PurposeThe purpose of this study was to establish that retinal pigment epithelial (RPE) cells take up indocyanine green (ICG) dye following systemic injection and that adaptive optics enhanced indocyanine green ophthalmoscopy (AO-ICG) enables direct visualization of the RPE mosaic in the living human eye.MethodsA customized adaptive optics scanning light ophthalmoscope (AOSLO) was used to acquire high-resolution retinal fluorescence images of residual ICG dye in human subjects after intravenous injection at the standard clinical dose. Simultaneously, multimodal AOSLO images were also acquired, which included confocal reflectance, nonconfocal split detection, and darkfield. Imaging was performed in 6 eyes of three healthy subjects with no history of ocular or systemic diseases. In addition, histologic studies in mice were carried out.ResultsThe AO-ICG channel successfully resolved individual RPE cells in human subjects at various time points, including 20 minutes and 2 hours after dye administration. Adaptive optics-ICG images of RPE revealed detail which could be correlated with AO dark-field images of the same cells. Interestingly, there was a marked heterogeneity in the fluorescence of individual RPE cells. Confirmatory histologic studies in mice corroborated the specific uptake of ICG by the RPE layer at a late time point after systemic ICG injection.ConclusionsAdaptive optics-enhanced imaging of ICG dye provides a novel way to visualize and assess the RPE mosaic in the living human eye alongside images of the overlying photoreceptors and other cells.

Background/aims-Tissue inhibitor of metalloproteinases-3 (TIMP-3) is normally synthesised by the retinal pigment epithelium (RPE) and deposited in Bruch's membrane. Mutations in the TIMP3 gene cause Sorsby's fundus dystrophy (SFD), which is characterised by thickening of Bruch's membrane, choroidal neovascularisation, and photoreceptor degeneration. To elucidate the role of TIMP-3 in human retinal degenerative diseases, we immunolocalised TIMP-3 in eyes with SFD caused by the Ser-181-Cys TIMP3 gene mutation or retinitis pigmentosa (RP; not caused by TIMP3 mutations). Methods-Standard light microscopic immunocytochemistry, including antigen retrieval, was used to localise TIMP-3 in paraYn sections of human eyes: two with SFD, three with diVerent genetic forms of RP, and two normal. (Br J Ophthalmol 1998;82:1329-1334 In Sorsby's fundus dystrophy (SFD), an autosomal dominant retinal degeneration, 1 extracellular deposits accumulate in Bruch's membrane, the five layered sheet of connective tissue that separates the retinal pigment epithelium (RPE) from its blood supply, the choriocapillaris.2 It was suggested 3 that these abnormal sub-RPE deposits interfere with transport of essential molecules from the choriocapillaris to the RPE, leading to dysfunction and death of the RPE cells and retinal photoreceptors. A serious complication of SFD is invasion of the thickened Bruch's membrane by newly formed, thin walled vessels derived from the choriocapillaris. These new vessels can grow into the subretinal space, causing exudative detachment of the RPE and photoreceptor demise. 4 Patients with SFD have mutations in exon 5 of the gene for TIMP-3 (tissue inhibitor of metalloproteinases-3).5-12 TIMP-3 is a member of a family of matrix metalloproteinase (MMP) inhibitors [13][14][15] thought to act as local regulators of matrix degradation by the MMPs. Recent studies have localised TIMP-3 protein to Bruch's membrane [16][17][18] and TIMP-3 mRNA to RPE cells, 17 19-21 and there is general consensus that the TIMP-3 in Bruch's membrane is synthesised and secreted by the RPE. It was suggested 5 6 22 that TIMP-3 normally functions for maintenance of the extracellular matrix (ECM) in Bruch's membrane and that a mutant TIMP3 gene product may lead to accumulated sub-RPE deposits in SFD by interfering with the normal balance between ECM deposition and degradation.Although many questions remain about the role of mutant TIMP-3 in the pathology of SFD, no animal models are yet available and the definitive histopathological study of SFD eyes 2 was performed before causative TIMP-3 mutations were identified. Using a recently developed monoclonal antibody (mAb) against human TIMP-3, 16 we have documented the distribution of TIMP-3 in human eyes with SFD caused by the Ser-181-Cys TIMP-3 mutation.We also wished to determine if TIMP-3 localisation in SFD was unique or might also be present in a dominant form of retinitis pigmentosa (RP) not caused by a TIMP-3 mutation but having sub-RPE deposits resembling those in SFD. 23 Finally, RPE...

Detachment of the neural retina from the retinal pigment epithelium induces photoreceptor degeneration. We studied the effects of this degeneration on the localization of two photoreceptor outer segment-specific integral membrane proteins, opsin and peripherin/rds, in rod photoreceptors. Results from laser scanning confocal microscopic and electron microscopic immunolocalization demonstrate that these two proteins, normally targeted to the newly-forming discs of the outer segments, accumulate in different sub-cellular compartments during photoreceptor degeneration: opsin immunolabeling increases throughout the photoreceptor cell's plasma membrane, while peripherin/rds immunolabeling occurs within cytoplasmic vesicles. The simplest hypothesis to explain our results is that these proteins are transported in different post-Golgi transport vesicles and separately inserted into the plasma membrane. More complex mechanisms involve having the two co-transported and then opsin finds its way into the plasma membrane but peripherin/rds does not, remaining behind in vesicles. Alternatively, both insert into the plasma membrane but peripherin/rds is recycled into cytoplasmic vesicles. We believe the data most strongly supports the first possibility. Although the transport pathways for these proteins have not been fully characterized, the presence of peripherin/rds-positive vesicles adjacent to the striated rootlet suggests a transport role for this cytoskeletal element. The accumulation of these proteins in photoreceptors with degenerated outer segments may also indicate that their rate of synthesis has exceeded the combined rates of their incorporation into newly forming outer segment disc membranes and their degradation. The accumulation may also provide a mechanism for rapid recovery of the outer segment following retinal reattachment and return of the photoreceptor cell to an environment favorable to outer segment regeneration.

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