Rabbit Retinal Pigment Epithelium Research Articles

Ex vivo cultivated retinal pigment epithelial cell transplantation for the treatment of rabbit corneal endothelial dysfunction

ObjectiveStem cell therapy is a promising strategy for the treatment of corneal endothelial dysfunction, and the need to find functional alternative seed cells of corneal endothelial cells (CECs) is urgent. Here, we determined the feasibility of using the retinal pigment epithelium (RPE) as an equivalent substitute for the treatment of corneal endothelial dysfunction.MethodsRPE cells and CECs in situ were obtained from healthy New Zealand male rabbits, and the similarities and differences between them were analyzed by electron microscopy, immunofluorescent staining, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Rabbit primary RPE cells and CECs were isolated and cultivated ex vivo, and Na+/K+-ATPase activity and cellular permeability were detected at passage 2. The injection of cultivated rabbit primary RPE cells, CECs and human embryonic stem cell (hESC)-derived RPE cells was performed on rabbits with corneal endothelial dysfunction. Then, the therapeutic effects were evaluated by corneal transparency, central corneal thickness, enzyme linked immunosorbent assay (ELISA), qRT-PCR and immunofluorescent staining.ResultsThe rabbit RPE cells were similar in form to CECs in situ and ex vivo, showing a larger regular hexagonal shape and a lower cell density, with numerous tightly formed cell junctions and hemidesmosomes. Moreover, RPE cells presented a stronger barrier and ionic pumping capacity than CECs. When intracamerally injected into the rabbits, the transplanted primary RPE cells could dissolve corneal edema and decrease corneal thickness, with effects similar to those of CECs. In addition, the transplantation of hESC-derived RPE cells exhibited a similar therapeutic effect and restored corneal transparency and thickness within seven days. qRT-PCR results showed that the expressions of CEC markers, like CD200 and S100A4, increased, and the RPE markers OTX2, BEST1 and MITF significantly decreased in the transplanted RPE cells. Furthermore, we have demonstrated that rabbits transplanted with hESC-derived RPE cells maintained normal corneal thickness and exhibited slight pigmentation in the central cornea one month after surgery. Immunostaining results showed that the HuNu-positive transplanted cells survived and expressed ZO1, ATP1A1 and MITF.ConclusionRPE cells and CECs showed high structural and functional similarities in barrier and pump characteristics. Intracameral injection of primary RPE cells and hESC-derived RPE cells can effectively restore rabbit corneal clarity and thickness and maintain normal corneal function. This study is the first to report the effectiveness of RPE cells for corneal endothelial dysfunction, suggesting the feasibility of hESC-derived RPE cells as an equivalent substitute for CECs.

The development of transplantation technique of 3D spheroids retinal pigment epithelium in the experiment on animals

Aim. This research is aimed to devise the technique for transplantation of 3D spheroids retinal pigment epithelium (RPE) in the experimental animal’s eyes (rabbits). Materials and methods . 3D spheroids of RPE for subsequent transplantation were created using agarose tablets (3D Petri Dishes, Microtissue, USA). The phenotype of the obtained cell cultures was studied by immunocytochemical tests (laser scanning confocal microscope “Fluo View FV10i”, Olympus, Japan). Vitrectomy - 2500 cuts per minute, vacuum 600 mm Hg (Alcon, Accurus, USA) was performed on all rabbits (n = 10). Then, we made retinotomy and injected spheroids in subretinal space (MicroDose injection kit 1 ml, Med One, USA). The following methods of control: ultrasound B-scan (Ultrasonic UD-6000, Tomey, Japan) and optical coherence tomography (OCT), (Askin Spectralis, Heidelberg engineering, Germany). Eyes were enucleated for histological examination on 7, 10, 14 and 20 days. Results. Immunocytochemical tests revealed preservation of the RPE epithelial phenotype in 3D spheroids. Clinical map was similar in all experimental animals - during the first 7 days after surgery we saw cystic edema and flat retinal detachment in the surgery area. As we observed, the retina was adjoining and retinal edema was decreasing. Also, on day 3, 7 and 10 on OCT we saw subretinal round conglomerates with a diameter of 60 to 80 µm - presumably RPE 3D spheroids. According to histological findings, there was observed adhesion of the RPE spheroids to the choroid with subsequent spreading and formation of new cell layer with the increase of observation periods. Conclusion. The proposed technology of cultivation of rabbit RPE with subsequent construction of 3D spheroids allows to preserve the epithelial phenotype of cells. The developed surgical technique of RPE transplantation is acceptable and can be used for further experimental studies to be implemented in clinical practice.

Первый опыт трансплантации 3D-сфероидов ретинального пигментного эпителия в эксперименте

Introduction . Available methods in the treatment of age-related macular degeneration (AMD) do not always lead to significant vision improvement. A new advanced method of AMD treatment is transplantation of retinal pigment epithelium (RPE) in the form of cell suspension or choroidal pigment complex. In our opinion, the most modern form of RPE transplant is a multicellular spheroid - the form of 3D cell culture in which cells are close to the conditions of native tissue. However, transplantation of 3D spheroids of RPE requires preclinical studies. Purpose . This research is aimed to devise the technique for transplantation of RPE 3D spheroids in the eyes of experimental animals (rabbits). Material and methods. 1. In vitro research phase. For immunocytochemical tests the 3D spheroids were explored on the 3rd, 7th, and 11th day of steroidogenesis (using the laser scanning confocal microscope «Fluo View FV10i», Olympus, Japan). The expression of epithelial markers (Alexa Fluor, Great Britain), such as: RPE65, ZO-1, Cytokeratin 8, 18, and Vimentin (the mesenchymal marker) was analyzed. 2. In vivo research phase. Vitrectomy (2500 cuts per minute, vacuum 600 mmHg), (Alcon, Accurus, USA) was performed on all rabbits (n=10). Then, a sharp cannula 39G was used to make a retinotomy above the central zone of retina, and spheroids (n=81) were injected (MicroDose injection kit 1 ml, Med One, USA) in subretinal space. The operation ended with the replacement of fluid into air and suturing scleral incision and the conjunctiva. The following methods of control were used: ultrasound B-scan (Ultrasonic UD-6000, Tomey, Japan) and optical coherence tomography (OCT) - (Askin Spectralis, Heidelberg engineering, Germany). Animals were taken out of the experiment on days 7, 10, 14 and 20 by air embolism. The eyeballs were enucleated for a subsequent histological examination. Results. 1. In vitro research phase. During immunocytochemical tests on the obtained 3D cultures, the presence of high expression of specific marker of retinal pigment epithelium RPE-65, also epithelial markers Cytokeratin 8, 18 and ZO-1 was noted. The expression of mesenchymal marker Vimentin was weak - that indicates the advantage of 3D cultivation of RPE cells to keep their phenotype. This indicates the advantage of 3D cultivation of epithelial cells to preserve their epithelial phenotype. 2. In vivo research phase. On day 1 during ultrasonic B-scanning in 6 rabbits there was observed a flat retinal detachment in the area of surgical intervention height up to 1 mm; in 4 rabbits there was detected adhesion of the membranes, detachment of retina was not visible. The picture of the morphological state during retinal OCT was similar in all experimental animals - during the first 7 days after surgery cystic edema was noted and also a flat retinal detachment in the surgery area. As we observed, the retina was attaching and retinal edema was decreasing. Also, on day 3, 7 and 10 we revealed subretinal round conglomerates with a diameter of 60 to 80 pm - presumably RPE 3D spheroids. No morphological changes of the retina were seen on day 14 and day 20. According to histological findings, there was found adhesion of the RPE spheroids to the choroid with subsequent spreading and formation of a new cell layer with an increase of follow-up periods. Conclusion. 1. The proposed technology of cultivation of rabbit RPE with subsequent construction of 3D spheroids allows to preserve the epithelial phenotype of cells, that is confirmed by immunocytochemical tests. 2. The developed surgical technique of RPE transplantation is acceptable, that is confirmed by the OCT and histological investigation. 3. The proposed surgical technique of subretinal transplantation of 3D spheroids of RPE is promising for further experimental studies to be implemented in clinical practice.

Hepatocyte growth factor promotes the proliferation of human embryonic stem cell derived retinal pigment epithelial cells.

Research that pertains to the molecular mechanisms involved in retinal pigment epithelial (RPE) development can significantly contribute to cell therapy studies. The effects of periocular mesenchymal cells on the expansion of RPE cells remain elusive. We have examined the possible proliferative role of hepatocyte growth factor (HGF) as a mesenchymal cell secretory factor against human embryonic stem cell derived RPE (hESC-RPE). We found that the conditioned medium of human mesenchymal stem cells from apical papilla and/or exogenous HGF promoted proliferation of the hESC-RPE cells as single cells and cell sheets, in addition to rabbit RPE sheets in vitro. Blockage of HGF signaling by HGF receptor inhibitor, PHA-665752, inhibited proliferation of hESC-RPE cells. However, differentiation of hESCs and human-induced pluripotent stem cells to a rostral fate and eye-field specification was unaffected by HGF. Our in vivo analysis showed HGF expression in periocular mesenchymal cells after optic cup formation in chicken embryos. Administration of HGF receptor inhibitor at this developmental stage in chicken embryos led to reduced eye size and disorganization of the RPE sheet. These findings suggested that HGF administration could be beneficial for obtaining higher numbers of hESC-RPE cells in human preclinical and clinical trials.

Inhibitory Effects of Plumbagin on Retinal Pigment Epithelial Cell Epithelial-Mesenchymal Transition In Vitro and In Vivo.

BackgroundThis study aimed to explore the effects of plumbagin (PLB) on epithelial-to-mesenchymal transition in retinal pigment epithelial (RPE) cells and in proliferative vitreoretinopathy (PVR) rabbit models.Material/MethodsRabbit RPE cells were exposed to various concentrations (0, 5, 15, and 25 μM) of PLB. Motility, migration, and invasion of PLB-treated cells were determined in vitro using Transwell chamber assays and scratch wound assays. The contractile ability was evaluated by cell contraction assay. Expression of matrix metalloproteinases (MMPs) and epithelial-mesenchymal transition (EMT) markers were assessed by western blotting. Furthermore, PLB was injected in rabbit eyes along with RPE cells after gas compression of the vitreous. The presence of PVR was determined by indirect ophthalmoscopy on days 1, 7, 14, and 21 after injection. Also, optical coherence tomography (OCT), ultrasound images, electroretinograms (ERG), and histopathology were used to assess efficacy and toxicity.ResultsPLB significantly inhibited the migration and invasion of RPE cells. The agent also markedly reduced cell contractive ability. Furthermore, PLB treatment resulted in the decreased expression of MMP-1, MMP2, α-SMA, and the protection of ZO-1. In addition, the PLB-treated eyes showed lower PVR grades than the untreated eyes in rabbit models. PLB exhibited a wide safety margin, indicating no evidence of causing retinal toxicity.ConclusionsPLB effectively inhibited the EMT of rabbit RPE cells in vitro and in the experimental PVR models. The results open new avenues for the use of PLB in prevention and treatment of PVR.

Effects of Curcumin on Epidermal Growth Factor in Proliferative Vitreoretinopathy

Background/Aims: Proliferative vitreoretinopathy (PVR) is a common refractory eye disease that causes blindness and occurs after retinal detachment or retinal reattachment. Epidermal growth factor (EGF) has been shown to play an important role in the migration and proliferation of retinal pigment epithelium (RPE) cells, which promote PVR. Curcumin inhibits RPE cell proliferation, but it is not known whether it participates in the formation of PVR. Curcumin regulates the biological functions of EGF, which plays important roles in the development of PVR. This study aimed to evaluate the effect of curcumin on the regulation of EGF in PVR. Methods: Rabbit RPE cells were cultured, and EGF expression was detected by immunocytochemistry. MTT assay was conducted to determine cell proliferation induced by different concentrations of EGF. Immunocytochemical staining was used to detect EGF expression after treatment with curcumin at varying concentrations. Real-time PCR (RT-PCR) and western blot analysis were used to detect the concentrations of EGF mRNA and protein after treatment with curcumin. After RPE cells and curcumin were injected into experimental rabbit eyes, the cornea, aqueous humor, lens, and vitreous opacity were observed and recorded simultaneously by indirect ophthalmoscopy, fundus color photography, and B-ultrasonography. The vitreous body was extracted, and the EGF content in the vitreous humor was measured by enzyme-linked immunosorbent assay (ELISA). Results: At each time point (24, 48, and 72 h), cell proliferation gradually increased with increasing EGF concentrations (0, 3, 6, and 9 ng/mL) in a dose-dependent manner. Cell proliferation between EGF concentrations of 9 and 12 ng/mL were no different, which suggested that 9 ng/mL EGF was the best concentration to use to stimulate RPE cell proliferation in vitro. Under all EGF concentrations (0, 3, 6, 9, and 12 ng/mL), RPE cell proliferation increased with time (from 24 to 72 h), suggesting a time–effect relationship. Curcumin downregulated EGF expression in RPE cells, which also indicated time–effect and dose–effect relationships. The best curcumin concentration for the inhibition of EGF expression was 15 µg/mL. RT-PCR and western blot analyses indicated that the EGF mRNA and expression of the protein in RPE cells treated with curcumin significantly decreased with time. Ocular examinations revealed that the vitreous opacity was lower and the proliferative membrane was thinner in the curcumin group compared with the control group. The PVR grade and the incidence of retinal detachment were significantly lower in the experimental group than in the control group. ELISA results showed that the EGF content in vitreous humor was higher in the control group than in the curcumin group. The curcumin and control groups were significantly different at each time point. Conclusion: Curcumin inhibited RPE cell proliferation by downregulating EGF and thus effectively inhibited the initiation and development of PVR.

Inhibition of curcumin on the expression of IL-1.BETA.-induced nuclear factor-.KAPPA.B-dependent inflammatory gene in rabbit RPE cells

Background Interleukin-1β (IL-1β) is an important inflammation-related factor in the initial stage of proliferative vitreoretinopathy (PVR). The previous research showed that curcumin can inhibit IL-1β-induced proliferation of rabbit retinal pigment epithelium (RPE) cells, but the anti-inflammatory mechanism and effect of curcumin are still undefined. Objective This study was to observe the migration of IL-1β-induced rabbit RPE cells, and evaluate the function and mechanism of inhibition of curcumin on IL-1β-induced inflammation of RPE cells. Methods Cultured rabbit RPE cells of generation 4 were used in this experiment.The cells were cultured in serum-free DMEM and 0, 0.1, 1.0 and 10.0 μg/L IL-1β were separately added in the medium for 24 hours.The expressions of cyclooxygenase-2 (COX-2) protein and mRNA in the cells were detected by Western blot and reverse transcription PCR to determine the optimal concentration of IL-1β.The cells were divided into IL-1β group and curcumin+ IL-1β group, and 1.0 μg/L IL-1 or 1.0 μg/L IL-1β combined with 10 μg/ml curcumin was respectively added into the medium for 24, 48 and 72 hours.The cells cultured by only serum-free medium served as the control group.Hematoxylin and eosin staining was conducted for the cells to count the number of cells migrating into the injured area under the optical microscope.The relative expression levels of COX-2 protein and mRNA in the cells were detected by Western blot and reverse transcription PCR, and the relative expression levels of nuclear factor (NF)-κBp65 and inhibitor of NF-κB-α (IκB-α) protein were also detected by Western blot assay.The expression intensity and location of NF-κBp65, IκB-α and COX-2 in the cells were detected by immunochemistry. Results RPE cells just isolated from the rabbit eyes were in round shape and abundant in melanin.The melanin significantly decreased in the fourth generations of RPE cells.The shape of cells became long and narrow, and net shaped distribution.Immunochemistry demonstrated the strong positive response of RPE cells for keratin (AE1/AE3). There were (31.93±1.21), (36.27±2.50) and (38.33±2.40) migratory cells in the control group after 24, 48 and 72 hours respectively.The number of migratory cells increased to 45.73±2.30, 71.13±1.92 and 80.60±1.71 in the IL-1β group, but obviously decreased to 13.13±2.20, 14.93±1.10 and 12.60±1.51 in the curcumin+ IL-1β group.A Significant increase in the migrating cell number was found in the IL-1β group compared with the control group and the curcumin+ IL-1β group in various time points (all at P<0.05). The relative expression levels of COX-2 protein and mRNA peaked in the 1.0 μg/L IL-1β group, so 1.0 μg/L of IL-1β was determined as the optimal concentration in the experiment.In 24, 48 and 72 hours after culture, the expression levels of COX-2 protein and mRNA in the cells were significantly lower in the curcumin+ IL-1β group than those in the control group (all at P<0.05). The relative expression level reached peak in NF-κBp65 protein and lowed bottom in IκB-α proteins at 48 hours after cultured in the IL-1β group, and the reverse trend was seen in the curcumin+ IL-1β group, with the significant differences between the two groups (both at P<0.05). Immunochemistry showed that NF-κBp65 was expressed strongly in the cell nuclei and cytoplasm in the IL-1β group and presented the weaker expression in the control group and the curcumin+ IL-1β group.Compared with the control group, the expression was weaker in IκB-α and stronger in COX-2 in the IL-1β group.In addition, the expression of IκB-α was enhanced and that of COX-2 was attenuated in the curcumin+ IL-1β group in comparison with the IL-1β group. Conclusions Curcumin inhibits the movement of rabbit RPE cells induced by IL-1β.IL-1β up-regulates the expression of COX-2 by activating NF-κB signal pathway, and curcumin plays an anti-inflammatory role by blocking this pathway. Key words: Curcumin/pharmacology; Retinal pigment epithelium/cytology; Cell migration/drug effect; Interleukin-1beta/antagonists i Cytokines/metabolism; Inflammation/etiology; Vitreoretinopathy, proliferative/drug therapy; Cells, cultured; Rabbits

Attenuated Glial Reaction in Experimental Proliferative Vitreoretinopathy Treated with Liposomal Doxorubicin

We investigated the therapeutic effect of liposomal doxorubicin (Lipo-dox) on experimental proliferative vitreoretinopathy (PVR). The toxicity of Lipo-dox was determined in vitro in cultured rabbit retinal pigment epithelium (RPE) cells by tetrazolium-based (MTT) assay for cell viability performed 48 and 96 hours after treatment, and in vivo by electroretinography and histopathology. The therapeutic effect of intravitreous injection of Lipo-dox was evaluated in a rabbit model of PVR induced by injection of rabbit RPE cells after gas compression of the vitreous. The presence of PVR was determined by indirect ophthalmoscopy on days 1, 7, 14, 21, and 28 after injection. Western blot and immunofluorescence studies were performed to evaluate the expression of the glial markers vimentin and glial fibrillary acidic protein (GFAP). A pharmacokinetic study also was performed and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS). The 50% inhibitory concentrations (IC₅₀) of doxorubicin (Doxo) and Lipo-dox in RPE cells were 0.01-0.1 and 0.1-1.0 μg/mL, respectively. Lipo-dox (10 μg/mL) did not reduce the amplitude reduction in the ERG study or produce obvious retinal toxicity. Lipo-dox still could be detected in the vitreous 7 days after injection. The Lipo-dox (10 μg/mL)-treated eyes showed lower grade PVR than did the untreated eyes. Lipo-dox also decreased the retinal expression levels of vimentin and GFAP. Lipo-dox can attenuate the severity of experimental PVR, and reduces the glial cell expression of intermediate filaments in PVR retinas. Lipo-dox has a wider safe dosage range and a longer half-life in the vitreous than does primary Doxo.

Chinese herbal medicine Lingqi Huangban Granule protects retinal pigment epithelial cells against oxidative stress-induced injury in vitro

To observe the protective effects of drug-contained serum of Lingqi Huangban Granule (LQHBG), a compound traditional Chinese herbal medicine, on oxidative stress-induced injury in rabbit retinal pigment epithelial (RPE) cells in vitro. The oxidative stress of rabbit RPE cells in vitro was induced with hydrogen peroxide (500μmol/L) and different concentrations of LQHBG were administered to rats to prepare medicated serum. RPE cells were randomized into normal control group (no hydrogen peroxide), model group (hydrogen peroxide), model plus serum group (hydrogen peroxide and 10% control serum), model plus low-dose LQHBG group (hydrogen peroxide and low-dose LQHBG-medicated serum) and model plus high-dose LQHBG group (hydrogen peroxide and high-dose LQHBG-medicated serum). Teminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and flow cytometry (FCM) were used to measure apoptosis of cultured rabbit RPE cells. Protein expressions of caspase-3 and Bcl-X(L) were observed by Western blot method. FCM results showed that the apoptotic rates of the normal control group, model group, control serum group and serum containing low- and high-dose LQHBG groups were (4.85±0.26)%, (20.02±1.37)%, (21.84±0.94)%, (13.56±0.55)%, and (8.58±0.39)%, respectively; compared with the model group, the apoptotic rates of RPE cells in the low- and high-dose LQHBG groups were obviously reduced in a dose-related manner (P<0.05). TUNEL results showed that nuclei of apoptotic cells were stained brown; the number of apoptotic cells in the low- and high-dose LQHBG groups was obviously less than that in the model group. The protein expression of caspase-3 was up-regulated in the model and control serum groups, which was higher than that in the high-dose LQHBG group (P<0.05). The protein expression of Bcl-X(L) was down-regulated in the model and control serum groups, which was lower than that in the low- and high-dose LQHBG groups (P<0.01). Drug-contained serum of LQHBG obviously reduces apoptosis and partly protects rabbit RPE cells from oxidative stress-induced injury. The protective function is due to an improvement in antioxidant abilities, down-regulation of the expression of caspase-3 and up-regulation of the expression of Bcl-X(L).

Differential expression and significance of complement C4b and transthyretin in proliferative vitreoretinopathy

To investigate the differential expression of complement C4b and transthyretin in proliferative vitreoretinopathy (PVR). It was a controlled experimental study. Human vitreous samples of 5 patients with PVR were analyzed by using two-dimensional gel electrophoresis and mass spectrometry, and the results were compared with those from normal control vitreous obtained from donor eyes. An in vivo model of PVR was created by intravitreous injection of cultured rabbit retinal pigment epithelial (RPE) cells. The vitreous of PVR models were analyzed by enzyme linked immunosorbent assay (ELISA) to confirm the proteomic results from the PVR patients. Seventy nine various proteins were expressed differently between PVR and normal vitreous, among which nine up-regulated proteins including complement C4b, transthyretin (TTR), and 7 albumins were identified by mass spectrometry. The up-regulation of complement C4b and TTR in PVR patients was also confirmed by ELISA. The concentration of complement C4b and TTR in normal vitreous were (20.18 ± 1.97) mg/L and (88.58 ± 8.84) mg/L respectively, in PVR patients were (38.1 ± 5.79) mg/L and (112.57 ± 6.89) mg/L respectively, difference significantly between these two groups (C4b: t = 11.54, TTR:t = 9.24; P < 0.05). Differences of complement C4b and TTR expression were observed between PVR and normal vitreous. These results have lead to the assumption that there is a connection between elevated concentrations of both complement C4b and TTR and the pathogenesis of PVR and further studies on the functions of these proteins are required.

The effect of curcumin on DNA content, mitochondrial transmembrane potential and calcium of rabbit cultured retinal pigment epithelial cells

To investigate the Change of DNA content (apoptosis rate), mitochondrial transmembrane potential (DeltaPsim) and calcium (Ca(2+)) of rabbit retinal pigment epithelial (RPE) cells cultured with curcumin. It was an experimental study. The RPE cells were dissociated from rabbit eyes and cultured. The RPE cells in the 4(th) passage were divided into 2 groups: curcumin group and control group (10% FBS-EMDM contains 0.05% dimethyl sulfoxide). The curcumin group contained 3 mass concentration: 10 mg/L, 15 mg/L and 20 mg/L. The MTT assay was used to evaluate the inhibition effect of RPE cells cultured with curcumin after 24 h, 48 h, 72 h and 96 h respectively. The IC(50) value in 24 h, 48 h, 72 h and 96 h were gotten by Linear Regression. Flow cytometry was performed to detect the change of DNA content (apoptosis rate), DeltaPsim and Ca(2+) of RPE cells cultured with curcumin (15 mg/L) after 8 h, 24 h, 48 h and 72 h respectively. RPE cells were significantly inhibited by curcumin in a dose dependent and time dependent manner. The IC(50) value of curcumin at 24 h, 48 h, 72 h and 96 h was 29.31 mg/L, 17.50 mg/L, 13.24 mg/L and 10.99 mg/L respectively. Ca(2+) was significantly increased at 8 h, 24 h, 48 h and 72 h after cultured with curcumin (15 mg/L) than that of the control group respectively (t = 7.50, 10.61, 20.74, 21.14, P < 0.01), and DeltaPsim was significantly decreased at 8 h, 24 h, 48 h and 72 h after cultured with curcumin (15 mg/L) than that of the control group respectively (t = 7.50, 11.74, 14.91, 15.29, P < 0.01). There was no change of DNA content in RPE cells at 8h after cultured with curcumin (15 mg/L), but significantly lower than that of the control group at 24 h, 48 h and 72 h respectively (t = 10.00, 14.68, 13.68, P < 0.01). The apoptosis of RPE cells induced by curcumin is caused by increase of Ca(2+) and decrease of DeltaPsim that causes decrease of DNA content. The RPE cells are significantly inhibited by curcumin, which may become a potential drug to prevent and treat proliferative vitreoretinopathy.

A Novel Rabbit Model for Studying RPE Transplantation

The goal of this project was to develop a model of retinal pigment epithelium (RPE) transplantation that permits extensive and reliable analysis of the transplants. Cultures of newborn rabbit RPE were evaluated by morphology, electrophysiology, and the expression of zonula occludens-1, cytokeratin, and the melanocyte marker S-100. Cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) were transplanted into the subretinal space of rabbits with a 30-gauge needle without making a conjunctival flap or sclerotomy. The transplants were examined by fundus photography, confocal scanning laser ophthalmoscopy (cSLO), optical coherence tomography (OCT), and angiography. At 2 months, the retina was examined histochemically. A 1-minute incubation at 37 degrees C with 20 muM CFDA-SE did not affect morphology or the expression of marker proteins. In coculture, the labeled cells integrated into monolayers that developed a normal transepithelial electrical resistance of 400 to 450 Omega . cm(-2). Dye was not transferred from labeled to nonlabeled RPE cells. Transplanted RPE was detectable for at least 2 months. Angiography demonstrated an intact blood-retinal barrier. The normal morphology of the retina and lack of debris in the subretinal space suggested that the transplanted RPE was functional. Primary cultures of newborn rabbit RPE were highly differentiated, even when labeled with CFDA-SE. Labeled cells were observed long-term in vitro and in vivo. This model can be used to examine how culture and transplantation protocols affect the reformation of a functional RPE monolayer. The similar size of rabbit and human eyes will facilitate the translation of these protocols to the bedside.

In vitro benzyl alcohol cytotoxicity: Implications for intravitreal use of triamcinolone acetonide

The aim of the study was to investigate the toxicity of benzyl alcohol (BA), the preservative in commercial triamcinolone acetonide (TA) suspensions, on retinal pigment epithelial (RPE) cells. Cultured RPE cells from a human cell line (ARPE-19) and from rabbits were exposed to the balanced salt solution (control) or BA (0.0225, 0.225, 0.9, 3 or 9 mg/mL) for 5, 30, 60, or 120 min. Morphological changes of RPE cells were evaluated by the trypan blue in situ staining. The proportions of dead cells were quantitatively measured by the trypan blue exclusion assay, and those of functional cells were assessed by a mitochondrial dehydrogenase assay. The mechanism of cytotoxicity was determined by the acridine orange/ethidium bromide staining and DNA laddering technique. Furthermore, ultrastructural changes were observed by transmission electron microscopy. The results showed that RPE cell damage was dose- and time-dependent. BA 0.225 mg/mL, the clinically relevant concentration in TA following intravitreal injection, caused ultrastructural damage and impaired human RPE cell function at 2 h; but BA 0.0225 mg/mL did not. BA 9.0 mg/mL, the concentration in commercial TA suspensions, was toxic within 5 min on each assay for both human and rabbit RPE cells. The major mechanism of cell death was necrosis. In conclusion, BA in commercial TA suspensions injected intravitreally (0.225–9 mg/mL) can damage RPE cells. Our in vitro study on benzyl alcohol cytotoxicity has significant clinical implications for intravitreal use of TA. We suggest that, before a commercial TA solution is used intravitreally, the vehicle should be removed to prevent damaging the RPE layer, particularly during macular hole surgery. Commercial development of a preservative-free TA suspension for intraocular use is urged.

Cyclin-dependent Kinase Inhibitor Roscovitine Induces Cell Cycle Arrest and Apoptosis in Rabbit Retinal Pigment Epithelial Cells

Cyclin-dependent kinases (CDKs) play essential roles in the intracellular control of the cell cycle. It has been postulated that roscovitine, a potent CDK2, CDK5, and CDC2 inhibitor, might inhibit cellular proliferation by arresting the cell cycle. This in vitro study investigated the antiproliferative and apoptotic effects of roscovitine in cultured rabbit retinal pigment epithelial (RPE) cells. Experiments using rabbit RPE from young pigmented rabbits were carried out using roscovitine dissolved in dimethylsulfoxide at concentrations ranging from 1 to 100 micromol. Cell proliferation was measured by an MTT assay. The cell cycle response of RPE cells to roscovitine was analyzed by flow cytometry of propidium iodide-stained nuclei. Proteins related to DNA damage in the RPE cells were then assayed by Western blot. Roscovitine inhibited proliferation of RPE cells in a dose-dependent manner. Cell cycle analysis after treatment demonstrated an accumulation of cells arrested in the S- and G2/M phases. Flow cytometry showed that 40 microM of roscovitine increased the cell population in the sub-G1 peak, which is considered a marker of cell death by apoptosis. Western blot analysis revealed Bcl-2 decreased and Bax increased after treatment of RPE cells with roscovitine. This study of the response of RPE cells to roscovitine demonstrated a bidirectional relationship between cell cycle control and apoptosis.

Dipeptide Monoester Ganciclovir Prodrugs for Transscleral Drug Delivery: Targeting the Oligopeptide Transporter on Rabbit Retina

The overall aim of this research work was to evaluate a series of dipeptide monoester prodrugs of an antiviral agent, ganciclovir (GCV), for oligopeptide transporter-targeted transscleral drug delivery to rabbit retina. The permeability and enzymatic hydrolysis of dipeptide monoester GCV prodrugs were evaluated using freshly excized rabbit retinal pigment epithelium (RPE)-choroidsclera (RCS) and sclera tissue preparations. Affinity and transport mechanism of these prodrugs and their translocation across RCS were investigated through competitive inhibition studies of [(3)H]glycylsarcosine with the prodrugs. The transport of glycylsarcosine was found to be saturable (K(m) = 1.21 +/- 0.41 mM, V(max) = 15.89 +/- 1.54 pmoles/min/cm(2)), pH, temperature, and energy dependant. Dipeptides, angiotensin converting enzyme inhibitors, and a beta-lactum antibiotic strongly inhibited the transport of glycylsarcosine indicating the functional presence of oligopeptide transport (OPT) system on the RPE. Dipeptide prodrugs (valine-valine-GCV, glycine-valine-GCV, and tyrosine-valine-GCV), and valine-GCV demonstrate a high enzymatic stability and affinity toward the retinal OPT system. The transport of the prodrugs was significantly inhibited ( approximately 50%) in the presence of glycylsarcosine. The rank order of scleral permeability was Gly-Val-GCV approximately GCV>Val-GCV>Tyr-Val-GCV approximately Val-Val-GCV. The RCS permeability values of Val-GCV (3.29 +/- 0.09 x 10(6)cm/s), Val-Val-GCV (4.14 +/- 0.33 x 10(6)cm/s), Gly-Val-GCV (3.40 +/- 0.47 x 10(6)cm/s) and Tyr-Val-GCV (3.08 +/- 0.29 x 10(6)cm/s) were two-fold higher than that of GCV (1.61 +/- 0.06 x 10(6)cm/s). The dipeptide monoester GCV prodrugs, owning to higher lipophilicity and OPT-mediated translocation across RPE, appear to be promising candidates in the treatment of ocular cytomegalovirus infections following an episcleral administration.

Effects of Topoisomerase II Inhibitors on Retinal Pigment Epithelium And Experimental Proliferative Vitreoretinopathy

The aim of this study was to compare the effect of several commercially available topoisomerase II inhibitors on the proliferation of retinal pigment epithelium (RPE) cells in vitro and to test the toxicity and efficacy of the inhibitor against experimental proliferative vitreoretinopathy (PVR). Three different topoisomerase II inhibitors (etoposide, doxorubicin, and daunorubicin) were tested in vitro. Rabbit RPE cells were cultured with or without the drugs at various concentrations. An MTT assay was used to determine the cell viability at 48 h and 96 h. Etoposide, a drug which showed a broad therapeutic range in vitro, was injected to the rabbit eye for the evaluation of the toxicity in vivo. Therapeutic effects of an intravitreal injection of etoposide were evaluated in an experimental PVR model induced by the intravitreal implantation of RPE cells in rabbits. All tested topoisomerase II inhibitors showed a significant reduction of cell viability in vitro. The slope of the dose-response curve was slowly declined for etoposide, and declined sharply for doxorubicin and daunorubicin. Therefore, etoposide was selected for further toxicity and efficacy studies in vivo. There was no significant change in b-wave amplitudes in the etoposide-injected eyes (0.02 mg, 10 microg/mL) after 2 weeks, but a significant reduction occurred in the etoposide-injected eyes (0.2 mg, 100 microg/mL). In the study of the experimental model of PVR, the rabbit eyes injected with RPE cells and etoposide (0.02 mg, 10 microg/mL) showed a significantly lower grading of PVR than that of the control eyes (injected RPE cells and PBS). These results indicate that etoposide would be an adjunctive for the prevention of PVR. Further pharmacokinetic study of the intravitreal injection of etoposide is required.

Diode Laser-Induced Mitosis in the Rabbit Retinal Pigment Epithelium

The induction of retinal pigment epithelium (RPE) proliferation without damaging the inner layers of the retina might be helpful in patients with RPE atrophic changes and degeneration. This study aimed to induce mitosis in the RPE of the rabbit after subthreshold photocoagulation with the diode laser. Twenty-five male Dutch rabbits received retinal photocoagulation using an 810-nm diode laser with different power settings and exposure times. The eyes were processed for light microscopy, electron transmission microscopy, and immunohistochemistry. Neither morphological alterations nor mitotic activity was found after 5-mJ energies. Retinal layers were not affected and RPE hyperplasia appeared in the treated areas associated with mitotic activity when 10 mJ was used. Mitosis induction and retinal damage appeared with 20, 50, and 100 mJ and were associated with ophthalmoscopic damage. The use of subthreshold 810-nm diode laser treatment may induce mitosis in the RPE without causing damage to the neighboring layers.

Selective and efficient culturing of retinal pigment epithelial cells using a feeder layer

The techniques to isolate and purify retinal pigment epithelial (RPE) cells from small piece of autologous tissues are extremely difficult, and it is important to develop an efficient cell culture technique for RPE cells. The purpose of this study was to investigate the effect of 3T3-J2 cells and conditioned medium from 3T3-J2 cells on the proliferation of cultured RPE cells. RPE cells from pigmented rabbits and a human RPE-derived cell line, ARPE-19, were used. First, the effects of co-culturing RPE cells with 3T3-J2 cells on the growth of the cells were analyzed. Second, the effects of the conditioned medium from 3T3-J2 cells on the proliferation of both types of cells were investigated. And third, the effects of the conditioned medium on RPE cell culture from a surgically removed choroidal neovascular (CNV) membrane were investigated. The 3T3-J2 cells increased the proliferation of both rabbit RPE cells and ARPE-19 cells. The number of rabbit RPE cells cultured in a mixture of the conditioned medium from 3T3-J2 cells was significantly higher than that in the reported optimal condition, and a similar tendency was observed for ARPE-19 cells. The results from enzyme-linked immunosorbent assay showed the presence of PDGF-AB, VEGF and IGF-I in the conditioned medium. The conditioned medium also promoted selective growth of human RPE cells from CNV. The results from this study present the conditions for efficient and selective culture of primary RPE cells.

Adenosine agonist regulation of outward active transport of fluorescein across retinal pigment epithelium in rabbits

To investigate the effect of an adenosine agonist, 2-5′- N-ethylcarboxamidoadenosine (NECA), on the outward active transport of fluorescein across the retinal pigment epithelium (RPE) in rabbits. High (5×10 −4–2×10 −3 m) and low (1×10 −5–1×10 −4 m) concentrations of NECA or phosphate buffered saline (PBS) were intravitreously injected into Dutch-belted rabbits. Sodium fluorescein was injected intravenously 180 min after NECA. Differential vitreous fluorophotometry was performed 3 hr after the sodium fluorescein injection and the vitreal fluorescein/fluorescein monoglucuronide (F/FG) ratio then was calculated. The F/FG ratios are inversely proportional to the outward active transport of fluorescein across the RPE. Retinal detachments were induced by injection of PBS into the subretinal space after the intravitreous injection of low- or high-dose NECA or PBS, and the size of the blebs was monitored. In eyes that received a low-dose injection of NECA, the F/FG ratio was higher compared with controls ( P<0·05); in eyes that received a high-dose intravitreal injection, the F/FG ratio was significantly lower compared with controls ( P<0·05). The effect of low-dose NECA on the F/FG ratio was suppressed by the A2 receptor antagonist, ZM241385, and the effect of high-dose NECA was suppressed by the A1 receptor antagonist, 8-cyclopentyl-1, 3-dipropylxanthine. The A3 receptor antagonist MRS1191 did not influence the effect of low- or high-dose NECA. Intravitreal injection of high-dose NECA enhanced the reabsorption of subretinal fluid compared with PBS; however, low-dose NECA inhibited reabsorption of subretinal fluid ( P<0·02 and 0·05, respectively). Intravitreous injection of high-dose NECA accelerates the active outward transport across the RPE via A1 receptors and low-dose NECA decelerates it via A2 receptors.

Transient immunosuppression stops rejection of virus-transduced enhanced green fluorescent protein in rabbit retina.

The expression of lentivirus-transduced enhanced green fluorescent protein (EGFP) was detectable in rabbit retinal pigment epithelium (RPE) within 3 to 5 days after subretinal injection of the vector. Within 2 to 3 weeks, EGFP-expressing cells were eliminated by rejection. In the current experiments, we monitor serum antibody titers for EGFP before and after transduction and determine whether systemic immunosuppression prevents recognition of EGFP by the immune system. While all control rabbits developed antibodies against EFGP and showed signs of rejection, no such evidence was observed with animals which received immunosuppression. One month of systemic immunosuppression permanently prevented rejection of RPE with EGFP expression. Fluorescence has been maintained for more than a year. If a control eye was injected with the same virus after terminating immunosuppression, both eyes showed signs of rejection. The lack of rejection is not due to tolerance but to a failure of the animals to detect the foreign protein. Detection must depend upon a brief window of time after surgery needed to introduce the vector, perhaps related to a concurrent but transient inflammation. This strategy may be useful in managing other types of rejection in the retina.