Joelle Hallak

Novel aspects of corneal angiogenic and lymphangiogenic privilege

In this article, we provide the results of experimental studies demonstrating that corneal avascularity is an active process involving the production of anti-angiogenic factors, which counterbalance the pro-angiogenic/lymphangiogenic factors that are upregulated during wound healing. We also summarize pertinent published reports regarding corneal neovascularization (NV), corneal lymphangiogenesis and corneal angiogenic/lymphangiogenic privilege. We outline the clinical causes of corneal NV, and discuss the angiogenic proteins (VEGF and bFGF) and angiogenesis regulatory proteins. We also describe the role of matrix metalloproteinases MMP-2, -7, and MT1-MMP, anti-angiogenic factors, and lymphangiogenic regulatory proteins during corneal wound healing. Established and potential new therapies for the treatment of corneal neovascularization are also discussed.

Risk Factors Associated with Incident Cataracts and Cataract Surgery in the Age-Related Eye Disease Study (AREDS): AREDS Report Number 32

OBJECTIVE To investigate potential risk factors associated with incident nuclear, cortical, and posterior subcapsular (PSC) cataracts and cataract surgery in participants in the Age-Related Eye Disease Study (AREDS). DESIGN Clinic-based prospective cohort study. PARTICIPANTS Persons (n = 4425) 55 to 80 years of age enrolled in a controlled clinical trial of antioxidant vitamins and minerals, AREDS, for age-related macular degeneration and cataract. METHODS Lens photographs were graded centrally for nuclear, cortical, and PSC opacities using the AREDS system for classifying cataracts. Type-specific incident cataracts were defined as an increase in cataract grade from none or mild at baseline to a grade of moderate at follow-up, also with a grade of at least moderate at the final visit, or cataract surgery. Cox regression analyses were used to assess baseline risk factors associated with type-specific opacities and cataract surgery. MAIN OUTCOME MEASURES Moderate cataract was defined as a grade of 4.0 or more for nuclear opacity, 10% or more involvement within the full visible lens for cortical opacity, and 5% or more involvement of the central 5-mm circle of the lens for PSC opacity. These were graded on baseline and annual lens photographs. RESULTS A clinic-based cohort of 4425 persons 55 to 80 years of age at baseline was followed up for an average of 9.8±2.4 years. The following associations were found: increasing age with increased risk of all types of cataract and cataract surgery; males with increased risk of PSC and decreased risk of cortical cataracts; nonwhite persons with increased risk of cortical cataract; hyperopia with decreased risk of PSC, nuclear cataract, and cataract surgery; Centrum (Wyeth Consumer Healthcare, Madison, NJ) use with decreased risk of nuclear cataract; diabetes with increased risk of cortical, PSC cataract, and cataract surgery; higher educational level with decreased risk of cortical cataract; and smoking with increased risk of cortical cataract and cataract surgery. Estrogen replacement therapy in female participants increased the risk of cataract surgery. CONCLUSIONS These findings largely are consistent with the results of previous studies, providing further evidence for possible modifiable risk factors for age-related cataract. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Corneal Neurotoxicity Due to Topical Benzalkonium Chloride

PURPOSE The aim of this study was to determine and characterize the effect of topical application of benzalkonium chloride (BAK) on corneal nerves in vivo and in vitro. METHODS Thy1-YFP+ neurofluorescent mouse eyes were treated topically with vehicle or BAK (0.01% or 0.1%). Wide-field stereofluorescence microscopy was performed to sequentially image the treated corneas in vivo every week for 4 weeks, and changes in stromal nerve fiber density (NFD) and aqueous tear production were determined. Whole-mount immunofluorescence staining of corneas was performed with antibodies to axonopathy marker SMI-32. Western immunoblot analyses were performed on trigeminal ganglion and corneal lysates to determine abundance of proteins associated with neurotoxicity and regeneration. Compartmental culture of trigeminal ganglion neurons was performed in Campenot devices to determine whether BAK affects neurite outgrowth. RESULTS BAK-treated corneas exhibited significantly reduced NFD and aqueous tear production, and increased inflammatory cell infiltration and fluorescein staining at 1 week (P < 0.05). These changes were most significant after 0.1% BAK treatment. The extent of inflammatory cell infiltration in the cornea showed a significant negative correlation with NFD. Sequential in vivo imaging of corneas showed two forms of BAK-induced neurotoxicity: reversible neurotoxicity characterized by axonopathy and recovery, and irreversible neurotoxicity characterized by nerve degeneration and regeneration. Increased abundance of beta III tubulin in corneal lysates confirmed regeneration. A dose-related significant reduction in neurites occurred after BAK addition to compartmental cultures of dissociated trigeminal ganglion cells. Although both BAK doses (0.0001% and 0.001%) reduced nerve fiber length, the reduction was significantly more with the higher dose (P < 0.001). CONCLUSION Topical application of BAK to the eye causes corneal neurotoxicity, inflammation, and reduced aqueous tear production.

Ocular Surface Extracellular DNA and Nuclease Activity Imbalance: A New Paradigm for Inflammation in Dry Eye Disease

PURPOSE We determined whether nucleases are deficient in the tear fluid of dry eye disease (DED) patients, and whether this causes extracellular DNA (eDNA) and neutrophil extracellular trap (NET) accumulation in the precorneal tear film, thus causing ocular surface inflammation. METHODS Exfoliated cells adhered to Schirmer test strips were collected on glass slides, and immunofluorescence confocal microscopy was used to evaluate neutrophils, eDNA, NETs, and their molecular components. Similar experiments were performed with mucoid films collected from the inferior conjunctival fornix or bulbar conjunctiva. We used quantitative PCR to evaluate eDNA signaling pathways and inflammatory cytokine expression. We also determined the amount of ocular surface eDNA and evaluated tear fluid nuclease activity. RESULTS eDNA, NETs, and neutrophils were present on the ocular surface in DED patients and abundant in mucoid films. NETs consisted of eDNA, histones, cathelicidin, and neutrophil elastase. Tear fluid nuclease activity was decreased significantly in DED patients, whereas the amount of eDNA on the ocular surface was increased significantly. Expression of genes downstream of eDNA signaling, such as TLR9, MyD88, and type I interferon, as well as the inflammatory cytokines interleukin-6 and tumor necrosis factor-α, was significantly increased in DED patients. CONCLUSIONS Extracellular DNA production and clearance mechanisms are dysregulated in DED. Nuclease deficiency in tear fluid allows eDNA and NETs to accumulate in precorneal tear film, and results in ocular surface inflammation. These findings point to novel therapeutic interventions in severe DED based on clearance of eDNA, NETs, and other molecular components from the ocular surface.

Peripapillary retinal nerve fiber layer thickness in sickle-cell hemoglobinopathies using spectral-domain optical coherence tomography

PURPOSE To determine whether patients with a sickle-cell hemoglobinopathy without glaucoma have peripapillary retinal nerve fiber layer (RNFL) thinning by spectral-domain optical coherence tomography. DESIGN Prospective study. METHODS All patients with a sickle cell hemoglobinopathy (sickle-cell disease, sickle-cell hemoglobin C disease, and sickle-cell thalassemia) and age-similar, race-matched controls underwent a comprehensive eye examination and spectral-domain optical coherence tomography of the macula and optic nerve head using the Heidelberg Spectralis (Heidelberg Engineering, Inc, Carlsbad, California, USA). Participants with prior retinal treatments (laser or surgery), diabetes mellitus, glaucoma, or other ocular diseases were excluded. The sickle-cell disease patients were grouped into those with focal macular thinning and those without. Those with macular thinning were grouped further into mild, moderate, and severe thinning groups based on temporal macular thickness. Analysis of variance testing and post hoc analysis with the Tukey test and Pearson correlation were performed to assess for peripapillary RNFL thickness differences among different groups. RESULTS One hundred fifty-one eyes of 88 sickle-cell patients and 55 eyes of 30 age-similar and race-matched (black) controls were included. Sickle-cell patient eyes with macular thinning (n = 81) had thinner mean peripapillary RNFL thicknesses in the nasal sector (P = .01) compared with non-sickle-cell control eyes and in the superotemporal sector (P = .01) compared with sickle-cell patient eyes without macular thinning (n = 70). In the severe macular thinning subgroup (n = 55), the mean peripapillary RNFL thickness was significantly thinner than that of controls (P < .05) in 6 of 7 sectors. There is a positive linear relationship between temporal macular thickness and global peripapillary RNFL thickness with a Pearson correlation coefficient of 0.60 (P < .0001). CONCLUSIONS Nonglaucomatous, black sickle-cell patients with focal macular thinning on spectral-domain optical coherence tomography have significantly thinner peripapillary RNFL than those without macular thinning or controls. The degree of thinning correlates with severity of temporal macular thinning. These patients may require different peripapillary RNFL thickness thresholds for future glaucoma evaluations.

Semaphorin 7a Links Nerve Regeneration and Inflammation in the Cornea

PURPOSE We determined Semaphorin 7a (Sema7a) localization and abundance in naive corneas and in corneas after nerve-transecting lamellar flap surgery, and determined the effect of Sema7a supplementation on corneal nerve regeneration and inflammation. METHODS Immunolocalization and Western blot analyses were performed to evaluate the abundance of Sema7a in naive corneas and corneas undergoing nerve regeneration after lamellar corneal surgery in thy1-YFP+ neurofluorescent mice. We used compartmental cultures of dissociated trigeminal ganglion cells to determine the effect of Sema7a exposure on neurite outgrowth in vitro. Finally, a Sema7a pellet was implanted under the corneal flap after lamellar transection surgery to determine the neuronal and inflammatory effects of Sema7a supplementation in vivo. RESULTS Sema7a was expressed in the corneal epithelium and stromal keratocytes, but was more abundant in the epithelium (74.3%) compared to the stroma (25.7%, P = 0.02). Sema7a expression was increased significantly in the cornea after lamellar corneal surgery and was localized to stromal cells near the regenerating nerve fronds. Exposure of trigeminal neurites to Sema7a (20 nM) in the side compartment increased neurite length significantly. The implanted Sema7a pellet increased significantly YFP+ inflammatory cell influx into the cornea as well as increased corneal nerve length. CONCLUSIONS Sema7a is expressed constitutively in the cornea, and potently stimulates nerve regeneration and inflammatory cell influx. Therefore, this immune semaphorin links nerve regeneration and inflammatory processes in the cornea.

CD11b+GR1+ Myeloid Cells Secrete NGF and Promote Trigeminal Ganglion Neurite Growth: Implications for Corneal Nerve Regeneration

PURPOSE We characterized fluorescent bone marrow cells (YFP(+) BMCs) in the thy1-YFP mouse and determine if they promote trigeminal ganglion (TG) cell neurite growth. METHODS Excimer laser annular keratectomy was performed in thy1-YFP mice, and corneas were imaged. BMCs were harvested from femur and tibia, and the expression of surface markers on YFP(+) BMCs was analyzed by flow cytometry. The immunosuppressive action of BMCs (YFP(+) and YFP(-)) was evaluated in an allogenic mixed lymphocyte reaction (MLR). Neurotrophic action of BMCs (YFP(+) and YFP(-)) was determined in compartmental and transwell cultures of dissociated TG cells. RESULTS Following annular keratectomy, YFP(+) BMCs infiltrated the cornea. YFP(+) BMCs shared surface markers (CD11b+Gr1+Ly6C+Ly6G-F4/80(low)) with monocytic myeloid-derived suppressor cells (MDSCs), had similar morphology, and suppressed T-cell proliferation in allogenic MLR in a dose-dependent manner. YFP(+) BMCs, but not YFP(-) BMCs, significantly increased growth of TG neurites in vitro. When cultured in a transwell with TG neurites, YFP(+) BMCs expressed neurotrophins and secreted nerve growth factor (NGF) in conditioned medium. YFP(+) BMCs that infiltrated the cornea maintained their phenotype and actions (neuronal and immune). CONCLUSIONS YFP(+) BMCs in thy1-YFP mice have immunophenotypic features of MDSCs. They secrete NGF and promote neuroregeneration. Their immunosuppressive and neurotrophic actions are preserved after corneal infiltration. These findings increase our understanding of the beneficial roles played by leukocyte trafficking in the cornea and may lead to therapeutic strategies that use NGF-secreting myeloid cells to repair diseased or injured neurons.

Depressive Symptoms in Patients With Dry Eye Disease: A Case-Control Study Using the Beck Depression Inventory.

Purpose: To measure depressive symptoms in patients with dry eye disease (DED) and controls using the Beck Depression Inventory (BDI) and to determine the association between depressive and DED symptoms. Methods: Fifty-three patients with DED and 41 controls were recruited to the study. DED symptoms were assessed using the Symptom Burden Tool and Ocular Surface Disease Index tool. Depressive symptoms were assessed using the BDI. Regression diagnostics were performed to detect outliers. Linear statistical models and polynomial regression were used to determine the relationship between depressive symptoms and DED symptoms. An independent t test was performed to determine differences in BDI scores between cases and controls. Scatter plots were generated and linear regression was used to estimate the association between scores. Logistic regression was used for the DED dichotomous outcome and depression status as exposure. Results: Regression models revealed that the association is linear more than quadratic or cubic. After adjusting for age, sex, race, and psychiatric medication, the regression coefficient between DED symptoms and depressive symptoms among DED cases was 1.22 (95% confidence interval, 0.27–2.18). DED symptom scores and depression scores were statistically significantly different between DED cases and controls. Adjusted logistic regression revealed an odds ratio of 2.79 (95% confidence interval, 0.96–8.12). Conclusions: This study provides further evidence regarding the association between DED and depression and their symptoms. Prospective studies are needed to understand the mechanisms underlying the association between symptoms of depression and symptoms of DED.

Symptom burden of patients with dry eye disease: A four domain analysis

Purpose To determine which sensory (symptom persistence and intensity) and reactive (activity and affective interference) domains of symptom analysis are essential for assessing symptom burden in dry eye disease (DED) patients. Methods A symptom domain tool was developed to investigate all four symptom domains in DED. In a cross-sectional pilot study, we administered the symptom burden tool and the Ocular Surface Disease Index (OSDI) questionnaire to 48 DED patients. Total and domain scores from the symptom burden tool and the OSDI were normalized to achieve comparability. Spearman correlation coefficients were calculated to measure the relationship between domains and subscales. Agreement between the symptom burden tool and OSDI was assessed by Bland-Altman plot. Assigned treatments were compared by symptom burden to determine whether treatment aggressiveness is linked to symptom intensity. Results There was high agreement between the symptom burden tool and the OSDI. Symptom persistence had a stronger correlation with affective interference (r  =  0.62 for the symptom burden tool and r = 0.73 for the OSDI) than activity interference (r = 0.58 for the symptom burden tool and r = 0.60 for the OSDI). Symptom intensity correlated weakly with affective interference (r = 0.38) and activity interference (r = 0.37) in the symptom burden tool (OSDI does not have a subscale for intensity). In patients with equal persistence of symptoms, those having high symptom intensity were receiving more aggressive treatment (66.7%) than those with lower symptom intensity (33.3%). Conclusions Persistence of symptoms correlates better with affective interference than activity interference. Intensity of symptoms may be important for treatment decisions.

Vision-related quality-of-life assessment using NEI VFQ-25 in patients after Boston keratoprosthesis implantation

Purpose: The aim of this study was to determine the impact of Boston keratoprosthesis (KPro) implantation on patient-reported visual function using the National Eye Institute Visual Function Questionnaire 25 (NEI VFQ-25). Methods: This is a prospective study of patients undergoing implantation of Boston KPro. The NEI VFQ-25 was used to assess vision-related quality of life preoperatively and postoperatively at 3 and 6 months and yearly thereafter. Baseline scores were compared with postoperative scores using paired t test. Stratified analysis was performed to compare results by visual acuity of the contralateral eye. Results: Twenty-four patients were included. Mean baseline NEI VFQ-25 overall score was 44.6. At the first follow-up time point, mean overall score was 70.0, representing a statistically significant change from baseline (P < 0.001). Breakdown of subcategories within VFQ-25 showed significant improvement in general vision, near and distance activities, social functioning, mental health, role difficulties, dependency, color vision, and peripheral vision (P < 0.05). This improvement was also observed when comparing baseline scores with postoperative scores at average follow-up of 16 months. Although a greater change in scores was observed in patients who had poor vision in the contralateral eye, patients with vision better than 20/200 in the nonsurgical eye showed statistically significant improvement in overall scores and subscale scores compared with baseline. Conclusions: The quality of life of patients who underwent KPro significantly improved postoperatively compared with their preoperative status. These results did not change when stratifying by visual acuity of the contralateral eye.