Morphometric Analysis of Retinal Ganglion Cell Axons in Normal and Glaucomatous Brown Norway Rats Optic Nerves.

Purpose: A reference atlas of optic nerve (ON) retinal ganglion cell (RGC) axons could facilitate studies of neuro-ophthalmic diseases by detecting subtle RGC axonal changes. Here we construct an RGC axonal atlas for normotensive eyes in Brown Norway rats, widely used in glaucoma research, and also develop/evaluate several novel metrics of axonal damage in hypertensive eyes. Methods: Light micrographs of entire ON cross-sections from hypertensive and normotensive eyes were processed through a deep learning-based algorithm, AxoNet2.0, to determine axonal morphological properties and were semiquantitatively scored using the Morrison grading scale (MGS) to provide a damage score independent of AxoNet2.0 outcomes. To construct atlases, ONs were conformally mapped onto an ON "template," and axonal morphometric data was computed for each region. We also developed damage metrics based on myelin morphometry. Results: In normotensive eyes, average axon density was ∼0.3 axons/µm2 (i.e., ∼80,000 axons in an ON). We measured axoplasm diameter, eccentricity, cross-sectional area, and myelin g-ratio and thickness. Most morphological parameters exhibited a wide range of coefficients of variation (CoV); however, myelin thickness CoV was only ∼2% in normotensive eyes. In hypertensive eyes, increased myelin thickness correlated strongly with MGS (P < 0.0001). Conclusions: We present the first comprehensive normative RGC axon morphometric atlas for Brown Norway rat eyes. We suggest objective, repeatable damage metrics based on RGC axon myelin thickness for hypertensive eyes. Translational Relevance: These tools can evaluate regional changes in RGCs and overall levels of damage in glaucoma studies using Brown Norway rats.

AxoNet 2.0: A Deep Learning-Based Tool for Morphometric Analysis of Retinal Ganglion Cell Axons.

Purpose: Assessment of glaucomatous damage in animal models is facilitated by rapid and accurate quantification of retinal ganglion cell (RGC) axonal loss and morphologic change. However, manual assessment is extremely time- and labor-intensive. Here, we developed AxoNet 2.0, an automated deep learning (DL) tool that (i) counts normal-appearing RGC axons and (ii) quantifies their morphometry from light micrographs. Methods: A DL algorithm was trained to segment the axoplasm and myelin sheath of normal-appearing axons using manually-annotated rat optic nerve (ON) cross-sectional micrographs. Performance was quantified by various metrics (e.g., soft-Dice coefficient between predicted and ground-truth segmentations). We also quantified axon counts, axon density, and axon size distributions between hypertensive and control eyes and compared to literature reports. Results: AxoNet 2.0 performed very well when compared to manual annotations of rat ON (R2 = 0.92 for automated vs. manual counts, soft-Dice coefficient = 0.81 ± 0.02, mean absolute percentage error in axonal morphometric outcomes < 15%). AxoNet 2.0 also showed promise for generalization, performing well on other animal models (R2 = 0.97 between automated versus manual counts for mice and 0.98 for non-human primates). As expected, the algorithm detected decreased in axon density in hypertensive rat eyes (P ≪ 0.001) with preferential loss of large axons (P < 0.001). Conclusions: AxoNet 2.0 provides a fast and nonsubjective tool to quantify both RGC axon counts and morphological features, thus assisting with assessing axonal damage in animal models of glaucomatous optic neuropathy. Translational Relevance: This deep learning approach will increase rigor of basic science studies designed to investigate RGC axon protection and regeneration.

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.

Using retinal function to define ischemic exclusion criteria for animal models of glaucoma.

Most animal models of glaucoma rely on induction of ocular hypertension (OHT), yet such models can suffer from high IOPs leading to undesirable retinal ischemia. Thus, animals with IOPs exceeding a threshold (e.g. > 60 mmHg) are often excluded from studies. However, due to the intermittent nature of IOP measurements, this approach may fail to detect ischemia. Conversely, it may also inappropriately eliminate animals with IOP spikes that do not induce ischemic damage. It is known that acute ischemia selectively impairs inner retinal function, which results in a reduced b-wave amplitude. Here, we explore the potential of using electroretinography (ERG) to detect ischemic damage in OHT eyes. 74 Brown Norway rats received a unilateral injection of magnetic microbeads to induce OHT, while contralateral eyes served as controls. IOP was measured every 2-3 days for 14 days after microbead injection. Retinal function was evaluated using dark-adapted bright flash ERG (2.1 log cd•s/m2) prior to, and at 7 and 14 days after, injection. We investigated two criteria for excluding animals: (IOP Criterion) a single IOP measurement > 60 mmHg; or (ERG Criterion) a b-wave amplitude below the 99.5% confidence interval for naïve eyes. 49 of 74 rats passed both criteria, 7 of 74 failed both, and 18 passed one criterion but not the other. We suggest that ERG testing can detect unwelcome ischemic damage in animal models of OHT. Since brief IOP spikes do not necessarily lead to ischemic retinal damage, and because extended periods of elevated IOP can be missed, such ERG-based criteria may provide more objective and robust exclusion criteria in future glaucoma studies.

Assessment of Visual and Retinal Function Following In Vivo Genipin-Induced Scleral Crosslinking.

Purpose: Genipin has been proposed as a possible neuroprotective therapy in myopia and glaucoma. Here, we aim to determine the effects of prolonged genipin-induced scleral stiffening on visual function. Methods: Eyes from Brown Norway rats were treated in vivo with either a single 15 mM genipin retrobulbar injection or sham retrobulbar injection and were compared to naïve eyes. Intraocular pressure, optomotor response, and electroretinograms were repeatedly measured over 4 weeks following retrobulbar injections to determine visual and retinal function. At 4 weeks, we quantified retinal ganglion cell axon counts. Finally, molecular changes in gene and protein expression were analyzed via real-time polymerase chain reaction (RT-PCR) and proteomics. Results: Retrobulbar injection of genipin did not affect intraocular pressure (IOP) or retinal function, nor have a sustained impact on visual function. Although genipin-treated eyes had a small decrease in retinal ganglion cell axon counts compared to contralateral sham-treated eyes (-8,558 ± 18,646; mean ± SD), this was not statistically significant (P = 0.206, n = 9). Last, we did not observe any changes in gene or protein expression due to genipin treatment. Conclusions: Posterior scleral stiffening with a single retrobulbar injection of 15 mM genipin causes no sustained deficits in visual or retinal function or at the molecular level in the retina and sclera. Retinal ganglion cell axon morphology appeared normal. Translational Significance: These results support future in vivo studies to determine the efficacy of genipin-induced posterior scleral stiffening to help treat ocular diseases, like myopia and 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.

AxoNet: A deep learning-based tool to count retinal ganglion cell axons.

In this work, we develop a robust, extensible tool to automatically and accurately count retinal ganglion cell axons in optic nerve (ON) tissue images from various animal models of glaucoma. We adapted deep learning to regress pixelwise axon count density estimates, which were then integrated over the image area to determine axon counts. The tool, termed AxoNet, was trained and evaluated using a dataset containing images of ON regions randomly selected from whole cross sections of both control and damaged rat ONs and manually annotated for axon count and location. This rat-trained network was then applied to a separate dataset of non-human primate (NHP) ON images. AxoNet was compared to two existing automated axon counting tools, AxonMaster and AxonJ, using both datasets. AxoNet outperformed the existing tools on both the rat and NHP ON datasets as judged by mean absolute error, R2 values when regressing automated vs. manual counts, and Bland-Altman analysis. AxoNet does not rely on hand-crafted image features for axon recognition and is robust to variations in the extent of ON tissue damage, image quality, and species of mammal. Therefore, AxoNet is not species-specific and can be extended to quantify additional ON characteristics in glaucoma and potentially other neurodegenerative diseases.

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.

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.

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.