Iris Pigment Epithelial Cells Research Articles

Experimental transplantation of autologous iris pigment epithelial cells to the subretinal space.

To investigate the cellular morphology in the subretinal space following transplantation of iris pigment epithelial (IPE) cells from the same eye. Following an iridectomy, fresh IPE cells were prepared and no culturing performed. After pars plana vitrectomy, a suspension of autologous IPE cells was injected into the subretinal space in 37 rabbits. The grafts were monitored by ophthalmoscopy and colour fundus photography. Rabbits were sacrificed at 1, 2, 3 and 6 months, respectively, and the eyes examined with light and electron microscopy. The grafted area retained the same configuration over 6 months but then appeared less pigmented. At 1-3 months, the IPE formed one or more contiguous layers on top of native RPE. At 6 months, cells compatible with grafted IPE were present in the subretinal space, often forming monolayer-like chains integrating with the native RPE. Depigmented cells of presumed IPE origin were seen and frequently in association with abundant melanin granules located in the apical portion of adjacent RPE cells. In such areas, large macrophage-like cells were observed. Transplanted IPE cells survived for up to 6 months in the subretinal space. Our observations suggest a scenario of remodelling of the cellular layers in the subretinal space over time where grafted IPE cells formed a compound layer with the native RPE. Transplantation of autologous IPE cells may have a potential as a treatment modality in selected cases of age-related macular degeneration.

Effects of drugs on cellular proliferation in cultured iris pigment epithelial cells and retinal pigment epithelial cells.

In this study, iris and retinal pigment epithelial cells were cultured from porcine and various drugs including methionine-enkephalin, isoproterenol, dibutyryl cAMP, endothelin-1, dexamethasone and phorbol 12-myristate 13-acetate (PMA) were used to investigate their effects on both cellular proliferation in cultured porcine iris and retinal pigment epithelial cells. Cellular proliferation was estimated with 3H-thymidine uptake. It is indicated that both pigment epithelial cells possess epithelial-like morphology and abundant pigment granules in cells obviously. Following the iris pigment epithelial cells being treated with endothelin-1, the 3H-thymidine uptake in the cells was increased to 126% as compared with the control. However, the cellular proliferation was decreased to 83% when the cells were treated with isoproterenol. In the case of methionine-enkephalin, dibutyryl cAMP, dexamethasone and phorbol 12-myristate 13-acetate (PMA), the thymidine uptake in the iris pigment epithelial cells was not affected by above drugs. In the retinal pigment epithelial cells, the 3H-thymidine uptakes were increased to 145% and 146% when the cells were incubated with methionine-enkephalin, and isoproterenol, respectively. In the presence of dibutyryl cAMP, dexamethasone and phorbol ester (PMA), the cellular proliferation was inhibited to 83%, 73% and 85% respectively. However, endothelin-1 did not affect the cellular proliferation in retinal pigment epithelial cells. These results show that the morphological shapes of iris pigment epithelial cells are similar to retinal pigment epithelial cells. However, the cellular proliferation in both cells may be regulated by distinct mechanisms.

Experimental transplantation of retinal and iris pigment epithelial cells into the subretinal space

A dysfunction of the retinal pigment epithelium (RPE) is the main cause for the development of age-related macular degeneration (ARMD) and visual loss in elderly people. For about 10 years experimental and clinical attempts to transplant RPE cells have been performed. The aims of this study were to elucidate the long term results of RPE allografts, to develop an atraumatic transplantation technique, and to explore the cellular response to RPE allografts, melanin granules, and autologous IPE cells. Surgery was performed on rabbits with a follow-up period of up to six months. After a pars plan vitrectomy, a subretinal bleb was created into which a suspension of RPE/IPE donor cells or melanin granules was injected. Pigmented RPE donor cells or preparations of melanin granules were implanted subretinally in albino rabbits. Pigmented rabbits were used in IPE transplantation. The eyes were monitored with ophthalmoscopy, fundus photography, light microscopy, electron microscopy and immunohistochemistry. Transplantation of suspensions of fresh pigmented RPE cells to the subretinal space in rabbits is feasible and induces virtually no complications when an atraumatic surgical procedure is used. The allograft forms a monolayer in conjunction with the native RPE and persists almost intact up to three months. At six months after transplantation, there was a cellular response exhibiting multilayers of cells, such as RPE and macrophages. Damage to adjacent photoreceptors in combination with melanin granules in the subretinal space indicates graft failure. No infiltration of lymphocytes was seen. Whether the cellular response was due to immunological or non-immunological mechanisms could not be determined from this experiment. Cyclosporine (CsA) could not prevent disintegration of the RPE transplant and graft failure. CsA was not capable of promoting graft survival as compared to the controls. The transplant seems to be disrupted either by immunological mechanisms that are not inhibited by CsA, or by non-immunologic events. Implantation of melanin granules to the subretinal space of albino rabbits induces a considerable phagocytic cellular response involving the host’s RPE, macrophages and glial cells. The migration of pigment-laden cells into the neural retina was frequently associated with focal photoreceptor damage. The cellular response was identical to that ensuing RPE cell transplantation. These findings support the concept that non-immunological events have a considerable influence on the outcome. In order to evaluate the impact of non-immunological mechanisms, a technique of transplanting fresh autologous IPE cells to the subretinal space of the same eye was developed. Grafted IPE cells were seen to survive for six months. There was a remodeling of the compound cellular layers in the subretinal space over time where grafted IPE cells joined the native RPE cells. The cellular response that developed exhibited macrophages, but no lymphocytes, and was in this respect similar to that observed following RPE transplantation. In RPE allografts, the photoreceptors appeared normal on light microscopy at three months, but at six months, the photoreceptors overlying the transplants generally exhibited pathological changes. In autologous IPE grafts, on the other hand, the photoreceptors displayed normal outer segment length and outer nuclear layer on top of grafted IPE cells. Focally, multilayers of both grafted IPE and RPE cells, together with macrophages, induce damage to adjacent photoreceptors as observed at 6 months. Cellular multilayers in the subretinal space, irrespective of genesis, are likely to have adverse effects on photoreceptors. The experiments using autologous IPE grafts show that non-immunogenic mechanisms have a decisive impact on the outcome of the transplant in the subretinal space. This thesis is based on the following original publications Crafoord S, Algvere PV, Seregard S, Dafgård Kopp EME (1999): Long-term outcome of RPE allografts to the subretinal space of rabbits. Acta Ophthalmol Scand 77: 247–254. Crafoord S, Algvere PV, Dafgård-Kopp E, Seregard S (2000): Cyclosporine treatment of RPE allografts in the rabbit subretinal space. Acta Ophthalmol Scand 78: 122–129. Crafoord S, Dafgård-Kopp E, Seregard S, Algvere PV (2000): Cellular migration into neural retina following implantation of melanin granules to the subretinal space. Graefe’s Arch Clin Exp Ophthalmol (2000) 238: 682–689. Crafoord S, Geng L, Seregard S, Algvere PV (2000): Transplantation of autologous iris pigment epithelial cells to the subretinal space: I. Morphological features (manuscript). V. Crafoord S, Geng L, Algvere PV (2000): Transplantation of autologous iris pigment epithelial cells to the subretinal space: II. Photoreceptor survival and consequences of cellular multilayers (manuscript). Author Sven Crafoord From: Division of Ophthalmology Department of Neuroscience and Locomotion Faculty of Health Sciences, E-581 85 Linköping Department of Ophthalmology Örebro Medical Centre Hospital SE-701 85 Örebro Department of Ophthalmology Karolinska Institutet St Erik’s Eye Hospital SE-112 82 Stockholm Sweden Accepted by: Faculty of Health Sciences Linköping University Sweden, 2000 ISBN 91-7219-586-X ISSN 0345-0082

Changes of phagocytic capacity in basic fibroblast growth factor-transfected iris pigment epithelial cells in rats

Purpose. To evaluate differences of phagocytic capacities in rat retinal pigment epithelial (RPE) cells, iris pigment epithelial (IPE) cells, and basic fibroblast growth factor (bFGF) cDNA-transfected IPE (bFGF-IPE) cells in vitro. Methods. The RPE cells and IPE cells were isolated from adult Long Evans rats’ eyes. The bFGF cDNA was transfected to the IPE using lipofection method. The bovine photoreceptor outer segments (POS) were isolated and labeled with Alexa 488 dye (fluorescent marker, EX: 494 nm, EM: 519 nm), and were applied to the cultured RPE, IPE, vector-IPE, and bFGF-IPE for 20 hours. The amount of phagocytosed POS in cells was photographed by fluorescence microscopy with fluorescein isothiocyanate (FITC) filter, and expressed as the ratio of the occupying area of fluorescence to the area of pigment epithelial cells in each observing field. Results. The phagocytic capacity of IPE and vector-IPE were about 70% of RPE, and that of bFGF-IPE increased to 150% of IPE. This increase was inhibited by pretreatment with anti-bFGF antibody to bFGF-IPE. Conclusions. IPE showed phagocytic capacity, but it reached only 70% of that in RPE. The stable expression of bFGF promoted its function.

Development and Cellular Functions of the Iris Pigment Epithelium

A number of studies have shown that transplantation of retinal pigment epithelial (RPE) cells to the subretinal space offers a promising treatment modality for retinal degenerative diseases. However, it is necessary to transplant autologous cells to avoid rejection; unfortunately, obtaining autologous RPE cells necessitates such traumatic surgical intervention as to make this approach irrelevant. It has been hypothesized that iris pigment epithelial (IPE) cells may be a possible substitute for RPE cells for transplantation into the subretinal space. The iris pigment epithelium, which has the same embryonic origin as retinal pigment epithelium, has not received much attention from visual scientists. Even though it forms a highly specialized tissue, it is not clear whether the iris pigment epithelium contributes critical functions to the health of the visual system. In vivo the IPE does not appear to have any of the functions characteristic of RPE; however, in vitro cultured IPE cells do acquire functions, such as specific phagocytosis of rod outer segments, that are characteristic of RPE cells, and have been shown to have the potential to carry out many functions characteristic of RPE cells, e.g., retinol metabolism. This review outlines the development and cellular functions of the IPE with special emphasis on the modulation of those functions that can allow the IPE cells to be transplanted to the subretinal space where they appear to acquire differentiated properties of retinal pigment epithelium (RPE).

Short-Time Application of Latanoprost Does Not Stimulate Melanogenesis in Bovine Ocular Melanin-Containing Cells in vitro

Topical use of latanoprost for glaucoma can lead to an increase in iris and eye lash pigmentation but the precise mechanism is unclear. To study the possible effect of this drug on ocular melanogenesis, we used cultures of bovine iris melanocytes, iris pigment epithelial cells, retinal pigment epithelial cells, and choroidal melanocytes. Latanoprost (at concentrations of 10^–8 and 10^–6 mol) was applied for 3 days, and cell numbers as well as melanin content were measured prior to and 10 days after exposure and compared to untreated controls. In none of the cell types examined a significant increase in melanin content or an increase in cell proliferation was observed. Additional treatment with the tyrosinase inhibitor α-methyl-p-tyrosine showed no significant effect either. Our results support the concept of a rather complex mechanism underlying the increased iris pigmentation after treatment with latanoprost.

Transplantation of iris pigment epithelium into the choroid slows down the degeneration of photoreceptors in the RCS rat.

Trophic factors [e.g. basic fibroblast growth factor (bFGF)] released by transplanted retinal pigment epithelial (RPE) cells are able to slow down the hereditary degeneration of the retina in the Royal College of Surgeons rat in sites distant from the site of transplantation where rod outer segment (ROS) phagocytic activity is not reconstituted by the transplants. To investigate whether iris pigmented epithelial (IPE) cells are also able to generate this rescue by trophic factors, we transplanted IPE cells from Long-Evans rats into the choroid and subretinal space of 17 young RCS rats. The eyes were enucleated after 6 months and prepared for light microscopy. Six age-matched RCS rats served as controls. Light microscope sections from the whole choroid, healthy choriocapillaris, transplanted cells and the maximum thickness of the choroid, and outer nuclear layer parameters were analyzed by computer-assisted morphometry. In transplanted animals photoreceptor cells were rescued from degeneration although the majority of the transplanted IPE cells were located in the choroid. In the non-transplanted group photoreceptors were absent. Transplantation of IPE cells slows down degeneration of the photoreceptors in the RCS rat. This photoreceptor-sparing effect by the IPE cells was observed even when the transplants were predominantly located within the choroid. The beneficial effect observed may be related to trophic factors possibly secreted by the transplanted IPE cells.

[In vitro culture of iris cells

The biological behaviour of iris cells in vitro was not yet completely investigated. For a more detailed study of the scope of cultivation of iris cells in vitro we isolated human iris pigment epithelium (IPE) cells and iris fibroblasts. For the purpose of this study iris cells were isolated from 19 donor eyes. A method was established for isolation and cultivation of IPE cells by means of fibronectin coating and the use of a special cell culture medium. Additionally, a method was developed for the selection of fibroblasts from iris stroma (IS) in vitro by means of fibronectin coating, passaging and proliferation in cell culture medium. The IPE and IS cells could be cultivated successfully. The IPE cells started to divide after a mean interval of 5.4 +/- 0.7 days in culture. The mitosis of IS cells was observed after 3.3 +/- 0.87 days in culture. Confluency of IPE cells was reached after 14.7 +/- 4.92 days and by IS cells after 8.1 +/- 1.45 days. Immunocytochemical staining using two antibodies for cytoceratin and one for human fibroblast showed that the IPE cell culture was pure and that the IS culture consisted of fibroblasts. Furthermore, electron microscopy of IPE and IS cultures confirmed the results of the immunocytochemical staining. The use of human IS and IPE cells in vitro has established a novel model for the research on iris cells. The model might possibly be applied in the research of metabolic structures and diseases of the iris.

Autologous iris pigment epithelial cell transplantation in monkey subretinal region

To establish autologous iris pigment epithelial (IPE) cell transplantation in monkey eyes. Autologous IPE cells from three monkeys were obtained by peripheral iridectomy, and cell culture was performed with autologous serum. The cultured cells were labeled with DiI, and transplantation was performed by transvitreal approach to the submacular region of each monkey. Fundus examination, photography, and fluorescein angiography were performed monthly. Histochemical analysis and electron microscopy were also performed six months after transplantation. Auto IPE cells grew well in auto serum. No fluorescein leakage or retinal thickening was observed after submacular transplantation. We demonstrated the presence of autologous IPE cells in the region 6 months after transplantation by histologic and electron microscopic examination. Electron microscopy also demonstrated that the transplanted cells appeared to be less pigmented and to have less mitochondria than did the host retinal pigment epithelial cells. However, some transplanted cells looked as if they embrace the photoreceptor outer segments. Autologous IPE cell culture of the monkey was established using auto serum. Six months after transplantation of the cultured IPE cells, we could still observe the cells in the region. This method may have potential use in human disease that requires autologous cell transplantation to prevent host-graft rejection, and to provide an alternative substance that functions like retinal pigment epithelium.

Autologous iris pigment epithelial cell transplantation in monkey subretinal region

Purpose. To establish autologous iris pigment epithelial (IPE) cell transplantation in monkey eyes. Methods. Autologous IPE cells from three monkeys were obtained by peripheral iridectomy, and cell culture was performed with autologous serum. The cultured cells were labeled with DiI, and transplantation was performed by transvitreal approach to the submacular region of each monkey. Fundus examination, photography, and fluorescein angiography were performed monthly. Histochemical analysis and electron microscopy were also performed six months after transplantation. Results. Auto IPE cells grew well in auto serum. No fluorescein leakage or retinal thickening was observed after submacular transplantation. We demonstrated the presence of autologous IPE cells in the region 6 months after transplantation by histologic and electron microscopic examination. Electron microscopy also demonstrated that the transplanted cells appeared to be less pigmented and to have less mitochondria than did the host retinal pigment epithelial cells. However, some transplanted cells looked as if they embrace the photoreceptor outer segments. Discussion. Autologous IPE cell culture of the monkey was established using auto serum. Six months after transplantation of the cultured IPE cells, we could still observe the cells in the region. This method may have potential use in human disease that requires autologous cell transplantation to prevent host-graft rejection, and to provide an alternative substance that functions like retinal pigment epithelium.

Auto iris pigment epithelial cell transplantation in patients with age-related macular degeneration: short-term results.

Autologous iris pigment epithelial cell transplantation was performed on patients with exudative age-related macular degeneration (AMD). Autologous IPE cell culture was performed using autologous serum after iridectomy in 7 patients with AMD. The cell suspensions (2 approximately 20 x 10(4) cells) were transplanted into the submacular lesion of individuals after removal of neovascular membranes. Subsequent ophthalmological examinations, including best corrected visual acuity and fluorescein or indocyanine green angiography, were performed. In addition, 15 patients with AMD, who underwent removal of neovascular membrane without transplantation, were evaluated as non randomized controls. Varying degrees of atrophy or defects of choriocapillaris and retinal pigment epithelium were observed in all of the patients. No cystoid macular edema or fluorescein leakage was observed after treatment, but window defects were present. No patient had decreased visual acuity. One treated patient developed mild subretinal fibrosis and an other patient developed mild preretinal fibrosis, however no difference was significant when compared with the control. In conclusion, the treatment resulted in no significant improvement in macular function, as compared with the control; however, no rejection or deterioration in visual acuity occurred up to the 13 month follow up.

Lipofuscin Accumulation in Iris Pigment Epithelial Cells Exposed to Photoreceptor Outer Segments

Lipofuscin accumulates in retinal pigment epithelial (RPE) cells with increasing age in response to phagocytotic degradation of worn-out disks of photoreceptor outer segments (POS). This study investigates the ability of iris pigment epithelial (IPE) cells to ingest POS and compares the phagocytotic capacity of RPE and IPE cells by measuring their amounts of lipofuscin-specific autofluorescence.IPE and RPE cells were isolated from the same calf eyes. After growing to confluence, primary cultures from 15 eyes (69 wells) were exposed to bovine POS daily, or kept as controls. After 1, 2 and 3 weeks, respectively, the cellular amounts of lipofuscin were quantified by a computerized static cytofluorometer system (Nikon Microphot SA with a Nikon p102 photometer). Lipofuscin-specific fluorescence (550 nm barrier filter eliminating nonspecific autofluorescence) of 100 randomly selected individual cells from each well was measured.Electron microscopy revealed phagosomes (containing POS disk membranes) and lipofuscin-like inclusions in the cytoplasm of RPE and IPE cells. In both IPE and RPE cultures, the POS-challenged cells had significantly higher (P<0.01) lipofuscin-specific fluorescence than the control cells. The fluorescence levels were higher in POS-challenged RPE cells as compared to IPE cells after 1 (P< 0.001), 2 (P<0.01) and 3 (P<0.001) weeks, respectively. The fluorescence level of POS-fed IPE cells at 3 weeks was 56% of that of RPE cells.IPE cells have the ability to phagocytose POS, resulting in an accumulation of lipofuscin intracellularly. The amount of lipofuscin was lower in IPE cells compared to RPE cells, probably reflecting the lower phagocytotic capacity of IPE.

Characterization of Iris Pigment Epithelial Cell for Auto Cell Transplantation

To establish auto iris pigment epithelial (IPE) transplantation, we characterized the properties of IPE cells and the method of culture using auto serum. Monkey and human IPE cells were obtained and cultured in several conditions, using auto, mouse, rabbit, bovine, or human serum. Immunocytochemical study was performed to confirm that the cells were epithelial in origin. The proliferation rate of the IPE was also calculated from fresh human IPE cells, which were obtained during filtering glaucoma surgery. Proliferation rate was also compared to that of retinal pigment epithelial (RPE) cells. Reverse-transcriptase and polymerase chain reaction for melanogenesis was performed, and the amount of pigment in the IPE cells was also calculated. Mouse and rabbit sera were not effective for the monkey IPE cell culture. Conversely, the cells grew well in the medium with auto, bovine, or human serum. Human IPE cells grew exponentially by the described methods and reached to 60,000 cells after about 4-5 weeks. When we compared them by proliferation rate, IPE cells were less proliferative than RPE cells. The gene expression for melanogenesis and the amount of pigment in the IPE gradually decreased through successive passages. Transplantation has been tried for the treatment of age-related macular degeneration using RPE from fetus or from eye bank eyes. However, focal rejection may play an important role in the clinical results. The establishment of auto IPE cell transplantation may improve the problem of rejection. In the present study, we established auto IPE cell culture using auto serum. The cultured IPE cell showed pigment epithelial cell properties until around five passages in both human and monkey.

Transplantation of autologous iris pigment epithelium to the subretinal space in rabbits.

Transplantation of autologous iris pigment epithelium (IPE) into the subretinal space has been suggested as one approach for the treatment of age-related macular degeneration. Autologous rabbit IPE cells were transplanted to the subretinal space to define the technique of transplantation and examine the survival of the transplanted cells. Autologous IPE cells were harvested by iridectomy and transplanted directly to the subretinal space of the fellow eye in 25 rabbits, using the parsplana approach. Animals were killed over a period of 5 months, and the retinas were examined morphologically by light and electron microscopy. Autologous IPE cells survived and formed a polarized monolayer above the retinal pigment epithelium in the subretinal space, with apical microvilli adjacent to photoreceptors. Fragments of phagocytosed photoreceptor rod outer segments were observed in phagosomes in the cytoplasm of IPE cells. Adjacent rod outer segments remained healthy throughout the experimental period. No signs of a cell-mediated immunologic response were observed. Our results show that in rabbits, autologous IPE cells transplanted to the subretinal space survive and do not adversely affect the photoreceptors. These results suggest that in humans, IPE cells might provide a substitute for retinal pigment epithelium cells as autologous transplants for the treatment of age-related macular degeneration.

Retinal pigment epithelial cells from Royal College of Surgeons dystrophic rats can take up melanin granules.

Many successful pigment epithelium transplantation studies involving pink-eyed Royal College of Surgeons (RCS) dystrophic rats showed highly pigmented transplanted cells forming a double layer with slightly pigmented cells, attached to Bruch's membrane. Since it is not clear whether transplanted pigmented cells can displace retinal pigment epithelial (RPE) host cells from Bruch's membrane, we suggested that RPE cells of RCS dystrophic rats can phagocytize melanin granules, possibly derived from perished transplanted cells. In a series of three experiments, RPE cells of nine pink-eyed, 2 1/2-month-old RCS dystrophic rats were isolated by trypsinization and mechanical dissection and cultivated in Dulbecco's modified Eagles' medium. These cells were then fed with melanin granules, isolated from bovine RPE cells, double-trypsinized after phagocytosis and viewed by light and electron microscopy. We also transplanted iris pigment epithelial (IPE) cells of 20-day-old Long-Evans rats into the subretinal space of pink-eyed RCS dystrophic rats of the same age, shown in light-microscopic photography after 42 days. Living RPE cells were heavily pigmented after feeding with isolated melanin granules in all three experiments as viewed by light microscopy. In addition, we identified melanin granules phagocytized by dystrophic RPE cells in electron microscopy. After transplantation of pigmented IPE cells into the subretinal space of pink-eyed RCS dystrophic rats' eyes, a layer of slightly pigmented cells was seen on Bruch's membrane below the transplanted IPE cells, shown in light microscopy. We have shown by phagocytosis assay that dystrophic RPE cells can take up melanin granules in vitro. Our results assume that pigmented cells in transplantation studies, found as a monolayer, attached to Bruch's membrane, cannot automatically be identified as transplanted cells. Instead, the possibility of perished transplanted cells serving as melanin donors for RPE host cells must be taken into consideration.

Transplantation of Autologous Iris Pigment Epithelium to the Subretinal Space in Rabbits

Background. Transplantation of autologous iris pigment epithelium (IPE) into the subretinal space has been suggested as one approach for the treatment of age-related macular degeneration. Autologous rabbit IPE cells were transplanted to the subretinal space to define the technique of transplantation and examine the survival of the transplanted cells. Methods. Autologous IPE cells were harvested by iridectomy and transplanted directly to the subretinal space of the fellow eye in 25 rabbits, using the parsplana approach. Animals were killed over a period of 5 months, and the retinas were examined morphologically by light and electron microscopy. Results. Autologous IPE cells survived and formed a polarized monolayer above the retinal pigment epithelium in the subretinal space, with apical microvilli adjacent to photoreceptors. Fragments of phagocytosed photoreceptor rod outer segments were observed in phagosomes in the cytoplasm of IPE cells. Adjacent rod outer segments remained healthy throughout the experimental period. No signs of a cell-mediated immunologic response were observed. Conclusions. Our results show that in rabbits, autologous IPE cells transplanted to the subretinal space survive and do not adversely affect the photoreceptors. These results suggest that in humans, IPE cells might provide a substitute for retinal pigment epithelium cells as autologous transplants for the treatment of age-related macular degeneration.

Cytokine gene expression after subretinal transplantation.

Transplantation study of neural retina, retinal pigment epithelial (RPE), or iris pigment epithelial (IPE) cells have been performed not only in animal model but in human age-related macular degeneration, and some of the findings reported with cystoid macular edema may have been due to graft rejection. In this investigation, we examined cytokine gene expression by reverse transcriptase-polymerase chain reaction at the transplanted subretinal space. Transplantation was performed in normal Royal College of Surgeon's rats using cultured human RPE and rat IPE. They were followed without immunosupression. Gene expression for melanogenesis of transplanted human RPE was observed only in the early days after transplantation. Rat interleukin (IL)-1alpha, -1beta1, -2, -6, interferon gamma, and tumor necrosis factor alpha (TNF alpha) genes were also expressed after the early days of transplantation. Cytokine expression was observed not only after cell transplantation but also after vehicle-only injection, which was considered a reaction to the surgical trauma. However, statistically significant amount of expressions of IL-1alpha, -1beta, and -6 were observed after the early days of transplantation of human RPE or IL-1alpha, -1beta, and TNF alpha of rat IPE, if we compare them to vehicle-only injection. These cytokines may play an important role for the local reaction after transplantation.

Expression of amiloride-sensitive sodium channel in rat eye.

To study the expression of the amiloride-sensitive sodium channel, a putative mechano-receptor in the rat eye, reverse transcriptase-polymerase chain reaction and in situ hybridization were done. The gene for the alpha subunit of the amiloride-sensitive sodium channel was shown by polymerase chain reaction to be expressed in mRNA isolated from the whole eye tissue. In situ hybridization demonstrated that the gene was expressed in basal layers of the corneal and conjunctival epithelium, ciliary epithelial cells, lens epithelial cells at the equator, retinal and iris pigment epithelial cells, ganglion cells and cells in the inner and outer nuclear layers of the retina. The results suggest that the amiloride-sensitive sodium channel plays a role in maintaining sodium balance as well as in possible mechanosensation in these ocular tissues.

The mRNA Expression of Cytokines and their Receptors in Cultured Iris Pigment Epithelial Cells: A Comparison with Retinal Pigment Epithelial Cells

It has been suggested that human iris pigment epithelial (IPE) cells isolated from iridectomized tissue could be used as autologous cells for transplantation into the subretinal space in diseases with dysfunctional retinal pigment epithelium (RPE). RPE cells synthesize a number of cytokines and their receptors which are important for its proper function. Nearly nothing is known about the capacity of IPE to synthesize cytokines or responding to them. To compare the mRNA expression of 36 cytokines or their receptors in cultured adult IPE cells and RPE cells we used semi-quantitative reverse transcription polymerase chain reactions (RT-PCR). Included in our assay were cytokines with known expression in RPE to get a broad basis for comparing IPE cells: basic fibroblast growth factor (bFGF or FGF-2), and one of its receptor (FGFR-1), epidermal growth factor (EGF), and its receptor EGF-R, transforming growth factor β(TGFβ), and its type III receptor TGFβ-R3, the platelet-derived growth factors and receptors (PDGF A, PDGF B, PDGF-Rα, PDGF-Rβ), tumor necrosis factor α(TNFα), and two receptors TNF-R1 and TNF-R2, insulin (INS) with receptor INS-R, insulin-like growth factors (IGF1, IGF2), and receptors (IGF1-R, IGF2-R), vascular endothelial growth factor (VEGF), and two receptors (VEGF-R1 or FLT-1 and VEGF-R2 or FLK-1), the receptor for VEGF-C: VEGF-R3 or FLK-4, interleukin 6 (IL6), and its receptor (IL6-R), nerve growth factor (NGF), interleukin 1α(IL1α), and a receptor (IL1-R). In addition, cytokines or their receptors not known to be expressed in RPE were included to widen our picture of cytokine gene expression in the eye: stem cell factor (SCF), its receptor (SCF-R), low-affinity nerve growth factor receptor p75 (p75(NGF-R), ciliary neutrothropic factor (CNTF), and its receptor (CNTF-R), glycoprotein 130 interleukin 6 transducer (gp130 (IL6-SD), leukemia inhibitory factor (LIF), and its receptor (LIF-R). Semi-quantitative expression data were obtained using series of fivefold dilutions of each cDNA and a fixed number of PCR cycles. The expression of RPE 65, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β2-microglobulin (B2MG) was used as a control for cellular origin, RNA quality and PCR conditions.With the exception of insulin and tumor necrosis factor αall other cytokines analysed and their receptors were expressed in both IPE and RPE cells, even though the levels varied. No qualitative or quantitative difference were observed in the mRNA expression level of 34 (94%) of the cytokines or receptors between IPE and RPE. In contrast, the mRNA expression level of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 [VEGF-RS (FLK-1)] was lower in IPE than in RPE cells. As an increased expression of VEGF in the RPE in maculae with age-related macular disease could be involved in its pathogenesis, a decreased expression of angiogenic growth factors in IPE cells could possibly be beneficial for the therapy of age-related maculopathy if indeed other tasks of non-functional RPE cells could be performed by IPE cells. The similarity of the mRNA expression pattern in 94% of the cytokines analyzed supports the assumption that IPE cells potentially can perform functions of RPE cells in the appropriate environment.

Iris Pigment Epithelial Cells of Long Evans Rats Demonstrate Phagocytic Activity

The phagocytic activities of iris pigment epithelial (IPE) cells and retinal pigment epithelial (RPE) cells of Long Evans rats towards latex beads and rod outer segments (ROS) were compared in vitro. IPE and RPE cells of Long Evans rats were isolated and pure cultures obtained. The cultures were incubated with latex beads, fixed, and analysed computer morphometrically, IPE and RPE cell cultures were also incubated with isolated ROS and examined using transmission electron microscopy. IPE cells were able to ingest latex beads. There was no significant difference between the number of latex particles phagocytized by IPE and RPE cells. After incubation with isolated ROS, IPE cells also recognized and ingested the ROS particles. However, the specific phagocytic capacity of IPE cells was 76% of that of RPE cells.The autologous IPE cells might have the potential to be used as an alternative to RPE cells for transplantation in the subretinal space.