Tear nanoDSF Denaturation Profile Is Predictive of Glaucoma.

Primary open-angle glaucoma (POAG) is a frequent blindness-causing neurodegenerative disorder characterized by optic nerve and retinal ganglion cell damage most commonly due to a chronic increase in intraocular pressure. The preservation of visual function in patients critically depends on the timeliness of detection and treatment of the disease, which is challenging due to its asymptomatic course at early stages and lack of objective diagnostic approaches. Recent studies revealed that the pathophysiology of glaucoma includes complex metabolomic and proteomic alterations in the eye liquids, including tear fluid (TF). Although TF can be collected by a non-invasive procedure and may serve as a source of the appropriate biomarkers, its multi-omics analysis is technically sophisticated and unsuitable for clinical practice. In this study, we tested a novel concept of glaucoma diagnostics based on the rapid high-performance analysis of the TF proteome by differential scanning fluorimetry (nanoDSF). An examination of the thermal denaturation of TF proteins in a cohort of 311 ophthalmic patients revealed typical profiles, with two peaks exhibiting characteristic shifts in POAG. Clustering of the profiles according to peaks maxima allowed us to identify glaucoma in 70% of cases, while the employment of artificial intelligence (machine learning) algorithms reduced the amount of false-positive diagnoses to 13.5%. The POAG-associated alterations in the core TF proteins included an increase in the concentration of serum albumin, accompanied by a decrease in lysozyme C, lipocalin-1, and lactotransferrin contents. Unexpectedly, these changes were not the only factor affecting the observed denaturation profile shifts, which considerably depended on the presence of low-molecular-weight ligands of tear proteins, such as fatty acids and iron. Overall, we recognized the TF denaturation profile as a novel biomarker of glaucoma, which integrates proteomic, lipidomic, and metallomic alterations in tears, and monitoring of which could be adapted for rapid non-invasive screening of the disease in a clinical setting.

Differences in Practitioner Experience, Practice Type, and Profession in Attitudes Toward Growing Contact Lens Practice.

OBJECTIVE: To investigate eye care practitioners' attitudes and perceptions toward potential interventions that can enhance contact lens (CL) practice across the world, and how this is influenced by their practice setting. METHODS: A self-administered, anonymized survey was constructed in English and then forward and backward translated into six more languages. The survey was distributed online via social media platforms and mailing lists involving reputed international professional bodies. RESULTS: In total, 2,222 responses from 27 countries with sufficient responses were analyzed (53% females, median age- 37 years). Most of the respondents were optometrists (81.9%) and 47.6% were from stand-alone/independent practices. Median working experience in CL prescribing was 11.0 years (IQR: 18.0, 4-22 years). Over two-third of them declared themselves to be very hopeful (22.9%) or hopeful (45.1%) about the future of their CL practice. Among the potential interventions proposed, continuous update of knowledge and skills and competently managing CL-related complications were rated the most important (median score: 9/10 for each). Practitioners working in national/regional retail chains expressed higher proactivity in recommending CLs (9/10) than those in local chains, hospitals, and universities (for all 8/10, P <0.05). National differences were also identified in eye care practitioner attitudes and perceptions ( P <0.05). CONCLUSIONS: The study provided important information to delineate a variety of elements characterizing CL practice across the world. These insights can serve as a basis to design strategies at national and international levels.

Possibilities of monitoring intraocular pressure in children using EASYTON transpalpebral tonometer.

PURPOSE: To compare the effectiveness of transpalpebral scleral tonometry (TPST) and corneal pneumotonometry in children, and assess the discomfort level when measuring intraocular pressure (IOP) by these methods. METHODS: TPST using EASYTON tonometer (Russia) and pneumotonometry using Reichert 7 Non-contact AutoTonometer (USA) have been sequentially performed on 84 eyes (42 children aged 5-14, ave. 9.3 ± 2.7), including 64 myopic eyes (-0.5 to 6.75D), 18 hyperopic eyes (+ 0.75 to + 3.75D), and 2 emmetropic eyes. We assessed tolerance to the procedure on a five-point scale using a questionnaire which listed several criteria: discomfort, presence of pain, fear or anxiety during the procedure, the child's resistance to measurement. RESULTS: EASYTON tonometry demonstrated repeatability of IOP indicators when measuring the same eye three times sequentially and almost the same IOP level in paired eyes of isometropic children. Pneumotonometry reveals a greater individual data variability and a more pronounced asymmetry of the paired eyes' indicators. IOP measured using the TPST was 18.3 ± 2.3 mmHg across the whole group, 18.2 ± 2.3 mmHg in myopic, and 18.5 ± 2.3 mmHg in hyperopic children. With pneumotonometry, the corresponding indicators were 17.1 ± 3.9 mmHg, 16.9 ± 3.8 mmHg, and 18.2 ± 4.0 mmHg. The average score for the TPST (4.64 ± 0.60 points) was significantly higher than that for pneumotonometry (3.85 ± 0.90 points) (p < 0.05). CONCLUSIONS: TPST provides broader possibilities for IOP control in pediatric practice, yielding more reliable and accurate results than pneumotonometry, eliminating the influence of corneal thickness and irregularity on the measurement result, and ensuring a calmer behavior and more comfort of children during the procedure.

Targeted Lipidomic Analysis of Aqueous Humor Reveals Signaling Lipid-Mediated Pathways in Primary Open-Angle Glaucoma.

Primary open-angle glaucoma (POAG) is characterized by degeneration of retinal ganglion cells associated with an increase in intraocular pressure (IOP) due to hindered aqueous humor (AH) drainage through the trabecular meshwork and uveoscleral pathway. Polyunsaturated fatty acids and oxylipins are signaling lipids regulating neuroinflammation, neuronal survival and AH outflow. Among them, prostaglandins have been previously implicated in glaucoma and employed for its treatment. This study addressed the role of signaling lipids in glaucoma by determining their changes in AH accompanying IOP growth and progression of the disease. Eye liquids were collected from patients with POAG of different stages and cataract patients without glaucoma. Lipids were identified and quantified by UPLC-MS/MS. The compounds discriminating glaucoma groups were recognized using ANCOVA and PLS-DA statistic approaches and their biosynthetic pathways were predicted by bioinformatics. Among 22 signaling lipids identified in AH, stage/IOP-dependent alterations in glaucoma were provided by a small set of mediators, including 12,13-DiHOME, 9- and 13-HODE/KODE, arachidonic acid and lyso-PAF. These observations correlated with the expression of cytochromes P450 (CYPs) and phospholipases A2 in the ocular tissues. Interestingly, tear fluid exhibited similar lipidomic alterations in POAG. Overall, POAG may involve arachidonic acid/PAF-dependent pathways and oxidative stress as evidenced from an increase in its markers, KODEs and 12,13-DiHOME. The latter is a product of CYPs, one of which, CYP1B1, is known as POAG and primary congenital glaucoma-associated gene. These data provide novel targets for glaucoma treatment. Oxylipin content of tear fluid may have diagnostic value in POAG.

Opportunities and threats to contact lens practice: A global survey perspective.

AIM: To understand the views of contact lens (CL) practitioners across the globe regarding what they perceive as opportunities and threats in CL practice. METHODS: A self-administered anonymised questionnaire, constructed in English and translated in six more languages, was distributed through reputed international professional bodies and academic institutions worldwide. The questionnaire included items on demographic characteristics, type of practice, and questions designed to explore practitioners' perspective on the future of their CL practice over the next five years. RESULTS: A total of 2408 valid responses were analysed. Multifocal CLs for presbyopia, CLs for myopia control, use of daily disposable (DD) CLs for occasional wear, and biocompatible materials to improve comfort were identified as promising areas of opportunities by practitioners (all 8/10). Respondents from North America, and Europe valued DDCLs for occasional wear moderately more favourable (Median: 9/10 for all) as compared to colleagues in Asia (Median: 8/10, p < 0.001), South America (Median: 8/10, p < 0.01), and Africa (Median: 8/10p < 0.01). Multifocal CLs for presbyopia was perceived as a better opportunity by practitioners in North America and Europe (Median: 9/10 for both), as well as in Australasia (Median: 8/10), in comparison to Asia, Africa, and Middle East (for all Median: 6/10, p < 0.001). Practitioners expressed concerns about the availability of CLs and CL prescriptions online without direct professional involvement (both 9/10). CONCLUSIONS: Overall, the most appealing opportunities for CL practice growth were identified in occasional use of DD CLs, biocompatible materials to reduce CL discomfort, multifocal CLs for presbyopia correction and management of myopia control with CLs. Lack of regulation in CL sales, especially online, seemed to be a constant threat. The insights from this study can be used to design targeted strategies to enhance CL practice across the globe and in specific geographical areas.

Terahertz scanning of the rabbit cornea with experimental UVB-induced damage: in vivo assessment of hydration and its verification.

SIGNIFICANCE: Water content plays a vital role in the normally functioning visual system; even a minor disruption in the water balance may be harmful. Today, no direct method exists for corneal hydration assessment, while it could be instrumental in early diagnosis and control of a variety of eye diseases. The use of terahertz (THz) radiation, which is highly sensitive to water content, appears to be very promising. AIM: To find out how THz scanning parameters of corneal tissue measured by an experimental setup, specially developed for in vivo contactless estimations of corneal reflectivity coefficient (RC), are related to pathological changes in the cornea caused by B-band ultraviolet (UVB) exposure. APPROACH: The setup was tested on rabbit eyes in vivo. Prior to the course of UVB irradiation and 1, 5, and 30 days after it, a series of examinations of the corneal state was made. At the same time points, corneal hydration was assessed by measuring RC. RESULTS: The obtained data confirmed the negative impact of UVB irradiation course on the intensity of tear production and on the corneal thickness and optical parameters. A significant (1.8 times) increase in RC on the 5th day after the irradiation course, followed by a slight decrease on the 30th day after it was revealed. The RC increase measured 5 days after the UVB irradiation course generally corresponded to the increase (by a factor of 1.3) of tear production. RC increase occurred with the corneal edema, which was manifested by corneal thickening (by 18.2% in the middle area and 17.6% in corneal periphery) and an increased volume of corneal tissue (by 17.6%). CONCLUSIONS: Our results demonstrate that the proposed approach can be used for in vivo contactless estimation of the reflectivity of rabbit cornea in the THz range and, thereby, of cornea hydration.

Alterations in proteome of human sclera associated with primary open-angle glaucoma involve proteins participating in regulation of the extracellular matrix.

Purpose: Primary open-angle glaucoma (POAG) is a common ocular disease, associated with abnormalities in aqueous humor circulation and an increase in intraocular pressure (IOP), leading to progressive optical neuropathy and loss of vision. POAG pathogenesis includes alterations of the structural properties of the sclera, especially in the optic nerve head area, contributing to the degeneration of the retinal ganglion cells. Abnormal sclera biomechanics hinder adequate compensation of IOP fluctuations, thus aggravating POAG progression. The proteomic basis of biomechanical disorders in glaucomatous sclera remains poorly understood. This study is aimed at revealing alterations in major scleral proteins, associated with POAG, at different stages of the disease and with different IOP conditions. Methods: Samples of sclera were collected from 67 patients with POAG during non-penetrating deep sclerectomy and from nine individuals without POAG. Scleral proteins were extracted with a strong lysis buffer, containing a combination of an ionic detergent, a chaotropic agent, and a disulfide reducing agent, and were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major scleral proteins were selected, subjected to in-gel digestion, and identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF)/TOF mass spectrometry (MS), coupled with tandem mass spectrometry (MS/MS). The specific POAG-associated alterations of the selected proteins were analyzed with SDS-PAGE and confirmed with western blotting of the scleral extracts, using the respective antibodies. The group of POAG-associated proteins was analyzed using Gene Ontology and genome-wide association study enrichment and protein-protein interaction network prediction. Results: A total of 11 proteins were identified, among which six proteins, namely, vimentin, angiopoietin-related protein 7, annexin A2, serum amyloid P component, serum albumin, and thrombospondin-4, were found to be upregulated in the sclera of patients with advanced and terminal POAG. In the early stages of the disease, thrombospondin-4 level was, on the contrary, reduced when compared with the control, whereas the concentration of vimentin varied, depending on the IOP level. Moreover, angiopoietin-related protein 7 manifested as two forms, exhibiting opposite behavior: The common 45 kDa form grew with the progression of POAG, whereas the 35 kDa (apparently non-glycosylated) form was absent in the control samples, appeared in patients with early POAG, and decreased in concentration over the course of the disease. Functional bioinformatics analysis linked the POAG-associated proteins with IOP alterations and predicted their secretion into extracellular space and their association with extracellular vesicles and a collagen-containing extracellular matrix. Conclusions: POAG is accompanied by alterations of the scleral proteome, which represent a novel hallmark of the disease and can reflect pathological changes in scleral biochemistry and biomechanics. The potential mechanisms underlying these changes relate mainly to the structure of the extracellular matrix, protein glycosylation, and calcium binding, and may involve fibroblast cytoskeleton regulation, as well as oxidative and inflammatory responses.

Comparative lipidomic analysis of inflammatory mediators in the aqueous humor and tear fluid of humans and rabbits.

INTRODUCTION: Ocular inflammation is a key pathogenic factor in most blindness-causing visual disorders. It can manifest in the aqueous humor (AH) and tear fluid (TF) as alterations in polyunsaturated fatty acids (PUFAs) and their metabolites, oxylipins, lipid mediators, which are biosynthesized via enzymatic pathways involving lipoxygenase, cyclooxygenase or cytochrome P450 monooxygenase and specifically regulate inflammation and resolution pathways. OBJECTIVES: This study aimed to establish the baseline patterns of PUFAs and oxylipins in AH and TF by their comprehensive lipidomic identification and profiling in humans in the absence of ocular inflammation and comparatively analyze these compounds in the eye liquids of rabbits, the species often employed in investigative ophthalmology. METHODS: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for qualitative and quantitative characterization of lipid compounds in the analyzed samples. RESULTS: A total of 28 lipid compounds were identified, including phospholipid derivatives and PUFAs, as well as 22 oxylipins. Whereas the PUFAs included arachidonic, docosahexaenoic and eicosapentaenoic acids, the oxylipins were derived mainly from arachidonic, linoleic and α-linolenic acids. Remarkably, although the concentration of oxylipins in AH was lower compared to TF, these liquids showed pronounced similarity in their lipid profiles, which additionally exhibited noticeable interspecies concordance. CONCLUSION: The revealed correlations confirm the feasibility of rabbit models for investigating pathogenesis and trialing therapies of human eye disorders. The identified metabolite patterns suggest enzymatic mechanisms of oxylipin generation in AH and TF and might be used as a reference in ocular inflammation studies.

Global trends in myopia management attitudes and strategies in clinical practice - 2019 Update.

PURPOSE: A survey in 2015 identified a high level of eye care practitioner concern about myopia with a reported moderately high level of activity, but the vast majority still prescribed single vision interventions to young myopes. This research aimed to update these findings 4 years later. METHODS: A self-administrated, internet-based questionnaire was distributed in eight languages, through professional bodies to eye care practitioners globally. The questions examined: awareness of increasing myopia prevalence, perceived efficacy of available strategies and adoption levels of such strategies, and reasons for not adopting specific strategies. RESULTS: Of the 1336 respondents, concern was highest (9.0 ±â€¯1.6; p < 0.001) in Asia and lowest (7.6 ±â€¯2.2; p < 0.001) in Australasia. Practitioners from Asia also considered their clinical practice of myopia control to be the most active (7.7 ±â€¯2.3; p < 0.001), the North American practitioners being the least active (6.3 ±â€¯2.9; p < 0.001). Orthokeratology was perceived to be the most effective method of myopia control, followed by pharmaceutical approaches and approved myopia control soft contact lenses (p < 0.001). Although significant intra-regional differences existed, overall, most practitioners did not consider single-vision distance under-correction to be an effective strategy for attenuating myopia progression (79.6 %), but prescribed single vision spectacles or contact lenses as the primary mode of correction for myopic patients (63.6 ±â€¯21.8 %). The main justifications for their reluctance to prescribe alternatives to single vision refractive corrections were increased cost (20.6 %) and inadequate information (17.6 %). CONCLUSIONS: While practitioner concern about myopia and the reported level of activity have increased over the last 4 years, the vast majority of eye care clinicians still prescribe single vision interventions to young myopes. With recent global consensus evidence-based guidelines having been published, it is hoped that this will inform the practice of myopia management in future.

Abnormal pigment epithelium-derived factor processing in progressive myopia.

Pigment Epithelium-Derived Factor (PEDF) is a secreted glycoprotein belonging to the family of non-inhibitory serpins. It is known, that in cases of complicated myopia, the content of PEDF in aqueous humor of the anterior chamber is significantly reduced. Here we examined a bulk of Tenon's capsule samples obtained from various groups of myopes, to examine PEDF processing in progressive myopia. We have analyzed the distribution of full length PEDF50 and its truncated form PEDF45 in the soluble and insoluble fractions extracted from Tenon's capsule of myopic and control (non-myopic) patients using SDS-polyacrylamide gel electrophoresis, as well as monitored the proteolytic degradation of PEDF ex vivo by enzyme-linked immunosorbent assay. These results were complemented by PEDF mRNA analysis in correspondent tissues by using qPCR and immunohistochemistry analysis of PEDF distribution in normal and myopic specimens. We found that in the Tenon's capsule of patients suffering from a high myopia the level of "soluble" 45 kDa PEDF reduced by 2-fold, while the content of "insoluble" 50 kDa form of PEDF was increased by 4-fold compared to controls. Excessive amount of PEDF50 in myopic specimens have been shown to correlate with the abrogated PEDF processing rather than with an increase of its expression. Moreover, immunohistochemical staining of the myopic Tenon's capsule tissue sections revealed the halo of deposited PEDF50 in the fibroblast extracellular space. These findings suggest that in myopia limited proteolysis of PEDF is altered or abrogated. Accumulation of full-length PEDF insoluble aggregates in the fibroblast intercellular space may affect cell survival and consequently causes the destructive changes in the extracellular matrix of the eye connective tissues. As a result, the abrogation of full-length PEDF normal processing can be an important mechanism leading to biomechanical destabilization of the scleral capsule and myopia progression.

Study of transmittance and reflectance spectra of the cornea and the sclera in the THz frequency range.

An adequate water balance (hydration extent) is one of the basic factors of normal eye function, including its external shells: the cornea and the sclera. Adequate control of corneal and scleral hydration is very important for early diagnosis of a variety of eye diseases, stating indications for and contraindications against keratorefractive surgeries and the choice of contact lens correction solutions. THz systems of creating images in reflected beams are likely to become ideal instruments of noninvasive control of corneal and scleral hydration degrees. This paper reports on the results of a study involving transmittance and reflectance spectra for the cornea and the sclera of rabbit and human eyes, as well as those of the rabbit eye, in the frequency range of 0.13 to 0.32 THz. The dependence of the reflectance coefficient of these tissues on water mass percentage content was determined. The experiments were performed on three corneas, three rabbit scleras, two rabbit eyes, and three human scleras. The preliminary results demonstrate that the proposed technique, based on the use of a continuous THz radiation, may be utilized to create a device for noninvasive control of corneal and scleral hydration, which has clear potential of broad practical application.

Rabbit Models of Ocular Diseases: New Relevance for Classical Approaches.

Over 100 million individuals are affected by irreversible visual impairments and blindness worldwide, while ocular diseases remain a challenging problem despite significant advances in modern ophthalmology. Development of novel drugs and drug delivery mechanisms, as well as advanced ophthalmological techniques requires experimental models including animals, capable of developing ocular diseases with similar etiology and pathology, suitable for future trials of new therapeutic approaches. Although experimental ophthalmology and visual research are traditionally performed on rodent models, these animals are often unsuitable for pre-clinical drug efficacy and safety studies, as well as for testing novel drug delivery approaches, e.g. controlled release of pharmaceuticals using intra-ocular implants. Therefore, rabbit models of ocular diseases are particularly useful in this context, since rabbits can be easily handled, while sharing more common anatomical and biochemical features with humans compared to rodents, including longer life span and larger eye size. This review provides a brief description of clinical, morphological and mechanistic aspects of the most common ocular diseases (dry eye syndrome, glaucoma, age-related macular degeneration, light-induced retinopathies, cataract and uveitis) and summarizes the diversity of current strategies for their experimental modeling in rabbits. Several applications of some of these models in ocular pharmacology and eye care strategies are also discussed.

Mitochondria-targeted antioxidant SkQ1 reverses glaucomatous lesions in rabbits.

Glaucoma is the main cause of irreversible blindness worldwide. This disease is characterized by apoptosis of retinal ganglion cells (RGC) and visual field loss that seems to be related to elevated intraocular pressure (IOP). Several lines of evidences have implicated the crucial role of mitochondrial dysfunction in the pathogenesis of glaucoma. Increased mitochondrial oxidative stress in RGC may underlie or contribute to susceptibility of RGC to apoptosis. In our work we (i) designed a rabbit model of chronic, moderately elevated IOP for studying glaucoma and (ii) demonstrated efficacy of mitochondria-targeted antioxidant SkQ1 as a tool to reverse several traits of experimental glaucoma induced by a series of injections of hydroxypropylmethylcellulose (HPMC) to the anterior chamber of the rabbit eye. It is shown that 6 months instillations of drops of 0.2.5-5 microM solution of SkQ1 normalize IOP and eye hydrodynamics and abolish an increase in lens thickness that accompanies glaucoma.

Sclera as the target tissue in progressive myopia.

UNLABELLED: Introduction: The aim of the study was to study the eye corneoscleral shell and connective tissue (CT) system in children with acquired and congenital progressive myopia, and to identify the informative diagnostic criteria of weakened supporting function of the sclera. MATERIAL AND METHODS: 155 children aged 9-17 were examined: 18 had emmetropia or hyperopia, 20--low, 32--moderate and 85--high myopia. 32 children had complicated myopia (CM) due to peripheral retinal degeneration (PRD). Corneal hysteresis (CH), scleral acoustic density (SAD), X-ray vertebral topography, plantography, joint hypermobility, serum cortisol (SC), and autonomic balance were measured. RESULTS: Corneal hysteresis, mmHg and SAD, rel. units (mean ± SE) were lower if myopia was higher: in low myopia, CH was 13.0 ± 0.3, in moderate myopia, 11.9 ± 0.3#, in high myopia, 10.7 ± 0.3#; SAD was resp. 215.9 ± 5.2, 204.9 ± 3.7# and 192.8 ± 5.8# (#: p < 0.05 with regard to low myopia). The lowest CH (10.3 ± 0.4) and SAD (186.5 ± 7.3) were found in acquired CM. Congenital myopia with PRD showed CH and SAD greater than in acquired CM (p < 0.05). Serum cortisol (nmol/L) in hyperopia was 335.8 ± 40.9 and dropped with higher myopia: in low myopia--290.7 ± 58.6, in moderate--250.9 ± 26.4, in high--243.9 ± 20.5. The lowest SC was found in acquired CM, consistent with CH and SAD. Connective tissue dysplasia progressed with higher myopia: it was found in 76.9% of children with low, 82.4% with moderate, 89.2% with high acquired myopia, and 91.7% with congenital myopia. Biomechanical defects of CT and hormonal imbalance were combined with vegetative nervous system (VNS) imbalance: in high myopia only 20.5% of children were eutonic, 61.5%--sympathicotonic and 17.5% parasympathicotonic. CONCLUSIONS: School age children with progressive myopia showed biomechanical abnormalities of the corneosclera, along with CT dysplasia, decreased SC and imbalanced VNS, more pronounced in acquired complicated myopia. Our findings contribute to the understanding of myopia pathogenesis and to the diagnostic/prognostic evaluation of myopic children, in particular when deciding whether sclera-strengthening intervention is needed.

Biomechanical and histological changes after corneal crosslinking with and without epithelial debridement.

PURPOSE: To test the biomechanical efficiency of corneal crosslinking with riboflavin without epithelial debridement (C3-R). SETTING: Moscow Helmholtz Research Institute of Eye Diseases, Moscow, Russia. METHODS: The left eyes of rabbits were crosslinked using standard crosslinking including epithelial removal (Group 1), using benzalkonium chloride-containing proxymetacaine eyedrops without epithelial removal (Group 2), or using preservative-free oxybuprocaine eyedrops without epithelial removal (Group 3). All left eyes received riboflavin solution and were irradiated with an ultraviolet-A double diode for 30 minutes (irradiance 3 mW/cm(2)). The animals were killed 1 day after crosslinking. Biomechanical and histological analyses were performed. RESULTS: Fourteen eyes were evaluated. There was a statistically significant increase in Young's modulus in Group 1 (102.45%) and in Group 2 (21.30%). In Group 3, no biomechanical changes were measured. Histology showed complete cell loss of keratocytes and endothelium in Group 1 and inhomogeneous keratocyte loss down to 200.0 microm in Group 2. No changes were observed in Group 3. CONCLUSIONS: Corneal crosslinking without epithelial debridement reduced the biomechanical effect by approximately one fifth compared with standard crosslinking, probably because of restricted and inhomogeneous stromal distribution of riboflavin. The cytotoxic damage was restricted to 200.0 microm stromal depth, which is an advantage over the standard method. Therefore, C3-R is not recommended for the routine treatment of keratoconus but primarily for cases with a corneal thickness less than 400.0 microm in which standard crosslinking cannot be used without serious risk to the endothelium.

Long-term biomechanical properties of rabbit sclera after collagen crosslinking using riboflavin and ultraviolet A (UVA).

PURPOSE: Scleral crosslinking by the photosensitizer riboflavin and ultraviolet A (UVA) has been shown to increase significantly the scleral biomechanical rigidity and might therefore become a possible sclera-based treatment modality for progressive myopia. In the present study, the long-term effect of the new crosslinking method on biomechanical properties was investigated in the rabbit sclera. METHODS: A 10 x 10 mm sector of the equatorial sclera of nine Chinchilla rabbit eyes was treated in vivo using a UVA double diode of 370 nm with a surface irradiance of 3 mW/cm(2) and application of 0.1% riboflavin-5-phosphate drops as photosensitizer for 30 min. Three days, 4 months and 8 months postoperatively, biomechanical stress-strain measurements of the treated scleral strips were performed and compared to contralateral control sclera using a microcomputer-controlled biomaterial tester. In addition, routine histological controls were performed. RESULTS: Following the crosslinking treatment, Young's modulus was increased by 320% after 3 days, 277% after 4 months and 502% after 8 months, and ultimate stress by 341% after 3 days, 131% after 4 months and 213.8% after 8 months versus the controls. The decrease in ultimate strain was between 24% and 44.8%. On histology, no tissue damage was detected. CONCLUSION: Our new method of scleral collagen crosslinking proved very effective and constant over a time interval of up to 8 months in increasing the scleral biomechanical strength. Therefore, the new treatment might become an option for strengthening scleral tissue in progressive myopia and other conditions associated with weakened sclera. There were no side-effects on the retina or retinal pigment epithelium. The new crosslinking treatment could now be tested in a suitable myopia model (like the tree shrew) and finally in human eyes.

Effects of poly(2-hydroxyethyl methacrylate) and poly(vinyl-pyrrolidone) hydrogel implants on myopic and normal chick sclera.

There has been generally little attention paid to the utilization of biomaterials as an anti-myopia treatment. The purpose of this study was to investigate whether polymeric hydrogels, either implanted or injected adjacent to the outer scleral surface, slow ocular elongation. White Leghorn (Gallus gallus domesticus) chicks were used at 2 weeks of age. Chicks had either (1) a strip of poly(2-hydroxyethyl methacrylate) (pHEMA) implanted monocularly against the outer sclera at the posterior pole, or (2) an in situ polymerizing gel [main ingredient: poly(vinyl-pyrrolidone) (PVP)] injected monocularly at the same location. Some of the eyes injected with the polymer were fitted with a diffuser or a -10D lens. In each experiment, ocular lengths were measured at regular intervals by high frequency A-scan ultrasonography, and chicks were sacrificed for histology at staged intervals. No in vivo signs of either orbital or ocular inflammation were observed. The pHEMA implant significantly increased scleral thickness by the third week, and the implant became encapsulated with fibrous tissue. The PVP-injected eyes left otherwise untreated, showed a significant increase in scleral thickness, due to increased chondrocyte proliferation and extracellular matrix deposition. However, there was no effect of the PVP injection on ocular elongation. In eyes wearing optical devices, there was no effect on either scleral thickness or ocular elongation. These results represent "proof of principle" that scleral growth can be manipulated without adverse inflammatory responses. However, since neither approach slowed ocular elongation, additional factors must influence scleral surface area expansion in the avian eye.

Long-term biomechanical properties of rabbit cornea after photodynamic collagen crosslinking.

PURPOSE: Photodynamic riboflavin/ultraviolet-A (UVA)-induced collagen cross-linking, which increases the biomechanical stiffness of the human cornea by about 300%, has been introduced recently as a possible treatment for progressive keratoconus. The present study was undertaken to evaluate the longterm biomechanical effects of this new cross-linking treatment as a necessary prerequisite to its clinical success. METHODS: The corneas of the left eyes of nine male rabbits were cross-linked. The contralateral eyes served as controls. After removal of the central 7 mm of the epithelium, the corneas were treated with the photosensitizer riboflavin and UVA irradiation for 30 mins with an irradiance of 3 mW/cm(2) using a 370-nm UVA double diode. Groups of three animals were killed immediately after treatment and at 3 and 8 months, respectively. Biomechanical stress-strain measurements were performed using a microcomputer-controlled biomaterial tester on 4 x 10-mm corneal strips. RESULTS: Corneal thickness in the treated rabbit cornea was 408 +/- 20 microm. A constant and significant increase in ultimate stress (of 69.7-106.0%), Young's modulus of elasticity (of 78.4-87.4%) and a decrease in ultimate strain (of 0.57-78.4%) were found over a time period of up to 8 months after cross-linking treatment. CONCLUSIONS: Riboflavin/UVA-induced collagen cross-linking leads to a longterm increase in biomechanical rigidity which remains stable over time. These data support our previous longterm clinical observations and give hope that this new treatment will halt progressive keratoconus definitively.

Long-term biomechanical properties after collagen crosslinking of sclera using glyceraldehyde.

PURPOSE: Chemical crosslinking by glyceraldehyde has been shown to increase significantly the biomechanical rigidity of sclera. It might therefore become an option for a sclera-based treatment of progressive myopia. The present pilot study was designed to test the long-term biomechanical efficiency of the new crosslinking method. METHODS: Six Chinchilla rabbits were treated with sequential sub-Tenon's injections of 0.15 ml 0.5 m glyceraldehyde, which were given in the supero-nasal quadrant of the right eye (OD) five times over 14 days. The rabbits were killed 4 months and 8 months after crosslinking treatment, respectively. Biomechanical stress-strain measurements of scleral strips from the treatment area were performed and compared to non-treated contralateral control sclera using a microcomputer-controlled biomaterial testing device. In addition, the eyes were examined histologically by light microscopy to evaluate possible side-effects. RESULTS: Following the crosslinking treatment, the ultimate stress was 10.2 +/- 2.3 MPa after 4 months and 8.5 +/- 2.2 MPa after 8 months versus 2.4 +/- 0.3 MPa in the controls (increases of 325% and 254.17%, respectively); Young's modulus was 104.6 +/- 13.7 MPa after 4 months and 53.2 +/- 5.2 MPa after 8 months versus 9.6 +/- 1.3 MPa in the controls (increases of 989.6% and 554.17%, respectively); and ultimate strain was 15.8 +/- 1.5% after 4 months and 24.1 +/- 0.7% after 8 months versus 38.4 +/- 4.6% in the controls (decreases of 58.84% and 37.24%, respectively). Histologically, no side-effects were found. CONCLUSION: Our new method of scleral collagen crosslinking proved very efficient in increasing scleral biomechanical strength over a period of up to 8 months. Glyceraldehyde can be applied easily by sequential parabulbar injections. Before clinical application in myopic patients, a study in an animal myopia model is recommended.

Crosslinking of scleral collagen in the rabbit using glyceraldehyde.

PURPOSE: To strengthen rabbit sclera in vivo using chemical crosslinking with glyceraldehyde for a scleral-based treatment of progressive myopia. SETTING: Department of Ophthalmology, Martin-Luther-University, Halle, Germany. METHODS: Five chinchilla rabbits were treated with sequential sub-Tenon injections of 0.15 mL 0.5 M glyceraldehyde into the superonasal quadrant of the right eye 5 times during 14 days. The rabbits were humanely killed and biomechanical stress-strain measurements of scleral strips from the treatment area were performed and compared with nontreated contralateral control sclera using a microcomputer-controlled biomaterial tester. The treated eyes were examined histologically by light microscopy to exclude possible adverse effects. RESULTS: Following the crosslinking treatment, the ultimate stress was 15.8 MPa +/- 6.0 (SD) versus 3.1 +/- 0.3 MPa in the controls (increase of 409.7%; P<.02), the Young modulus was 129.6 +/- 53.7 MPa versus 11.5 +/- 1.8 MPa in the controls (increase of 1027%, P<.01), and ultimate strain was 19.8% +/- 2.6% MPA versus 38.2% +/- 5.1% MPA in the controls (decrease of 48.2% P<.05). Histologically, mild side effects were found in the peripheral cornea adjacent to the treatment area, with some inflammatory infiltrate and moderate loss of keratocytes. CONCLUSIONS: Glyceraldehyde crosslinking of scleral collagen increased the scleral biomechanical rigidity efficiently. Glyceraldehyde can be easily applied by sequential parabulbar injections. There were no side effects on the retina, so the new method might become a treatment modality for strengthening scleral tissue to prevent progressive myopia.