatorial to retro-equatorial region in association with longer axial length suggests a region of enlargement of Bruch's membrane in the equatorial to retro-equatorial area in association with axial elongation. The finding may be of interest to elucidate the process of emmetropization/myopization.

Polyethylene glycol induced mouse model of retinal degeneration

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness. This study was done to characterize dry AMD-like changes in mouse retinal pigment epithelium (RPE) and retina after polyethylene glycol (PEG) treatment. We injected male C57BL/6 mice subretinally with PBS, 0.025, 0.25, 0.5 and 1.0 mg of PEG-400 and the animals were sacrificed on day 5. Eyes were harvested and processed for histological analysis. In all other experiments 0.5 mg PEG was injected and animals were sacrificed on days 1, 3, 5 or 14. Paraffin, 5 μm and plastic, 1 μm and 80 nm sections were used for further analysis. Subretinal injection of 0.5 mg PEG induced a 32% reduction of outer nuclear layer (ONL) thickness, 61% decrease of photoreceptor outer and inner segment length, 49% decrease of nuclear density in the ONL and 31% increase of RPE cell density by day 5 after injection. The maximum level of TUNEL positive nuclei in the ONL (6.8 + 1.99%) was detected at day 5 after PEG injection and co-localized with Casp3act. Histological signs of apoptosis were observed in the ONL by light or electron microscopy. Degeneration of RPE cells was found in PEG injected eyes. Gene expression data identified several genes reported to be involved in human AMD. C3, Cfi, Serping1, Mmp9, Htra1 and Lpl were up-regulated in PEG injected eyes compared to PBS controls. PEG leads to morphological and gene expression changes in RPE and retina consistent with dry AMD. This model will be useful to investigate dry AMD pathogenesis and treatment.

Quantitative autofluorescence and cell density maps of the human retinal pigment epithelium.

Lipofuscin (LF) accumulation within RPE cells is considered pathogenic in AMD. To test whether LF contributes to RPE cell loss in aging and to provide a cellular basis for fundus autofluorescence (AF) we created maps of human RPE cell number and histologic AF. Retinal pigment epithelium-Bruch's membrane flat mounts were prepared from 20 donor eyes (10 ≤ 51 and 10 > 80 years; postmortem: ≤4.2 hours; no retinal pathologies), preserving foveal position. Phalloidin-binding RPE cytoskeleton and LF-AF (488-nm excitation) were imaged at up to 90 predefined positions. Maps were assembled from 83,330 cells in 1470 locations. From Voronoi regions representing each cell, the number of neighbors, cell area, and total AF intensity normalized to an AF standard was determined. Highly variable between individuals, RPE-AF increases significantly with age. A perifoveal ring of high AF mirrors rod photoreceptor topography and fundus-AF. Retinal pigment epithelium cell density peaks at the fovea, independent of age, yet no net RPE cell loss is detectable. The RPE monolayer undergoes considerable lifelong re-modeling. The relationship of cell size and AF, a surrogate for LF concentration, is orderly and linear in both groups. Autofluorescence topography differs distinctly from the topography of age-related rod loss. Digital maps of quantitative AF, cell density, and packing geometry provide metrics for cellular-resolution clinical imaging and model systems. The uncoupling of RPE LF content, cell number, and photoreceptor topography in aging challenges LF's role in AMD.

Functional and morphological analysis of the subretinal injection of retinal pigment epithelium cells

Replacement of retinal pigment epithelium (RPE) cells by transplantation is a potential treatment for some retinal degenerations. Here, we used a combination of invasive and noninvasive methods to characterize the structural and functional consequences of subretinal injection of RPE cells. Pigmented cells from primary cultures were injected into albino mice. Recovery was monitored over 8 weeks by fundus imaging, spectral domain optical coherence tomography (sdOCT), histology, and electroretinography (ERG). sdOCT showed that retinal reattachment was nearly complete by 1 week. ERG response amplitudes were reduced after injection, with cone-mediated function then recovering better than rod function. Photoreceptor cell loss was evident by sdOCT and histology, near the site of injection, and is likely to have been the main cause of incomplete recovery. With microscopy, injected cells were identified by the presence of apical melanosomes. They either established contact with Bruch's membrane, and thus became part of the RPE monolayer, or were located on the apical surface of the host's cells, resulting in apposition of the basal surface of the injected cell with the apical surface of the host cell and the formation of a series of desmosomal junctions. RPE cell density was not increased, indicating that the incorporation of an injected cell into the RPE monolayer was concomitant with the loss of a host cell. The transplanted and remaining host cells contained large vacuoles of ingested debris as well as lipofuscin-like granules, suggesting that they had scavenged the excess injected and host cells, and were stressed by the high digestive load. Therefore, although significant functional and structural recovery was observed, the consequences of this digestive stress may be a concern for longer-term health, especially where RPE cell transplantation is used to treat diseases that include lipofuscin accumulation as part of their pathology.