Madoka Yoshida

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.

Iris pigment epithelial cell transplantation for degenerative retinal diseases

The transplantation of different types of cells into the eye to treat retinal diseases has advanced in the past 20 years. One of the types of cells used for transplantation is the iris pigment epithelial (IPE) cell, because autologous IPE cells are easily obtained and their properties are similar to those of retinal pigment epithelial (RPE) cells and retinal cells. IPE cells are transplanted as; freshly isolated or cultured cells to replace defective or diseased RPE cells, genetically modified IPE cells for delivering target molecules to the retina or RPE, and retinal progenitor cells. IPE cells have also been transplanted for non-retinal disorders. The survival of the transplanted cells in the host is an important factor for the success of transplantation. Autologous IPE cells have been found in the transplanted subretinal space and were able to phagocytose rod outer segments even 6 months after transplantation. Allogeneic and xenogenic cells will not remain in the region longer than autologous cells. Allogenic cells transplanted into the subretinal space are rejected in humans. Thus, we have transplanted cultured autologous IPE cells in 56 patients with age-related macular degeneration. The long-term results (more than 2 years with a maximum of 8 years) showed that the visual acuity (VA) was significantly improved over the pre-transplantation VA, although a slight decrease of VA was observed 2 weeks after the transplantation. One patient showed a vasculitis-like lesion. IPE cells that were transduced with neurotrophic factors by plasmid or viral vectors have also been transplanted in animals. We have transduced several neurotrophic factor genes into IPE cells with a plasmid vector, adeno-associated virus, or adenovirus. Transplantation of these transduced IPE cells into the subretinal space rescued photoreceptor cells from several types of photoreceptor toxicities. In addition, transduction of a gene into the IPE cells suppressed the systemic dissemination of the viral genome. The neuroprotective effects of the IPE cells were different for the different types of neurotrophic factor, and some of the neurotrophic factors may enhance systemic immune reaction after transplantation. IPE cells have also been used as retinal progenital cells because they originate from the same cell lines that give rise to the neural retina and RPE cells. The transduction of the photoreceptor-related homeobox gene was reported to induce photoreceptor phenotypes in IPE cells. Furthermore, transplantations of IPE cells have been performed to treat central nervous system disorders. In this review, we summarize recent progress on IPE transplantation.

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.

Expression of Vasohibin, an Antiangiogenic Factor, in Human Choroidal Neovascular Membranes

PURPOSE To determine whether vasohibin, an antiangiogenic factor produced by vascular endothelial cells, is expressed in the choroidal neovascular (CNV) membranes obtained from human eyes with age-related macular degeneration (AMD) or polypoidal choroidal vasculopathy (PCV). DESIGN Retrospective, interventional case series. METHODS The medical charts of 21 eyes of 21 patients with AMD or PCV who underwent surgical removal of the CNV membrane were reviewed. The removed tissues were immunostained for von Willebrand Factor (vWF), vascular endothelial growth factor (VEGF), and vasohibin. The levels of the messenger ribonucleic acid of VEGF, VEGFR2, and vasohibin were determined by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) from the CNV membranes excised from nine AMD and nine PCV patients. RESULTS The patients were divided into three groups; four patients were placed in the most active group (Group H), 13 in the less active group (Group E), and four in the nonactive group (Group S). Immunohistochemistry showed that vasohibin, vWF, and VEGF were expressed in the vascular endothelial cells in the CNV membranes and in the polypoidal vessels. RT-PCR showed that there was a strong correlation between the level of expression of VEGFR2 and vasohibin (P = .0002). Eyes with a lower vasohibin-to-VEGF ratio tended to have larger subretinal hemorrhages or vitreous hemorrhages, whereas eyes with higher vasohibin-to-VEGF ratio had subretinal fibrosislike lesions. Statistical analysis of the vasohibin-to-VEGF ratio among the three groups was significant (P = .0209). CONCLUSIONS Vasohibin is expressed in human CNV membranes. Our results indicate that the vasohibin-to-VEGF ratio may be related with the activity of the CNV.

Molecular Genetic Analysis of ABCR Gene in Japanese Dry Form Age-Related Macular Degeneration

PURPOSE To explore whether the mutation in the retina-specific ATP-binding cassette transporter (ABCR) gene, the Stargardts disease gene, contributes to the prevalence of the dry form of age-related macular degeneration (dry AMD) in Japanese unrelated patients. METHODS Twenty-five Japanese unrelated patients with dry AMD who were diagnosed by fluorescein angiography and indocyanine green angiography were chosen as the dry AMD group. None of these cases had apparent choroidal neovascularization. To detect the mutations in the ABCR gene, genomic DNA was extracted from leukocytes of peripheral blood, and 26 exons of the ABCR gene were amplified by polymerase chain reaction (PCR). All the PCR products were then directly sequenced. When a mutation was detected, the occurrence of a mutation was compared between these AMD patients and the control group. RESULTS After direct sequencing, a point mutation in exon 29 was found in one of the 25 dry AMD patients. In addition, a polymorphism in exon 45 was found in two other patients, and three sequence variations in exon 23 were detected in all patients. The incidence in AMD patients in whom a mutation in exon 29 (4%) was detected was less than that in controls (5%). Screening of the intron-exon boundaries also led to the identification of intronic mutation in intron 33. CONCLUSION In this study we found no relationship between allelic variation in the ABCR gene and the prevalence of dry AMD in Japanese unrelated patients.

Visual function after removal of subretinal neovascular membranes in patients with age-related macular degeneration

Abstract.Background: Retinal pigment epithelial (RPE) cells have been transplanted to replace the RPE cells lost after surgical excision of choroidal neovascular membranes (CNV) associated with age-related macular degeneration. The purpose of this study was to analyze the visual function of eyes with altered RPE after surgical excision of choroidal neovascular membranes (CNV) associated with age-related macular degeneration, and to determine the effect of proliferated or migrated RPE cells on visual function. Methods: Forty-seven patients with age-related macular degeneration underwent excision of CNVs following vitrectomy and tamponade with sulfur hexafluoride (SF6) or silicone oil. The appearance of pre- or subretinal fibrosis and pigmentation of the lesion was considered to indicate proliferation and migration of RPE cells. Microperimetry was also performed. Results: A significant correlation was found between the size of CNVs removed by surgery and the size estimated by indocyanine green angiography (P=0.0126). The mean number of RPE cells lost was estimated at 1.52×104. Pre- or subretinal fibrosis or pigmentation was observed in 37 patients (75.5%). The number of eyes with fibrosis was significantly higher in eyes with silicone oil tamponade than with SF6 tamponade (P=0.0016). A statistically significant correlation was not found between the presence of fibrosis or pigmentation and the postoperative visual acuity. Not all patients used the area of pigmentation for fixation, and microperimetry showed that some of the patients had scotomas in well-pigmented areas. Conclusions: Fibrosis and pigmentation after excision of CNVs may not always indicate normal function in these areas. These observations are especially relevant for transplantation of pigment epithelial cells in the future.

Evaluation of Indocyanine Green Toxicity to Rat Retinas

Purpose: To investigate the toxicity of indocyanine green (ICG) on retinal cells using cultured retinal pigment epithelium (RPE) cells and the effects of intravitreous injection of ICG into rat eyes. Methods: Cultured RPE cells were exposed to various concentrations of ICG for 2 min, a viability assay was performed 1 day after exposure. For an in vivo study, 5 µl of ICG (5 or 25 mg/ml) were injected into the vitreous cavity of rat eyes, which were examined 1, 3 and 7 days after the injection by histological and glutamine synthetase (GS) immunohistological evaluation. Results: Viabilities of RPE cells were decreased dependent on the ICG dose. In the histological evaluation, we observed differences of effects of ICG between the central retinal area and the peripheral area. ICG injection caused degeneration of all retinal layers in the central retinal area. GS immunoreactivities decreased by ICG injection, which corresponded to an area of severe destruction. Conclusion: A high concentration of ICG may cause toxic effects on retinal cells. Mueller cell dysfunction may play some role in the retinal toxicity caused by ICG.

Adult-onset Foveomacular Vitelliform Dystrophy with Retinal Folds

BACKGROUND Adult-onset foveomacular vitelliform dystrophy is characterized by a solitary, oval, slightly elevated, yellowish subretinal lesion of the fovea. We examined a patient with adult-onset foveomacular vitelliform dystrophy with stellate retinal folds by optical coherence tomography and scanning laser ophthalomoscopy. CASE A 58-year-old Japanese woman with a complaint of decreased vision in her right eye was diagnosed as having adult-onset foveomacular vitelliform dystrophy. OBSERVATIONS Ophthalmoscopic examination revealed a yellowish lesion of one-third disc diameter in size at the fovea in the right eye. Fluorescein angiography demonstrated an irregular block of choroidal fluorescence corresponding to the yellowish lesion, which was surrounded by stellate retinal folds. Optical coherence tomography images showed a steep elevation of the retinal pigment epithelium (RPE) as a focally protruded reflective band over an optically clear space. Scanning laser ophthalmoscopy provided morphologic enhancement in the specifically affected layers of the macula. Using an argon green laser, band-shaped bright reflexes were seen in the right fovea. The helium-neon laser revealed a bright patch corresponding to the yellowish lesion over the fovea, which was surrounded by stellate retinal folds. The diode laser revealed a bright patch corresponding to the yellowish lesion. CONCLUSION The stellate retinal folds of this patient were considered to be caused by the steep elevation of the RPE with an extracellular accumulation of the vitelliform deposits.

Clinical Features of Bilateral Acute Idiopathic Maculopathy

PURPOSE To describe the clinical course of bilateral acute idiopathic maculopathy (BAIM), and to analyze its pathophysiology. CASE A 33-year-old Japanese woman presented with a sudden, severe, bilateral visual disturbance following a flu-like illness. She was examined by fluorescein angiography (FA), indocyanine green angiography (IA), scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), and multifocal electroretinography (mfERG). OBSERVATIONS A diagnosis of BAIM was made in this patient based on typical ophthalmoscopic features, which included a pathognomonic yellowish-white foveal lesion. FA demonstrated a breakdown of the outer blood-retinal barrier, with the size and location corresponding to the white lesion, and IA disclosed a choroidal circulatory disturbance. SLO demonstrated that the deep retinal and choroidal layers were disorganized, and OCT showed retinal edema. Electrophysiological dysfunction was detected by mfERGs. After steroid therapy, the patients visual acuity recovered to normal. The pooling of fluorescein dye and the OCT-determined retinal edema were resolved. However, the physiological dysfunction detected by mfERGs remained. CONCLUSIONS We conclude that the major abnormality in BAIM is an alteration of the retinal pigment epithelium causing severe edema.

Two types of optical coherence tomographic images of retinal pigment epithelial detachments with different prognosis

Aims: To report the optical coherence tomographic (OCT) findings in retinal pigment epithelium (RPE) detachments. Methods: 24 eyes were analysed by OCT and by fluorescein (FAG) and/or indocyanine green angiography. Results: RPE detachments were classified by the OCT images into those with a partial or total highly reflective line in the area of the detachment (nine eyes), and those that showed the same reflex in the detached area as in other parts (15 eyes). The nine eyes had irregular hyperfluorescence by FAG, significantly larger detachments, and lower visual acuity than the 15 eyes. Conclusion: The OCT showed that two types of RPE detachments are associated with eyes with different characteristics.