Evaluation of Spatially Targeted Scleral Stiffening on Neuroprotection in a Rat Model of Glaucoma.

Purpose: Scleral stiffening may protect against glaucomatous retinal ganglion cell (RGC) loss or dysfunction associated with ocular hypertension. Here, we assess the potential neuroprotective effects of two treatments designed to stiffen either the entire posterior sclera or only the sclera adjacent to the peripapillary sclera in an experimental model of glaucoma. Methods: Rat sclerae were stiffened in vivo using either genipin (crosslinking the entire posterior sclera) or a regionally selective photosensitizer, methylene blue (stiffening only the juxtaperipapillary region surrounding the optic nerve). Ocular hypertension was induced using magnetic microbeads delivered to the anterior chamber. Morphological and functional outcomes, including optic nerve axon count and appearance, retinal thickness measured by optical coherence tomography, optomotor response, and electroretinography traces, were assessed. Results: Both local (juxtaperipapillary) and global (whole posterior) scleral stiffening treatments were successful at increasing scleral stiffness, but neither provided demonstrable neuroprotection in hypertensive eyes as assessed by RGC axon counts and appearance, optomotor response, or electroretinography. There was a weak indication that scleral crosslinking protected against retinal thinning as assessed by optical coherence tomography. Conclusions: Scleral stiffening was not demonstrated to be neuroprotective in ocular hypertensive rats. We hypothesize that the absence of benefit may in part be due to RGC loss associated with the scleral stiffening agents themselves (mild in the case of genipin, and moderate in the case of methylene blue), negating any potential benefit of scleral stiffening. Translational Relevance: The development of scleral stiffening as a neuroprotective treatment will require the identification of better tolerated stiffening protocols and further preclinical testing.

Individual-Specific Modeling of Rat Optic Nerve Head Biomechanics in Glaucoma.

Glaucoma is the second leading cause of blindness worldwide and is characterized by the death of retinal ganglion cells (RGCs), the cells that send vision information to the brain. Their axons exit the eye at the optic nerve head (ONH), the main site of damage in glaucoma. The importance of biomechanics in glaucoma is indicated by the fact that elevated intraocular pressure (IOP) is a causative risk factor for the disease. However, exactly how biomechanical insult leads to RGC death is not understood. Although rat models are widely used to study glaucoma, their ONH biomechanics have not been characterized in depth. Therefore, we aimed to do so through finite element (FE) modeling. Utilizing our previously described method, we constructed and analyzed ONH models with individual-specific geometry in which the sclera was modeled as a matrix reinforced with collagen fibers. We developed eight sets of scleral material parameters based on results from our previous inverse FE study and used them to simulate the effects of elevated IOP in eight model variants of each of seven rat ONHs. Within the optic nerve, highest strains were seen inferiorly, a pattern that was consistent across model geometries and model variants. In addition, changing the collagen fiber direction to be circumferential within the peripapillary sclera resulted in more pronounced decreases in strain than changing scleral stiffness. The results from this study can be used to interpret data from rat glaucoma studies to learn more about how biomechanics affects RGC pathogenesis in glaucoma.

Biomechanical properties of the rat sclera obtained with inverse finite element modeling.

It is widely accepted that biomechanics plays an important role in glaucoma pathophysiology, but the mechanisms involved are largely unknown. Rats are a common animal model of glaucoma, and finite element models are being developed to provide much-needed insight into the biomechanical environment of the posterior rat eye. However, material properties of rat ocular tissues, including the sclera, are currently unknown. Since the sclera plays a major role in posterior ocular biomechanics, our goal was to use inverse finite element modeling to extract rat scleral material properties. We first used digital image correlation to measure scleral surface displacement during whole-globe inflation testing. We modeled the sclera as a nonlinear material with embedded collagen fibers and then fit modeled displacements to experimental data using a differential evolution algorithm. Subject-specific models were constructed in which 3 parameters described the stiffness of the ground substance and collagen fibers in the posterior eye, and 16 parameters defined the primary orientation and alignment of fibers within eight scleral sub-regions. We successfully extracted scleral material properties for eight rat eyes. Model displacements recreated general patterns of the experimental displacements but did not always match local patterns. The fiber directions and fiber concentration parameters were highly variable, but on average, fibers were aligned circumferentially and were more aligned in the peripapillary sclera than in the peripheral sclera. The material properties determined here will be used to inform future finite element models of the rat posterior eye with the goal of elucidating the role of biomechanics in glaucoma pathophysiology.

Factors affecting optic nerve head biomechanics in a rat model of glaucoma.

Glaucoma is the leading cause of irreversible blindness and is characterized by the death of retinal ganglion cells, which carry vision information from the retina to the brain. Although it is well accepted that biomechanics is an important part of the glaucomatous disease process, the mechanisms by which biomechanical insult, usually due to elevated intraocular pressure (IOP), leads to retinal ganglion cell death are not understood. Rat models of glaucoma afford an opportunity for learning more about these mechanisms, but the biomechanics of the rat optic nerve head (ONH), a primary region of damage in glaucoma, are only just beginning to be characterized. In a previous study, we built finite-element models with individual-specific rat ONH geometries. Here, we developed a parametrized model of the rat ONH and used it to perform a sensitivity study to determine the influence that six geometric parameters and 13 tissue material properties have on rat optic nerve biomechanical strains due to IOP elevation. Strain magnitudes and patterns in the parametrized model generally matched those from individual-specific models, suggesting that the parametrized model sufficiently approximated rat ONH anatomy. Similar to previous studies in human eyes, we found that scleral properties were highly influential: the six parameters with highest influence on optic nerve strains were optic nerve stiffness, IOP, scleral thickness, the degree of alignment of scleral collagen fibres, scleral ground substance stiffness and the scleral collagen fibre uncrimping coefficient. We conclude that a parametrized modelling strategy is an efficient approach that allows insight into rat ONH biomechanics. Further, scleral properties are important influences on rat ONH biomechanics, and additional efforts should be made to better characterize rat scleral collagen fibre organization.

Sustained scleral stiffening in rats after a single genipin treatment.

Scleral stiffening has been proposed as a therapy for glaucoma and myopia. Previous in vivo studies have evaluated the efficacy of scleral stiffening after multiple treatments with a natural collagen crosslinker, genipin. However, multiple injections limit clinical translatability. Here, we examined whether scleral stiffening was maintained after four weeks following a single genipin treatment. Eyes from brown Norway rats were treated in vivo with a single 15 mM genipin retrobulbar injection, sham retrobulbar injection, or were left naive. Eyes were enucleated either 1 day or four weeks post-injection and underwent whole globe inflation testing. We assessed first principal Lagrange strain of the posterior sclera using digital image correlation as a proxy for scleral stiffness. Four weeks post-injection, genipin treatment resulted in a 58% reduction in scleral strain as compared to controls (p = 0.005). We conclude that a single in vivo injection of genipin effectively stiffened rat sclera for at least four weeks which motivates further functional studies and possible clinical translation of genipin-induced scleral stiffening.

A Methodology for Individual-Specific Modeling of Rat Optic Nerve Head Biomechanics in Glaucoma.

Glaucoma is the leading cause of irreversible blindness and involves the death of retinal ganglion cells (RGCs). Although biomechanics likely contributes to axonal injury within the optic nerve head (ONH), leading to RGC death, the pathways by which this occurs are not well understood. While rat models of glaucoma are well-suited for mechanistic studies, the anatomy of the rat ONH is different from the human, and the resulting differences in biomechanics have not been characterized. The aim of this study is to describe a methodology for building individual-specific finite element (FE) models of rat ONHs. This method was used to build three rat ONH FE models and compute the biomechanical environment within these ONHs. Initial results show that rat ONH strains are larger and more asymmetric than those seen in human ONH modeling studies. This method provides a framework for building additional models of normotensive and glaucomatous rat ONHs. Comparing model strain patterns with patterns of cellular response seen in studies using rat glaucoma models will help us to learn more about the link between biomechanics and glaucomatous cell death, which in turn may drive the development of novel therapies for glaucoma.

Estimating Human Trabecular Meshwork Stiffness by Numerical Modeling and Advanced OCT Imaging.

Purpose: The purpose of this study was to estimate human trabecular meshwork (hTM) stiffness, thought to be elevated in glaucoma, using a novel indirect approach, and to compare results with direct en face atomic force microscopy (AFM) measurements. Methods: Postmortem human eyes were perfused to measure outflow facility and identify high- and low-flow regions (HF, LF) by tracer. Optical coherence tomography (OCT) images were obtained as Schlemm's canal luminal pressure was directly manipulated. TM stiffness was deduced by an inverse finite element modeling (FEM) approach. A series of AFM forcemaps was acquired along a line traversing the anterior angle on a radially cut flat-mount corneoscleral wedge with TM facing upward. Results: The elastic modulus of normal hTM estimated by inverse FEM was 70 ± 20 kPa (mean ± SD), whereas glaucomatous hTM was slightly stiffer (98 ± 19 kPa). This trend was consistent with TM stiffnesses measured by AFM: normal hTM stiffness = 1.37 ± 0.56 kPa, which was lower than glaucomatous hTM stiffness (2.75 ± 1.19 kPa). None of these differences were statistically significant. TM in HF wedges was softer than that in LF wedges for both normal and glaucomatous eyes based on the inverse FEM approach but not by AFM. Outflow facility was significantly correlated with TM stiffness estimated by FEM in six human eyes (P = 0.018). Conclusions: TM stiffness is higher, but only modestly so, in glaucomatous patients. Outflow facility in both normal and glaucomatous human eyes appears to associate with TM stiffness. This evidence motivates further studies to investigate factors underlying TM biomechanical property regulation.

Quantification of the efficacy of collagen cross-linking agents to induce stiffening of rat sclera.

The concept of scleral stiffening therapies has emerged as a novel theoretical approach for treating the ocular disorders glaucoma and myopia. Deformation of specific regions of the posterior eye is innately involved in the pathophysiology of these diseases, and thus targeted scleral stiffening could resist these changes and slow or prevent progression of these diseases. Here, we present the first systematic screen and direct comparison of the stiffening effect of small molecule collagen cross-linking agents in the posterior globe, namely using glyceraldehyde, genipin and methylglyoxal (also called pyruvaldehyde). To establish a dose-response relationship, we used inflation testing to simulate the effects of increasing intraocular pressure in freshly harvested rat eyes stiffened with multiple concentrations of each agent. We used digital image correlation to compute the mechanical strain in the tissue as a metric of stiffness, using a novel treatment paradigm for screening relative stiffening by incubating half of each eye in cross-linker and using the opposite half as an internal control. We identified the doses necessary to increase stiffness by approximately 100%, namely 30 mM for glyceraldehyde, 1 mM for genipin and 7 mM for methylglyoxal, and we also identified the range of stiffening it was possible to achieve with such agents. Such findings will inform development of in vivo studies of scleral stiffening to treat glaucoma and myopia.

System for Rapid, Precise Modulation of Intraocular Pressure, toward Minimally-Invasive In Vivo Measurement of Intracranial Pressure.

Pathologic changes in intracranial pressure (ICP) are commonly observed in a variety of medical conditions, including traumatic brain injury, stroke, brain tumors, and glaucoma. However, current ICP measurement techniques are invasive, requiring a lumbar puncture or surgical insertion of a cannula into the cerebrospinal fluid (CSF)-filled ventricles of the brain. A potential alternative approach to ICP measurement leverages the unique anatomy of the central retinal vein, which is exposed to both intraocular pressure (IOP) and ICP as it travels inside the eye and through the optic nerve; manipulating IOP while observing changes in the natural pulsations of the central retinal vein could potentially provide an accurate, indirect measure of ICP. As a step toward implementing this technique, we describe the design, fabrication, and characterization of a system that is capable of manipulating IOP in vivo with <0.1 mmHg resolution and settling times less than 2 seconds. In vitro tests were carried out to characterize system performance. Then, as a proof of concept, we used the system to manipulate IOP in tree shrews (Tupaia belangeri) while video of the retinal vessels was recorded and the caliber of a selected vein was quantified. Modulating IOP using our system elicited a rapid change in the appearance of the retinal vein of interest: IOP was lowered from 10 to 3 mmHg, and retinal vein caliber sharply increased as IOP decreased from 7 to 5 mmHg. Another important feature of this technology is its capability to measure ocular compliance and outflow facility in vivo, as demonstrated in tree shrews. Collectively, these proof-of-concept demonstrations support the utility of this system to manipulate IOP for a variety of useful applications in ocular biomechanics, and provide a framework for further study of the mechanisms of retinal venous pulsation.

Pressure and shear stress in trabecular bone marrow during whole bone loading.

Skeletal adaptation to mechanical loading is controlled by mechanobiological signaling. Osteocytes are highly responsive to applied strains, and are the key mechanosensory cells in bone. However, many cells residing in the marrow also respond to mechanical cues such as hydrostatic pressure and shear stress, and hence could play a role in skeletal adaptation. Trabecular bone encapsulates marrow, forming a poroelastic solid. According to the mechanical theory, deformation of the pores induces motion in the fluid-like marrow, resulting in pressure and velocity gradients. The latter results in shear stress acting between the components of the marrow. To characterize the mechanical environment of trabecular bone marrow in situ, pore pressure within the trabecular compartment of whole porcine femurs was measured with miniature pressure transducers during stress-relaxation and cyclic loading. Pressure gradients ranging from 0.013 to 0.46 kPa/mm were measured during loading. This range was consistent with calculated pressure gradients from continuum scale poroelastic models with the same permeability. Micro-scale computational fluid dynamics models created from computed tomography images were used to calculate the micromechanical stress in the marrow using the measured pressure differentials as boundary conditions. The volume averaged shear stress in the marrow ranged from 1.67 to 24.55 Pa during cyclic loading, which exceeds the mechanostimulatory threshold for mesenchymal lineage cells. Thus, the loading of bone through activities of daily living may be an essential component of bone marrow health and mechanobiology. Additional studies of cell-level interactions during loading in healthy and disease conditions will provide further incite into marrow mechanobiology.