Morphologic observations of retinal pigment epithelial proliferation and neovascularization in the rabbit

Neovascularization and proliferation of the retinal pigment epithelium (RPE) was induced in the rabbit by subretinal injection of vitreous without rupture of Bruch's membrane. New vessels developed between the layer of RPE and photoreceptor outer segments, but were enveloped in proliferating RPE. For this reason they were occult; no fluorescein leakage was visible by angiography. The vessels were identified only by histologic examinations. Endothelial cell budding was the initial stage of vessel development, first seen two weeks after injection. The new vessels grew from the choriocapillaris, penetrated Bruch's membrane, and spread into the subretinal space, despite the absence of subretinal fluid. Fenestrations with diaphragms were found in the endothelial walls during the earliest stages of vessel formation, and were also present in the fully matured vessels. Intermediate junctional complexes were frequently observed among the endothelial cells. During maturation of these plexi, junctions changed from open to putative tight junctions. The mature vessels were ultimately completely enveloped by collagen and RPE cells. Our results show that all new vessels in this animal model have the morphologic characteristics of choriocapillaris. We assume that they leak fluorescein, as does the choriocapillaris, but that the dye has no opportunity to pool in the subretinal space and thus cannot be seen during angiography.     

Newly-formed subretinal vessels. Fine structure and fluorescein leakage

The ultrastructure of experimentally induced newly formed subretinal vessels was correlated with the amount of fluorescein leakage demonstrated by the neovascular membranes during angiography. The membranes that demonstrated leakage contained subretinal vessels with a fenestrated endothelial wall and intermediate interendothelial cell junctions. As these subretinal plexi matured, they stopped demonstrating leakage. This involution process was accompanied by the formation of focal interendothelial tight junctions; however, loss of fenestrations was not observed. The membranes that never demonstrated fluorescein leakage also contained fenestrated subretinal vessels at both early and late stages of development; and their interendothelial junctions showed similar maturation from open to focal tight junctions. Thus all subretinal vessels had "leaky" morphology strongly resembling that of the normal choriocapillaris, whether they demonstrated fluorescein leakage or not. The authors conclude that newly formed subretinal vessels retain the characteristics of the choriocapillaris from which they are believed to proliferate; they have the potential to leak fluorescein at all stages of their development. The absence of fluorescein leakage during angiography cannot always be correlated with the absence of "leaky" morphology.     

Pathogenesis of laser-induced choroidal subretinal neovascularization.

The early stages (1 day to 3 weeks) in the development of laser-induced choroidal subretinal neovascularization were studied in the monkey eye. Histopathology revealed that the intense laser beam disrupted the choroid/Bruch's membrane/retinal pigment epithelium (RPE) complex and initiated a repair process. Although all lesions received the same energy density, the initial choroidal wound varied among the lesions: in some, the necrotic choroid was surrounded by hemorrhagic retinal detachment with RPE denudation; in others, the necrotic choroid was surrounded only by minimal damage to the RPE monolayer. Formation of the choroidal wound was followed by an inflammatory response. Later, newly formed choroidal tissue filled the wound and continued to proliferate towards the subretinal space. RPE cells from the edges of the wound proliferated over the newly formed subretinal tissue and closed the wound. In lesions with a large area of damaged RPE, coverage of the wound was slow; fluid accumulated in the subretinal space, and the lesions demonstrated pooling of fluorescein on angiography (leaky lesions). In lesions with minimal damage to RPE monolayer, closure of the wound was rapid, and the proliferating choroidal tissue did not reach the subretinal space. There was no subretinal fluid accumulation and no pooling of fluorescein on angiography (nonleaky lesions). Our results indicate that both the amount of damage of the choroid/Bruch's membrane/RPE complex and the ability of RPE cells around the damaged area to proliferate and restore the continuity of the RPE layer determine the evolution of newly formed choroidal fibrovascular tissue into a subretinal membrane with or without pooling.     

Natural history of choroidal neovascularization after surgical induction in an animal model

Purpose: To study an expanded time course of surgically induced choroidal neovascularization (CNV) in a porcine model applying fluorescence angiography and immunohistology. Methods: Twenty‐two porcine eyes underwent vitrectomy, a retinal bleb was raised and the detached retina perforated using endodiathermy. Bruch’s membrane was perforated with a retinal perforator at a site where the overlying neuroretina was normal. Eyes were enucleated in a time interval between 30 min and 42 days after the perforation, and the pigs were subsequently killed. Immediately prior to enucleation, fundus photographs and fluorescein angiograms were obtained. Sections of paraffin‐embedded eyes were immunohistochemically stained. Results: On fluorescein angiography, membranes aged 14 days or less exhibited leakage in 10/11 cases while the remaining showed persistent staining. The propensity to leak diminished with time and only 1/3 of the oldest membranes leaked. In eyes enucleated immediately after surgery, neuroretinas overlying the induced lesions were intact without apparent atrophy of cells. At day 3, macrophages and myofibroblasts formed membrane‐like structures in the subretinal space. At day 7, the outer surface of the membrane was covered by retinal pigment epithelium (RPE) cells and the neuroretinas had suffered damage in the form of outer segment loss. In the time period 14–42 days, the CNV membrane became completely enveloped by RPE cells. The degree of membrane vascularization increased with time and was at its maximum after 42 days. Intact outer segments were identified over the oldest membranes. Conclusion: The formation of surgical CNV membranes followed the normal reparatory pathway and the degree of vascularization of CNV membranes continued to increase during the 42 days. However, propensity to leak diminished with time. We believe that this was because of the fact that RPE cells completely enveloped older membrane and thus prevented leakage from the newly formed vessels. Photoreceptor outer segments, which had atrophied after 7 days, were able to regenerate over CNV membranes and could be identified again after 42 days.     

Morphologic fluorescein angiographic, and light microscopic features of experimental choroidal neovascularization

We induced choroidal neovascularization in the rhesus monkey by impoverishing the blood supply to the inner retina and producing defects in Bruch's membrane by photocoagulation. Fourteen of 46 eyes undergoing photocoagulation developed neovascular fronds which were identified and categorized by histopathologic examination and fluorescein angiography. All new vessels gained access to the retina through defects in Bruch's membrane at the site of photocoagulation marks. In eight eyes the new vessels remained localized to the immediate vicinity of photocoagulation marks. In four eyes neovascular fronds infiltrated the subretinal space for distances up to 6 disk diameters from the point of entry into the retina. In the two eyes choroidovitreal neovascular complexes developed but rapidly regressed shortly after gaining the vitreous cavity. Fluorescein angiography demonstrated that all neovascular fronds were grossly incompetent to dye but that formed feeding channels had some degree of integrity. Light microscopic studies showed the proliferating networks to be composed of capillaries with well-formed basement membranes and more mature vessels with the basic structure of choroidal arteries and veins.     

Experimental subretinal neovascularization in the rabbit

Subretinal neovascularization (SRN) in the rabbit was induced by subretinal injection of vitreous without rupture of Bruch's membrane. Eight of 26 eyes developed SRN. The incidence of SRN rose from 33% to 57% in a period of 4–40 weeks. Because of the absence of any fluorescein angiographic indication of SRN, these occult new vessels were identified by light and transmission electron microscopy. Histological examination showed that these newly formed vessels are composed of continuous capillaries with the morphologic characteristics of choriocapillaris, including diaphragmed fenestrations, basement membranes, and junctional complexes. The new vessels originated from the choriocapillaris and penetrated through Bruch's membrane into the subretinal space, where they were associated with the degenerated sensory retina and proliferating glial and/or RPE cells. This experiment provides a model of SRN without breaks in Bruch's membrane.     

Dye laser photocoagulation treatment for experimental subretinal neovascularization. 2. Histopathological findings of unsuccess lesions

We observed the histopathological process in unhealed lesions following photocoagulation treatment for experimental subretinal neovascularization (SRN) in rhesus monkey eyes. Long-lasting SRNs were produced experimentally by the method which we previously reported. These SRNs were treated by a 590nm dye laser beam, and were examined clinically and histopathologically. Lesions in which insufficient photocoagulation treatment was performed showed serous fluid accumulation and fluorescein angiography revealed remaining neovascularization. In these lesions abundant patent neovascularization was seen with proliferation of spindle-shaped fibroblast-like retinal pigment epithelial (RPE) cells in the subretinal space. These metaplastic RPE cells produced matrix and abundant collagen fiber around the cells. The above results showed that insufficient photocoagulation induced neovascularization and proliferation of RPE cells in the subretinal space and promoted the formation of fibrovascular membrane.     

Comparative study of experimental choroidal neovascularization by optical coherence tomography and histopathology

PURPOSE: To study the development, progression, and regression of experimental choroidal neovascularization (CNV) by correlating the cross-sectional images from sequential optical coherence tomography (OCT) with histopathologic sections of the same retinal regions. METHODS: Laser photocoagulation was performed in the posterior pole of the eye of 4 rhesus monkeys to induce CNV. Funduscopy, fluorescein angiography (FAG), and OCT were performed on day 1 and weekly for 13 weeks. Histological serial sections of CNV tissue were compared to corresponding OCT images. RESULTS: In the developmental stage of CNV, the CNV was observed by OCT as a nodular high reflex area continuing from the highly reflective retinal pigment epithelium (RPE). Histopathological studies showed that the CNV was composed of tightly packed proliferated RPE and immature vascular endothelial cells. In the active stage, OCT revealed a thick multi-layered high reflex area under the sensory retina. This high reflex area corresponded with the CNV membrane that consisted of newly formed blood vessels with wide vascular lumens and proliferated spindle-shaped RPE cells. In the regressive stage, OCT revealed a dome-like, white-colored highly reflective layer continuing from the RPE layer with moderate reflection beneath the layer. Histopathologically, the neovascular tissue was enveloped by mono-layered, cuboidal-shaped RPE cells with melanin granules. CONCLUSION: Optical coherence tomography images clearly demonstrated the positional relationship between the CNV and the RPE. Optical coherence tomography imaging provides information on the CNV which complements conventional examinations by funduscopy and FAG.     

A role of the retinal pigment epithelium in the involution of subretinal neovascularization]

We clarified a role of the retinal pigment epithelium (RPE) in the regression of experimentally induced subretinal neovascularization (SRN) in monkey. Eight eyes of 5 rhesus monkeys were used in this study. Two weeks after intense krypton laser photocoagulation to the posterior pole of the fundus, 0.5M l-ornithine hydrochloride solution 0.03 ml was injected intravitreously for the purpose of selective RPE damage. After ornithine injection, SRN continued without any evidence of spontaneous regression over 8 weeks following photocoagulation. Histopathologically, SRN developed with wide lumen in the subretinal space accompanied with serous detachment of the sensory retina, and new vessels were not enveloped completely by the proliferating RPE cells. We already showed that experimentally induced subretinal neovascularizations naturally regress spontaneously by envelopment of RPE cells 5 to 8 weeks after photocoagulation. Our results suggested that SRN persist actively without regression due to incomplete enclosure by RPE by selective damage of RPE at the active stage of SRN. We have confirmed that the RPE cells played an important role at the involution stage of SRN.     

Spontaneous involution of subfoveal neovascularization

We studied five patients who maintained or spontaneously regained significant central vision despite the presence of a subfoveal neovascular membrane. Sequential photographs and fluorescein angiograms showed a pattern of development common to these patients and not observed in patients who lose all central vision. The pattern involves formation of a pigmented ring around the subfoveal neovascular membrane followed by alteration of the membrane from one that leaks fluorescein to one that stains but does not leak. This pattern is associated with gradual resorption of subretinal fluid and apparent limitation of subretinal hemorrhage and fibrosis. This course suggests the occurrence of spontaneous involution of some subfoveal neovascular membranes and implicates the retinal pigment epithelium in this process.     

Selective retina therapy in patients with central serous chorioretinopathy

Background Central serous chorioretinopathy (CSC) is a disease with a localized breakdown of the outer blood–retinal barrier located within the retinal pigment epithelium (RPE) causing subretinal fluid accumulation. Selective retina therapy (SRT) is a new, minimally invasive laser technology that has been designed to selectively target the RPE. SRT spares retinal tissue.Methods Twenty-seven eyes of 27 patients with active CSC were treated with SRT using a pulsed double-Q-switched Nd-YLF prototype laser (λ=527 nm, t=1.7 μs). At baseline, best-corrected visual acuity was determined and fluorescein angiography and optical coherence tomography were performed. The patients were followed for up to 3 months.Results After 4 weeks 85.2% of patients showed complete resolution of subretinal fluid and in 96.3% there was no leakage visible on fluorescein angiography. After 3 months 100% of patients demonstrated no subretinal fluid and 100% of patients had no leakage activity on fluorescein angiography. Visual acuity, 20/40 at baseline, improved to 20/28 after 4 weeks and to 20/20 after 3 months.Conclusion SRT is a safe and effective treatment for active CSC. Especially if the RPE leak is located close to the fovea, SRT is the favoured therapeutic option. We recommend earlier treatment of patients with acute CSC in order to prevent development of chronic changes due to CSC with irreversible anatomical and functional damage. SRT might be considered as a first-line treatment for active CSC.     

The treatment of serous macular detachment secondary to choroidal melanomas and nevi

The authors successfully treated with laser photocoagulation five patients who had a serous macular detachment secondary to leakage from a pigmented choroidal tumor. Three patients had dye leakage on fluorescein angiography from a choroidal neovascular membrane. The subretinal fluid resolved in all three patients after the neovascular membrane was obliterated by either krypton red (2 patients) or argon green (1 patient) laser photocoagulation. The other two patients had prominent leakage from a localized choroidal neovascular membrane as well as mild diffuse leakage over the entire tumor. Focal argon laser treatment to the neovascular membrane alone did not cause permanent resolution of the subretinal fluid. The fluid did resolve, however, after the entire area of diffuse leakage was retreated with photocoagulation. In one of the patients who received both focal and confluent laser treatment, the choroidal tumor grew in a collar-button fashion through Bruch's membrane at the original site of focal treatment. This eye was enucleated, and results of histopathologic examination showed a choroidal melanoma.     

Photodynamic therapy with verteporfin in mallatia leventinese

OBJECTIVE: To describe a case of a patient with documented genetic mallatia leventinese who developed a classic choroidal neovascular membrane and underwent photodynamic therapy (PDT) with verteporfin (Visudyne; CIBA Vision Corp., Duluth, GA). DESIGN: Interventional case report. INTERVENTION: The patient underwent a complete ophthalmologic evaluation and fluorescein angiography. Photodynamic therapy with verteporfin was performed. MAIN OUTCOME MEASURES: Clinical and angiographic records were analyzed for evidence of changes in visual acuity, clinically evident subretinal fluid and the extent of fluorescein leakage from choroidal neovascularization (CNV). RESULTS: Three weeks after treatment, a fluorescein angiogram showed closure of the neovascular membrane, no evident subretinal fluid was seen, and visual acuity had improved from 20/60- to 20/40. Nine weeks after the application, fluorescein angiography demonstrated a microscopic hyperfluorescent spot at the site of the previously active CNV at which a small area of shallow subretinal fluid was observed, and visual acuity was 20/50. Thirty-four weeks after PDT, visual acuity was 20/60, subretinal fluid resolved, and fluorescein angiography did not show any further changes. CONCLUSIONS: Photodynamic therapy with verteporfin may be considered as a possible treatment in patients with mallatia leventinese who develop classic CNV.     

Long-term effects of short-term retinal bleb detachments in rabbits

PURPOSE: To examine the effects of saline-induced bleb detachments in rabbit retina. METHODS: Retinal bleb detachments were produced by the injection of 50 microl of balanced salt solution (BSS) into the subretinal space of one eye of each of six rabbits using a glass pipette with a flat tip, 50 microm in diameter. The retina was examined by biomicroscopy, scanning laser ophthalmoscopy (SLO), auto-fluorescence and simultaneous fluorescein and indocyanine green (ICG) angiography. Histological examination was carried out at 1, 2, 3 and 4 months after surgery. RESULTS: All rabbits showed leakage of fluorescein for at least a day after detachment, but within 1 month the leakage ceased. ICG staining developed gradually at the level of the RPE or Bruch's membrane near sites of previous staining. Lipofuscin fluorescence also developed gradually around areas of staining. Histology revealed the source of the excessive lipofuscin to be in the RPE layer, especially in cells migrating away from Bruch's membrane. CONCLUSIONS: Short-term bleb detachments cause a transient breakdown in the blood-retinal barrier, long-term ICG staining at or deep to the RPE layer, hyperlipofuscinosis and migration of the RPE. The abnormal lipofuscin accumulation is apparent on fluorescence ophthalmoscopy and can be confused with markers such as green fluorescent protein.     

Long-term outcome of RPE allografts to the subretinal space of rabbits.

PURPOSE: To determine the long-term RPE allograft survival in the subretinal space using suspensions of RPE cells and atraumatic transplantation surgery. METHODS: Nineteen albino rabbits were transplanted with suspensions of pigmented RPE cells from brown rabbits. Following pars plana vitrectomy, the RPE cell suspension was injected through a small retinotomy using a glass micropipette into the subretinal space under microscopic control. No immunosuppression was used. The eyes were monitored by biomicroscopy, color fundus photography, and fluorescein angiography. Rabbits were sacrificed at 1, 3 and 6 months, respectively, and the eyes processed for light and electron microscopy, using monoclonal antibodies for identifying macrophages. RESULTS: Transplanted RPE cells were present in the subretinal space in all eyes at 6 months. There was no fluorescein leakage. Generally, the RPE allograft formed a monolayer, but focal fragmentation and disruption with dispersion of melanin pigment occurred. Foci of multilayers of cells in the subretinal space, containing large macrophages, were associated with adjacent photoreceptor damage. There was no infiltration of lymphocytes but macrophages and glial cells were contiguous to the transplant. Cells harboring intracytoplasmatic melanin pigment were observed in the neural retina. CONCLUSION: Transplantation of RPE cell suspensions to the subretinal space generally forms a monolayer that persists at 6 months. However, in areas of multilayers of RPE cells and macrophages, graft failure occurs in combination with adjacent photoreceptor damage. Graft failure is not associated with the infiltration of lymphocytes, but other mechanisms seem to occur.     

Allogenic fetal retinal pigment epithelial cell transplant in a patient with geographic atrophy

PURPOSE: To test the hypothesis that healthy fetal retinal pigment epithelium (RPE) can rescue the remaining viable RPE and choriocapillaries and thereby the photoreceptors in non-neovascular age-related macular degeneration (ARMD) (geographic atrophy [GA]). METHODS: A 65-year-old legally blind woman with non-neovascular ARMD underwent fetal RPE transplantation. Best-corrected visual acuity testing, detailed fundus examination, fundus photography, fluorescein angiography, scanning laser ophthalmoscope macular perimetry, and humoral and cellular immune response testing were performed. A suspension of RPE was infused into the subretinal space through a retinotomy along the superotemporal arcade at the edge of the area of GA. The patient did not take systemic immunosuppressants. RESULTS: The patient's vision remained unchanged for 5 months after the surgery. Fluorescein angiography after transplantation showed leakage and staining at the level of the outer retina. There was progressive subretinal fibrosis in the area of the transplant. Immune response studies showed a weakly positive mixed lymphocyte response against phosducin and rhodopsin. CONCLUSION: Although it is surgically feasible to transplant fetal RPE to the subretinal space of patients with GA, such an allogenic RPE transplant without immunosuppression leads to leakage on fluorescein angiography and eventual fibrosis. A very weak immune response against proteins associated with photoreceptors is also of concern.     

Role of psychological stress and the hypothalamicpituitary -adrenal axis in the pathophysiology of central serous chorioretinopathy

Central serous chorioretinopathy (CSC) is characterized by serous detachment of the sensory retina as a consequence of the focal leakage of fluid from the choriocapillaries to subretinal space through a defect of the retinal pigment epithelium (RPE). The exact cause of CSC has not well unknown. Psychological stress is thought to contribute to CSC, but the physiologic mechanisms are unclear. It is hypothesized that psychological stress can induce CSC through the mechanism of the hypothalamic-pituitary-adrenal (HPA) system. Psychological stress can adversely affect HPA axis and causes glucocorticoid levels to elevate. Increased glucocorticoids constrict choroid vessels, which leads to ischemia of choroids and damage vascular endothelial cells, thus causing vasopermeability to increase. RPE dysfunction will occur as a result of abnormalities in the choroidal circulation. The large molecules including protein may enter the subretinal space through the damaged vessels and RPE.     

Juxtapapillary subretinal pigment epithelial polypoid pseudocysts associated with unilateral tilted optic disc: case report with clinicopathologic correlation

OBJECTIVE: To describe the unique preoperative appearance, successful postoperative clinical course, and histopathologic features of a cluster of progressively enlarging pseudocysts that arose at the temporal margin of a unilateral tilted optic disc. STUDY DESIGN: Case report. METHODS: Clinical observation, color fundus photography, fluorescein angiography, and optical coherence tomography, as well as routine histologic and immunohistochemical studies of tissue removed by subretinal surgery. RESULTS: Subretinal surgical excision of the lesions resulted in retinal reattachment with improved postoperative visual acuity. Histologic examination disclosed a cluster of fluid-filled polypoid pseudocysts lined by small vessels of choroidal origin lying beneath the basement membrane of the overlying retinal pigment epithelium (RPE). CONCLUSIONS: We postulate that buds of small vessels of choroidal origin grew through or around the edge of Bruch's membrane at the temporal margin of the tilted optic disc and then passed under the juxtapapillary RPE. Ensuing leakage of proteinaceous fluid from these vessels eventuated in formation of a cluster of polypoid pseudocysts and subsequent localized papillomacular retinal separation with visual loss. The lesions were amenable to subretinal surgical removal with restoration of visual acuity.     

Slow leakage from the retinal pigment epithelium (ooze) in age-related macular degeneration

The authors describe a condition called retinal pigment epithelial (RPE) ooze in patients with age-related macular degeneration. Patients with ooze have a neurosensory retinal detachment, a slowly progressive indiscrete leakage of fluorescein dye from the level of the retinal pigment epithelium, and no other signs of choroidal neovascularization. Sixteen patients with RPE ooze were followed for a mean of 4.5 years without treatment. Nine patients had a single round or oval area of leakage. In seven of these patients, choroidal neovascular membranes developed, with moderate to poor visual outcomes. The other seven patients had multiple smaller areas of leakage from the level of the RPE. None of these patients had a choroidal neovascular membrane, and all had resolution of the subretinal fluid with good final visual acuities. Patients with discrete round or oval RPE oozes appear to be at a high risk for the development of more definite choroidal neovascular membranes and vision loss.