Retinal and Choroidal Pathologies in Aged BALB/c Mice Following Systemic Neonatal Murine Cytomegalovirus Infection.

Although pathologies associated with acute virus infections have been extensively studied, the effects of long-term latent virus infections are less well understood. Human cytomegalovirus, which infects 50% to 80% of humans, is usually acquired during early life and persists in a latent state for the lifetime. The purpose of this study was to determine whether systemic murine cytomegalovirus (MCMV) infection acquired early in life disseminates to and becomes latent in the eye and if ocular MCMV can trigger in situ inflammation and occurrence of ocular pathology. This study found that neonatal infection of BALB/c mice with MCMV resulted in dissemination of virus to the eye, where it localized principally to choroidal endothelia and pericytes and less frequently to the retinal pigment epithelium (RPE) cells. MCMV underwent ocular latency, which was associated with expression of multiple virus genes and from which MCMV could be reactivated by immunosuppression. Latent ocular infection was associated with significant up-regulation of several inflammatory/angiogenic factors. Retinal and choroidal pathologies developed in a progressive manner, with deposits appearing at both basal and apical aspects of the RPE, RPE/choroidal atrophy, photoreceptor degeneration, and neovascularization. The pathologies induced by long-term ocular MCMV latency share features of previously described human ocular diseases, such as age-related macular degeneration.

Retinal and choroidal capillaries contribution to age-related macular degeneration (AMD) phenotypes in murine models of the disease.

Mouse models of age-related macular degeneration (AMD) such as Ccl2-/- and Ccl2-/-/Cx3cr1-/- have not yet been fully characterized ultrastructurally. Although we have previously shown extranuclear DNA (enDNA) leakage into the cytoplasm and damaged mitochondria in the retinal pigment epithelium (RPE) of these AMD mouse models, little is known about the state of their vascular capillaries of the retina and choroid. Our ultrastructural survey shows that the aberrations were not restricted to the RPE cells, but also extended to the vasculature of the retina and choroid. Their endothelial aberrations included cytoplasmic degeneration, pyknotic DNA, hypertrophic nuclei, and loss of fenestration in addition to duplication of basement membrane and loss of density in Bruch's membrane. Moreover, the state of the vasculature in the mutant mice models suggests that the capillaries could also be active contributors to the pathological findings seen in AMD. The goal of this study is to gain insights into the early events of AMD that may lead to a better understanding of AMD's pathogenesis, improve our preventative measures, and formulate designed therapeutic regimens that are tailored to target the initial pathological events.Abbreviations: AMD: age-related macular degeneration; BM: Bruch's membrane; DPC: degenerate pericyte; EN: endothelial nucleus; enDNA: extranuclear DNA; GCL: ganglion cell layer; HEN: hypertrophic endothelial nucleus; IPL: inner plexiform layer; NFL: nerve fiber layer; OPL: outer plexiform layer; RBC: red blood cell; RPE: retinal pigment epithelium; SNPs: Single nucleotide polymorphisms.

Ultrastructural changes in the choriocapillaris of N-methyl-N-nitrosourea-induced retinal degeneration in C57BL/6 mice.

N-methyl-N-nitrosourea (MNU) is known to cause apoptosis of photoreceptor cells and changes in retinal pigment epithelium (RPE). However, the changes in choriocapillaris, which nourishes photoreceptor cells by diffusing tissue fluid through RPE, have not been reported in detail. Therefore, we studied the ultrastructural transformation in and around the choriocapillaris to characterize the interdependence between choriocapillaris and surrounding tissue components in a mouse model. Seven-week-old male C57BL/6 mice were given a single intraperitoneal injection of MNU (60 mg/kg of body weight). Perfusion-fixed eyeballs were examined chronologically using immunohistochemistry and electron microscopy until the photoreceptor cells were lost. Sequential ultrastructural changes were observed in photoreceptor cells, RPE, Bruch's membrane, choriocapillaris, and choroidal melanocytes after an MNU injection. The lumens of the choriocapillaris narrowed following dilation, and the vascular endothelium showed structural alterations. When the photoreceptor cells were completely lost, the choriocapillaris appeared to be in a recovery process. Our results suggest that transport abnormality through Bruch's membrane and structural changes in the choroid might have influenced the morphology of choriocapillaris. The thin wall of the choriocapillaris appears to be the cause of the vulnerability with its altered morphology.

Density of PAS positive patterns in uveal melanoma: Correlation with vasculogenic mimicry, gene expression class, BAP-1 expression, macrophage infiltration, and risk for metastasis.

Purpose: Periodic acid-Schiff (PAS) positive patterns of vasculogenic mimicry (VM) have been associated with poor prognosis in uveal melanoma (UM). We examined these patterns with digital image analysis and transmission electron microscopy, and correlated them with BAP-1 expression, gene expression class, macrophage infiltration, and metastatic disease in full tumor cross-sections and intratumor regions. Methods: Thirty-two enucleated eyes with UM were stained immunohistochemically (BAP-1, laminin, CD31, and CD68) and with PAS without hematoxylin counterstain. Retrospective data on gene expression class and patient survival were retrieved. Tumor sections were digitally scanned and analyzed with the QuPath Bioimage analysis software, and imaged with transmission electron microscopy. Results: The mean area proportion covered by CD31, laminin, and PAS positive patterns in tumor cross-sections was 0.9% (SD 0.6), 3.0% (SD 1.9), and 8.4% (SD 5.9), respectively. PAS density was statistically significantly greater in tumors with gene expression class 2 (p=0.02). The cumulative 5-year metastasis-free survival decreased for each quartile of increased PAS density (1.0, 0.75, 0.40, and 0.17, p=0.004). Forty percent of the tumors had heterogeneous BAP-1 expression. Intratumor regions with low BAP-1 expression were more likely to harbor VM (p<0.0001), and had statistically significantly greater PAS density (p<0.0001) and number of CD68 positive cells (p=0.01). Conclusions: PAS positive patterns in UM are composed of a mixture of blood vessels and extracellular matrix (ECM), including VM. Increased density of PAS positive patterns correlated with gene expression class and metastasis, and colocated to tumor regions with macrophage infiltration and low BAP-1 expression.

Ultrastructural analysis of submacular choriocapillaris and its transport systems in AMD and aged control eyes.

The choriocapillaris is the source of nutrients and oxygen for photoreceptors, which consume more oxygen per gram of tissue than any other cell in the body. The purpose of this study was to evaluate and compare the ultrastructure of the choriocapillaris and its transport systems in patients with and without age-related macular degeneration (AMD). Ultrastructural changes were also evaluated in subjects that were homozygous for polymorphisms in high risk CFH alleles (Pure 1) only or homozygous only for high risk ARMS2/HTRA1 (Pure 10) alleles. Tissue samples were obtained from the macular region of forty male (n = 24) and female (n = 16) donor eyes and prepared for ultrastructural studies with transmission electron microscopy (TEM). The average age of the aged donors was 74 ±â€¯7.2 (n = 30) and the young donors 31.7 ±â€¯11.25 (n = 10). There was no significant difference in average ages between the adult groups. TEM images of the capillaries in the choriocapillaris (CC) were taken at 4,000X and 25,000X and used to measure the area of endothelial cell somas, the number of fenestrations, and area of caveolae within the endothelial cells per length of Bruchs membrane (BrMb). The Student t-test and Wilcoxon sum rank test were used to determine significant differences. There was no significant difference between young subjects and aged controls in any of the morphological criteria assessed. There was a significant decrease in the number of fenestrations/mm of BrMb in atrophic areas of GA eyes (p = 0.007) when compared with aged control eyes. A significant increase was found in the caveolae area as a percent of the endothelial cell soma of capillaries from GA subjects as compared with the controls (p = 0.03). Loss of capillary segments in choriocapillaris was also evident, especially in areas of geographic atrophy and CNV. In eyes from patients with sequence variations, the capillary endothelial cells often appeared degenerative and exhibited atypical fenestrations and pericytes covering the blood vessels. Subjects that were homozygous for polymorphisms in high risk CFH alleles only had more fenestrations/mm of BrMb than subjects that were homozygous only for high risk ARMS2/HTRA1 alleles (p = 0.04), while the latter had greater caveolae area/endothelial cell area than the former (p = 0.007). This study demonstrated an attenuation of CC and a significant decline in the two major transport systems in CC endothelial cells in AMD. This may contribute to drusen deposition, nutrient transport, and vision loss in AMD subjects.

Histomorphometric analysis of the choroid of donkeys, buffalos, camels and dogs.

AIM: The present study was carried out to investigate the morphological and histomorphometric characters of choroid in donkeys, buffalos, camels and dogs. RESULTS: The findings of the study revealed that, macroscopically, the choroid was consisted of two areas in all studied animals, except in camel which consists of one area. Histologically, the choroid consists of five layers. Interestingly, the anterior borders of all investigated animals were free of pigments except in camel. Morphometric analysis revealed significant species differences in the mean total thickness of the choroid and its different layers. In addition, significant differences were also found between the ratios of the means of different layers to the total thickness of the choroid. CONCLUSION: In conclusion, these variations might be related to the different lifestyles and visual behavior of the investigated animals.

RESIDUAL CHOROIDAL VESSELS IN ATROPHY CAN MASQUERADE AS CHOROIDAL NEOVASCULARIZATION ON OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY: Introducing a Clinical and Software Approach.

PURPOSE: To present a postprocessing approach in optical coherence tomography angiography (OCTA) to facilitate the visualization and interpretation of lesions in age-related macular degeneration with coexisting atrophy and choroidal neovascularization (CNV). METHODS: This retrospective study included 32 eyes of 26 patients with atrophy and treated CNV and 8 eyes with treatment-naive geographic atrophy. En face optical coherence tomography slabs highlighting atrophy were pseudocolored and merged with the corresponding OCTA. Cross-sectional optical coherence tomography and postprocessed OCTA were analyzed to identify CNV and normal choroidal vessels in relationship to the atrophy. We correlate the OCTA findings with those in a donor eye with treatment-naive geographic atrophy studied with transmission electronic microscopy. RESULTS: Medium-sized choroidal vessels were displaced anteriorly in areas of atrophy in all 40 eyes (100%), visualized in the choriocapillaris slab in all eyes, and in the outer retinal slab in 30 of 40 eyes (75.0%). Cross-sectional OCTA was used to confirm the presence of CNV. Postprocessing successfully highlighted the CNV and distinguished it from choroidal vessels in atrophy. Donor eye transmission electronic microscopy confirmed the anterior displacement of medium-sized choroidal vessels in geographic atrophy. CONCLUSION: The anterior displacement of larger choroidal vessels in atrophy requires clinician vigilance to avoid misinterpreting these vessels as CNV on en face OCTA. Our proposed postprocessing approach offers a potential solution to facilitate the interpretation of en face OCTA in these cases. In the absence of other tools, clinicians are encouraged to rely on the location of flow relative to Bruch membrane on cross-sectional OCTA flow images.

The hyaloid vasculature facilitates basement membrane breakdown during choroid fissure closure in the zebrafish eye.

A critical aspect of vertebrate eye development is closure of the choroid fissure (CF). Defects in CF closure result in colobomas, which are a significant cause of childhood blindness worldwide. Despite the growing number of mutated loci associated with colobomas, we have a limited understanding of the cell biological underpinnings of CF closure. Here, we utilize the zebrafish embryo to identify key phases of CF closure and regulators of the process. Utilizing Laminin-111 as a marker for the basement membrane (BM) lining the CF, we determine the spatial and temporal patterns of BM breakdown in the CF, a prerequisite for CF closure. Similarly, utilizing a combination of in vivo time-lapse imaging, ß-catenin immunohistochemistry and F-actin staining, we determine that tissue fusion, which serves to close the fissure, follows BM breakdown closely. Periocular mesenchyme (POM)-derived endothelial cells, which migrate through the CF to give rise to the hyaloid vasculature, possess distinct actin foci that correlate with regions of BM breakdown. Disruption of talin1, which encodes a regulator of the actin cytoskeleton, results in colobomas and these correlate with structural defects in the hyaloid vasculature and defects in BM breakdown. cloche mutants, which entirely lack a hyaloid vasculature, also possess defects in BM breakdown in the CF. Taken together, these data support a model in which the hyaloid vasculature and/or the POM-derived endothelial cells that give rise to the hyaloid vasculature contribute to BM breakdown during CF closure.

Quick-freeze/deep-etch electron microscopy visualization of the mouse posterior pole.

The mouse is one of the most commonly used mammalian systems to study human diseases. In particular it has been an invaluable tool to model a multitude of ocular pathologies affecting the posterior pole. The aim of this study was to create a comprehensive map of the ultrastructure of the mouse posterior pole using the quick-freeze/deep-etch method (QFDE). QFDE can produce detailed three-dimensional images of tissue structure and macromolecular moieties, without many of the artifacts introduced by structure-altering post-processing methods necessary to perform conventional transmission electron microscopy (cTEM). A total of 18 eyes from aged C57BL6/J mice were enucleated and the posterior poles were processed, either intact or with the retinal pigment epithelium (RPE) cell layer removed, for imaging by either QFDE or cTEM. QFDE images were correlated with cTEM cross-sections and en face images through the outer retina. Nicely preserved outer retinal architecture was observed with both methods, however, QFDE provided excellent high magnification imaging, with greater detail, of the apical, central, and basal planes of the RPE. Furthermore, key landmarks within Bruch's membrane, choriocapillaris, choroid and sclera were characterized and identified. In this study we developed methods for preparing the outer retina of the mouse for evaluation with QFDE and provide a map of the ultrastructure and cellular composition of the outer posterior pole. This technique should be applicable for morphological evaluation of mouse models, in which detailed visualization of subtle ocular structural changes is needed or in cases where post-processing methods introduce unacceptable artifacts.

Retinoic acid receptor signaling regulates choroid fissure closure through independent mechanisms in the ventral optic cup and periocular mesenchyme.

Retinoic acid receptor (RAR) signaling is required for morphogenesis of the ventral optic cup and closure of the choroid fissure, but the mechanisms by which this pathway regulates ventral eye development remain controversial and poorly understood. Although previous studies have implicated neural crest-derived periocular mesenchyme (POM) as the critical target of RA action in the eye, we show here that RAR signaling regulates choroid fissure closure in zebrafish by acting on both the ventral optic cup and the POM. We describe RAR-dependent regulation of eight genes in the neuroepithelial cells of the ventral retina and optic stalk and of six genes in the POM and show that these ventral retina/optic stalk and POM genes function independently of each other. Consequently, RAR signaling regulates ventral eye development through two independent, nonredundant mechanisms in different ocular tissues. Furthermore, the identification of two cohorts of genes implicated in ventral eye morphogenesis may help to elucidate the genetic basis of ocular coloboma in humans.

Effect of Methyl-CpG binding domain protein 2 (MBD2) on AMD-like lesions in ApoE-deficient mice.

The role of methyl-CpG binding domain protein 2 (MBD2) in an ApoE-deficient mouse model of age-related macular degeneration (AMD) was investigated. Eight-week-old Mbd2/ApoE double deficient (Mbd2(-/-) ApoE(-/-)) mice (n=12, 24 eyes, experimental group) and MBD2 (wt) ApoE(-/-) mice (n=12, 24 eyes, control group) were fed on Western-type diet for 4 months. The mice were sacrificed, and total serum cholesterol levels were analyzed and Bruch's membrane (BM) of the eyes was removed for ultrastructural observation by transmission electron microscopy. Moreover, intercellular adhesion molecule 1 (ICAM-1) immunoreactivities were evaluated by fluorescence microscopy in sections of the eyes in both groups for further understanding the function mechanism of MBD2. There was no significant difference in the total serum cholesterol levels between control group and experimental group (P>0.05). Transmission electron microscopy revealed that AMD-like lesions, various vacuoles accumulated on BM, notable outer collagenous layer deposits and dilated basal infoldings of retinal pigment epithelium (RPE) were seen in both groups, and the BM in control group was significantly thickened as compared with experimental group (P<0.05). Fluorescence micrographs exhibited the expression of ICAM-1 in choroid was higher in control group than in experimental group. We are led to conclude that MBD2 gene knockout may lead to accumulation of more deposits on the BM and influence the pathogenesis of AMD via triggering endothelial activation and inflammatory response in choroid, improving microcirculation, and reducing lipid deposition so as to inhibit the development of AMD-like lesions. Our study helps to provide a new therapeutic approach for the clinical treatment of AMD.

The regulatory roles of apoptosis-inducing factor in the formation and regression processes of ocular neovascularization.

The role of apoptosis in the formation and regression of neovascularization is largely hypothesized, although the detailed mechanism remains unclear. Inflammatory cells and endothelial cells both participate and interact during neovascularization. During the early stage, these cells may migrate into an angiogenic site and form a pro-angiogenic microenvironment. Some angiogenic vessels appear to regress, whereas some vessels mature and remain. The control mechanisms of these processes, however, remain unknown. Previously, we reported that the prevention of mitochondrial apoptosis contributed to cellular survival via the prevention of the release of proapoptotic factors, such as apoptosis-inducing factor (AIF) and cytochrome c. In this study, we investigated the regulatory role of cellular apoptosis in angiogenesis using two models of ocular neovascularization: laser injury choroidal neovascularization and VEGF-induced corneal neovascularization in AIF-deficient mice. Averting apoptosis in AIF-deficient mice decreased apoptosis of leukocytes and endothelial cells compared to wild-type mice and resulted in the persistence of these cells at angiogenic sites in vitro and in vivo. Consequently, AIF deficiency expanded neovascularization and diminished vessel regression in these two models. We also observed that peritoneal macrophages from AIF-deficient mice showed anti-apoptotic survival compared to wild-type mice under conditions of starvation. Our data suggest that AIF-related apoptosis plays an important role in neovascularization and that mitochondria-regulated apoptosis could offer a new target for the treatment of pathological angiogenesis.

In-vivo investigation of laser-induced choroidal neovascularization in rat using spectral-domain optical coherence tomography (SD-OCT).

PURPOSE: This study investigated the in-vivo formation process of laser-induced choroidal neovascularization (CNV) in rat using high-resolution spectral-domain optical coherence tomography (SD-OCT), and compared the results to histological methods. METHODS: Brown Norway rats (n = 60, 6-8 weeks of age) received 532-nm diode laser photocoagulation. SD-OCT and fluorescein angiography (FA) were performed in vivo 2, 5, 7, 14, and 21 days post-laser application. Haematoxylin and eosin (H&E) staining and immunohistochemistry for CD31, phosphorylated vascular endothelial factor receptor 2 (pVEGFR2) were conducted at each time point to observe the CNV in vitro. Choroidal flatmount preparations were observed using a confocal laser scanning microscope (CLSM) and a scanning electron microscope (SEM). RESULTS: SD-OCT monitored the longitudinal morphological changes of laser-induced CNV. CNV reached its maximal size on day 7, and began a gradual reduction on day 14. FA revealed similar dynamic changes in leakage. CNV thickness, as assessed by SD-OCT, was consistent with H&E-stained sections at each time point. CLSM and SEM revealed the details of the fibrovascular membrane. CD31 and pVEGFR2 expression supported the results of SD-OCT and histology. CONCLUSIONS: SD-OCT was a convenient and reliable tool for the imaging of the CNV formation process and quantification of the lesion size in vivo.

Tapetal dysplasia in a Swedish Vallhund dog.

OBJECTIVE: To describe the gross, histopathological, and ultrastructural findings in a dog with bilateral tapetal dysplasia. PROCEDURES: The globes of a 15-year-old neutered male Swedish Vallhund dog with a ventrally displaced tapetum in both eyes were fixed in 10% formalin and submitted to the Comparative Ocular Pathology Laboratory of Wisconsin for histological evaluation. Sections were stained with hematoxylin and eosin, Masson's trichrome, and Melan-A immunohistochemistry (IHC), and tissues were subsequently processed for transmission electron microscopy. RESULTS: Bilateral fundic and gross examination revealed a tapetal fundus inferior to the optic nerve head (ONH) and a nontapetal fundus with mild scattering of tapetal tissue superior to the ONH. Histologically, there was decreased pigmentation of the retinal pigment epithelium with only a few melanin granules in the peripheral retina. The affected tapetum was relatively acellular and fibrous with occasional tapetal cells scattered throughout the inner choroid or displaced into the vascular outer choroid. Special stains revealed that the tapetum was mostly composed of collagen (Masson's trichrome) and failed to express Melan-A (IHC) unlike a normal canine control tapetum. Ultrastructurally, the tapetum was markedly dysplastic both superior and inferior to the ONH with no uniformly arranged tapetal cells. The few cells identified within the tapetum contained irregularly arranged and disorganized electron-dense structures within their cytoplasm, which were interpreted as dysplastic tapetal rodlets. CONCLUSIONS: Based on microscopic and ultrastructural findings, this is the first report of tapetal dysplasia in a dog.

Formation of immune complexes and thrombotic microangiopathy after intravitreal injection of bevacizumab in the primate eye.

BACKGROUND: In this study, the effect of intravitreal injection of bevacizumab on choroidal blood vessels was examined in primate eyes. METHODS: Four Cynomolgus monkeys received an intravitreal injection of 1.25 mg bevacizumab. The eyes were enucleated on days 1, 4, 7 and 14. For each animal, one eye was embedded in paraffin whereas the other eye was embedded for electron microscopy. Seven untreated or PBS (phosphate buffered saline)-injected monkeys were used as controls. RESULTS: Thrombotic microangiopathy was found in the choriocapillaris and choroidal vessels of all eight injected eyes. Acute microangiopathy was characterized ultrastructurally as swelling of the endothelium, loss of fenestrations and complete collapse of the capillaries, and was commonly observed in bevacizumab-treated eyes. Quantitative analysis showed reduction of the lumina of the choriocapillaris in the eyes of three of the monkeys. Bevacizumab was frequently localized inside the blood vessels, often filling the entire breadth of the vessels, and formed clusters with blood cells. Death of photoreceptors occurred in two monkeys. CONCLUSIONS: This study indicate that intravitreal injection of bevacizumab in monkeys induces activation of platelets, degranulation of thrombocytes and neutrophils, formation of immune complexes, thrombotic microangiopathy and alteration of the blood flow in choroidal vessels.

An essential role for RPE-derived soluble VEGF in the maintenance of the choriocapillaris.

Clinical and experimental observations indicate a role for VEGF secreted by the retinal pigment epithelium (RPE) in the maintenance of the choriocapillaris (CC). VEGF in mice is produced as three isoforms, VEGF120, VEGF164, and VEGF188, that differ in their ability to bind heparan sulfate proteoglycan. RPE normally produces the more soluble isoforms, VEGF120 and VEGF164, but virtually no VEGF188, reflecting the fact that molecules secreted by the RPE must diffuse across Bruch's membrane (BrM) to reach the choriocapillaris. To determine the role of RPE-derived soluble VEGF on the choriocapillaris survival, we used mice that produce only VEGF188. VEGF188/188 mice exhibited normal choriocapillaris development. However, beginning at 7 months of age, we observed a progressive degeneration characterized by choriocapillaris atrophy, RPE and BrM abnormalities, culminating in areas of RPE loss and dramatic choroidal remodeling. Increased photoreceptor apoptosis in aged VEGF188/188 mice led to a decline in visual acuity as detected by electroretinogram (ERG). These changes are reminiscent of geographic atrophy (GA) and point to a role for RPE-derived VEGF in the maintenance of the choriocapillaris.

Multilayer subwavelength gratings or sandwiches with periodic structure shape light reflection in the tapetum lucidum of taxonomically diverse vertebrate animals.

Eye shine in the dark has attracted many researchers to the field of eye optics, but the initial studies of subwavelength arrangements in tapetum began only with the development of electronic microscopy at the end of the 20th century. As a result of a number of studies, it was shown that the reflective properties of the tapetum are due to their specialized cellular subwavelength microstructure (photonic crystals). These properties, together with the mutual orientation of the crystals, lead to a significant increase in reflection, which, in turn, enhances the sensitivity of the eye. In addition, research confirmed that optical mechanisms of reflection in the tapetum are very similar even for widely separated species. Due to progress in the field of nano-optics, researchers now have a better understanding of the main principles of this phenomenon. In this review, we summarize electron microscopic and functional studies of tapetal structures in the main vertebrate classes. This allows data on the microstructure of the tapetum to be used to improve our understanding of the visual system.

The eye of the tongue sole Cynoglossus bilineatus (Lacepède, 1802) (Teleostei: Pleuronectiformes).

We report the ocular features of the tongue sole, Cynoglossus bilineatus (Lacepède, 1802), a marine, bottom-dwelling flatfish. In this species, both eyes are located juxtaposed on the same side of the flat head. Histology revealed the sclera to be fibrous (collagenous) in nature. The choroid possesses the choriocapillaris, and adjacent to it, 3-4 rows of iridophores with stacks of cytoplasmic platelets. No choroidal gland is present. The retinal pigment epithelium (RPE) contains scanty melanin granules. Its vitread half is modified into a dense tapetum with lipid spheres (about 0.34 µm in diameter). In juveniles, the tapetal spheres arise by budding from the smooth endoplasmic reticulum of the RPE. There are blood vessels within the retina; the vitreal vessels penetrate the retina and ramify close to the level of the outer limiting membrane. The vessels are capillaries in nature. The photoreceptor layer contains abundant rods, and twin cones and single cones, being arranged into square mosaics. The optic disc is non-pleated and shows pan- cytokeratin immunopositivity, which is related to the bundled cytokeratin filaments detected in astrocytes by electron microscopy. The retinal tapetum and choroidal iridophores help the species to live in a muddy bottom having dim-light environment. The lack of a choroidal gland, hypoxic aquatic condition and presence of a dense retinal tapetum (that limits O2 transport to the photoreceptors) appear to have favored the proliferation of vitreal vessels within the retina in this species. The fibrous sclera has probably arisen to provide structural support to the eye in migration from the lateral to the dorsal aspect of the head during larval metamorphosis.

Long-Term Ultrastructural Outcomes of Autologous Transplantation of Retinal Pigment Epithelium-Partial Thickness Choroid (RPE-PTC) Sheet in Rabbits.

BACKGROUND: To observe the ultrastructural outcomes of autologous transplantation of retinal pigment epithelium-partial-thickness choroidal (RPE-PTC) sheets in rabbits after 6 months. METHODS: Eighteen pigmented rabbits were used in this study. Among them, nine rabbits were used for autologous transplantation of RPE-PTC sheets. Tissue sections were observed under a transmission electron microscope for one, three, and six months after transplantation, respectively. RESULTS: One, three, and six months after the autologous transplantation of RPE-PTC sheets, the inner and outer segments of photoreceptor cells were arranged regularly, and the connection between the inner and outer segments was normal. The inner structure of the RPE cells and tight junctions among them remained normal. Phagocytosis of outer segment of photoreceptor cells could also be observed in RPE cells. The structure of the Bruch's membrane appeared loose, rather than being dense as normal, and it was undulated after one and three months, while it became dense after six months. The graft and the bed were healed well, the boundary was unclear, and the graft was vascularized after one, three, and six months, respectively. CONCLUSIONS: Our findings revealed that the RPE-PTC sheets could quickly rebuild blood vessels, thereby maintaining the normal physiological functions of RPE cells, as well as the survival and functional status of photoreceptor cells for a long-time.