Lucian V. Del Priore

Reattachment to a substrate prevents apoptosis of human retinal pigment epithelium

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

Retinal pigment epithelial cell transplantation after subfoveal membranectomy in age-related macular degeneration: Clinicopathologic correlation

PURPOSE To report the histopathology after retinal pigment epithelial cell transplantation and subfoveal membranectomy in age-related macular degeneration. METHODS An 85-year-old white woman with bilateral choroidal neovascularization underwent subfoveal membranectomy combined with transplantation of a sheet of human adult retinal pigment epithelium (retinal pigment epithelium) under the foveal center in the right eye. The patient was immunosuppressed postoperatively with prednisone, cyclosporine, and azathioprine. The patient died from congestive heart failure 114 days after surgery. RESULTS A patch of hyperpigmentation was visible at the transplant site under the foveola after surgery. Mound-like clusters of individual round, large densely pigmented cells were present in the subretinal space and outer retina in this area. There was loss of the photoreceptor outer segments and native retinal pigment epithelium in the center of the transplant bed, with disruption of the outer nuclear layer predominantly over regions of multilayered pigmented cells. Cystic spaces were present in the inner and outer retina. A residual intra-Bruchs membrane component of the original choroidal neovascular complex was present under the transplant site. CONCLUSIONS The transplant site contained clusters of round, pigmented cells that did not form a uniform monolayer in most areas. The morphology at the transplant site is consistent with the lack of visual improvement seen after surgery in this patient.

Retinal Pigment Epithelial Debridement as a Model for the Pathogenesis and Treatment of Macular Degeneration

PURPOSE To determine the effects of the absence of the retinal pigment epithelium on the choriocapillaris and outer retina by performing retinal pigment epithelial cell debridement with mitomycin C to inhibit cell proliferation pharmacologically in the porcine eye. METHODS A pars plana vitrectomy was performed in 12 eyes, and two neurosensory retinal detachments per eye were created by injecting 10(-3) mg/ml mitomycin C and 0.25% edetic acid into the subretinal space. Twenty minutes later, the retinal pigment epithelium was debrided, and the retina was reattached with a fluid-gas exchange. RESULTS Bruchs membrane was devoid of native retinal pigment epithelium, and the choriocapillaris was patent immediately after debridement. No proliferation of the retinal pigment epithelium occurred 1 week after debridement, and choriocapillaris atrophy was present beneath areas of Bruchs membrane that were devoid of retinal pigment epithelium. Four weeks postsurgery, choriocapillaris atrophy persisted in all debrided blebs, although unpigmented retinal pigment epithelium repopulated portions of Bruchs membrane in one of three blebs. Outer retinal atrophy was present in areas of Bruchs membrane with no retinal pigment epithelium and no choriocapillaris 4 weeks postsurgery. The choriocapillaris was patent in areas of mitomycin C injection without debridement. CONCLUSION Absence of the retinal pigment epithelium leads to atrophy of the choriocapillaris within 1 week after surgery. This finding provides an animal model to study transplantation of retinal pigment epithelium onto bare patches of Bruchs membrane in age-related macular degeneration and other diseases and provides insight into the pathogenesis of nonexudative age-related macular degeneration.

Correlation between Scanning Laser Ophthalmoscope Microperimetry and Anatomic Abnormalities in Patients with Subfoveal Neovascularization

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.

Treatment of Persistent Glaucoma Secondary to Periocular Corticosteroids

PURPOSE To describe two patients with uveitis who developed increased intraocular pressure that was unresponsive to maximum medical therapy eight and 13 months after periocular injection of triamcinolone acetonide. METHODS Excised periocular tissue was analyzed for corticosteroid activity by gas chromatography and mass spectrometry. RESULTS Excision of the periocular tissue, which contained visible triamcinolone acetonide, resulted in a normal intraocular pressure within 14 days in both patients. Analysis of the excised tissue disclosed residual corticosteroid in one of the two patients. CONCLUSION Removal of periocular tissue containing injected corticosteroids may facilitate the management of patients developing increased intraocular pressure unresponsive to maximum medical therapy.

Histopathologic Examination of Vascular Patterns in Subfoveal Neovascular Membranes

BACKGROUND Subfoveal neovascular membranes cause significant visual loss in age-related macular degeneration (AMD) and the ocular histoplasmosis syndrome. The frequency of post-laser treatment persistence or recurrence of subfoveal membranes in AMD is as high as 51%. The reason for the high incidence of failure after laser treatment is unknown. The authors performed a histopathologic study of subfoveal membranes to determine the distribution of blood vessels within the neovascular complex, and to see if the blood vessel pattern would provide insight into the reason for laser treatment failure. METHODS The authors used light microscopy to examine serial sections of subfoveal membranes from six patients (4 with AMD, 2 with the ocular histoplasmosis syndrome). The data from this examination were used to create detailed two-dimensional vascular maps of each membrane. RESULTS The authors found that subfoveal membranes from patients with AMD and the ocular histoplasmosis syndrome, whether occurring de novo or after laser treatment, have a nonuniform distribution of blood vessels, and that large areas which include the membrane margin may be avascular. CONCLUSIONS Using current laser treatment protocols, it is likely that avascular or poorly perfused peripheral areas of the neovascular complex would be left untreated after laser photocoagulation. Partial treatment of the neovascular complex may contribute to the high rate of post-laser treatment persistence or recurrence of subfoveal membranes.

Experimental and Surgical Aspects of Retinal Pigment Epithelial Cell Transplantation

Abstract OBJECTIVES: To discuss the current status of experimental studies on transplantation of the retinal pigment epithelium (RPE), and surgical results on the excision of subfoveal choroidal new vessel membranes in age-related macular degeneration (AMD) and presumed ocular histoplasmosis (POHS). STUDY DESIGN: Experimental study on transplantation of the RPE in organ culture and in vivo, and results of a prospective series on surgical excision of choroidal neovascularization. SETTING: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine. PATIENTS: Patients with subfoveal neovascular membranes secondary to AMD and POHS. MAIN OUTCOME MEASURES: Histological results on transplantation of the RPE onto Bruchs membrane in experimental animal models, and visual results on surgical excision of subfoveal choroidal neovascularization in patients with AMD and POHS. RESULTS: We have demonstrated that the RPE monolayer can heal after surgical debridement in the experimental animal, and retinal recovery occurs after healing of the RPE monolayer. We have demonstrated that RPE can reattach to healthy Bruchs membrane in vitro, and have performed RPE transplantation into the subretinal space in vivo. CONCLUSIONS: We are currently involved in a multifaceted approach aimed at RPE transplantation and surgical reconstruction of the photoreceptor-RPE-Bruchs membrane interface. We are hopeful that our approach will be applicable to the clinical problem of subfoveal choroidal new vessel membranes in AMD and POHS, in addition to the broad applicability of RPE transplantation to patients with diseases arising from primary RPE dysfunction.

Uveal Effusion Syndrome Associated With Primary Pulmonary Hypertension and Vomiting

PURPOSE We treated a 69-year-old woman with bilateral uveal effusions associated with primary pulmonary hypertension, congestive heart failure, and vomiting. METHODS The patient underwent ophthalmoscopic, echocardiographic, and fluorescein angiographic examinations. RESULTS Increased venous pressure caused by congestive heart failure and vomiting was implicated as the cause of bilateral uveal effusions. The uveal effusions responded well to systemic furosemide. CONCLUSION Primary pulmonary hypertension with right-sided congestive heart failure and vomiting can lead to the development of uveal effusions.

Perfusion of the Subfoveal Choriocapillaris Affects Visual Recovery After Submacular Surgery in Presumed Ocular Histoplasmosis Syndrome

PURPOSE To determine the relationship between the visual result and perfusion of the subfoveal choriocapillaris after surgical excision of subfoveal neovascularization in presumed ocular histoplasmosis syndrome. METHODS We reviewed the records of 38 eyes of 37 patients with gradable postoperative fluorescein angiograms and color photographs after surgical excision of a subfoveal neovascular membrane in presumed ocular histoplasmosis syndrome. The postoperative photographs and fluorescein angiograms were graded in a masked fashion for the presence of perfusion of the subfoveal choriocapillaris. We used preoperative and postoperative best-corrected visual acuities to determine the correlation between postoperative perfusion of the subfoveal choriocapillaris and both final visual acuity and visual improvement after surgery. RESULTS After surgery, the subfoveal choriocapillaris was perfused in 24 of the 38 eyes (63%) and nonperfused in 14 (37%). Best-corrected visual acuity improved by at least 2 Snellen lines in 17 of the 24 perfused eyes (71%) and two of the 14 nonperfused eyes (14%) (P = .0089). Best-corrected visual acuity of 20/100 or better was achieved in 18 of the perfused eyes (75%) and four nonperfused eyes (29%) (P = .0076). CONCLUSION Both final visual acuity and improvement in visual acuity were correlated with postoperative perfusion of the subfoveal choriocapillaris in patients with presumed occular histoplasmosis syndrome. Development of techniques to maintain or reestablish perfusion of the subfoveal choriocapillaris after surgery may improve visual outcome in these eyes.

Retinal Pigment Epithelial Transplantation in the Management of Subfoveal Choroidal Neovascularization

Clinical and laboratory evidence suggests that transplantation of the retinal pigment epithelium (RPE) may prove beneficial in the treatment of patients with subfoveal choroidal neovascularization. In this report, we review the clinical results of surgical excision of subfoveal choroidal neovascularization in the presumed ocular histoplasmosis syndrome (POHS), age-related macular degeneration (AMD), and other disorders. We show that postoperative atrophy of the subfoveal choriocapillaris, which is present in 90% of AMD eyes and 37% of POHS eyes, is an important prognostic factor in visual recovery after subfoveal surgery. Because the RPE is frequently removed with the choroidal neovascular membrane, atrophy of the subfoveal choriocapillaris may be caused by absence of the RPE. In view of these considerations, we developed an animal model to understand the effects of RPE removal on the overlying retina and underlying choriocapillaris. These experiments show that atrophy of the choriocapillaris develops in ...