Unilateral choroidal folds

Choroidal folds are wave-like formations in Bruch's membrane, the retinal pigment epithelium and the choriocapillaris. They may be idiopathic or associated with a variety of ocular disorders including orbital tumors, inflammatory conditions, hypotony and hyperopia. Patients with idiopathic folds tend to be asymptomatic, while those individuals with acutely acquired folds tend to experience visual disturbances and metamorphopsia. The following case report presents a patient diagnosed with unilateral choroidal folds secondary to hyperopia. The clinical appearance, etiology, differential diagnosis, and management of choroidal folds are discussed.     

Fine structure of the retinal epithelium and tapetum lucidum of the opossum (didelphis virginiana)

The fine structure of the retinal epithelium has been studied by electron microscopy in the opossum (Didelphis virginiana). The retinal epithelium, over most of the retina, is typical of that in other vertebrates and consists of a single layer of heavily pigmented, cuboidal cells. These cells display extensive basal (scleral) infoldings and numerous apical (vitreal) processes which enclose photoreceptor outer segments. A semicircular area of the retinal epithelium in the superior fundus is further specialized as a tapetum lucidum. The reflecting material consists of a large quantity of lipoidal spheres scattered throughout the epithelial cells. Centrally in the tapetal area very few or no melanosomes are found, indicating a non-occlusible tapetum. Peripherally in the tapetum, the epithelial cells contain both reflecting material and melanosomes. As in the non-tapetal area, the epithelial cells of the tapetum display large amounts of smooth endoplasmic reticulum and numerous mitochondria. Bruch's membrane everywhere displays the usual pentalaminate structure described for most vertebrates. The choriocapillaris is also typical, in that numerous fenestrations are present in the endothelium bordering Bruch's membrane.     

Identification of a synergistic interaction between endothelial cells and retinal pigment epithelium

The retinal pigment epithelium located between the neurosensory retina and the choroidal vasculature is critical for the function and maintenance of both the photoreceptors and underlying capillary endothelium. While the trophic role of retinal pigment epithelium on choroidal endothelial cells is well recognized, the existence of a reciprocal regulatory function of endothelial cells on retinal pigment epithelium cells remained to be fully characterized. Using a physiological long‐term co‐culture system, we determined the effect of retinal pigment epithelium‐endothelial cell heterotypic interactions on cell survival, behaviour and matrix deposition. Human retinal pigment epithelium and endothelial cells were cultured on opposite sides of polyester transwells for up to 4 weeks in low serum conditions. Cell viability was quantified using a trypan blue assay. Cellular morphology was evaluated by H&E staining, S.E.M. and immunohistochemistry. Retinal pigment epithelium phagocytic function was examined using a fluorescent bead assay. Gene expression analysis was performed on both retinal pigment epithelium and endothelial cells by quantitative PCR. Quantification of extracellular matrix deposition was performed on decellularized transwells stained for collagen IV, fibronectin and fibrillin. Our results showed that presence of endothelial cells significantly improves retinal pigment epithelium maturation and function as indicated by the induction of visual cycle‐associated genes, accumulation of a Bruch's membrane‐like matrix and increase in retinal pigment epithelium phagocytic activity. Co‐culture conditions led to increased expression of anti‐angiogenic growth factors and receptors in both retinal pigment epithelium and endothelial cells compared to monoculture. Tube‐formation assays confirmed that co‐culture with retinal pigment epithelium significantly decreased the angiogenic phenotype of endothelial cells. These findings provide evidence of critical interdependent interactions between retinal pigment epithelium and endothelial cell involved in the maintenance of retinal homeostasis.     

Autophagy and Exosomes in the Aged Retinal Pigment Epithelium: Possible Relevance to Drusen Formation and Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of loss of central vision in the elderly. The formation of drusen, an extracellular, amorphous deposit of material on Bruch''s membrane in the macula of the retina, occurs early in the course of the disease. Although some of the molecular components of drusen are known, there is no understanding of the cell biology that leads to the formation of drusen. We have previously demonstrated increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we found that drusen in AMD donor eyes contain markers for autophagy and exosomes. Furthermore, these markers are also found in the region of Bruch''s membrane in old mice. By in vitro modeling increased mtDNA damage induced by rotenone, an inhibitor of mitochondrial complex I, in the RPE, we found that the phagocytic activity was not altered but that there were: 1) increased autophagic markers, 2) decreased lysosomal activity, 3) increased exocytotic activity and 4) release of chemoattractants. Exosomes released by the stressed RPE are coated with complement and can bind complement factor H, mutations of which are associated with AMD. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients and may be associated with the pathogenesis of AMD in the elderly.     

Correlations between Photodegradation of Bisretinoid Constituents of Retina and Dicarbonyl Adduct Deposition

Non-enzymatic collagen cross-linking and carbonyl adduct deposition are features of Bruch''s membrane aging in the eye, and disturbances in extracellular matrix turnover are considered to contribute to Bruch''s membrane thickening. Because bisretinoid constituents of the lipofuscin of retinal pigment epithelial (RPE) cells are known to photodegrade to mixtures of aldehyde-bearing fragments and small dicarbonyls (glyoxal (GO) and methylglyoxal (MG)), we investigated RPE lipofuscin as a source of the reactive species that covalently modify protein side chains. Abca4^−/− and Rdh8^−/−/Abca4^−/− mice that are models of accelerated bisretinoid formation were studied and pre-exposure of mice to 430 nm light enriched for dicarbonyl release by bisretinoid photodegradation. MG protein adducts were elevated in posterior eyecups of mutant mice, whereas carbonylation of an RPE-specific protein was observed in Abca4^−/− but not in wild-type mice under the same conditions. Immunolabeling of cryostat-sectioned eyes harvested from Abca4^−/− mice revealed that carbonyl adduct deposition in Bruch''s membrane was accentuated. Cell-based assays corroborated these findings in mice. Moreover, the receptor for advanced glycation end products that recognizes MG and GO adducts and glyoxylase 1 that metabolizes MG and GO were up-regulated in Abca4^−/− mice. Additionally, in acellular assays, peptides were cross-linked in the presence of A2E (adduct of two vitamin A aldehyde and ethanolamine) photodegradation products, and in a zymography assay, reaction of collagen IV with products of A2E photodegradation resulted in reduced cleavage by the matrix metalloproteinases MMP2 and MMP9. In conclusion, these mechanistic studies demonstrate a link between the photodegradation of RPE bisretinoid fluorophores and aging changes in underlying Bruch''s membrane that can confer risk of age-related macular degeneration.     

Zinc deficiency leads to lipofuscin accumulation in the retinal pigment epithelium of pigmented rats

Background

Age-related macular degeneration (AMD) is associated with lipofuscin accumulation whereas the content of melanosomes decreases. Melanosomes are the main storage of zinc in the pigmented tissues. Since the elderly population, as the most affected group for AMD, is prone to zinc deficit, we investigated the chemical and ultrastructural effects of zinc deficiency in pigmented rat eyes after a six-month zinc penury diet.

Methodology/Principal Findings

Adult Long Evans (LE) rats were investigated. The control animals were fed with a normal alimentation whereas the zinc-deficiency rats (ZD-LE) were fed with a zinc deficient diet for six months. Quantitative Energy Dispersive X-ray (EDX) microanalysis yielded the zinc mole fractions of melanosomes in the retinal pigment epithelium (RPE). The lateral resolution of the analysis was 100 nm. The zinc mole fractions of melanosomes were significantly smaller in the RPE of ZD-LE rats as compared to the LE control rats. Light, fluorescence and electron microscopy, as well as immunohistochemistry were performed. The numbers of lipofuscin granules in the RPE and of infiltrated cells (Ø>3 µm) found in the choroid were quantified. The number of lipofuscin granules significantly increased in ZD-LE as compared to control rats. Infiltrated cells bigger than 3 µm were only detected in the choroid of ZD-LE animals. Moreover, the thickness of the Bruch''s membrane of ZD-LE rats varied between 0.4–3 µm and thin, rangy ED1 positive macrophages were found attached at these sites of Bruch''s membrane or even inside it.

Conclusions/Significance

In pigmented rats, zinc deficiency yielded an accumulation of lipofuscin in the RPE and of large pigmented macrophages in the choroids as well as the appearance of thin, rangy macrophages at Bruch''s membrane. Moreover, we showed that a zinc diet reduced the zinc mole fraction of melanosomes in the RPE and modulated the thickness of the Bruch''s membrane.     

Agents that bind annexin A2 suppress ocular neovascularization

TM601 is a synthetic polypeptide with sequence derived from the venom of the scorpion Leiurus quinquestriatus that has anti‐neoplastic activity. It has recently been demonstrated to bind annexin A2 on cultured tumor and vascular endothelial cells and to suppress blood vessel growth on chick chorioallantoic membrane. In this study, we investigated the effects of TM601 in models of ocular neovascularization (NV). When administered by intraocular injection, intravenous injections, or periocular injections, TM601 significantly suppressed the development of choroidal NV at rupture sites in Bruch's membrane. Treatment of established choroidal NV with TM601 caused apoptosis of endothelial cells and regression of the NV. TM601 suppressed ischemia‐induced and vascular endothelial growth factor‐induced retinal NV and reduced excess vascular permeability induced by vascular endothelial growth factor. Immunostaining with an antibody directed against TM601 showed that after intraocular or periocular injection, TM601 selectively bound to choroidal or retinal NV and co‐localized with annexin A2, which is undetectable in normal retinal and choroidal vessels, but is upregulated in endothelial cells participating in choroidal or retinal NV. Intraocular injection of plasminogen or tissue plasminogen activator, which like TM601 bind to annexin A2, also suppressed retinal NV. This study supports the hypothesis that annexin A2 is an important target for treatment of neovascular diseases and suggests that TM601, through its interaction with annexin A2, causes suppression and regression of ocular NV and reduces vascular leakage and thus may provide a new treatment for blinding diseases such as neovascular age‐related macular degeneration and diabetic retinopathy. J. Cell. Physiol. 225: 855–864, 2010. © 2010 Wiley‐Liss, Inc.     

Esterified Cholesterol Is Highly Localized to Bruch's Membrane, as Revealed by Lipid Histochemistry in Wholemounts of Human Choroid

Accumulation of neutral lipids in Bruch''s membrane (BrM) is a major age change in human retina and contributes to the formation of extracellular lesions associated with age-related macular degeneration. We developed a BrM–choroid wholemounting technique suitable for reliable staining and evaluated different fluorescent lipid dyes for topographic semiquantitative analysis of BrM lipids. Thin BrM–choroid complexes with partially stripped choroid from 10 aged donor eyes were prepared with an optimized wholemounting technique. Preparation quality was monitored by examining 1-μm-thick sections of representative samples. The staining patterns of Nile Red, BODIPY 493/503, filipin for unesterified cholesterol (UC-F), filipin for esterified cholesterol (EC-F), and Oil Red O in wholemounts were compared with their staining patterns in chorioretinal sections, using wide-field epi-fluorescence microscopy. Wholemounts exhibited optimal flatness on the BrM side. Reduced tissue thickness allowed reliable dye penetration and staining of BrM. Only EC-F was with high specificity localized to BrM and demonstrated an intense and distinct granular staining pattern not previously appreciated in chorioretinal sections. All other lipid dyes also stained choroidal or retinal tissue intensely. No dye provided perfect characteristics in regard to representing all neutral lipid classes present in BrM or to fluorescence intensity. Nevertheless, only EC-F was highly localized to BrM with a specific granular pattern. Because direct assays indicate that esterified cholesterol is abundantly present in BrM, we consider EC-F the most valuable choice for analyzing neutral lipid deposits in human BrM. (J Histochem Cytochem 57:731–739, 2009)     

Viewing Ageing Eyes: Diverse Sites of Amyloid Beta Accumulation in the Ageing Mouse Retina and the Up-Regulation of Macrophages

Background

Amyloid beta (Aβ) accumulates in the ageing central nervous system and is associated with a number of age-related diseases, including age-related macular degeneration (AMD) in the eye. AMD is characterised by accumulation of extracellular deposits called drusen in which Aβ is a key constituent. Aβ activates the complement cascade and its deposition is associated with activated macrophages. So far, little is known about the quantitative measurements of Aβ accumulation and definitions of its relative sites of ocular deposition in the normal ageing mouse.

Methodology/Principal Findings

We have traced Aβ accumulation quantitatively in the ageing mouse retina using immunohistochemistry and Western blot analysis. We reveal that it is not only deposited at Bruch''s membrane and along blood vessels, but unexpectedly, it also coats photoreceptor outer segments. While Aβ is present at all sites of deposition from 3 months of age, it increases markedly from 6 months onward. Progressive accumulation of deposits on outer segments was confirmed with scanning electron microscopy, revealing age-related changes in their morphology. Such progress of accumulation of Aβ on photoreceptor outer segments with age was also confirmed in human retinae using immunohistochemistry. We also chart the macrophage response to increases in Aβ showing up-regulation in their numbers using both confocal laser imaging of the eye in vivo followed by in vitro immunostaining. With age macrophages become bloated with cellular debris including Aβ, however, their increasing numbers fail to stop Aβ accumulation.

Conclusions

Increasing Aβ deposition in blood vessels and Bruch''s membrane will impact upon retinal perfusion and clearance of cellular waste products from the outer retina, a region of very high metabolic activity. This accumulation of Aβ may contribute to the 30% reduction of photoreceptors found throughout life and the shortening of those that remain. The coating of Aβ on outer segments may also have an impact upon visual function with age.     

Modification of native collagen with cell‐adhesive peptide to promote RPE cell attachment on Bruch's membrane

Current efforts to reverse loss of visual function due to Age‐related Macular Degeneration point to the restoration of the Retinal Pigment Epithelial (RPE) layer. Restoration of the RPE layer involves replacing lost RPE cells as well as addressing the degeneration of the underlying Bruch's membrane (BM). To advance the potential of using donor BM, we present a strategy to achieve specific and controllable modification of the inner collagenous layer (ICL) of the Bruch's membrane. In particular, interaction between a collagen binding peptide (CBP) sequence with exposed collagen fibers on the ICL surface is utilized to anchor bioactive molecules. Here, a cell‐adhesion sequence is added to the collagen binding sequence to promote attachment and survival of ARPE‐19. First, the binding specificity of the CBP sequence is verified with a fluorescent binding assay. Subsequently, the effect of modification using the peptide is studied qualitatively using confocal fluorescent imaging and quantitatively through a cell proliferation assay. Results of these experiments indicate that the peptide sequence binds specifically to collagen fibers. Additionally, modification using the peptide enhanced cell adhesion, allowing large uniform cell networks to be formed on the surface. Furthermore, modification with the peptide also delayed the onset of apoptosis on adherent cells. Biotechnol. Bioeng. 2009;102: 1723–1729. © 2008 Wiley Periodicals, Inc.     

Directional protein secretion by the retinal pigment epithelium: roles in retinal health and the development of age‐related macular degeneration

The structural and functional integrity of the retinal pigment epithelium (RPE) is fundamental for maintaining the function of the neuroretina. These specialized cells form a polarized monolayer that acts as the retinal–blood barrier, separating two distinct environments with highly specialized functions: photoreceptors of the neuroretina at the apical side and Bruch's membrane/highly vascularized choriocapillaris at the basal side. The polarized nature of the RPE is essential for the health of these two regions, not only in nutrient and waste transport but also in the synthesis and directional secretion of proteins required in maintaining retinal homoeostasis and function. Although multiple malfunctions within the RPE cells have been associated with development of age‐related macular degeneration (AMD), the leading cause of legal blindness, clear causative processes have not yet been conclusively characterized at the molecular and cellular level. This article focuses on the involvement of directionally secreted RPE proteins in normal functioning of the retina and on the potential association of incorrect RPE protein secretion with development of AMD. Understanding the importance of RPE polarity and the correct secretion of essential structural and regulatory components emerge as critical factors for the development of novel therapeutic strategies targeting AMD.     

Choriocapillaris and Choroidal Microvasculature Imaging with Ultrahigh Speed OCT Angiography

We demonstrate in vivo choriocapillaris and choroidal microvasculature imaging in normal human subjects using optical coherence tomography (OCT). An ultrahigh speed swept source OCT prototype at 1060 nm wavelengths with a 400 kHz A-scan rate is developed for three-dimensional ultrahigh speed imaging of the posterior eye. OCT angiography is used to image three-dimensional vascular structure without the need for exogenous fluorophores by detecting erythrocyte motion contrast between OCT intensity cross-sectional images acquired rapidly and repeatedly from the same location on the retina. En face OCT angiograms of the choriocapillaris and choroidal vasculature are visualized by acquiring cross-sectional OCT angiograms volumetrically via raster scanning and segmenting the three-dimensional angiographic data at multiple depths below the retinal pigment epithelium (RPE). Fine microvasculature of the choriocapillaris, as well as tightly packed networks of feeding arterioles and draining venules, can be visualized at different en face depths. Panoramic ultra-wide field stitched OCT angiograms of the choriocapillaris spanning ∼32 mm on the retina show distinct vascular structures at different fundus locations. Isolated smaller fields at the central fovea and ∼6 mm nasal to the fovea at the depths of the choriocapillaris and Sattler''s layer show vasculature structures consistent with established architectural morphology from histological and electron micrograph corrosion casting studies. Choriocapillaris imaging was performed in eight healthy volunteers with OCT angiograms successfully acquired from all subjects. These results demonstrate the feasibility of ultrahigh speed OCT for in vivo dye-free choriocapillaris and choroidal vasculature imaging, in addition to conventional structural imaging.     

Bioengineered Bruch's-like extracellular matrix promotes retinal pigment epithelial differentiation

In the eye, the retinal pigment epithelium (RPE) adheres to a complex protein matrix known as Bruch's membrane (BrM). The aim of this study was to provide enriched conditions for RPE cell culture through the production of a BrM-like matrix. Our hypothesis was that a human RPE cell line would deposit an extracellular matrix (ECM) resembling BrM. The composition and structure of ECM deposited by ARPE19 cells (ARPE19-ECM) was characterized. To produce ARPE19-ECM, ARPE19 cells were cultured in the presence dextran sulphate. ARPE19-ECM was decellularized using deoxycholate and characterized by immunostaining and western blot analysis. Primary human RPE and induced pluripotent stem cells were seeded onto ARPE19-ECM or geltrex coated surfaces and examined by microscopy or RT-PCR. Culture of ARPE19 cells with dextran sulphate promoted nuclear localization of SOX2, formation of tight junctions and deposition of ECM. ARPE19 cells deposited ECM proteins found in the inner layers of BrM, including fibronectin, vitronectin, collagens IV and V as well as laminin-alpha-5, but not those found in the middle elastic layer (elastin) or the outer layers (collagen VI). ARPE19-ECM promoted pigmentation in human RPE and pluripotent stem cell cultures. Expression of RPE65 was significantly increased on ARPE19-ECM compared with geltrex in differentiating pluripotent stem cell cultures. ARPE19 cells deposit ECM with a composition and structure similar to BrM in the retina. Molecular cues present in ARPE19-ECM promote the acquisition and maintenance of the RPE phenotype. Together, these results demonstrate a simple method for generating a BrM-like surface for enriched RPE cell cultures.     

Measuring polarization changes in the human outer retina with polarization‐sensitive optical coherence tomography

Morphological changes in the outer retina such as drusen are established biomarkers to diagnose age‐related macular degeneration. However, earlier diagnosis might be possible by taking advantage of more subtle changes that accompany tissues that bear polarization‐altering properties. To test this hypothesis, we developed a method based on polarization‐sensitive optical coherence tomography with which volumetric data sets of the macula were obtained from 10 young (<25 years) and 10 older (>54 years) subjects. All young subjects and 5 of the older subjects had retardance values induced by the retinal pigment epithelium and Bruch's membrane (RPE‐BM) complex that were just above the noise floor measurement (5°‐13° at 840 nm). In contrast, elevated retardance, up to 180°, was observed in the other 5 older subjects. Analysis of the degree of polarization uniformity (DOPU) demonstrates that reduced DOPU (<0.4) in the RPE is associated with elevated double pass phase retardation (DPPR) below the RPE‐BM complex, suggesting that the observed elevated DPPR in older subjects is the result of increased scattering or polarization scrambling. Collectively, our measurements show that the outer retina can undergo dramatic change in its polarization properties with age, and in some cases still retain its clinically normal appearance.      

Developmental distribution of collagen IV isoforms and relevance to ocular diseases

Type IV collagens are the most abundant proteins in basement membranes. Distinct genes encode each of six isoforms, α1(IV) through α6(IV), which assemble into one of three characteristic heterotrimers. Disease-causing mutations in each of the six genes are identified in humans or mice and frequently include diverse ocular pathogenesis that encompass common congenital and progressive blinding diseases, such as optic nerve hypoplasia, glaucoma, and retinal degeneration. Understanding where and when collagen IV molecules are expressed is important because it defines limits for the location and timing of primary pathogenesis. Although localization of collagen IV isoforms in developed human eyes is known, the spatial and temporal distribution of type IV collagens throughout ocular development has not been determined in humans or in mice. Here, we use isoform-specific monoclonal antibodies to systematically reveal the localization of all six collagen IV isoforms in developing mouse eyes. We found that α1(IV) and α2(IV) always co-localized and were ubiquitously expressed throughout development. α3(IV) and α4(IV) also always co-localized but in a much more spatially and temporally specific manner than α1(IV) and α2(IV). α5(IV) co-localized both with α3(IV)/α4(IV), and with α6(IV), consistent with α5(IV) involvement in two distinct heterotrimers. α5(IV) was present in all basement membranes except those of the vasculature. α6(IV) was not detected in vasculature or in Bruch's membrane, indicating that α5(IV) in Bruch's membrane is part of the α3α4α5 heterotrimer. This comprehensive analysis defines the spatial and temporal distribution of type IV collagen isoforms in the developing eye, and will contribute to understanding the mechanisms underlying collagen IV-related ocular diseases that collectively lead to blindness in millions of people worldwide.     

Intraocular microablation of choroidal tissue by a 308 nm AIDA excimer laser for RPE-transplantation in patients with age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the western nations beyond 50 years of age. The most frequent cause for severe visual loss is the growth of neovascular membrances from the choroid into the subretinal space. This usually results in irreversible degeneration of the overlying retina. Surgical removal of the membrane is feasible, however, usually results in functional loss of apposing retinal photoreceptors since retinal pigment epithelial (RPE) cells are removed concurrently due to their tight adherence to the neovascular complex. Therefore, various attempts have been undertaken to fill the resulting RPE cell defect with either heterologous or autologous RPE cell transplants. So far cell survival, function and subsequent visual function has been disappointing. To minimize trauma and resulting dedifferentiation harvesting in the eye and transplantation in whole sheets and without temporary removal from the eyes would be desirable. This may be achieved by isolating grafts consisting of choroid, Bruch's membrance and RPE cells from the peripheral retina and transplantation of this graft under the neurosensory retina after removal of the choroidal neovascularization. However, the choroidal component of such a graft would be expected to interfere with diffusion of metabolites to and from the retina. Therefore, outcome would be expected to be better if the choroidal tissue would be removed before translocation. In preclinical experiments we used a 308 nm UV AIDA excimer laser to microablate choroidal tissue from such a graft in human donor eyes.     

Parapapillary Atrophy: Histological Gamma Zone and Delta Zone

Background

To examine histomorphometrically the parapapillary region in human eyes.

Methodology/Principal Findings

The histomorphometric study included 65 human globes (axial length:21–37 mm). On anterior-posterior histological sections, we measured the distance Bruch''s membrane end (BME)-optic nerve margin (“Gamma zone”), BME-retinal pigment epithelium (RPE) (“Beta zone”), BME-beginning of non-occluded choriocapillaris, and BME-beginning of photoreceptor layer. “Delta zone” was defined as part of gamma zone in which blood vessels of at least 50 µm diameter were not present over a length of >300 µm. Beta zone (mean length:0.35±0.52 mm) was significantly (P = 0.01) larger in the glaucoma group than in the non-glaucomatous group. It was not significantly (P = 0.28) associated with axial length. Beta zone was significantly (P = 0.004) larger than the region with occluded choriocapillaris. Gamma zone (mean length:0.63±1.25 mm) was associated with axial length (P<0.001;r² = 0.73) with an increase starting at an axial length of 26.5 mm. It was not significantly (P = 0.24) associated with glaucomatous optic neuropathy. Delta zone (present only in eyes with axial length of ≥27 mm) was associated with axial length (P = 0.001) and scleral flange length (P<0.001) but not with glaucoma (P = 0.73).

Conclusions/Significance

Parapapillary gamma zone (peripapillary sclera without overlying choroid, Bruch''s membrane and deep retinal layers) was related with axial globe elongation and was independent of glaucoma. Delta zone (no blood vessels >50 µm diameter within gamma zone) was present only in highly axially elongated globes and was not related with glaucoma. Beta zone (Bruch''s membrane without RPE) was correlated with glaucoma but not with globe elongation. Since the region with occluded choriocapillaris was smaller than beta zone, complete loss of RPE may have occurred before complete choriocapillaris closure.     

An in vitro model of the back of the eye for studying retinal pigment epithelial-choroidal endothelial interactions

At the back of the eye, the outermost cell layer of the retina, the pigmented epithelium, lies against a basement membrane that is adjacent to the choroidal vessels that supply the outer sensory retina. During pathogenesis, these interfaces become damaged, and the homeostatic balance between the retinal pigment epithelium (RPE) and the choroidal vessels becomes disrupted, leading to choroidal neovascularization and blindness. To study the cell interactions at the back of the eye, we have used a coculture system in which a stable RPE monolayer has been cultured on a transwell insert and placed over a collagen gel sandwich into which choroidal endothelial cells (CECs) have been seeded. RPE cells have been stimulated by an inflammatory cytokine, interleukin-1 (IL-1beta), and the ability of the underlying choroidal endothelium to form vascular tubes has been tested. IL-1beta stimulation of the RPE insert increased the number of tubes formed by CECs in the gel as early as 3 d. By 7 d, tubes began to regress. Both IL-8 and monocyte chemotactic protein-1 (MCP-1) were found to be secreted in greater amounts in stimulated RPE. Because MCP-1 is also a chemokine for monocytes, which in turn secrete angiogenic factors, monocytes were added to the upper surface of the choroidal gel sandwich and then incubated with the stimulated RPE insert as above. By day 7, more tubes formed and there was no regression over the experimental time period. The versatility of this model has been illustrated in that both RPE and CECs can be cultured in a more natural construct and their molecular interactions tested by physiologically altering one cell type and not the other.     

Electron Microscopy of the Tapetum Lucidum of the Cat

The fine structure of the tapetum of the cat eye has been investigated by electron microscopy. The tapetum is made up of modified choroidal cells, seen as polygonal plates grouped around penetrating blood vessels which terminate in the anastomosing capillary network of the choriocapillaris. The tapetal cells are rectangular in cross-section, set in regular brick-like rows, and attain a depth of some thirty-five cell layers in the central region. This number is gradually reduced peripherally, and is replaced at the margin of the tapetum by normal choroidal tissue. The individual cells are packed with long slender rods 0.1 µ by 4 to 5 µ. The rods are packed in groups and with their long axes oriented roughly parallel to the plane of the retinal surface. Each cell contains several such groups. Cells at the periphery or in the outer layers of the tapetum are frequently seen to contain both tapetal rods and melanin granules, the latter typical of the choroidal melanocytes. Also melanocyte granules may have intermediate shapes. These observations plus the similar density of the two inclusions lead to the belief that the tapetal rods may be melanin derivatives. A fibrous connective tissue layer lies between the tapetum and the retina. The subretinal capillary network, the choriocapillaris, rests on this layer and is covered by the basement membrane of the retinal epithelium. The cytoplasm of the retinal epithelium exhibits marked absorptive modifications where it comes in contact with the vessels of the choriocapillaris. This fibrous layer and the basement membrane of the retinal epithelium apparently comprise the structural elements of Bruch's membrane.