Validating a Sclera-Based Smartphone Application for Screening Jaundiced Newborns in Ghana.

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.

Cytosine-functionalized bioinspired hydrogels for ocular delivery of antioxidant transferulic acid.

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.

Scleral Lens Sensor for Ocular Electrolyte Analysis.

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+ , Ca2+ , Mg2+ , and Zn2+ 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 Ca2+ 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 Mg2+ 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 Zn2+ 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.

Ocular swabs on exhumed bodies: An alternative to the collection of "classical" tissue samples in forensic genetics.

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.

Microstructure-based numerical simulation of the mechanical behaviour of ocular tissue.

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.

Quantification of Scleral Biomechanics and Collagen Fiber Alignment.

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.

Characterization of liposomal carriers for the trans-scleral transport of Ranibizumab.

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.

Effects of collagen microstructure and material properties on the deformation of the neural tissues of the lamina cribrosa.

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.

Collagen Architecture of the Posterior Pole: High-Resolution Wide Field of View Visualization and Analysis Using Polarized Light Microscopy.

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.

Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques.

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.

The contribution of glycosaminoglycans to the mechanical behaviour of the posterior human sclera.

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.

Preclinical assessment of scleral lens as a reservoir-based ocular therapeutic system.

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.

Study of optical clearing in polarization measurements by Monte Carlo simulations with anisotropic tissue-mimicking models.

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.

Quantitative mapping of collagen fiber alignment in thick tissue samples using transmission polarized-light microscopy.

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.

Microfluidic-based non-enzymatic glycation enhances cross-linking of human scleral tissue compared to conventional soaking.

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 mm2 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 + DR7 group) and 30 days (BSS + DR30 group) at room temperature, and 7 days in a microfluidic chip (BSS + DR + µF7 group). The BSS + DR7 and BSS + DR30 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 + µF7 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.

Age estimation based on aspartic acid racemization in human sclera.

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.

Effect of flaxseed on choroid-sclera complex thickness and on LDL oxidation in the sclera, choroid and retina of diet-induced hypercholesterolaemic rabbits.

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.

Elastic properties of human posterior eye.

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.

Optical characteristics of the cornea and sclera and their alterations under the effect of nondestructive 1.56-µm laser radiation.

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.

Structural and biomechanical effects of photooxidative collagen cross-linking with photosensitizer riboflavin and 370 nm UVA light on human corneoscleral tissues.

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.