Ultrastructural Details of Epiretinal Membrane Foveoschisis.

INTRODUCTION: The aim of this study was to describe differences in the vitreomacular interface (VMI) in idiopathic epiretinal membrane (ERM) foveoschisis compared to macular pseudohole (MPH) and lamellar macular hole (LMH). METHODS: We analysed surgically excised epiretinal material and internal limiting membrane (ILM) specimens obtained from 16 eyes of 16 patients with ERM foveoschisis (6 eyes), MPH (5 eyes), and LMH (5 eyes) during standard pars plana vitrectomy (PPV) with membrane peeling. The three entities were classified according to the newly introduced optical coherence tomography (OCT) terminology. Transmission electron microscopy (TEM) was used to describe the ultrastructural features. RESULTS: We found fibrocellular epiretinal tissues in all samples analysed. However, the cell and collagen composition of the VMI differed between groups. Eyes with ERM foveoschisis were characterized by a higher number of cells, multilayered membranes, and thick strands of vitreous collagen embedding the major cell types of myofibroblasts compared to MPH. Eyes with MPH also showed a predominance of myofibroblasts, but these were located directly on the ILM with no collagen between the cells and the ILM. Eyes with LMH showed a thick, multilayered epiretinal proliferation consisting mainly of non-tractional glial cells, corresponding to hypodense epiretinal proliferation on OCT. Eyes with ERM foveoschisis and MPH were more likely to have incomplete PVD compared to LMH in terms of posterior hyaloid status. DISCUSSION/CONCLUSION: Tractional ERMs in eyes with ERM foveoschisis and MPH differ in their ultrastructure. The main difference is in the amount and topographical distribution of vitreous collagen. However, the epiretinal cell types are predominantly myofibroblasts in both entities. This highlights the importance of distinguishing ERM foveoschisis from both MPH and LMH in terms of pathogenesis and surgical peeling procedures.

Enzymatic vitreolysis using reengineered Vibrio mimicus-derived collagenase.

Purpose: Collagen is a key player contributing to vitreoelasticity and vitreoretinal adhesions. Molecular reorganization causes spontaneous weakening of these adhesions with age, resulting in the separation of the posterior hyaloid membrane (PHM) from the retina in what is called complete posterior vitreous detachment (PVD). Incomplete separation of the posterior hyaloid or tight adherence or both can lead to retinal detachment, vitreomacular traction syndrome, or epiretinal membrane formation, which requires surgical intervention. Pharmacological vitrectomy has the potential of avoiding surgical vitrectomy; it is also useful as an adjunct during retinal surgery to induce PVD. Previously studied enzymatic reagents, such as collagenase derived from Clostridium histolyticum, are nonspecific and potentially toxic. We studied a novel collagenase from Vibrio mimicus (VMC) which remains active (VMA), even after deletion of 51 C-terminal amino acids. To limit the activity of VMA to the vitreous cavity, a fusion construct (inhibitor of hyaluronic acid-VMA [iHA-VMA]) was made in which a 12-mer peptide (iHA, which binds to HA) was fused to the N-terminus of VMA. The construct was evaluated in the context of PVD. Methods: VMA and iHA-VMA were expressed in Escherichia coli, purified, and characterized with gelatin zymography, collagen degradation assay, fluorescamine-based assay, and cell-based assays. Two sets of experiments were performed in New Zealand albino rabbits. Group A (n = 10) received iHA-VMA, while group B (n = 5) received the equivalent dose of VMA. In both groups, saline was injected as a control in the contralateral eyes. Animals were monitored with indirect ophthalmoscopy, optical coherence tomography (OCT), and B-scan ultrasonography. Retinal toxicity was assessed with hematoxylin and eosin (H&E) staining of retinal tissue. Results: The activity of iHA-VMA and VMA was comparable and 65-fold lower than that of C. histolyticum collagenase Type IV. In the iHA-VMA group, all the rabbits (n = 10) developed PVD, with complete PVD seen in six animals. No statistically significant histomorphological changes were seen. In the VMA group, four of the five rabbits developed complete PVD; however, retinal morphological changes were seen in two animals. Conclusions: iHA-VMA displays targeted action confined to the vitreous and shows potential for safe pharmacologic vitreolysis.

Analysis of opticin binding to collagen fibrils identifies a single binding site in the gap region and a high specificity towards thin heterotypic fibrils containing collagens II, and XI or V/XI.

Opticin is a class III member of the extracellular matrix small leucine-rich repeat protein/proteoglycan (SLRP) family found in vitreous humour and cartilage. It was first identified associated with the surface of vitreous collagen fibrils and several other SLRPs are also known to bind collagen fibrils and it some cases alter fibril morphology. The purpose of this study was to investigate the binding of opticin to the collagen II-containing fibrils found in vitreous and cartilage. Electron microscopic studies using gold labelling demonstrated that opticin binds vitreous and thin cartilage collagen fibrils specifically at a single site in the gap region of the collagen D-period corresponding to the e2 stain band; this is the first demonstration of the binding site of a class III SLRP on collagen fibrils. Opticin did not bind thick cartilage collagen fibrils from cartilage or tactoids formed in vitro from collagen II, but shows high specificity for thin, heterotypic collagen fibrils containing collagens II, and XI or V/XI. Vitreous collagen fibrils from opticin null and wild-type mice were compared and no difference in fibril morphology or diameter was observed. Similarly, in vitro fibrillogenesis experiments showed that opticin did not affect fibril formation. We propose that when opticin is bound to collagen fibrils, rather than influencing their morphology it instead hinders the binding of other molecules to the fibril surfaces and/or act as an intermediary bridge linking the collagen fibrils to other non-collagenous molecules.

Hyaluronic acid coating of gold nanoparticles for intraocular drug delivery: Evaluation of the surface properties and effect on their distribution.

Due to the unique anatomical structure of the eye, ocular drug delivery is a promising delivery route for the treatment of several ocular diseases, such as the ocular neovascularization that contributes to diabetic retinopathy. This disease is triggered by inflammation, retinal ischemia, and/or deposits of advanced-glycation end-products (AGEs), as well as increased levels of vascular endothelial growth factor (VEGF), interleukins, or reactive oxygen species (ROS). Gold has unique antioxidant and antiangiogenic properties and can inhibit angiogenic molecules. Furthermore, gold nanoparticles (GNPs) are not only biocompatible, they are easy to synthesize, they absorb and scatter visible light, and they can be made with precise control over size and shape. GNPs are an excellent candidate for ocular drug delivery because they can be conjugated to an extraordinarily diverse array of different biomolecules, and surface functionalization can improve the mobility of GNPs across the physiological barriers of the eye, such as the vitreous humour or the inner limiting membrane. For this purpose, we employed low molecular weight hyaluronan (HA) to increase the mobility of the nanoparticles as well as target them to HA receptors that are expressed in different cells of the eye. In this study, the combination of gold and HA enhanced the stability of the whole carrier and promoted their distribution across ocular tissues and barriers to reach the retina. Moreover, analysis in vitro, ex vivo, and in ovo revealed the protective and antiangiogenic effect of GNPs as inhibitors of AGEs-mediated- retinal pigment epithelial cell death and neovascularization. We demonstrated that conjugation with HA enhances GNP stability and distribution due to a specific CD44 receptor interaction. The capacity of HA-GNPs to distribute through the vitreous humour and their avidity for the deeper retinal layers ex vivo, suggest that HA-GNPs are a promising delivery system for the treatment of ocular neovascularization and related disorders.

Non-reversible tissue fixation retains extracellular vesicles for in situ imaging.

Extracellular vesicles (EVs) are secreted nanosized particles with many biological functions and pathological associations. The inability to image EVs in fixed tissues has been a major limitation to understanding their role in healthy and diseased tissue microenvironments. Here, we show that crosslinking mammalian tissues with formaldehyde results in significant EV loss, which can be prevented by additional fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for visualization of EVs in a range of normal and cancer tissues.

Bush-like integrin filament networks associated with hyaloid vasculature in murine neonate eyes.

The vitreous of perinatal mice temporarily develops a unique vascular system, called the vasa hyaloidea propria (VHP). Observations showed the vessels possessed an extracellular matrix including the basement membrane in their entire length. Immunostaining of whole mount preparations of VHP with integrin ß1 antibody displayed a bush-like network consisting of long and straight fibers which were associated with the VHP but extended apart from the blood vessels. Electron microscopically, each fiber was composed of a bundle of thin filaments different from collagen fibrils. Macrophages associated with the VHP appeared to be arrested by the integrin bushes. The integrin bushes fragmented and disappeared by postnatal day 10, just before the regression of the VHP. Macrophages were involved in the digestion and clearance of integrin bushes. The vitreous integrin bushes appear to provide a scaffold for architectural maintenance of the hyaloid vessels and macrophages.

Diffusion through the ex vivo vitreal body - Bovine, porcine, and ovine models are poor surrogates for the human vitreous.

The human vitreous humour is a complex gel structure whose composition and physical properties can vary considerably from person to person and also change with age. To date, the viscoelastic properties of the human vitreous gel has not been thoroughly investigated and despite many years of intensive research, an ideal vitreous substitute remains a challenge. Understanding the physical structure and properties of the vitreous is of fundamental and therapeutic interest, providing a clear insight into diffusion and transport of administered ophthalmic drug molecules into the vitreous. A number of mammalian surrogates, mainly bovine, porcine and ovine vitreous humours have been used in the literature as a means of studying ophthalmic drug transport and diffusion. In this study, the mechanical, physical and rheological properties of ovine, porcine, and bovine surrogates were investigated and compared to human vitreous. In addition, a bespoke Franz cell construct was used to compare the diffusion of a model drug (fluorescein) through vitreous samples. Despite the similarity in rheological properties between bovine, porcine and human vitreous samples, diffusion of fluorescein through the different vitreous samples revealed great differences in values of steady-state flux and diffusion coefficient. In addition, a first-generation vitreous mimic, composed of 4.5 mg/mL hyaluronic acid with complex viscosity of 0.3 ±â€¯0.01 Pa has been evaluated and was demonstrated to be a better mimic of the human vitreous than the mammalian samples investigated.

Effects of enzymatic degradation on dynamic mechanical properties of the vitreous and intravitreal nanoparticle mobility.

Intravitreal mobility of nanocarriers may have implications on the efficacy of the encapsulated drug in the treatment of vitreo-retinal diseases, with any changes in the integrity of the vitreous microstructure influencing nanoparticle biodistribution. This study investigated enzymatically digested vitreous models to mimic the aging eye. Collagenase, hyaluronidase, or trypsin was employed to selectively digest the structural components of the vitreous. Physical properties of digested bovine vitreous were initially assessed via texture analysis and oscillatory shear testing. Morphological changes in bovine vitreous microstructure were visualized by scanning electron microscopy and diffusion dynamics of hyaluronic acid coated nanoparticles through degraded porcine and bovine vitreous were examined using fluorescence spectroscopy and multiple particle tracking microscopy, respectively. After enzymatic treatment, the vitreous liquefied and its dynamic mechanical properties significantly changed with a decrease in stiffness and an increase in damping capacity. Micrographs confirmed specific digestion of each of the structural vitreous components. Furthermore, enzymatic degradation reduced steric hindrance and enhanced convective flow within the vitreous, resulting in increased intravitreal nanoparticle mobility which could alter the drug pharmacokinetics.

Ultrastructural and histopathologic findings after pars plana vitrectomy with a new hypersonic vitrector system. Qualitative preliminary assessment.

PURPOSE: Preliminary assessment of a new prototype ultrasound-based hypersonic vitrector (HV) by qualitatively examining the histopathological changes in the retina and vitreous body after pars plana vitrectomy (PPV) and its ability to fragment vitreous collagen. METHODS: Fourteen porcine cadaveric eyes, 20 eyes in live swine and six human cadaveric eyes underwent PPV using the HV or a pneumatic guillotine vitrector (GV). An additional 4 porcine crystalline lenses were touched with either the HV or GV for 1 minute. Following PPV, human vitreous was removed and processed for electron microscopy (EM). Eyes and lenses were fixed and sectioned for light microscopy (LM). RESULTS: There were no macroscopic retinal or optic nerve defects associated with either HV or GV PPVs. Cadaveric retinal specimens showed separation of the inner limiting membrane (ILM) and vacuolization and fragmentation at the nerve fiber layer (NFL) and the ganglion cell layer (GCL). ILM fragmentation and separation were found after PPV in live swine with both vitrectors. Small disruptions of the posterior capsule or structural lens defects were found after HV touch. The EM analysis revealed more fragmentation of human vitreous collagen fibrils after HV compared to GV PPV. CONCLUSIONS: LM and EM analysis of retina, vitreous, and crystalline lens after PPV showed similar morphological changes using the HV or the GV. Vitreous fragmentation appeared more effective with the HV. Overall this study suggests that the HV may be a promising new technology. More work is needed to quantitatively assess its safety and efficacy.

Microarchitecture of the Vitreous Body: A High-Resolution Optical Coherence Tomography Study.

PURPOSE: To report novel vitreous body microarchitecture findings using high-resolution spectral-domain optical coherence tomography (HR-SD-OCT). DESIGN: Prospective, cross-sectional study. METHODS: Horizontal and vertical retinal cross-sectional images that were 10 mm long were acquired from 17 eyes of 17 young healthy volunteers using HR-SD-OCT with enhanced vitreous imaging (EVI). Images were acquired through the fovea, upper vessel arcade, and lower vessel arcade. RESULTS: Three new findings on vitreous body microarchitecture were found. First, material located between the retina and posterior vitreous cortex was easily detected in 90% of upper and lower vessel arcade scans. Most scans contained hyperreflective dots and multilayered hyperreflective lines around the detached vitreous cortex. Second, a lamellar structure was observed in the vitreous body in 70%-80% of all scans, excluding vertical scans of the upper arcade vessel area. Third, tubular zones of hypodensity were detected in >80% of scans, excluding horizontal scans of the macula. Interestingly, the location of tubular zones of hypodensity seemed to correspond with the location of retinal vessels. Subject age, refractive error, and axial length were not significantly different in scans with and without material between the retina and vitreous, lamellar structures, and tubular zones of hypodensity. CONCLUSIONS: The microarchitecture of the vitreous body can be visualized using HR-SD-OCT and EVI.

Characterization of equine hyalocytes: their immunohistochemical properties, morphologies and distribution.

In horse, the characterizations of hyalocytes under the steady state are still unclear. Therefore, we investigated characterizations of hyalocytes in normal equine eyes by their immunohistochemical phenotype, histomorphology and distribution. Thirty-one eyes from 18 horses, divided into 4 groups (G) by age, were used: early (G1) and late gestation (G2) fetuses, 1- to 3-year-old (G3) and 8- to 24-year-old (G4) horses. Equine hyalocytes were histologically classified into 4 types, and they immunohistochemically expressed MHC II and CD163. Hyalocytes were detected on and/or around ciliary processes and pars plana in G2, G3 and G4, but were not located on retina and optic papilla. A significant increase in distribution was found between G2 and both G3 and G4, and the largest distribution was found at ciliary processes in these groups. Equine hyalocytes were characterized as residential ocular macrophage and MHC II antigen-bearing cell, accompanied by a pleomorphic appearance and located in the contiguous ciliary body. Our data provided characterizations of hyalocytes in normal equine eyes and may well contribute to improving the understanding of pathogenesis of equine ocular disease.

Vitreomacular Attachment Ultrastructure and Histopathological Correlation.

PURPOSE: The ultrastructural anatomy of the vitreomacular interface in young human donor eyes and animal eyes is explored using scanning electron microscopy (SEM) to determine its relationship with the formation of the perimacular ridge from abusive head trauma, as well as macular hole formation, vitreomacular traction syndrome, and preretinal hemorrhage. MATERIALS AND METHODS: SEM is used to image the posterior poles of 23 human donor eyes, as well as several cow, dog, monkey, pig, and rabbit eyes for vitreomacular interface anatomy. We examined autopsy eyes from abusive head trauma histopathologically. RESULTS: Two rings of thick, circumferential, vitreous attachment at the area centralis are found. An inner ring at the fovea, R1, and an outer ring at the perifoveal region, R2, are both observed in eyes from donors < 30 years of age; comparatively, in eyes from donors > 30 years, only R2 is present (p<0.001). R2 is found with unique elliptical shape in Cynomolgus monkey. Macula, R1, and R2 are not detected in cow, dog, pig, or rabbit eyes. CONCLUSIONS: The vitreomacular ring attachments found in donor eyes correspond anatomically with the perimacular ridge found histopathologically in abusive head trauma, and likely correlates with the macular hole, vitreomacular traction syndrome, and preretinal hemorrhage. Vitreomacular interface anatomy in the monkey, but not the cow, dog, pig, or rabbit, demonstrates some anatomical similarity to that of the human, consistent with species differences regarding the area centralis.

The Ultrastructural Localization of Type II, IV, and VI Collagens at the Vitreoretinal Interface.

BACKGROUND: The vitreoretinal interface is the border of the cortical vitreous and the inner surface of the retina. The adhesion of the cortical vitreous to the ILM, namely vitreoretinal adhesion, involves a series of complex molecular adhesion mechanisms and has been considered as an important pathogenic factor in many vitreoretinal diseases. The presence of type VI collagen at the vitreoretinal interface and its possible interaction with collagens and glycoproteins indicates that type VI collagen may contribute to the vitreoretinal adhesion. PURPOSE: To clarify the ultrastructural location of type VI collagen and its relationship to type II and IV collagens at the vitreoretinal interface. METHODS: The ultrastructural localization of type II, IV and VI collagens in the adult human vitreoretinal interface of five donor eyes was evaluated by transmission electron microscopy using immunogold labeling. RESULTS: In the pre-equatorial region, we observed densely packed vitreous lamellae with a partly intraretinal course containing type II and VI collagens, reticular structures containing type IV and VI collagens and a thin inner limiting membrane (ILM) containing type IV and VI collagens in a linear distribution pattern. From the anterior to the posterior retina, the linear pattern of type IV and VI collagen labeling gradually became more diffusely present throughout the entire thickness of the ILM. CONCLUSIONS: The presence of type VI collagen in vitreous lamellae penetrating the ILM into the superficial retina suggests that type VI collagen may be involved in the organization of vitreous fibers into lamellae and in the adhesion of the vitreous fibers to the retina. The close relation of type VI to type IV collagen in the ILM suggests that type VI collagen is an important collagen type in the ILM. The topographic variations of type IV and VI collagens in the different regions of the ILM suggest a regional heterogeneity of the ILM. The reticular labeling pattern of type IV and VI collagens observed in the anterior vitreous are highly similar to labeling patterns of blood vessel walls. In the anterior vitreous, they may represent remnants of the regressed embryonic hyaloid blood vessel system. Their presence is in support of the theory on interactive remodeling of the developing vitreous as opposed to the main stream theory of displacement and compression of the primary by the secondary vitreous.

In Vivo Assessment of Pharmacologic Vitreolysis in Rabbits With the Digital Fluoroscopy System.

PURPOSE: The purpose of this study was to demonstrate the efficacy of the digital fluoroscopy system (DFS) for the in vivo assessment of pharmacologically induced posterior vitreous detachment (PVD) and vitreous liquefaction in a rabbit model. METHODS: Twenty eyes from 10 New Zealand white rabbits were divided into 5 groups. In each group, one rabbit received an intravitreal injection of 2.0 U plasmin in the right eye and 0.5 U plasmin in the left eye. Intravitreal injection of 0.1 mL balanced salt solution (BSS) was given in the right eye, and no injection was given in the left eye of another rabbit used as a control. Intraocular fluid dynamics were assessed by the DFS, using a contrast agent in each group at different time intervals (6 hours, 12 hours, 1 day, 3 days, and 7 days). After rabbits were killed, both eyes were enucleated. Scanning electron microscopy was used to confirm the morphological alterations of the vitreoretinal interface as observed in the DFS. RESULTS: Complete PVD was observed after 12 hours with 2.0 U plasmin injection, whereas complete PVD was observed only after 3 days in eyes injected with 0.5 U plasmin. Eyes that received BSS injection or did not receive an injection failed to show complete PVD even after 7 days. Complete vitreous liquefaction was observed after 7 days with 2.0 U plasmin injection, but no eyes with 0.5 U plasmin or BSS injection showed complete liquefaction. We could clearly confirm the presence of PVD and the degree of vitreous liquefaction by using DFS. CONCLUSIONS: Digital fluoroscopy system appears to be a useful tool for the evaluation of pharmacological vitreolysis in rabbits with clear in vivo visualization of PVD and vitreous liquefaction.

[The vitreous and the macula].

The macula is a site where various vitreoretinal disorders occur. In 1983 we started to observe the retinal surface of postmortem eyes with a scanning electron microscope (SEM). We investigated the anatomy of the vitreous in postmortem eyes by slit lamp biomicroscopy. The novel anatomy of the premacular vitreous led us to conduct a clinical study of vitreomacular interface diseases. In 1997, time domain optical coherence tomography(OCT) became available which facilitated visualization of the vitreoretinal interface. Swept source OCT which was introduced in 2012 can depict liquefied lacunae in the vitreous. It enabled us to elucidate the mechanism of vitreoretinal diseases. I. SEM revealed the remnants of vitreous cortex at fovea with high incidence (44%), which suggests strong vitreoretinal attachment at the fovea and vitreous cortex origin of the epiretinal membrane. II. We studied the anatomy of the vitreous in postmortem eyes. The vitreous of bisected eye balls was stained by fluorescein and immersed in water and observed by slit-lamp biomicroscopy. We discovered a "posterior precortical vitreous pocket (PPVP)" in adult eyes without posterior vitreous detachment (PVD). III. We performed clinical study in various vitreoretinal diseases based on the novel vitreous anatomy and explained their mechanism. 1. In diabetic retinopathy, ring shaped fibrovascular tissue surrounding the macula is formed along the outer margin of the PPVP. Although PVD progresses outside the PPVP, its posterior wall remains attached to the retina, which causes macular traction or cystoid macular edema. 2. In eyes with idiopathic epimacular membrane (IEM), detached vitreous cortex had an oval defect corresponding to the IEM. Posterior wall of the PPVP that is premacular vitreous cortex appeared to be the framework of IEM. 3. During vitrectomy for macular hole, premacular round defect appears when PVD is created. The residual cortex on the macula is fibrous membrane with elasticity. The tangential contraction of premacular cortex may generate anterior traction to the fovea, which leads to macular hole. IV. Using time domain OCT, we demonstrated the evolution of macular hole, myopic foveoschisis and lamellar macular hole. After 2007, we investigated age related changes of vitreoretinal interface by spectral domain OCT V. We demonstrated whole structure of the PPVP using swept source OCT. PPVP is a boat shaped premacular liquefied space which has a connecting channel to Cloquet's canal. PPVP develops during childhood. Visualization of vitreous structure proved that our previous assumptions are reasonable. Although the physiological function of the PPVP is unclear, we speculate that the aqueous flows into the PPVP though Cloquet's canal and the connecting channel.

Preservation of the structure of enzymatically-degraded bovine vitreous using synthetic proteoglycan mimics.

PURPOSE: Vitreous liquefaction and subsequent posterior vitreous detachment can lead to several sight-threatening diseases, including retinal detachment, macular hole and macular traction syndrome, nuclear cataracts, and possibly, open-angle glaucoma. In this study, we tested the ability of three novel synthetic chondroitin sulfate proteoglycan mimics to preserve the structure and physical properties of enzymatically-degraded bovine vitreous. METHODS: Chondroitin sulfate proteoglycan mimics, designed to bind to type II collagen, hyaluronic acid, or both, were applied to trypsin- or collagenase-treated bovine vitreous in situ and in vitro. Rheology and liquefaction tests were performed to determine the physical properties of the vitreous, while Western blots were used to detect the presence and degradation of soluble collagen II (α1). Deep-etch electron microscopy (DEEM) identified the ultrastructure of mimic-treated and untreated enzyme-degraded bovine vitreous. RESULTS: Proteoglycan mimics preserved the physical properties of trypsin-degraded bovine vitreous and protected against vitreous liquefaction. Although the collagen-binding mimic maintained the physical properties of collagenase-treated vitreous, liquefaction still occurred. Western blots indicated that the mimic provided only marginal protective ability against soluble collagen degradation. Deep-etch electron microscopy, however, showed increased density and isotropy of microstructural components in mimic-treated vitreous, supporting the initial result that vitreous structure was preserved. CONCLUSIONS: Proteoglycan mimics preserved bovine vitreous physical properties after enzymatic degradation. These compounds may be useful in delaying or preventing the pathological effects of age-related, or enzymatically-induced, degradation of the vitreous body.

Penetration of silicate nanoparticles into the corneal stroma and intraocular fluids.

PURPOSE: The aim of this study was to investigate transmission of topical silicate nanoparticles (SiNPs) through the corneal stroma, anterior chamber, and vitreous fluids by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and inductively coupled plasma atomic emission spectrometry (ICP-AES), respectively. METHODS: SiNPs with a mean diameter of 40.6 ± 5.6 nm determined by dynamic light scattering were used in this study. The permeability of SiNPs was examined across isolated corneal buttons over a 30-minute period. To visualize the transport and diffusion of nanoparticles through the corneal tissue, SiNPs were applied over the corneal surface and evaluated at 5 and 30 minutes after SiNPs loading for SEM and 15 minutes for TEM. Sections of 10-µm thickness were cut and visualized using SEM. TEM study was performed on 70- to 90-nm-thick sections. ICP-AES was used to determine the concentration of SiNPs. RESULTS: The determined range of synthesized SiNPs by dynamic light scattering was 40 nm (41.9 ± 5.6 nm). Transmission of SiNPs through the corneal stroma was shown successfully with electron microscopic (SEM and TEM) images. The ICP-AES results revealed SiNPs in the anterior chamber and vitreous fluid. CONCLUSIONS: Topical administration of SiNPs, as a noninvasive, and available modality with acceptable penetration through the corneal stroma and deep into the intraocular fluids including the anterior chamber and vitreous cavity, may be considered as a suitable alternative to invasive intravitreal injection of other expensive antineovascularization agents.

Ultrastructural changes of the vitreoretinal interface during long-term follow-up after removal of the internal limiting membrane.

PURPOSE: To investigate long-term ultrastructural changes in the retina after internal limiting membrane (ILM) peeling through the examination of morphologic changes 3 years after vitrectomy in cynomolgus monkeys. DESIGN: Laboratory investigation. METHODS: Pars plana vitrectomy was performed, followed by ILM peeling, in 2 primate eyes. Ultrastructural changes were investigated using light microscopy and transmission and scanning electron microscopy 3 years after ILM peeling. RESULTS: The remaining posterior vitreous and ILM-peeled areas were clearly recognized after the long-term follow-up. The exposed Müller cell processes were partially damaged, while regenerative spindle-shaped Müller cell processes developed, covering most of the retina. Notably, the nerve fiber layer was found to be uncovered and exposed to the vitreous space owing to misdirection of glial wound healing in some parts. In these areas, glial wound healing occurred beneath the nerve fiber layer. Although the glial cells covered the damaged areas, there was no apparent ILM regeneration in the shape of a continuous flat sheet, with the exception of accumulated deposits of basement membrane materials. CONCLUSIONS: Although the retinal structures were well preserved after ILM peeling, ILM peeling resulted in mild damage to the vitreoretinal interface, which was not completely restored even after 3 years. The multilinear shape of the exposed nerve fiber may explain the previously reported dissociated optic nerve fiber layer appearance. The glial cells produced basement membrane materials around their processes, although they did not restore the ILM as a flat sheet.

Vitreomacular interface and outer foveal microstructure in fellow eyes of patients with unilateral macular holes.

PURPOSE: To investigate the relationship between the vitreomacular interface and the integrity of the photoreceptor microstructures in the normal fellow eyes of patients with unilateral macular holes. METHODS: Retrospective observational case series. Fifty-five normal fellow eyes of 55 patients with unilateral macular holes were enrolled in the study. All patients underwent complete ophthalmologic examination including best-corrected visual acuity, slit-lamp biomicroscopy, fundus photography, and spectral domain optical coherence tomography at initial and follow-up visits. The features of the vitreomacular interface were graded based on spectral domain optical coherence tomography findings. RESULTS: At the initial visit, 28 of 55 eyes (51%) had vitreomacular attachments with or without perifoveal posterior vitreous detachment. On their initial visit, a triangular elevation of the cone outer segment tips line was identified in 11 of 18 eyes (61%) with perifoveal posterior vitreous detachment across all quadrants with persistent attachment to the fovea. Conversely, none of the remaining 37 eyes with the other stages of posterior vitreous detachment showed any abnormalities. Over a mean follow-up period of 18 months (range, 12-24 months), the elevation of the cone outer segment tips line resolved after spontaneous vitreomacular separation without macular holes in 3 eyes, remained unchanged in 6 eyes, and showed progression to a full-thickness macular hole in 2 eyes. CONCLUSION: These findings suggest that an elevation of the cone outer segment tips line in the normal fellow eyes of patients with macular holes is caused by the focal traction of the vitreous at the foveal center. This is considered to be an important primary change observed in the macular tissue in full-thickness macular hole formation.