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Blast-induced traumatic brain injury is the signature injury of modern military conflicts. To more fully understand the effects of blast exposure, we placed rats in different holder configurations, exposed them to blast overpressure, and assessed the degree of eye and brain injury. Anesthetized Long-Evans rats received blast exposures directed at the head (63 kPa, 195 dB-SPL) in either an "open holder" (head and neck exposed; n = 7), or an "enclosed holder" (window for blast exposure to eye; n = 15) and were compared to non-blast exposed (control) rats (n = 22). Outcomes included optomotor response (OMR), electroretinography (ERG), and spectral domain optical coherence tomography (SD-OCT) at 2, 4, and 6 months post-blast, and cognitive function (Y-maze) at 3 months. Spatial frequency and contrast sensitivity were reduced in ipsilateral blast-exposed eyes in both holders (p 0.01), while contralateral eyes showed greater deficits with the enclosed holder (p 0.05). Thinner retinas (p 0.001) and reduced ERG a- and b- wave amplitudes (p 0.05) were observed for both ipsilateral and contralateral eyes with the enclosed, but not the open, holder. Rats in the open holder showed cognitive deficits compared to rats in the enclosed holder (p 0.05). Overall, the animal holder configuration used in blast exposure studies can significantly affect outcomes. Enclosed holders may cause secondary damage to the contralateral eye by concussive injury or blast wave reflection off the holder wall. Open holders may damage the brain via rapid head movement (contrecoup injury). These results highlight additional factors to be considered when evaluating patients with blast exposure or developing models of blast injury.

The sclera is believed to biomechanically influence eye size, facilitating the excessive axial elongation that occurs during myopigenesis. Here, we test the hypothesis that the sclera will be remodeled and exhibit altered biomechanics in the mouse model of form-deprivation (FD) myopia, accompanied by altered retinoid concentrations, a potential signaling molecule involved in the process.Male C57 Bl/6J mice were subjected to unilateral FD (n = 44 eyes), leaving the contralateral eye untreated (contra; n = 44). Refractive error and ocular biometry were measured in vivo prior to and after 1 or 3 weeks of FD. Ex vivo measurements were made of scleral biomechanical properties (unconfined compression: n = 24), scleral sulfated glycosaminoglycan (sGAG) content (dimethylmethylene blue: n = 18, and immunohistochemistry: n = 22), and ocular all-trans retinoic acid (atRA) concentrations (retina and RPE + choroid + sclera, n = 24). Age-matched naïve controls were included for some outcomes (n = 32 eyes).Significant myopia developed after 1 (-2.4 ± 1.1 diopters [D], P0.001) and 3 weeks of FD (-4.1 ± 0.7 D, P = 0.025; mean ± standard deviation). Scleral tensile stiffness and permeability were significantly altered during myopigenesis (stiffness = -31.4 ± 12.7%, P0.001, and permeability = 224.4 ± 205.5%, P0.001). Total scleral sGAG content was not measurably altered; however, immunohistochemistry indicated a sustained decrease in chondroitin-4-sulfate and a slower decline in dermatan sulfate. The atRA increased in the retinas of eyes form-deprived for 1 week.We report that biomechanics and GAG content of the mouse sclera are altered during myopigenesis. All scleral outcomes generally follow the trends found in other species and support a retina-to-sclera signaling cascade underlying mouse myopigenesis.

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.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.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.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.The development of scleral stiffening as a neuroprotective treatment will require the identification of better tolerated stiffening protocols and further preclinical testing.

Determining tissue biomechanical material properties from mechanical test data is frequently required in a variety of applications. However, the validity of the resulting constitutive model parameters is the subject of debate in the field. Parameter optimization in tissue mechanics often comes down to the “identifiability” or “uniqueness” of constitutive model parameters; however, despite advances in formulating complex constitutive relations and many classic and creative curve-fitting approaches, there is currently no accessible framework to study the identifiability of tissue material parameters. Our objective was to assess the identifiability of material parameters for established constitutive models of fiber-reinforced soft tissues, biomaterials, and tissue-engineered constructs and establish a generalizable procedure for other applications. To do so, we generated synthetic experimental data by simulating uniaxial tension and compression tests, commonly used in biomechanics. We then fit this data using a multi-start optimization technique based on the nonlinear least-squares method with multiple initial parameter guesses. We considered tendon and sclera as example tissues, using constitutive models that describe these fiber-reinforced tissues. We demonstrated that not all the model parameters of these constitutive models were identifiable from uniaxial mechanical tests, despite achieving virtually identical fits to the stress-stretch response. We further show that when the lateral strain was considered as an additional fitting criterion, more parameters are identifiable, but some remain unidentified. This work provides a practical approach for addressing parameter identifiability in tissue mechanics.

Background Astronauts undergoing long-duration spaceflight are exposed to numerous health risks, including Spaceflight-Associated Neuro-Ocular Syndrome (SANS), a spectrum of ophthalmic changes that can result in permanent loss of visual acuity. The etiology of SANS is not well understood but is thought to involve changes in cerebrovascular flow dynamics in response to microgravity. There is a paucity of knowledge in this area; in particular, cerebrospinal fluid (CSF) flow dynamics have not been well characterized under microgravity conditions. Our study was designed to determine the effect of simulated microgravity (head-down tilt [HDT]) on cerebrovascular flow dynamics. We hypothesized that microgravity conditions simulated by acute HDT would result in increases in CSF pulsatile flow. Methods In a prospective cohort study, we measured flow in major cerebral arteries, veins, and CSF spaces in fifteen healthy volunteers using phase contrast magnetic resonance (PCMR) before and during 15° HDT. Results We found a decrease in all CSF flow variables [systolic peak flow (p = 0.009), and peak-to-peak pulse amplitude (p = 0.001)]. Cerebral arterial average flow (p = 0.04), systolic peak flow (p = 0.04), and peak-to-peak pulse amplitude (p = 0.02) all also significantly decreased. We additionally found a decrease in average cerebral arterial flow (p = 0.040). Finally, a significant increase in cerebral venous cross-sectional area under HDT (p = 0.005) was also observed. Conclusions These results collectively demonstrate that acute application of −15° HDT caused a reduction in CSF flow variables (systolic peak flow and peak-to-peak pulse amplitude) which, when coupled with a decrease in average cerebral arterial flow, systolic peak flow, and peak-to-peak pulse amplitude, is consistent with a decrease in cardiac-related pulsatile CSF flow. These results suggest that decreases in cerebral arterial inflow were the principal drivers of decreases in CSF pulsatile flow.

PurposeTo evaluate the effects of negative periocular pressure (NPP), and concomitant intraocular pressure (IOP) lowering, on the biomechanics of the optic nerve head (ONH) and cornea.MethodsWe developed a validated finite element (FE) model of the eye to compute tissue biomechanical strains induced in response to NPP delivered using the Multi-Pressure Dial (MPD) system. The model was informed by clinical measurements of IOP lowering and was based on published tissue properties. We also conducted sensitivity analyses by changing pressure loads and tissue properties.ResultsApplication of -7.9 mmHg NPP decreased strain magnitudes in the ONH by c. 50% while increasing corneal strain magnitudes by c. 25%. Comparatively, a similar increase in corneal strain was predicted to occur due to an increase in IOP of 4 mmHg. Sensitivity studies indicated that NPP lowers strain in the ONH by reducing IOP and that these effects persisted over a range of tissue stiffnesses and spatial distributions of NPP.ConclusionsNPP is predicted to considerably decrease ONH strain magnitudes. It also increases corneal strain but to an extent expected to be clinically insignificant. Thus, using NPP to lower IOP and hence decrease ONH mechanical strain is likely biomechanically beneficial for glaucoma patients.Translational RelevanceThis study provides the first description of how NPP affects ONH biomechanics and explains the underlying mechanism of ONH strain reduction. It complements current empirical knowledge about the MPD system and guides future studies of NPP as a treatment for glaucoma.

Measurement of intraocular pressure (IOP) is a standard procedure in ophthalmic research in animals, specifically in glaucoma research, and the control of IOP is essential during certain veterinary ophthalmic surgeries. We evaluated the effect of isoflurane on IOP in the clinically healthy laboratory rabbits and tested a way to minimize the alteration of IOP during isoflurane anesthesia. After measurement of the baseline IOP in each eye of 9 awake New Zealand white rabbits, animals were anesthetized by using either: (1) isoflurane without premedication, (2) a combination of ketamine and xylazine, or (3) isoflurane inhalation after an injection of ketamine–xylazine premedication. Isoflurane led to a sustained increase in IOP of approximately 12 mm Hg. In contrast, ketamine and xylazine decreased IOP by nearly 5 mm Hg (all values compared with baseline measurements in awake, unrestrained animals). The observed decrease in IOP after ketamine–xylazine anesthesia is consistent with anesthetic effects generally seen during anesthesia in other studies. The increased IOP after isoflurane anesthesia in rabbits in this study was an unexpected result that appears to be specific to this combination of anesthetic and animal species. Premedication with ketamine–xylazine diminished the effect of isoflurane inhalation on IOP. These results should be considered in the design of ophthalmic research studies using rabbits and in intraocular surgery where IOP stability is desired.

Background/Objectives Spaceflight associated neuro-ocular syndrome (SANS), a health risk related to long-duration spaceflight, is hypothesized to result from a headward fluid shift that occurs with the loss of hydrostatic pressure gradients in weightlessness. Shifts in the vascular and cerebrospinal fluid compartments alter the mechanical forces at the posterior eye and lead to flattening of the posterior ocular globe. The goal of the present study was to develop a method to quantify globe flattening observed by magnetic resonance imaging after spaceflight. Subjects/Methods Volumetric displacement of the posterior globe was quantified in 10 astronauts at 5 time points after spaceflight missions of ~6 months. Results Mean globe volumetric displacement was 9.88 mm3 (95% CI 4.56–15.19 mm3, p 3 (95% CI 3.73–14.27 mm3, p = 0.001) at R + 30 days; 6.53 mm3 (95% CI 1.24–11.83 mm3, p 3 (95% CI −0.96 to 9.86 mm3, p = 0.12) at R + 180 days; and 7.21 mm3 (95% CI 1.82–12.60 mm3, p Conclusions There was a consistent inward displacement of the globe at the optic nerve, which had only partially resolved 1 year after landing. More pronounced globe flattening has been observed in previous studies of astronauts; however, those observations lacked quantitative measures and were subjective in nature. The novel automated method described here allows for detailed quantification of structural changes in the posterior globe that may lead to an improved understanding of SANS.

Lysyl oxidase like-1 (LOXL1), a vital crosslinking enzyme in elastin fiber maintenance, is essential for the stability and strength of elastic vessels and tissues. Variants in the LOXL1 locus associate with a dramatic increase in risk of exfoliation syndrome, a systemic fibrillopathy, which often presents with ocular hypertension and exfoliation glaucoma. We examined the role of LOXL1 in conventional outflow function, the prime regulator of intraocular pressure. Using Loxl1(−/−), Loxl1(+/−) and Loxl1(+/+) mice, we observed an inverse relationship between LOXL1 expression and intraocular pressure, which worsened with age. Elevated intraocular pressure in Loxl1(−/−) mice was associated with a larger globe, decreased ocular compliance, increased outflow facility, extracellular matrix abnormalities, and dilated intrascleral veins, yet no dilation of arteries or capillaries. Interestingly, in living Loxl1(−/−) mouse eyes, Schlemm’s canal was less susceptible to collapse when challenged with acute elevations in intraocular pressure, suggesting elevated episcleral venous pressure. Thus, LOXL1 expression is required for normal intraocular pressure control, while ablation results in altered extracellular matrix repair/homeostasis and conventional outflow physiology. Dilation of Schlemm’s canal and distal veins, but not arteries, is consistent with key structural and functional roles for elastin in low-pressure vessels subjected to cyclical mechanical stress.

The iris is a muscular organ whose deformations can cause primary angle-closure glaucoma (PACG), a leading cause of blindness. PACG risk assessment does not consider iridial biomechanical factors, despite their expected influence on iris deformations. Here we exploited an existing biometric data set consisting of near-infrared movies acquired during the pupillary light reflex (PLR) as a unique resource to study iris biomechanics. The PLR caused significant (>100%) and essentially spatially uniform radial strains in the iris in vivo, consistent with previous findings. Inverse finite element modeling showed that sphincter muscle tractions were c. 5-fold greater than iridial stroma stiffness (range 4- to 13-fold, depending on sphincter muscle size). This muscle traction is greater than has been previously estimated, which may be due to methodological differences and/or to different patient populations in our study (European descent) vs. previous studies (Asian); the latter possibility is of particular interest due to differential incidence rates of PACG in these populations. Our methodology is fast and inexpensive and may be a useful tool in understanding biomechanical factors contributing to PACG.

Optic nerve head (ONH) biomechanics is centrally involved in the pathogenesis of glaucoma, a blinding ocular condition often characterized by elevation and fluctuation of the intraocular pressure and resulting loads on the ONH. Further, tissue viscoelasticity is expected to strongly influence the mechanical response of the ONH to mechanical loading, yet the viscoelastic mechanical properties of the ONH remain unknown. To determine these properties, we conducted micromechanical testing on porcine ONH tissue samples, coupled with finite element modeling based on a mixture model consisting of a biphasic material with a viscoelastic solid matrix. Our results provide a detailed description of the viscoelastic properties of the porcine ONH at each of its four anatomical quadrants (i.e., nasal, superior, temporal, and inferior). We showed that the ONH's viscoelastic mechanical response can be explained by a dual mechanism of fluid flow and solid matrix viscoelasticity, as is common in other soft tissues. We obtained porcine ONH properties as follows: matrix Young's modulus E=1.895[1.056,2.391] kPa (median [min., max.]), Poisson's ratio ν=0.142[0.060,0.312], kinetic time-constant τ=214[89,921] sec, and hydraulic permeability k=3.854×10

Glaucoma is the leading cause of global irreversible blindness, necessitating research for new, more efficacious treatment options than currently exist. Trabecular meshwork (TM) cells play an important role in the maintenance and function of the aqueous outflow pathway, and studies have found that there is decreased cellularity of the TM in glaucoma. Regeneration of the TM with stem cells has been proposed as a novel therapeutic option by several reports over the last few decades. Stem cells have the capacity for self-renewal and the potential to differentiate into adult functional cells. Several types of stem cells have been investigated in ocular regenerative medicine: tissue specific stem cells, embryonic stem cells, induced pluripotent stem cells, and adult mesenchymal stem cells. These cells have been used in various glaucoma animal models and ex vivo models and have shown success in IOP homeostasis and TM cellularity restoration. They have also demonstrated stability without serious side effects for a significant period of time. Based on current knowledge of TM pathology in glaucoma and existing literature regarding stem cell regeneration of this tissue, we propose a human clinical study as the next step in understanding this potentially revolutionary treatment paradigm. The ability to protect and replace TM cells in glaucomatous eyes could change the field forever.

Optic nerve head (ONH) biomechanics is centrally involved in the pathogenesis of glaucoma, a blinding ocular condition often characterized by elevation and fluctuation of the intraocular pressure and resulting loads on the ONH. Further, tissue viscoelasticity is expected to strongly influence the mechanical response of the ONH to mechanical loading, yet the viscoelastic mechanical properties of the ONH remain unknown. To determine these properties, we conducted micromechanical testing on porcine ONH tissue samples, coupled with finite element modeling based on a mixture model consisting of a biphasic material with a viscoelastic solid matrix. Our results provide a detailed description of the viscoelastic properties of the porcine ONH at each of its four anatomical quadrants (i.e., nasal, superior, temporal, and inferior). We showed that the ONH's viscoelastic mechanical response can be explained by a dual mechanism of fluid flow and solid matrix viscoelasticity, as is common in other soft tissues. We obtained porcine ONH properties as follows: matrix Young's modulus E = 1.895 [ 1.056 , 2.391 ] kPa (median [min., max.]), Poisson's ratio ν = 0.142 [ 0.060 , 0.312 ] , kinetic time-constant τ = 214 [ 89 , 921 ] sec, and hydraulic permeability k = 3.854 × 10 − 1 [ 3.457 × 10 − 2 , 9.994 × 10 − 1 ] mm4/(N.sec). These values can be used to design and fabricate physiologically appropriate ex vivo test environments (e.g., 3D cell culture) to further understand glaucoma pathophysiology. Statement of significance Optic nerve head (ONH) biomechanics is an important aspect of the pathogenesis of glaucoma, the leading cause of irreversible blindness. The ONH experiences time-varying loads, yet the viscoelastic behavior of this tissue has not been characterized. Here, we measure the time-dependent response of the ONH in porcine eyes and use mechanical modeling to provide time-dependent mechanical properties of the ONH. This information allows us to identify time-varying stimuli in vivo which have timescales matching the characteristic response times of the ONH, and can also be used to design and fabricate ex vivo 3D cultures to study glaucoma pathophysiology in a physiologically relevant environment, enabling the discovery of new generations of glaucoma medications focusing on neuroprotection.

Glaucoma is the leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP), the primary risk factor for glaucoma, is thought to induce abnormally high strains in optic nerve head (ONH) tissues, which ultimately result in retinal ganglion cell damage and vision loss. The mechanisms by which excessive deformations result in vision loss remain incompletely understood. The ability of computational and in vitro models of the ONH to provide insight into these mechanisms, in many cases, depends on our ability to replicate the physiological environment, which in turn requires knowledge of tissue biomechanical properties. The majority of mechanical data published to date regarding the ONH has been obtained from tensile testing, yet compression has been shown to be the main mode of deformation in the ONH under elevated IOP. We have thus tested pig and rat ONH tissue using unconfined cyclic compression. The material constants C(1), obtained from fitting the stress vs. strain data with a neo-Hookean material model, were 428 [367, 488] Pa and 64 [53, 76] Pa (mean [95% Confidence Interval]) for pig and rat optic nerve head, respectively. Additionally, we investigated the effects of strain rate and tissue storage on C1 values. These data will inform future efforts to understand and replicate the in vivo biomechanical environment of the ONH.

Glaucoma is the second leading cause of blindness in the world. Disease progression is associated with reduced cellularity in the trabecular meshwork (TM), a fluid drainage tissue in the anterior eye. A promising therapy seeks to deliver stem cells to the TM to regenerate the tissue and restore its function. However, like many stem cell-based regenerative therapies, preclinical development relies heavily on histology to evaluate outcomes. To expedite clinical translation, we are developing an ultrasound/photoacoustic (US/PA) imaging platform for longitudinal tracking of stem cells in the anterior eye. Methods: Mesenchymal stem cells (MSCs) were labeled with gold nanospheres in vitro and injected through the cornea into the anterior chamber of ex vivo porcine eyes. Physiological pressure was imposed to mimic in vivo conditions. AuNS-labeled MSCs were injected through the cornea while single-wavelength US/PA images were acquired. At 5 hours post-injection, three-dimensional multi-wavelength US/PA datasets were acquired and spectroscopic analysis was used to detect AuNS-labeled MSCs. US/PA results were compared to fluorescent microscopy. Results: The US/PA imaging platform was able to provide real-time monitoring of the stem cell injection and distinguish AuNS-labeled MSCs from highly absorbing background tissues in the anterior segment. Conclusion: Our US/PA imaging approach can inform preclinical studies of stem cell therapies for glaucoma treatment, motivating further development of this theranostic imaging tool for ophthalmic applications.

Spaceflight is known to cause ophthalmic changes in a condition known as spaceflight-associated neuro-ocular syndrome (SANS). It is hypothesized that SANS is caused by cephalad fluid shifts and potentially mild elevation of intracranial pressure (ICP) in microgravity. Head-down tilt (HDT) studies are a ground-based spaceflight analogue to create cephalad fluid shifts. Here, we developed non-invasive magnetic resonance imaging (MRI)-based techniques to quantify ophthalmic structural changes under acute 15° HDT. We specifically quantified: (i) change in optic nerve sheath (ONS) and optic nerve (ON) cross-sectional area, (ii) change in ON deviation, an indicator of ON tortuosity, (iii) change in vitreous chamber depth, and (iv) an estimated ONS Young's modulus. Under acute HDT, ONS cross-sectional area increased by 4.04 mm 2 (95% CI 2.88–5.21 mm 2 , p < 0. 000), while ON cross-sectional area remained nearly unchanged (95% CI −0.12 to 0.43 mm 2 , p = 0.271). ON deviation increased under HDT by 0.20 mm (95% CI 0.08–0.33 mm, p = 0.002). Vitreous chamber depth decreased under HDT by −0.11 mm (95% CI −0.21 to −0.03 mm, p = 0.009). ONS Young's modulus was estimated to be 85.0 kPa. We observed a significant effect of sex and BMI on ONS parameters, of interest since they are known risk factors for idiopathic intracranial hypertension. The tools developed herein will be useful for future analyses of ON changes in various conditions.

Purpose Re-cellularization of the trabecular meshwork (TM) using stem cells is a potential novel treatment for ocular hypertension associated with glaucoma. To assess the therapeutic efficacy of this approach, improved in vivo and ex vivo models of TM pathophysiology are needed. Here, we investigate whether oxidative stress, induced by hydrogen peroxide (H2O2), can model glaucomatous ocular hypertension in the readily available porcine anterior segment organ culture model. Methods The impact of H2O2 on TM cell viability and function was first evaluated in vitro using primary porcine TM cells. Oxidative stress was then induced by H2O2 infusion into perfused porcine anterior segments. Trabecular meshwork function was assessed by tracking matrix metalloproteinase (MMP) activity and the ability of the preparation to maintain intraocular pressure (IOP) homeostasis after a flow challenge (doubled fluid infusion rate). Finally, the TM was evaluated histologically. Results H2O2 treatment resulted in a titratable reduction in cellularity across multiple primary TM cell donor strains. In organ culture preparations, H2O2-treated eyes showed impaired IOP homeostasis (i.e., IOPs stabilized at higher levels after a flow challenge vs. control eyes). This result was consistent with reduced MMP activity and TM cellularity; however, damage to the TM microstructure was not histologically evident in anterior segments receiving H2O2. Conclusions Titrated H2O2 infusion resulted in TM cellular dysfunction without destruction of TM structure. Thus, this porcine organ culture model offers a useful platform for assessing trabecular meshwork therapies to treat ocular hypertension associated with glaucoma.

Glucocorticoids are widely used as an ophthalmic medication. A common, sight-threatening adverse event of glucocorticoid usage is ocular hypertension, caused by dysfunction of the conventional outflow pathway. We report that netarsudil, a rho-kinase inhibitor, decreased glucocorticoid-induced ocular hypertension in patients whose intraocular pressures were poorly controlled by standard medications. Mechanistic studies in our established mouse model of glucocorticoid-induced ocular hypertension show that netarsudil both prevented and reduced intraocular pressure elevation. Further, netarsudil attenuated characteristic steroid-induced pathologies as assessed by quantification of outflow function and tissue stiffness, and morphological and immunohistochemical indicators of tissue fibrosis. Thus, rho-kinase inhibitors act directly on conventional outflow cells to prevent or attenuate fibrotic disease processes in glucocorticoid-induced ocular hypertension in an immune-privileged environment. Moreover, these data motivate the need for a randomized prospective clinical study to determine whether netarsudil is indeed superior to first-line anti-glaucoma drugs in lowering steroid-induced ocular hypertension.

The biomechanical properties of the cornea and sclera are important in the onset and progression of multiple ocular pathologies and vary substantially between individuals, yet the source of this variation remains unknown. Here we identify genes putatively regulating corneoscleral biomechanical tissue properties by conducting high-fidelity ocular compliance measurements across the BXD recombinant inbred mouse set and performing quantitative trait analysis. We find seven cis-eQTLs and non-synonymous SNPs associating with ocular compliance, and show by RT-qPCR and immunolabeling that only two of the candidate genes, Smarce1 and Tns4, showed significant expression in corneal and scleral tissues. Both have mechanistic potential to influence the development and/or regulation of tissue material properties. This work motivates further study of Smarce1 and Tns4 for their role(s) in ocular pathology involving the corneoscleral envelope as well as the development of novel mouse models of ocular pathophysiology, such as myopia and glaucoma.

Summary Controlling intraocular pressure (IOP) remains the mainstay of glaucoma therapy. The trabecular meshwork (TM), the key tissue responsible for aqueous humor (AH) outflow and IOP maintenance, is very sensitive to mechanical forces. However, it is not understood whether Piezo channels, very sensitive mechanosensors, functionally influence AH outflow. Here, we characterize the role of Piezo1 in conventional AH outflow. Immunostaining and western blot analysis showed that Piezo1 is widely expressed by TM. Patch-clamp recordings in TM cells confirmed the activation of Piezo1-derived mechanosensitive currents. Importantly, the antagonist GsMTx4 for mechanosensitive channels significantly decreased steady-state facility, yet activation of Piezo1 by the specific agonist Yoda1 did not lead to a facility change. Furthermore, GsMTx4, but not Yoda1, caused a significant increase in ocular compliance, a measure of the eye's transient response to IOP perturbation. Our findings demonstrate a potential role for Piezo1 in conventional outflow, likely under pathological and rapid transient conditions., Graphical abstract, Highlights • Piezo1 is functionally expressed in the TM, the most important tissue controlling IOP • Suppression of mechanosensitive channel leads to a significant decrease in facility • Our data suggest a role for Piezo in pathological situations and rapid IOP transients, Ophthalmology; Cellular Physiology; Biomechanics

This chapter outlines basic concepts for quantifying, understanding and comparing mechanical forces and material properties, with a particular emphasis on the relevance of these concepts to ocular biomechanics. We introduce the concepts of stress, strain, elastic modulus, and viscoelasticity. Additionally, we examine several simple equations used to describe the mechanical environment of the eye at an organ level. We conclude with a discussion of the ways in which cells perceive and respond to their mechanical environment.

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.

Glaucoma is the leading cause of irreversible blindness. Current treatments use drugs or surgery to reduce intraocular pressure (IOP). In this study, a drug‐free, nonsurgical method is developed that lowers IOP for 4 months without requiring daily patient adherence. The approach involves expanding the suprachoroidal space (SCS) of the eye with an in situ‐forming hydrogel injected using a microneedle. This study tests the hypothesis that SCS expansion increases the drainage of aqueous humor from the eye via the unconventional pathway, which thereby lowers IOP. SCS injection of a commercial hyaluronic acid (HA) hydrogel reduces the IOP of normotensive rabbits for more than 1 month and an optimized HA hydrogel formulation enables IOP reduction for 4 months. Safety assessment by clinical ophthalmic examinations indicate the treatment is well tolerated. Histopathology shows minor hemorrhage and fibrosis at the site of injection. Further analysis by ultrasound biomicroscopy demonstrates a strong correlation of IOP reduction with SCS expansion. Outflow facility measurements show no difference in pressure‐dependent outflow by the conventional pathway between treated and untreated eyes, supporting the hypothesis. In conclusion, SCS expansion with an in situ‐forming hydrogel can enable extended IOP reduction for treating ocular hypertension and glaucoma without drugs or surgery., The study reports a novel strategy for treating ocular hypertension and glaucoma with a drug‐free, nonsurgical procedure. The expansion of the suprachoroidal space with an in situ‐forming hyaluronic acid hydrogel administered using a microneedle reduced intraocular pressure of normotensive New Zealand white rabbits for approximately four months after a single injection without significant complications.

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 naive 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.

Nonarteritic anterior ischemic optic neuropathy (NAION) has been associated with a thickened choroid at the optic nerve head (ONH). Here, we use computational modeling to better understand how choroidal expansion and choroidal geometry influence tissue deformation within the ONH relative to intraocular pressure (IOP) and intracranial pressure (ICP) effects.Using a model of the posterior eye that included the sclera, peripapillary sclera, annular ring, pia mater, dura mater, neural tissues, Bruch's membrane, choroid, and lamina cribrosa, we examined how varying material properties of ocular tissues influenced ONH deformations under physiological and supra-physiological, or "pathological," conditions. We considered choroidal expansion (c. 35 µL of expansion), elevated IOP (30 mm Hg), and elevated ICP (20 mm Hg), and calculated peak strains in the ONH relative to a baseline condition representing an individual in the upright position.Supra-physiological choroidal expansion had the largest impact on strains in the prelaminar neural tissue. In addition, compared to a tapered choroid, a "blunt" choroid insertion at the ONH resulted in higher strains. Elevated IOP and ICP caused the highest strains within the lamina cribrosa and retrolaminar neural tissue, respectively.Acute choroidal expansion caused large deformations of the ONH and these deformations were impacted by choroid geometry. These results are consistent with the concept that compartment syndrome due to the choroid geometry and/or expansion at the ONH contributes to NAION. Prolonged deformations due to supra-physiological loading may induce a mechanobiological response or ischemia, highlighting the potential impact of choroidal expansion on biomechanical strains in the ONH.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

A subset of long-duration spaceflight astronauts have experienced ophthalmic abnormalities, collectively termed spaceflight-associated neuro-ocular syndrome (SANS). Little is understood about the pathophysiology of SANS; however, microgravity-induced alterations in intracranial pressure (ICP) due to headward fluid shifts is the primary hypothesized contributor. In particular, potential changes in optic nerve (ON) tortuosity and ON sheath (ONS) distension may indicate altered cerebrospinal fluid dynamics during weightlessness. The present longitudinal study aims to provide a quantitative analysis of ON and ONS cross-sectional areas, and ON deviation, an indication of tortuosity, before and after spaceflight. Ten astronauts undergoing ~6-month missions on the International Space Station (ISS) underwent high-resolution magnetic resonance imaging (MRI) preflight and at five recovery time points extending to 1 year after return from the ISS. The mean changes in ON deviation, ON cross-sectional area, and ONS cross-sectional area immediately post flight were −0.14 mm (95% CI: −0.36 to 0.08, Bonferroni-adjusted P = 1.00), 0.13 mm2 (95% CI −0.66 to 0.91, Bonferroni-adjusted P = 1.00), and −0.22 mm2 (95% CI: −1.78 to 1.34, Bonferroni-adjusted P = 1.00), respectively, and remained consistent during the recovery period. Terrestrially, ONS distension is associated with increased ICP; therefore, these results suggest that, on average, ICP was not pathologically elevated immediately after spaceflight. However, a subject diagnosed with optic disc edema (Frisen Grade 1, right eye) displayed increased ONS area post flight, although this increase is relatively small compared to clinical populations with increased ICP. Advanced quantitative MRI-based assessment of the ON and ONS could help our understanding of SANS and the role of ICP.

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 naive 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.

Glucocorticoids are widely used as an ophthalmic medication. A common, sight-threatening adverse event of glucocorticoid usage is ocular hypertension, caused by dysfunction of the conventional outflow pathway. We report that netarsudil, a rho-kinase inhibitor, rapidly reversed glucocorticoid-induced ocular hypertension in patients whose intraocular pressures were uncontrolled by standard medications. Mechanistic studies in our established mouse model of glucocorticoid-induced ocular hypertension show that netarsudil both prevented and reversed intraocular pressure elevation. Further, netarsudil reversed characteristic steroid-induced pathologies as assessed by quantification of outflow function and tissue stiffness, and morphological and immunohistochemical indicators of tissue fibrosis. Thus, rho-kinase inhibitors act directly on conventional outflow cells to efficaciously prevent or reverse fibrotic disease processes in glucocorticoid-induced ocular hypertension. These data motivate a novel indication for these agents to prevent or treat ocular hypertension secondary to glucocorticoid administration, and demonstrate the antifibrotic effects of rho-kinase inhibitors in an immune-privileged environment.

Figure illustrating the fiber directions assigned to the peripapillary sclera proper and scleral sling.

Derivation of the poroelastic theory used to obtain material properties of sclera in A Biphasic Approach for Characterizing Tensile, Compressive, and Hydraulic Properties of the Sclera, provided for convenience.

Figure illustrating convergence of outcome measure standard deviation with increasing number of simulations. This shows that the parameter space was sampled sufficiently.

Figure illustrating the mesh and boundary conditions on an ONH model iteration.

Purpose Rodent and primate models are commonly used in glaucoma research; however, both have their limitations. The tree shrew (Tupaia belangeri) is an emerging animal model for glaucoma research owing in part to having a human-like optic nerve head anatomy, specifically a collagenous load-bearing lamina. However, the anterior segment anatomy and function have not been extensively studied in the tree shrew. Thus, the purpose of this study was to provide the first detailed examination of the anterior segment anatomy and aqueous outflow facility in the tree shrew. Methods Aqueous outflow dynamics were measured in five ostensibly normal eyes from three tree shrews using the iPerfusion system over a range of pressures. Gross histological assessment and immunohistochemistry were performed to characterize anterior segment anatomy and to localize several key molecules related to aqueous outflow. Results Anterior segment anatomy in tree shrews is similar to humans, demonstrating a scleral spur, a multilayered trabecular meshwork and a circular Schlemm's canal with a single lumen. Average outflow facility was 0.193 µL/min/mm Hg (95% confidence interval, 0.153–0.244), and was stable over time. Outflow facility was more similar between contralateral eyes (approximately 5% average difference) than between eyes of different animals. No significant dependence of outflow facility on time or pressure was detected (pressure–flow nonlinearity parameter of 0.01 (95% % confidence interval, −0.29 to 0.31 CI µL/min/mm Hg). Conclusions These studies lend support to the usefulness of the tree shrew as a novel animal model in anterior segment glaucoma and pharmacology research. The tree shrew's cost, load-bearing collagenous lamina cribrosa, and lack of washout or anterior chamber deepening provides a distinct experimental and anatomic advantage over the current rodent and nonhuman primate models used for translational research.

Glaucoma is the leading cause of irreversible vision loss, and reducing elevated intraocular pressure is currently the only effective clinical treatment. The trabecular meshwork is the main resistance site for aqueous outflow that maintains intraocular pressure. In this study, we transplanted human trabecular meshwork stem cells (TMSCs) intracamerally into mice that received laser photocoagulation over a 180° arc of the trabecular meshwork. TMSCs preferentially homed and integrated to the laser-damaged trabecular meshwork region and expressed differentiated cell markers at 2 and 4 weeks. Laser-induced inflammatory and fibrotic responses were prevented by TMSC transplantation with simultaneous ultrastructure and function restoration. Cell affinity and migration assays and elevated expression of CXCR4 and SDF1 in laser-treated mouse trabecular meshwork suggest that the CXCR4/SDF1 chemokine axis plays an important role in TMSC homing. Our results suggest that TMSCs may be a viable candidate for trabecular meshwork refunctionalization as a novel treatment for glaucoma.

Alterations in stiffness of the trabecular meshwork (TM) may play an important role in primary open-angle glaucoma (POAG), the second leading cause of blindness. Specifically, certain data suggest an association between elevated intraocular pressure (IOP) and increased TM stiffness; however, the underlying link between TM stiffness and IOP remains unclear and requires further study. We here first review the literature on TM stiffness measurements, encompassing various species and based on a number of measurement techniques, including direct approaches such as atomic force microscopy (AFM) and uniaxial tension tests, and indirect methods based on a beam deflection model. We also briefly review the effects of several factors that affect TM stiffness, including lysophospholipids, rho-kinase inhibitors, cytoskeletal disrupting agents, dexamethasone (DEX), transforming growth factor-β(2) (TGF-β(2)), nitric oxide (NO) and cellular senescence. We then describe a method we have developed for determining TM stiffness measurement in mice using a cryosection/AFM-based approach, and present preliminary data on TM stiffness in C57BL/6J and CBA/J mouse strains. Finally, we investigate the relationship between TM stiffness and outflow facility between these two strains. The method we have developed shows promise for further direct measurements of mouse TM stiffness, which may be of value in understanding mechanistic relations between outflow facility and TM biomechanical properties.

The biomechanical environment within the optic nerve head (ONH) is complex and is likely directly involved in the loss of retinal ganglion cells (RGCs) in glaucoma. Unfortunately, our understanding of this process is poor. Here we describe factors that influence ONH biomechanics, including ONH connective tissue microarchitecture and anatomy; intraocular pressure (IOP); and cerebrospinal fluid pressure (CSFp). We note that connective tissue factors can vary significantly from one individual to the next, as well as regionally within an eye, and that the understanding of ONH biomechanics is hindered by anatomical differences between small-animal models of glaucoma (rats and mice) and humans. Other challenges of using animal models of glaucoma to study the role of biomechanics include the complexity of assessing the degree of glaucomatous progression; and inadequate tools for monitoring and consistently elevating IOP in animal models. We conclude with a consideration of important open research questions/challenges in this area, including: (i) Creating a systems biology description of the ONH; (ii) addressing the role of astrocyte connective tissue remodeling and reactivity in glaucoma; (iii) providing a better characterization of ONH astrocytes and non-astrocytic constituent cells; (iv) better understanding the role of ONH astrocyte phagocytosis, proliferation and death; (v) collecting gene expression and phenotype data on a larger, more coordinated scale; and (vi) developing an implantable IOP sensor.