Morphology Of Retinal Pigment Epithelium Cells 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.

An in vivo model of focal light emitting diode-induced cone photoreceptor phototoxicity in adult pigmented mice: Protection with bFGF

PurposeTo develop a model of focal injury by blue light-emitting diode (LED)-induced phototoxicity (LIP) in pigmented mouse retinas and to study the effects on cone, Iba-1+ cells and retinal pigment epithelium (RPE) cell populations after administration of basic fibroblast growth factor (bFGF) and minocycline, alone or combined. MethodsIn anesthetized dark-adapted adult female pigmented C57BL/6 mice, left pupils were dilated and the eye exposed to LIP (500 lux, 45 s). The retina was monitored longitudinally in vivo with SD-OCT for 7 days (d). Ex vivo, the effects of LIP and its protection with bFGF (0.5 μg) administered alone or combined with minocycline (45 mg/kg) were studied in immunolabeled arrestin-cone outer segments (a+OS) and quantified within a predetermined fixed-size circular area (PCA) centered on the lesion in flattened retinas at 1, 3, 5 or 7d. Moreover, Iba-1+ cells and RPE cell morphology were analysed with Iba-1 and ZO-1 antibodies, respectively. ResultsLIP caused a focal lesion within the superior-temporal retina with retinal thinning, particularly the outer retinal layers (116.5 ± 2.9 μm to 36.8 ± 6.3 μm at 7d), and with progressive diminution of a+OS within the PCA reaching minimum values at 7d (6218 ± 342 to 3966 ± 311). Administration of bFGF alone (4519 ± 320) or in combination with minocycline (4882 ± 446) had a significant effect on a+OS survival at 7d and Iba-1+ cell activation was attenuated in the groups treated with minocycline. In parallel, the RPE cell integrity was progressively altered after LIP and administration of neuroprotective components had no restorative effect at 7d. ConclusionsLIP resulted in progressive outer retinal damage affecting the OS cone population and RPE. Administration of bFGF increased a+OS survival but did not prevent RPE deterioration.

Chronic exposure to tumor necrosis factor alpha induces retinal pigment epithelium cell dedifferentiation

BackgroundThe retinal pigment epithelium (RPE) is a monolayer of pigmented cells with important barrier and immuno-suppressive functions in the eye. We have previously shown that acute stimulation of RPE cells by tumor necrosis factor alpha (TNFα) downregulates the expression of OTX2 (Orthodenticle homeobox 2) and dependent RPE genes. We here investigated the long-term effects of TNFα on RPE cell morphology and key functions in vitro.MethodsPrimary porcine RPE cells were exposed to TNFα (at 0.8, 4, or 20 ng/ml per day) for 10 days. RPE cell morphology, phagocytosis, barrier- and immunosuppressive-functions were assessed.ResultsChronic (10 days) exposure of primary RPE cells to TNFα increases RPE cell size and polynucleation, decreases visual cycle gene expression, impedes RPE tight-junction organization and transepithelial resistance, and decreases the immunosuppressive capacities of the RPE. TNFα-induced morphological- and transepithelial-resistance changes were prevented by concomitant Transforming Growth Factor β inhibition.ConclusionsOur results indicate that chronic TNFα-exposure is sufficient to alter RPE morphology and impede cardinal features that define the differentiated state of RPE cells with striking similarities to the alterations that are observed with age in neurodegenerative diseases such as age-related macular degeneration.

Chronic exposure to TNFα impairs RPE barrier and immunosuppressive functions

PurposeThe retinal pigment epithelium (RPE) is a monolayer of pigmented cells with important roles in the outer blood‐retinal barrier and subretinal immune suppression. Failure of RPE functions and inflammation have both been hypothesized to play a role in the pathophysiology of age related macular degeneration (AMD). We here investigated the long‐term effects of TNFα on RPE morphology and function in vitro.MethodsPrimary porcine RPE cells were cultivated until confluence, then recombinant TNFα was added daily in the culture medium (at 0.8, 4, 20 or 100 ng/ml=C1,C2,C3 and C4) for 10 days. RPE cell morphology and gene expression, barrier, phagocytosis and immunosuppressive functions were assessed.ResultsCell morphology and gene expression:TNFα (i) decreased cell number (3653.6,3428,3227,2791 and 2020 cells/mm2 respectively for control,C1,C2,C3 and C4, all p < 0.01); (ii) increased cell size (+5.3,+12.6,+13.9 and +9.5% of control for C1,C2,C3 and C4, all p < 0.05); (iii) increased the % of multinucleated cells (5.7,7.7,9.4,9.9 and 15.9% of multinucleated cells for control,C1,C2,C3 and C4, all p < 0.05); (iv) and decreased OTX2 (a major RPE gene) expression (‐11.1, ‐19.7, ‐52 and ‐82.9% of control for C1,C2,C3 and C4, all p < 0.05). Barrier function: Stimulation by TNFα (i) disturbed Zonula Occludens 1 cellular distribution and actin F distribution and (ii) significantly decreased RPE transepithelial resistance in a dose‐dependent manner (‐70, ‐88.5 and ‐90.8% of control for C2,C3 and C4, p < 0.05). Immunossupressive function: 10 day pre‐stimulation with ‐TNFα significantly decreased RPE capacity to induce monocyte death after 24 h of co‐culture (p < 0.05).ConclusionsChronic exposure to TNFα deteriorates major RPE functions that are essential to visual function and might play a key role in the pathophysiology of AMD.

Fast subcellular optical coherence photoacoustic microscopy for pigment cell imaging.

We developed a fast ultrahigh resolution optical coherence photoacoustic microscopy (FU-OCPAM) system by combining two complementary imaging modes of optical coherence microscopy (OCM) and photoacoustic microscopy (PAM) for cellular/subcellular imaging. The system used optical scanning to realize fast imaging speed and provided ultrahigh resolution of 1.24 and 0.59 μm for OCM and PAM, respectively. We imaged the retinal pigment epithelium (RPE) to demonstrate the subcellular imaging capability of the FU-OCPAM system. The OCM and PAM images clearly showed the RPE cell morphology and reflected the complementary optical properties of scattering and absorption. A quantitative analysis of the RPE cells was made based on photoacoustic (PA) signals. The cell area mainly ranged from 80 to 300 μm2, and had a linear relationship with the sum intensity of PA signals which mainly reflected the melanin content of the cells. The morphology and the PA signal could be used to identify qualitatively and quantitatively the aging and healthy states of the RPE cells. The results show the potential applications in studying the real-time cellular response to external stimulations and the progress of aging and diseases at the cellular level with FU-OCPAM.

Eliminating residual iPS cells for safety in clinical application.

Eliminating residual iPS cells for safety in clinical application.

A modified histoimmunochemistry-assisted method for in situ RPE evaluation

We have developed a method for morphological evaluation of retinal pigment epithelium (RPE) with minimal perturbation of RPE-Bruch's membrane-choriocapillaris complex (RBCC). To prepare RBCC, the anterior segments of the eye were removed. The remaining eye cup was radially cut into 4 to 6 pieces from periphery to the optic nerve head. Each piece was carefully dissected into 3 parts: sclera, RBCC and neurosensory retina. RBCC flatmount with RPE monolayer facing up was formed by several relaxing radial cuts. After immuno-staining with tissue specific markers, the RBCC could be distinguished as the superficial RPE monolayer and the underneath choriocapillaris layers by fluorescence microscopy. The density, distribution, and morphology of RPE cells varied among species. This method may have brought several advantages for RPE screening over other means, because of its straight forward approach, minimal manipulation of samples, plus there is no requirement for bleaching, which result in high efficiency for result readout. For a complete morphological study of RPE in situ, this method may be combined with other methods, such as cryosections, scanning electron microscopy, etc.

A modified histoimmunochemistry-assisted method for in situ RPE evaluation

We have developed a method for morphological evaluation of retinal pigment epithelium (RPE) with minimal perturbation of RPE-Bruch’s membranechoriocapillaris complex (RBCC). To prepare RBCC, the anterior segments of the eye were removed. The remaining eye cup was radially cut into 4 to 6 pieces from periphery to the optic nerve head. Each piece was carefully dissected into 3 parts: sclera, RBCC and neurosensory retina. RBCC flatmount with RPE monolayer facing up was formed by several relaxing radial cuts. After immuno-staining with tissue specific markers, the RBCC could be distinguished as the superficial RPE monolayer and the underneath choriocapillaris layers by fluorescence microscopy. The density, distribution, and morphology of RPE cells varied among species. This method may have brought several advantages for RPE screening over other means, because of its straight forward approach, minimal manipulation of samples, plus there is no requirement for bleaching, which result in high efficiency for result readout. For a complete morphological study of RPE in situ, this method may be combined with other methods, such as cryosections, scanning electron microscopy, etc.

Drusen are associated with local and distant disruptions to human retinal pigment epithelium cells

Drusen are extra-cellular deposits that form between the retinal pigment epithelium (RPE) and Bruch's membrane (BM). Numerous and/or confluent drusen are a significant risk factor for age-related macular degeneration (AMD). Here, using whole mounted human RPE preparation we show that RPE cell morphology changes in association with drusen. These changes included an increase in cell size and distortion in the regularity of their distribution. Further, although binucleation is relatively rare in human RPE, there was a marked increase in the number of binucleated RPE cell associated with individual druse. Surprisingly many of these changes were found at distances up to 400μm from drusen.

Two-Photon–Excited Fluorescence Imaging of Human RPE Cells with a Femtosecond Ti:Sapphire Laser

To record the distribution and spectrum of human retinal pigment epithelial cell lipofuscin (LF) by two-photon-excited fluorescence (TPEF) and confocal laser scanning microscopy. Ex vivo TPEF imaging of the human retinal pigment epithelium (RPE) of human donor eyes was conducted with a multiphoton laser scanning microscope that employs a femtosecond Ti:sapphire laser as an excitation laser source. The spectrum of autofluorescence of LF granules was analyzed with a confocal laser scanning microscope coupled to a UV argon laser. TPEF examination allowed for imaging of RPE cell morphology and intracellular distribution of LF granules with high-contrast and submicrometer resolution. Variations in cell size and shape as well as in autofluorescence spectra of individual LF granules were recorded. The typical diameter of LF granules was found to be below 1 mum, with some RPE cells possessing larger granules. Remarkably, enhanced blue-green autofluorescence was observed from these larger LF granules. TPEF imaging represents a novel tool for the investigation of morphologic and spectral characteristics of human RPE cells. Spectral variations of individual LF granules may indicate differences in the complex molecular composition. Compared to conventional single-photon excited autofluorescence, TPEF with a tunable laser source allows for reduced photo damage and deeper sensing depth. It may help to elucidate further the pathophysiological role of LF accumulation as a common downstream pathogenetic pathway in retinal diseases. With the proof of principle from this ex vivo study, further work is now planned to evaluate the safety of TPEF RPE imaging in RPE cultures and animal models.

Characterization of retinal pigment epithelial cells cultured on microporous filters.

Retinal pigment epithelium (RPE) cultured on microporous filter supports was compared to RPE cultured on plastic and evaluated for features characteristic of RPE in vivo. RPE cells grown on filters were cuboidal, formed junctional complex structures between cells, and had elaborate microvilli and basal infoldings similar to RPE in vivo, while RPE grown on plastic also formed intercellular junctions but appeared squamous and had few microvilli and basal infoldings. RPE grown on filters or plastic secreted an extracellular matrix at the basal surface and ingested isolated rat rod outer segments at the apical surface. RPE grown on filters coated with laminin or fibronectin became confluent more rapidly than RPE grown on uncoated filters, while RPE grown at the same density on filters coated with collagen type I did not become confluent. The laminin and fibronectin coatings did not alter the RPE cell morphology; however, cells seeded on collagen-coated filters grew in large disorganized clusters. RPE grown on laminin-coated filters formed functional tight junctions as evidenced by the capacity of RPE monolayers to prevent the bulk flow of medium and the passage of trypan blue across the filter. Radiolabeled sucrose and inulin were used to measure the paracellular flux through the tight junctions between cells. The passage of these tracers was linear over time, with the lower molecular weight tracer, sucrose, passing through the monolayer more readily than inulin. Values for the flux of radiolabeled bovine serum albumin across RPE monolayers fell between values for sucrose and inulin. The results from these studies show that RPE monolayers cultured on laminin-coated filters maintain a morphology similar to that of RPE in vivo, are capable of ingesting rod outer segments, and form a selectively permeable barrier to various tracers. This culture system should be useful for studies of transepithelial transport, secretion, endocytosis and exocytosis that require independent control of the extracellular environment at the apical and basolateral cell surfaces.