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The anterior segment of the eye consists of the cornea, iris, ciliary body, crystalline lens, and aqueous humor outflow pathways. Together, these tissues are essential for the proper functioning of the eye. Disorders of vision have been ascribed to defects in all of them; some disorders, including glaucoma and cataract, are among the most prevalent causes of blindness in the world. To characterize the cell types that compose these tissues, we generated an anterior segment cell atlas of the human eye using high-throughput single-nucleus RNA sequencing (snRNAseq). We profiled 195,248 nuclei from nondiseased anterior segment tissues of six human donors, identifying >60 cell types. Many of these cell types were discrete, whereas others, especially in the lens and cornea, formed continua corresponding to known developmental transitions that persist in adulthood. Having profiled each tissue separately, we performed an integrated analysis of the entire anterior segment, revealing that some cell types are unique to a single structure, whereas others are shared across tissues. The integrated cell atlas was then used to investigate cell type-specific expression patterns of more than 900 human ocular disease genes identified through either Mendelian inheritance patterns or genome-wide association studies.

Objectives: Myopia is the most common refractive vision disorder. In recent years, several studies have suggested that the alteration of the exosomal protein levels in the aqueous humor (AH) is associated with the development of several eye diseases. Therefore, we aimed to explore the exosomal protein profile of the AH from myopia patients. Methods: Exosomes were isolated from the AH. The quality, concentration, and size distribution of exosomes for each patient were measured using nanoparticle tracking analysis system. Then, the exosomal proteins were purified and digested by trypsin for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Results: There was no significant difference observed between the myopia and control when comparing the concentration and size distribution of exosomes in the AH for each sample. Based on LC-MS/MS analysis, myopia patients had higher and more complex exosomal peptide content. We found two proteins that were common in AH exosomes and eight proteins that were highly expressed in the myopia group. Conclusions: Our results provide pioneering findings for the exploration of the exosomal protein profile in myopia development. Further studies may provide significant information for the diagnosis, clinical treatment, and prognosis of myopia., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Purpose: The aim of this study was to describe the cellular infiltrate in aqueous and vitreous samples of patients with uveitis analysed by multiparametric flow cytometry., Methods: This is a retrospective analysis of aqueous and vitreous samples analysed by flow cytometry for diagnostic purposes, in cases of masquerade syndromes and infectious and non-infectious uveitis. Data collected included demographics, anatomical classification of uveitis, phenotypic diagnosis, anterior chamber cells grading, vitreous haze and time of follow-up since presentation to sample obtained., Results: Thirty-one samples (17 aqueous and 14 vitreous fluids) from 31 patients, 18 men, were analysed. The mean age at the time of sample collection was 60.23±17.03 years. The most frequent anatomical classification was panuveitis (14 of 31). T cells accounted for the main cellular component in the majority of the samples (10 of 13 aqueous samples; 7 of 14 in vitreous samples). CD4:CD8 ratios ranged from 0.21 to 16.3 in the case of aqueous samples and from 0.5 to 9.7 in the case of vitreous samples., Discussion: Flow cytometry analysis of aqueous and vitreous samples from patients with uveitis could provide insight into the pathogenesis of human uveitis and help develop accurate animal models which better mimic human disease., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: Conventional wisdom posits that aqueous humor leaves the eye by passive bulk flow without involving energy-dependent processes. However, recent studies have shown that active processes, such as cell contractility, contribute to outflow regulation. Here, we examine whether inhibiting cellular metabolism affects outflow facility in mice., Methods: We measured outflow facility in paired enucleated eyes from C57BL/6J mice using iPerfusion. We had three Experimental Sets: ES1, perfused at 35°C versus 22°C; ES2, perfused with metabolic inhibitors versus vehicle at 35°C; and ES3, perfused at 35°C versus 22°C in the presence of metabolic inhibitors. Inhibitors targeted glycolysis and oxidative phosphorylation (2-deoxy-D-glucose, 3PO and sodium azide). We also measured adenosine triphosphate (ATP) levels in separate murine anterior segments treated like ES1 and ES2., Results: Reducing temperature decreased facility by 63% [38%, 78%] (mean [95% confidence interval (CI)], n = 10 pairs; P = 0.002) in ES1 after correcting for changes in viscosity. Metabolic inhibitors reduced facility by 21% [9%, 31%] (n = 9, P = 0.006) in ES2. In the presence of inhibitors, temperature reduction decreased facility by 44% [29%, 56%] (n = 8, P < 0.001) in ES3. Metabolic inhibitors reduced anterior segment adenosine triphosphate (ATP) levels by 90% [83%, 97%] (n = 5, P<<0.001), but reducing temperature did not affect ATP., Conclusions: Inhibiting cellular metabolism decreases outflow facility within minutes. This implies that outflow is not entirely passive, but depends partly on energy-dependent cellular processes, at least in mice. This study also suggests that there is a yet unidentified mechanism, which is strongly temperature-dependent but metabolism-independent, that is necessary for nearly half of normal outflow function in mice.

Uveitis is common in cats, and is often a feature of feline infectious peritonitis (FIP). We evaluated 3 tools for detection of feline coronavirus (FCoV) in aqueous humor: 1) a 7b gene reverse-transcription real-time PCR ( 7b -RT-rtPCR) assay to detect FCoV RNA, 2) a spike gene mutation RT-rtPCR ( S -RT-rtPCR) assay to detect 2 point mutations in the spike gene of FCoV in cats positive by 7b -RT-rtPCR, and 3) immunocytochemistry (ICC) for detection of FCoV antigen in aqueous humor macrophages. We studied 58 cats, including 31 cats with FIP and 27 control cats. FIP was excluded by postmortem examination and negative immunohistochemistry (IHC). Aqueous humor samples obtained postmortem were assessed using 7b -RT-rtPCR in all cats, and positive samples were evaluated with S -RT-rtPCR. ICC evaluation of aqueous humor samples from 36 of the 58 cats was done using an avidin-biotin complex method and monoclonal anti-FCoV IgG 2A. Sensitivity, specificity, and negative and positive predictive values were calculated including 95% CIs. 7b -RT-rtPCR had a specificity of 100.0% (95% CI: 87.2-100.0) and sensitivity of 35.5% (95% CI: 19.2-54.6). Specificity of S -RT-rtPCR could not be determined because there were no FCoV 7b -RT-rtPCR-positive samples in the control group. Sensitivity of S -RT-rtPCR was 12.9% (95% CI 3.6-29.8). Sensitivity and specificity of ICC were 62.5% (95% CI: 40.6-81.2) and 80.0% (95% CI: 44.4-97.5), respectively. The combination of 7b -RT-rtPCR and IHC could be useful in diagnosing FIP; S -RT-rtPCR did not add value; and ICC of aqueous humor samples cannot be recommended for the diagnosis of FIP.

Purpose: To measure the concentrations of growth factors in the aqueous humor of patients with congenital cataract and to investigate the biological effects of a selected cytokine (fibroblast growth factor 4 [FGF4]) on cell proliferation, migration, and transformation., Methods: In the aqueous humor obtained from 55 eyes with congenital cataract and 55 eyes with age-related cataract, 40 growth factors were screened and selected cytokines were confirmed with enzyme-linked immunosorbent assays. After the addition of various concentrations of FGF4 (0, 2.5, 15, or 50 ng/mL) to the incubation medium, cellular functions were evaluated., Results: The concentration of FGF4 was significantly higher in the aqueous humor of patients with congenital cataract than in that of patients with age-related cataract. The human SRA01/04 lens epithelial cell line was treated with FGF4 and the cell proliferation increased significantly both dose- and time-dependently. The wound healing assay and Transwell migration assay revealed a significant increase in the migration capacity of the SRA01/04 cell line treated with 15 or 50 ng/mL of FGF4 compared with that of control cells. The intensity of immunofluorescent staining for α-smooth muscle actin increased significantly in the SRA01/04 cell line when treated with FGF4. Cytoskeletal protein (F-actin) staining showed that changes of cell morphology were induced in primary lens epithelial cells by FGF4., Conclusions: This study provides a comprehensive profile of growth factors in congenital cataract. FGF4 induced cellular changes, and may have utility as a biomarker to predict the formation of visual axis opacification. [J Pediatr Ophthalmol Strabismus. 2020;57(3):159-168.]., (Copyright 2020, SLACK Incorporated.)

Alzheimer's disease (AD) is an ageing-related neurodegenerative disease characterized and diagnosed by deposition of insoluble amyloid-β (Aβ) plaques in the brain. The plaque accumulation in the brain directly affects reduced levels of Aβ in cerebrospinal fluid (CSF) and blood, as Aβ can freely transport the blood-brain barrier, and clinical investigations have suggested these two biofluids as promising samples for in vitro diagnosis. Given that the human eye structurally resembles the brain and Aβ accumulation often observed in the ocular region of AD patients, in this study, we examined aqueous humor Aβ as another possible surrogate biomarker. First, using the acute Aβ-infused AD mouse model by injecting Aβ to the CSF in intracerebroventricular region of normal ICR mice, we investigated whether Aβ concentration in the aqueous humor in AD models is positively correlated with the concentration in the CSF. Then, we examined the correlation of aqueous humor Aβ levels with increased plaque deposition in the brain and reduced Aβ levels in both CSF and blood in adult and aged 5XFAD Alzheimer transgenic mice. Collectively, the synthetic Aβ injected into CSF immediately migrate to the aqueous humor, however, the age-dependently reducing pattern of Aβ levels in CSF and blood was not observed in the aqueous humor., Competing Interests: The authors have declared that no competing interests exist.

Purpose: To describe a case of photoreceptor outer segment glaucoma (Schwartz-Matsuo syndrome) with electron microscopic evidence of photoreceptor outer segments in the trabecular meshwork (TM)., Design: This is a clinicopathologic case report., Participant: A 48-year-old Filipino man., Methods: Specimens of aqueous humor and TM in a clinical case of Schwartz-Matsuo syndrome were examined by electron microscopy., Main Outcome Measures: Electron photomicroscopy., Results: Electron microscopy showed evidence of retinal photoreceptor outer-segments in both an aqueous humor and a TM specimen., Conclusion: Schwartz-Matsuo syndrome is associated with the presence of photoreceptor outer segments in the TM.

Introduction: Intraocular lymphoma is rare. There are very few studies on intraocular lymphoma published from Asian countries., Objective: To report our case series of intraocular lymphoma patients from a tertiary eye centre in Singapore., Subjects and Methods: Nine patients with intraocular lymphoma managed between January 2005 and December 2014 were identified from Ocular Autoimmune Systemic Inflammatory Infectious Study (OASIS) database. Demographic characteristics, clinical presentation, investigations performed and outcomes recorded., Results: There were almost equal distribution between males (four patients) and females (five patients) with mean age of presentation was 60.3 years. Five patients had bilateral involvement and vitreo-retina was the most common site of infiltration. All of our patients had central nervous system involvement although four of them had presented with ocular manifestations initially. Anterior chamber fluid cytology, as a less invasive alternative to vitreous analysis was proven to be useful. The time from ocular presentation to diagnosis of ocular lymphoma was variable; from one day to 18 months. Mortality in our study group was 55% with death occurring 1 month to 8 years from diagnosis of intraocular lymphoma., Conclusion: Intraocular lymphoma is a masquerade syndrome that mimics chronicuveitis and poses a diagnostic challenge. The diagnosis is often delayed and despite the eventual diagnosis, the disease prognosis is poor even with aggressive treatment., (© NEPjOPH.)

Regulation of cellular volume is an essential process to balance volume changes during cell proliferation and migration or when intracellular osmolality increases due to transepithelial transport. We previously characterized the key role of volume-regulated anion channels (VRAC) in the modulation of the volume of trabecular meshwork (TM) cells and, in turn, the aqueous humour (AH) outflow from the eye. The balance between the secretion and the drainage of AH determines the intraocular pressure (IOP) that is the major casual risk factor for glaucoma. Glaucoma is an ocular disease that causes irreversible blindness due to the degeneration of retinal ganglion cells. The recent identification of Leucine-Rich Repeat-Containing 8 (LRRC8A-E) proteins as the molecular components of VRAC opens the field to elucidate their function in the physiology of TM and glaucoma. Human TM cells derived from non-glaucomatous donors and from open-angle glaucoma patients were used to determine the expression and the functional activity of LRRC8-mediated channels. Expression levels of LRRC8A-E subunits were decreased in HTM glaucomatous cells compared to normotensive HTM cells. Consequently, the activity of VRAC currents and volume regulation of TM cells were significantly affected. Impaired cell volume regulation will likely contribute to altered aqueous outflow and intraocular pressure.

Uveal melanoma (UM) is the major intraocular malignancy in adults, of which the molecular biology is still unknown. Therefore, this study was designed to determine the aqueous concentrations of angiogenic, inflammatory, and chemotactic cytokines in eyes with UM.Aqueous humor samples were collected from 38 patients with UM and 22 patients undergoing cataract surgery. Interleukin 6, 8 (IL-6, IL-8, respectively), interferon-inducible protein-10 (IP-10), placental growth factor1 (PIGF1), regulated on activation, normal T Cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), nerve growth factor-beta (NGF-β), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and vascular endothelia growth factor A (VEGF-A) were assessed by multiplex bead assay.In the study group, significantly higher concentrations of IL-6 (P = .006), IL-8 (P = .018), IP-10 (P = .004), RANTES (P = .008), MCP-1 (P = .02), NGF-β (P = .013), EGF (P < .001), PIGF1 (P = .01), bFGF (P = .016), and VEGF (P = .017) were measured, when compared with the control group.Several angiogenic, inflammatory, and chemotactic cytokines are highly expressed in the aqueous humor of the UM eyes, which provides new insights into the pathophysiology of UM and could be potential targets for treatment.

Purpose : To determine aqueous humor CD4+/CD8+ T-lymphocyte ratio changes in sarcoid and non-sarcoid uveitis with anterior chamber involvement. Methods : The case-control study includes 61 patients with either anterior uveitis, intermediate uveitis with anterior spill, or panuveitis. A total of 21 of them were categorized as sarcoid uveitis and 40 as non-sarcoid uveitis according to diagnostic criteria. CD4+/CD8+ ratio in the aqueous humor was determined using flow cytometry. Results : Significantly higher CD4+/CD8+ ratio in the aqueous humor was observed in patients with sarcoid uveitis (6.3 ± 1.4; mean ± SEM) compared to non-sarcoid uveitis (1.6 ± 0.1; mean ± SEM). Whole blood CD4+/CD8+ ratio was not elevated in subjects with sarcoid and non-sarcoid uveitis. Aqueous humor CD4+/CD8+ ratio >3.5 was observed to be associated with sarcoid uveitis (OR 38, 95% CI 7.0-205.2). Conclusion : Increased aqueous humor CD4+/CD8+ ratio in sarcoid uveitis. Immunophenotyping of localized lymphocytosis in aqueous humor could be utilized as an additional confirmatory marker for ocular sarcoidosis.

Tumor-derived cell-free DNA (cfDNA) has biomarker potential; therefore, this study aimed to identify cfDNA in the aqueous humor (AH) of retinoblastoma eyes and correlate somatic chromosomal copy-number alterations (SCNA) with clinical outcomes, specifically eye salvage. AH was extracted via paracentesis during intravitreal injection of chemotherapy or enucleation. Shallow whole-genome sequencing was performed using isolated cfDNA to assess for highly recurrent SCNAs in retinoblastoma including gain of 1q, 2p, 6p, loss of 13q, 16q, and focal MYCN amplification. Sixty-three clinical specimens of AH from 29 eyes of 26 patients were evaluated; 13 eyes were enucleated and 16 were salvaged (e.g., saved). The presence of detectable SCNAs was 92% in enucleated eyes versus 38% in salvaged eyes ( P = 0.006). Gain of chromosome 6p was the most common SCNA found in 77% of enucleated eyes, compared with 25% of salvaged eyes ( P = 0.0092), and associated with a 10-fold increased odds of enucleation (OR, 10; 95% CI, 1.8-55.6). The median amplitude of 6p gain was 1.47 in enucleated versus 1.07 in salvaged eyes ( P = 0.001). The presence of AH SCNAs was correlated retrospectively with eye salvage. The probability of ocular salvage was higher in eyes without detectable SCNAs in the AH ( P = 0.0028), specifically 6p gain. This is the first study to correlate clinical outcomes with SCNAs in the AH from retinoblastoma eyes, as such these findings indicate that 6p gain in the aqueous humor is a potential prognostic biomarker for poor clinical response to therapy. Implications: The correlation of clinical outcomes and SCNAs in the AH identified in the current study requires prospective studies to validate these finding before SCNAs, like 6p gain, can be used to predict clinical outcomes at diagnosis. Mol Cancer Res; 16(11); 1701-12. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Purpose: To compare lymphocyte populations present within inflamed eyes in two rat models of autoimmune uveitis., Methods: Experimental autoimmune uveitis (EAU) and primed mycobacterial uveitis (PMU) were initiated in Lewis rats. Aqueous and vitreous were collected at peak inflammation (PMU at day 2, EAU at day 14). The number of cells in the aqueous and vitreous was determined and compared for each eye and between the two models. Intraocular CD-19+ B cells, CD3+ T cells, and CD4+ or CD8+ T-cell subpopulations were identified by flow cytometry and compared between EAU and PMU., Results: The median number of cells/mL collected from PMU aqueous (7.98 × 107 cells/mL), was not significantly different from the number of cells collected from EAU aqueous (1.61 × 107 cells/mL, P = 0.94). EAU aqueous contains a significantly larger mononuclear population (median 61%, interquartile range [IQR] 44%-67%) than PMU (median 9%, IQR 8%-10% [P < 0.0001]). Within the mononuclear population, EAU and PMU aqueous demonstrate similar proportions of CD3+, CD4+ T cells. However, EAU has a larger CD3+, CD8+, T-cell population than PMU, and this population also demonstrates co-expression of CD45R. B cells comprise a significantly larger median percentage of cells in EAU aqueous (median 18%, IQR 15%-20%) compared to PMU (median 13%, IQR 9%-15%, P = 0.006)., Conclusions: Flow cytometry analysis of intraocular lymphocytes from EAU and PMU identifies similarities and differences between the T-cell and B-cell populations present at peak inflammation. Complementary animal models that have well-defined mechanistic differences will improve our ability to test potential new therapies and bring meaningful advances into clinical practice for patients with uveitis.

Purpose: To assess the ability of swept-source (SS) optical coherence tomography (OCT) of the anterior segment (AS) to measure anterior chamber (AC) inflammation (both flare and cells) objectively. To compare OCT-derived inflammatory indices with standard techniques., Design: Prospective evaluation of a diagnostic test., Participants: Patients diagnosed with anterior uveitis (active or inactive) and controls., Methods: Participants underwent an AC inflammation evaluation including: clinical cell and flare grading and laser flare photometry (LFP). Uveitis patients were divided into active or inactive uveitis status according to clinical grading. Anterior segment SS-OCT scans were obtained for each participant. Tomographic images were analyzed to count the AC cells, and to calculate to absolute measurements of aqueous signal intensity. The absolute values were compared with the signal measured by the scan outside the eye, generating an optical density ratio (aqueous-to-air relative intensity [ARI] index). Correlations between OCT-derived AC inflammatory indexes and LFP, clinical grading, participant category (active or inactive uveitis, control), age, gender, and central corneal thickness (CCT) were assessed., Main Outcome Measures: Correlation between OCT-derived AC inflammatory indexes (ARI index and AC cells on OCT) and standard clinical techniques (LFP, clinical cell grading)., Results: Two hundred thirty-seven eyes (70 active uveitis, 97 inactive uveitis, and 70 controls) were included. Anterior chamber cells count on OCT did not differ between inactive uveitis and controls, but was significantly higher in active uveitis compared to the other categories (both P < 0.0001). All groups had different LFP (all P < 0.0001). Active uveitis had significantly higher ARI index compared with inactive uveitis and controls (both P < 0.0001). Interobserver agreement (intraclass correlation coefficient) for ARI index was 0.78. The ARI index correlated positively with age (P = 0.043) and negatively with CCT (P = 0.006). The ARI index correlated with LFP in the active uveitis group (P < 0.0001), but not in the others. Anterior chamber cells on OCT increased among all cell clinical grades (P < 0.0001). The ARI index increased among all flare clinical grades (P < 0.005)., Conclusions: Anterior segment SS-OCT could be used for a comprehensive assessment of AC inflammation, providing objective measurements of inflammatory cells and aqueous flare., (Copyright © 2017 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Importance: Retinoblastoma (Rb) is one of the first tumors to have a known genetic etiology. However, because biopsy of this tumor is contraindicated, it has not been possible to define the effects of secondary genetic changes on the disease course., Objective: To investigate whether the aqueous humor (AH) of Rb eyes has sufficient tumor-derived DNA to perform genetic analysis of the tumor, including DNA copy number alterations., Design, Setting, and Participants: This investigation was a case series study at a tertiary care hospital (Children's Hospital Los Angeles) with a large Rb treatment center. Cell-free DNA (cfDNA) was isolated from 6 AH samples from 3 children with Rb, including 2 after primary enucleation and 1 undergoing multiple intravitreous injections of melphalan for vitreous seeding. Samples were taken between December 2014 and September 2015., Main Outcomes and Measures: Measurable levels of nucleic acids in the AH and identification of tumor-derived DNA copy number variation in the AH. The AH was analyzed for DNA, RNA, and micro-RNA using Qubit high-sensitivity kits. Cell-free DNA was isolated from the AH, and sequencing library protocols were optimized. Shallow whole-genome sequencing was performed on an Illumina platform, followed by genome-wide chromosomal copy number variation profiling to assess the presence of tumor DNA fractions in the AH cfDNA of the 3 patients. One child's cfDNA from the AH and tumor DNA were subjected to Sanger sequencing to isolate the RB1 mutation., Results: Six AH samples were obtained from 3 Rb eyes in 3 children (2 male and 1 female; diagnosed at ages 7, 20, and 28 months). A corroborative pattern between the chromosomal copy number variation profiles of the AH cfDNA and tumor-derived DNA from the enucleated samples was identified. In addition, a nonsense RB1 mutation (Lys→STOP) from 1 child was also identified from the AH samples obtained during intravitreous injection of melphalan, which matched the tumor sample postsecondary enucleation. Sanger sequencing of the AH cfDNA and tumor DNA with polymerase chain reaction primers targeting RB1 gene c.1075A demonstrated this same RB1 mutation., Conclusions and Relevance: In this study evaluating nucleic acids in the AH from Rb eyes undergoing salvage therapy with intravitreous injection of melphalan, the results suggest that the AH can serve as a surrogate tumor biopsy when Rb tumor tissue is not available. This novel method will allow for analyses of tumor-derived DNA in Rb eyes undergoing salvage therapy that have not been enucleated.

Purpose: To evaluate the effect of filtering surgery using the EX-PRESS device on intraocular pressure (IOP), flare count, anterior chamber (AC) depth, and the corneal endothelium in primary open-angle glaucoma (POAG), including normal-tension glaucoma (NTG)., Patients and Methods: The study comprised 63 consecutive patients with POAG or NTG. They underwent complete ophthalmologic examinations, including measurement of visual acuity (VA), IOP, flare count, AC depth, and cell density of the corneal endothelium (CE). Two outcomes were used for surgical success: outcome 1 was defined as a ≥20% reduction in the IOP from the preoperative value, with an IOP ≥ 5 mmHg but ≤18 mmHg, and outcome 2 was defined as a ≥20% reduction in the IOP, with an IOP ≥ 5 mmHg but ≤15 mmHg., Results: The IOP significantly decreased from 17.7 preoperatively to 12.4 mmHg postoperatively (P < 0.0001). The success rates for outcome 1 at 24 months were 81.3 and 46.7% for POAG and NTG, respectively (P = 0.0065); the corresponding success rates for outcome 2 were 79.4 and 46.7% (P = 0.0120). The flare count significantly increased on postoperative day 1 (P = 0.0002) but recovered to insignificant levels by day 3 (P = 0.0686). A transient decrease in AC depth was observed by 4 weeks after surgery (P = 0.0017). The CE changed from 2529 preoperatively to 2428/mm 2 at 24 months (P = 0.0089)., Conclusion: The advantage of filtering surgery using the EX-PRESS device includes reduced inflammation, and the disadvantages include loss of the CE and reduced effectiveness of NTG.

Uveitis, an intraocular inflammatory disease, occurs mostly in young people and can result in the loss of socioeconomic capabilities. Silibinin has been shown to exert anti-inflammatory effects in human retinal pigment epithelial (RPE) cells. The present study investigated the anti-inflammatory effect of silibinin pretreatment on endotoxin-induced uveitis (EIU) in rats and the mechanisms by which it exerts these effects. Uveitis was induced via injection of lipopolysaccharides (LPS) into Lewis rats. Twenty-four hours after the LPS injection, histological examination showed that silibinin decreased inflammatory cell infiltration in the anterior segment of the eyes of LPS-treated rats. Analyses of the aqueous humor showed that silibinin decreased cell infiltration, protein concentration, nitric oxide (NO), and prostaglandin (PG)-E2 production. Western blot analysis indicated that silibinin decreased the expression of inducible NO synthase (iNOS), cyclooxygenase (COX-2), and phosphorylated IkB in the iris-ciliary body (ICB). Immunohistochemistry showed that silibinin decreased intercellular adhesion molecule (ICAM-1) expression in the ICB. In addition, western blot analysis showed that silibinin attenuated the expression of iNOS, COX-2, ICAM-1, and nuclear p65 in LPS-treated RAW cells. In conclusion, silibinin pretreatment prevents EIU and the subsequent production of proinflammatory mediators and ICAM-1, at least in part, by blocking the NF-κB-dependent signaling pathway both in vivo and in vitro. These effects may contribute to the silibinin-mediated preventive effects on intraocular inflammatory diseases such as acute uveitis.

Aim: To determine if preoperative treatment with a topical non-steroidal anti-inflammatory drug (NSAID) lowers the concentration of intraocular interleukin (IL)-12 and the incidence of postoperative macular edema in patients with non-proliferative diabetic retinopathy undergoing cataract surgery., Methods: A total of 55 patients were randomized to diclofenac (n=27) or placebo (n=28). Patients receiving diclofenac started preoperative treatment with 0.1% topical diclofenac four times a day 7 days before cataract surgery and the therapy was discontinued 30 days after surgery. Patients in the control group were administered placebo 7 days preoperatively and a standard postoperative therapy with 0.1% topical dexamethasone four times a day for 30 days after surgery. All patients received postoperative antibiotic prophylaxis with tobramycin eye drops four times daily for 30 days. Seven days before the cataract surgery, on the day of surgery, and 1, 7, 30, and 90 days after surgery, central foveal thickness (CFT) was measured with optical coherence tomography (OCT) and the aqueous humor was sampled at the beginning of cataract surgery for the analysis of IL-12 concentration. Due to loss to follow-up and insufficient aqueous humor samples, the data of 3 patients treated with diclofenac and 8 patients receiving placebo were not analyzed., Results: The aqueous humor IL-12 concentration was significantly lower in the diclofenac group than in the placebo group (t=-2.85, p=0.007). The diclofenac group had a significantly smaller increase in CFT after phacoemulsification (F=13.57, p<0.001)., Conclusion: Patients preoperatively treated with diclofenac had significantly lower intraocular levels of IL-12 and a lower increase in CFT, which indicates that a combination of preoperative and postoperative treatment with a topical NSAID may lower the incidence of postoperative macular edema in patients with diabetic retinopathy.

Purpose: Canonical Wnt signaling is associated with glaucoma pathogenesis and intraocular pressure (IOP) regulation. Our goal was to gain insight into the influence of non-pigmented ciliary epithelium (NPCE)-derived exosomes on Wnt signaling by trabecular meshwork (TM) cells. The potential impact of exosomes on Wnt signaling in the ocular drainage system remains poorly understood., Methods: Exosomes isolated from media collected from cultured NPCE cells by differential ultracentrifugation were characterized by dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), and nanoparticle tracking analysis (NTA), sucrose density gradient migration and transmission electron microscopy (TEM). The cellular target specificity of the NPCE-derived exosomes was investigated by confocal microscopy-based monitoring of the uptake of DiD-labeled exosomes over time, as compared to uptake by various cell lines. Changes in Wnt protein levels in TM cells induced by NPCE exosomes were evaluated by Western blot., Results: Exosomes derived from NPCE cells were purified and detected as small rounded 50-140 nm membrane vesicles, as defined by DLS, NTA, TRPS and TEM. Western blot analysis indicated that the nanovesicles were positive for classic exosome markers, including Tsg101 and Alix. Isolated nanoparticles were found in sucrose density fractions typical of exosomes (1.118-1.188 g/mL sucrose). Using confocal microscopy, we demonstrated time-dependent specific accumulation of the NPCE-derived exosomes in NTM cells. Other cell lines investigated hardly revealed any exosome uptake. We further showed that exosomes induced changes in Wnt signaling protein expression in the TM cells. Western blot analysis further revealed decreased phosphorylation of GKS3β and reduced β-catenin levels. Finally, we found that treatment of NTM cells with exosomes resulted in a greater than 2-fold decrease in the level of β-catenin in the cytosolic fraction. In contrast, no remarkable difference in the amount of β-catenin in the nuclear fraction was noted, relative to the control., Conclusions: The data suggest that NPCE cells release exosome-like vesicles and that these nanoparticles affect canonical Wnt signaling in TM cells. These findings may have therapeutic relevance since canonical Wnt pathway is involved in intra-ocular pressure regulation. Further understanding of NPCE-derived exosome-responsive signaling pathways may reveal new targets for pharmacological intervention within the drainage system as a target for glaucoma therapy.

The aim of this study has been to determine whether eye chambers constitute part of the normal migratory pathway of naive CD4+ and CD8+ T cells in mouse and if natural CD4+CD25+Foxp3+ and CD8+CD25+Foxp3+ regulatory T cells are present within these eye compartments. To this aim, the cells obtained from aqueous humor (AH) of normal mice were phenotyped in terms of the expression CD4, CD8, CD25, CD127 and transcription factor Foxp3. The mean percentage of CD8+ T cells in the total AH lymphocyte population was as high as 28.69%; the mean percentage of CD8high and CD8low cells in this population was 34.09% and 65.91%, respectively. The presence of cells with the regulatory phenotype, i.e. CD25+Foxp3+ cells, constituted only 0.32% of CD8+ T cell subset. Regarding the expression of CD25, AH CD8+ T cells were an exceptional population in that nearly 85% of these cells expressed this molecule without concomitant Foxp3 expression. Despite having this phenotype, they should not be viewed as activated cells because most of them co-expressed CD127, which indicates that they are naive lymphocytes. With regard to the markers applied in the present research, CD8+CD25+CD127+Foxp3- T cells represent the most numerous subset of AH CD8+ cells. The results suggest that eye chambers in mice are an element in the normal migratory pathway of naive CD8+ T cells. The study presented herein demonstrated only trace presence of CD4+ cells in the eye chambers, as the mean percentage of these cells was just 0.56. Such selective and specific homing of CD8+ and CD4+ cells to the eye chambers is most clearly engaged in the induction and maintenance of ocular immune privilege., Competing Interests: The authors have declared that no competing interests exist.

A 33-year-old black South African male patient was referred to our uveitis service with a 5-year history of panuveitis refractory to treatment. He had an associated history of recurrent mouth and genital ulcers, predating the uveitis history by about 2 years. The measurement of his intraocular CD4/CD8 ratio helped make a diagnosis of Behçet's disease uveitis.

Different optical coherence tomography (OCT), both time-domain OCT and spectral-domain OCT, has been used to evaluate anterior chamber cells in previous studies. Recently, commercial swept-source OCT was available. The comparison among three kinds of OCTs have not been done before. Here, we compared their feasibility in discerning aqueous cells in vitro. The peripheral blood was diluted to eight different cell concentrations and was packed into plastic-film bag. Each sample was tested using different scan modes of three OCTs. It was easy to discern aqueous cells from background noise in both Visante AS-OCT and RTVue XR Avanti OCT, but difficult in Casia SS-1000 OCT. The measured blood cell densities in high-resolution corneal cross-sectional scan of Visante AS-OCT, cornea line scan and 3D cornea scan of RTVue XR Avanti OCT were highly correlated with the actual blood cell concentration (Pearson correlation=0.991, 0.989, and 0.993 respectively). The mean adjusted cell densities of cornea line scan (10.46±2.87 spots mm -2 ) and 3D cornea scan (11.01±2.47 spots mm -2 ) of RTVue XR Avanti OCT were significantly larger than that of Visante AS-OCT (1.55±1.75 spots mm -2 ) (P<0.01). Our study demonstrated that RTVue XR Avanti OCT, a spectral-domain OCT, is superior to other two OCTs in evaluating aqueous cells.

We evaluated the anti-inflammatory effect of tyrosol (Tyr) on endotoxin-induced uveitis (EIU) in rats. EIU was induced in male Lewis rats by subcutaneous injection of lipopolysaccharide (LPS). Tyr (10, 50 or 100 mg/kg) was intravenously injected 2 hr before, simultaneously and 2 hr after LPS injection. The aqueous humor (AqH) was collected 24 hr after LPS injection; the infiltrating cell number, protein concentration, and tumor necrosis factor (TNF)-α, prostaglandin (PG)-E2 and nitric oxide (NO) levels were determined. Histopathologic examination and immunohistochemical studies for nuclear factor (NF)-κB, inhibitor of κB (IκB)-α, cyclooxygenase (COX)-2 and inducible NO synthase (iNOS) in the iris-ciliary body (ICB) were performed at 3 or 24 hr after LPS injection. To further clarify the anti-inflammatory effects, RAW264.7 macrophages were stimulated with LPS in the presence or absence of Tyr. Tyr reduced, in a dose-dependent manner, the infiltrating cell number, protein concentration, and TNF-α, PGE2 and NO levels in AqH and improved histopathologic scores of EIU. Tyr also inhibited LPS-induced COX-2 and iNOS expression, IκB-α degradation and nuclear translocation of activated NF-κB in ICB. Tyr significantly suppressed inflammatory mediator production in the culture medium and COX-2 and iNOS expression and activated NF-κB translocation in LPS-stimulated RAW264.7 cells. These results suggest that Tyr suppresses ocular inflammation of EIU by inhibiting NF-κB activation and subsequent proinflammatory mediator production.

Primary open angle glaucoma (POAG) is an optic neuropathy and an irreversible blinding disease. The etiology of glaucoma is not known but numerous risk factors are associated with this disease including aging, elevated intraocular pressure (IOP), race, myopia, family history and use of steroids. In POAG, the resistance to the aqueous humor drainage is increased leading to elevated IOP. Lowering the resistance and ultimately the IOP has been the only way to slow disease progression and prevent vision loss. The primary drainage pathway comprising of the trabecular meshwork (TM) is made up of relatively large porous beams surrounded by extracellular matrix (ECM). Its juxtacanalicular tissue (JCT) or the cribriform meshwork is made up of cells embedded in dense ECM. The JCT is considered to offer the major resistance to the aqueous humor outflow. This layer is adjacent to the endothelial cells forming Schlemm's canal, which provides approximately 10% of the outflow resistance. The ECM in the TM and the JCT undergoes continual remodeling to maintain normal resistance to aqueous humor outflow. It is believed that the TM is a major contributor of ECM proteins and evidence points towards increased ECM deposition in the outflow pathway in POAG. It is not clear how and from where the ECM components emerge to hinder the normal aqueous humor drainage. This review focuses on the involvement of the ECM in ocular hypertension and glaucoma and the mechanisms by which various ocular hypotensive drugs, both current and emerging, target ECM production, remodeling, and deposition., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Myeloid/NK cell precursor acute leukemia (MNKL) is a rare type of leukemia, and ocular complications have not previously been reported. We now report a patient with MNKL who developed intraocular infiltrates during follow-up., Methods and Results: A 13-year-old boy diagnosed with MNKL developed left eye pain 3 months after starting treatment. Examination of the left eye revealed a visual acuity of counting fingers at 20 cm, ciliary hyperemia, small corneal keratic precipitates, hypopyon, grade 4 vitreous opacities, and an obscured fundus. The differential diagnosis was between an opportunistic infection associated with immunodeficiency and an intraocular leukemic cell infiltrate. Therefore, a sample of aqueous humor was aspirated. Multiplex PCR/broad-range PCR of the aqueous humor was below detection limits for viruses, bacteria, and fungi. Flow cytometry (FCM) detected NK-related CD56-positive cells, thus leading to a diagnosis of ocular infiltrates due to MNKL. With treatment of the ocular infiltrates by consolidation systemic chemotherapy including intrathecal methotrexate (MTX), there was clearing of the vitreous opacities; and optic disc swelling, retinal hemorrhages, exudates, and protuberant lesions were now seen. With the addition of local radiation therapy to the eye, there was a dramatic treatment response, with regression of the optic disc findings and retinal lesions, and an improved visual acuity of 1.5., Conclusion: We encountered the first case of MNKL in which ocular infiltrates developed during follow-up. Multiplex PCR and FCM of the aqueous humor were useful in rapidly distinguishing leukemic cell infiltrates from an opportunistic infection. This case highlights the usefulness of intrathecal MTX and local radiotherapy in treating ocular infiltrates in patients with MNKL., Competing Interests: The authors have no conflicts of interest to disclose.

Purpose: Epiretinal membrane (ERM) is a frequent macular complication in patients with retinitis pigmentosa (RP). The etiology of ERM formation in RP is largely unknown. The purpose of this study was to investigate the association between aqueous flare, a surrogate index of intraocular inflammation, and ERM secondary to RP., Methods: We retrospectively studied a total of 206 eyes of 117 patients who were diagnosed with typical RP. Aqueous flare values were measured consecutively in 2012 and 2013 using a laser flare cell meter. Spectral-domain optical coherence tomography images and fundus photographs taken on the same day of the aqueous flare measurements were analyzed for ERM detection., Results: The mean values of aqueous flare, age, and frequency of male sex were significantly higher in the RP patients with ERM compared with the RP patients without ERM (P < 0.0001, P = 0.007, and P = 0.004, respectively). After adjustment for age and sex, the eyes in the highest quartile of aqueous flare had significantly higher odds of having ERM than those in the lowest quartile (odds ratio [OR], 2.68; 95% confidence interval [CI], 1.04-6.93), and the linear trend across flare levels was significant (P = 0.005). In addition, each 1-log-transformed increase in flare values was associated with an elevation of the likelihood of having ERM (OR, 2.59; 95% CI, 1.33-5.06)., Conclusions: Our analysis demonstrated that elevated aqueous flare is associated with ERM secondary to RP, suggesting that inflammation may be implicated in the pathogenesis of ERM formation in RP.

The trabecular meshwork (TM) tissue controls drainage of aqueous humor from the anterior chamber of the eye primarily by regulating extracellular matrix (ECM) remodeling by matrix metalloproteinases (MMPs). Glaucomatous TM tissue is stiffer than age-matched controls, which may be due to alterations in ECM cross-linking. In this study, we used genipin or beta-aminopropionitrile (BAPN) agents to induce or inhibit matrix cross-linking, respectively, to investigate the effects on outflow resistance and ECM remodeling. Treatment with BAPN increased outflow rates in perfused human and porcine anterior segments, whereas genipin reduced outflow. Using a fluorogenic peptide assay, MMP activity was increased with BAPN treatment, but reduced with genipin treatment. In genipin-treated TM cells, Western immunoblotting showed a reduction of active MMP2 and MMP14 species and the presence of TIMP2-MMP14 higher molecular weight complexes. BAPN treatment increased collagen type I mRNA and protein levels, but genipin reduced the levels of collagen type I, tenascin C, elastin and versican. CD44 and fibronectin levels were unaffected by either treatment. Collectively, our results show that matrix cross-linking has profound effects on outflow resistance and ECM composition and are consistent with the emerging paradigm that the stiffer the ECM, the lower the aqueous outflow facility through the TM.

Purpose: To determine the feasibility and accuracy of spectral-domain optical coherence tomography (SD-OCT) based grading of anterior chamber cell, using aqueous sampling as a standard, in a rabbit model of anterior uveitis., Methods: Adult Dutch-belted rabbits were preimmunized with M. tuberculosis (Tb) H37RA antigen, 1 week prior to induction of anterior uveitis with an intracameral injection of Tb antigen. The anterior chamber was imaged with SD-OCT, followed by a slit lamp examination. Two independent, trained graders recorded their estimate of anterior chamber cell count using the Standardization of Uveitis Nomenclature (SUN) scores for each eye prior to performing an anterior chamber tap to determine the aqueous cell density using a hemocytometer. Using the aqueous cell density as a standard, correlation with SD-OCT counts were compared to those with SUN scores., Results: Overall, SD-OCT correlated well with the hemocytometer counts (Spearman coefficient = 0.53, P < 0.001) compared with SUN grading and hemocytometer counts (Spearman coefficient = 0.02, P = 0.88). The correlation improved to 0.65 (P < 0.001) when we excluded eyes with corneal thickness ≥ 470 μm. Eyes with corneal thickness ≥ 470 μm exhibited the greatest degree of ocular inflammation and corneal opacity., Conclusions: In our rabbit model, SD-OCT grading of anterior chamber cell correlated significantly better with aqueous cell counts, compared to traditional slit lamp grading. Spectral-domain optical coherence tomography grading of anterior chamber cell may be a good alternative to SUN grading. Although SUN grading remains the clinical gold standard, alternative quantitative methods to assess ocular inflammation could provide insight into disease mechanism and aid in measuring treatment response.

Purpose: To investigate aqueous flare change patterns in retinal vein occlusions (RVO) following intravitreal application of dexamethasone implant. To evaluate the correlations between central macular thickness (CMT), best corrected visual acuity (BCVA) and aqueous flare values., Methods: This prospective, interventional pilot study included 40 eyes of 40 patients with macular edema associated with RVO. All patients received intravitreal injection of dexamethasone implant at baseline visit. Full ophthalmologic examination, CMT and aqueous flare measurements were performed prior to injection and repeated at months 1, 3, and 5. Aqueous flare, BCVA and CMT changes between visits were assessed. Further statistical analysis was performed to investigate correlations between these three parameters., Results: The patients consist of 23 (57.5%) men and 17 (42.5%) women with a mean age of 65.7 ± 6.2 years. Of the 40 eyes, 24 (60%) had branch retinal vein occlusion and 16 (40%) had central retinal vein occlusion. BCVA (log MAR) was significantly correlated with aqueous flare at each visit (r = 0.436, p  = 0.005; r = 0.349, p = 0.027; r = 0.357, p = 0.024; r = 0.551, p = 0.000, respectively). Flare values were also correlated with CMT measurements in 3rd and 5th months (r = 0.324, p = 0.042; r = 0.556, p = 0.000, respectively). Comparing the percentage change in CMT and flare values over time, the change in CMT from baseline to 3rd and 5th months (r = 0.441, p = 0.004, r = 0.448, p = 0.004), from 1st month to 3rd and 5th months (r = 0.441, p = 0.004; r = 0.461, p = 0.003) and from 3rd month to 5th month were directly correlated with aqueous flare changes (r = 0.584, p = 0.000). Twenty-two eyes (55%) required second dexamethasone implant application at 5th month., Conclusion: Considering the inflammatory base of RVO, dexamethasone implants effectively decreased the flare values in the anterior chamber. The CMT change was directly correlated with aqueous flare values.

Objective: To investigate the safety and efficacy of cyclosporine A sustained release from modified intraocular lens for preventing posterior capsular opacification (PCO) in rabbit eyes., Methods: Forty-five New Zealand albino rabbits undergoing phacoemulsification in their right eyes were randomly and equally divided into three groups. Group A had implanted original IOL, group B had implanted PLGA-IOL(IOL coated with polylactide-glycoli acid), and group C had implanted CsA-PLGA-IOL (CsA loaded PLGA-IOL). All the 45 eyes were examined by a slit-lamp microscope. The intraocular pressures were recorded. Anterior chamber flare and aqueous humor cells were graded at different time point after surgery. The concentrations of CsA in the aqueous humor and blood were determined by high performance liquid chromatography. Anterior segment tissue was histologically examined. Wet posterior capsules were weighed. PCO was graded 6 months later., Results: The mean concentrations of CsA in group C at 2 h,1 d,3 d,7 d,14 d,30 d,60 d after operation were (11.47±2.42) mg/L, (10.30±2.15) mg/L, (6.71±1.45) mg/L, (4.81±1.16) mg/L, (6.11±0.84) mg/L, (2.53±0.77) mg/L, (0.86±0.28) mg/L. The concentrations of CsA in blood were undetectable. During the early days after operation, the reactions of the anterior chamber in group A and B were more severe than group C. The initial appearance of PCO in group C was much later than in the other two groups, and the grade of PCO in group C was much lower than the other two groups. The mean weights of wet posterior capsules in group A(312.86±52.91) mg and B(310.64±62.42) mg were much heavier than that of group C(56.93 ± 24.24) mg. Histological observation showed that there was remarkably less accumulation of lens materials on the posterior capsules in group C than in the other two groups. No toxic actions were found in intraocular tissues in group C., Conclusion: Our study suggested that Cyclosporin A modified intraocular lens could effectively and safely prevent the formation and development of posterior capsular opacification in rabbit eyes for a relatively long term.

Aqueous humor levels of cytokines and growth/inflammatory factors were measured in 38 patients with macular edema who had major branch retinal vein occlusion (BRVO) or macular BRVO and were treated with intravitreal ranibizumab injection (IRI). Patients with recurrence of macular edema received further IRI as needed. Aqueous humor levels of vascular endothelial growth factor (VEGF), soluble VEGF receptor-1 (sVEGFR-1), and other cytokines/factors were measured. Compared with major BRVO, macular BRVO was associated with lower aqueous humor levels of sVEGFR-1, its ligands (VEGF and placental growth factor), and other growth/inflammatory factors (platelet-derived growth factor-AA, monocyte chemotactic protein-1, soluble intercellular adhesion molecule-1, interleukin-6, and interleukin-8). The mean number of IRI over 6 months was significantly lower in the macular BRVO group than in the major BRVO group. These findings suggest that macular BRVO requires fewer IRI than major BRVO and is associated with lower aqueous humor levels of various growth/inflammatory factors and cytokines., (© 2016 S. Karger AG, Basel.)

To assess the accuracy of standard clinical grading of aqueous flare in uveitis according to the Standardization of Uveitis Nomenclature consensus, and compare the results with the readings of the laser flare meter, Kowa 500. Two examiners clinically graded the flare in 110 eyes. The flare was then measured using the Kowa laser flare meter. Twenty-nine eyes were graded as anterior chamber flare +2; for 18 of these, the clinicians were in agreement, the rest differed by the order of one grade. The range of the laser flare meter for these eyes was 5.2-899.1 photons/ms. The median value was 41.4. Seventy-four eyes were graded with flare +1. Agreement was established in 51 of these eyes. Disagreement for the rest was again by the order of 1, and the flare meter range was 1.1-169.9 photons/ms, median value 18.4. For the clinical measure of flare 0, the clinicians disagreed on three out of five eyes. The flare meter readings ranged from 2.5 to 14.1 photons/ms, median value 9.9. Only two eyes were graded with flare +3 and there was one step disagreement on both of them. We found little evidence of association between the flare readings and intraocular pressure or age. Our findings suggest that clinical evaluation of aqueous flare is subjective. Compared with the Kowa laser flare meter's numeric readings, the discrepancies observed indicate that clinical grading is an approximate science. The laser flare meter provides an accurate, reproducible, non-invasive assessment of aqueous flare that can prove valuable in research and clinical decisions.

Purpose: To investigate the correlation between aqueous flare values and central visual function in patients with retinitis pigmentosa (RP)., Design: Retrospective, observational case series., Methods: We retrospectively studied 160 patients diagnosed with typical RP and 59 control subjects. Aqueous flare values were measured by laser flare cell meter. The relationships between aqueous flare and best-corrected visual acuity (VA) and mean deviation (MD) of static perimetry tests were analyzed in RP patients., Results: The aqueous flare values were significantly higher in the RP patients compared to the control subjects (10.6 ± 7.9 vs 5.0 ± 2.1 photon counts per millisecond [pc/ms], P < .0001). In the RP patients, the aqueous flare values were negatively correlated with VA (r = 0.359, P < .0001) and MD (r = -0.330, P < .0001). Age-subgroup analysis showed a significant correlation between aqueous flare and VA in the RP patients' 40s, 50s, and 60s and between aqueous flare and MD in the 30s, 40s, 50s, and 60s. The RP patients with MD values ≥-15 decibels (dB) showed significantly higher levels of aqueous flare than those with MD values <-15 dB (12.0 ± 6.2 vs 8.7 ± 5.8, P = .0001)., Conclusions: Aqueous flare is increased in RP patients and negatively correlates with central visual function. These results suggest a close relationship between inflammation and central vision loss in RP., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 μl. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

Aqueous humor (AH) is a dynamic intraocular fluid that supports the vitality of tissues that regulate intraocular pressure. We recently discovered that extracellular nanovesicles called exosomes are a major constituent of AH. Exosomes function in extracellular communication and contain proteins and small RNA. Our goal was to characterize the physical properties of AH exosomes and their exosomal RNA (esRNA) content. We isolated exosomes from human AH collected during cataract surgery from five patients using serial ultracentrifugation. We measured the size and concentration of AH exosomes in solution using nanoparticle tracking analysis. We found a single population of vesicles having a mean size of 121 ± 11 nm in the unprocessed AH. Data show that centrifugation does not significantly affect the mean particle size (121 ± 11 nm versus 124 ± 21 nm), but does impact the final number of exosomes in solution (87% loss from the unprocessed AH; n = 5). We extracted esRNA from the pooled human AH samples using miRCURY RNA isolation kit from Exiqon. The quality of extracted esRNA was evaluated using Agilent Bioanalyzer 2100 and was used to generate a sequencing library for small RNA sequencing with Illumina MiSeq sequencer. More than 10 different miRNAs were identified; abundant species included miR-486-5p, miR-204, and miR-184. We found that the majority of extracellular vesicles in the AH were in the exosome size range, suggesting that miRNAs housed within exosomes may function in communication between AH inflow and outflow tissues., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Purpose: The differential diagnosis of a patient presenting with anterior uveitis is broad and can present a diagnostic challenge. In this study, we evaluate the characteristic findings of inflammatory cells on optical coherence tomography (OCT) both in vitro and in vivo., Methods: Blood from two healthy volunteers was prepared using standardized methods for cell sorting with a flow cytometer (FASCAria). Neutrophils, lymphocytes, monocytes, and red blood cells were placed in suspension and scanned with a 26-kHz Fourier-domain OCT system (RTVue) with 5-μm axial resolution. Custom software algorithms were used to identify cells based on their reflectance distribution. These algorithms were then applied to OCT images obtained from uveitis patients with active anterior chamber inflammation., Results: On OCT images the cells appeared as hyperreflective spots. In vitro, cell reflectance was statistically significantly different between all of the cell types (neutrophils, monocytes, lymphocytes, and red blood cells, P < 0.001, Mann-Whitney test). In vivo, the relationship between underlying disease and cell type imaged on OCT was highly statistically significant, with human leukocyte antigen (HLA)-B27-associated uveitis patients having a predominantly polymorphonuclear pattern on OCT and sarcoidosis and inflammatory bowel disease patients having a predominantly mononuclear pattern on OCT (P < 0.001, Fisher's exact test)., Conclusions: These in vitro and in vivo data demonstrate the potential of OCT to evaluate cells in the anterior chamber of patients noninvasively. Optical coherence tomography may be a useful adjunct to guide the diagnosis and treatment of ocular inflammatory conditions., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)

Objectives: To evaluate the efficacy of intravitreal carboplatin plus bevacizumab in refractory retinoblastoma., Methods: Perspective study.Eleven patients (11 eyes) with the diagnosis of refractory retinoblastoma were enrolled in Department of Ophthalmology of Peking University People's Hospital from June 2013 to March 2014. They underwent intravitreal carboplatin plus bevacizumab every 4 weeks, an average of 4.5 times of treatment.Observe for 3 months after the last treatment. Aqueous humor was taken for cytological and VEGF detection and retinal funds were taken photos for observation.Statistical analyses between experimental group and control group and before and after intravitreal injection within experimental group were performed with independent samples t test., Results: Tumor in vitreous cavity reduced significantly in seven patients, however, poor control in four cases, and three of them were recurrent after first-line treatment. Cytology detection for aqueous humor showed no tumor cells in all of them. Aqueous VEGF of patients with retinoblastoma (60.65 ± 6.20) was significantly higher than the control group (21.98 ± 6.91). The difference was statistically significant (t = 13.80, P < 0.01). And the aqueous VEGF content decreased significantly after treatment (t = 2.12, P < 0.05)., Conclusion: Intravitreal carboplatin plus bevacizumab, is a relatively safe, effective treatment for refractory retinoblastoma, however, ineffective for recurrent tumor.

Purpose: To compare anterior chamber cell detection after phacoemulsification between Fourier-domain optical coherence tomography (OCT) and slitlamp biomicroscopy., Design: Observational prospective comparative case series., Setting: Dr. Agarwal's Eye Hospital and Eye Research Centre, Chennai, India., Methods: Eyes with preoperative nuclear opalescence (NO) grades 1 to 3 that had phacoemulsification with posterior chamber intraocular lens implantation were included. Slitlamp biomicroscopy and Fourier-domain OCT (Cirrus HD) of the anterior chamber were performed 1 day postoperatively. The results were evaluated, and a correlation analysis between the results and preoperative NO, effective phacoemulsification time (EPT), postoperative central corneal thickness (CCT), and intraocular pressure (IOP) was performed., Results: The preoperative NO grade was 1 in 20 eyes, 2 in 55 eyes, and 3 in 42 eyes. Anterior chamber grading was possible by slitlamp biomicroscopy and Fourier-domain OCT in 106 eyes (90.5%) and 117 eyes (100.0%), respectively. A positive correlation between slitlamp biomicroscopy and OCT grading was noted (R(2) = 0.986, P=.000). Slitlamp grading was not possible in 11 eyes (9.4%) with corneal edema (mean CCT 754 μm ± 44.2 [SD]); OCT detected cells in all 11 eyes. There was a difference in slitlamp grading and OCT grading in 7 eyes (6.6%). There was a significant correlation between the OCT cell count and the EPT (P=.000) and NO (P=.000)., Conclusion: Fourier-domain OCT was as reliable as slitlamp biomicroscopy in assessing postoperative anterior chamber cells., (Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

Background: Phacolytic glaucoma is induced by lens protein or macrophages that have leaked through a macroscopically intact anterior lens capsule. Here, we report a case of phacolytic glaucoma with anterior lens capsule disruptions visualized by scanning electron microscopy (SEM)., Case Presentation: A 71-year-old man was referred to our institute for increased intraocular pressure (IOP) in the right eye. Slit-lamp biomicroscopic examination revealed corneal edema, the presence of inflammatory cells and iridescent crystalline in the anterior chamber, and a hypermature cataract in the right eye. Despite treatment with topical glaucoma medication (0.15% brimonidine, 1% brinzolamide/0.5% timolol, and 0.03% bimatoprost) and systemic mannitol, his IOP remained uncontrolled. Light microscopy was used to examine the aqueous humor obtained via anterior chamber paracentesis and the anterior lens capsule obtained via intracapsular cataract extraction (ICCE), which revealed that the anterior lens capsule was intact. However, SEM revealed full-thickness disruptions in the anterior lens., Conclusion: This is the first reported case of phacolytic glaucoma with disruptions of the anterior lens capsule confirmed by SEM.

Purpose: To compare clinical findings and the aqueous levels of inflammatory mediators between toxic anterior segment syndrome (TASS) and endotoxin-induced uveitis (EIU) animal models and to evaluate the efficacy of systemic steroid pretreatment in both animal models., Methods: Rats were used in this study. Ortho-phthalaldehyde solution was injected into the anterior chamber to produce TASS (n=30), and lipopolysaccharide was injected into one hind footpad to produce EIU (n=30). Clinical findings were evaluated under slit-lamp examination, and the aqueous levels of prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNF-α) were measured 24 hours after these procedures. Twelve of the rats in each animal model were pretreated with intraperitoneal injection of dexamethasone for 4 days before the development of TASS and EIU., Results: Corneal haze scores were significantly higher for TASS than EIU, but clinical scores for anterior uveitis were not different between the two animal models. Although aqueous levels of PGE2 were markedly increased in both animal models, PGE2 levels were significantly higher for TASS than for EIU. However, an increase in aqueous levels of TNF-α was observed only in EIU. Dexamethasone pretreatment reduced the corneal haze score and clinical score for anterior uveitis in both animal models and inhibited the increase in aqueous levels of PGE2 and TNF-α., Conclusions: Prostaglandin E2 and TNF-α in aqueous humor seem to be regulated differently in animal models of TASS and EIU. However, dexamethasone pretreatment improved the clinical findings and inhibited the increases in PGE2 and TNF-α in both animal models., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Green tea extract (GTE) ingested by rats exerted anti-oxidative activities in various ocular tissues as shown in our previous studies. The present work investigated anti-inflammatory effects of GTE on endotoxin-induced uveitis (EIU). EIU was generated in adult rats by a footpad injection of 1 mg/kg lipopolysaccharide (LPS). Oral administration of GTE (550 mg/kg) was given one, two or four times after LPS injection. Twenty-four hours later, LPS produced severe hyperemia and edema in the iris. Immunocytochemical examinations showed an accumulation of infiltrating cells in the aqueous humor that were immunopositive for cluster of differentiation 43 (CD43) and CD68, markers for leucocytes and macrophages, respectively. Analyses of the aqueous humor showed an increase in pro-inflammatory mediators including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). GTE treatments improved the clinical manifestations and reduced infiltrating cells and protein exudation in the aqueous humor, which were not observed under half dose of GTE (275 mg/kg). The number of CD68 positive macrophages residing in the iris and ciliary was also reduced. GTE suppressed production of TNF-α, IL-6 and MCP-1 in the aqueous humor, which was associated with a down-regulation of LPS receptor complex subunits, Toll-like receptor 4 (TLR-4) and CD14, and suppression of nuclear factor-kappa Bp65 (NF-κBp65) in the iris and ciliary body. Our findings show that GTE is a potent anti-inflammatory agent against the inflammation of EIU, and suggest a potential use in treatment of acute uveitis.

Purpose: To investigate the effect of pressure/flow on the barrier function and protein expression of normal and senescent porcine aqueous humor plexus (AAP) cells, which are the porcine equivalent of human Schlemm's canal endothelial cells., Methods: AAP cells were grown for 2 weeks in physiological (5% O2) or hyperoxic conditions (40% O2) to model cell senescence. Control and senescent AAP cells were subjected to control and elevated hydrostatic pressure gradient of 10 mm Hg for 72 hours. Hydraulic conductivity (HC) and transendothelial electric resistance (TEER) were measured. The expressions of senescence-associated β-galactosidase and DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) were monitored, and the protein expression profile was analyzed by Western blot., Results: After 14 days of hyperoxia, AAP cells stained positive for 8-OHdG and β-galactosidase. Pressure elevation/flow resulted in significant increase of HC in control cells (from 1.37 ± 0.12 to 1.64 ± 0.18 μL/mm Hg/min/cm(2), P < 0.05), but not in senescent cells (1.15 ± 0.17 and 1.08 ± 0.10 μL μL/mm Hg/min/cm(2)). TEER changes were consistent with the HC results. Western blot analysis showed that the expression level of myosin light chain, claudin-5, and VE-cadherin significantly reduced under pressure elevation in control cells but not in senescent cells., Conclusions: AAP cells are mechano-sensitive; however, cell senescence rendered the cells less responsive to mechanical stimulus, which may have pathological consequences.

Purpose: Structures of the aqueous humor drainage tract are contractile, although the tract is not entirely composed of muscle. We characterized the mouse aqueous drainage tract by immunolabeling contractile markers and determined whether profiling these markers within the tract distinguished its key structures of the trabecular meshwork (TM) and ciliary muscle (CM)., Methods: Enucleated eyes from pigmented C57BL/6 (n=8 mice) and albino BALB/c (n=6 mice) mice were processed for cryo- and formalin-fixed paraffin-embedded sectioning. Immunofluorescence labeling was performed for the following: (a) filamentous actin (using fluorescence-conjugated phalloidin), representing a global contractile marker; (b) α-smooth muscle actin (α-SMA), caldesmon, and calponin, representing classic smooth muscle epitopes; and (c) nonmuscle myosin heavy chain, representing a nonmuscle contractile protein. Tissue labeling was identified by confocal microscopy and analyzed quantitatively. Hematoxylin and eosin staining provided structural orientation., Results: A small portion of the TM faced the anterior chamber; the rest extended posteriorly alongside Schlemm's canal (SC) within the inner sclera. Within the drainage tract, filamentous actin labeling was positive in TM and CM. α-SMA and caldesmon labeling was seen primarily along the CM, which extended from the anterior chamber angle to its posterior termination beyond the SC near the retina. Low intensity, patchy α-SMA and caldesmon labeling was seen in the TM. Myosin heavy chain immunoreactivity was primarily found in the TM and calponin was primarily observed in the CM. C57BL/6 and BALB/c comparison showed that pigment obscured fluorescence in the ciliary body., Conclusions: Our strategy of profiling contractile markers distinguished mouse aqueous drainage tract structures that were otherwise indistinguishable by hematoxylin and eosin staining. The mouse TM was seen as an intervening structure between SC, a part of the conventional drainage tract, and CM, a part of the unconventional drainage tract. Our findings provide important insights into the structural and functional organization of the mouse aqueous drainage tract and a basis for exploring the role of contractility in modulating aqueous outflow.

Purpose: To evaluate the effect of a heparin-coated foldable intraocular lens (IOL) on postoperative flare in high-risk patients., Setting: Vienna Institute for Research in Ocular Surgery, Department of Ophthalmology, Hanusch Hospital, Vienna, Austria., Design: Randomized clinical trial., Methods: This study included patients with diabetes mellitus (DM), pseudoexfoliation syndrome (PXF), or both scheduled for cataract surgery in both eyes. One eye received a coated IOL (Polylens EC-1YH PAL) and the other eye, an uncoated control IOL (Polylens EC-1Y PAL). Aqueous flare measurements were taken using a laser flare meter (FM-600, Kowa) preoperatively and 1 hour, 1 day, 3 weeks, and 3 months postoperatively. Visual acuity and anterior (ACO) and posterior (PCO) capsule opacification were assessed, and IOL centration and tilt were measured with a Purkinje meter., Results: Eighty eyes of 40 patients were included in the study; 64 eyes of 32 patients completed the study. The mean flare values (photons/ms) preoperatively and 1 hour, 1 day, 3 weeks, and 3 months postoperatively were 6.98 ± 3.71 (SD), 19.14 ± 18.90, 16.62 ± 25.33, 10.33 ± 9.70, 8.74 ± 4.64, respectively, with the coated IOL and 8.65 ± 5.73, 22.08 ± 16.23, 13.44 ± 13.71, 7.50 ± 4.11, and 7.03 ± 3.70, respectively, with the control IOL. No significant differences were found in flare, visual acuity, ACO, PCO, tilt, or centration between the coated IOL and the control IOL at any timepoint., Conclusion: No significant differences in flare or capsule performance were found between the coated IOL and control IOL., (Copyright © 2013 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

Purpose: To compare the merits and limitations of hematoxylin-eosin (HE) and methyl violet staining for displaying ghost cells from vitreous or aqueous humor., Methods: A specimen containing ghost cells was adjusted to five different concentrations: (12 x 10(4), 10 x 10(4), 8 x 10(4), 6 x 10(4) and 4 x 10(4) cells/ml) and subjected to smearing and methyl violet and HE staining. The staining results were observed by light microscopy., Results: The ghost cells were readily observed at a cell density of > 8 x 10(4) cells/ml with methyl violet staining, but only a few cells were occasionally seen at lower cell densities. In contrast, ghost cells were seen at all cell densities with HE staining., Conclusion: Methyl violet staining is more rapid and simpler for the identification of ghost cells, but its staining color more readily fades, the slides cannot be stored, and it is only effective at a cell density of > 8 x 10(4) cells/ml. In contrast, HE staining is more time-consuming but it can display cell morphology and distinguish cell components more explicitly and slides can be permanently stored. HE staining has advantages over methyl violet staining in detecting the ghost cells when the concentration is < 8 x 10(4) cells/ml.