Cornea

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Ding J, Wirostko B, Sullivan DA. Human growth hormone promotes corneal epithelial cell migration in vitro. Cornea 2015;34(6):686-92.Abstract

PURPOSE: Corneal wound healing is a highly regulated process that requires the proliferation and migration of epithelial cells and interactions between epithelial cells and stromal fibroblasts. Compounds that can be applied topically to the ocular surface and that have the capability of activating corneal epithelial cells to proliferate and/or migrate would be useful to promote corneal wound healing. We hypothesize that human growth hormone (HGH) will activate signal transducers and activators of transcription-5 (STAT5) signaling and promote corneal wound healing by enhancing corneal epithelial cell and fibroblast proliferation and/or migration in vitro. The purpose of this study was to test these hypotheses. METHODS: We studied cell signaling, proliferation, and migration using an immortalized human corneal epithelial cell line and primary human corneal fibroblasts in vitro. We also examined whether insulin-like growth factor-1 (IGF-1), a hormone known to mediate many of HGH's growth promoting actions, may play a role in this effect. RESULTS: We show that HGH activates STAT5 signaling and promotes corneal epithelial cell migration in vitro. The migratory effect requires an intact communication between corneal epithelia and fibroblasts and is not mediated by IGF-1. CONCLUSIONS: HGH may represent a topical therapeutic to promote corneal epithelial wound healing. This warrants further investigation.

Ding J, Sullivan DA. The effects of insulin-like growth factor 1 and growth hormone on human meibomian gland epithelial cells. JAMA Ophthalmol 2014;132(5):593-9.Abstract
IMPORTANCE: A phase 1 study has reported that dry eye disease is the most common adverse effect of human exposure to the antibody figitumumab, an anticancer drug that prevents insulin-like growth factor 1 (IGF-1) from binding to its receptor. We hypothesized that the mechanism underlying this effect is the inhibition of IGF-1 action in epithelial cells of the meibomian gland. OBJECTIVES: To test the hypothesis that IGF-1 stimulates meibomian gland function in vitro and to examine whether growth hormone, a closely related hormone of IGF-1, has the same effect. DESIGN, SETTING, AND MATERIAL: Immortalized human meibomian gland epithelial cells were cultured in the presence or the absence of IGF-1, growth hormone, and an IGF-1 receptor-blocking antibody. Signaling pathways, cell proliferation, neutral lipid staining, and a key protein involved in lipid biogenesis were evaluated. INTERVENTION: Application of IGF-1 and growth hormone to human meibomian gland epithelial cells. MAIN OUTCOMES AND MEASURES: Immunoblotting, cell counting, and neutral lipid staining. RESULTS Insulin-like growth factor 1 activated the phosphoinositol 3-kinase/Akt and forkhead box O1 pathways (showing a dose-dependent effect on immunoblotting), stimulated cellular proliferation (about 1.8-fold increase in cell number), increased sterol regulatory element-binding protein 1 expression (about 3-fold increase on immunoblotting), and promoted lipid accumulation in human meibomian gland epithelial cells (about 2-fold increase in lipid staining). These IGF-1 actions, which may be blocked by cotreatment with the anti-IGF-1 antibody, were accompanied by inconsistent effects on extracellular signal-regulated kinase phosphorylation. We were not able to demonstrate activation of Akt, forkhead box O1, extracellular signal-regulated kinase, Janus kinase 2, or signal transducers and activators of transcription 5, induced cell proliferation, or lipid accumulation in these cells by growth hormone application. CONCLUSIONS AND RELEVANCE: Our results support the hypothesis that IGF-1 acts on human meibomian gland epithelial cells and may explain why treatment with figitumumab, the IGF-1 inhibitor, causes dry eye disease. Ophthalmic care for dry eye disease may be needed when patients with cancer undergo treatment with drugs that inhibit IGF-1 action.
Ding J, Sullivan DA. Aging and dry eye disease. Exp Gerontol 2012;47(7):483-90.Abstract
Dry eye disease is a prevalent eye disorder that in particular affects the elderly population. One of the major causes of dry eye, meibomian gland dysfunction (MGD), shows increased prevalence with aging. MGD is caused by hyperkeratinization of the ductal epithelium of meibomian gland and reduced quantity and/or quality of meibum, the holocrine product that stabilizes and prevents the evaporation of the tear film. Of note, retinoids which are used in current anti-aging cosmetics may promote the development of MGD and dry eye disease. In this review, we will discuss the possible mechanisms of age-related MGD.
Ding J, Kam WR, Dieckow J, Sullivan DA. The influence of 13-cis retinoic acid on human meibomian gland epithelial cells. Invest Ophthalmol Vis Sci 2013;54(6):4341-50.Abstract
PURPOSE: Meibomian gland dysfunction (MGD) is a primary cause of dry eye disease. One of the risk factors for MGD is exposure to 13-cis retinoic acid (13-cis RA), a metabolite of vitamin A. However, the mechanism is not well understood. We hypothesize that 13-cis RA inhibits cell proliferation, promotes cell death, alters gene and protein expressions, and attenuates cell survival pathways in human meibomian gland epithelial cells. METHODS: To test our hypotheses, immortalized human meibomian gland epithelial cells were cultured with or without 13-cis RA for varying doses and time. Cell proliferation, cell death, gene expression, and proteins involved in proliferation/survival and inflammation were evaluated. RESULTS: We found that 13-cis RA inhibited cell proliferation, induced cell death, and significantly altered the expression of 6726 genes, including those involved in cell proliferation, cell death, differentiation, keratinization, and inflammation, in human meibomian gland epithelial cells. Further, 13-cis RA also reduced the phosphorylation of Akt and increased the generation of interleukin-1β and matrix metallopeptidase 9. CONCLUSIONS: Exposure to 13-cis RA inhibits cell proliferation, increases cell death, alters gene expression, changes signaling pathways, and promotes inflammatory mediator and protease expression in meibomian gland epithelial cells. These effects may be responsible, at least in part, for the 13-cis RA-related induction of MGD.
Dohlman TH, Di Zazzo A, Omoto M, Hua J, Ding J, Hamrah P, Chauhan SK, Dana R. E-Selectin Mediates Immune Cell Trafficking in Corneal Transplantation. Transplantation 2016;100(4):772-80.Abstract

BACKGROUND: Immune rejection continues to threaten all tissue transplants. Here we sought to determine whether platelet (P)- and endothelial (E)-selectin mediate T cell recruitment in corneal transplantation and whether their blockade can reduce T cell graft infiltration and improve long-term corneal allograft survival. METHODS: In a murine model of allogeneic corneal transplantation, we used PCR and immunohistochemistry to investigate expression of P- and E-selectin in rejected versus accepted allografts and lymph node flow cytometry to assess expression of selectin ligands by effector T cells. Using P- and E-selectin neutralizing antibodies, we evaluated the effect of blockade on CD4 T cell recruitment, as well as the effect of anti-E-selectin on long-term allograft survival. RESULTS: The P- (93.3-fold, P < 0.05) and E-selectin (17.1-fold, P < 0.005) are upregulated in rejected versus accepted allogeneic transplants. Type 1 T helper cells from hosts with accepted and rejected grafts express high levels of P-selectin glycoprotein ligand 1 and glycosylated CD43. In vivo blockade of P (0.47 ± 0.03, P < 0.05) and E selectin (0.49 ± 0.1, P < 0.05) reduced the number of recruited T cells compared with IgG control (0.98 ± 0.1). Anti-E-selectin reduced the number of mature antigen-presenting cells trafficking to lymphoid tissue compared with control (6.96 ± 0.9 vs 12.67 ± 0.5, P < 0.05). Anti-E-selectin treatment delayed graft rejection and increased survival compared with control, although this difference did not reach statistical significance. CONCLUSIONS: In a model of corneal transplantation, P- and E-selectin mediate T cell recruitment to the graft, E-selectin mediates APC trafficking to lymphoid tissue, and blockade of E-selectin has a modest effect on improving long-term graft survival.

Dohlman TH, Singh RB, Dana R. Advances in the Medical Management of Neurotrophic Keratitis. Semin Ophthalmol 2021;36(4):335-340.Abstract
Neurotrophic Keratitis (NK) is a degenerative disorder of the cornea characterized by decreased or absent sensory corneal innervation, corneal epitheliopathy and impaired healing.The clinical presentation of NK can range from persistent epithelial defects to corneal perforation and management is often both challenging and protracted. Historically, the management of NK has consisted of non-specific strategies to facilitate corneal epithelial healing such as lubrication, bandage contact lenses and tarsorrhaphy. Recent advances in the development of therapeutics for NK have provided new and efficacious targeted strategies for its management.In this article, we review recombinant human nerve growth factor (Cenegermin), currently approved for clinical use in the United States and Europe, as well as other promising therapeutic options that are in pre-clinical development such as thymosine β4, connexin43 inhibitors, and artificial extracellular matrix components.
Dohlman TH, Omoto M, Hua J, Stevenson W, Lee S-M, Chauhan SK, Dana R. VEGF-trap Aflibercept Significantly Improves Long-term Graft Survival in High-risk Corneal Transplantation. Transplantation 2015;99(4):678-86.Abstract

BACKGROUND: Graft failure because of immune rejection remains a significant problem in organ transplantation, and lymphatic and blood vessels are important components of the afferent and efferent arms of the host alloimmune response, respectively. We compare the effect of antihemangiogenic and antilymphangiogenic therapies on alloimmunity and graft survival in a murine model of high-risk corneal transplantation. METHODS: Orthotopic corneal transplantation was performed in hemevascularized and lymph-vascularized high-risk host beds, and graft recipients received subconjunctival vascular endothelial growth factor (VEGF)-trap, anti-VEGF-C, sVEGFR-3, or no treatment, beginning at the time of surgery. Fourteen days after transplantation, graft hemeangiogenesis and lymphangiogenesis were evaluated by immunohistochemistry. The frequencies of Th1 cells in regional lymphoid tissue and graft-infiltrating immune cells were evaluated by flow cytometry. Long-term allograft survival was compared using Kaplan-Meier curves. RESULTS: VEGF-trap significantly decreased graft hemangiogenesis as compared to the control group and was most effective in reducing the frequency of graft-infiltrating immune cells. Anti-VEGF-C and sVEGFR3 significantly decreased graft lymphangiogenesis and lymphoid Th1 cell frequencies as compared to control. VEGF-trap (72%), anti-VEGF-C (25%), and sVEGFR-3 (11%) all significantly improved in the 8-week graft survival compared to control (0%), although VEGF-trap was significantly more effective than both anti-VEGF-C (P < 0.05) and sVEGFR-3 (P < 0.05). CONCLUSION: In a clinically relevant model of high-risk corneal transplantation in which blood and lymphatic vessels are present and treatment begins at the time of transplantation, VEGF-trap is significantly more effective in improving long-term graft survival as compared to anti-VEGF-C and sVEGFR-3, but all approaches improve survival when compared to untreated control.

Dohlman TH, Lertsuwanroj B, D'Amico DJ, Ciralsky JB, Kiss S. Evaluation of signs and symptoms of ocular surface disease after intravitreal injection. Acta Ophthalmol 2019;97(8):e1154-e1156.
Dohlman TH, McSoley M, Amparo F, Carreno-Galeano T, Wang M, Dastjerdi M, Singh RB, Coco G, Di Zazzo A, Shikari H, Saboo U, Sippel K, Ciralsky J, Yoo SH, Sticca M, Wakamatsu TH, Murthy S, Hamrah P, Jurkunas U, Ciolino JB, Gomes JAP, Perez VL, Yin J. Bevacizumab in High-Risk Corneal Transplantation: A Pilot Multicenter Prospective Randomized Control Trial. Ophthalmology 2022;
Dohlman C. The Boston Keratoprosthesis-The First 50 Years: Some Reminiscences. Annu Rev Vis Sci 2022;8:1-32.Abstract
Millions of people worldwide are bilaterally blind due to corneal diseases including infectious etiologies, trauma, and chemical injuries. While corneal transplantation can successfully restore sight in many, corneal graft survival decreases in eyes with chronic inflammation and corneal vascularization. Additionally, the availability of donor cornea material can be limited, especially in underdeveloped countries where corneal blindness may also be highly prevalent. Development of methods to create and implant an artificial cornea (keratoprosthesis) may be the only option for patients whose eye disease is not suitable for corneal transplantation or who live in regions where corneal transplantation is not possible. The Boston Keratoprosthesis (B-KPro) is the most commonly implanted keratoprosthesis worldwide, having restored vision in thousands of patients. This article describes the initial design of the B-KPro and the modifications that have been made over many years. Additionally, some of the complications of surgical implantation and long-term care challenges, particularly complicating inflammation and glaucoma, are discussed.
Domenech-Estarellas EA, Mamata H, Luo ZK. Targeted steroid ointment application to the lid margins in ocular graft-versus-host disease associated blepharitis treatment. Ocul Surf 2021;21:348-350.
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Eghrari AO, Vasanth S, Gapsis BC, Bison H, Jurkunas U, Riazuddin AS, Gottsch JD. Identification of a Novel TCF4 Isoform in the Human Corneal Endothelium. Cornea 2018;37(7):899-903.Abstract
PURPOSE: Alternative splice isoforms of TCF4, a gene implicated in Fuchs corneal dystrophy, have been identified in multiple human tissues outside of the eye. The aim of this study was to identify the transcriptional profile of TCF4 in the corneal endothelium. METHODS: We extracted RNA from the donor corneal endothelium and performed rapid amplification of cDNA ends. We tested the expression pattern of 1 newly identified isoform (7b) in a panel of cDNA derived from multiple human tissues and included cDNA from corneal endothelial (CE) and retinal pigment epithelial cell lines. To further delineate differential expression of TCF4 splice variants that span CTG18.1, we analyzed expression of 6 alternative splice isoforms that are transcribed from either exon 2 or 3 in RNA extracted from the corneal endothelium of 3 normal donors and a CE cell line. RESULTS: We identified 11 different isoforms in control CE tissue, including 1 isoform (7b) not reported previously. This isoform is enriched specifically in the corneal endothelium and placenta compared with other tissues in a panel of human cDNA. CONCLUSIONS: We demonstrate the complex expression profile of TCF4 in the human corneal endothelium and reveal expression of alternative splice variants of TCF4.
Eguchi A, Inomata T, Nakamura M, Nagino K, Iwagami M, Sung J, Midorikawa-Inomata A, Okumura Y, Fujio K, Fujimoto K, Miura M, Akasaki Y, Shokirova H, Hirosawa K, Kuwahara M, Zhu J, Dana R, Murakami A, Kobayashi H. Heterogeneity of eye drop use among symptomatic dry eye individuals in Japan: large-scale crowdsourced research using DryEyeRhythm application. Jpn J Ophthalmol 2021;65(2):271-281.Abstract
PURPOSE: To determine eye drop type and usage frequency and investigate risk factors for no eye drop use in individuals with symptomatic dry eye (DE) in Japan. STUDY DESIGN: Crowdsourced observational study. METHODS: This study was conducted using the DryEyeRhythm smartphone application between November 2016 and September 2019. Data collected included the type and frequency of eye drop use, demographics, medical history, lifestyle, and self-reported symptoms. Symptomatic DE was defined as an Ocular Surface Disease Index total score of ≥ 13. Risk factors for no eye drop use were identified using multivariate logistic regression analyses. RESULTS: Among 2619 individuals with symptomatic DE, 1876 did not use eye drops. The most common eye drop type was artificial tears (53.4%), followed by hyaluronic acid 0.1% (33.1%) and diquafosol sodium 3% (18.7%). Risk factors (odds ratio [95% confidence interval]) for no eye drop use were age (0.97 [0.97-0.98]), body mass index (1.04 [1.01-1.07]), brain disease (0.38 [0.15-0.98]), collagen disease (0.30 [0.13-0.68]), mental illness other than depression and schizophrenia (0.65 [0.45-0.93]), cataract surgery (0.12 [0.02-0.59]), ophthalmic surgery other than cataract and laser-assisted in situ keratomileusis (0.55 [0.34-0.88]), current (0.47 [0.38-0.57]) or past (0.58 [0.43-0.77]) contact lens use, >8 h screen exposure time (1.38 [1.05-1.81]), <6 h (1.24 [1.01-1.52]) and >9 h (1.34 [1.04-1.72]) sleep time, and water intake (0.97 [0.94-0.98]). CONCLUSION: Many participants with symptomatic DE did not use optimized eye drop treatment and identified risk factors for no eye drop use. The DryEyeRhythm application may help improve DE treatment.
Elbasiony E, Cho W, Mittal SK, Chauhan SK. Suppression of lipopolysaccharide-induced corneal opacity by hepatocyte growth factor. Sci Rep 2022;12(1):494.Abstract
Keratitis induced by bacterial toxins, including lipopolysaccharide (LPS), is a major cause of corneal opacity and vision loss. Our previous study demonstrates hepatocyte growth factor (HGF) promotes epithelial wound healing following mechanical corneal injury. Here, we investigated whether HGF has the capacity to suppress infectious inflammatory corneal opacity using a new model of LPS-induced keratitis. Keratitis, induced by two intrastromal injections of LPS on day 1 and 4 in C57BL/6 mice, resulted in significant corneal opacity for up to day 10. Following keratitis induction, corneas were topically treated with 0.1% HGF or PBS thrice daily for 5 days. HGF-treated mice showed a significantly smaller area of corneal opacity compared to PBS-treated mice, thus improving corneal transparency. Moreover, HGF treatment resulted in suppression of α-SMA expression, compared to PBS treatment. HGF-treated corneas showed normalized corneal structure and reduced expression of pro-inflammatory cytokine, demonstrating that HGF restores corneal architecture and immune quiescence in corneas with LPS-induced keratitis. These findings offer novel insight into the potential application of HGF-based therapies for the prevention and treatment of infection-induced corneal opacity.
Elhusseiny AM, Eleiwa TK, Yacoub MS, George J, ElSheikh RH, Haseeb A, Kwan J, Elsaadani IA, Abo Shanab SM, Solyman O, Saeed HN. Relationship between screen time and dry eye symptoms in pediatric population during the COVID-19 pandemic. Ocul Surf 2021;22:117-119.
Elhusseiny AM, Saeed HN. Corneal opacification in Sanjad-Sakati syndrome. Am J Ophthalmol Case Rep 2022;26:101503.
Elhusseiny AM, Saeed HN. Posterior Polymorphous Corneal Dystrophy in a Pediatric Population. Cornea 2022;41(6):734-739.Abstract
PURPOSE: The aim of this study was to evaluate the clinical and topographic features of posterior polymorphous corneal dystrophy (PPCD) in children aged 15 years or younger with a long-term follow-up. Retrospective case series. METHODS: A retrospective chart review of patients who were diagnosed with PPCD at Boston Children's Hospital from 1999 to 2020 was performed. Data collected included age at the time of diagnosis, slit lamp findings, cycloplegic refraction, best-corrected visual acuity, central corneal thickness, specular microscopy, and corneal topography findings whenever available. RESULTS: Twenty-seven eyes of 19 patients were included (11 unilateral and 8 bilateral cases). Ten patients were girls (52.6%). Left eye was affected in 14 eyes. The mean age at the time of diagnosis was 8.5 ± 3.3 years, with a mean follow-up of 5.3 years. In unilateral cases, there was a statistically significant difference in the endothelial cell density (P = 0.01), coefficient variation (P = 0.03), and hexagonality (P = 0.01) between the affected and the contralateral unaffected eyes. The mean best-corrected visual acuity at initial presentation was 0.8 ± 0.2 compared with 0.9 ± 0.08 in unaffected eyes (P = 0.04). The mean astigmatism was higher in the affected eye (+1.7 diopters) compared with (+1.00) the unaffected eye (P = 0.07). At initial presentation, 7 of 27 eyes had amblyopia, which resolved, either partially or completely, in 5 eyes after treatment. CONCLUSIONS: PPCD can present early in children with astigmatism and anisometropic amblyopia. A careful slit lamp examination for children presenting with anisoastigmatism is necessary to diagnose PPCD. Contrary to adults, presentation is often unilateral. Such patients should be followed up regularly with cycloplegic retinoscopy to prevent and treat refractive amblyopia if present.
Emami-Naeini P, Dohlman TH, Omoto M, Hattori T, Chen Y, Lee HS, Chauhan SK, Dana R. Soluble vascular endothelial growth factor receptor-3 suppresses allosensitization and promotes corneal allograft survival. Graefes Arch Clin Exp Ophthalmol 2014;252(11):1755-62.Abstract
PURPOSE: To investigate the effect of VEGF-C and VEGF-D blockade via soluble VEGFR-3 (sVEGFR-3) on T cell allosensitization, corneal neovascularization, and transplant survival. METHODS: Corneal intrastromal suture placement and allogeneic transplantation were performed on BALB/c mice to evaluate the effect of sVEGFR-3 on corneal neovascularization. Soluble VEGFR-3 trap was injected intraperitoneally to block VEGF-C/D (every other day starting the day of surgery). Immunohistochemical staining of corneal whole mounts was performed using anti-CD31 (PECAM-1) and anti-LYVE-1 antibodies to quantify the levels of hem- and lymphangiogenesis, respectively. Mixed lymphocyte reaction (MLR) was performed to assess indirect and direct host T cell allosensitization and the frequencies of IFN-γ-producing T cells in the draining lymph nodes were assessed using flow cytometry. Graft opacity and survival was evaluated by slit-lamp biomicroscopy. RESULTS: Treatment with sVEGFR-3 resulted in a significant blockade of lymphangiogenesis 2 weeks post-transplantation and significantly prolonged corneal allograft survival compared to the control group at 8 weeks post-transplantation (87.5 % vs. 50 %), and this was associated with significant reduction in the frequencies of allosensitized T cells and decreased frequencies of IFN-γ-producing CD4 T cells. CONCLUSIONS: Soluble VEGFR-3 suppresses corneal lymphangiogenesis and allograft rejection and may offer a viable therapeutic modality for corneal neovascularization and corneal transplantation.
English JT, Norris PC, Hodges RR, Dartt DA, Serhan CN. Identification and Profiling of Specialized Pro-Resolving Mediators in Human Tears by Lipid Mediator Metabolomics. Prostaglandins Leukot Essent Fatty Acids 2017;117:17-27.Abstract

Specialized pro-resolving mediators (SPM), e.g. Resolvin D1, Protectin D1, Lipoxin A₄, and Resolvin E1 have each shown to be active in ocular models reducing inflammation. In general, SPMs have specific agonist functions that stimulate resolution of infection and inflammation in animal disease models. The presence and quantity of SPM in human emotional tears is of interest. Here, utilizing a targeted LC-MS-MS metabololipidomics based approach we document the identification of pro-inflammatory (Prostaglandins and Leukotriene B₄) and pro-resolving lipid mediators (D-series Resolvins, Protectin D1, and Lipoxin A₄) in human emotional tears from 12 healthy individuals. SPMs from the Maresin family (Maresin 1 and Maresin 2) were not present in these samples. Principal Component Analysis (PCA) revealed gender differences in the production of specific mediators within these tear samples as the SPMs were essentially absent in these female donors. These results indicate that specific SPM signatures are present in human emotional tears at concentrations known to be bioactive. Moreover, they will help to further appreciate the mechanisms of production and action of SPMs in the eye, as well as their physiologic roles in human ocular disease resolution.

Erickson S, Sullivan AG, Barabino S, Begovic E, Benitez-Del-Castillo JM, Bonini S, Borges JS, Brzheskiy V, Bulat N, Cerim A, Craig P, Cușnir V, Cușnir V, Cușnir V, Doan S, Dülger E, Farrant S, Geerling G, Goldblum D, Golubev S, Gomes JAP, González-Méijome JM, Grupcheva CN, Gündüz UÖmür, Horwath-Winter J, Källmark F, Karanadze N, Karcic HH, Karcic S, Kontadakis G, Messmer EM, Mrugacz M, Murphy C, O'Leary OE, Procopciuc V, Pult H, Raus P, Şahin A, Setälä N, Stanila A, Stanila DM, Utheim TP, Vehof J, Versura P, Villani E, Willcox MDP, Wolffsohn JS, Zagórski Z, Zoega GMár, Sullivan DA, Sullivan DA, Gomes JAP, Versura P, Willcox MDP. TFOS European ambassador meeting: Unmet needs and future scientific and clinical solutions for ocular surface diseases. Ocul Surf 2020;Abstract
The mission of the Tear Film & Ocular Surface Society (TFOS) is to advance the research, literacy, and educational aspects of the scientific field of the tear film and ocular surface. Fundamental to fulfilling this mission is the TFOS Global Ambassador program. TFOS Ambassadors are dynamic and proactive experts, who help promote TFOS initiatives, such as presenting the conclusions and recommendations of the recent TFOS DEWS II™, throughout the world. They also identify unmet needs, and propose future clinical and scientific solutions, for management of ocular surface diseases in their countries. This meeting report addresses such needs and solutions for 25 European countries, as detailed in the TFOS European Ambassador meeting in Rome, Italy, in September 2019.

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