Human Vitreous Research Articles

Proteomic insight into the molecular function of the vitreous.

The human vitreous contains primarily water, but also contains proteins which have yet to be fully characterized. To gain insight into the four vitreous substructures and their potential functions, we isolated and analyzed the vitreous protein profiles of three non-diseased human eyes. The four analyzed substructures were the anterior hyaloid, the vitreous cortex, the vitreous core, and the vitreous base. Proteins were separated by multidimensional liquid chromatography and identified by tandem mass spectrometry. Bioinformatics tools then extracted the expression profiles, signaling pathways, and interactomes unique to each tissue. From each substructure, a mean of 2,062 unique proteins were identified, with many being differentially expressed in a specific substructure: 278 proteins were unique to the anterior hyaloid, 322 to the vitreous cortex, 128 to the vitreous base, and 136 to the vitreous core. When the identified proteins were organized according to relevant functional pathways and networks, key patterns appeared. The blood coagulation pathway and extracellular matrix turnover networks were highly represented. Oxidative stress regulation and energy metabolism proteins were distributed throughout the vitreous. Immune functions were represented by high levels of immunoglobulin, the complement pathway, damage-associated molecular patterns (DAMPs), and evolutionarily conserved antimicrobial proteins. The majority of vitreous proteins detected were intracellular proteins, some of which originate from the retina, including rhodopsin (RHO), phosphodiesterase 6 (PDE6), and glial fibrillary acidic protein (GFAP). This comprehensive analysis uncovers a picture of the vitreous as a biologically active tissue, where proteins localize to distinct substructures to protect the intraocular tissues from infection, oxidative stress, and energy disequilibrium. It also reveals the retina as a potential source of inflammatory mediators. The vitreous proteome catalogues the dynamic interactions between the vitreous and surrounding tissues. It therefore could be an indirect and effective method for surveying vitreoretinal disease for specific biomarkers.

Increased Vitreous Heme Oxygenase Activity is Associated with Proliferative Diabetic Retinopathy

Heme oxygenase is an enzyme having three isoforms and is responsible for breakdown of heme into iron, carbon monoxide and bilirubin. There is no study related to heme oxygenase levels in human vitreous. So in this study our objectives were to measure heme oxygenase (HO) enzyme activity in vitreous and plasma of patients with proliferative diabetic retinopathy (PDR) and compare with the HO levels of patients with macular hole (MH). All protocols were approved by the Institution’s Ethics Board. Blood and vitreous samples were collected from 15 patients with PDR and 11 from macular hole who underwent vitrectomy surgery. HO enzyme activity was measured in vitreous and plasma samples by spectrophotometric method. In the PDR group, vitreous HO was 8.03 ± 1.95 pmol of bilirubin/mg protein which is significantly higher when compared to the MH group, 0.67 ± 0.2 pmol of bilirubin/mg protein. However, there was no difference in their HO activity in plasma, between the groups. Total Heme oxygenase enzyme activity is elevated in vitreous of PDR. Vitreous HO activity positively correlated with already reported adiponectin levels in these patients.

Characteristic of ocular safety profile of triamcinolone made in China

To characterize the safety profile of triamcinolone acetonide made in China (Transton) and triamcinolone acetonide acetate (Tongyong) for their ocular application. Experimental study. In vitro cell viability assay was performed on 3 types of human ocular cells to evaluate the cytotoxicity of the simulated vitreal concentrations (from a 1:15 dilution as if injected into 1.5 ml of rabbit vitreous to 1:50 dilution as if injected into 5 ml of human vitreous) of Transton and Tongyong using MTT method.In vivo 28 guinea pigs, randomly divided into four groups, were used for evaluating either intravitreal 6 µl of the two types of triamcinolone suspension or 18 µl of their supernatant. Following the injections, the eyes were monitored by biomicroscopy, ophthalmoscopy, tonometry, electroretinography, and histology. The Dunnett's test was used to analyze in vitro cell viability. Paired sample t-test and Generalized Estimating Equations (GEE) were respectively used to compare electroretinography data and intraocular pressure between experimental eyes and the control eyes. The undiluted supernatant of Transton and Tongyong was toxic to human scleral fibroblasts when compared with their control groups (MTT values = 0.046 ± 0.036 and 0.044 ± 0.05 versus 0.367 ± 0.106 and 0.413 ± 0.128) (P < 0.01) or with the BSS group (0.368 ± 0.106 and 0.441 ± 0.137) (P < 0.01). 1:15 or greater dilution of supernatant of Transton and Tongyong did not show cytotoxicity on cultured human retinal pigment epithelium cells or Müller cell (P > 0.05), but 1:15 dilution of Tongyong supernatant showed cytotoxicity on the Müller cells (MTT value = 0.366 ± 0.062 versus 0.417 ± 0.042 for BSS) (P = 0.03). In vivo, neither intravitreal 6 µl (0.25 mg, equivalent to 4 mg in 0.1 ml for human eyes) of the suspension nor 18 µl (equivalent resultant preservative concentration to intravitreal 4 mg in 0.1 ml for rabbit eyes) of the supernatant of Transton and Tongyong showed ocular toxicity. The equivalent dose (6 µl) to 4 mg in 0.1 ml intravitreal suspension for human eye or equivalent resultant preservative concentration to 0.1 ml intravitreal suspension for rabbit eye is found safe in guinea pig eyes.

Bone morphogenetic protein 2: A potential new player in the pathogenesis of diabetic retinopathy

Diabetic retinopathy (DR) is one of the most common complications of diabetes mellitus. Vision loss in DR principally occurs due to breakdown of the blood–retinal barrier (BRB), leading to macular edema, retinal detachment and inner retinal and vitreous hemorrhage. Several growth factors have been shown to play crucial role in the development of these vascular changes; however, the cellular and molecular mechanisms of DR are not yet fully revealed. In the current study we investigated the role of bone morphogenetic protein-2 (BMP2) in DR. We examined the changes in the protein levels of BMP2 in human vitreous and retina in addition to the mouse retina of streptozotocin-induced diabetes. To detect the source of BMP2 during diabetes, human retinal endothelial cells (hRECs) were subjected to high glucose (HG) for 5 days and levels of BMP2 protein were analyzed in conditioned media of these cells relative to control. We also evaluated the effect of BMP2 on the levels of VEGF in cultured rat Müller cells (rMC1). In addition, we tested the pro-inflammatory effects of BMP2 by examining its effect on leukocyte adhesion to cultured hRECs, and levels of adhesion molecules and cytokines production. Finally, the effect of different concentrations of BMP2 on permeability of confluent monolayer of hRECs was evaluated using FITC-Dextran flux permeability assay and by measuring Transcellular Electrical Resistance (TER) using Electric Cell-substrate Impedance Sensing (ECIS).Our results show, for the first time, the up-regulation of BMP2 in diabetic human and mouse retinas in addition to its detection in vitreous of patients with proliferative DR (72 ± 7 pg/ml). In vitro, hRECs showed upregulation of BMP2 in HG conditions suggesting that these cells are a potential source of BMP2 in diabetic conditions. Furthermore, BMP2 induced VEGF secretion by Müller cells in-vitro; and showed a dose response in increasing permeability of cultured hRECs. Meanwhile, BMP2 pro-inflammatory effects were recognized by its ability to induce leukocyte adhesion to the hRECs, intercellular adhesion molecule-1 (ICAM-1) and upregulation of interleukin-6 and 8 (IL-6 and IL-8). These results show that BMP2 could be a contributing growth factor to the development of microvascular dysfunction during DR via enhancing both pro-angiogenic and inflammatory pathways. Our findings suggest BMP2 as a potential therapeutic target to prevent/treat DR.

New eye phantom for ophthalmic surgery.

In this work, we designed and realized a new phantom able to mimic the principal mechanical, rheological, and physical cues of the human eye and that can be used as a common benchmark to validate new surgical procedures, innovative vitrectomes, and as a training system for surgeons. This phantom, in particular its synthetic humor vitreous, had the aim of reproducing diffusion properties of the natural eye and can be used as a system to evaluate the pharmacokinetics of drugs and optimization of their dose, limiting animal experiments. The eye phantom was built layer-by-layer starting from the sclera up to the retina, using low cost and easy to process polymers. The validation of the phantom was carried out by mechanical characterization of each layer, by diffusion test with commercial drugs into a purposely developed apparatus, and finally by a team of ophthalmic surgeons. Experiments demonstrated that polycaprolactone, polydimethylsiloxane, and gelatin, properly prepared, are the best materials to mimic the mechanical properties of sclera, choroid, and retina, respectively. A polyvinyl alcohol-gelatin polymeric system is the best for mimicking the viscosity of the human humor vitreous, even if the bevacizumab half-life is lower than in the human eye.

Soluble opsin is present in human vitreous

The purpose of this research project was to evaluate if intravitreal opsins are present in human vitreous liquid which is, so far, unknown. Therefore a pilot study was conducted including 22 vitreal samples which were harvested at the beginning of a standard 23-gauge three-port pars plana vitrectomy for macular pucker, diabetic vitreous hemorrhage or vitreal floater removal as well as macular hole closure or vitreomacular traction relief from the central vitreous body. No adverse events or serious side effects occurred. All samples were immediately stabilized by human albumin and arginine and subsequently frozen. Short-wavelength cone opsin concentrations were analyzed by enzyme immune essay (EIA) with anti-proteolytic 400 mM arginine, pH 8.7, in the antigen capture phase. Intravitreal short-wavelength cone opsins were detected in all analyzed samples and respective concentrations ranged at levels of 157 pg/ml ± 73 pg/ml (MV ± SD; range: 27 pg/m–286 pg/ml). Eyes with MP/MH/DVH/VMT and VF exhibited intravitreal short-wavelength cone opsin concentrations of 189 pg/ml ± 68 pg/ml (range: 72 pg/ml–286 pg/ml)/96 pg/ml ± 39 pg/ml (range: 50 pg/ml–138 pg/ml)/126 pg/ml ± 88 pg/ml (range: 27 pg/ml–198 pg/ml)/224 pg/ml and 121 pg/ml. Further studies will quantify the intravitreal opsin pattern of all visual opsins and compare these concentrations between different vitreoretinal diseases. This in turn might offer a better pathophysiological understanding and new diagnostic and therapeutic strategies for various eye pathologies. As a hypothesis, soluble opsins might be a biomarker for retinal damage comparable to creatinine for kidney damage.

Computational Model for Oxygen Transport and Consumption in Human Vitreous

Previous studies that measured liquefaction and oxygen content in human vitreous suggested that exposure of the lens to excess oxygen causes nuclear cataracts. Here, we developed a computational model that reproduced available experimental oxygen distributions for intact and degraded human vitreous in physiologic and environmentally perturbed conditions. After validation, the model was used to estimate how age-related changes in vitreous physiology and structure alter oxygen levels at the lens. A finite-element model for oxygen transport and consumption in the human vitreous was created. Major inputs included ascorbate-mediated oxygen consumption in the vitreous, consumption at the posterior lens surface, and inflow from the retinal vasculature. Concentration-dependent relations were determined from experimental human data or estimated from animal studies, with the impact of all assumptions explored via parameter studies. The model reproduced experimental data in humans, including oxygen partial pressure (Po2) gradients (≈15 mm Hg) across the anterior-posterior extent of the vitreous body, higher oxygen levels at the pars plana relative to the vitreous core, increases in Po2 near the lens after cataract surgery, and equilibration in the vitreous chamber following vitrectomy. Loss of the antioxidative capacity of ascorbate increases oxygen levels 3-fold at the lens surface. Homogeneous vitreous degeneration (liquefaction), but not partial posterior vitreous detachment, greatly increases oxygen exposure to the lens. Ascorbate content and the structure of the vitreous gel are critical determinants of lens oxygen exposure. Minimally invasive surgery and restoration of vitreous structure warrant further attention as strategies for preventing nuclear cataracts.

Retinal pigment epithelium cells produce VEGF in response to oxidized phospholipids through mechanisms involving ATF4 and protein kinase CK2

Oxidized phospholipids (OxPLs) are pleiotropic lipid mediators known to induce proangiogenic and proinflammatory cellular effects that are increasingly recognized to be involved in a number of physiologic and pathologic processes in the retina. Immunohistochemical studies have detected OxPLs in retinal structures, such as retinal pigment epithelium (RPE) or photoreceptor cells. This study analyzed whether OxPLs could play a role in upregulation of VEGF, which is a cause of pathological neovascularization characteristic of eye diseases such as age-related macular degeneration. We confirmed accumulation of OxPLs in the eye using reversed-phase liquid chromatography coupled to mass spectrometry. Multiple species of oxidized phosphatidylcholines (OxPCs) were detected in human vitreous, including biologically active fragmented species POVPC, PGPC, PONPC and PAzPC. In in vitro experiments human fetal RPE and primary RPE cells were stimulated with OxPLs. Primary RPE cells were transfected with small interfering RNAs targeting ATF4. mRNA levels of VEGF in fetal and primary RPE cells were determined by real-time quantitative PCR. VEGF protein concentrations were measured in culture medium by ELISA. We found that OxPCs and other classes of OxPLs upregulated the expression of VEGF in fetal and primary RPE cells, which critically depended on ATF4. In addition, upregulation of VEGF in primary RPE cells was blocked by a chemical inhibitor of protein kinase CK2 known to suppress induction of ATF4 and VEGF by OxPLs. Our data show that different species of OxPLs, which are present in the human eye are capable of stimulating expression of VEGF in fetal and primary RPE cells via ATF4-dependent mechanisms.

In-depth mass spectrometric mapping of the human vitreous proteome

Mapping of proteins involved in normal eye functions is a prerequisite to identify pathological changes during eye disease processes. We therefore analysed the proteome of human vitreous by applying in-depth proteomic screening technologies. For ethical reasons human vitreous samples were obtained by vitrectomy from “surrogate normal patients” with epiretinal gliosis that is considered to constitute only negligible pathological vitreoretinal changes. We applied different protein prefractionation strategies including liquid phase isoelectric focussing, 1D SDS gel electrophoresis and a combination of both and compared the number of identified proteins obtained by the respective method. Liquid phase isoelectric focussing followed by SDS gel electrophoresis increased the number of identified proteins by a factor of five compared to the analysis of crude unseparated human vitreous. Depending on the prefractionation method proteins were subjected to trypsin digestion either in-gel or in solution and the resulting peptides were analysed on a UPLC system coupled online to an LTQ Orbitrap XL mass spectrometer. The obtained mass spectra were searched against the SwissProt database using the Mascot search engine. Bioinformatics tools were used to annotate known biological functions to the detected proteins. Following this strategy we examined the vitreous proteomes of three individuals and identified 1111 unique proteins. Besides structural, transport and binding proteins, we detected 261 proteins with known enzymatic activity, 51 proteases, 35 protease inhibitors, 35 members of complement and coagulation cascades, 15 peptide hormones, 5 growth factors, 11 cytokines, 47 receptors, 30 proteins of visual perception, 91 proteins involved in apoptosis regulation and 265 proteins with signalling activity. This highly complex mixture strikingly differs from the human plasma proteome. Thus human vitreous fluid seems to be a unique body fluid. 262 unique proteins were detected which are present in all three patient samples indicating that these might represent the constitutive protein pattern of human vitreous. The presented catalogue of human vitreous proteins will enhance our understanding of physiological processes in the eye and provides the groundwork for future studies on pathological vitreous proteome changes.

THE EFFECTS OF AFLIBERCEPT ON THE VIABILITY AND METABOLISM OF OCULAR CELLS IN VITRO

To investigate the effects of the vascular endothelial growth factor-neutralizing agent aflibercept on primary cultures of human trabecular meshwork cells (hTMC), human scleral fibroblasts (hFibro), and a retinal pigment epithelial cell line (ARPE-19). Various concentrations of aflibercept were incubated with confluent cell cultures for 24 hours. Ranibizumab was used as an active control for comparison. Assays of cellular metabolism (MTT assay) and cell viability (calcein dye uptake) were performed. Compared with untreated controls (100% live), a 24-hour exposure to 1 mg/mL aflibercept had no significant effect on cell viability in hTMC (100.1 ± 1.7%), hFibro (102.4 ± 2.4%), or ARPE-19 (99.3 ± 3.9%) cells. Aflibercept vehicle controls also had no detrimental effect. Aflibercept (1 mg/mL) had no statistically significant effect on metabolic activity in hTMC (84.3 ± 10.2%), hFibro (102.7 ± 4.3%), and ARPE-19 (104.6 ± 12.6%) cells. When compared side-by-side in ARPE-19 cells, aflibercept and the anti-vascular endothelial growth factor agent ranibizumab had no toxicity at the highest concentration tested (1 mg/mL). The authors' data reveal that concentrations of aflibercept in the range expected to occur in the human vitreous after intraocular injection are not harmful in an in vitro cell assay.

Ranibizumab Is a Potential Prophylaxis for Proliferative Vitreoretinopathy, a Nonangiogenic Blinding Disease

Proliferative vitreoretinopathy (PVR) exemplifies a disease that is difficult to predict, lacks effective treatment options, and substantially reduces the quality of life of an individual. Surgery to correct a rhegmatogenous retinal detachment fails primarily because of PVR. Likely mediators of PVR are growth factors in vitreous, which stimulate cells within and behind the retina as an inevitable consequence of a breached retina. Three classes of growth factors [vascular endothelial growth factor A (VEGF-A), platelet-derived growth factors (PDGFs), and non-PDGFs (growth factors outside of the PDGF family)] are relevant to PVR pathogenesis because they act on PDGF receptor α, which is required for experimental PVR and is associated with this disease in humans. We discovered that ranibizumab (a clinically approved agent that neutralizes VEGF-A) reduced the bioactivity of vitreous from patients and experimental animals with PVR, and protected rabbits from developing disease. The apparent mechanism of ranibizumab action involved derepressing PDGFs, which, at the concentrations present in PVR vitreous, inhibited non-PDGF-mediated activation of PDGF receptor α. These preclinical findings suggest that available approaches to neutralize VEGF-A are prophylactic for PVR, and that anti-VEGF-based therapies may be effective for managing more than angiogenesis- and edema-driven pathological conditions.

Epithelial-mesenchymal transdifferentiation of retinal pigment epithelial cells induced by vitreous humor in vitro

Objective To observe the effect of epithelial-mesenchymal transdifferentiation (EMT) of human retinal pigment epithelial (RPE) cells induced by vitreous humor in vitro.Methods The third to fifth passage cultured RPE cells were divided into two groups of treatment by 10％ serum-containing Dulbecco minimum essential medium (DMEM)/F12 medium (group A),or the same medium supplemented with 25％ human vitreous (group B).The morphological changes were observed with a phase contrast microscrope.Cell migration,invasion and contractility were tested using a scratch wound assay,Transwell invasion assay and collagen gel contraction analysis.The expression levels of α-smooth muscle actin (SMA)and Snail1 were detected by reverse transcriptase polymerase chain reaction (RT-PCR).Results The RPE cells in group A were flat and gathered together.The RPE cells in group B grew as a fan-shaped projection at one edge and cone-shaped tail at the opposite edge,or spindle-shaped,and appeared to separate.In group A,filamentous actin distributed mainly at the margin of the cells with the distribution an oval shape.In group B,filamentous actin reorganized and formed fan-like flat pseudopodia at one edge of the cells.Compared to group A,the migration and invasion of the cells increased significantly (t=14.190,22.630;P＜0.05),but contractility decreased remarkably (t=6.221,P＜0.05) in group B.Compared to group A,the expression level of Snail1 mRNA increased significantly (t=3.218,P=0.032),but the expression level of α-SMA mRNA decreased (t=3.990,P=0.016).Conclusions Vitreous humor can induce the EMT of RPE cells.Increasing cell migration,cell invasion,and expression of Snail1 mRNA as well as up-regulated cells' contractility and expression of α-SMA mRNA may be the mechanism. Key words: Pigment epithelium of eye/growth & development; Cell, cultured; Cell transdifferentiation

Measuring the Effect of Pars Plana Vitrectomy on Vitreous Oxygenation Using Magnetic Resonance Imaging

To study the effect of pars plana vitrectomy (PPV) on vitreous oxygenation (pO2) using magnetic resonance imaging (MRI). Patients due to undergo PPV for either macular hole or epiretinal membrane were recruited. MRI scanning was performed 1 week before and at least 3 months after PPV. MRI T1 mapping was performed using an inversion recovery-true fast imaging with steady-state precession (TrueFISP) sequence at several inversion times, from a single slice positioned through the center of both eyes in the axial oblique plane. Additional phantom data were measured in porcine vitreous, to define the relationships between T1 relaxation times and balanced salt solution (BSS), to simulate human vitreous and aqueous, respectively, for a suitable pO2 range (5-70 mm Hg). Pre-PPV pO2 was also measured intraoperatively using a polarographic oxygen probe. Eleven participants (age range 59-84) were recruited; two declined the post-PPV scan. Corrected T1 times indicated that the mean (±SD) pO2 increased significantly following PPV, from 13.2 ± 5.8 to 34.5 ± 8.0 mm Hg (P < 0.001). In the nonsurgical (control) eye, pO2 did not change significantly from the first to second MRI scan (13.7 ± 7.8 vs. 16.3 ± 8.7 mm Hg, P = 0.239). Mean pO2 measured intraoperatively was 7.2 ± 0.6 mm Hg (n = 10). These results confirm that vitrectomy substantially increases vitreous pO2. MRI is a noninvasive technique that can be used to study vitreous oxygenation in both vitrectomized and nonvitrectomized eyes.

Regression activity that is naturally present in vitreous becomes ineffective as patients develop proliferative diabetic retinopathy

The realisation that targeting agents in the vitreous is an effective approach to treating patients with diabetic retinopathy (DR) has increased awareness that changes in the composition/bioactivity of the vitreous is a contributor to the pathogenesis of DR. The overall goal of this study was to test the hypothesis that the vitreous has regression activity, and that lysophosphatidic acid (LPA) contributes to such activity. LPA is a bioactive phospholipid present in many biological fluids, and has been recently appreciated for its ability to promote regression of blood vessels. Vitreous-mediated regression was monitored on tubes organised from primary retinal endothelial cells or neovessels that sprouted from retinal explants. LPA was quantified radioenzymatically. Bovine and human vitreous promoted regression of retinal explant vessels and of tubes organised from primary retinal endothelial cells. LPA was a substantial component of this regression activity. Comparing the regression activities of vitreous from patients with different stages of DR revealed that, as patients developed proliferative diabetic retinopathy (PDR), vitreous lost its ability to promote regression, even though the amount of LPA did not change. The underlying mechanism was a PDR-vitreous-mediated insensitivity to LPA, which could be overcome pharmacologically. Our findings suggest that a decline in the responsiveness to regression factors such as LPA, which are naturally present in the vitreous, contributes to the pathogenesis of PDR.

Proteomic analysis of human vitreous associated with idiopathic epiretinal membrane

Proteomic analysis of human vitreous associated with idiopathic epiretinal membrane

Pharmacokinetics of Ocriplasmin in Vitreous

Ocriplasmin contains the active moiety of plasmin enzyme. At a physiologic pH, ocriplasmin is highly proteolytic and autolytic, limiting its duration of activity. Specific inhibitors of plasmin are present in the vitreous under normal and disease conditions and could affect its activity. Each may contribute to its mode of action. Degradation characteristics were determined in porcine, human vitreous, and PBS under reducing conditions with different incubation periods between 0 and 24 hours on SDS-PAGE Tris-glycine gels. Residual activity was determined by spectrophotometry of p-nitroaniline release through hydrolysis of L-pyroglutamyl-L-phenylalanyl-L-lysine-p-nitroaniline hydrochloride. The presence of endogenous inactivators of ocriplasmin in human vitreous was determined in a series of vitreous samples using an ELISA specific for alpha(2)-antiplasmin, antithrombin, and antitrypsin. Degradation productions from autolysis are similar between vitreous and PBS with a significant prolongation of the effect in vitreous. Both follow a nonlinear pattern over time. The degradation corresponds best to a second-order kinetic process. The resulting rate constants were 207 ± 60 M(-1) s(-1) in PBS, 81 ± 15 M(-1) s(-1) in porcine vitreous, and 195 M(-1) s(-1) in human vitreous natural inhibitors were identified in samples of donor vitreous. Amounts differed significantly between samples, which may help explain the observed variability in human subjects. Ocriplasmin is autolytic in vitreous. Biologic activity extends to several days following injection. The exact duration will vary based on the presence and concentration of serine protease inhibitors.

Human Vitreous: MR Imaging of Oxygen Partial Pressure

To develop a magnetic resonance (MR) imaging approach to noninvasively image quantitative Po(2) in the human vitreous. Human studies were approved by the institutional review board with informed consent obtained from all subjects and were HIPAA compliant. Animal studies were performed with animal care committee approval. An MR imaging method to measure the longitudinal relaxation rate, or R1, of water was implemented with a 3.0-T MR imager. R1 was calibrated in water phantoms at multiple Po(2) and temperature conditions (n = 10) and in ex vivo animal vitreous (n = 2). Vitreous Po(2) was imaged in three human volunteers (age range, 26-28 years) in multiple sessions on separate days to evaluate reproducibility. The effects of temperature and ambient air were evaluated by acquiring data with the eye open and closed. Statistical analysis consisted of t tests, with P less than .05 indicating significant difference. Calibrations of phantoms and ex vivo vitreous yielded an R1 association with oxygen of 0.209 sec(-1) + Po(2) ⋅ 2.07 × 10(-4) sec(-1)/mm Hg at 37°C, and an association with temperature (Δ[1/R1]/ΔTemperature) of 0.106 sec/°C ± 0.009 (standard deviation). A difference in R1 was found between the phantoms and vitreous. If uncorrected, vitreal Po(2) would be significantly overestimated (P < .001). In vivo human vitreous Po(2) maps were spatially heterogeneous, with a whole vitreous Po(2) of 16.7 mm Hg ± 6.5 (eye closed). Measurements between open and closed eyes showed spatially dependent R1 differences, which translated to temperature differences of 0.34°-0.83°C across the eye. This study established an MR imaging protocol to image quantitative vitreous Po(2) noninvasively and evaluated effects from vitreal macromolecules, temperature gradients, and ambient air on vitreal Po(2) values. Measurement of vitreous Po(2) with MR imaging has the potential to be used to study eye diseases noninvasively.

Physiological vitreous changes may contribute to the pathogenesis of macular degeneration

Editor, There is accumulating evidence that the vitreomacular interface, vitreomacular traction and mechanical stress might be risk factors for the development of age-related macular degeneration (AMD): vitreomacular adhesion may promote exudative AMD, total posterior vitreous detachment (PVD) as well as vitrectomy have been found to be protective against the development of AMD (Krebs et al. 2011; Schulze et al. 2012). The vitreous cortex touches the retinal surface, and developmental and age-related remodelling of the vitreous as well as the presence of a bursa premacularis has been described before (Worst & Los 1995). Los (1997) described in her PhD thesis that the human vitreous shows perimacular concentric circles of longitudinal cisterns as well as a bursa premacularis. Moreover, the perpendicular pillar-like cisterns undergo changes in time. More than 100 autopsy eyes, studied by Worst, were derived from patients above the age of 50 (mean age: 70); the findings of Los, described in her thesis, were based upon the examination of more than one hundred donor eyes from younger patients, with varying ages. All autopsy eyes were studied with courtesy of the Corneal Eye Bank in Amsterdam, and the anatomical studies were performed by the Jan Worst Research Group in Haren, the Netherlands. However, as it is technically very difficult to reveal and to photograph intact intravitreal structures in their three-dimensional organization after injection dye into fresh, unfixed vitreous bodies suspended in a balanced salt solution (BBS) – several specimens got damaged during preparation or were even lost!– it is impossible to give the exact number of eyes in which the typical perimacular elongated cistern-like structures and the bursa premacularis could be visualized (see Fig. 1A). With informed consent, clinical data were collected from ten patients above the age of 50 during triamcinolone-assisted vitrectomy in the Alkmaar Medical Center in Alkmaar, the Netherlands. The human vitreous shows in vivo identical perimacular concentric circles of longitudinal cisterns as well as a bursa premacularis (see Fig. 1B and C). Based upon these identical anatomical aspects of the vitreous and the location of perimacular and macular lesions in AMD, we hypothesize that there is a relationship: as such retinal and choroidal lesions geographically correspond with the location of the longitudinal vitreal cisterns and the bursa premacularis, they can be interpreted as mechanical effects that may progress to AMD, depending on the magnitude and direction of vector forces during eye rotations. The usual chronic course of AMD with exacerbations and remissions and often progressive, bilateral appearance are suggestive of intermittent traumatic effects. Physiological vitreous changes and eye rotations occur in everyone, but vitreous structures will only touch the retina in the presence of elongated cisterns and/or significant vitreous movements, which process might be facilitated by vitreomacular adhesions. Our hypothesis that mechanical stress might contribute to the pathogenesis of AMD is supported by the clinical findings of others, who used spectral domain optical coherence tomography to evaluate the vitreoretinal interface: a positive relationship between a persistent attachment of the posterior vitreous and early signs of AMD was found (Schulze et al. 2012) as well as a complete correspondence between the adhesion and subretinal neovascularization (Krebs et al. 2011). We realize that observational and anatomical, experimental studies have their limitations, and further histopathological as well as case–control studies are required to confirm our hypothesis. Bursa premacularis filled with ink in an autopsy eye (A) and clinically after injection of triamcinolone during pars plana vitrectomy (B and C). Area of Martegiani on the left of the bursa in the autopsy eye (A). The bursa premacularis in the center is surrounded by distended cisterns of the circulus petaliformis: these structures are thought to make contact with the retinal surface periodically. We thank Peter J. de Lint, vitreoretinal surgeon in the Medical Center Alkmaar; the Corneal Eye Bank, Amsterdam; the Jan Worst Research Group, Haren; the ophthalmic photography units of the VU University Medical Center, Amsterdam; and the Medical Center Alkmaar, the Netherlands.

Ultrasound imaging velocimetry of the human vitreous

Knowledge of vitreous motion in response to saccades is a prerequisite for understanding vitreous rheology. Purpose of present paper is to introduce Ultrasound Image Velocimetry of the human eye, measure scleral and vitreous velocity fields and test the reproducibility of the proposed technique. Twelve patients with varying diagnosis underwent Ocular Dynamic Ultrasound; scleral angular velocity (VS) was measured by 2 different operators and reproducibility calculated. Squared velocity of the vitreous (E), which is representative of kinetic energy per unit mass, was computed from velocity. The time evolution of the energy of the vitreous was described by its spatial average (ES), whereas spatial distribution was described by its time average (ET). Peak and average ES, the ratio Kp of the peak of the spatially averaged kinetic energy per unit mass to the maximum squared scleral angular velocity, vitreous motion onset time (TO) and vitreous motion decay time (TD) were also defined. Inter-operator reproducibility coefficient was 0.043 and correlation between operators was significant. VS, peak and average ES, Kp ratio and TD differed among patients but not among operators. VS correlated with ES and TD. ES and TD but not VS, were significantly different in patients with Posterior Vitreous Detachment. Patients with retinal detachment showed significantly higher VS and ES. Kp was inversely correlated to age and refraction. Measures proved accurate and reproducible. E is related to VS, retinal traction and mechanical stimulation. Identified variables varied with age, refraction pathologic conditions.

FURTHER CHARACTERIZATION OF OCULAR SAFETY PROFILE OF COMMERCIALLY AVAILABLE PRESERVED AND PRESERVATIVE-FREE TRIAMCINOLONE ACETONIDE

To characterize the safety profile of triamcinolone acetonide (TA) for intraocular application. In vitro cell viability assay was performed on 2 types of human ocular cells to evaluate the cytotoxicity of the simulated different vitreal concentrations (from a 1:15 dilution as if injected into 1.5 mL of rabbit vitreous to 1:50 dilution as if injected into 5 mL of human vitreous) of preservative and excipient (supernatant) from Kenalog-40. In vivo 35 guinea pigs were used for evaluating either a dose of intravitreal triamcinolone acetonide (Kenalog-40 and Triesence) or the supernatant of Kenalog-40. The animal eyes were monitored by biomicroscopy, ophthalmoscopy, tonometry, electroretinography, and histology. A ≥ 1:15 dilution of triamcinolone acetonide supernatant from Kenalog-40 did not show cytotoxicity on cultured human pigment epithelial (retinal pigment epithelium) cells or Müller cells. In vivo, neither intravitreal 6 μL (0.248 mg, equivalent to 4 mg in 0.1 mL for human eyes) nor 18 μL (0.744 mg, equivalent to 4 mg in 0.1 mL for rabbit eyes and equivalent to 12 mg in 0.1 mL for human eyes) of triamcinolone acetonide suspension showed ocular toxicity. No significant difference was noted between Kenalog-40 and Triesence clinically and histopathologically. The equivalent triamcinolone acetonide doses to 0.1 mL (4 or 12 mg) intravitreal injection for human eye were found safe in guinea pig eyes. No significant difference was noted for 0.1 mL intravitreal injection between Kenalog-40 and Triesence.