Comparison of tight junction permeability for albumin in iris pigment epithelium and retinal pigment epithelium in vitro

Background: The degenerative retinal diseases are one of the major causes of visual loss in the western world. Although heterologous RPE transplants rescue the photoreceptors in the dystrophic rat model, rejection remains a major limiting factor. Given the common embryonic origin, iris pigment epithelial (IPE) cells might be able to take over the functions of retinal pigment epithelial (RPE) cells, serving as an autologous graft for transplantation and thereby preventing rejection. One of the main functions of RPE cells is the generation of tight junctions which form the outer blood-retinal barrier. In this study we compared the tight junction permeabilities of IPE and RPE cells isolated from Long Evans rats by measuring their albumin clearances. Methods: IPE and RPE cells were cultured on semipermeable filter supports with and without the addition of 0.02% ethylene diaminetetraacetic acid (EDTA). At selected intervals, the albumin clearances of the IPE and RPE cells were measured spectrophotometrically and compared. The morphology of the cells was compared using electron microscopy and fluorescent labeling. Results: IPE and RPE cells both restricted the passage of albumin in vitro. After the modulation of tight junctions with 0.02% EDTA, the clearance increased in both types of cells in a similar fashion. The morphology of tight junctions was visualized with electron microscopy. Conclusion: These results indicate that the functional barrier for macromolecules is similar in IPE and RPE cells in vitro. This raises the possibility that IPE cells would form tight junctions in the subretinal space, thereby substituting for the blood-retinal barrier normally formed by RPE cells.     

Transforming growth factor-β regulates human retinal pigment epithelial cell phagocytosis by influencing a protein kinase C-dependent pathway

Background: Transforming growth factor- (TGF-) plays an important role in the pathogenesis of many ocular diseases, including proliferative vitreoretinopathy. We examined the effect of TGF- on the phagocytosis of rod outer segments by retinal pigment epithelium (RPE), which is a major function of RPE, and investigated the dependence of this effect on the protein kinase C (PKC) pathway. Methods: Phagocytotic uptake of fluoresceinated bovine rod outer segments was determined by flow cytometry. RPE cells were treated with TGF-1 or TGF-2 and their effects on phagocytosis were examined. The effects of various PKC inhibitors (calphostin C, staurosporine, and extended exposure to phorbol 12-myristate 13-acetate, PMA) and a stimulator (brief exposure to PMA) on RPE phagocytosis was evaluated. Results: Both TGF-1 and TGF-2 up-regulated RPE phagocytosis and PMA abolished the upregulating effect of TGF-. In contrast, PKC inhibition by staurosporine and calphostin C resulted in increased phagocytosis. A combination of TGF- and PKC inhibitor treatment did not produced any additive effect on phagocytosis. Conclusion: We concluded that TGF- up-regulates human RPE phagocytosis, but that this effect is counteracted by PKC activation. It is possible that this TGF--induced effect is due, in part, to a negative modulation of the PKC-dependent pathway.     

Transplantation of fetal retinal pigment epithelium in age-related macular degeneration with subfoveal neovascularization

Background: Age-related macular degeneration (ARMD) is caused by abnormal retinal pigment epithelium (RPE) and may be complicated by choroidal neovascularization. The object of treatment would be to replace the diseased RPE with normal human RPE. Method: Five patients with ARMD (preoperative visual acuity 0.08–0.2) underwent removal of subretinal fibrovascular membranes using pars plana vitrectomy techniques. Human fetal RPE (15–17 weeks gestational age) was cultured and transplanted as a monolayer patch into the subretinal space. Transplants were followed by funduscopy and fluorescein angiography. Macular function was assessed using scanning laser ophthalmoscopic (SLO) microperimetry. Results: Three RPE transplants were placed in the fovea; two were placed parafoveally. All transplants have survived for 3 months. They have grown and increased in size covering part of the epithelial defect caused by removal of the fibrovascular membrane. SLO microperimetry indicated that visual function was present in four of the transplants at 1 month but in only two at 3 months after surgery. Function over the transplants, especially those in the fovea, was compromised by cystoidlike macular edema. Conclusions: Human fetal RPE transplants survive well in the macula for as long as 3 months. They are capable of growing to cover epithelial defects caused by removal of subretinal neovascular membranes. The causes for development of macular edema in transplants directly in the fovea warrant further evaluation.     

Interpretation of high-pass resolution perimetry with a probability plot

Background: The interpretation of high-pass resolution perimetry (HRP) fields can be difficult. An age-related probability plot was derived from a known data base to determine whether this improved specificity and sensitivity in early glaucoma detection Methods: Forty glaucoma patients with minor field loss and 40 normals of equivalent age and sex underwent HRP. All had previous Humphrey field data available for comparison. The detection of previously confirmed field defects by HRP was examined using different parameters. Results: Using the age-corrected 95% confidence levels gave the best results, with sensitivity of 82.5% (33/40) and specificity of 85% (34/40). Using the contour plot yielded lower sensitivity (67.5%), while subjectively eyeballing the ring printout gave higher sensitivity (90%), but led to poor specificity (72.5%). Conclusion: The use of a probability plot enhances the performance of HRP. A certain proportion of cases may be missed using one criterion alone, but combining the information with other indices can increase the yield.The authors have no proprietary interest in the equipment and technique described herein     

Reattachment to a substrate prevents apoptosis of human retinal pigment epithelium

Background: Epithelial cells generally fail to survive in suspension. Harvesting human retinal pigment epithelium (RPE) for transplantation may separate the cells from their extracellular matrix and induce apoptosis. We investigated whether reattachment of RPE to a substrate will prevent apoptosis. Methods: Second-passage human RPE cells were plated onto tissue culture plastic precoated with extracellular matrix, fibronectin or laminin, uncoated tissue culture plastic, untreated plastic and untreated plastic coated with 4% agarose. Reattachment rates were determined for each substrate 24 h after plating. The TUNEL technique was used to determine apoptosis rates in attached cells, unattached cells and the entire cell population. Results: Attachment rates were as follows: ECM-coated tissue culture plastic fibronectin-coated tissue culture plastic laminin-coated tissue culture plastic uncoated tissue culture plastic untreated plastic agarose-coated untreated plastic. Apoptosis rates for the entire cell population increased as the RPE cell attachment rate decreased. The proportion of apoptotic cells in the entire population was inversely related to the percent attached cells (r = -0.95). Conclusion: Reattachment of harvested RPE to a substrate decreased the rate of RPE apoptosisin vitro. RPE cells which are removed from their substrate prior to transplantation must reattach rapidly to a substrate to prevent apoptosis.Presented in part at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 1996     

The effect of nasolacrimal occlusion on drug-induced mydriasis

Background: Nasolacrimal occlusion has been shown to improve the efficacy of some topically applied ocular drugs. The aim of this study was to investigate the effect of nasolacrimal occlusion on tropicamide-induced mydriasis. Methods: We compared pupillary dilatation by 0.125% tropicamide with and without nasolacrimal occlusion in 40 healthy volunteers. Results: Analysis of variance with repeated measures failed to show any advantage due to nasolacrimal occlusion in drug-induced mydriasis. Conclusion: Nasolacrimal occlusion did not increase the mydriasis obtained with 0.125% tropicamide.     

Influence of oxygen free radicals and free radical scavengers on the growth behaviour and oxidative tissue damage of bovine retinal pigment epithelium cells in vitro

Purpose: This investigation was carried out to ascertain whether oxygen free radicals can influence the growth behaviour and consecutive lipid peroxidation of retinal pigment epithelium (RPE) cells in vitro and whether scavengers can counteract these effects. Methods: The experimental model was based on calf RPE cells. Hypoxanthine/xanthine oxidase (HX/XO) and superoxide dismutase/catalase (SOD/CAT) served as the radical generating system and scavengers, respectively. The components were tested alone and in combination. Lipid peroxides were determined in culture supernatants by a thiobarbituric acid assay. Results: Concentrations of up to 100 mol/1 of HX alone and 500/1000 U of XO alone, as well as the application of the scavengers without the radical generating system (HX/XO), had no effect. Doserelated reduction of cell growth and increase of lipid peroxidation were found with HX/XO treatment (single dose of 500 and 1000 U/ml 24 h after seeding). After application of 500 or 1000 U/ml of XO, CAT, when given alone (1200 U/ ml), counteracted the effect of the radicals on cell growth and lipid peroxidation; SOD (300 U/ml) had no effect. A combination of SOD and CAT was no better than the effect of CAT alone. Conclusion: The prevention of radical-induced reduction of cell growth and lipid peroxidation by scavengers supports trials of therapy using antioxidants and/or free radical scavengers for various ocular syndromes with RPE involvement.     

Iris pigment epithelium transplantation

Background: Iris pigment epithelium (IPE) cells and retinal pigment epithelium (RPE) cells possess the same embryonic origin. It is also known that the pigmented epithelial cells in the eye have a high transdifferentiation potential. In this study we transplanted IPE cells into the subretinal space of albino Royal College of Surgeons (RCS) rats and evaluated their influence on the degeneration of the photoreceptors. Methods: IPE cells of Long Evans rats were isolated and pure cultures were obtained. The isolated cells were transplanted into the subretinal space of RCS rats. Light microscopic and morphometric analysis were carried out. Results: The IPE transplants survived in the subretinal space and attached themselves to the Bruch's membrane. The transplanted cells were able to delay the degeneration of the photoreceptors for up to 3 months. Conclusion: These results suggest that IPE cells could be successfully transplanted and survive in the subretinal space. In the transplanted eyes the photoreceptors were preserved for a period of 3 months. Further studies are needed to explore the capability of IPE cells to assume the main functions of RPE cells in the subretinal space and their potential in the therapy of selective degenerative diseases of the retina.     

Retinal pigment epithelial cells: autocrine and paracrine stimulation of extracellular matrix contraction

Background: This study was carried out to examine the biological activity of contraction promoters produced by dedifferentiating retinal pigment epithelial cells (RPE) and to evaluate the importance of autocrine and paracrine effects within a semi-closed environment like the vitreal cavity. Methods: RPE at different stages of dedifferentiation in culture were examined for their ability (a) to generate tractional forces in vitro, with and without serum stimulation, and (b) to produce and release contraction-stimulating proteins. Autocrine versus paracrine effects of cell-secreted promoters were tested by using RPE or human dermal fibroblasts (HDF) as target cells. The contraction-stimulating activity of the cell-secreted promoters was partially characterized and compared to the activity of defined promoters. Results: Our study confirmed that RPE can synthesize and secrete cell-contraction-promoting factor(s) active in stimulating the development of tractional forces by RPE as well as HDF. The quantity of biological activity secreted per cell decreases with progressive dedifferentiation, yet the responsiveness of the cell to contraction promoters increases. The contraction promoter(s) synthesized by RPE is partially distinct from the promote rs in serum, TGF-1 and 2, IGF-1, ET-1 and PDGF. The contraction-promoting effects of the RPE product(s) can be completely blocked by staurosporine. Conclusion: Dedifferentiation of RPE is characterized by increasing capacity to generate tractional forces and decreasing synthetic capacity. RPE within a semi-closed system like the vitreal cavity can, theoretically, act both as promoting and active component of traction-related events (tractional retinal detachment).     

Ocular findings in Japanese women with nevus of Ota

Background: Nevus of Ota is common in Japanese women, but most patients are not examined ophthalmologically. Methods: We performed ophthalmologic examinations on 16 Japanese women who had had bluish pigmentation in the periorbital region, sclera, and conjunctiva since birth. Results: Fifteen patients had unilateral involvement, and one had bilateral lesions. The visual acuities were good, and the intraocular pressures were within normal range. All patients had a negative family history. Three patients had light pigmentation in the optic disc in the affected eye. Conclusion: We believe that optic disc pigmentation associated with nevus of Ota, as found in these three patients, may be common but have been rarely described.     

Transplantation of RPE in age-related macular degeneration: Observations in disciform lesions and dry RPE atrophy

A study was carried out to investigate whether human RPE allografts are tolerated or rejected in the subretinal space and to determine the feasibility of RPE transplantation in subjects with age-related macular degeneration (AMD). Methods: Patches of human fetal RPE (13–20 weeks of gestational age) were transplanted into the subretinal space of five patients after surgical removal of subfoveal fibrovascular membranes, and to four subjects with dry geographic atrophy. Suspensions of RPE cells were transplanted to four other patients with nonexudative AMD. Results were evaluated with clinical ophthalmological examination, SLO microperimetry and fluorescein angiography over 8–20 months. Results: In disciform lesions, RPE transplants developed macular edema and fluorescein leakage concomitant with gradual reduction of visual acuity, implying host-graft rejection, over 1–6 months. In geographic atrophy, three of four transplants showed little change in shape and size after 12 months (one transplant was slowly rejected). In non-exudative AMD, RPE suspension transplants showed no evidence of rejection and were associated with the disappearance of drusen; visual acuity remained stable and SLO microperimetry confirmed retinal function over the transplanted area. Conclusion: Human RPE allografts are not invariably rejected in the subretinal space without immunosuppression. The rejection rate is lower in nonexudative than in neovascular AMD. An intact blood-retinal barrier is likely to protect against rejection. It is technically feasible to transplant human RPE into the submacular space without adversely affecting visual function in nonexudative AMD over relatively long periods of timeThis work was supported by grants from the Swedish Medical Research Council (B96-12X-11561-01A) and the Crown Princess Margareta Foundation, Stockholm, Sweden.     

Anterior chamber inflammation after transconjunctival cryosurgery

Background: Inflammation caused by transconjunctival cryotherapy for prophylactic retinal detachment surgery was measured in various conditions. Methods: Thirty-four eyes of 28 patients with peripheral retinal lesions predisposing to retinal detachment were studied by laser flare cell meter before and after treatment. Results: The mean flare value for 34 eyes was 4.06 ± 1.45 photon counts/ms before surgery and 5.72 ± 2.52 pc/ms after surgery (p < 0.05). Flare value was elevated at 1, 2, and 3 weeks after treatment, peaking at 2 weeks (p < 0.05), and normal again at 4 weeks. There were no significant differences in flare increase between eyes with and without retinal breaks, eyes with and without limited retinal detachment, eyes with myopia more and less than – 8.0 D, and eyes with a treatment area limited to one quadrant and extending over more than one quadrant. Conclusion: Transconjunctival cryosurgery caused mild inflammation in the anterior chamber of the eye for 3 weeks. The inflammation was not affected by the presence of retinal break or limited retinal detachment, the degree of myopia, or the extent of the treatment area.     

Iatrogenic choroidal neovascularisation following argon laser photocoagulation for choroidal malignant melanoma

Background: Choroidal neovascularisation is known to occur following photocoagulation for choroidal melanomas. Its occurrence rate, possible causes and clinical impact were studied. Methods: Post-treatment fluorescein angiograms were reviewed from 18 patients who had received argon laser photocoagulation as sole treatment of their small choroidal melanomas, to look for choroidal neovascularisation. Where it was found an assessment of its clinical impact was made. Results: choroidal neovascularisation was found in 50% of cases. Choroidoretinal neovascularisation, found in five patients, caused vitreous haemorrhage in one patient but was otherwise benign. Choroidovitreal neovascularisation was found in four patients. It occurred early and altered their clinical management. Three of these patients had a vitreous haemorrhage, one of whom also suffered a retinal detachment. The three diabetic patients in the series all developed aggressive choroidovitreal neovascularisation. Tumour size, tumours location and number of treatment sessions did not appear to affect the occurrence of choroidal reovascularisation, nor did other medical or ocular conditions except for diabetes. Conclusion: Choroidal neovascularisation occurs commonly after melanoma photocoagulation. Although sometimes benign, it can be aggressive, particularly in diabetic patients, in whom it might be better to consider different forms of tumour treatment.     

Endophthalmitis after Lasiodiplodia theobromae corneal abscess

Background:Lasiodiplodia theobromae is an exceptional cause of human keratomycosis. Patient: We treated a 53-year-old man with fungal keratitis, which had been treated with topical betamethasone and gentamicin for 1 month, and endophthalmitis due toLasiodiplodia theobromae. Despite intensive systemic, topical and intravitreal fungicidal treatment, enucleation had to be performed. Results: The vitreous aspirate cultures were negative as of the second amphotericin intravitreal injection. However, histology revealed that the fungus was present in the cornea, ciliary body, iris and retina. Conclusion: The use of topical steroids may worsen the outcome of the infection.     

β-Galactosidase transgene expression in transplanted rabbit retinal pigment epithelial cells in vivo

Background: Intraocular transplantation of genetically modified cells that release a particular substance could have a major impact on the treatment of various ocular diseases. We studied the expression of the reporter gene -galactosidase (lacZ) in transplanted retinal pigment epithelial (RPE) cells in vivo Methods: RPE cells from pigmented rabbits were transduced with the -galactosidase gene in a retroviral vector. Cells were then assayed for gene expression and transplanted subretinally into the eyes of New Zealand White rabbits. RPE cells that were transduced with a similar vector without the -galactosidase gene were used as controls. Rabbits were killed on days 1, 7, and 21 and the eyes processed for transmission electron microscopy Results: Neomycin-resistant rabbit RPE cells that showed -galactosidase activity were generated within 2–5 weeks. After transplantation, viable RPE cells that expressed the transgene and that phagocytosed rod outer segments were observed on days 1, 7, and 21 Conclusions: The results show that generation of genetically modified RPE cells is feasible and that the transplanted cells remain viable and continue to express the transgene in the subretinal space of the host animal for at least 21 days. Transplantation of such genetically modified RPE cells could provide a new tool for studying retinal diseases and, potentially, for correcting metabolic abnormalities in retinal degenerations and dystrophies.     

Early experience with intravenous immunoglobulin treatment in Wegener's granulomatosis with ocular involvement

Background: Pooled intravenous gammaglobulin (IVIg) was reported to be effective in the treatment of Wegener's granulomatosis (WG). No reports have been made on the effects of this new treatment on ocular manifestations of WG. Method: IVIg treatment was given to two patients suffering from WG with ocular involvement after several other treatment regimes had failed. Results: Although the systemic disease was under control, the ocular symptoms of both patients worsened during and after IVIg treatment. In one case an adverse effect consisting of retinal vasculitis was noted on two occasions. Conclusion: Although beneficial effects of IVIg treatment on WG have been previously described, the two cases with ocular involvement presented here did not reveal any positive response. Paradoxical and unpredictable reactions cannot be ruled out. Thus, patients treated with IVIg should be closely surveyed by an ophthalmologist.     

The spectrum of eye disease in children with AIDS due to vertically transmitted HIV disease: Clinical findings, virology and recommendations for surveillance

Background: Eye disease in children infected with HIV is uncommon, but there is little information on which children require screening or surveillance and the management of those with retinitis. Methods: We examined 12 children with symptomatic HIV disease (of a cohort of 98 HIV-positive children) using indirect ophthalmoscopy. When retinitis was found it was documented photographically at the time of diagnosis and on follow-up. Results: Four cases of infective retinitis were diagnosed: three with probable cytomegalo-virus retinitis and one with progressive outer retinal necrosis. Conclusion: Detection of reactivation is difficult and we recommend fundus photography under general anaesthesia wherever possible. Children with other end-organ disease, symptomatic eye disease and advanced HIV disease with severe immunosuppression require ophthalmological surveillance.     

Precision of ultrasonic estimates of choroidal melanoma regression

Background: Important prognostic information may be gained from knowledge of the volume and, over time, the change in volume of intraocular tumours such as choroidal melanomas. Methods: The precision and time consumption of three different ultrasonographic methods were evaluated. Seven choroidal melanomas were analysed after placement of a ruthenium plaque and again after tumour regression had occurred. Perpendicular ultrasound B-scans were printed and analysed by overlay grid counting or outline tracing of tumour structures to calculate the volume. These two methods, using only two perpendicular scans, were compared with a micro-computer-controlled ultrasonographic three-dimensional rotation scanning system, where each tumour was manually outlined in 20 revolving scan planes. Results: The three-dimensional volume scanning method was the most precise, but also the most demanding in hardware and time consumption. Conclusion: Increased precision and less observer-dependent estimation of shrinkage rate after radiotherapy is available at the cost of sophisticated equipment.     

Ophthalmological and neuro-ophthalmological involvement in Churg-Strauss syndrome: a case report

Background: It is well known that different types of eye involvement may develop during the course of systemic vasculitides. Methods: We report here a case of Churg-Strauss syndrome (allergic granulomatous angiitis) characterized by the presence of multiple ophthalmological and neuro-ophthalmological lesions, i.e., mononeuritis of the fourth cranial nerve, multifocal choroidal ischaemia, and bilateral ischaemic optic neuropathy. Results: Ischaemic lesions in the posterior ciliary plexus and chorio-retinal circulation, which appeared simultaneously after a phase of disease activity, were documented. Conclusion: The simultaneous occurrence of multiple ocular features in a patient with Churg-Strauss syndrome suggests that regional vasculitis may be the pathological mechanism underlying the multiple ophthalmological lesions in this disorder.     

Angiogenesis in early choroidal neovascularization secondary to age-related macular degeneration

Background: The morphological features of angiogenesis in early choroidal neovascularization secondary to age-related macular degeneration are yet to be fully described. Methods: Six eyes from five patients which on clinical and histological examination showed advanced age-related macular degeneration and early choroidal neovascularization have been studied by transmission electron microscopy. Results: Pre-existing choroidal capillaries and venules showed changes which included endothelial cell budding, pericyte enlargement, endothelial cell sprout formation and the development of intrachoroidal new vessels. In one case, an endothelial cell sprout continuous with an intrachoroidal vessel penetrated Bruch's membrane. Examination of early subretinal pigment epithelial new vessels showed them to spread between the inner layers of Bruch's membrane within the space usually occupied by the basal linear deposit and drusen. New vessel formation took place in blind pouches at the margins of new vessel networks, either in the absence of pericytes or in the presence of mainly myofibroblast-like pericytes. Conclusion: This ultrastructural study describes two phases of new vessel growth associated with the onset of choroidal neovascularization secondary to age-related macular degeneration. The initial intrachoroidal phase appears to be a low-turnover form of neovascularization which may lead to new vessels penetrating Bruch's membrane. Extensive subretinal pigment epithelial neovascularization, on the other hand, results from a high-turnover phase of neovascularization characterized by extensive endothelial cell proliferation and migration. Pericyte phenotypic changes associated with these different phases of neovascularization appear to relate to the dynamics of angiogenesis taking place in each process.