Elevation of Tear MMP-9 Concentration as a Biomarker of Inflammation in Ocular Pathology by Antibody Microarray Immunodetection Assays.

Matrix metalloproteinases are a family of enzymes fundamental in inflammatory processes. Between them, MMP-9 is up-regulated during inflammation; thus, its quantification in non-invasive fluids is a promising approach for inflammation identification. To this goal, a biomarker quantification test was developed for ocular inflammation detection using anti-MMP-9 antibody microarrays (AbMAs). After validation with eight healthy control tear samples characterized by ELISA, 20 samples were tested from individuals diagnosed with ocular inflammation due to: cataracts, glaucoma, meibomian gland dysfunction, allergy, or dry eye. Concentration values of tear MMP-9 were obtained for each sample, and 12 patients surpassed the pathological threshold (30 ng/mL). A significant elevation of MMP-9 concentration in the tears of glaucoma patients compared with healthy controls was observed. In order to evaluate the diagnostic ability, an ROC curve analysis was performed using our data, determining the optimal threshold for the test at 33.6 ng/mL of tear MMP-9. In addition, a confusion matrix was applied, estimating sensitivity at 60%, specificity at 88%, and accuracy at 68%. In conclusion, we demonstrated that the AbMAs system allows the quantification of MMP-9 in pathologies that involve inflammation of the ocular surface.

Validation of the SITA faster strategy for the management of glaucoma.

PURPOSE: To evaluate the SITA Faster strategy (SFR) in normal subjects and glaucoma patients. METHODS: This randomized, controlled, cross-sectional study included 236 Visual fields (VFs) obtained from 59 subjects, grouped as controls, mild, moderate and severe glaucoma patients. All examinations were performed using the HFA-3 perimeter with the 24/2 program, adopting two strategies: the SITA Standard (SS) and SFR. Glaucoma severity was studied using continuous (mean defect -MD- and visual field index -VFI) and categorical (scoring systems from the AGIS and CIGTS studies) perimetric indices. RESULTS: There were no differences in the reliability indices obtained with both strategies when controls were compared to glaucoma patients. SFR significantly reduced the VF duration, saving 68% and 59.1% in glaucoma patients (60.8% in mild, 62.9% in moderate and 48.7% in severe glaucoma: p = 0.0004). The test duration was correlated with glaucoma severity, especially for the SFR (Spearman rho = - 0.88, - 0.82, 0.87 and 0.85 for the VFI, MD, AGIS and CIGTS, respectively). Although both strategies were correlated, the SS showed a greater severity of the defect in glaucoma patients (SS vs SFR VFI = 94.5 vs. 95, MD = - 3.17 vs. - 2.81, AGIS = 6 vs. 5, CIGTS = 4.85 vs. 3.41: p = 0.001). There was strong agreement between strategies, although this was lower in advanced glaucoma. CONCLUSIONS: The SFR strategy constitutes an adequate strategy to monitor glaucoma, especially in mild and moderate cases. Its speed allows more VFs to be assessed, as recommended in clinical practice guidelines.

Changes in tear biomarker levels in keratoconus after corneal collagen crosslinking.

Purpose: The purpose of this work was to analyze the expressions of matrix metalloproteinase 9 (MMP-9), calcyclin (S100A6), and cystatin S (CST4) in the tears of keratoconus (KC) patients. The correlations between the expressions of these proteins and the values of various ocular surface parameters were examined after accelerated corneal crosslinking (A-CXL) with pulsed ultraviolet light. Methods: This prospective, observational study enrolled patients with different grades of KC, scheduled to undergo the A-CXL procedure, as well as healthy subjects. Tear samples were analyzed by employing customized antibody microarray assays for MMP-9, S100A6, and CST4 proteins. The keratometry readings at the maximum keratometry (Kmax) and the simulated keratometry (SimK) values were obtained for examining the postoperative evolution of corneal topography. The state of the ocular surface was evaluated using the results of the Ocular Surface Disease Index (OSDI) questionnaire, tear osmolarity (OSM) test, Schirmer test (SCH), Tear Break Up Time (TBUT), tear clearance (CLR), and fluorescein (FLUO) and lissamine green (LG) corneal staining. Results: A total of 18 patients (22 eyes) and 10 healthy subjects were studied. The concentrations of MMP-9 and S100A6 decreased in tears, from 104.5 ± 78.98 ng/ml and 350.20 ± 478.08 ng/ml before the surgery to 48.7 ± 24.20 ng/ml and 55.70 ± 103.62 ng/ml, respectively, after 12 months of follow up. There were no changes in the CST4 concentration after 12 months of follow up (2202.75 ± 2863.70 versus 2139.68 ±2719.89 ng/ml). When the patients were divided into three groups according to the evolutive stage of KC, the trends for the three biomarkers in each group were the same as in the general group. Basal concentrations of MMP-9 and S100A6 from healthy subjects and KC patients were compared. The levels of MMP-9 and S100A6 in tears were (9.8 ± 5.11 and 104.55 ± 78.98 ng/ml, p<0.01; and 11.35 ± 3.18 and 350.26 ± 478.06 ng/ml, respectively, p<0.01). This was not the case for CST4, which did not exhibit statistically significant differences between the two groups (2261.94 ± 510.65 and 2176.73 ± 2916.27 ng/ml respectively, p=0.07). Conclusions: A-CXL promoted a decrease in the concentrations of MMP-9 and S100A6 in the tear film. This effect may be related to the restoration of corneal homeostasis and the consequent repair of the tissue damage caused by KC. Moreover, the A-CXL treatment did not produce lasting alterations in the ocular surface, and the values of the evaluated clinical parameters did not change significantly.

Subconjunctival PRGF Fibrin Membrane as an Adjuvant to Nonpenetrating Deep Sclerectomy: A 2-Year Pilot Study.

PURPOSE: To evaluate the potential role of the autologous PRGF (plasma rich in growth factors) fibrin membrane in tissue regeneration after glaucoma filtering surgery. MATERIALS AND METHODS: Ten patients with medically uncontrolled primary open-angle glaucoma underwent nonpenetrating deep sclerectomy and were treated with PRGF fibrin membrane as adjuvant. Intraocular pressure reduction was the primary outcome. This variable was measured preoperatively and also at each follow-up visit. Secondary outcomes included the number of antiglaucoma medications, anterior segment optical coherence tomography bleb examination, photographic bleb evaluation, and subjective clinical symptomatology evaluation. RESULTS: The surgical technique showed a significant reduction (p < 0.05) in intraocular pressure in relation to preoperative values at each time of the study, decreasing from 23.3 ± 6.4 to 15.2 ± 4.6 mm Hg at 2 years. Furthermore, the number of antiglaucoma medications consumed showed a significant reduction at the end point of the study compared with the preoperative situation. Optical coherence tomography and photographic filtering bleb variables experienced a progressive reduction during the follow-up. Subjective symptoms showed a reduction from 8.3 ± 4.5 to 4.2 ± 5.3 at 2 years. CONCLUSIONS: PRGF-Endoret treatment could promote ocular surface regeneration after glaucoma surgery, enhancing the surgery success rates and reducing the need for postoperative medications. It is important to highlight that this is a preliminary study and some large clinical studies are necessary to verify these results.

Label-free LC-MS/MS quantitative analysis of aqueous humor from keratoconic and normal eyes.

PURPOSE: The etiology of keratoconus (KC) and the factors governing its progression are not well understood. It has been suggested that this disease might be caused by biochemical alterations in the cornea; changes in the expression profiles of human aqueous humor (hAH) proteins have been observed in some diseases. To gain a new insight into the molecular mechanisms of KC pathology, we examined the hAH proteomes of those in the advanced stages of this disease. We used a high-throughput mass spectrometry approach to compare hAH protein expression in patients with KC and in control subjects. METHODS: Aqueous humor samples were acquired from five keratoconus patients during keratoplasty surgery and from five myopic control subjects during phakic intraocular lens implantation. Quantitative mass spectrometry analysis using spectral counting was performed to determine the relative amounts of hAH proteins in the samples from KC patients and control individuals. RESULTS: All KC patients included in the study presented severe keratoconus (K2 >52 D), and slit-lamp examination revealed microfolds in Descemet's membrane, without clinical signs of hydrops. We found significant differences between the expression levels of 16 proteins in the two groups. In KC samples, seven proteins were overexpressed and nine were underexpressed in comparison with the control group. Gene ontology analysis revealed that these deregulated proteins are implicated in several biologic processes, such as the regulation of proteolysis, responses to hypoxia, and responses to hydrogen peroxide, among others. CONCLUSIONS: The protein expression profiles in hAH from KC patients and myopic control subjects differ significantly. This result suggests that some components of the hAH proteome are involved in this disease. Further in-depth analysis of the hAH proteome should provide a better understanding of the mechanisms governing the pathophysiology of KC.

Comparison of Cardiovascular Risk and Events among Spanish Patients with and without Ocular Pseudoexfoliation.

The purpose of this study was to calculate and compare individual cardiovascular risk (CVR) and the development of cardiovascular events and mortality in patients with and without ocular pseudoexfoliation (PEX). A cohort study was carried out to compare two groups of patients who underwent cataract surgery: patients with (n = 99) and without PEX (n = 239). The CVR factors were recorded for all the subjects, and CVR was calculated for each individual using ERICE risk assessment charts. After a six-year follow-up, the cardiovascular events and mortality rates were compared between the two groups. The mean CVR was 36.41% in the PEX group and 33.72% in the non-PEX group (p = 0.13). High blood pressure was detected in significantly more PEX patients (71.4%) than non-PEX patients (58.6%, p = 0.035), yet no differences were found in the other CVR factors. The prevalence of cardiovascular events in the PEX and non-PEX patients was 17.1% and 12.5%, respectively (p = 0.26), with 5% of patients in the PEX group and 5% in the non-PEX group suffering an acute coronary event (p = 0.9). Moreover, 12% of the PEX patients and 7.5% of the non-PEX patients experienced a stroke (p = 0.17), and the six-year mortality rate was 29.3% in the PEX group and 25.9% in the non-PEX group (p = 0.52). PEX was associated with high blood pressure in our cohort of patients, although cardiovascular risk was not shown to be higher in this group. In addition, patients with PEX did not experience more cardiovascular events or have a higher mortality rate than patients without PEX during the period studied.

Treatment of Open-Angle Glaucoma and Ocular Hypertension with Preservative-Free Tafluprost/Timolol Fixed-Dose Combination Therapy: Results from the VISIONARY Study Population in Spain.

Purpose: Data are presented from ophthalmology clinics in Spain participating in the VISIONARY study, examining the effectiveness, tolerability, and safety of the preservative-free tafluprost (0.0015%) and timolol (0.5%) fixed-dose combination (PF tafluprost/timolol FC) in the treatment of OAG and OHT. Methods: An observational, multicenter prospective study examined treatment outcomes following a switch to PF tafluprost/timolol FC in adult OAG/OHT patients demonstrating insufficient response to beta-blocker or prostaglandin analog (PGA) monotherapy. Primary end point was mean change in intraocular pressure (IOP) from baseline at month 6. Changes in the severity of ocular signs and symptoms were also assessed. Results: Overall, 92 patients (51.1% female) were included. Mean (standard deviation) age was 68.3 (12.1) years. Mean IOP was reduced from 21.9 mmHg at baseline to 16.7 mmHg at month 6 (22.3% decrease; P < 0.0001). Significant IOP reductions were observed at weeks 4 and 12 (P < 0.0001). Baseline PGA and beta-blocker users demonstrated mean month 6 IOP reductions of 5.5 mmHg (23.5%; P < 0.001) and 3.5 mmHg (14.6%; P = 0.029), respectively. Severity of conjunctival hyperemia, dry eye, irritation, itching, foreign body sensation, and eye pain was significantly reduced. Three treatment-related adverse events were reported, all were nonserious and mild/moderate in severity. Conclusion: In real-world clinical practice, PF tafluprost/timolol FC treatment provided significant IOP reductions over 6 months and was well tolerated among OAG/OHT patients showing poor response to PGA or beta-blocker monotherapy. IOP-lowering efficacy and improvements in ocular signs and symptoms were evident from week 4 and maintained over the 6-month study period. Trial Registration: European Union electronic Register of Post-Authorisation Studies (EU PAS) register number EUPAS22204.

Supraciliary versus intrascleral implantation with hema implant (Esnoper V-2000) in deep sclerectomy: a multicenter randomized controlled trial.

PURPOSE: To compare the supraciliary versus intrascleral implantation of the hema implant (Esnoper V-2000) in terms of the efficacy and safety in nonpenetrating deep sclerectomy (NPDS). PATIENTS AND METHODS: Prospective, randomized, unmasked, competitive and multicenter clinical trial. Eighty-three eyes from 83 patients suffering from open-angle glaucoma (40 males, 43 females) were enrolled and followed up for 12 months. Main outcome measures were best-corrected visual acuity (BCVA), intraocular pressure (IOP), perimetry (mean defect, MD and Visual Field Index, VFI), pachymetry, number of antiglaucoma medications and analysis of blebs according Moorfields Bleb Grading. RESULTS: The IOP was significantly reduced in both groups from 23.74 ± 6.9 mmHg (implant sutured to the sclera, group 1) and 23.46 ± 6.47 mmHg (implant placed in the suprachoroidal space, group 2) to 15.43 ± 4.27 mmHg (p < 0.001) and 14.62 ± 3.64 mmHg (p < 0.001), respectively. There were no statistically significant differences in mean IOP values between the groups a year after the surgery (p = 0.581). BCVA did not show statistical differences in comparison with baseline (p = 0.09, group 1; p = 0.42, group 2). The mean number of antiglaucoma medications was reduced in both groups from 2.58 ± 0.04 and 2.68 ± 0.02 before the surgery to 0.32 ± 0.76 and 0.24 ± 0.66 after surgery. CONCLUSION: Nonpenetrating deep sclerectomy using hema implant (Esnoper V-2000) is safe and effective regardless of the positioning of the implant. We achieved IOP decrease and reduction in antiglaucoma medications during the first year after surgery without significant differences between both techniques.

Custom RT-qPCR-array for glaucoma filtering surgery prognosis.

Excessive subconjunctival scarring is the main reason of failure of glaucoma filtration surgery. We analyzed conjunctival and systemic gene expression patterns after non penetrating deep sclerectomy (NPDS). To find expression patterns related to surgical failure and their correlation with the clinical outcomes. This study consisted of two consecutive stages. The first was a prospective analysis of wound-healing gene expression profile of six patients after NPDS. Conjunctival samples and peripheral blood samples were collected before and 15, 90,180, and 360 days after surgery. In the second stage, we conducted a retrospective analysis correlating the late conjunctival gene expression and the outcome of the NPDS for 11 patients. We developed a RT-qPCR Array for 88 key genes associated to wound healing. RT-qPCR Array analysis of conjunctiva samples showed statistically significant differences in 29/88 genes in the early stages after surgery, 20/88 genes between 90 and 180 days after surgery, and only 2/88 genes one year after surgery. In the blood samples, the most important changes occurred in 12/88 genes in the first 15 days after surgery. Correspondence analyses (COA) revealed significant differences between the expression of 20/88 genes in patients with surgical success and failure one year after surgery. Different expression patterns of mediators of the bleb wound healing were identified. Examination of such patterns might be used in surgery prognosis. RT-qPCR Array provides a powerful tool for investigation of differential gene expression wound healing after glaucoma surgery.

Ocular Surface Disease Parameters After Collagen Cross-Linking for Keratoconus.

PURPOSE: To analyze the variations in tear quantity and quality after corneal collagen cross-linking (CXL) in patients with keratoconus. METHODS: This is a prospective observational study of 24 eyes with keratoconus that underwent epithelium-off CXL. The variables studied were tear film osmolarity, subjective symptoms (Ocular Surface Disease Index), tear breakup time, Schirmer test (SCH) score, tear clearance, fluorescein staining, and lissamine green staining. Variables were determined preoperatively and 3, 6, and 12 months after CXL. RESULTS: All the studied variables remained stable after 1-year follow-up (no significant difference in comparison with baseline values; Mann-Whitney U test). There was a positive paired correlation between the SCH score and tear clearance at 3; 0.532 (P < 0.01), 6; 0.434 (P = 0.04), and 12 months; 0.675 (P < 0.01). There was no correlation between the SCH score and tear breakup time, apart from a positive correlation at 12 months; 0.601 (P = 0.05). A negative correlation was found between osmolarity and the SCH score at 3; -0.589 (P < 0.01) and 12 months; -0.049 (P = 0.04). The Ocular Surface Disease Index did not correlate with any of the studied variables (Spearman test). CONCLUSIONS: CXL, at least during the first postoperative year, does not modify the parameters currently used to evaluate tear film function. There is no correlation between objective and subjective parameters in the analysis of the tear film function.

Changes in tear protein profile in patients with conjunctivochalasis.

PURPOSE: To compare the protein profiles of tears from normal volunteers and patients with conjunctivochalasis (CCH), with a view to identifying proteins whose expression is altered in this pathology. METHODS: Tears from 8 normal subjects and 6 patients with CCH were analyzed by 2-dimensional electrophoresis. Total protein from tears was separated in the first dimension by isoelectric focusing, and the second dimension was carried out using 8%-16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel images were analyzed using Progenesis SameSpot software. Those spots of interest were manually cut out from the gels, and the corresponding proteins were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF). Expression levels of proteins that had been found to be significantly altered were further verified by Western blot. RESULTS: Approximately 250 spot proteins were detected in the whole tear proteome. Twenty-four spots were significantly upregulated in CCH compared with that in controls. Eleven protein spots were identified, which included proteins belonging to the S100 family (A8, A9, A4; 2.44, 1.71, and 2.82 fold upregulation, respectively), guanosine triphosphate-binding protein 2 (1.95 fold), l-lactate dehydrogenase A-like 6B (2.32 fold), fatty acid-binding protein (2.01 fold), keratin type I cytoskeletal 10 (1.81 fold), glutathione S-transferase P (2.27 fold), peroxiredoxin-1, peroxiredoxin-5 (1.79- and 1.92 fold, respectively), and cullin-4B+ glyceraldehyde 3-phosphate dehydrogenase (1.96 fold). CONCLUSIONS: We have identified a group of proteins, which is upregulated in CCH tears. Although some of them, such as S100A4, S100A8, and peroxiredoxin-5, are markers of inflammation and oxidative processes, monitoring their levels in CCH might be useful for assessing the severity and progression of the disease.

Needle-guided intrascleral fixation of posterior chamber intraocular lens for aphakia correction.

We present a technique for the surgical correction of aphakia that allows intrascleral fixation of a posterior chamber intraocular lens (IOL) without sutures. The technique is useful in situations in which one haptic has to be fixated and capsule support is adequate for fixation of the second haptic. The haptic is externalized with a 25-gauge needle; no surgical instrumentation other than that needed for conventional cataract surgery is used. The technique is particularly appropriate for 3-piece IOLs with flexible haptics.