Effect of anti-VEGF on retinal blood flow in diabetic mice using laser speckle flowgraphy.

PURPOSE: To assess intra- (repeatability) and inter-observer (reproducibility) variability of laser speckle flowgraphy (LSFG) for retinal blood flow (RBF) measurement in 20 eyes of wild type (C57BL/6J) mice and effect of intravitreal Aflibercept on RBF in optic nerve head (ONH) region of 10 eyes of Ins2 (Akita) diabetic mice. METHODS: 'Mean blur rate (MBR)' was measured for all quadrants of tissue area (MT), vessel (MV) and total area (MA) of ONH region. Changes in MT were analysed at each timepoint. Repeatability was evaluated by measuring MBR variability without changing mouse head position, and reproducibility after resetting mouse head position by another operator. Coefficient of repeatability (CR) through Bland-Altman plot method coefficient of variation (COV) and Intraclass correlation coefficient (ICC) was calculated. Intravitreal Aflibercept (1 µg) was administered to Akita eyes and intraocular pressure (IOP) was measured using a tonometer at baseline, day 7, 14, 21 and 28 post-injection. Hurvich and Tsai's criterion was used. RESULTS: Coefficient of repeatability values of repeatability and reproducibility for all quadrants were within limits of agreement. Reliability was excellent (ICC 0.98-0.99) and reproducibility was moderate to excellent (ICC 0.64-0.96). There was a non-significant IOP increase in all Akita eyes at Day 28 (p > 0.05), and significant increase in MT in all quadrants at Day 21 and superior, inferior and temporal quadrants at Day 28 (p < 0.05). CONCLUSION: Laser speckle flowgraphy demonstrates excellent repeatability and moderate to excellent reproducibility in measuring RBF. Intravitreal Aflibercept injection results in a significant increase in MT up to 28 days post-injection without significant increase in IOP.

Fluorescein Labeled Leukocytes for in vivo Imaging of Retinal Vascular Inflammation and Infiltrating Leukocytes in Laser-Induced Choroidal Neovascularization Model.

Purpose: To study the effect of anti-VEGF treatment on retinal inflammation in a laser-induced CNV rodent model.Methods: Leukocytes labeled with 1% sodium fluorescein were injected into the laser-induced CNV (wild type C57BL/6) mice at days 4 (baseline), 7, 14, and 19. At baseline intravitreally 3 mice received 1× PBS, and 3 mice received anti-VEGF. FFA, OCT, and SLO were performed at each time point to assess the CNV pathophysiology and inflammatory response.Results: Fluorescein leakage, SRF, and leukocyte infiltration were observed at baseline in both the groups before injection. From days 7 to 19, leukocyte infiltration and SRF were noted in the 1× PBS group, but limited or no SRF and leukocyte infiltration was observed in the anti-VEGF group.Conclusions: Leukocyte infiltration was established as an in vivo imaging inflammatory marker and along with FFA and OCT showed response to anti-VEGF therapy in laser-induced CNV model.

Cytokine Profiling in Patients With Exudative Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy.

Purpose: The purpose of this study was to investigate the cytokine profiles in plasma and aqueous humor of patients with choroidal neovascularization (CNV) due to exudative AMD and polypoidal choroidal vasculopathy (PCV). Methods: In this cross-sectional study, 16 patients clinically diagnosed with AMD, 18 patients with PCV, and 50 age- and sex-matched cataract patients without AMD/PCV (controls) were enrolled. Study subjects were treatment naïve, and 200 µL undiluted aqueous humor and 5 mL peripheral venous blood were collected from the study subjects. Clinical samples were analyzed for 41 different cytokines by Luminex bead-based multiplex assay. Cytokines concentrations with detection rates of 50% or more were included for the analysis, and the differences in plasma and aqueous humor cytokines levels between each group were analyzed. Results: The age of the patients with AMD and PCV was 70.62 ± 10.15 (mean ± SD) and 71.48 ± 9.08 years, respectively, and that in the control group was 62.8 ± 10.67 years. Aqueous humor cytokines growth-regulated oncogene (GRO), macrophage-derived chemokine (MDC), and macrophage inflammatory protein (MIP)-1α were significantly higher in AMD patients than controls (all P < 0.04), and GRO, MDC, MIP-1α, IL-8, IFN-γ-inducible protein 10, and monocyte chemotactic protein levels were significantly higher in PCV patients than controls (all P < 0.03). Soluble CD40 ligand and platelet-derived growth factor-AA levels were higher in plasma of healthy controls compared with AMD subjects. No significant differences in cytokine levels were observed between AMD and PCV patients for both plasma and aqueous humor. Conclusions: In AMD and PCV patients, our data suggest that the pathologic changes are primarily driven by dysregulation of local immune factors in the eye, whereas the plasma cytokine levels are not elevated.

Non-Occlusive Retinal Vascular Inflammation and Role of Red Blood Cell Deformability in Birdshot Chorioretinopathy.

Purpose: To investigate differences in red blood cell (RBC) deformability between birdshot chorioretinopathy (BCR) subjects and matched controls, and to postulate its relationship with lack of vascular occlusion in BCR. Methods: In a single center, prospective, non-randomized mechanistic study, blood samples were collected from eight healthy controls and nine BCR patients, and subjected to biochemical and hematological tests, as well as RBC indices assessment using dual-beam optical tweezers. Results: The mean age of the controls was 52.37 ± 10.70 years and BCR patients was 53.44 ± 12.39 years. Initial cell size (Io) for the controls was 8.48 ± 0.25 µm and 8.87 ± 0.31 µm for BCR RBCs (p = 0.014). The deformability index (DI) for the controls was 0.066 ± 0.02 and that for BCR RBCs was 0.063 ± 0.03 (p = 0.441). Conclusion: There was no statistically significant difference in DI between RBCs from BCR and healthy controls. This may explain the rare occurrence of retinal vascular occlusion despite the underlying vasculitic pathophysiology of BCR.

Dataset of plasma and aqueous humor cytokine profiles in patients with exudative age related macular degeneration and polypoidal choroidal vasculopathy.

In this report the data was obtained from a prospective case-control study with a sample size of sixteen patients with exudative age related macular degeneration (AMD) due to choroidal neovascularization (CNV) and eighteen patients with polypoidal choroidal vasculopathy (PCV) and fifty controls (cataract patients without any other ocular diseases). Luminex bead based multiplex assay with a panel of 41 analytes was used to study the cytokine levels in plasma and aqueous humor.

Surface characteristics and antimicrobial properties of modified catheter surfaces by polypyrogallol and metal ions.

Catheter associated infections (CAIs) are the major cause of nosocomial infections leading to increased morbidity, mortality rates and economical loss. Though the antibiotic coated surface modified catheters are reported to be effective in preventing CAIs, presence of sub-lethal concentrations of antibiotics in long term instilled catheters poses a risk of development and spread of drug resistant microbial strains. Herein, we have developed an antibiotic-free alternative strategy to coat catheter surfaces using pyrogallol (PG) and metal ions (Ag+/Mg2+). Surface characteristics, antimicrobial and anti-biofilm properties with hemocompatibility of the coated catheters were studied. Structural characteristics of coated catheters were similar to the uncoated catheters with improved wettability. All the coated catheters with PG and different PG/metal ion combinations exhibited broad spectrum antibacterial activity. Catheters coated with PG/metal ions combination showed effective antibiofilm properties against MRSA strains. None of the coated catheters showed any significant hemolysis for rabbit erythrocytes. In addition, polypyrogallol (pPG) coating attenuated the hemolytic properties of silver without altering the antimicrobial properties. The inherent antimicrobial properties of the coating agent along with antimicrobial metal ions broaden the application landscape which includes coating of other medical devices, clean room construction and development of antimicrobial surfaces. The chemical formulation can also be used to design antiseptic solutions to prevent healthcare associated infections.

Assessment of flow dynamics in retinal and choroidal microcirculation.

Alterations in ocular blood flow have been implicated in mechanisms that lead to vision loss in patients with various ocular disorders such as diabetic retinopathy, glaucoma, and age-related macular degeneration. Assessment of retinal and choroidal blood flow is also a window to evaluate systemic diseases that affect microvasculature. Quantification and qualification of the blood flow in the retina and choroid help us understand pathophysiology, stratify disease risk, and monitor disease progression in these disorders. Multiple methods are used by researchers for assessment of blood flow, but a gold standard is lacking. We review commonly used methods, both invasive and noninvasive, for evaluation of blood flow, including intravital microscopy, laser Doppler velocimetry, laser Doppler flowmetry, laser interferometry, confocal scanning laser Doppler flowmetry, laser speckle flowgraphy, Doppler optical coherence tomography, blue-field entoptic simulation, retinal vessel caliber assessment, optical coherence tomography angiography, retinal function imaging, color Doppler imaging, and scanning laser ophthalmoscope angiogram. As technology evolves, better evaluation of blood flow in various ocular and systemic diseases will likely bring new perspectives into clinical practice and translate to better diagnosis and treatment.

Incidence of Endophthalmitis after Intravitreal Injections: Risk Factors, Microbiology Profile, and Clinical Outcomes.

PURPOSE: To report the incidence and characteristics of endophthalmitis after intravitreal injections (IVI) of antivascular endothelial growth factor agents and triamcinolone acetonide. METHODS: Patients' medical records were retrospectively reviewed from January 2009 to June 2016, and the incidence, risk factors, clinical and microbiological characteristics of post-IVI endophthalmitis were evaluated. RESULTS: The total number of intravitreal injections given, which included ranibizumab, bevacizumab, and triamcinolone acetonide, was 20,566, of which 27 cases developed endophthalmitis, giving an overall incidence of 0.131%. Significant reduction (p < 0.003) in incidence of endophthalmitis was observed in patients who received prefilled compounded bevacizumab injections (0.050%) compared to multiple bevacizumab injections from a single vial (0.235%). In the triamcinolone acetonide group, the incidence was 0.26%. Staphylococcus species were isolated from 18 cases (67%), and all strains were sensitive to vancomycin. CONCLUSIONS: Adherence to strict aseptic protocols and use of prefilled compounded bevacizumab injections reduces the rate of post-IVI endophthalmitis.

Zika Virus and the Eye.

Zika virus (ZIKV), a mosquito-borne flavivirus, is the latest global health concern. Transmission is mainly via Aedes mosquitoes and the infection can be diagnosed on molecular or serologic testings. It typically causes a mild self-remitting illness of low-grade fever, maculopapular rash, and myalgia, but when severe, it is associated with neurological deficits and congenital structural defects. Ocular manifestations are usually mild like nonpurulent conjunctivitis in adults, though it may be linked to uveitis, maculopathy, and hypertensive iridocyclitis. Ocular signs seem to be more significant in congenital ZIKV-macular pigment mottling, neuroretinal atrophy with macular involvement, iris coloboma, and changes in retinal vasculature are noted in infants with infected mothers. Risk factors include ZIKV infection in first trimester and smaller cephalic diameter at birth. Hence, ophthalmic examination in newborns is now recommended. Currently, prevention and active surveillance are integral as there is no known vaccine, and treatment is only symptomatic.

Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles.

Tear film is a complex mixture of lipids, proteins and minerals which covers the external surface of the eye, thereby providing lubrication, nutrition and protection to the underlying cells. Analysis of tears is an emerging area for the identification of biomarkers for the prediction, diagnosis, and prognosis of various ocular diseases. Tears are easily accessible and their collection is non-invasive. Therefore, advancing technologies are gaining prominence for identification of multiple analytes in tears to study changes in protein or metabolite composition and its association with pathological conditions. Tear cytokines are ideal biomarkers for studying the health of the ocular surface and also help in understanding the mechanisms of different ocular surface disorders like dry eye disease and vernal conjunctivitis. Bead based multiplex assays have the capability of detecting multiple analytes in a small amount of sample with a higher sensitivity. Here we describe a standardized protocol of tear sample collection, extraction and analysis of cytokine profiling using a bead based multiplex assay.

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Dataset of longitudinal analysis of tear cytokine levels, CD4, CD8 counts and HIV viral load in dry eye patients with HIV infection.

The data presented in this article shows the longitudinal analysis of tear fluid cytokine profiles, blood CD4 and CD8 counts and HIV viral load in 34 dry eye patients with HIV infection during the HAART therapy. Clinical samples were collected from HIV patients with dry eye disease at the time of presentation to the clinic (visit 1), three months (visit 2) and 6 months (visit 3) after the presentation. At each time point tear samples were evaluated for 41 cytokines using Luminex bead based multiplex assay and blood samples were tested for HIV viral load and CD4 and CD8 counts.

Dataset of tear film cytokine levels in dry eye disease (DED) patients with and without HIV infection.

The tear film cytokine profiling data in this article was obtained from a prospective case-control study with a sample size of 34 dry eye disease (DED) patients with HIV infection and 32 DED patients without HIV infection, see "A distinct cytokines profile in tear film of dry eye disease (DED) patients with HIV infection" (R. Agrawal, P.K. Balne, A. Veerappan, V.B. Au, B. Lee, E. Loo, A. Ghosh, L. Tong, S.C. Teoh, J. Connolly, P. Tan, 2016) [1]. Tear samples were collected from all the subjects using Schirmer׳s strips and cytokine profiling was done using the Luminex bead based multiplex assay with a panel of 41 analytes. The cytokine level differences in each group of subjects were analyzed using logistic regression models.

A distinct cytokines profile in tear film of dry eye disease (DED) patients with HIV infection.

PURPOSE: To investigate the tear cytokine profile in HIV patients with dry eye disease (DED) and study the association between the severity of ocular inflammatory complications and tear cytokines levels. We postulate that HIV-mediated inflammation may be the underlying pathogenic mechanism for HIV-associated DED. METHODS: The current prospective case-control study compared tear film cytokine profiles in DED patients with HIV infection (n=34) and age/gender-matched DED patients without HIV infection [controls (n=32)]. Participants were recruited from tertiary referral eye care centre and communicable disease clinics, Singapore. Ocular surface health was documented using tear film, Schirmer's test, corneal staining, and conjunctival injection measurements. Tear samples were collected using Schirmer's strips and analysed for the levels of 41 cytokines using Luminex bead assay. Logistic regression models were performed to determine correlation and significance. RESULTS: Among the 41 cytokines analysed, statistically significant differences were observed in the mean values of epithelial growth factor (EGF), growth related oncogene (GRO) and interferon gamma-induced protein 10 (IP-10). EGF and IP-10 levels were higher and GRO levels were lower in the tears of DED patients with HIV infection compared to DED patients without HIV infection. No significant association was found between varying levels of ocular surface parameters and cytokine concentrations in HIV patients with DED (p>0.05). CONCLUSIONS: EGF and IP-10 were significantly elevated and GRO levels were lower in the tear profile of HIV patients with DED compared to immunocompetent patients with DED. This study suggests a novel cytokine driven paradigm for ocular inflammatory complications of HIV infection. Additional studies in large organised cohorts can validate the results.

Dataset of aqueous humor cytokine profile in HIV patients with Cytomegalovirus (CMV) retinitis.

The data shows the aqueous humor cytokine profiling results acquired in a small cohort of 17 HIV patients clinically diagnosed with Cytomegalovirus retinitis using the FlexMAP 3D (Luminex®) platform using the Milliplex Human Cytokine® kit. Aqueous humor samples were collected from these patients at different time points (pre-treatment and at 4-weekly intervals through the 12-week course of intravitreal ganciclovir treatment) and 41 cytokine levels were analyzed at each time point. CMV DNA viral load was assessed in 8 patients at different time points throughout the course of ganciclovir treatment. The data described herein is related to the research article entitled "Aqueous humor immune factors and cytomegalovirus (CMV) levels in CMV retinitis through treatment - The CRIGSS study" (Iyer et al., 2016) [1]. Cytokine levels against the different time points which indicate the response to the given treatment and against the CMV viral load were analyzed.

Aqueous humor immune factors and cytomegalovirus (CMV) levels in CMV retinitis through treatment - The CRIGSS study.

PURPOSE: This study aims to perform comprehensive longitudinal immune factor analysis of aqueous humor in relation to the aqueous CMV viral load and systemic CD4 counts during treatment of patients with co-infection of HIV and CMVR. METHODS: Aqueous humor samples were collected from 17 HIV-positive patients with CMVR scheduled to undergo weekly intravitreal ganciclovir therapy as part of the prospective CMV Retinitis Intravitreal Ganciclovir Singapore Study (CRIGSS) over the course of 1year. Full data across all the 4 time points was obtained and analyzed for CMV DNA viral load, 41 cytokine and chemokine factors using real-time PCR with the FlexMAP 3D (Luminex®) platform and assessed using the Milliplex Human Cytokine® kit. RESULTS: The following immune factors (Spearman correlation coefficient r value in parenthesis, p<0.05) showed strong correlation with CMV DNA load in the aqueous - MCP-1 (0.80, IFN-g (0.83), IP-10 (0.82), IL-8 (0.81), fractalkine (0.73), RANTES (0.68) - while the following showed moderate correlation - PDGF-AA (0.58), Flt-3L (0.59) and G-CSF (0.53). Only PDGF-AA revealed a statistically significant negative correlation with serum CD4 levels (r=-0.74). CONCLUSION: Immune factors that correlate with intraocular CMV DNA load are identified. They are indicative of a Th1 and monocyte-macrophage mediated response, and exhibit a decreasing trend longitudinally through the course of treatment. These factors may be an important new consideration in individualizing the treatment of patients with CMVR.

Polymerase chain reaction evaluation of infectious multifocal serpiginoid choroiditis.

PURPOSE: To study infectious agents associated with multifocal serpiginoid choroiditis (MSC) based on polymerase chain reaction (PCR) evaluation and specific anti-microbial therapy. METHODS: Retrospective review of medical records. RESULTS: Thirteen patients with MSC were evaluated with PCR for the following organisms: Mycobacterium tuberculosis (MTB), herpes simplex virus 1 (HSV 1), varicella zoster virus (VZV), and cytomegalovirus (CMV). Nine (69.2%) were PCR positive for one or more organisms. Seven (53.8%) were positive for MTB, 3 (23.1%) for CMV (1 positive for both MTB and CMV), and 1 (7.6%) for both HSV 1 and MTB. All 13 patients received anti-TB therapy and corticosteroids. Nine patients completed 6 months follow-up; 6 resolved completely, 2 continued to have active lesions, while 1 CMV PCR-positive patient required additional valgancyclovir therapy. CONCLUSIONS: TB is the most important etiology for MSC in endemic countries. The role of herpes viruses in MSC remains unclear and needs further investigation.