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Purpose: To induce a posterior vitreous detachment (PVD) in porcine and human cadaver eyes in vitro with Dispase (Gibco, Grand Island, NY)., Methods: Dispase (0.5 mL) was injected into the vitreous cavity of enucleated porcine (0.05-25 U/mL) and human (5 U/mL) eyes. After incubation at 37 degrees C for 15-120 minutes, the globes were hemisected and the extent of PVD was graded as complete, partial, or absent. The structural integrity of the retina was estimated by measuring the elastic constant and maximal stretching before fracture. Retinal cell viability was determined by an intracellular esterase assay. Light, transmission, and scanning electron microscopy were performed to examine the ultrastructure of the vitreoretinal interface., Results: After 15 minutes, a partial or total PVD was present in 7/10, 8/10, or 9/10 enucleated porcine eyes incubated with 1, 5, or 10 U/mL Dispase, respectively, versus 3/10 control eyes (P < 0.05). A partial or complete PVD was present in 3/5, 4/5, 5/5, or 14/15 porcine eyes after 15, 30, 60, or 120 minutes of treatment with 0.1 U/mL Dispase, respectively. Similarly, 19/20 human cadaver eyes developed a complete and 1/20 an incomplete PVD after incubation with 5 U/mL Dispase for 15 minutes. Microscopy demonstrated that Dispase cleaved the attachment of the posterior hyaloid to the internal limiting membrane with minimal damage to the inner retina. Retinal cell viability and the mechanical properties of the retina were similar for Dispase-treated and control eyes., Conclusion: Dispase disrupts the attachment of the posterior hyaloid to the inner limiting membrane with minor morphologic changes in the inner retina. The enzyme may be useful in removing cortical vitreous during vitreous surgery.

Objective: To establish the technical feasibility and safety of photoreceptor transplantation in retinitis pigmentosa., Methods: A sheet of human photoreceptor cells was harvested from 2 human cadaveric eyes with a vibratome and transplanted into the subretinal spaces of 2 patients with advanced retinitis pigmentosa and visual acuity of no light perception by means of submacular surgery techniques. Preoperative and postoperative electrophysiologic testing, fundus photography, fluorescein angiography, and scanning laser ophthalmoscopy were performed., Results: Twelve months after photoreceptor transplantation, the visual acuity of each patient remained no light perception. The temporal edge of the retinotomy in 1 patient was folded but was not associated with a retinal detachment. The patients were not immunosuppressed, and there was no evidence of rejection of the allogeneic transplant. Cystoid macular edema, uveitis, and macular pucker were not observed., Conclusion: A sheet of adult human photoreceptor cells can be harvested from human cadaveric eyes and safely transplanted to the subretinal spaces of patients with retinitis pigmentosa without systemic immunosuppression.

Purpose: To describe a method for the harvesting and storing of intact viable sheets of adult human retinal pigment epithelial (RPE) cells., Methods: Adult human RPE cells were harvested as intact sheets from 21 cadaver eyes, using the enzyme Dispase. The sheets were embedded in 50% gelatin containing 300 mM sucrose and stored at 4 degrees C. The viability of the cells, as well as their ability to proliferate in vitro, was studied for 96 hours after harvesting. Light microscopy (LM), transmission (TEM) and scanning electron microscopy (SEM) were performed to determine the integrity and ultrastructural features of the cells. Microbiologic culture of the harvested sheets was performed to exclude contamination., Results: LM, TEM and SEM showed intact RPE cells with well-developed microvilli, basal infoldings and intercellular connections. The initial viability of intact RPE sheets was 86%, with a progressive decline in viability with increased storage time. Cells harvested within 24 hours after death maintained greater viability than those harvested after 24 hours (p < 0.05). Harvested RPE cells were free of microbial contamination and rapidly proliferated when cultured in vitro., Conclusion: Intact sheets of adult human RPE can be isolated using the enzyme Dispase. The cells appeared suitable for retinal transplantation if harvested within 24 hours of death and maintained 82% viability for as long as 48 hours if stored at 4 degrees C.

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 apoptosis in vitro. RPE cells which are removed from their substrate prior to transplantation must reattach rapidly to a substrate to prevent apoptosis.

Purpose: The purpose of the study is to identify the anatomic abnormalities associated with an absolute scotoma and the location and stability of fixation in patients with subfoveal neovascularization in age-related macular degeneration, presumed ocular histoplasmosis syndrome, and other disorders., Methods: Scanning laser ophthalmoscope microperimetry was superimposed on color fundus photographs and fluorescein angiograms of 21 eyes with subfoveal neovascular membranes secondary to age-related macular degeneration (14 eyes) and presumed ocular histoplasmosis syndrome (7 eyes). The authors determined the location and the area occupied by the absolute scotoma and each of the following subretinal lesions: subretinal hemorrhage, neurosensory retinal detachment, retinal pigment epithelial (RPE) atrophy, RPE hyperplasia, atrophy of the choriocapillaris, hard exudates, and the subfoveal neovascular membrane. The area of absolute scotoma determined by scanning laser ophthalmoscope microperimetry was superimposed on the anatomic lesions. The authors calculated the relative risk ratio (RR) of an absolute scotoma occurring in regions corresponding to each anatomic abnormality, and determined the preferred location and stability of fixation in each eye., Results: An absolute scotoma was present in areas of chorioretinal scar (RR = 107.61), RPE atrophy (RR = 9.97), subretinal hemorrhage (RR = 2.88), and the neovascular membrane (RR = 1.86). Fixation was stable in all patients with presumed ocular histoplasmosis syndrome but only 29% of patients with age-related macular degeneration. Fifty-five percent of patients with stable fixation fixated over an area of RPE hyperplasia., Conclusion: The relative risk of an absolute scotoma is highest over areas of chorioretinal scars, RPE atrophy, subretinal hemorrhage, and the neovascular membrane. Fixation is more stable in patients with subfoveal neovascularization from presumed ocular histoplasmosis syndrome than with age-related macular degeneration and frequently is present over an area of RPE hyperplasia.

Background: Transplantation of the retinal pigment epithelium (RPE) involves delivering donor RPE onto a bare area of host Bruch's membrane that is surrounded by a confluent monolayer of RPE. We investigated the effects of different plating densities and the presence of an adjacent confluent monolayer on the growth characteristics and final morphology of pig RPE in vitro., Methods: In single-well experiments, porcine RPE were plated in 24-well plates at densities varying from 1 to 75 cells/mm2. Triplicate plates were counted on the 3rd and 10th days after plating and at confluence. A multiwell chamber was built to allow cells plated at different densities to be bathed with conditioned media from adjoining wells., Results: In single-well experiments, plating at low densities increased the time to reach confluence and resulted in fewer, larger and more fusiform RPE at confluence. In multiwell experiments, the growth rate of cells plated at low density decreased as the amount of high-density medium increased in communicating wells and led to smaller, rounder cells at confluence. The presence of low-density RPE in adjoining wells increased the growth rate of RPE plated at high density and produced fewer, larger and more fusiform cells at confluence. Newly plated RPE grew more slowly when confluent monolayers of RPE were present in adjoining wells., Conclusions: Plating density is a critical factor in determining the growth rate and the final morphology of RPE in tissue culture. The presence of a neighboring confluent monolayer of RPE inhibits the growth rate of newly plated RPE in vitro.

We studied morphometrically the color transparencies and fluorescein angiograms of 5 patients with six exudative retinal arterial macroaneurysms. Our aim was to express the dependence of the exudate morphology upon the location of the macroaneurysm with an algebraic polynomial analysis. We derived the second-degree conical equations of the exudate curves and computed the location of their cardinal descriptive parameters. The relationship between the site of the macroaneurysms and the computed hypothetical parameters of their exudate curves revealed that the structural features of the exudates are dependent upon the distance of the macroaneurysm to the foveola, the gravitational force and the clearing capacity of the venous net. The point where the macroaneurysm develops approximately 3 mm from the center of the macula seems to be a 'crucial point'. Macroaneurysms located closer than this point may cause the exudates that occupy the fovea and create severe visual loss.

Twenty-one macroaneurysms and related vessels of 19 patients were evaluated morphometrically. Macroaneurysms were classified into two groups as hemorrhagic and exudative in terms of their major clinical sign. Average diameters of the macroaneurysms were arranged in a Gaussian distribution curve (mean and standard deviation: 281.60 +/- 57.28 micrometers). Regarding the distribution curve based on this data macroaneurysm can be defined as being greater than 109.76 micrometers. Macroaneurysms were most frequent on the superotemporal vessels (52.38%), followed by the inferotemporal (38.10%), inferonasal (4.76%) and superonasal vessels. Average diameter (r = +0.68, p = 0.0006) and area (r = +0.71, p = 0.0003) of the macroaneurysms were significantly correlated to the diameter of the relevant arterial segments. The distribution of the macroaneurysms in respect to arterial bifurcation (chi 2 = 18.762, p = 0.0003) and arteriovenous crossings (chi 2 = 8.286, p = 0.0405) were nonrandom with macroaneurysms clustering near this points. Hemorrhagic macroaneurysms were significantly closer to the optic disc (p < 0.01) and were located on relatively larger arterioles (p < 0.01). They were also more circular (p < 0.01) in shape and greater in area (p < 0.01) and diameter (p < 0.01) than the exudative ones. These findings suggest that the location of the macroaneurysm is closely related to its clinical appearance.

To compare the initial and follow-up discernible glaucomatous neuroretinal rim (NRR) and parapapillary chorioretinal changes in patients with primary open-angle glaucoma (POAG) and exfoliative glaucoma (EG), we analyzed 40 optic discs from 40 patients with POAG, 40 optic discs from 40 patients with EG and 20 optic discs from 20 normal subjects. The mean intraocular pressure was higher in the EG group when compared with POAG (p < 0.001). Although the mean disc areas in both groups were not significantly different, the mean initial NRR area to disc area ratio was significantly smaller in patients with EG (p < 0.001). At the initial diagnosis the most prominent NRR defects of the patients with POAG were at the inferotemporal and superotemporal sectors, followed by the temporal and nasal sectors. However, the NRR in the EG group was noticed to decrease diffusely without such a sectorial preference. There was no significant difference between the mean loss of the NRR to disc area ratio in both groups at the end of the follow-up period of 1 year (p > 0.05). The values of parapapillary chorioretinal atrophy areas in POAG and EG patients were not significantly different from each other, neither at the initial examination nor during the follow-up period (p > 0.05). Those results suggest that high intraocular pressure in eyes with EG may constitute a major risk for rapid and progressive glaucomatous optic disc damage.