Predicting dry eye using non-invasive techniques of tear film surface assessment

PURPOSE. To measure tear film surface quality in healthy and dry eye subjects using three noninvasive techniques of tear film quality assessment and to establish the ability of these noninvasive techniques to predict dry eye. METHODS. Thirty four subjects participated in the study, and were classified as dry eye or normal, based on standard clinical assessments. Three non-invasive techniques were applied for measurement of tear film surface quality: dynamic-area high-speed videokeratoscopy (HSV), wavefront sensing (DWS) and lateral shearing interferometry (LSI). The measurements were performed in both natural blinking conditions (NBC) and in suppressed blinking conditions (SBC). RESULTS. In order to investigate the capability of each method to discriminate dry eye subjects from normal subjects, the receiver operating curve (ROC) was calculated and then the area under the curve (AUC) was extracted. The best result was obtained for the LSI technique (AUC=0.80 in SBC and AUC=0.73 in NBC), which was followed by HSV (AUC=0.72 in SBC and AUC=0.71 in NBC). The best result for DWS was AUC=0.64 obtained for changes in vertical coma in suppressed blinking conditions, while for normal blinking conditions the results were poorer. CONCLUSIONS. Non-invasive techniques of tear film surface assessment can be used for predicting dry eye and this can be achieved in natural blinking as well as suppressed blinking conditions. In this study, LSI showed the best detection performance, closely followed by the dynamic-area HSV. The wavefront sensing technique was less powerful, particularly in natural blinking conditions.

Diagnosing dry eye with dynamic-area high-speed videokeratoscopy

Dry eye syndrome is one of the most commonly reported eye health conditions. Dynamic-area highspeed videokeratoscopy (DA-HSV) represents a promising alternative to the most invasive clinical methods for the assessment of the tear film surface quality (TFSQ), particularly as Placido-disk videokeratoscopy is both relatively inexpensive and widely used for corneal topography assessment. Hence, improving this technique to diagnose dry eye is of clinical significance and the aim of this work. First, a novel ray-tracing model is proposed that simulates the formation of a Placido image. This model shows the relationship between tear film topography changes and the obtained Placido image and serves as a benchmark for the assessment of indicators of the ring’s regularity. Further, a novel block-feature TFSQ indicator is proposed for detecting dry eye from a series of DA-HSV measurements. The results of the new indicator evaluated on data from a retrospective clinical study, which contains 22 normal and 12 dry eyes, have shown a substantial improvement of the proposed technique to discriminate dry eye from normal tear film subjects. The best discrimination was obtained under suppressed blinking conditions. In conclusion,this work highlights the potential of the DA-HSV as a clinical tool to diagnose dry eye syndrome.

Corneal nerves in refractive surgery and dry eye

This study aimed to investigate the morphology and function of corneal sensory nerves in 1) patients after corneal refractive surgery and 2) patients with dry eye due to Sjögren's syndrome. A third aim was to explore the possible correlation between cytokines detected in tears and development of post-PRK subepithelial haze. The main methods used were tear fluid ELISA analysis, corneal in vivo confocal microscopy, and noncontact esthesiometry. The results revealed that after PRK a positive correlation exists between the regeneration of subbasal nerves and the thickness of regenerated epithelium. Pre- or postoperative levels of the tear fluid cytokines TGF-β1, TNF-α, or PDGF-BB did not correlate with the development of corneal haze objectively estimated by in vivo confocal microscopy 3 months after PRK. After high myopic LASIK, a discrepancy between subjective dry eye symptoms and objective signs of dry eye was observed. The majority of patients reported ongoing dry eye symptoms even 5 years after LASIK, although no objective clinical signs of dry eye were apparent. In addition, no difference in corneal sensitivity was observed between these patients and controls. Primary Sjögren's syndrome patients presented with corneal hypersensitivity, although their corneal subbasal nerve density was normal. However, alterations in corneal nerve morphology (nerve sprouting and thickened stromal nerves) and an increased number of antigen-presenting cells among subbasal nerves were observed, implicating the presence of an ongoing inflammation. Based on these results, the relationship between nerve regeneration and epithelial thickness 3 months after PRK appears to reflect the trophic effect of corneal nerves on epithelium. In addition, measurement of tear fluid cytokines may not be suitable for screening patients for risk of scar (haze) formation after PRK. Presumably, at least part of the symptoms of "LASIK-associated dry eye" are derived from aberrantly regenerated and abnormally functioning corneal nerves. Thus, they may represent a form of corneal neuropathy or "phantom pain" rather than conventional dry eye. Corneal nerve alterations and inflammatory findings in Sjögren's syndrome offer an explanation for the corneal hypersensitivity or even chronic pain or hyperalgesia often observed in these patients. In severe cases of disabling chronic pain in patients with dry eye or after LASIK, when conventional therapeutic possibilities fail to offer relief, consultation of a physician specialized in pain treatment is recommended.

Interrelations between Dry Eye Syndrome and Tear Fluid Phospholipid Transfer Protein

The simplified model of human tear fluid (TF) is a three-layered structure composed of a homogenous gel-like layer of hydrated mucins, an aqueous phase, and a lipid-rich outermost layer found in the tear-air interface. It is assumed that amphiphilic phospholipids are found adjacent to the aqueous-mucin layer and externally to this a layer composed of non-polar lipids face the tear-air interface. The lipid layer prevents evaporation of the TF and protects the eye, but excess accumulation of lipids may lead to drying of the corneal epithelium. Thus the lipid layer must be controlled and maintained by some molecular mechanisms. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. The aim of this thesis was to investigate the presence and molecular mechanisms of lipid transfer proteins in human TF. The purpose was also to study the role of these proteins in the development of dry eye syndrome (DES). The presence of TF PLTP and CETP was studied by western blotting and mass spectrometry. The concentration of these proteins was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. To study the molecular mechanisms involved in PLTP mediated lipid transfer Langmuir monolayers and asymmetrical flow field-flow fractionation (AsFlFFF) was used. Ocular tissue samples were stained with monoclonal antibodies against PLTP to study the secretion route of PLTP. Heparin-Sepharose affinity chromatography was used for PLTP pull-down experiments and co-eluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. To study whether PLTP plays any functional role in TF PLTP-deficient mice were examined. The activity of PLTP was also studied in dry eye patients. PLTP is a component of normal human TF, whereas CETP is not. TF PLTP concentration was about 2-fold higher than that in human plasma. Inactivation of PLTP by heat treatment or immunoinhibition abolished the phospholipid transfer activity in tear fluid. PLTP was found to be secreted from lacrimal glands. PLTP seems to be surface active and is capable of accepting lipid molecules without the presence of lipid-protein complexes. The active movement of radioactively labeled lipids and high activity form of PLTP to acceptor particles suggested a shuttle model of PLTP-mediated lipid transfer. In this model, PLTP physically transports lipids between the donor and acceptor. Protein-protein interaction assays revealed ocular mucins as PLTP interaction partners in TF. In mice with a full deficiency of functional PLTP enhanced corneal epithelial damage, increased corneal permeability to carboxyfluorescein, and decreased corneal epithelial occludin expression was demonstrated. Increased tear fluid PLTP activity was observed among human DES patients. These results together suggest a scavenger property of TF PLTP: if the corneal epithelium is contaminated by hydrophobic material, PLTP could remove them and transport them to the superficial layer of the TF or, alternatively, transport them through the naso-lacrimal duct. Thus, PLTP might play an integral role in tear lipid trafficking and in the protection of the corneal epithelium. The increased PLTP activity in human DES patients suggests an ocular surface protective role for this lipid transfer protein.

Lacrimal Proline Rich 4 (LPRR4) Protein in the Tear Fluid Is a Potential Biomarker of Dry Eye Syndrome

Dry eye syndrome (DES) is a complex, multifactorial, immune-associated disorder of the tear and ocular surface. DES with a high prevalence world over needs identification of potential biomarkers so as to understand not only the disease mechanism but also to identify drug targets. In this study we looked for differentially expressed proteins in tear samples of DES to arrive at characteristic biomarkers. As part of a prospective case-control study, tear specimen were collected using Schirmer strips from 129 dry eye cases and 73 age matched controls. 2D electrophoresis (2DE) and Differential gel electrophoresis (DIGE) was done to identify differentially expressed proteins. One of the differentially expressed protein in DES is lacrimal proline rich 4 protein (LPRR4). LPRR4 protein expression was quantified by enzyme immune sorbent assay (ELISA). LPRR4 was down regulated significantly in all types of dry eye cases, correlating with the disease severity as measured by clinical investigations. Further characterization of the protein is required to assess its therapeutic potential in DES.

Punctal plug: a medical device to treat dry eye syndrome and for sustained drug delivery to the eye

Punctal plugs (PPs) are miniature medical implants that were initially developed for the treatment of dry eyes. Since their introduction in 1975, many PPs made from different materials and designs have been developed. PPs, albeit generally successful, suffer from drawbacks such as epiphora and suppurative canaliculitis. To overcome these issues intelligent designs of PPs were proposed (e.g. SmartPLUG™ and Form Fit™). PPs are also gaining interest among pharmaceutical scientists for sustaining drug delivery to the eye. This review aims to provide an overview of PPs for dry eye treatment and drug delivery to treat a range of ocular diseases. It also discusses current challenges in using PPs for ocular diseases.

A novel cyclosporin a aqueous formulation for dry eye treatment: in vitro and in vivo evaluation.

PURPOSE: The aim of the present study was the in vitro and in vivo evaluation of a novel aqueous formulation based on polymeric micelles for the topical delivery of cyclosporine A for dry eye treatment. METHODS: In vitro experiments were carried out on primary rabbit corneal cells, which were characterized by immunocytochemistry using fluorescein-labeled lectin I/isolectin B4 for the endothelial cells and mouse monoclonal antibody to cytokeratin 3+12 for the epithelial ones. Living cells were incubated for 1 hour or 24 hours with a fluorescently labeled micelle formulation and analyzed by fluorescence microscopy. In vivo evaluations were done by Schirmer test, osmolarity measurement, CyA kinetics in tears, and CyA ocular distribution after topical instillation. A 0.05% CyA micelle formulation was compared to a marketed emulsion (Restasis). RESULTS: The in vitro experiments showed the internalization of micelles in the living cells. The Schirmer test and osmolarity measurements demonstrated that micelles did not alter the ocular surface properties. The evaluation of the tear fluid gave similar CyA kinetics values: AUC = 2339 ± 1032 min*μg/mL and 2321 ± 881.63; Cmax = 478 ± 111 μg/mL and 451 ± 74; half-life = 36 ± 9 min and 28 ± 9 for the micelle formulation and Restasis, respectively. The ocular distribution investigation revealed that the novel formulation delivered 1540 ± 400 ng CyA/g tissue to the cornea. CONCLUSIONS: The micelle formulation delivered active CyA into the cornea without evident negative influence on the ocular surface properties. This formulation could be applied for immune-related ocular surface diseases.

The epidemiology of dry eye in Melbourne, Australia

OBJECTIVE: To describe the epidemiology of dry eye in the adult population of Melbourne, Australia. DESIGN: A cross-sectional prevalence study. PARTICIPANTS: Participants were recruited by a household census from two of nine clusters of the Melbourne Visual Impairment Project, a population-based study of age-related eye disease in the 40 and older age group of Melbourne, Australia. Nine hundred and twenty-six (82.3% of eligible) people participated; 433 (46.8%) were male. They ranged in age from 40 to 97 years, with a mean of 59.2 years. MAIN OUTCOME MEASURES: Self-reported symptoms of dry eye were elicited by an interviewer-administered questionnaire. Four objective assessments of dry eye were made: Schirmer's test, tear film breakup time, rose bengal staining, and fluorescein corneal staining. A standardized clinical slit-lamp examination was performed on all participants. Dry eye for the individual signs or symptoms was defined as: rose bengal > 3, Schirmers < 8, tear film breakup time < 8, > 1/3 fluorescein staining, and severe symptoms (3 on a scale of 0 to 3). RESULTS: Dry eye was diagnosed as follows: 10.8% by rose bengal, 16.3% by Schirmer's test, 8.6% by tear film breakup time, 1.5% by fluorescein staining, 7.4% with two or more signs, and 5.5% with any severe symptom not attributed to hay fever. Women were more likely to report severe symptoms of dry eye (odds ratio [OR] = 1.85; 95% confidence limits [CL] = 1.01, 3.41). Risk factors for two or more signs of dry eye include age (OR = 1.04; 95% CL = 1.01, 1.06), and self-report of arthritis (OR = 3.27; 95% CL = 1.74, 6.17). These results were not changed after excluding the 21 people (2.27%) who wore contact lenses. CONCLUSIONS: These are the first reported population-based data of dry eye in Australia. The prevalence of dry eye varies by sign and symptom.

Clinical Treatment of Dry Eye Using 0.03% Tacrolimus Eye Drops

Purpose: To report the clinical outcome of the treatment of dry eyes using 0.03% tacrolimus eye drops (olive oil + tacrolimus 0.03%) (Ophthalmos, Sao Paulo, Brazil). Methods: Sixteen eyes of 8 patients with Sjogren syndrome dry eyes (age, 51.13 +/- 9.45 years) were enrolled in this study (prospective noncontrolled interventional case series). Patients were instructed to use topical 0.03% tacrolimus eye drops twice a day (every 12 hours) in the lower conjunctival sac. Schirmer I test, break-up time, corneal fluorescein, and rose bengal staining score were performed in all patients 1 day before, and 14, 28, and 90 days after treatment with 0.03% tacrolimus eye drops. Results: The average fluorescein staining and rose bengal staining scores improved statistically significantly after 14 days of treatment and improved even more after 28 and 90 days. The average Schirmer I test did not improve statistically significantly after 28 days of treatment, although we did observe a significant improvement after 90 days of treatment with 0.03% tacrolimus eye drops. The average break-up time did not improve statistically after 14 days of treatment, although we observed a significant improvement after 28 and 90 days of treatment with 0.03% tacrolimus eye drops. Conclusions: Topical 0.03% tacrolimus eye drops successfully improved tear stability and ocular surface status in patients with dry eyes.

Fluorophotometry is a sensitive method/technique to evaluate epithelial permeability in murine experimental dry eye

Purpose. Fluorophotometry is a well validated method for assessing corneal permeability in human subjects. However, with the growing importance of basic science animal research in ophthalmology, fluorophotometry’s use in animals must be further evaluated. The purpose of this study was to evaluate corneal epithelial permeability following desiccating stress using the modified Fluorotron Master™. ^ Methods. Corneal permeability was evaluated prior to and after subjecting 6-8 week old C57BL/6 mice to experimental dry eye (EDE) for 2 and 5 days (n=9/time point). Untreated mice served as controls. Ten microliters of 0.001% sodium fluorescein (NaF) were instilled topically into each mouse’s left eye to create an eye bath, and left to permeate for 3 minutes. The eye bath was followed by a generous wash with Buffered Saline Solution (BSS) and alignment with the Fluorotron Master™. Seven corneal scans using the Fluorotron Master were performed during 15 minutes (1 st post-wash scans), followed by a second wash using BSS and another set of five corneal scans (2nd post-wash scans) during the next 15 minutes. Corneal permeability was calculated using data calculated with the FM™ Mouse software. ^ Results. When comparing the difference between the Post wash #1 scans within the group and the Post wash #2 scans within the group using a repeated measurement design, there was a statistical difference in the corneal fluorescein permeability of the Post-wash #1 scans after 5 days (1160.21±108.26 vs. 1000.47±75.56 ng/mL, P<0.016 for UT-5 day comparison 8 [0.008]), but not after only 2 days of EDE compared to Untreated mice (1115.64±118.94 vs. 1000.47±75.56 ng/mL, P>0.016 for UT-2 day comparison [0.050]). There was no statistical difference between the 2 day and 5 day Post wash #1 scans (P=.299). The Post-wash #2 scans demonstrated that EDE caused a significant NaF retention at both 2 and 5 days of EDE compared to baseline, untreated controls (1017.92±116.25, 1015.40±120.68 vs. 528.22±127.85 ng/mL, P<0.05 [0.0001 for both]). There was no statistical difference between the 2 day and 5 day Post wash #2 scans (P=.503). The comparison between the Untreated post wash #1 with untreated post wash #2 scans using a Paired T-test showed a significant difference between the two sets of scans (P=0.000). There is also a significant difference between the 2 day comparison and the 5 day comparison (P values = 0.010 and 0.002, respectively). ^ Conclusion. Desiccating stress increases permeability of the corneal epithelium to NaF, and increases NaF retention in the corneal stroma. The Fluorotron Master is a useful and sensitive tool to evaluate corneal permeability in murine dry eye, and will be a useful tool to evaluate the effectiveness of dry eye treatments in animal-model drug trials.^