Redefining the ontogeny of hyalocytes as yolk sac-derived tissue-resident macrophages of the vitreous body.

BACKGROUND: The eye is a highly specialized sensory organ which encompasses the retina as a part of the central nervous system, but also non-neural compartments such as the transparent vitreous body ensuring stability of the eye globe and a clear optical axis. Hyalocytes are the tissue-resident macrophages of the vitreous body and are considered to play pivotal roles in health and diseases of the vitreoretinal interface, such as proliferative vitreoretinopathy or diabetic retinopathy. However, in contrast to other ocular macrophages, their embryonic origin as well as the extent to which these myeloid cells might be replenished by circulating monocytes remains elusive. RESULTS: In this study, we combine transgenic reporter mice, embryonic and adult fate mapping approaches as well as parabiosis experiments with multicolor immunofluorescence labeling and confocal laser-scanning microscopy to comprehensively characterize the murine hyalocyte population throughout development and in adulthood. We found that murine hyalocytes express numerous well-known myeloid cell markers, but concomitantly display a distinct immunophenotype that sets them apart from retinal microglia. Embryonic pulse labeling revealed a yolk sac-derived origin of murine hyalocytes, whose precursors seed the developing eye prenatally. Finally, postnatal labeling and parabiosis established the longevity of hyalocytes which rely on Colony Stimulating Factor 1 Receptor (CSF1R) signaling for their maintenance, independent of blood-derived monocytes. CONCLUSION: Our study identifies hyalocytes as long-living progeny of the yolk sac hematopoiesis and highlights their role as integral members of the innate immune system of the eye. As a consequence of their longevity, immunosenescence processes may culminate in hyalocyte dysfunction, thereby contributing to the development of vitreoretinal diseases. Therefore, myeloid cell-targeted therapies that convey their effects through the modification of hyalocyte properties may represent an interesting approach to alleviate the burden imposed by diseases of the vitreoretinal interface.

Hyalocytes-guardians of the vitreoretinal interface.

Originally discovered in the nineteenth century, hyalocytes are the resident macrophage cell population in the vitreous body. Despite this, a comprehensive understanding of their precise function and immunological significance has only recently emerged. In this article, we summarize recent in-depth investigations deciphering the critical role of hyalocytes in various aspects of vitreous physiology, such as the molecular biology and functions of hyalocytes during development, adult homeostasis, and disease. Hyalocytes are involved in fetal vitreous development, hyaloid vasculature regression, surveillance and metabolism of the vitreoretinal interface, synthesis and breakdown of vitreous components, and maintenance of vitreous transparency. While sharing certain resemblances with other myeloid cell populations such as retinal microglia, hyalocytes possess a distinct molecular signature and exhibit a gene expression profile tailored to the specific needs of their host tissue. In addition to inflammatory eye diseases such as uveitis, hyalocytes play important roles in conditions characterized by anomalous posterior vitreous detachment (PVD) and vitreoschisis. These can be hypercellular tractional vitreo-retinopathies, such as macular pucker, proliferative vitreo-retinopathy (PVR), and proliferative diabetic vitreo-retinopathy (PDVR), as well as paucicellular disorders such as vitreo-macular traction syndrome and macular holes. Notably, hyalocytes assume a significant role in the early pathophysiology of these disorders by promoting cell migration and proliferation, as well as subsequent membrane contraction, and vitreoretinal traction. Thus, early intervention targeting hyalocytes could potentially mitigate disease progression and prevent the development of proliferative vitreoretinal disorders altogether, by eliminating the involvement of vitreous and hyalocytes.

In vitro generation of primary cultures of human hyalocytes.

Purpose: A growing number of studies on animal models have demonstrated that some ocular diseases are the result of the interaction between hyalocytes and the ocular inflammatory setting. Endogenous and exogenous substances might alter the structure and behavior of hyalocytes that can contribute to the pathogenesis of some ocular diseases. Obtaining primary cultures of human hyalocytes could help understand the role of these cells in response to different treatments. Methods: Hyalocytes were isolated from eyes of 54 patient volunteers subjected to vitrectomy for different clinical reasons. By testing different matrices and growth media, we reproducibly generated primary cultures of hyalocytes that we characterized morphologically and biologically, basally and upon treatment with several agents (basic fibroblast growth factor (bFGF), transforming growth factor beta 1 (TGF-ß), platelet-derived growth factor subunit-BB (PDGF-BB), ascorbic acid, dexamethasone, and hydrogen peroxide). Results: We were able to generate primary cultures from vitreous human samples, growing the cells on collagen-coated plates in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum; primary cells expressed the hyalocyte markers. Specific cytoskeletal modifications were observed upon treatment with bFGF, TGF-ß, PDGF-BB, ascorbic acid, dexamethasone, and hydrogen peroxide. Only bFGF was able to promote cell growth and hyaluronic acid production. Conclusions: We describe for the first time the generation and the characterization of primary cultures of human hyalocytes from living donors. Although human hyalocytes share some characteristics with animal hyalocytes, human hyalocytes have their own features typical of the species, confirming how important human experimental models are for investigating human pathologies and their treatments.

Evidence for the activation of pyroptotic and apoptotic pathways in RPE cells associated with NLRP3 inflammasome in the rodent eye.

BACKGROUND: Age-related macular degeneration (AMD) is a devastating eye disease causing irreversible vision loss in the elderly. Retinal pigment epithelium (RPE), the primary cell type that is afflicted in AMD, undergoes programmed cell death in the late stages of the disease. However, the exact mechanisms for RPE degeneration in AMD are still unresolved. The prevailing theories consider that each cell death pathway works independently and without regulation of each other. Building upon our previous work in which we induced a short burst of inflammasome activity in vivo, we now investigate the effects of prolonged inflammasome activity on RPE cell death mechanisms in rats. METHODS: Long-Evans rats received three intravitreal injections of amyloid beta (Aß), once every 4 days, and were sacrificed at day 14. The vitreous samples were collected to assess the levels of secreted cytokines. The inflammasome activity was evaluated by both immunohistochemistry and western blot. The types of RPE cell death mechanisms were determined using specific cell death markers and morphological characterizations. RESULTS: We found robust inflammasome activation evident by enhanced caspase-1 immunoreactivity, augmented NF-κB nuclear translocalization, increased IL-1ß vitreal secretion, and IL-18 protein levels. Moreover, we observed elevated proteolytic cleavage of caspase-3 and gasdermin D, markers for apoptosis and pyroptosis, respectively, in RPE-choroid tissues. There was also a significant reduction in the anti-apoptotic factor, X-linked inhibitor of apoptosis protein, consistent with the overall changes of RPE cells. Morphological analysis showed phenotypic characteristics of pyroptosis including RPE cell swelling. CONCLUSIONS: Our data suggest that two cell death pathways, pyroptosis and apoptosis, were activated in RPE cells after exposure to prolonged inflammasome activation, induced by a drusen component, Aß. The involvement of two distinct cell death pathways in RPE sheds light on the potential interplay between these pathways and provides insights on the future development of therapeutic strategies for AMD.

Immune Profiling of T Cells Infiltrating Vitreous Humor in Tubercular Uveitis.

PURPOSE: To assess cellular composition and local cytokine response in vitreous humor of tubercular uveitis. METHODS: Cells were collected from vitreous cassettes and peripheral blood of 8 tubercular uveitis and 5 control subjects, undergoing vitrectomy and analyzed by flow cytometry for cellular composition, activation status, proinflammatory cytokine expression, and uptake of TLR9 ligand, CpG ODN 2216. RESULTS: CD3 + T cells with equal proportion of CD4+ and CD8 + T cells formed major fraction of infiltrating cells. The vitreous humor showed higher expression of recent activation marker, CD69, and proinflammatory cytokines, IFN-γ and IL-17A, in CD4 + T cells as compared to peripheral blood. Lastly, intraocular CD4 + T cells showed reduced uptake of ODN 2216 than peripheral blood. CONCLUSIONS: Our results indicate that local antigenic stimuli trigger T cell infiltration and activation of CD4 + T cells that are hyporesponsive to TLR9 stimulation. These infiltrating T cells might be responsible in further aggravating ocular inflammation.

Acute Retinal Necrosis Caused by the Zoster Vaccine Virus.

We report acute retinal necrosis caused by the vaccine Oka strain following immunization of a 78-year-old woman with live zoster vaccine. Whole genome sequencing confirmed the ocular vOka strain to be derived from the vaccine and excluded the presence of new mutations or recombination with wild-type Varicella zoster virus.

Effects of mechanical stress and vitreous samples in retinal pigment epithelial cells.

In rhegmatogenous retinal detachment (RRD), scattered RPE cells from the basement membrane into the vitreous cavity undergo an epithelial mesenchymal transition (EMT) and form the intraocular fibrous membrane in response to vitreous fluid. We investigated whether exposure to vitreous samples was associated with EMT-associated signals and mesenchymal characters. Human vitreous samples were collected from patients with RRD, epiretinal membrane (ERM), or macular hole (MH). We evaluated the effects of vitreous on ARPE-19 cells in suspension cultures using poly 2-hydroxyethyl methacrylate-coated dishes and three-dimensional (3D) Matrigel cultures. We found that exposure to vitreous samples did not induce morphological changes or accelerate wound closure in monolayers. Several samples showed increased phosphorylation of Smad2 and nuclear translocation of nuclear factor-κB. Mechanical stress triggered an elevation of phosphorylation levels in Smad2. In addition, exposure to vitreous fluid increased the phosphorylation of p38 mitogen-activated protein kinase in cell suspension cultures after mechanical stress. Moreover, ARPE-19 cells showed a stellate invasive phenotype in 3D Matrigel cultures with vitreous samples. In this study, we demonstrated that mechanical stress and vitreous were associated with EMT-associated signals and invasive phenotypes in 3D cultures but not in monolayers. These results have important implications for the role of vitreous humor in the induction of EMT and intraocular fibrosis.

p19(Arf) limits primary vitreous cell proliferation driven by PDGF-B.

Arf encodes an important tumor suppressor, p19(Arf), which also plays a critical role to control hyperplasia in the primary vitreous during mouse eye development. In the absence of Arf, mice are born blind and display a phenotype closely mimicking severe forms of the human eye disease, persistent hyperplastic primary vitreous (PHPV). In this report, we characterize p19(Arf) expression in perivascular cells that normally populate the primary vitreous and express the Arf promoter. Using a new ex vivo model, we show that these cells respond to exogenous Tgfß, despite being isolated at a time when Tgfß has already turned on the Arf promoter. Treatment of the cells with PDGF-B ligand doubles the population of cells in S-phase and ectopic expression of Arf blunts that effect. We show this effect is mediated through Pdgfrß as expression of Arf represses expression of Pdgfrß mRNA and protein to approximately 60%. p53 is not required for Arf-dependent blockade of PDGF-B driven proliferation and repression of Pdgfrß protein as ectopic expression of Arf is still able to inhibit the 2-fold increase in the S-phase fraction of cells upon treatment with PDGF-B. Finally, induction of mature miR-34a, a microRNA previously identified to be regulated by p19(Arf) does not depend on p53 while the expression of the primary transcript does require p53. These data corroborate that, as in vivo, p19(Arf) functions to inhibit PDGF-B driven proliferation ex vivo.

CFH Y402H polymorphism is associated with elevated vitreal GM-CSF and choroidal macrophages in the postmortem human eye.

PURPOSE: Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in people 50 years of age or older in developed countries. The homozygous CC genotype in the complement factor H (CFH) Y402H single nucleotide polymorphism (SNP; rs1061170) is widely recognized as a risk factor for the development of AMD. In this study, we examined vitreal levels of granulocyte macrophage colony-stimulating factor (GM-CSF), a hematopoietic cytokine, and macrophages in the choroid of postmortem human eyes genotyped for the CFH Y402H SNP. METHODS: Twenty-two pairs of postmortem, non-diseased, human donor eyes were obtained. The vitreous and retinal tissues of the left eyes were collected for GM-CSF level measurement and CFH Y402H genotyping, respectively. The right eyes were paraffin-embedded and sectioned for immunohistochemistry using a macrophage and microglia marker, CD68. Cell cultures of RPE cells were stimulated with complement C3a, C5a, 4-hydroxynonenal (HNE), or tumor necrosis factor alpha (TNF-α), and GM-CSF expression was measured with a suspension assay or quantitative PCR. RESULTS: Eyes genotyped with the CC or the CT risk variant of the CFH Y402H SNP showed significantly increased levels of GM-CSF in the vitreous compared to eyes with the protective TT variant (mean ± standard error of mean, 607.54±85.83 pg/ml or 656.32±15.20 pg/ml versus 286.69±81.96 pg/ml, p<0.05). The choroid of eye tissues genotyped with the CC variant showed higher levels of CD68 immunoreactivity than the tissues genotyped with the TT variant (p<0.05). The GM-CSF levels detected in the supernatant of RPE cells in culture treated with HNE or TNF-α were significantly higher compared to the non-treated control (145.88±5.06 pg/ml and 149.32±3.76 pg/ml versus 123.27±4.05 pg/ml, p<0.05). Furthermore, the gene expression of GM-CSF detected in the lysate of RPE cells stimulated with complement C3a or C5a showed significantly increased fold changes compared to the non-treated control (C3a: 2.38±0.31 fold, p<0.05; C5a: 2.84±0.54 fold, p<0.01). CONCLUSIONS: Our data showed a relationship between the CFH Y402H polymorphism and GM-CSF levels in the vitreous and accumulation of choroidal macrophages in the postmortem eye. These data suggest that the at-risk variant of the CFH gene may contribute to the dysregulation of proinflammatory cytokines locally in the eye.

Hyalocytes in idiopathic epiretinal membranes: a correlative light and electron microscopic study.

PURPOSE: To describe characteristics of epiretinal cells at the vitreoretinal interface by correlative light and electron microscopy (CLEM). METHODS: Epiretinal membrane (ERM) specimens and internal limiting membrane (ILM) specimens were harvested by sequential peeling during vitrectomy from 27 eyes with idiopathic epiretinal gliosis, and processed for CLEM. Intraoperatively, the presence of posterior vitreous detachment (PVD) was documented. We used anti-vimentin, anti-α-smooth muscle actin (α-SMA), and anti-CD45 as primary antibodies. A fluorescein-tagged immunonanogold cluster was used as secondary antibody and visualized under the fluorescence and transmission electron microscope. RESULTS: We demonstrated CD45-positive cells specifically labelled at their plasma membranes with ultrastructural features known for hyalocytes, such as oval nucleus with marginal chromatin, vacuoles, dense granules, and thin cytoplasmic protrusions. CD45-positive cells were mostly located on a thick layer of native vitreous collagen. They were covered by newly formed collagen strands with multilayered proliferation of myofibroblasts. We also demonstrated immunoreactivity for vimentin and alpha-SMA. Cell fragments with positive labelling for α-SMA and vimentin were not only found on the vitreal side of the ILM, but also on the retinal side. CONCLUSIONS: By CLEM, the majority of CD45-positive cells in epiretinal cell proliferation were characterized as hyalocytes. In the context of anomalous PVD and vitreoschisis, ultrastructural features and topographic localization of hyalocytes suggest that these cells play a significant role in ERM formation. CLEM enables a more accurate characterization of epiretinal cell proliferation, and therefore, contributes to a better understanding of the pathogenesis of diseases at the vitreoretinal interface.

In vitrothree-dimensional volumetric printing of vitreous body models using decellularized extracellular matrix bioink.

Hyalocytes, which are considered to originate from the monocyte/macrophage lineage, play active roles in vitreous collagen and hyaluronic acid synthesis. Obtaining a hyalocyte-compatible bioink during the 3D bioprinting of eye models is challenging. In this study, we investigated the suitability of a cartilage-decellularized extracellular matrix (dECM)-based bioink for printing a vitreous body model. Given that achieving a 3D structure and environment identical to those of the vitreous body necessitates good printability and biocompatibility, we examined the mechanical and biological properties of the developed dECM-based bioink. Furthermore, we proposed a 3D bioprinting strategy for volumetric vitreous body fabrication that supports cell viability, transparency, and self-sustainability. The construction of a 3D structure composed of bioink microfibers resulted in improved transparency and hyalocyte-like macrophage activity in volumetric vitreous mimetics, mimicking real vitreous bodies. The results indicate that our 3D structure could serve as a platform for drug testing in disease models and demonstrate that the proposed printing technology, utilizing a dECM-based bioink and volumetric vitreous body, has the potential to facilitate the development of advanced eye models for future studies on floater formation and visual disorders.

Human vitreous: MR imaging of oxygen partial pressure.

PURPOSE: To develop a magnetic resonance (MR) imaging approach to noninvasively image quantitative Po(2) in the human vitreous. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Cell-mediated contraction of vitreous explants from chicken embryo: possibility of screening for therapeutic agents against proliferative vitreoretinal diseases.

PURPOSE: We aimed to establish a novel screening system for identifying potential therapeutic agents for treating proliferative vitreoretinal diseases (PVDs). In this study, we focused on vitreous explants from chicken embryos and evaluated the usefulness of quantitatively analyzing the effects of potential candidates on cell-mediated vitreous contraction, which leads to blindness in PVDs. METHODS: Vitreous explants were extracted from 19-day-old embryonic chickens and then incubated with retinal Müller cells or endothelial cells to permit cell adhesion. After cell adhesion occurred, we examined the effect of the attached cells on the wet weight of vitreous explants as an index of vitreous contraction. We also performed hematoxylin and eosin staining to characterize the cell morphology on the vitreous surface. RESULTS: Contraction of the vitreous explants was observed after cell adhesion of not only retinal Müller cells but also endothelial cells. We confirmed the adhesion of these cells on vitreous explants and estimated the number of adherent cells with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. The cells on the vitreous surface presented an elongated fibroblast-like phenotype. Integrin was found to be a receptor involved in cell adhesion on the vitreous surface. DISCUSSION: Our results suggest that vitreous explants from chicken embryos may be novel useful tools for screening antiadhesion therapeutic agents in PVDs. This preliminary study must be validated with human vitreous and human retinal pigment epithelial cells.

Human adipose-derived mesenchymal stem cells can survive and integrate into the adult rat eye following xenotransplantation.

BACKGROUND: Novel threads of discovery provide the basis for optimism for the development of a stem-cell-based strategy for the treatment of retinal blindness. Accordingly, achievement to suitable cell source with potential-to-long-term survival and appropriate differentiation can be an effective step in this direction. METHODS: After derivation of human adipose-derived mesenchymal stem cells (HAD-MSCs), they were stably transfected with a vector containing Turbo-green fluorescent protein (GFP) and JRed to be able to trace them after transplantation. Labeled HAD-MSCs were transplanted into the intact adult rat eye and their survival, integration, and migration during 6 months post-transplantation were assessed. RESULTS: The transplanted cells were traceable in the rat vitreous humor (VH) up until 90 days after transplantation, with gradual reduction in numbers, their adhesion and expansion capacity after recovery. These cells were also integrated into the ocular tissues. Nonetheless, some of the implanted cells succeeded to cross the blood-retina barrier (BRB) and accumulate in the spleen with time. CONCLUSIONS: The survival of the HAD-MSCs for a period of 90 days in VH and even longer period of up to 6 months in other eye tissues makes them a promising source to be considered in regenerative medicine of eye diseases. However, the potency of crossing the BRB by the implanted cells suggests that use of HAD-MSCs must be handled with extreme caution.

[The effect of OPTC-shRNA on the bovine retinal pigment epithelial cells and hyalocytes co-culture collagen gel contraction system].

OBJECTIVE: To investigate the OPTC-shRNA inhibiting effect on the opticin expression by the bovine hyalocytes and retina pigment epithelial (RPE) cells co-culture collagen gel contraction system. METHODS: Experimental study. The OPTC-shRNA expression vector was designed and transfected into bovine RPE cells cultured in vitro. The relative expression and the inhibition rate of the opticin protein were measured by Western blot on days 3, 5 and 7. An in vitro cells co-culture bovine type I collagen gel contraction assay was constructed consisting of the hyalocytes and RPE cells. Six groups were established in this experiments:OPTC-shRNA plasmid transfected RPE cells and hyalocytes (group A), empty plasmid transfected RPE cells and hyalocytes (group B), non-transfected RPE cells and hyalocytes (group C), non-transfected RPE cells (group D), only hyalocytes (group E), and no cells (group F). The collagen gel contractile activities of these groups were compared by One-way ANOVA, SNK-q tests and regression analysis;and the influence of the hyalocytes density variance on the collagen gel contraction in groups A, B and C were also analyzed. RESULTS: The OPTC-shRNA expression vector with significant inhibition effect was constructed and transfected into bovine RPE cells successfully. The results of Western blot analysis showed that the inhibitory rates on the opticin expression on days 3, 5 and 7 were (83.91 ± 2.88), (84.71 ± 4.27) and (82.85 ± 2.72)%, respectively. Furthermore, the differences among days 3, 5 and 7 were insignificant (F = 1.15, P > 0.05). On day 3, the gel contraction rates for the sub-groups with various hyalocytes densities (2×10(7), 1×10(8) and 5×10(8)/L) in groups A, B and C were: group A: (23.52 ± 2.08), (56.00 ± 1.02), (61.62 ± 1.73)%; group B: (16.56 ± 2.01), (36.41 ± 1.33), (49.56 ± 1.75)%; group C: (15.75 ± 1.37), (37.45 ± 1.14), (48.45 ± 1.97)%. The gel contraction rates for groups D and E were (12.18 ± 0.95)% and (10.95 ± 0.93)%, respectively; no gel contraction was observed in group F. Pairwise comparisons of the gel contraction rates were performed by SNK-q test among groups A, B and C for various hyalocyte densities. In the 2×10(7)/L cell density group, the differences between groups A and B or C were significant (q = 11.38, 2.72, respectively, P both < 0.05), the differences between B and C were insignificant (q = 1.34, P > 0.05). In the 1×10(8)/L cell density group, the differences between groups A and B or C were significant (q = 8.83, 46.22, respectively, P both < 0.05), the differences between B and C were insignificant (q = 1.34, P > 0.05). In the 5×10(8)/L cell density group, the differences between groups A and B or C were significant (q = 48.83, 46.22, respectively, P both < 0.05), the differences between groups B and C were insignificant (q = 1.74, P > 0.05). Pairwise comparisons of the sub-groups with different hyalocyte densities in groups A, B and C (comparisons of 2×10(7)/L and 1×10(8)/L, 2×10(7)/L and 5×10(8)/L, 2×10(7)/L and 2×10(7)/L, respectively), the differences were all significant (group A:q = -55.97, -65.66, -9.69, respectively; group B: q = -34.53, -57.41, -22.88, respectively; group C: q = -41.94, -63.19, -21.25, P all < 0.05). Furthermore, the regression analysis was performed between the hyalocyte density and the collage gel contraction rates in each group. The results showed that there was a positive correlation between the gel contraction rates of the co-culture collagen gel contraction system and its hyalocyte density (groups A, B, C: r = 0.919, 0.981, 0.937, respectively, P all < 0.05). Pairwise comparison of groups D and E, D and F, E and F by SNK-q test revealed q = 54.87, 49.33, 5.54, respectively, P all < 0.05. CONCLUSION: Opticin is capable of regulating the contraction of bovine hyalocytes and RPE cells co-culture collagen gel.

Efficient vitreolysis by combining plasmin and sulfur hexafluoride injection in a preclinical study in rabbit eyes.

PURPOSE: To investigate the efficacy of plasmin and sulfur hexafluoride (SF(6)) on the vitreoretinal junction, as well as the long-term safety in the eye and effect on the recipient's general health after application in the eye. METHODS: The study design included four groups of rabbits with three animals in each group. Group 1 received an intravitreal injection (IVI) of plasmin and SF(6) in the right eye; group 2 received an IVI of plasmin in the right eye; group 3 received an IVI of SF(6) in the right eye; and group 4 received an IVI of balanced salt solution in the right eye, which served as a normal control. Long-term safety (up to approximately three months) after plasmin and/or SF(6) injection was evaluated morphologically by clinical examination, histology, and immunohistochemistry, and functionally by electroretinograms (ERGs). General health evaluations after intravitreal injection included the assessment of weight gain, food intake, body temperature, and complete blood count analysis. RESULTS: Plasmin plus SF(6) injection resulted in complete posterior vitreous detachment (PVD), whereas plasmin or SF(6) injection alone resulted in only partial PVD. Balanced salt solution did not induce PVD. Eighty days after intravitreal injection, there were no major differences among the eyes of the three groups of animals compared with the normal control animals upon clinical evaluation, or regarding retinal morphology and ERGs. The lenses examined remained clear for up to 80 days following the intravitreal injection of plasmin plus SF(6), except one eye in the plasmin-treated group. ERGs decreased transiently one week after intravitreal injection in groups 1 through 3, but animals recovered fully to normal status afterward. General health was not affected after the injection of plasmin plus SF(6). CONCLUSIONS: Efficient vitreoretinal separation could be achieved, and an acceptable long-term safety profile was noted after plasmin plus SF(6) injection in the eye. No major ocular toxicity or systemic toxicity was found after the injection of plasmin plus SF(6). These results provide good support for the future clinical use of plasmin plus SF(6) for treatment of a variety of vitreoretinopathies.

Quantification and structure elucidation of in vivo bevacizumab modification in rabbit vitreous humor after intravitreal injection.

Off-label and intravitreal use of bevacizumab, a recombinant immunoglobulin against VEGF, has been practiced widely for ophthalmic treatments. However, longitudinal data of its intravitreal status is unavailable due to a lack of reliable methods for bevacizumab determination. Thus its pharmacokinetics and pharmacodynamics are uncertain. We developed and validated a high performance liquid chromatographic method to determine bevacizumab in vitreous humor and utilized a novel strategy to assess in vivo temporal binding changes by affinity chromatography. Mass spectrometry and tandem mass spectrometry detection were used for structural evaluation. The coefficient of variation (CV) for intrabatch imprecision varied from 0.5 to 14.3% and for interbatch imprecision from 1.9 to 11.6%. The linearity was over 0.9982, lower limit of quantification 1.95 µg, recoveries over 95%, and accuracy between 90 and 112% over the range of 1.95-250 µg of bevacizumab in 100 µL of vitreous humor. Blank vitreous humor showed no interference peak. It was stable at room temperature for 5 h. Bevacizumab elimination in the vitreous followed first order kinetics with half-life as 5.7 days and elimination rate as 0.1221 day(-1). Peptide mapping and tandem mass spectrometry revealed structural modifications of the in vivo bevacizumab mainly on the heavy chain in both variable and constant regions 7 days after intravitreal injection. Minor changes were also discovered on the light chain. Affinity chromatography showed significant affinity changes in samples 21 days after intravitreal injection. The changes were consistent with structural modifications as found in endothelial cell migration assays results. In conclusion, we have established a robust chromatographic method for determination of bevacizumab and strategies with affinity chromatography and molecular mass detection that revealed bevacizumab structural and possible functional changes in vitreous.

Photodynamic properties of vital dyes for vitreoretinal surgery.

The purpose of this study was to evaluate photodynamic properties of indocyanine green (ICG), brilliant blue G (BBG) and trypan blue (TB) as currently used vital dyes for chromovitrectomy. Under consideration of intraoperative illumination intensities and dye concentrations, a simulative in vitro investigation was set up. Therefore, standardized dilutions of original ICG, BBG and TB vials were irradiated at a wavelength of 366 nm with an intensity of 14 µW/cm2 between 0 and 48 h. After this, all samples were measured spectroscopically in a 220- to 750-nm bandwidth. Analyzing the vital dyes over the time course, an exponential photolysis was observed for ICG, whereas BBG and TB presented photostable properties. Regarding ICG, 5% of the concentration was degraded to toxic metabolites every 20 min. For this reason, our study provides evidence that intraocular dye concentrations and modern endoillumination systems alone cannot fully prevent ICG photodegradation.

Hyalocytes: essential cells of the vitreous cavity in vitreoretinal pathophysiology?

PURPOSE: To review the present understanding of hyalocytes. METHODS: A review of recent studies that investigated the roles of hyalocytes in the pathophysiology of the vitreous cavity. RESULTS: Studies on immunocytochemistry and chimeric mice with green fluorescent protein transgenic mice show that hyalocytes belong to the monocyte/macrophage lineage and derive from bone marrow. The effects of hyalocytes on the vitreous cavity environment can be divided into three categories: synthesis of extracellular matrix, regulation of the vitreous cavity immunology, and modulation of inflammation. In noninflamed eyes, vitreous cavity is an immune-privileged site that is maintained by a system called vitreous cavity-associated immune deviation, in which hyalocytes play the role of antigen-presenting cells. However, cultured hyalocytes proliferate in response to inflammatory molecules and secrete vascular endothelial growth factor and urokinase-type plasminogen activator. A collagen gel embedded with hyalocytes contracts over time, which is enhanced by transforming growth factor-ß but is inhibited by Rho kinase inhibitor. These results suggest that hyalocytes can be an exacerbating factor in inflamed eyes. Clinically, hyalocytes are frequently found in the surgically removed specimens of epiretinal membrane or proliferative vitreoretinopathy. CONCLUSION: Elucidating the properties of hyalocytes is important to understand the biology of vitreous cavity and to develop novel treatments for vitreoretinal diseases.

Intravitreal cell-based production of glucagon-like peptide-1.

BACKGROUND: To examine the efficacy and safety of an intravitreal cell-based production of glucagon-like peptide-1 (GLP-1) by intravitreally implanted and encapsulated cells. METHODS: The experimental study included 12 Sprague-Dawley rats. Four cell beads with a diameter of 600 µm were intravitreally implanted. Each bead contained 3,000 GLP-1-secreting cells, which were encapsulated by a barium cross-linked sodium alginate matrix. At baseline and at each of the follow-up examinations at Day 3, Day 7, and Day 14, 4, 3, 3, and 2 animals, respectively, were killed. The concentration of active GLP-1 in the vitreous body samples was determined by enzyme-linked immunosorbent assay. The retinas were histologically examined. RESULTS: The active GLP-1 concentration in the vitreous samples increased significantly after baseline (<5 pM) to a peak at Day 3 (287 ± 196 pM) and at Day 7 (238 ± 55 pM), before it decreased at Day 14 (70 ± 8 pM). The histologic examinations did not show signs of apoptosis or tissue destruction. CONCLUSION: The intravitreal application of beads containing alginate-encapsulated cells producing GLP-1 resulted in an intraocular production of GLP-1 with a significant increase in the intraocular GLP-1 concentration, without observed cytotoxic effects. An intravitreal cell-based drug therapy with GLP-1 appears feasible.