Time-course of experimental choroidal neovascularization in Dutch-Belted rabbit: clinical and histological evaluation

In order to study the pathogenesis of choroidal neovascularization (CNV) and explore a suitable animal model for assessing anti-angiogenic agents, rabbit CNV was induced by subretinal injection of a cocktail containing endotoxin and growth-factor, incorporated in Heparin-sepharose beads. The presence and development of CNV lesions was visualized by fluorescein angiography and quantified by image analysis. The time-course of experimental CNV was evaluated clinically and histologically, with some lesions followed up to 3-years. The suitability of this model for drug evaluation was appraised by the systemic administration of dexamethasone. The experimental results suggest two subsets of CNV, primary and secondary, observed in all treated eyes. Primary CNV, defined as neovascularization extending into the subretinal space and associated with injury to Bruch's membrane at the time of injection, was visible in 100% of eyes by 2-weeks and stable by 3-months. Secondary CNV, defined as neovascularization extending into the sub RPE space away from the initial injection related injury, became visible as early as 2-weeks in some of eyes and developed in 100% of eyes by 8-months. Both primary and secondary CNV were maintained and demonstrated leakage throughout the entire observation period. Atrophy of primary retinal pigmented epithelium (RPE) cells and hyper-proliferation of secondary RPE cells were observed in tissue sections with CNV lesions. The formation and growth of primary and secondary CNV were significantly inhibited by dexamethasone. This study indicates that a reproducible and quantitative model of rabbit CNV has been established utilizing subretinal administration of endotoxin and growth-factor. Studies of the stages of experimental CNV both clinically and histologically indicated an intimate relationship between CNV, macrophages and RPE. Furthermore, the inhibition observed with dexamethasone points to the possibility of being able to evaluate effective means of pharmacological intervention.     

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.     

Peripapillary subretinal neovascularization in retinoblastoma

Background Peripapillary subretinal neovascularization (PSRNV) is a rare type of choroidal neovascularization. Herein we report a case of retinoblastoma complicating PSRNV, and discuss the histopathological findings. Methods A 1-year-old male underwent enucleation of his right eyeball based on the clinical diagnosis of bilateral retinoblastoma after chemotherapy. Results There was a mass arising from the retina showing highly calcified and necrotic retinoblastoma. The peripapillary region revealed neovascular membrane extending from the optic nerve head to the subretinal space. The membrane included retinal pigment epithelial (RPE) cells and glial cells, as well as endothelial cells. Immunohistochemistry revealed cytokeratin 18-positive RPE cells situated beneath glial fibrillary acidic protein-positive glial cells and their processes. The neovascular membrane did not have a connection with vessels arising from the optic nerve head. There were multiple mound foci made up of proliferated RPE cells in the globe. Conclusion These results suggest that migration of RPE cells and glial cells plays a crucial role in the pathogenesis of PSRNV, which might be directly or indirectly mediated by retinoblastoma.     

Choroidal neovascularization can digest Bruch's membrane. A prior break is not essential

A novel form of retinal phototoxicity has facilitated investigation of the effect of retinal pigment epithelial (RPE) damage on the choriocapillaris of the rat. In response to isolated RPE injury, the choriocapillaris buds, digests the endothelial basement membrane, and projects pseudopodia that erode Bruch's membrane. The development of subretinal neovascularization in one of the rats demonstrates that breaks in Bruch's membrane may result from this lytic process and that neither degeneration of Bruch's membrane nor a preexisting break is essential for subretinal neovascularization to occur.     

Choroidal neovascularization in long-standing case of Vogt-Koyanagi-Harada disease

The eyes in a case of Vogt-Koyanagi-Harada disease (VKH) with long-standing uveitis for 26 years after the onset were studied histopathologically. It was found that typical granulomatous inflammation was persistent in the uveal tract and the choroidal neovascularization occurred in the peripheral fundus accompanied by proliferation of the retinal pigment epithelial cells (RPE). Some of the new vessels under the pigment epithelium extended into the vitreous. It was concluded that the ocular inflammation of VKH was essentially granulomatous even in this long-standing case. Disappearance of choroidal melanocytes, existence of epithelioid cells containing pigment granules, and accumulation of lymphocytes and plasma cells in the lesion indicated that the inflammation was an autoimmune reaction against uveal melanocytes, although the trigger initiating the disease remains unknown. It was further concluded that the peripheral fundus as well as the peripapillary and macular areas was a predilected site for choroidal neovascularization in chronic uveitis. The choroidal neovascularization may develop in such a way that the uveal inflammation damages the Bruch's membrane and choriocapillaris and consequently causes retinal ischemia, thus stimulating the endothelium of the choriocapillaris and the overlying RPE to proliferate. There is a close relation between choroidal neovascularization and proliferation of RPE. Choroidal neovascularization may cause reactive proliferation of the RPE and vice versa.     

A model of subretinal neovascularization in the pigmented rat

We produced krypton laser photocoagulation lesions of mild to moderate whiteness in the posterior retinas of one eye of 23 pigmented rats, and identical appearing argon laser burns in the fellow eyes. We observed foci of subretinal neovascularization, histopathologically markedly similar to that which occurs in several human retinal diseases, in the krypton laser treated eyes of 6 of the 14 rats that were followed for one to three months after photocoagulation. No such lesions were observed in the argon laser treated fellow eyes, nor in krypton or argon laser treated eyes examined earlier than one month after photocoagulation. The photocoagulation damaged only the choriocapillaris, the retinal pigment epithelium (RPE), and the photoreceptor layer. In the acute lesions, we did not observe ruptures in Bruch's membrane. The neovascularization was surrounded by multiple layers of RPE cells, a histopathologic finding that has also been reported in some human eyes with subretinal neovascularization in age-related macular degeneration. These observations suggest that the RPE cells may be modifying the proliferative behavior of adjacent choroidal capillaries. This model differs from previous models of subretinal neovascularization in primates, and may be useful for additional studies of this important pathological process.     

Surgical induction of choroidal neovascularization in a porcine model

Purpose To develop a reproducible surgical technique for the induction of choroidal neovascularization (CNV) in the subretinal space of porcine eyes and to analyse the resulting CNV clinically and histologically. Methods Two different modifications of a surgical technique previously described were compared with the original method. In ten porcine eyes retinal pigment epithelial (RPE) cells were removed using a silicone tipped cannula, in ten porcine eyes Bruch’s membrane was perforated once with a retinal perforator without prior RPE removal and in ten eyes RPE removal was followed by a single perforation of Bruch’s membrane. Fifteen of the eyes, five from each group, were enucleated 30 minutes after surgery, while the remaining eyes were enucleated after 14 days. Prior to enucleation, at day 14, fundus photographs and fluorescein angiograms were obtained. Eyes were examined by light microscopy and by immunohistochemical staining. In addition to these 30 eyes, two eyes underwent surgery with the purpose of subsequent scanning electron microscopic (SEM) examination. Results In eyes enucleated immediately after surgery neuroretinas overlying the induced lesions were intact without apparent atrophy of cells regardless of the surgical technique applied. The process of RPE removal was found to induce breaks in Bruch’s membrane and both the size and the number of breaks varied between eyes. CNV membranes were identified in 15 of 15 eyes enucleated after 14 days. CNV membranes induced by perforation of Bruch’s membrane without prior RPE removal were significantly thicker than membranes from eyes undergoing both RPE removal and Bruch’s perforation (p = 0.03) and also thicker than membranes from eyes with only RPE-removal (p < 0.01). CNV membranes from eyes with perforation of Bruch’s membrane without prior RPE removal had a higher cellular content and were more richly vascularized and also exhibited the highest propensity to leak in fluorescense angiograms. Conclusion All three surgical techniques were capable of inducing CNV, but the one applying perforation of Bruch’s membrane without RPE removal was easier to reproduce and involved fewer variables than the techniques utilizing RPE removal. The presence of RPE cells seems to affect both the morphology and cellular composition of induced CNV. The authors have no commercial interest in this study or in funding organizations. Full control of all primary data remains with the authors, and we agree to allow Graefe’s Archive for Clinical and Experimental Ophthalmology to review these data if requested.     

Lysosomal enzyme cytochemistry of human RPE, Bruch's membrane and drusen

Twenty-five human eyes of various ages from eye bank donors and surgical enucleations were obtained for ultrastructural cytochemical demonstration of acid phosphatase (AcPase) and arylsulfatase B (ASB) in the retinal pigment epithelium (RPE) and Bruch's membrane. Results with post-mortem (less than 10 hr) tissues were comparable to those of fresh specimens. Vigorous reactivity was demonstrated in lysosomes of RPE and choriocapillary endothelium but no reactive sites were found in Bruch's membrane, although many lysosome-like dense bodies occurred in eyes greater than 20 yr of age. Granular drusen of 30-70-yr-olds contained no reactive bodies. In eyes greater than 80 years old blebs of RPE basal cytoplasm protruding into Bruch's membrane contained reactive lysosomes. We conclude that the RPE ordinarily does not extrude or exocytose active lysosomes (ie, phagolysosomes, other secondary lysosomes, residual bodies, lipofuscin) or lysosomal enzymes. Aged RPE, however, extrudes cytoplasm with active lysosomes into Bruch's membrane. The possible impact of this process on the extracellular connective tissue is discussed, particularly with regard to age-related deterioration of Bruch's membrane and neovascularization.     

Pathogenesis of lesions in late age-related macular disease

To summarize the evidence that exists concerning the pathogenesis of lesions in late age-related macular disease (AMD), we reviewed both experimental evidence and clinical observations that address these problems. There is good evidence that choroidal neovascularization is due to a change in the balance of growth factors derived from the retinal pigment epithelial basolateral plasma membrane domain (RPE). Retinal angiomatous proliferation may also have a similar pathogenesis involving the apical domain. Detachment of the RPE is likely to be a consequence of increased resistance of Bruch's membrane to water flow due to deposition of lipids. Geographic atrophy is preceded by accumulation of autofluorescent material in the RPE and possible causal relationships between the two have been demonstrated. There is increasing understanding concerning the sequence of events that lead to those lesions causing loss of central vision in AMD. Therapeutic approaches that address the underlying mechanisms are more likely to succeed than current treatment options. Such an approach has already been initiated in the management of choroidal neovascularization.     

Clinicopathological findings of retinal angiomatous proliferation

Background Neovascular membranes obtained from surgical excision of neovascularization for retinal angiomatous proliferation (RAP) were examined histopathologically in an attempt to elucidate the pathogenic mechanism of RAP. Methods Nine eyes of eight patients (mean age, 79±6 years) who underwent neovascularization excision were studied. Three eyes had stage II with RPE detachment, six had stage III. Immunohistochemical studies were performed to identify von Willebrand factor, vascular endothelial factor (VEGF), CD68 and hypoxia inducible factors (HIF-1 alpha and HIF-2 alpha). Results Multiple soft drusen were present in the macular area in all patients. In one stage II eye, we observed intraretinal neovascularization as a VEGF-positive mass, CD68-positive macrophage migration and HIF expression. In another stage II eye, neovascularization had extended above the RPE, while VEGF-positive fibroblasts were observed below the RPE. Therefore, in stage II, neither angiographic nor histopathological examinations identified choroidal neovascularization. In one phase III eye, angiography demonstrated choroidal neovascularization and chorioretinal anastomosis. Histopathologically, chorio-retinal communication was observed in the region where the RPE was destroyed, and VEGF-positive neovascularization was also seen below the RPE. Conclusions The findings of multiple drusen in elderly patients together with macrophage migration and HIF expression surrounding VEGF-positive retinal neovascularization suggest ischemic and inflammatory factors to be associated with the development and progression of RAP.     

Bruch's membrane and its significance for retinopexy following laser irradiation

Retinopexy may be produced by either of two mechanisms following transpupillary irradiation of the retina with the Nd:YAG laser. Which of these mechanisms is operative will depend on the degree of disruption produced within the elastic component of Bruch's membrane. Small defects in the elastic component of Bruch's membrane are soon completely bridged by regenerated retinal pigment epithelium (RPE) cells. However, there is a risk of subretinal neovascularization in such defects. Large defects in the elastic component of Bruch's membrane are not completely bridged by regenerating RPE cells before proliferating Müller cells invade this region and spread from it, mushroom-like, within the choroid. In this case subretinal neovascularization does not occur. In the former instance (small defects) retinopexy is achieved by a process of interdigitation between the microvilli of Müller and RPE cells; and in the latter (large defects) via a press-stud-like anchorage of Müller cells within the choroid. It has not yet been established whether these two retinopexy phenomena, observed after irradiation of rabbit retinas, occur in the same way in man.     

The role of retinal pigment epithelium in the involution of subretinal neovascularization

The extravascular milieu around laser-induced experimental subretinal neovascularization (SRN) was studied during the evolution of the neovascular membrane from its early leaky stage to its late involuted stage. When the first signs of visible leakage appeared on angiography, newly formed vessels were spread in the subretinal space around the break in Bruch's membrane, fluid was accumulating in the subretinal space, and retinal pigment epithelial (RPE) cells were proliferating in a papillary pattern around the newly formed vessels; the RPE proliferation began with the undamaged cells at the edges of the laser injury. With further maturation, the RPE continued to envelope the subretinal vessels. This RPE proliferation was associated with the disappearance of fluid between the enveloped vessels and the sensory retina, and the gradual cessation of fluorescein leakage during angiography. At the end of the involution process, when the neovascular membrane no longer demonstrated any leakage, the subretinal vessels were found to be tightly enveloped by RPE cells, and no fluid separated them from the sensory retina. The authors' results suggest that involution of the neovascular membrane with maturation, as demonstrated by the cessation of visible fluorescein leakage, is the result of RPE proliferation that tightly envelopes the newly formed vessels and probably resorbs the previously accumulated subretinal fluid, as well as preventing its further accumulation in the subretinal space.     

Optical coherence tomographic findings of idiopathic polypoidal choroidal vasculopathy

BACKGROUND AND OBJECTIVE: To identify the histological level of abnormal vessels associated with idiopathic polypoidal choroidal vasculopathy (IPCV), we examined IPCV with Optical Coherence Tomography (OCT). PATIENTS AND METHODS: Fourteen patients diagnosed with IPCV were examined with Indocyanine green (ICG) angiography and OCT. RESULTS: ICG angiography demonstrated branching vascular networks with polypoidal dilatations at the terminals beneath the retinal pigment epithelium (RPE). OCT showed dome-like elevation of the RPE, and moderate reflex or nodular appearance were seen beneath the RPE. CONCLUSION: The abnormal vessel associated with IPCV is supposed to be choroidal neovascularization with polypoidal dilatations at the terminals between Bruch's membrane and RPE. We consider that this disease is a peculiar form of age-related macular degeneration.     

The extracellular matrix of human retinal pigment epithelial cells in vivo and its synthesis in vitro

The production of extracellular matrix material by retinal pigment epithelium (RPE) may influence or mediate some of the many important functions of this tissue. Using immunohistochemical staining techniques, the extracellular matrix surrounding the RPE in vivo and the components produced by RPE in vitro have been investigated. Frozen sections of eye bank eyes showed antigen specific staining for collagen types I, III, and IV, and for fibronectin and laminin in Bruch's membrane and surrounding the RPE. Only very slight staining of the membrane was seen with antiserum against type II collagen, and there was no staining for type V collagen. Specific staining was demonstrated for the four collagens and glycoproteins in the extracellular matrix of RPE cells grown in culture. Once again, there was no staining for type V collagen. This study demonstrates that the RPE is capable of producing many of the components of the extracellular matrix found in Bruch's membrane and surrounding the RPE in vivo. This function may be important in the maintenance of a physical barrier to subretinal neovascularization, and may also play a role in such pathologic states as proliferative vitreoretinopathy.     

Retinal pigment epithelium tears following intravitreal ranibizumab therapy

Two patients with choroidal neovascularization secondary to age-related macular degeneration (AMD) developed a retinal pigment epithelial (RPE) tear following intravitreal injection of ranibizumab. One patient developed the RPE tear within 2 weeks of the injection, the other within 6 weeks of a second injection. Both patients presented with vision loss of one line at diagnosis of the RPE tear. During long-term follow-up, visual acuity improved in one patient by one line and deteriorated in the second patient by three lines. RPE tears may occur after intravitreal injection of ranibizumab in patients with neovascular AMD, probably because of the rapid regression of the fibrovascular membrane.     

Impaired RPE survival on aged submacular human Bruch's membrane

Resurfacing of diseased or iatrogenically damaged Bruch's membrane with healthy retinal pigment epithelium (RPE) has been proposed as adjunctive treatment for age-related macular degeneration (AMD). The purpose of this study was to determine whether cultured fetal human RPE cells can attach and differentiate on aged submacular human Bruch's membrane. Bruch's membrane was debrided to expose native RPE basement membrane, the superficial inner collagenous layer directly below the RPE basement membrane, or the deep inner collagenous layer. These are three surfaces that transplanted RPE cells will encounter in situ. Approximately 3146 cultured fetal RPE cells mm(-2) were seeded onto these three surfaces and grown in organ culture for 1, 7, or 14 days. Explants were bisected and examined histologically or analyzed with a scanning electron microscope. RPE nuclear density was measured on stained sections. Morphology and cell density were compared to cells seeded onto bovine corneal endothelial cell-extracellular matrix (BCE-ECM). In situ submacular RPE nuclear density was also measured in tissue sections of donor eyes ranging from 18 weeks gestation to 88 years of age to determine the effect of age on RPE density. Compared to cells seeded onto BCE-ECM at similar density, RPE cell coverage and cellular morphology on aged submacular human Bruch's membrane was poor at all time points. In contrast to cells on BCE-ECM, RPE cell density on Bruch's membrane decreased with time. In general, cell morphology on all three Bruch's membrane surfaces worsened by day-7 compared to day-1. Although some cells were more pigmented on RPE basement membrane and the deep inner collagenous layer at day-7, poor cellular morphology indicated the remaining cells were not well differentiated. At day-14, the cells were uniform and cuboidal on BCE-ECM, with cell density similar to that at day-7 and similar to in situ density of young donors (相似文献   

Age related macular degeneration surgery

Age related macular degeneration (ARMD) is a disease in which the retinal pigment epithelium (RPE) is damaged in the central retinal area. In the exudative form, the vision loss is due to choroidal neovascularization, while in the nonexudative or atrophic form, there is a vision loss because of the retinal pigment epithelium atrophy. Treatment which proved to be efficient in lowering the risk of severe vision loss in the exudative form includes laser photocoagulation, photodynamic therapy, transpupilary thermotherapy and as surgical treatment, the controversial subretinal membrane extraction. In all these situations the RPE is damaged by the disease itself and by the therapeutic procedure too. Retinal translocation is a surgical procedure that intends to remove the neurosensory retina from an area with damaged RPE to an healthy RPE area, through a 360 degrees retinotomy or through a limited one. This paper present some ARMD cases treated by subretinal membrane extraction and one by limited retinal translocation. The question which is still remaining is which are the risks and benefits for the following treatment procedures: laser photocoagulation, photodynamic therapy, transpupilary thermotherapy and surgical approach?     

Pathologic features of senile macular degeneration

Senile macular degeneration (SMD) has several morphologic forms that may exist singly or in various combinations. Patients with drusen are at an increased risk to develop SMD. Types of drusen that have been recognized clinically and histopathologically include hard or nodular, soft, glistening or calcified, and diffuse. Hard drusen are probably a consequence of extrusion of material from one or a cluster of retinal pigment epithelial (RPE) cells. Soft drusen are a sign of more widespread disease of the RPE, develop in eyes with hard drusen, and represent small areas of serous detachment of the RPE and the thickened inner aspect of Bruch's membrane. RPE areolar atrophy can occur in the setting of hard and soft drusen and in larger RPE detachments. From the morphologic point of view, we propose that the process leading to disciform scar formation in SMD begins with thickening of the inner aspect of Bruch's membrane due to production of abnormal basement membrane by the RPE. This thickened area is weakly attached and allows the development of localized detachments (soft drusen). These localized detachments become confluent into large detachments of the RPE. Choroidal neovascularization occurs in association with diffuse and soft drusen and larger serous RPE detachments. Bleeding from neovascular tissue leads to disciform scar formation.     

Focal adhesion kinase signaling pathway participates in the formation of choroidal neovascularization and regulates the proliferation and migration of choroidal microvascular endothelial cells by acting through HIF-1 and VEGF expression in RPE cells

Choroidal neovascularization (CNV) is one of the most frequent causes of severe and progressive vision loss, while its pathogenesis is still poorly understood. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, plays a crucial role in linking signals initiated by both the extracellular matrix (ECM) and soluble signaling factors and controls essential cellular processes. Extensive evidence has shown that FAK is activated in angiogenic response. This study aims to investigate the effect of FAK on CNV formation. The Brown-Norway (BN) rats underwent laser rupture of Bruch's membrane to induce CNV and were then killed at 1, 3, 7, and 14 days following laser injury. Immunofluorescence and Western blot were processed to detect FAK protein. Retinal pigment epithelial (RPE) cells were cultured under hypoxia and RNA interference (RNAi) technique was used to knock down the FAK gene in RPE cells. Expression of hypoxia inducible factor-1 (HIF-1α) and vascular endothelial growth factor (VEGF) in RPE cells were investigated by RT-PCR and Western blot. Two kinds of coculture models were used to observe the effects of specific blockade of FAK in RPE cells on the proliferation and migration of choroidal microvascular endothelial cells (CECs), respectively. FAK was highly expressed in the rat RPE-choroid tissue after photocoagulation. In vitro experiment showed that FAK was involved in hypoxia signaling in cultured RPE cells. The absence of FAK effectively reduced the expression of hypoxia-induced HIF-1α and VEGF in RPE cells, resulting in the inhibition of proliferation and migration of CECs. Our results suggest that FAK pathway activation plays a role in the development of CNV, and regulates the proliferation and migration of CECs by acting through HIF-1 and then up-regulating the expression of the angiogenic factor VEGF in RPE cells. It is reasonable to propose that FAK siRNA will potentially provides a means to attenuate the strong stimuli for neovascularization in CNV-dependent disorders, which could present a therapeutically relevant strategy for the inhibition of CNV.     

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.