Search

Objectives: Reducing the burden of bilirubin-induced neurologic complications in low-resource countries requires reliable and accessible screening tools. We sought to optimize and validate a sclera-based smartphone application, Neonatal Scleral-Conjunctival Bilirubin (neoSCB), for screening neonatal jaundice., Methods: Using a cross-sectional design, consecutive eligible infants (aged 0-28 days, in the hospital, not critically ill) were enrolled in Ghana from March 2019 to April 2020. Jaundice screening was performed with neoSCB (Samsung Galaxy S8) to quantify SCB and JM-105 (Dräger) for transcutaneous bilirubin (TcB). Screening values were compared with total serum bilirubin (TSB) measured at the point of care., Results: Overall, 724 infants participated in the optimization and validation phases of the study. The analysis for validation included 336 infants with no previous treatment of jaundice. Single neoSCB image captures identified infants with TSB >14.62 mg/dL (250 μmol/L) with reasonably high sensitivity, specificity, and receiver operating characteristic area under the curve at 0.94 (95% confidence interval [CI], 0.91 to 0.97), 0.73 (95% CI, 0.68 to 0.78), and 0.90, respectively. These findings were comparable to the sensitivity and specificity of JM-105 (0.96 [95% CI, 0.90 to 0.99] and 0.81 [95% CI, 0.76 to 0.86], respectively). The TcB/TSB had a larger correlation coefficient (r = 0.93; P < .01) than SCB/TSB (r = 0.78; P < .01). Performance of both devices was lower in infants with previous phototherapy (n = 231)., Conclusions: The diagnostic performance of neoSCB was comparable to JM-105 and is a potential, affordable, contact-free screening tool for neonatal jaundice., (Copyright © 2022 by the American Academy of Pediatrics.)

Contact lenses (CLs) are being pointed out as feasible platforms for controlled delivery of ophthalmic drugs. Bioinspired strategies may endow CLs with affinity for a given drug by mimicking its physiological receptor using adequate functional monomers and tuning their conformation in the space through the molecular imprinting technology. However, there are some active substances, such as efficient antioxidant agents, that cannot be used as templates because they degrade during polymerization or even hinder the polymerization itself. Therefore, the development of CLs able to sustain the release of antioxidants for the prevention and/or treatment of several age-related and light-induced eye diseases has not been explored yet. Searching for an alternative bioinspired strategy, the present work relies on the fact that some drugs owe their therapeutic action to their ability to interact with nucleotides that build up DNA and RNA. Thus, the aim of this work was to design hydrogels functionalized with the nitrogenous base cytosine for the controlled uptake and release of transferulic acid (TA) having a complementary chemical structure in terms of hydrogen bonding and π-π stacking ability. Hydrogels were prepared from mixtures of 2-hydroxyethyl methacrylate (HEMA), glycidyl methacrylate (GMA) and ethyleneglycolphenylether methacrylate (EGPEM). GMA was used as a bridge to immobilize cytosine after hydrogel synthesis, while EGPEM was added to reinforce hydrophobic interactions with TA. The hydrogels were characterized in terms of suitability to be used as CLs (swelling, light transmission, mechanical properties, biocompatibility) and capability to host TA and sustain its release in lachrymal fluid while maintaining the antioxidant activity. Relevantly, the bioinspired CLs favored TA accumulation in cornea and sclera tissues.

The quantitative analysis of tear analytes in point-of-care settings can enable early diagnosis of ocular diseases. Here, a fluorescent scleral lens sensor is developed to quantitatively measure physiological levels of pH, Na + , K + , Ca 2+ , Mg 2+ , and Zn 2+ ions. Benzenedicarboxylic acid, a pH probe, displays a sensitivity of 0.12 pH units within pH 7.0-8.0. Crown ether derivatives exhibit selectivity to Na + and K + ions within detection ranges of 0-100 and 0-50 mmol L -1 , and selectivities of 15.6 and 8.1 mmol L -1 , respectively. A 1,2 bis(o-aminophenoxy)ethane-N,N,-N',N'-tetraacetic-acid-based probe allows Ca 2+ ion sensing with 0.02-0.05 mmol L -1 sensitivity within 0.50-1.25 mmol L -1 detection range. 5-Oxazolecarboxylic acid senses Mg 2+ ions, exhibiting a sensitivity of 0.10-0.44 mmol L -1 within the range of 0.5-0.8 mmol L -1 . The N-(2-methoxyphenyl)iminodiacetate Zn 2+ ion sensor has a sensitivity of 1 µmol L -1 within the range of 10-20 µmol L -1 . The fluorescent sensors are subsequently multiplexed in the concavities of an engraved scleral lens. A handheld ophthalmic readout device comprising light-emitting diodes (LEDs) and bandpass filters is fabricated to excite as well as read the scleral sensor. A smartphone camera application and an user interface are developed to deliver quantitative measurements with data deconvolution. The ophthalmic system enables the assessment of dry eye severity stages and the differentiation of its subtypes., (© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We present the results obtained on DNA extracted from ocular (scleral/corneal) swabs collected from exhumed bodies at different times of burial. To our knowledge, there are no publications in the scientific forensic literature dealing with sclera/cornea as a source of DNA in the forensic laboratory. The obtained results demonstrate that cornea/sclera swabbing might be a promising alternative to the sampling of other tissues for DNA extraction even in highly putrefied bodies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This paper aims to present a novel full-eye biomechanical material model that incorporates the characteristics of ocular tissues at microstructural level, and use the model to analyse the age-related stiffening in tissue behaviour. The collagen content in ocular tissues, as obtained using X-ray scattering measurements, was represented by sets of Zernike polynomials that covered both the cornea and sclera, then used to reconstruct maps of collagen fibril magnitude and orientation on the three-dimensional geometry of the eye globe. Fine-mesh finite-element (FE) models with eye-specific geometry were built and supported by a user-defined material model (UMAT), which considered the regional variation of fibril density and orientation. The models were then used in an iterative inverse modelling study to derive the material parameters that represent the experimental behaviour of ocular tissues from donors aged between 50 and 90 years obtained in earlier ex vivo studies. Sensitivity analysis showed that reducing the number of directions that represented the anisotropy of collagen fibril orientation at each X-ray scattering measurement point from 180 to 16 would have limited and insignificant effect on the FE solution (0.08%). Inverse analysis resulted in material parameters that provided a close match with experimental intraocular pressure-deformation behaviour with a root mean square of error between 3.6% and 4.3%. The results also demonstrated a steady increase in mechanical stiffness in all ocular regions with age. A constitutive material model based on distributions of collagen fibril density and orientation has been developed to enable the accurate representation of the biomechanical behaviour of ocular tissues. The model offers a high level of control of stiffness and anisotropy across ocular globe, and therefore has the potential for use in planning surgical and medical procedures.

The stiffness of the sclera is important in several ocular disorders, and there is hence a need to quantify the biomechanical properties of this tissue. Here, we present two methods for measuring the stiffness of scleral ocular tissues: ocular compliance testing and digital image correlation strain mapping. In tandem with these approaches, we provide two methods to spatially quantify the anisotropic alignment of collagen fibers making up the sclera, using second harmonic generation microscopy and small-angle light scattering. Together, these approaches allow specimen-specific measurement of tissue stiffness and collagen alignment, which are key factors in determining how the eye responds to mechanical loads.

Age-related macular degeneration (AMD) is a leading cause of blindness in the modern world. The standard treatment regimen for neovascular AMD is the monthly/bimonthly intravitreal injection of anti-VEGF agents such as ranibizumab or aflibercept. However, these repeated invasive injections can lead to sight-threatening complications. Sustained delivery by encapsulation of the drug in carriers is a way to reduce the frequency of these injections. Liposomes are biocompatible, non-toxic vesicular nanocarriers, which can be used to encapsulate therapeutic agents to provide sustained release. The protein encapsulation was performed by a modified dehydration-rehydration (DRV) method. The liposomes formed were characterized for size, zeta potential, encapsulation efficiency, stability, in vitro release, and ex vivo release profiles. In addition, the localization of the liposomes themselves was studied ex vivo. Entrapment-efficiency of ranibizumab into 100-nm liposomes varied from 14.7 to 57.0%. Negatively-charged liposomes prepared from DPPC-DPPG were found to have the slowest release with a low initial burst release compared to the rest of liposomal formulations. The ex vivo protein release was found to slower than the in vitro protein release for all samples. In conclusion, the DPPC-DPPG liposomes significantly improved the encapsulation and release profile of ranibizumab.

It is widely considered that intraocular pressure (IOP)-induced deformation within the neural tissue pores of the lamina cribrosa (LC) contributes to neurodegeneration and glaucoma. Our goal was to study how the LC microstructure and mechanical properties determine the mechanical insult to the neural tissues within the pores of the LC. Polarized light microscopy was used to measure the collagen density and orientation in histology sections of three sheep optic nerve heads (ONH) at both mesoscale (4.4μm) and microscale (0.73μm) resolutions. Mesoscale fiber-aware FE models were first used to calculate ONH deformations at an IOP of 30mmHg. The results were then used as boundary conditions for microscale models of LC regions. Models predicted large insult to the LC neural tissues, with 95th percentile 1st principal strains ranging from 7 to 12%. Pores near the scleral boundary suffered significantly higher stretch compared to pores in more central regions (10.0±1.4% vs. 7.2±0.4%; p=0.014; mean±SD). Variations in material properties altered the minimum, median, and maximum levels of neural tissue insult but largely did not alter the patterns of pore-to-pore variation, suggesting these patterns are determined by the underlying structure and geometry of the LC beams and pores. To the best of our knowledge, this is the first computational model that reproduces the highly heterogeneous neural tissue strain fields observed experimentally., Statement of Significance: The loss of visual function associated with glaucoma has been attributed to sustained mechanical insult to the neural tissues of the lamina cribrosa due to elevated intraocular pressure. Our study is the first computational model built from specimen-specific tissue microstructure to consider the mechanics of the neural tissues of the lamina separately from the connective tissue. We found that the deformation of the neural tissue was much larger than that predicted by any recent microstructure-aware models of the lamina. These results are consistent with recent experimental data and the highest deformations were found in the region of the lamina where glaucomatous damage first occurs. This study provides new insight into the complex biomechanical environment within the lamina., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Purpose: The purpose of this study was to leverage polarized light microscopy (PLM) to visualize the collagen fiber architecture of posterior pole and optic nerve head with micrometer-scale resolution and to identify and quantify major organizational components., Methods: Eight sheep posterior poles were cryosectioned and imaged using PLM. Collagen fiber orientation was determined by using custom scripts, and the resulting orientation maps were inspected and quantified to identify major structural elements and tested for differences in mean fiber orientation and anisotropy, using linear mixed effect models., Results: Images revealed an intricate organization of collagen fibers in the posterior pole. In the lamina cribrosa, interweaving fibers formed large knots and wrapped around nerve fiber pores, with beam insertions into the scleral canal wall that were either narrow and straight or wide. In the peripapillary sclera, three significantly different (P < 0.0001) components were identified: fibers oriented circumferentially proximal to the canal, radially in the innermost sclera, and unaligned with interweaving fibers. The radial fibers were between 60 and 180 μm thick, extending at least 3 mm from the canal., Conclusions: PLM revealed structural aspects of the lamina cribrosa and sclera that may have important biomechanical roles but that were previously unreported or not characterized quantitatively. In the lamina cribrosa, these roles included wide and narrow beam insertions and details of collagen fibers interweaving and wrapping around the pores. In the sclera, we described regions of circumferential, radial, and unaligned "random" fibers. Although there is consensus that circumferential fibers protect neural tissues by resisting canal expansion, the role of the radial fibers remains unclear.

This work presents an optospectroscopic characterization technique for soft tissue microstructure using site-matched confocal Raman microspectroscopy and polarized light microscopy. Using the technique, the microstructure of soft tissue samples is directly observed by polarized light microscopy during loading while spatially correlated spectroscopic information is extracted from the same plane, verifying the orientation and arrangement of the collagen fibers. Results show the response and orientation of the collagen fiber arrangement in its native state as well as during tensile and compressive loadings in a porcine sclera model. An example is also given showing how the data can be used with a finite element program to estimate the strain in individual collagen fibers. The measurements demonstrate features that indicate microstructural reorganization and damage of the sclera's collagen fiber arrangement under loading. The site-matched confocal Raman microspectroscopic characterization of the tissue provides a qualitative measure to relate the change in fibrillar arrangement with possible chemical damage to the collagen microstructure. Tests and analyses presented here can potentially be used to determine the stress-strain behavior, and fiber reorganization of the collagen microstructure in soft tissue during viscoelastic response., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Bacterial keratitis is a sight threatening infection of the cornea which remains one of the most important potential complications of contact lens use. If the corneal ulcer is small, peripheral with no impending perforation present, intensive monotherapy with fluoroquinolones could be used. Therefore, a study was conducted with the objective to provide pharmacological data of the intra-ocular diffusion after administration of Ofloxacin using a scleral lens reservoir, as well as an evaluation of surface tolerability in rabbits., Materials and Methods: Samples of corneas, aqueous humor and vitreous were collected to measure the drug levels of Ofloxacin using High Performance Liquid Chromatography. The corneas were examined by electron microscopy scanning and the eyeballs by light polarizing microscopy in order to evaluate surface tolerability., Results: Ofloxacin levels found in the aqueous humor and cornea were higher than those previously reported. The mean Ofloxacin corneal levels exceeded the MIC (Minimum Inhibitory Concentration) for which 90% of isolates are indicated for all bacteria implicated in keratitis., Conclusion: To our knowledge, this is the first preclinical study assessing local tolerance and intra-ocular diffusion of Ofloxacin after administration using a scleral lens reservoir., (Copyright © 2016 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

We evaluated nano-structural and chemical changes in human scleral collagen caused by non-enzymatic glycation using AFM, Raman spectroscopy, and microfluidics. Twenty 8 × 2 mm 2 scleral strips (n = 5, each) were divided into four groups of pure sclera tissues (control group) and sclera tissues with incubation (1 hr in BSS and ribose) and preservation (23 hr in 90% ethanol) for 7 days (BSS + DR 7 group) and 30 days (BSS + DR 30 group) at room temperature, and 7 days in a microfluidic chip (BSS + DR + µF 7 group). The BSS + DR 7 and BSS + DR 30 groups were incubated in a mixture of balanced salt solution (BSS) and 0.2 M D-ribose in PBS, pH 7.4 containing 0.1% sodium azide, while the BSS + DR + µF 7 group was incubated in the same solutions supplied by two inlet reservoirs from a microfluidic chip. The scleral tissues incubated in the microfluidic environment showed a clear irregular parallel arrangement of collagen fibrils with tangled fibrils. A Raman shift was observed at 919 cm -1 in the glycation groups. Non-enzymatic glycation led to an increased in the density of scleral stromal collagen. Our method using non-enzymatic glycation in a microfluidic environment successfully induced collagen cross-linking. These in vitro results suggested that glycation can be used to strengthen connective tissues. SCANNING 38:421-426, 2016. © 2016 Wiley Periodicals, Inc., (© Wiley Periodicals, Inc.)

We conducted Monte Carlo simulations based on anisotropic sclera-mimicking models to examine the polarization features in Mueller matrix polar decomposition (MMPD) parameters during the refractive index matching process, which is one of the major mechanisms of optical clearing. In a preliminary attempt, by changing the parameters of the models, wavelengths, and detection geometries, we demonstrate how the depolarization coefficient and retardance vary during the refractive index matching process and explain the polarization features using the average value and standard deviation of scattering numbers of the detected photons. We also study the depth-resolved polarization features during the gradual progression of the refractive index matching process. The results above indicate that the refractive index matching process increases the depth of polarization measurements and may lead to higher contrast between tissues of different anisotropies in deeper layers. MMPD-derived polarization parameters can characterize the refractive index matching process qualitatively.

Immersion optical clearing makes it possible to use transmission polarized-light microscopy for characterization of thick (200 to 2000  μm) layers of biological tissues. We discuss polarization properties of thick samples in the context of the problem of characterization of collagen fiber alignment in connective tissues such as sclera and dermis. Optical chirality caused by azimuthal variations of the macroscopic (effective) optic axis of the medium across the sample thickness should be considered in polarization mapping of thick samples of these tissues. We experimentally evaluate to what extent the optical chirality affects the measurement results in typical situations and show under what conditions it can be easily taken into account and does not hinder, but rather helps, in characterization of collagen fiber alignment.

We characterized the structural and mechanical changes after experimental digestion of sulfated glycosaminoglycans (s-GAGs) in the human posterior sclera, using ultrasound thickness measurements and an inflation test with three-dimensional digital image correlation (3D-DIC). Each scleral specimen was first incubated in a buffer solution to return to full hydration, inflation tested, treated in a buffer solution with chondroitinase ABC (ChABC), then inflation tested again. After each test series, the thickness of eight locations was measured. After enzymatic treatment, the average scleral thickness decreased by 13.3% (p < 0.001) and there was a stiffer overall stress-strain response (p < 0.05). The stress-strain response showed a statistically significant increase in the low-pressure stiffness, high-pressure stiffness and hysteresis. Thus, s-GAGs play a measurable role in the mechanical behaviour of the posterior human sclera., (© 2016 The Author(s).)

Age estimation based on racemization of aspartic acid residues (AAR) in permanent proteins has been established in forensic medicine for years. While dentine is the tissue of choice for this molecular method of age estimation, teeth are not always available which leads to the need to identify other suitable tissues. We examined the suitability of total tissue samples of human sclera for the estimation of age at death. Sixty-five samples of scleral tissue were analyzed. The samples were hydrolyzed and after derivatization, the extent of aspartic acid racemization was determined by gas chromatography. The degree of AAR increased with age. In samples from younger individuals, the correlation of age and D-aspartic acid content was closer than in samples from older individuals. The age-dependent racemization in total tissue samples proves that permanent or at least long-living proteins are present in scleral tissue. The correlation of AAR in human sclera and age at death is close enough to serve as basis for age estimation. However, the precision of age estimation by this method is lower than that of age estimation based on the analysis of dentine which is due to molecular inhomogeneities of total tissue samples of sclera. Nevertheless, the approach may serve as a valuable alternative or addition in exceptional cases.

The aim of the present study was to evaluate the effect of flaxseed on choroid-sclera complex thickness and on LDL oxidation in the sclera, choroid and retina of diet-induced hypercholesterolaemic rabbits. New Zealand male albino rabbits (n 21) were divided into two groups: group 1 (G1; n 11), fed a hypercholesterolaemic diet, and group 2 (G2; n 10), fed a hypercholesterolaemic diet enriched with flaxseed flour. The serum concentrations of total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol, TAG and fasting blood glucose were determined at the start of the experiment and on the day of killing (8th week). Choroid and sclera samples were subjected to haematoxylin-eosin (HE) staining and histomorphometric and immunohistochemical analyses with the anti-oxidised LDL antibody. Sensory retina samples were subjected to an immunohistochemical analysis with the primary monoclonal nitrotyrosine antibody. At the end of the experiment, a significant increase was observed in TC and LDL-C concentrations in G1 rabbits when compared with G2 rabbits (P= 0·008 and P= 0·02, respectively). HE staining revealed a significant increase in choroid-sclera complex thickness in G1 rabbits when compared with G2 rabbits (P< 0·001). Immunohistochemical analysis of choroid and sclera samples with the anti-oxidised LDL marker revealed a significant increase in immunoreactivity in G1 rabbits when compared with G2 rabbits (P< 0·001). Immunohistochemical analysis of sensory retina samples with the anti-nitrotyrosine marker revealed a significant increase in immunoreactivity in G1 rabbits when compared with G2 rabbits (P= 0·002). Flaxseed reduced the choroid-sclera complex thickness of diet-induced hypercholesterolaemic rabbits and the expression of oxidised LDL in the choroid-sclera complex as well as the expression of nitrotyrosine in the sensory retina.

This study examines the elastic properties of the human posterior retina, choroid, and sclera. Twenty-four human eyes from 30- to 74-year-old donors were obtained from an eye bank. Vertically and horizontally oriented tissue strips of the retina, choroid, and sclera (ideally n = 12 in each group) were harvested from the posterior eyes. Their thicknesses were estimated optically. The samples were stretched at 1 mm/s in 37°C saline. Stress and strain were obtained from the mechanical tests, and then the transition stress, transition strain, toe modulus, and heel modulus were calculated. Statistical analysis was performed for comparison between groups. Linear regression analyses were used to explore the relationship between the mechanical parameters and age. We found that the stress-strain relationship of the retina, choroid, and sclera were nonlinear. Except for the retinal transition strain (p = 0.0124), no statistical difference was found between the vertical and horizontal meridian in the mechanical parameters (p > 0.05). Furthermore, weak relationship was observed between some of the mechanical parameters and the donors' age. Our results suggest that there is significant anisotropy in the retina, and mechanical properties of each layer may change with age., (© 2013 Wiley Periodicals, Inc.)

This study quantitatively investigated the immediate effects of a photooxidative collagen cross-linking treatment with photosensitizer riboflavin (RF) and 370 nm UVA light in in vitro human corneoscleral collagen fibrils using histology, thickness, scanning electron microscopy, and atomic force microscopy analyses. Twenty 8 x 2 mm corneoscleral strips were dissected sagittally from donor tissue using a scalpel. Four parameters were investigated, including the density, thickness, adhesion force, and stiffness of corneoscleral tissues before and after the collagen cross-linking treatment. The RFUVA-catalyzed collagen cross-linking treatment led to an increase in the density of both corneal (8%) and scleral (23%) stromal collagens. However, there was no difference in corneoscleral thickness. Furthermore, RFUVA-catalyzed collagen cross-linking treatment led to an increased biomechanical response of corneosclera: 25 and 8% increases in corneoscleral stiffness, and 24 and 22% increases in corneoscleral adhesion force. The collagen cross-linking treatment through RF-sensitized photoreaction may cause structural and biomechanical changes in the collagen fibril network of the cornea and the sclera. This is due to narrowing of the interfibrillar spacing and the stromal edema.

High success rates in clinical trials on keratoconic corneas suggest the possibility of efficient treatment against myopic progression. This study quantitatively investigated the in vitro ultrastructural effects of a photooxidative collagen cross-linking treatment with photosensitizer riboflavin and UVA light in human corneo-scleral collagen fibrils. A total of 30.8 × 2 mm corneo-scleral strips from donor tissue were sagittally dissected using a scalpel. The five analytic parameters namely fibril density, fibril area, corneo-scleral thickness, fibril diameter, and fibril arrangement were investigated before and after riboflavin-UVA-catalyzed collagen cross-linking treatment. Collagen cross-linking effects were measured at the corneo-scleral stroma and were based on clinical corneal cross-linking procedures. The structural response levels were assessed by histology, digital mechanical caliper measurement, scanning electron microscopy, and atomic force microscopy. Riboflavin-UVA-catalyzed collagen cross-linking treatment led to an increase in the area, density, and diameters of both corneal (110, 112, and 103 %) and scleral (133, 133, and 127 %) stromal collagens. It also led to increases in corneal (107 %) and scleral (105 %) thickness. Collagen cross-linking treatment through riboflavin-sensitized photoreaction may cause structural property changes in the collagen fibril network of the cornea and sclera due to stromal edema and interfibrillar spacing narrowing. These changes were particularly prominent in the sclera. This technique can be used to treat progressive keratoconus in the cornea as well as progressive myopia in the sclera. Long-term collagen cross-linking treatment of keratoconic and myopic progression dramatically improves weakened corneo-scleral tissues.

Optical properties of cornea and sclera of the eye and their alterations under the effect of 1.56-μm laser radiation are studied. The laser settings corresponded to the laser treatment regimens used (1) to correct the shape of the cornea and change the refraction of the eye and (2) to improve the hydraulic permeability of the sclera in glaucoma cases. A fiber-optical system to investigate the dynamics of the reflected and transmitted scattered laser radiation and a setup with a double integrating sphere to determine the optical properties of the ocular tissues on the basis of the Monte-Carlo simulation of the propagation of light was used. When the radiation characteristics corresponded to the treatment regimens for correcting the shape of the cornea, no noticeable changes were detected in its optical properties. When irradiating the sclera in conditions corresponding to the treatment regimens for improving its hydraulic permeability, the optical characteristics of the tissue showed definite changes. The results obtained as to the dynamics of the optical signals during the course of laser irradiation of the cornea and sclera create prerequisites for designing test systems to be used with novel medical laser techniques for correcting visual abnormalities.

Background/purpose: Although scleral contact lenses are prescribed with increasing frequency, little is known about their long-term effects on ocular physiology. The main goal of this paper is to predict values of oxygen transmissibility of scleral lens systems by applying the concept of resistors in series to parameters characteristic of current scleral lenses. A second aim is to find the maximal lens and post-lens tear layer thickness combinations above which hypoxia-induced corneal swelling would be found., Methods: Theoretical calculations were used to predict the oxygen transmissibility of scleral lens systems, considering several material permeabilities (Dks 100-170), varying lens thicknesses (250-500 μm), the known tear permeability (Dk of 80) and expected post-lens tear layer thicknesses (100-400 μm). The Holden-Mertz Dk/t criteria of 24 Fatt units for the central cornea and the Harvitt-Bonanno criteria of 35 Fatt units for the limbal area were used as reference points., Results: Our calculations of oxygen transmissibility, with varying tear layer and lens thicknesses, ranged from 10 to 36.7 at the scleral lens centers and from 17.4 to 62.6 at the peripheries. Our calculations of maximum central lens thicknesses show a practical range of 250-495 μm, in conjunction with a post-lens tear layer thickness of 100-250 μm., Conclusion: Our computations show that most modern scleral lenses, with recommended fitting techniques, should lead to some level of hypoxia-induced corneal swelling. Recommendations are made to minimize hypoxia-induced corneal swelling: highest Dk available (>150) lens with a maximal central thickness of 250 μm and fitted with a clearance that does not exceed 200 μm., (Copyright © 2012 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Purpose: To examine the presence and distribution of proteoglycan (PG) core proteins in the adult human retina, choroid, and sclera., Methods: Postmortem human eye tissue was dissected into Bruch's membrane/choroid complex, isolated Bruch's membrane, or neurosensory retina. PGs were extracted and partially purified by anion exchange chromatography. Trypsinized peptides were analyzed by tandem mass spectrometry and PG core proteins identified by database search. The distribution of PGs was examined by immunofluorescence microscopy on human macular tissue sections., Results: The basement membrane PGs perlecan, agrin, and collagen-XVIII were identified in the human retina, and were present in the internal limiting membrane, blood vessel walls, and Bruch's membrane. The hyalectans versican and aggrecan were also detected. Versican was identified in Bruch's membrane, while aggrecan was distributed throughout the retina, choroid, and sclera. The cartilage link protein HAPLN1 was abundant in the interphotoreceptor matrix and sclera, while HAPLN4 (brain link protein 2) was found throughout the retina and choroid. The small leucine-rich repeat PG (SLRP) family members biglycan, decorin, fibromodulin, lumican, mimecan, opticin, and prolargin were present, with different patterns of distribution in the retina, choroid, and sclera., Conclusions: A combination of proteomics and immunohistochemistry approaches has provided for the first time a comprehensive analysis of the presence and distribution of PG core proteins throughout the human retina, choroid, and sclera. This complements our knowledge of glycosaminoglycan chain distribution in the human eye, and has important implications for understanding the structure and functional regulation of the eye in health and disease.

Nanostructured lipid carriers (NLC) have been developed for sustained release of triamcinolone acetonide (TA), a corticosteroid commonly indicated for macular edema, neovascularization, and other ocular inflammatory disorders. TA-NLC were prepared by high-pressure homogenization and characterized for in vitro release by dialysis bag. Ex vivo permeation profile was assessed using rabbit sclera isolated and mounted in Franz diffusion cells. TA-NLC were placed in episcleral donor compartment and choroidal side was perfused with HEPES buffer. Tissue sections underwent drug wash-out, following analysis by validated RP-HPLC of drug content and perfused fractions collected over 24 hours. Drug release followed one-order kinetics and permeability studies confirmed that TA is able to diffuse across rabbit sclera in sustained profile, following zero-order kinetics. Strong tissue binding was observed, providing a drug depot. These findings are of potential use when designing future TA therapy strategies for ocular diseases of posterior segment., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The purpose of this study was to quantify the melanin pigment content in sclera, choroid-RPE, and retina, three tissues encountered during transscleral drug delivery to the vitreous, in human, rabbit, monkey, minipig, and dog models. Strain differences were assessed in NZW × NZR F1 and Dutch belted rabbits and Yucatan and Gottingen minipigs. The choroid-RPE and retina tissues were divided into central (posterior pole area) and peripheral (away from posterior pole) regions while the sclera was analyzed without such division. Melanin content in the tissues was analyzed using a colorimetric assay. In all species the rank order for pigment content was: choroid-RPE >retina ≥ sclera, except in humans, where scleral melanin levels were higher than retina and central choroid. The melanin content in a given tissue differed between species. Further, while the peripheral tissue pigment levels tended to be generally higher compared to the central regions, these differences were significant in human in the case of choroid-RPE and in human, monkey, and dogs in the case of retina. Strain difference was observed only in the central choroid-RPE region of rabbits (NZW × NZR F1 >Dutch Belted). Species, strain, and regional differences exist in the melanin pigment content in the tissues of the posterior segment of the eye, with Gottingen minipig being closest to humans among the animals assessed. These differences in melanin content might contribute to differences in drug binding, delivery, and toxicity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Aim: To investigate a possible association between the biomechanical load and unload behaviour and the elastin content of the sclera canal ring (SCR) and a superiorly localized sclera ring (SPS) in the porcine eye., Methods: Two sclera rings were trephined from each of 40 porcine eyes, one containing the SCR and the other an SPS. The load and the unload curves were measured in the extension range of 0-2.0 mm by a biomaterial tester. Hysteresis was determined from the area enclosed by the loading and unloading curve. Histochemical staining with resorcin-fuchsin and morphometric analysis of paraffin-embedded sections of both rings were performed to detect the area occupied by elastin fibres., Results: At 1 mm extension, the mean load of the SCR was 0.89 ± 0.22 N and that of the SPS 1.13 ± 0.19 N, which was not significantly different between both rings (p > 0.05). Mean hysteresis in the SCR was 1.55 ± 0.30 N × mm and 1.90 ± 0.18 N × mm in the SPS, which was significantly different between both rings (p = 0.01). Mean sclera thickness was 986 μm in the SCR (range: 900-1,060 μm) and 971 μm in the SPS (range: 800-1,200 μm) without a statistically significant difference between both sclera rings (p = 0.78). The area occupied by elastin fibres was 15.5 ± 3.4% in the SCR and 4.5 ± 1.5% in the SPS, which was significantly different between both rings (p = 0.0001)., Conclusion: Hysteresis in the SCR was significantly lower than in the SPS, indicating a higher elasticity of the SCR in the porcine eye. This effect could be explained by a higher content of elastin in the surrounding ring of the peripapillary optic nerve head providing reversible contraction in cases of intra-ocular pressure variations., (Copyright © 2011 S. Karger AG, Basel.)

Purpose: Previous work has suggested a major role of scleral biomechanics in the pathogenesis of glaucoma. Since fiber orientation in connective tissues is a key determinant of tissue biomechanics, experimental characterization of scleral fiber orientation is needed to fully understand scleral biomechanics. This is a report of baseline experimental measurements of fiber orientation in whole normal rat scleras., Methods: Twenty ostensibly normal Norway brown rat eyes were fixed in 4% paraformaldehyde. The scleras were cleaned of intra- and extraorbital tissues and dissected into five patches, and each patch was glycerol treated to maximize its transparency. Fiber orientation was measured using small-angle light scattering (SALS). Scattering patterns were analyzed to extract two microstructural parameters at each measurement location-the preferred fiber orientation and the degree of alignment-yielding a fiber orientation map for each sclera., Results: Rat sclera is structurally anisotropic with several consistent features. At the limbus, fibers were highly aligned and organized primarily into a distinct ring surrounding the cornea. In the equatorial region, the fibers were primarily meridionally aligned. In the posterior and peripapillary region, the scleral fibers were mostly circumferential but less aligned than those in the anterior and equatorial regions., Conclusions: Circumferential scleral fibers may act as reinforcing rings to limit corneal and optic nerve head deformations, whereas equatorial meridional fibers may either provide resistance against extraocular muscle forces or limit globe axial elongation.

The goal of the investigation was to study the changes of scleral matrix collagen in progressing primary open-angle glaucoma (POAG). In scleral samples of 16 patients aged 52-81 years with different stages of POAG amino acid composition, collagen content and cross-linking ratio characterizing tissue rigidity were estimated. Collagen I was found to be the dominant type in sclera of glaucomatous and normal eyes, though as POAG progressing the content and cross-linking increase, that underlies the scleral rigidity increase and permeability decrease. Similar ratio of collagen cross-linking with age and glaucoma progression give evidence of specific pathologic changes of the dominant matrix component, that may be one of significant pathogenic factor of POAG.

This study examined the structures and the elastic and viscous properties of human scleral collagen fibrils by atomic force microscopy (AFM). Sample preparation was performed to minimize the sources of artifacts for further imaging. To observe the morphological and property characteristics of human scleral surfaces, AFM was used as a microscopic tool. The AFM topography, phase shift and deflection images of the dehydrated scleral collagen fibrils were obtained. The visco-elasticity of collagen fibrils was determined from the force-distance curves of the AFM. Inspection of the fibril surface in high resolution showed that the D-period spacing along the collagen fibrils was clearly evident. The fibril diameter over a scan size of 5 x 5 microm2 was 145.22 +/- 17.78 nm (n = 178) ranging from 98 to 220 nm, and the D-periodicity was 69.14 +/- 14.15 nm (n = 189), which is similar to the normal 67 nm D-periodicity. Force-distance analysis indicated that human scleral collagen had comparatively high adhesion force and elasticity, to protect the eye from external trauma and to withstand the expansive force made by the intraocular pressure.

In this study, the organization of collagen fibrils within the sclera of the eye was investigated using the 7 keV hard X-ray microscope of the Pohang light source and compared to images from electron and atomic force microscopy. From the captured X-ray images, individual collagen fibrils were observed clearly in a spatial resolution much better than 100 nm, both in longitudinal sections and in transverse sections. Some of the collagen fibrils showed evidence of axial periodicity. In some regions of the samples, we could see cross-bridge like structures between adjacent collagen fibrils. The X-ray microscope also allowed the observation of keratocytes and the lamella structure of the scleral stroma. The X-ray microscope has some unique advantages in the nano-scale imaging of bio-samples relative to other established imaging techniques., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

It has been shown that the collagen content and denaturation temperature in scleral tissue increase during the development of glaucoma whereas the fluorescence intensity does not change. These effects may be related to the disturbance of collagen catabolism.

This study manipulates both clear cornea and opaque sclera by two dehydration processes for revealing the relationship between altered tissue structures and change in optical functions. In contrast to the high levels of light scattering in dehydrated tissues by critical point dry, a simple dehydration at 4-8 °C effectively and significantly improved their visible-light transmission, even in the sclera, with accompanying dense fiber packing. Further improvement in visible-light transmission, from 40-50% to 80-90%, has been achieved by flatting tissue surface with cover glasses during dehydration at low temperature. Such optical clearing of sclera by dehydration is reversible. However, chemical cross-linking effectively stabilizes their densely packed microscopic structures and visible-light transmission at over 50% irreversibly, even at wet conditions. Interestingly, the repetition of both low temperature dehydration/cross-linking treatments effectively reduced the required amounts of cross-linking reagents to keep a high transparency. Wet transparent cross-linked sclera can also show a characteristic strong tensile strength. Furthermore, rabbit corneal epithelium has regenerated on the transparent sclera with cross-linking in vitro., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Purpose: Pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) is a new, broad-spectrum agent that has an inhibition effect on the proliferation, migration, and collagen contraction of human Tenon's fibroblasts, and thus modulating the wound healing process of glaucoma filtering surgical site. This study investigated the pharmacokinetics of topically administered pirfenidone (0.5%) in rabbit eyes., Methods: Pirfenidone solution (50 μl) was instilled into the rabbit's conjunctival sac. The rabbits were quickly sacrificed at 2, 5, 8, 10, 15, 20, 30, 60, 90, and 120 min after the administration and ocular tissues were obtained. The concentrations of pirfenidone in conjunctiva, sclera, cornea, aqueous humor, and vitreous were determined by high performance liquid chromatography., Results: After topical administration, there was wide distribution and fast clearance of pirfenidone among the various ocular tissues. The mean maximum concentrations (C(max)) of pirfenidone in cornea, conjunctiva, sclera, aqueous humor, and vitreous were 9.64 mg/g, 9.62 mg/g, 2.13 mg/g, 34.88 mg/l and 0.52 mg/l, respectively. The half-life for these tissues was 18.26, 34.16, 15.71, 70.91, and 39.48 min, respectively., Conclusions: Measurable concentrations of pirfenidone are achieved in ocular tissues after topical application in rabbit model. Topical administration of pirfenidone may be an effective approach for modulation of wound healing responses in glaucoma filtration surgical site.

Multiphoton excitation laser scanning microscopy, relying on the simultaneous absorption of two or more photons by a molecule, is one of the most exciting recent developments in biomedical imaging. Thanks to its superior imaging capability of deeper tissue penetration and efficient light detection, this system becomes more and more an inspiring tool for intravital bulk tissue imaging. Two-photon excitation microscopy including 2-photon fluorescence and second harmonic generated signal microscopy is the most common multiphoton microscopic application. In the present review we take diverse ocular tissues as intravital samples to demonstrate the advantages of this approach. Experiments with registration of intracellular 2-photon fluorescence and extracellular collagen second harmonic generated signal microscopy in native ocular tissues are focused. Data show that the in-tandem combination of 2-photon fluorescence and second harmonic generated signal microscopy as two-modality microscopy allows for in situ co-localization imaging of various microstructural components in the whole-mount deep intravital tissues. New applications and recent developments of this high technology in clinical studies such as 2-photon-controlled drug release, in vivo drug screening and administration in skin and kidney, as well as its uses in tumourous tissues such as melanoma and glioma, in diseased lung, brain and heart are additionally reviewed. Intrinsic emission two-modal 2-photon microscopy/tomography, acting as an efficient and sensitive non-injurious imaging approach featured by high contrast and subcellular spatial resolution, has been proved to be a promising tool for intravital deep tissue imaging and clinical studies. Given the level of its performance, we believe that the non-linear optical imaging technique has tremendous potentials to find more applications in biomedical fundamental and clinical research in the near future.

The present investigation was first to study scleral fibronectin (FN) levels in primary open-angle glaucoma, by applying an immunohistochemical study. The enhanced accumulation of this glycoprotein was found in the deep and inner scleral layers. Active FN expression was ascertained to correspond to the scleral morphological changes characteristic for glaucoma (garneting of collagenous fibers, accumulation of free glycosamine glycans) and to be of focal nature. The findings show the role of FN in the mechanisms of scleral destruction in primary open-angle glaucoma.

A simple, selective, and sensitive LC-MS/MS method was developed for the simultaneous extraction and determination of eight beta-blockers (atenolol, sotalol, nadolol, pindolol, timolol, metoprolol, betaxolol and propranolol) in various bovine eye tissues including sclera, choroid-RPE, retina, and vitreous. The analytes were extracted by liquid-liquid extraction after samples were alkalinized with 2% NaOH solution in water. The chromatographic separation was performed on a Hypersil-ODS C18 column (100 mm x 2.1 mm, 3.9 microm) using a gradient mixture of (A) 5 mM ammonium formate in water (pH 3.5 adjusted with formic acid) and (B) acetonitrile:methanol (75:25) containing 0.02% triethyl amine (pH 4.0; adjusted with formic acid) as mobile phase at a flow rate of 0.4 ml/min. The compounds were ionized in the positive electrospray ionization (ESI) mode and detected in the multiple reaction monitoring (MRM) mode. The average recoveries in all four eye tissues for all beta-blockers were >82%, except for sotalol (>51%). The matrix effect for beta-blockers ranged from 81 to 110% in the four eye tissues. This analytical method was validated and applied successfully for simultaneous quantification of the beta-blockers in sclera after tissue exposure using cassette dosing method. The calibration curve was linear in the range of 10-2000 ng/ml for all analytes, with the correlation coefficient >0.996. Intra-day and inter-day precision (% CV) was less than 15%, and accuracy ranged from 85 to 110% for all analytes. Scleral uptake was the lowest for sotalol and atenolol, two hydrophilic beta-blockers, and the highest for propranolol, a lipophilic beta-blocker.

The dynamics of the eyeball, most notably the changes in intraocular pressure, need to be stabilized optically to prevent noticeable changes in image quality. This control depends on the rheological properties of the eyeball coats and how the elasticity of the cornea, sclera, and limbus vary relative to one another. Nonlinear finite element modeling shows that image quality can be preserved over a range of elastic moduli. For intraocular pressure variations from 8 to 40 mm Hg, optical image stability is best for an elastic secant modulus of the cornea of 0.267 MPa. Optimal quality is achieved when the elastic moduli of the limbus and sclera are, respectively, 3.6 and 4 times that of the corneal modulus.

The paper aims at the evaluation of prospects for using glyceraldehyde as a cross-linking agent for the scleral tissue. Stability parameters (denaturation temperature, Young's modulus, ultimate tensile stress, proteolytic resistance) and analytical parameter (fluorescence intensity) were determined during the glycation process of isolated rabbit sclera. The analysis of fluorescence spectral characteristic provided information about some glycation products. The glyceraldehyde treatment was resulted in a significant increase in thermal stability, proteolytic resistance and improvement of biomechanical characteristics (Young's modulus, ultimate tensile stress). Unique properties of the reaction between scleral collagen and glyceraldehyde are observed at short cross-linking times. The appearance of intermediate collagen fraction with lowest thermal and proteolytic stability was detected.

We present here the results of protein extraction from different ocular regions using different detergents. Extraction strategies used to determine optimal protein extraction included: pressure cycling and aqueous-organic phase extraction in combination with electrophoretic fractionation for anterior, posterior, and peripapillary sclera. Detergent extraction of proteins from freshly enucleated porcine eyes (n = 8) showed significant differences for different eye regions. Protein yield ranged from 2.3 to 50.7 mug protein/mg for different ocular tissues, with the lens yielding the most protein. ASB-14 and Triton X-100 provided the best protein yields (n = 10) for anterior and posterior sclera. The spectrophotometric measurements for ASB-14 were not consistent with SDS-PAGE densitometry. A combination of 0.5% Triton X-100, 0.5% Tween-20, and 0.1% Genapol C-100 was found optimal for extraction from sclera. Proteins from different regions of the eye are best extracted with different detergents. The pressure cycling technology provided superior extraction compared to the other methods. Additional aqueous-organic phase partitioning enables superior fractionation when compared to SDS-PAGE alone. Organic phase fractionation is compatible with MS and allowed identification of 34, 71, and 77 proteins respectively from anterior, posterior, and peripapillary sclera. The extraction strategy may affect the final outcome in protein profiling by MS or by other methods.

It has been shown by biochemical analysis and differential scanning calorimetry that the connective tissue formed around a transplant as a result of sclero-reinforcing interference (capsula) is similar to intact sclera. The main component of newly formed capsules is collagen I whose fibers have a perfect structure and the amount of cross-links sufficient to provide normal thermomechanical properties. A fraction of collagen having thermally labile "immature" cross-links in capsules formed around the transplant impregraned with Panaxal has been detected by differential scanning calorimetry. It was suggested that fibroblasts in tissues of these capsules have a high synthetic activity.

To characterize the in situ distribution of actin in Schlemm's canal endothelium (SCE) and juxtacanalicular tissue (JCT) cells in glaucomatous human eyes, and compare to the distribution in normal eyes. Fresh human eye bank eyes were perfused and fixed at pressure (n=27 normal eyes and 22 confirmed glaucomatous eyes). Schlemm's canal was opened by microdissection and outflow tissues were labelled for confocal microscopy to visualize F-actin, nuclei, laminin and/or CD31. Images were acquired in Z-series from the inner wall of Schlemm's canal, juxtacanalicular tissue and outer corneoscleral meshwork. In normal eyes, inner wall Schlemm's canal endothelial (SCE) cells showed a dense peripheral F-actin band, as previously described. JCT cells showed a more random and amorphous F-actin distribution. In glaucoma eyes, peripheral F-actin bands were less common in inner wall SCE cells; instead, F-actin was more centrally located within the cell and appeared "tangled". These actin tangles were also prominent in JCT cells of glaucoma eyes. Glaucoma eyes also demonstrated structures with features of cross-linked actin networks (CLANs), and more frequent occurrence of punctuate actin concentrations. There was a significant degree of heterogeneity, with some regions from glaucomatous eyes appearing normal and vice versa. F-actin architecture in human outflow pathway cells in situ differs between normal and glaucoma eyes, with glaucomatous tissue showing a more "disordered" actin architecture overall. Some of these changes are likely due to effects secondary to administration of anti-glaucoma medications. Most of the changes that we observed could potentially affect the biomechanical properties of the outflow pathway tissues in glaucoma, but their role in the pathogenesis of ocular hypertension remains unclear.

Good visual acuity requires that the axial length of the ocular globe is matched to the refractive power of the cornea and lens to focus the images of distant objects onto the retina. During the growth of the juvenile eye, this is achieved through the emmetropization process that adjusts the ocular axial length to compensate for the refractive changes that occur in the anterior segment. A failure of the emmetropization process can result in either excessive or insufficient axial growth, leading to myopia or hyperopia, respectively. Emmetropization is mainly regulated by the retina, which generates two opposite signals: "GO/GROW" signals to increase axial growth and "STOP" signals to block it. The presence of GO/GROW and STOP signals was investigated by a proteomics analysis of the retinas from chicken with experimental myopia and hyperopia. Of 18 differentially expressed proteins that were identified, five displayed an expression profile corresponding to GO/GROW signals, and two corresponded to STOP signals. Western blotting confirmed that apolipoprotein A-I (apoA-I) has the characteristics of a STOP signal both in the retina as well as in the fibrous sclera. In accordance with this, intraocular application of the peroxisome proliferator-activated receptor alpha agonist GW7647 resulted in up-regulation of apoA-I levels and in a significant reduction of experimental myopia. In conclusion, using a comprehensive functional proteomics analysis of chicken ocular growth models we identified targets for ocular growth control. The correlation of elevated apoA-I levels with reduced ocular axial growth points toward a functional relationship with the observed morphological changes of the eye.

Purpose: To characterize the in situ distribution of actin in Schlemm's canal endothelium (SCE) and juxtacanalicular tissue (JCT) cells in glaucomatous human eyes, and compare to the distribution in normal eyes., Methods: Fresh human eye bank eyes were perfused and fixed at pressure (n=27 normal eyes and 22 confirmed glaucomatous eyes). Schlemm's canal was opened by microdissection and outflow tissues were labelled for confocal microscopy to visualize F-actin, nuclei, laminin and/or CD31. Images were acquired in Z-series from the inner wall of Schlemm's canal, juxtacanalicular tissue and outer corneoscleral meshwork., Results: In normal eyes, inner wall Schlemm's canal endothelial (SCE) cells showed a dense peripheral F-actin band, as previously described. JCT cells showed a more random and amorphous F-actin distribution. In glaucoma eyes, peripheral F-actin bands were less common in inner wall SCE cells; instead, F-actin was more centrally located within the cell and appeared 'tangled'. These actin tangles were also prominent in JCT cells of glaucoma eyes. Glaucoma eyes also demonstrated structures with features of cross-linked actin networks (CLANs), and more frequent occurrence of punctuate actin concentrations. There was a significant degree of heterogeneity, with some regions from glaucomatous eyes appearing normal and vice versa., Conclusion: F-actin architecture in human outflow pathway cells in situ differs between normal and glaucoma eyes, with glaucomatous tissue showing a more 'disordered' actin architecture overall. Some of these changes are likely due to effects secondary to administration of anti-glaucoma medications. Most of the changes that we observed could potentially affect the biomechanical properties of the outflow pathway tissues in glaucoma, but their role in the pathogenesis of ocular hypertension remains unclear.

Purpose: The purpose of this work was to demonstrate the use of the combined imaging modality of multiphoton autofluorescence and second-harmonic generation (SHG) microscopy in obtaining spectrally resolved morphologic features of the cornea, limbus, conjunctiva, and sclera in whole, ex vivo porcine eyes., Methods: The 780-nm output of a femtosecond, titanium-sapphire laser was used to induce broadband autofluorescence (435-700 nm) and SHG (390 nm) from various regions of the surface of ex vivo porcine eyes. A water-immersion objective was used for convenient imaging of the curved surface of the eye., Results: Multiphoton autofluorescence was useful in identifying cellular structures of the different domains of the ocular surface, and the SHG signal can be used to resolve collagen organization within the cornea stroma and sclera of ex vivo porcine eyes., Conclusions: Multiphoton autofluorescence and SHG microscopy have been demonstrated to be an effective technique for resolving, respectively, the cellular and collagen structures within the ocular surface of ex vivo porcine eyes. SHG imaging resolved the difference in structural orientations between corneal and sclera collagen fibers. Specifically, the corneal collagen is organized in a depth-dependent fashion, whereas the scleral collagen is randomly packed. Because this technique does not require histologic preparation procedures, it has the potential to be applied for in vivo studies with minimal disturbance to the eye.

Transscleral delivery of triamcinolone acetonide into the vitreous using sub-Tenon's injections may be a safer alternative to reduce the sight-threatening complications of direct intravitreal injections. However, sub-Tenon's injections have demonstrated low and poorly sustained vitreous drug levels in animal studies. To improve our understanding of the clearance mechanisms of corticosteroids, we evaluated vitreous drug levels following sub-Tenon's injection of triamcinolone acetonide in rabbits with selective elimination of conjunctival lymphatic/blood vessels and the choroid. Pigmented rabbits were given a sub-Tenon's injection of a preservative-free triamcinolone acetonide formulation of either a 10- or 20-mg dose in the superotemporal quadrant. The effect eliminating both conjunctival and choroidal clearance was evaluated by injecting the drug, followed by immediate euthanasia, effectively terminating both lymph and blood flow in the conjunctiva and choroid. To inhibit only the clearance from conjunctival lymphatics/blood vessels of a sub-Tenon's injection of triamcinolone acetonide, a group of rabbits had a 'conjunctival window' created by incising an 7 mmx7 mmx7 mm square through the conjunctiva to bare sclera in the superotemporal quadrant. To eliminate only the clearance of drug from the choroidal circulation, cryotherapy was performed in another group of rabbits creating a chorioretinal scar in the superotemporal quadrant. Following the sub-Tenon's drug injection, the eyes were enucleated in all groups after 3 hr and vitreous drug levels were measured with HPLC. In normal animals, a 10-mg sub-Tenon's injection showed no detectable vitreous drug levels; however, a 20-mg injection showed positive vitreous drug levels. This suggested that collectively, the transscleral clearance mechanisms inhibiting delivery into the vitreous may be saturated with a drug depot that has a higher release rate. A 10-mg sub-Tenon's drug depot was able to deliver drug into the vitreous when both the conjunctival and choroidal drug clearance was eliminated by euthanizing the animal immediately following the drug injection. In rabbits that had only a 'conjunctival window', selectively eliminating conjunctival drug clearance, vitreous drug levels were detected. However, in rabbits that had only cryotherapy, selectively eliminating choroidal drug clearance, vitreous drug levels were not detected suggesting that the conjunctival lymphatics/blood vessels may be an important barrier to the transscleral delivery of triamcinolone acetonide. Variability in the vitreous drug levels between rabbits in each group precluded statistical testing. In summary, the rabbit appeared to demonstrate saturable ocular barriers to transscleral delivery of triamcinolone acetonide into the vitreous following a sub-Tenon's injection. The results suggested that the conjunctival lymphatics/blood vessels may be an important barrier to the delivery of triamcinolone acetonide to the vitreous in this rabbit model. The barrier location and clearance abilities of the ocular tissues are important to consider when developing a successful transscleral drug delivery system. Animal models, retaining the dynamics of blood and lymph flow, may improve the basic understanding of the ocular barriers involved with transscleral drug transport and warrants further investigation.

Recently discovered endogenous opioid peptides such as nociceptin are known to modulate neurotransmitter release of primary afferent neurons (especially substance P, SP) and they have also been demonstrated in peripheral nerve fibres. The aim of this study was to investigate the opioid peptidergic innervation of the anterior eye segment and to compare it with the innervation pattern of SP in order to shed light on the functional relationship between these peptides. Anterior eye segments of 20 rat eyes were cut in a tangential plane and the sections stained with antibodies against SP, nociceptin, nocistatin, endomorphin 1 and 2, leu-enkephalin and met-enkephalin. Sections of the spinal cord or brain were used as positive controls. Numerous SP-immunoreactive nerve fibres were found in the conjunctiva, cornea, episclera, trabecular meshwork, iris and ciliary body. A weak staining for met-enkephalin and leu-enkephalin could only be found in the iris and anteriormost ciliary body. Nerve fibres immunoreactive for nociceptin, nocistatin, and endomorphin 1 or 2 could not be detected in any part of the anterior eye segment. It is tempting to speculate that the opioid peptidergic innervation of the anterior ciliary body may play a role in the modulation of intraocular inflammation.

The purpose of the present study was to compare the localization of prostaglandin E(2) receptor subtypes in normal human and mouse ocular tissues. Paraffin embedded sections of normal human and mouse (129 Sv/Ev) eyes were treated with EP(1), EP(2), EP(3) and EP(4) specific antibodies and subsequently incubated with Alexa Fluor secondary antibody (Ex/Em=555/571) to detect the presence of EP receptor proteins. Fluorescence of the localized antibodies was visualized in a Carl Zeiss Microscope (Axiovert 200) and photographed using Carl Zeiss Axiocam camera. In mice EP(1) and EP(3) receptor subtypes were only moderately expressed, EP(3) receptor expression being almost negligible. In human cornea and iris ciliary body, EP(1) and EP(3) receptors were prominently expressed. EP(4) receptor was expressed moderately in human and mouse ocular tissues. EP(2) receptor was the most prominently and abundantly expressed receptor in both human and mouse ocular tissues. It is concluded that the pattern of the distribution of EP receptor subtypes in the ocular tissues are similar in human and mouse. Thus, 129 Sv/Ev strains of mice would make an appropriate animal model for studying the ocular pathophysiological roles of prostaglandin receptor agonists.

The circadian signaling molecule, melatonin, is produced by pinealocytes and retinal photoreceptors. In the retina, melatonin is thought to diffuse into the inner retina to act as a paracrine signal of darkness by binding to specific receptors in retinal neurons. The retinal cell locations of the Mel1a and Mel1c melatonin receptor types have been reported, but the localization of the Mel1b receptor, which is the most highly expressed melatonin receptor type in the retina, is unknown. To determine the cellular distribution of Mel1b melatonin receptor protein in the Xenopus laevis retina and other ocular tissues, polyclonal antibodies were raised against a peptide fragment of the X. laevis Mel1b receptor. Western blot analysis of several ocular tissues revealed the presence of one or more immunoreactive bands in the sclera, cornea, lens, retinal pigment epithelium (RPE)/choroid, and neural retina. In the neural retina, the major immunoreactive bands displayed electrophoretic mobilities corresponding to approximately 35, 42, 45, and 80 Kd. Sections of X. laevis eyes were analyzed by immunocytochemistry and confocal microscopy, in combination with antibodies against the Mel1a melatonin receptor, a rod photoreceptor-specific protein, opsin, and two amacrine cell-specific markers, tyrosine hydroxylase (TOH; dopaminergic cells) and glutamic acid decarboxylase (GAD; GABA-ergic cells). Mel1b immunoreactivity was localized to the apical membranes of RPE cells, and punctate Mel1b immunoreactivity was observed in both rod and cone photoreceptor inner segments. Presumptive horizontal cells that ramify in the outer plexiform layer (OPL) were immunoreactive for Mel1b, and were exclusive of the Mel1a immunoreactivity present in the OPL. Neither TOH nor GAD co-localized with the Mel1b immunoreactivity that was present in the inner plexiform layer (IPL), suggesting that Mel1b is not expressed in dopaminergic or GABA-ergic amacrine cells. Mel1b immunoreactivity was observed in ganglion cells of the retina, a population of cells covering the outer surface of the outer fibrous layer of the sclera, and in lens fibers located in the outer regions of the lens. These results suggest that melatonin may influence retinal function by binding to receptors on RPE and photoreceptor cells, and by acting on neurons of the inner retina that do not use dopamine or GABA as a neurotransmitter. Furthermore, melatonin may bind to receptors on cells located in the sclera and lens, perhaps to modify the growth or function of these ocular tissues.

Although disease of the sclera is unusual, when it occurs it can rapidly destroy both the eye and vision. However, normally the sclera provides an opaque protective coat for the intraocular tissues and a stable support during variations in internal pressure and eye movements, which would otherwise perturb the visual process through distortion of the retina and the lens/iris diaphragm. This stability, which is vital for clear vision is made possible by the organisation and viscoelastic properties of scleral connective tissue. Microscopically, the sclera displays distinct concentric layers including, from outside, Tenon's capsule, episclera, the scleral stroma proper and lamina fusca, melding into underlying choroid. Two sites exhibit specialised structure and function: the perilimbal trabecular meshwork, through which aqueous filters into Schlemm's canal, and the lamina cribrosa, which permits axons of the optic nerve to exit the posterior sclera. Throughout, sclera is densely collagenous, the stroma consisting of fibrils with various diameters combining into either interlacing fibre bundles or defined lamellae in outer zones. Scleral fibrils are heterotypic structures made of collagen types I and III, with small amounts of types V and VI also present. Scleral elastic fibres are especially abundant in lamina fusca and trabecular meshwork. The interfibrillar matrix is occupied by small leucine-rich proteoglycans, decorin and biglycan, containing dermatan and dermatan/chondroitin sulphate glycosaminoglycans, together with the large proteoglycan, aggrecan, which also carries keratan sulphate sidechains. Decorin is closely associated with the collagen fibrils at specific binding sites situated close to the C-terminus of the collagen molecules. Proteoglycans influence hydration, solute diffusion and fluid movement through the sclera, both from the uvea and via the trabecular meshwork. As the sclera is avascular, nutrients come from the choroid and vascular plexi in Tenon's capsule and episclera, where there is an artery to artery anastomosis in which blood oscillates, rather than flows rapidly. This predisposes to the development of vasculitis causing a spectrum of inflammatory conditions of varying intensity which, in the most severe form, necrotising scleritis, may destroy all of the structural and cellular components of the sclera. Scleral cells become fibroblastic and the stroma is infiltrated with inflammatory cells dominated by macrophages and T-lymphocytes. This process resembles, and may be concurrent with, systemic disease affecting other connective tissues, particularly the synovial joints in rheumatoid arthritis. Current views support an autoimmune aetiology for scleritis. Whilst the role of immune complexes and the nature of initial pro-inflammatory antigen(s) remain unknown, the latter may reside in scleral tissue components which are released or modified by viral infection, injury or surgical trauma.