Heterogenous thinning of peripapillary tissues occurs early during high myopia development in juvenile tree shrews.

Myopia is an independent risk factor for glaucoma, but the link between both conditions remains unknown. Both conditions induce connective tissue remodeling at the optic nerve head (ONH), including the peripapillary tissues. The purpose of this study was to investigate the thickness changes of the peripapillary tissues during experimental high myopia development in juvenile tree shrews. Six juvenile tree shrews experienced binocular normal vision, while nine received monocular -10D lens treatment starting at 24 days of visual experience (DVE) to induce high myopia in one eye and the other eye served as control. Daily refractive and biometric measurements and weekly optical coherence tomography scans of the ONH were obtained for five weeks. Peripapillary sclera (Scl), choroid-retinal pigment epithelium complex (Ch-RPE), retinal nerve fiber layer (RNFL), and remaining retinal layers (RRL) were auto-segmented using a deep learning algorithm after nonlinear distortion correction. Peripapillary thickness values were quantified from 3D reconstructed segmentations. All lens-treated eyes developed high myopia (-9.8 ± 1.5 D), significantly different (P < 0.001) from normal (0.69 ± 0.45 D) and control eyes (0.76 ± 1.44 D). Myopic eyes showed significant thinning of all peripapillary tissues compared to both, normal and control eyes (P < 0.001). At the experimental end point, the relative thinning from baseline was heterogeneous across tissues and significantly more pronounced in the Scl (-8.95 ± 3.1%) and Ch-RPE (-16.8 ± 5.8%) when compared to the RNFL (-5.5 ± 1.6%) and RRL (-6.7 ± 1.8%). Furthermore, while axial length increased significantly throughout the five weeks of lens wear, significant peripapillary tissue thinning occurred only during the first week of the experiment (until a refraction of -2.5 ± 1.9 D was reached) and ceased thereafter. A sectorial analysis revealed no clear pattern. In conclusion, our data show that in juvenile tree shrews, experimental high myopia induces significant and heterogeneous thinning of the peripapillary tissues, where the retina seems to be protected from profound thickness changes as seen in Ch-RPE and Scl. Peripapillary tissue thinning occurs early during high myopia development despite continued progression of axial elongation. The observed heterogeneous thinning may contribute to the increased risk for pathological optic nerve head remodeling and glaucoma later in life.

Longitudinal Changes of Bruch's Membrane Opening, Anterior Scleral Canal Opening, and Border Tissue in Experimental Juvenile High Myopia.

Purpose: To investigate the relative positional changes between the Bruch's membrane opening (BMO) and the anterior scleral canal opening (ASCO), and border tissue configuration changes during experimental high myopia development in juvenile tree shrews. Methods: Juvenile tree shrews were assigned randomly to two groups: binocular normal vision (n = 9) and monocular -10 D lens treatment starting at 24 days of visual experience to induce high myopia in one eye while the other eye served as control (n = 12). Refractive and biometric measurements were obtained daily, and 48 radial optical coherence tomography B-scans through the center of the optic nerve head were obtained weekly for 6 weeks. ASCO and BMO were segmented manually after nonlinear distortion correction. Results: Lens-treated eyes developed high degree of axial myopia (-9.76 ± 1.19 D), significantly different (P < 0.001) from normal (0.34 ± 0.97 D) and control eyes (0.39 ± 0.88 D). ASCO-BMO centroid offset gradually increased and became significantly larger in the experimental high myopia group compared with normal and control eyes (P < 0.0001) with an inferonasal directional preference. The border tissue showed a significantly higher tendency of change from internally to externally oblique configuration in the experimental high myopic eyes in four sectors: nasal, inferonasal, inferior, and inferotemporal (P < 0.005). Conclusions: During experimental high myopia development, progressive relative deformations of ASCO and BMO occur simultaneously with changes in border tissue configuration from internally to externally oblique in sectors that are close to the posterior pole (nasal in tree shrews). These asymmetric changes may contribute to pathologic optic nerve head remodeling and an increased risk of glaucoma later in life.

Modelling eye lengths and refractions in the periphery.

PURPOSE: To create a simplified model of the eye by which we can specify a key optical characteristic of the crystalline lens, namely its power. METHODS: Cycloplegic refraction and axial length were obtained in 60 eyes of 30 healthy subjects at eccentricities spanning 40° nasal to 40° temporal and were fitted with a three-dimensional parabolic model. Keratometric values and geometric distances to the cornea, lens and retina from 45 eyes supplied a numerical ray tracing model. Posterior lens curvature (PLC) was found by optimising the refractive data using a fixed lens equivalent refractive index ( n eq ). Then, n eq was found using a fixed PLC. RESULTS: Eccentric refractive errors were relatively hyperopic in eyes with central refractions ≤-1.44 D but relatively myopic in emmetropes and hyperopes. Posterior lens power, which cannot be measured directly, was derived from the optimised model lens. There was a weak, negative association between derived PLC and central spherical equivalent refraction. Regardless of refractive error, the posterior retinal curvature remained fixed. CONCLUSIONS: By combining both on- and off-axis refractions and eye length measurements, this simplified model enabled the specification of posterior lens power and captured off-axis lenticular characteristics. The broad distribution in off-axis lens power represents a notable contrast to the relative stability of retinal curvature.

Nonlinear distortion correction for posterior eye segment optical coherence tomography with application to tree shrews.

We propose an empirical distortion correction approach for optical coherence tomography (OCT) devices that use a fan-scanning pattern to image the posterior eye segment. Two types of reference markers were used to empirically estimate the distortion correction approach in tree shrew eyes: retinal curvature from MRI images and implanted glass beads of known diameter. Performance was tested by correcting distorted images of the optic nerve head. In small animal eyes, our purposed method effectively reduced nonlinear distortions compared to a linear scaling method. No commercial posterior segment OCT provides anatomically correct images, which may bias the 3D interpretation of these scans. Our method can effectively reduce such bias.

Scleral crosslinking using genipin can compromise retinal structure and function in tree shrews.

Scleral crosslinking using genipin has been identified as a promising treatment approach for myopia control. The efficacy of genipin to alter biomechanical properties of the sclera has been shown in several animal models of myopia but its safety profile remains unclear. In this safety study, we aim to investigate the effect of scleral crosslinking using retrobulbar injections of genipin on retinal structure and function at genipin doses that were shown to be effective in slowing myopia progression in juvenile tree shrews. To this end, three or five retrobulbar injections of genipin at 0 mM (sham), 10 mM, or 20 mM were performed in one eye every other day. Form deprivation myopia was induced in the injected eye. We evaluated retinal function using full-field electroretinography and retinal structure using in vivo optical coherence tomography imaging and ex vivo histology. The optical coherence tomography results revealed significant thinning of the peripapillary retinal nerve fiber layer in all genipin treated groups including the lowest dose group, which showed no significant treatment effect in slowing myopia progression. In contrast, inducing form deprivation myopia alone and in combination with sham injections caused no obvious thinning of the retinal nerve fiber layer. Electroretinography results showed a significant desensitizing shift of the b-wave semi-saturation constant in the sham group and the second highest genipin dose group, and a significant reduction in b-wave maxima in the two highest genipin dose groups. The ex vivo histology revealed noticeable degeneration of photoreceptors and retinal pigment epithelium in one of two investigated eyes of the highest genipin dose group. While scleral crosslinking using genipin may still be a feasible treatment option for myopia control, our results suggest that repeated retrobulbar injections of genipin at 10 mM or higher are not safe in tree shrews. An adequate and sustained delivery strategy of genipin at lower concentrations will be needed to achieve a safe and effective scleral crosslinking treatment for myopia control in tree shrews. Caution should be taken if the proposed treatment approach is translated to humans.

Modeling the biomechanics of the lamina cribrosa microstructure in the human eye.

Glaucoma is among the leading causes of blindness worldwide that is characterized by irreversible damage to the retinal ganglion cell axons in the lamina cribrosa (LC) region of the optic nerve head (ONH), most often associated with elevated intraocular pressure (IOP). The LC is a porous, connective tissue structure that provides mechanical support to the axons as they exit the eye and the biomechanics of the LC microstructure likely play a crucial role in protecting the axons passing through it. There is a limited knowledge of the IOP-driven biomechanics of the LC microstructure, primarily due to its small size and the difficulty with imaging the LC both in vitro and in vivo. We present finite element (FE) models of three human eye posterior poles that include the LC microstructure and interspersed neural tissues (NT) composed of retinal axons that are constructed directly from segmented, binary images of the LC. These models were used to estimate the stresses and strains in the LC and NT for an acute IOP elevation from 0 to 45 mmHg and compared with identical models except that the LC was represented as a homogenized continuum material with either homogeneous isotropic neo-Hookean properties or heterogeneous properties derived from local connective tissue volume fraction (CTVF) and predominant LC beam orientation. Stresses and strains in the LC and NT microstructure were investigated, and results were compared against those from the models wherein the LC was represented as a homogenized continuum. The regionalized volumetric average stresses and strains showed that the microstructural model yielded similar patterns to our prior approach using an LC continuum representation with mapped LC CTVF/anisotropy, but the microstructural modeling approach allows analysis of the stresses and strains in the LC and NT separately. As expected, the LC beams carried most of the IOP load in the microstructural models but exhibited less strain, while the encapsulated NT exhibited lower stresses and much higher strains. Results also revealed that the continuum models underestimate the maximum strains in the LC beams and NT by a factor of 2-3. Microstructural modeling should provide greater insight into the biomechanical factors driving damage to the axons (NT) and LC connective tissue remodeling that occur in glaucoma. The methods presented are ideal for modeling any structure with a complex microstructure composed of different materials, such as trabecular bone, lung, and tissue engineering scaffolds such as decellularized LC. Matlab code for mesh generation from a segmented image stack of the microstructure is included as Supplemental Material. STATEMENT OF SIGNIFICANCE: Glaucoma is among the leading causes of blindness worldwide that is characterized by axon damage in the lamina cribrosa (LC) region of the eye. We present a new approach for finite element modeling the entire eye-specific 3D LC microstructure and the interspersed neural tissues, incorporated into an eye-specific posterior eye model that provides appropriate boundary and loading conditions. Results are presented for three human donor eyes, showing that prior modeling approaches underestimate the stresses and strains in the laminar microstructure. We constructed models from image stacks of the segmented microstructure (Matlab code included) using an approach that is ideal for modeling any structure with a complex microstructure composed of different materials, such as trabecular bone, lung, and tissue engineering scaffolds.

How chromatic cues can guide human eye growth to achieve good focus.

The postnatal growing eye uses visual cues to actively control its own axial elongation to achieve and maintain sharp focus, a process termed emmetropization. The primary visual cue may be the difference in image sharpness as sensed by the arrays of short- and long-wavelength sensitive cone photoreceptors caused by longitudinal chromatic aberration: Shorter wavelengths focus in front of longer wavelengths. However, the sparse distribution of short-wavelength sensitive cones across the retina suggests that they do not have sufficient spatial sampling resolution for this task. Here, we show that the spacing of the short-wavelength sensitive cones in humans is sufficient for them, in conjunction with the longer wavelength cones, to use chromatic signals to detect defocus and guide emmetropization. We hypothesize that the retinal spacing of the short-wavelength sensitive cones in many mammalian species is an evolutionarily ancient adaption that allows the efficient use of chromatic cues in emmetropization.

Effect of different preconditioning protocols on the viscoelastic inflation response of the posterior sclera.

Preconditioning by repeated cyclic loads is routinely used in ex vivo mechanical testing of soft biological tissues. The goal of preconditioning is to achieve a steady and repeatable mechanical response and to measure material properties that are representative of the in vivo condition. Preconditioning protocols vary across studies, and their effect on the viscoelastic response of tested soft tissue is typically not reported or analyzed. We propose a methodology to systematically analyze the preconditioning process with application to inflation testing. We investigated the effect of preconditioning on the viscoelastic inflation response of tree shrew posterior sclera using two preconditioning protocols: (i) continuous cyclic loading-unloading without rest and (ii) cyclic loading-unloading with 15-min rest between cycles. Posterior scleral surface strain was measured using three-dimensional Digital Image Correlation (3D-DIC). We used five variables of characterizing features of the stress-strain loop curve to compare the two preconditioning protocols. Our results showed protocol-dependent differences in the tissue response during preconditioning and at the preconditioned state. Incorporating a resting time between preconditioning cycles significantly decreased the number of cycles (10.5 ± 2.9 cycles vs. 3.1 ± 0.5 cycles, p < 0.001) but increased the total time (15.8 ± 4.4 min vs. 51.2 ± 8.3 min, p < 0.001) needed to reach preconditioned state. At the preconditioned state, 2 of 5 characteristic variables differed significantly between protocols: hysteresis loop area (difference=0.023 kJ/m3, p = 0.0020) and elastic modulus at high IOPs (difference=24.0 MPa, p = 0.0238). Our results suggest that the analysis of the preconditioning process is an essential part of inflation experiments and a prerequisite to properly characterize the tissue viscoelastic response. Furthermore, material properties obtained at the preconditioned state can be impacted by the resting time used during preconditioning and may not be directly compared across studies if the resting time varies by 15 min between studies. STATEMENT OF SIGNIFICANCE: Although applying a preconditioning protocol by repeated cyclic loads is common practice in ex vivo mechanical characterization of soft tissues, the tissue response is typically not reported or analyzed, and the protocol's potential effect on the response remains unclear. This is partially caused by lack of a standardized methodology to precondition soft tissues. We present the first systematic analysis of two representative preconditioning protocols used during inflation testing in ocular biomechanics. Our results show protocol-dependent differences in the viscoelastic response during the preconditioning process and at the preconditioned state. Consequently, the analysis of the preconditioning response represents an essential part of mechanical testing and a prerequisite to properly characterize the tissue viscoelastic response. The effect of preconditioning on the preconditioned state response must be considered when comparing results across studies with different preconditioning protocols.

Effect of Scleral Crosslinking Using Multiple Doses of Genipin on Experimental Progressive Myopia in Tree Shrews.

Purpose: To evaluate the effect of scleral crosslinking (SXL) on slowing experimental progressive myopia in tree shrew eyes using sub-Tenon's injections of genipin (GEN) at different concentrations and number of injections. Methods: Three or five sub-Tenon's injections of GEN at 0 mM (sham), 10 mM, or 20 mM were performed in one eye every other day starting at 18 days of visual experience. Form deprivation (FD) myopia was induced in the injected eye between 24 and 35 days of visual experience; the fellow eye served as control. Tree shrews were randomly assigned to five experimental groups: FD (n = 8); FD + 5 × sham injections (n = 6); FD + 3 × GEN injections at 10 mM (n = 6) and 20 mM (n = 6); and FD + 5 × GEN injections at 20 mM (n = 6). Refractive state and ocular dimensions were measured daily. Results: Compared with the FD group, the sham-injected group showed a transient effect on slowing vitreous chamber elongation. With increasing GEN dose, SXL had an increasing treatment effect on slowing vitreous chamber elongation and myopia progression. In addition, SXL led to a dose-dependent shortening of the aqueous chamber depth and corneal thickening. Lens thickening was observed in the group with the highest concentration. Conclusions: We have shown that SXL using GEN can slow axial elongation and myopia progression in tree shrews. The extent of this treatment effect was dose dependent. Several unexpected effects were observed (corneal thickening, decrease of the anterior chamber depth, and lens thickening), which require further optimization of the GEN delivery approach before clinical consideration. Translational Relevance: The results of this preclinical study suggest that scleral crosslinking using genipin can slow myopia progression.

Histologic validation of optical coherence tomography-based three-dimensional morphometric measurements of the human optic nerve head: Methodology and preliminary results.

PURPOSE: To compare the three-dimensional (3D) morphology of the deep load-bearing structures of the human optic nerve head (ONH) as revealed in vivo by spectral domain optical coherence tomography (SDOCT) with ex vivo quantitative 3D histology. METHODS: SDOCT imaging of the ONH was performed in six eyes from three brain-dead organ donors on life-support equipment awaiting organ procurement (in vivo conditions). Following organ procurement (ex vivo conditions), the eyes were enucleated and underwent a pars plana vitrectomy followed by pressurization to physiologic IOP and immersion fixation. Ex vivo ONH morphology was obtained from high-fidelity episcopic fluorescent 3D reconstruction. Morphologic parameters of the observed ONH canal geometry and peripapillary choroid, as well as the shape, visibility and depth of the lamina cribrosa were compared between ex vivo and in vivo measurements using custom software to align, scale, and manually delineate the different regions of the ONH. RESULTS: There was significant correspondence between in vivo and ex vivo measurements of the depth and shape of the lamina cribrosa, along with the size and shape of Bruch's membrane opening (BMO) and anterior scleral canal opening (ASCO). Weaker correspondence was observed for choroidal thickness; as expected, a thinner choroid was seen ex vivo due to loss of blood volume upon enucleation (-79.9%, p < 0.001). In addition, the lamina was shallower (-32.3%, p = 0.0019) and BMO was smaller ex vivo (-3.38%, p = 0.026), suggesting post mortem shrinkage of the fixed tissue. On average, while highly variable, only 31% of the anterior laminar surface was visible in vivo with SDOCT (p < 0.001). CONCLUSIONS: Morphologic parameters by SDOCT imaging of the deep ONH showed promising correspondence to histology metrics. Small but significant shrinkage artifact, along with large effects of exsanguination of the choroid, was seen in the ex vivo reconstructions of fixed tissues that may impact the quantification of ex vivo histoarchitecture, and this should be considered when developing models and biomarkers based on ex vivo imaging of fixed tissue. Lack of visibly of most of the lamina surface in SDOCT images is an important limitation to metrics and biomarkers based on in vivo images of the ONH deep tissues.

A novel tree shrew model of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective therapy. Animal models effectively reproducing IPF disease features are needed to study the underlying molecular mechanisms. Tree shrews are genetically, anatomically, and metabolically closer to humans than rodents or dogs; therefore, the tree shrew model presents a unique opportunity for translational research in lung fibrosis. Here we demonstrate that tree shrews have in vivo and in vitro fibrotic responses induced by bleomycin and pro-fibrotic mediators. Bleomycin exposure induced lung fibrosis evidenced by histological and biochemical fibrotic changes. In primary tree shrew lung fibroblasts, transforming growth factor beta-1 (TGF-ß1) induced myofibroblast differentiation, increased extracellular matrix (ECM) protein production, and focal adhesion kinase (FAK) activation. Tree shrew lung fibroblasts showed enhanced migration and increased matrix invasion in response to platelet derived growth factor BB (PDGF-BB). Inhibition of FAK significantly attenuated pro-fibrotic responses in lung fibroblasts. The data demonstrate that tree shrews have in vivo and in vitro fibrotic responses similar to that observed in IPF. The data, for the first time, support that the tree shrew model of lung fibrosis is a new and promising experimental animal model for studying the pathophysiology and therapeutics of lung fibrosis.

Analysis of the effects of finite element type within a 3D biomechanical model of a human optic nerve head and posterior pole.

BACKGROUND AND OBJECTIVE: Biomechanical stresses and strains can be simulated in the optic nerve head (ONH) using the finite element (FE) method, and various element types have been used. This study aims to investigate the effects of element type on the resulting ONH stresses and strains. METHODS: A single eye-specific model was constructed using 3D delineations of anatomic surfaces in a high-resolution, fluorescent, 3D reconstruction of a human posterior eye, then meshed using our simple meshing algorithm at various densities using 4- and 10-noded tetrahedral elements, as well as 8- and 20-noded hexahedral elements. A mesh-free approach was used to assign heterogeneous, anisotropic, hyperelastic material properties to the lamina cribrosa, sclera and pia. The models were subjected to elevated IOP of 45 mmHg after pre-stressing from 0 to 10 mmHg, and solved in the open-source FE package Calculix; results were then interpreted in relation to computational time and simulation accuracy, using the quadratic hexahedral model as the reference standard. RESULTS: The 10-noded tetrahedral and 20R-noded hexahedral elements exhibited similar scleral canal and laminar deformations, as well as laminar and scleral stress and strain distributions; the quadratic tetrahedral models ran significantly faster than the quadratic hexahedral models. The linear tetrahedral and hexahedral elements were stiffer compared to the quadratic element types, yielding much lower stresses and strains in the lamina cribrosa. CONCLUSIONS: Prior studies have shown that 20-noded hexahedral elements yield the most accurate results in complex models. Results show that 10-noded tetrahedral elements yield very similar results to 20-noded hexahedral elements and so they can be used interchangeably, with significantly lower computational time. Linear element types did not yield acceptable results.

Technical advance: The use of tree shrews as a model of pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease with a high morbidity and mortality. Some of the mechanisms of fibrosis development have been described using rodent models; however, the relevance of findings in these animal models is difficult to assess. New innovative models are needed that closely mimic IPF disease pathology. METHODS: To overcome this unmet need of investigating IPF with a relevant model, we utilized tree shrews, which are genetically, anatomically, and metabolically similar to primates and humans. Using human antibodies and primers, we investigated the role of macrophage phenotypic switching in normal and IPF subjects and bleomycin-injured tree shrews. RESULTS: Bronchoalveolar lavage (BAL) cells from tree shrews expressed human markers, and there was recruitment of monocyte-derived macrophages (MDMs) to the lung in IPF subjects and bleomycin-injured tree shrews. MDMs were polarized to a profibrotic phenotype in IPF and in bleomycin-injured tree shrews. Resident alveolar macrophages (RAMs) expressed proinflammatory markers regardless of bleomycin exposure. Tree shrews developed bleomycin-induced pulmonary fibrosis with architectural distortion in parenchyma and widespread collagen deposition. CONCLUSION: The profibrotic polarization of macrophages has been demonstrated to be present in IPF subjects and in fibrotic mice. Although the lung macrophages have long been considered to be homogeneous, recent evidence indicates that these cells are heterogeneous during multiple chronic lung diseases. Here, we show new data that indicate a critical and essential role for macrophage-fibroblast crosstalk promoting fibroblast differentiation and collagen production. in the development and progression of fibrosis. The current data strongly suggest development of therapeutics that attenuate of the profibrotic activation of MDMs may mitigate macrophage-fibroblast interaction. These observations demonstrate that tree shrews are an ideal animal model to investigate the pathogenesis of IPF as they are genetically, anatomically, and metabolically closer to humans than the more commonly used rodent models.

Connective Tissue Remodeling in Myopia and its Potential Role in Increasing Risk of Glaucoma.

Myopia and glaucoma are both increasing in prevalence and are linked by an unknown mechanism as many epidemiologic studies have identified moderate to high myopia as an independent risk factor for glaucoma. Myopia and glaucoma are both chronic conditions that lead to connective tissue remodeling within the sclera and optic nerve head. The mechanobiology underlying connective tissue remodeling differs substantially between both diseases, with different homeostatic control mechanisms. In this article, we discuss similarities and differences between connective tissue remodeling in myopia and glaucoma; selected multi-scale mechanisms that are thought to underlie connective tissue remodeling in both conditions; how asymmetric remodeling of the optic nerve head may predispose a myopic eye for pathological remodeling and glaucoma; and how neural tissue deformations may accumulate throughout both pathologies and increase the risk for mechanical insult of retinal ganglion cell axons.

A Mesh-Free Approach to Incorporate Complex Anisotropic and Heterogeneous Material Properties into Eye-Specific Finite Element Models.

Commercial finite element modeling packages do not have the tools necessary to effectively incorporate the complex anisotropic and heterogeneous material properties typical of the biological tissues of the eye. We propose a mesh-free approach to incorporate realistic material properties into finite element models of individual human eyes. The method is based on the idea that material parameters can be estimated or measured at so called control points, which are arbitrary and independent of the finite element mesh. The mesh-free approach approximates the heterogeneous material parameters at the Gauss points of each finite element while the boundary value problem is solved using the standard finite element method. The proposed method was applied to an eye-specific model a human posterior pole and optic nerve head. We demonstrate that the method can be used to effectively incorporate experimental measurements of the lamina cribrosa micro-structure into the eye-specific model. It was convenient to define characteristic material orientations at the anterior and posterior scleral surface based on the eye-specific geometry of each sclera. The mesh-free approach was effective in approximating these characteristic material directions with smooth transitions across the sclera. For the first time, the method enabled the incorporation of the complex collagen architecture of the peripapillary sclera into an eye-specific model including the recently discovered meridional fibers at the anterior surface and the depth dependent width of circumferential fibers around the scleral canal. The model results suggest that disregarding the meridional fiber region may lead to an underestimation of local strain concentrations in the retina. The proposed approach should simplify future studies that aim to investigate collagen remodeling in the sclera and optic nerve head or in other biological tissues with similar challenges.

Scleral structure and biomechanics.

As the eye's main load-bearing connective tissue, the sclera is centrally important to vision. In addition to cooperatively maintaining refractive status with the cornea, the sclera must also provide stable mechanical support to vulnerable internal ocular structures such as the retina and optic nerve head. Moreover, it must achieve this under complex, dynamic loading conditions imposed by eye movements and fluid pressures. Recent years have seen significant advances in our knowledge of scleral biomechanics, its modulation with ageing and disease, and their relationship to the hierarchical structure of the collagen-rich scleral extracellular matrix (ECM) and its resident cells. This review focuses on notable recent structural and biomechanical studies, setting their findings in the context of the wider scleral literature. It reviews recent progress in the development of scattering and bioimaging methods to resolve scleral ECM structure at multiple scales. In vivo and ex vivo experimental methods to characterise scleral biomechanics are explored, along with computational techniques that combine structural and biomechanical data to simulate ocular behaviour and extract tissue material properties. Studies into alterations of scleral structure and biomechanics in myopia and glaucoma are presented, and their results reconciled with associated findings on changes in the ageing eye. Finally, new developments in scleral surgery and emerging minimally invasive therapies are highlighted that could offer new hope in the fight against escalating scleral-related vision disorder worldwide.

Matching the LenStar optical biometer to A-Scan ultrasonography for use in small animal eyes with application to tree shrews.

We describe an analysis strategy to obtain ultrasonography-matched axial dimensions of small animal eyes using the LenStar biometer. The LenStar optical low-coherence reflectometer is an attractive device for animal research due to its high precision, non-invasiveness, and the ability to measure the axial dimensions of cornea, anterior chamber, lens, vitreous chamber, and axial length. However, this optical biometer was designed for clinical applications in human eyes and its internal analysis provides inaccurate values when used on small eyes due to species-dependent differences in refractive indices and relative axial dimensions. The LenStar uses a near infrared light source to measure optical path lengths (OPLs) that are converted by the LenStar's EyeSuite software into geometrical lengths (GLs) based on the refractive indices and axial dimensions of the human eye. We present a strategy that extracts the OPLs, determines refractive indices specific for the small animal eye of interest and then calculates corrected GLs. The refractive indices are obtained by matching the LenStar values to ultrasonography values in the same eyes. As compared to ultrasounography, we found that the internal calculations of the LenStar underestimate the axial dimensions of all ocular compartments of the tree shrew eye: anterior segment depth by 6.17±4.50%, lens thickness by 1.37±3.06%, vitreous chamber depth by 29.23±2.35%, and axial length by 10.62±1.75%. Using tree shrew-specific refractive indices, the axial dimensions closely matched those measured by ultrasonography for each compartment. Our analysis strategy can be easily translated to other species by obtaining a similar paired data set using ultrasonography and LenStar, and applying our step by step procedures.

Experimental myopia increases and scleral crosslinking using genipin inhibits cyclic softening in the tree shrew sclera.

PURPOSE: Myopia progression is thought to involve biomechanical weakening of the sclera, which leads to irreversible deformations and axial elongation of the eye. Scleral crosslinking has been proposed as a potential treatment option for myopia control by strengthening the mechanically weakened sclera. The biomechanical mechanism by which the sclera weakens during myopia and strengthens after crosslinking is not fully understood. Here, we assess the effect of lens-induced myopia and exogenous crosslinking using genipin on the inelastic mechanical properties of the tree shrew sclera measured by cyclic tensile tests. METHODS: Cyclic tensile tests were performed on 2-mm wide scleral strips at physiological loading conditions (50 cycles, 0-3.3 g, 30 s cycle-1 ). Two scleral strips were obtained from each eye of juvenile tree shrews exposed to two different visual conditions: normal and 4 days of monocular -5 D lens wear to accelerate scleral remodelling and induce myopia. Scleral strips were mechanically tested at three alternative conditions: immediately after enucleation; after incubation in phosphate buffered saline (PBS) for 24 h at 37°C; and after incubation for 24 h in PBS supplemented with genipin at a low cytotoxicity concentration (0.25 mm). Cyclic softening was defined as the incremental strain increase from one cycle to the next. RESULTS: -5D lens treatment significantly increased the cyclic softening response of the sclera when compared to contralateral control eyes (0.10% ± 0.029%, mean ± standard error, P = 0.037). Exogenous crosslinking of the lens treated sclera significantly decreased the cyclic softening response (-0.12% ± 0.014%, P = 2.2 × 10-5 ). Contrary to all other groups, the genipin-cross-linked tissue did not exhibit cyclic softening significantly different from zero within the 50-cycle test. CONCLUSIONS: Results indicated that cyclic tensile loading leads to an inelastic, cyclic softening of the juvenile tree shrew sclera. The softening rate increased during lens-induced myopia and was diminished after genipin crosslinking. This finding suggests that axial elongation in myopia may involve a biomechanical weakening mechanism that increased the cyclic softening response of the sclera, which was inhibited by scleral crosslinking using genipin.

Multi-Scale Modeling of Vision-Guided Remodeling and Age-Dependent Growth of the Tree Shrew Sclera During Eye Development and Lens-Induced Myopia.

The sclera uses unknown mechanisms to match the eye's axial length to its optics during development, producing eyes with good focus (emmetropia). A myopic eye is too long for its own optics. We propose a multi-scale computational model to simulate eye development based on the assumption that scleral growth is controlled by genetic factors while scleral remodeling is driven by genetic factors and the eye's refractive error. We define growth as a mechanism that changes the tissue volume and mass while remodeling involves internal micro-deformations that are volume-preserving at the macroscale. The model was fitted against longitudinal refractive measurements in tree shrews of different ages and exposed to three different visual conditions: (i) normal development; (ii) negative lens wear to induce myopia; and (iii) recovery from myopia by removing the negative lens. The model was able to replicate the age- and vision-dependent response of the tree shrew experiments. Scleral growth ceased at younger age than scleral remodeling. The remodeling rate decreased as the eye emmetropized but increased at any age when a negative lens was put on. The predictive power of the model was investigated by calculating the susceptibility to scleral remodeling and the response to form deprivation myopia in tree shrews. Both predictions were in good agreement with experimental data that were not used to fit the model. We propose the first model that distinguishes scleral growth from remodeling. The good agreement of our results with experimental data supports the notion that scleral growth and scleral remodeling are two independently controlled mechanisms during eye development.

A Novel Organ Culture Model to Quantify Collagen Remodeling in Tree Shrew Sclera.

Increasing evidence suggests that unknown collagen remodeling mechanisms in the sclera underlie myopia development. We are proposing a novel organ culture system in combination with two-photon fluorescence imaging to quantify collagen remodeling at the tissue- and lamella-level. Tree shrew scleral shells were cultured up to 7 days in serum-free media and cellular viability was investigated under: (i) minimal tissue manipulations; (ii) removal of intraocular tissues; gluing the eye to a washer using (iii) 50 µL and (iv) 200 µL of cyanoacrylate adhesive; (v) supplementing media with Ham's F-12 Nutrient Mixture; and (vi) culturing eyes subjected to 15 mmHg intraocular pressure in our new bioreactor. Two scleral shells of normal juvenile tree shrews were fluorescently labeled using a collagen specific protein and cultured in our bioreactor. Using two-photon microscopy, grid patterns were photobleached into and across multiple scleral lamellae. These patterns were imaged daily for 3 days, and tissue-/lamella-level strains were calculated from the deformed patterns. No significant reduction in cell viability was observed under conditions (i) and (v). Compared to condition (i), cell viability was significantly reduced starting at day 0 (condition (ii)) and day 3 (conditions (iii, iv, vi)). Tissue-level strain and intralamellar shear angel increased significantly during the culture period. Some scleral lamellae elongated while others shortened. Findings suggest that tree shrew sclera can be cultured in serum-free media for 7 days with no significant reduction in cell viability. Scleral fibroblasts are sensitive to tissue manipulations and tissue gluing. However, Ham's F-12 Nutrient Mixture has a protective effect on cell viability and can offset the cytotoxic effect of cyanoacrylate adhesive. This is the first study to quantify collagen micro-deformations over a prolonged period in organ culture providing a new methodology to study scleral remodeling in myopia.