Gabriele Thumann

Subretinally transplanted embryonic stem cells rescue photoreceptor cells from degeneration in the RCS rats.

The Royal College of Surgeons (RCS) rat is an animal model for retinal degeneration such as the age-related macular degeneration. The RCS rat undergoes a progressive retinal degeneration during the early postnatal period. A potential treatment to prevent this retinal degeneration is the transplantation into the subretinal space of cells that would replace functions of the degenerating retinal pigment epithelium (RPE) cells or may form neurotrophic factors. In this study we have investigated the potential of subretinally transplanted embryonic stem cells to prevent the genetically determined photoreceptor cell degeneration in the RCS rat. Embryonic stem cells from the inner cell mass of the mouse blastocyst were allowed to differentiate to neural precursor cells in vitro and were then transplanted into the subretinal space of 20-day-old RCS rats. Transplanted and sham-operated rats were sacrificed 2 months following cell transplantation. The eyes were enucleated and photoreceptor degeneration was quantified by analyzing and determining the thickness of the outer nuclear layer by light and electron microscopy. In the eyes transplanted with embryonic cells up to 8 rows of photoreceptor cell nuclei were observed, whereas in nontreated control eyes the outer nuclear layer had degenerated completely. Transplantation of embryonic stem cells appears to delay photoreceptor cell degeneration in RCS rats.

One year follow up of macular translocation with 360 degree retinotomy in patients with age related macular degeneration

Aim: To evaluate the benefits of macular translocation with 360 degree retinotomy in patients with exudative age related macular degeneration (ARMD). Methods: A consecutive interventional case series was performed on patients who underwent macular translocation between June 1997 and January 2000 at the department of ophthalmology, University of Aachen, Germany. A retrospective pilot study was set up with a minimum follow up of 12 months in 39 consecutive patients with subfoveal choroidal neovascularisation secondary to ARMD. The surgical technique included pars plana vitrectomy, induction of retinal detachment, 360 degree retinotomy, removal of the choroidal neovascular membranes (CNVM), macular translocation, peripheral laser retinopexy, and silicone oil endotamponade. Results: 18 patients showed predominantly occult CNVM, six patients had predominantly classic CNVM, and 15 showed subretinal haemorrhage. At the 12 month follow up 13 patients (33%) showed an improvement in visual acuity of more than three lines (logMAR scale), 18 patients (46%) retained stable visual acuity with a change of equal or less than three lines (logMAR scale), and eight patients (21%) showed a decrease in visual acuity of more than three lines (logMAR scale). Recurrence of CNVM was observed in three (8%) eyes at 5–11 months postoperatively. Other complications included proliferative vitreoretinopathy with retinal detachment (n=10), peripheral epiretinal membranes (n=9), macular pucker (n=2), corneal decompensation (n=2), and hypotony (n=11). 18 patients (46%) complained about persistent diplopia. Conclusion: Macular translocation surgery is able to maintain or improve distant vision in the majority of patients with exudative ARMD. Proliferative vitreoretinopathy and diplopia are the two major complications. A prospective randomised controlled trial comparing macular translocation with observation for patients with the occult form of exudative ARMD may be justified.

Natural history of choroidal neovascularization induced by vascular endothelial growth factor in the primate.

Abstract Background: A new model of choroidal neovascularization (CNV) has been developed in the primate by implanting vascular endothelial growth factor (VEGF)-impregnated microspheres in the subretinal space. Methods: CNV was induced in Macaca mulatta monkeys by implanting VEGF-impregnated gelatin microspheres in the subretinal space. Progression of CNV was followed for 24 weeks after surgery using fluorescein angiography. Eyes were enucleated at various time points, and lesions were evaluated for evidence of CNV by light microscopy and by immunohistochemical staining. Results: CNV developed in 12 (92%) of 13 eyes. Fluorescein leakage was first observed in the 2nd postoperative week and was apparent for the following 12 weeks. CD31 staining for endothelial cells was first observed at day 7 and was evident for the following 8 weeks. Glial fibrillary acidic protein staining revealed a glial adhesion between the proliferative membrane and the retina at 6 weeks after implantation. Smooth muscle actin-positive cells were found a + 2 weeks and remained prominent for at least the next 6 weeks. Cytokeratin-positive retinal pigment epithelial (RPE) cells, first identified in the proliferative membrane at day 3, predominated throughout the growth of the membrane. Macrophages (RAM-II positive) were present at day 3 but were no longer observed after day 7. Conclusion: In monkeys, subretinal implantation of VEGF-impregnated gelatin microspheres leads to the development of CNV. Early, disciform and reparative stages of CNV were observed, similar to those seen in humans. This model will be useful for studying the pathogenesis of CNV and for evaluating potential treatment strategies.

The in vitro and in vivo behaviour of retinal pigment epithelial cells cultured on ultrathin collagen membranes.

The transplantation of pigment epithelial cells as a therapeutic modality for retinal degeneration requires that the transplanted cells form a monolayer in the subretinal space that will establish communication with photoreceptors. Since previous studies have shown that transplanted cells in suspension do not form a monolayer, it will be necessary to transplant preformed pigment epithelial cell monolayers at the location of the exposed photoreceptors. To establish cell monolayers, retinal pigment epithelial (RPE) cells were cultured on ultrathin collagen membranes. Cells were examined for morphology, for characteristics of differentiation and viability. Membrane degradation and long-term biocompatibility in vivo were assessed following subconjunctival and subretinal implantation in rabbits. These studies have shown that RPE cells adhere, proliferate, form monolayers, and acquire differentiated properties on a collagen membrane that has features similar to Bruchs membrane. Membranes transplanted subconjunctivally and subretinally exhibit excellent biocompatibility without any evidence of inflammation or rejection. RPE cells cultured on collagen membranes acquire differentiated characteristics similar to those of RPE cells in vivo and form complete monolayers that are amenable to be transplanted to the subretinal space. The collagen membranes are non-toxic and do not elicit any rejection or inflammatory response when implanted subconjunctivally or subretinally in rabbits.

Phagocytosis of rod outer segments by human iris pigment epithelial cells in vitro.

Abstract · Background: We set out to evaluate the growth potential of human iris pigment epithelial (hIPE) cells in vitro, to establish whether these cells acquire the ability to phagocytose rod outer sgments (ROS) and to compare the phagocytic activity of hIPE to that of human retinal pigment epithelial (hRPE) cells. · Methods: hIPE and hRPE cells were isolated and cultured from human donor eyes and surgical specimens and growth characteristics were analyzed. HIPE and hRPE of an eye of a 46-year-old donor were used for the phagocytosis assay. Phagocytosis was evaluated by adding ROS isolated from porcine retina to cultures of hIPE and hRPE, which had been labeled with the pH-sensitive fluorescent dye, carboxy-SNAFL. After 4 h the number of ingested ROS was counted with a light microscope. For each cell type phagosomes in 500 cells were counted. The epithelial characteristics of the cells used in this study were evidenced by their morphology. · Results: Morphologically cultured hIPE are indistinguishable from the hRPE cultured from the same donor eye and show a similar pattern of cytokeratin distribution. Cultured hIPE acquire the ability to phagocytose ROS at a level slightly lower than hRPE; hIPE contained 0.76 phagosomes per cell, hRPE 0.99 phagosomes per cell. · Conclusion: The morphology of hIPE in culture and the acquisition of the phagocytic phenotype indicate that these cells have the ability to differentiate into cells that have characteristics in common with hRPE. The acquisition of phagocytic activity suggests that it is feasible to culture hIPE from surgical iridectomies and that these cultured cells can be transplanted into the subretinal space in individuals with retinal degenerations.

Quantitative Morphologic and Functional Evaluation of the Optic Nerve Head in Chronic Open-Angle Glaucoma

Glaucoma leads to morphologic changes of the optic nerve head and to functional defects. Morphologic changes in the three-dimensional surface structure of the optic nerve head at its entrance site into the globe can be examined by laser scanning tomography. The standard technique for evaluating functional defects in glaucoma is static computerized perimetry. We compared these two techniques to determine which is more sensitive for follow-up of glaucomatous damage of the optic nerve head. If decreased function is presumed to precede imminent cell death, visual field analysis should be the more sensitive method, as cell death results in absolute defects of the visual field. However, the neuronal networks do not necessarily function in this way. In the case of loss of individual elements in the neuronal network, the complex linkages, even at the retinal level, are able to maintain functions and compensate for loss of function, which means that visual field defects would not be prominent. If the damage increases with time and is accompanied by a progressive loss of ganglion cells, however, compensation is eventually no longer possible, and the functional defects then become measurable by visual field analysis. Thus, morphologic absolute changes may be more prominent than visual field defects in the early stages of glaucoma. To evaluate the quantitative relationship between morphometrically measurable defects of the optic nerve head and measurable functional defects, we first examined the visual field with static computerized perimetry and then evaluated the surface structure with a laser scanning tomograph in 90 patients with chronic open-angle glaucoma, 10 patients with ocular hypertension, and 10 patients without any eye disease. Based on the 95th percentile of the standardized rim/disk area ratio, we calculated the relative rim area loss and correlated this with the mean defect in visual field analysis. The scatterplot shows an exponential curve. In the early stages of glaucoma, visual field defects were less prominent than morphologic absolute changes; 40% of the neuroretinal rim area is lost by glaucomatous optic nerve damage before first defects in visual field analysis appear. In late stages of glaucoma, changes in perimetry are more prominent than those observed with biomorphometry. These results show that in the follow-up of patients with early stages of glaucomatous damage, special attention should be given to morphologic absolute changes. In patients with advanced glaucoma, progress of the damage should be observed by repeated functional, rather than morphologic, examinations. It is important to keep in mind, however, that the sensitivity of any method is dependent on technology. One reason why functional tests may not be as sensitive as morphologic examination in observing patients with early stages of glaucoma may simply be that functional tests are not yet sensitive enough to detect early damage.

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.

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).

Novel organotypic culture model of adult mammalian neurosensory retina in co-culture with retinal pigment epithelium.

PURPOSE The purpose of this study was to assess survival of adult mammalian neurosensory retina cultured in contact with the layer of a choroid-retinal pigment epithelium (RPE) explant. METHODS The entire adult porcine neurosensory retina and RPE-choroid layer were placed in tissue culture by juxtaposing both tissues in their original orientation. Culture of the neurosensory retina alone and freshly prepared retina were used as control. After 3 days in culture retinal explants were fixed and processed for immunohistochemistry and TUNEL technique. RESULTS We observed limited nuclei loss and significant reduction in apoptotic cells in nuclear cell layers (GCL, INL, and ONL) and decreased Muller cell hypertrophy in retina-RPE cultures compared to retinal cultures alone. In addition, cultures were characterized by reduced upregulation of GFAP, vimentin as well as S100 and increased glutamine synthetase expression. CONCLUSIONS As any tissue culture model, retinal tissue culture is a short-term system and since degenerative processes begin quite early it may be a good model to investigate degenerative processes in the retina. However, our model of culture of retina adjacent to the RPE-choroid layer improves the maintenance of neural retina as evidenced by reduced apoptosis in nuclear cell layers (GCL, INL, and ONL) and reduced gliosis as indicated by the diminished expression of glial-specific proteins and increased glutamine synthetase compared to cultures of retina alone. Thus the retina-RPE-choroid culture system can enable the evaluation of interactions between RPE and neural retina, the role of signaling molecules as well the effect of pharmaceuticals on retinal biology.

A comparison between IV paracetamol and IV metamizol for postoperative analgesia after retinal surgery

ABSTRACT Objective: To assess clinical efficacy of IV paracetamol 1 g and IV metamizol 1 g on a 24‐h dosing schedule in this randomized, double-blinded, placebo-controlled study of 38 ASA physical status I–III patients undergoing retinal surgery. Research design and methods: General anaesthesia using remifentanil, propofol, and desflurane was performed for surgery. The patients were randomly allocated to three groups, receiving infusions of paracetamol 1 g/100 mL (Para Group), of metamizol 1 g/100 mL (Meta Group), or of 100 mL of saline solution as placebo control (Plac Group) 30 min before arrival in the recovery area and every 6 h up to 24 h postoperatively. All patients had unrestricted access to intravenous opioid rescue medication. Main outcome measures: The primary efficacy variables were pain scores at rest over 30 h postoperatively analysed by using repeated ANOVA measurement. Secondary efficacy variables were pain scores on coughing, also analysed by repeated ANOVA measurement. Results: Five patients in the Plac Group and one patient in the Meta Group interrupted the study protocol. Regarding pain scores at rest, Mauchly-test of sphericity was significant ( p = 0.03). For the time effects a significant result was detected ( p < 0.001). The main effect between the three treatment groups was significantly different ( p = 0.01). The Bonferroni adjusted pair wise comparisons between the Plac Group and the Para Group showed a significant difference in favour of IV paracetamol ( p = 0.024; mean difference 14.8; 95% CI 1.6–28.0), between the Plac Group and the Meta Group in favour of IV metamizol ( p = 0.025; mean difference 14.4; 95% CI 1.5–27.4), and no significant difference between the Para Group and the Meta Group ( p = 1.0; mean difference 0.4; 95% CI –12.8 to 13.6). Pain scores on coughing showed a significant different main effect between the three treatment groups ( p = 0.022). The Bonferroni adjusted pair wise comparisons between the Plac Group and the Para Group showed a significant difference in favour of IV paracetamol ( p = 0.032; mean difference 17.9; 95% CI 1.3–34.6), a difference, though not reaching statistical significance, in favour of IV metamizol between the Plac Group and the Meta Group ( p = 0.081; mean difference 15.0; 95% CI –1.4 to 31.4), and no significant difference between the Para Group and the Meta Group ( p = 1.0; mean difference 2.9; 95% CI –13.8 6 to 19.6). None of the patients experienced itching; one patient in the Meta Group developed a mild erythema. There was no statistical difference in the incidence of nausea (Plac vs. Para Group: p = 0.94, Plac vs. Meta Group: p = 0.98, Para vs Meta Group: p = 0.95) or vomiting (Plac vs. Para Group: p = 0.73, Plac vs. Meta Group: p = 0.85, Para vs Meta Group: p = 0.86) between the groups. Patients in the Plac Group experienced significantly more often sedation than patients in the Meta Group ( p = 0.049). There was a trend of higher sedation in the Plac Group than in the Para Group, which did not reach statistical significance ( p = 0.07). There was no difference in sedation between the Meta and the Para Groups ( p = 0.84). Conclusion: IV paracetamol 1 g has a similar analgesic potency as IV metamizol 1 g for postoperative analgesia after retinal surgery.