Atomic force microscopy (AFM) is a valuable tool for assessing mechanical properties of biological samples, but interpretations of measurements on whole tissues can be difficult due to the tissue’s highly heterogeneous nature. To overcome such difficulties and obtain more robust estimates of tissue mechanical properties, we describe an AFM force mapping and data analysis pipeline to characterize the mechanical properties of cryosectioned soft tissues. We assessed this approach on mouse optic nerve head and rat trabecular meshwork, cornea, and sclera. Our data show that the use of repeated measurements, outlier exclusion, and log-normal data transformation increases confidence in AFM mechanical measurements, and we propose that this methodology can be broadly applied to measuring soft tissue properties from cryosections.

Purpose Electroretinography (ERG) is used to assess retinal function in ophthalmology clinics and animal models of ocular disease; however, analyzing ERG waveforms can be a time-intensive process with interobserver variability. We developed ERGAssist, an automated approach, to perform non-subjective and repeatable feature identification (“marking”) of the ERG waveform. Methods The automated approach denoised the recorded waveforms and then located the b-wave after applying a lowpass filter. If an a-wave was present, the lowpass filter wave was also used to help locate the a-wave, which was considered the initial large negative response after the flash stimuli. Oscillatory potentials (OPs) were found using a bandpass filter on the denoised waveform. We used two cohorts. One was a Coherence cohort that consisted of ERGs with eight dark-adapted and three light-adapted stimuli in Brown Norway rats (−6 to 1.5 log cd·s/m2). The Verification cohort consisted of control and diabetic (DM) Long Evans rats. We examined retinal function using a five-step dark-adapted protocol (−3 to 1.9 log cd·s/m2). Results ERGAssist showed a strong correlation with manual markings of ERG features in our Coherence dataset, including the amplitudes (a-wave: r2 = 0.99; b-wave: r2 = 0.99; OP: r2 = 0.92) and implicit times (a-wave: r2 = 0.96; b-wave: r2 = 0.90; OP: r2 = 0.96). In the Verification cohort, both approaches detected differences between control and DM animals and found longer OP implicit times (P < 0.0001) in DM animals. Conclusions These results provide verification of ERGAssist to identify features of the full-field ERG. Translational Relevance This ERG analysis approach can increase the rigor of basic science studies designed to investigate retinal function using full-field ERG. To aid the community, we have developed an open-source graphical user interface (GUI) implementing the methods presented.

Murine models are commonly used to study glaucoma, the leading cause of irreversible blindness. Glaucoma is associated with elevated intraocular pressure (IOP), which is regulated by the tissues of the aqueous outflow pathway. In particular, pectinate ligaments (PLs) connect the iris and trabecular meshwork (TM) at the anterior chamber angle, with an unknown role in maintenance of the biomechanical stability of the aqueous outflow pathway, thus motivating this study. We conducted histomorphometric analysis and optical coherence tomography-based finite element (FE) modeling on three cohorts of C57BL/6 mice: ‘young’ (2–6 months), ‘middle-aged’ (11–16 months), and ‘elderly’ (25–32 months). We evaluated the age-specific morphology of the outflow pathway tissues. Further, because of the known pressure-dependent Schlemm’s canal (SC) narrowing, we assessed the dependence of the SC lumen area to varying IOPs in age-specific FE models over a physiological range of TM/PL stiffness values. We found age-dependent changes in morphology of outflow tissues; notably, the PLs were more developed in older mice compared to younger ones. In addition, FE modeling demonstrated that murine SC patency is highly dependent on the presence of PLs, and that increased IOP caused SC collapse only with sufficiently low TM/PL stiffness values. Moreover, the elderly model showed more susceptibility to SC collapse compared to the younger models. In conclusion, our study elucidated the previously unexplored role of PLs in the aqueous outflow pathway, indicating their function in supporting TM and SC under elevated IOP.

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.

Glaucoma is the leading cause of irreversible blindness worldwide. Recently, estrogen deficiencies caused by early menopause, alterations in estrogen signaling via mutations in estrogen receptors, and polymorphisms along estrogen metabolic pathways have all been linked to an increased risk of developing glaucoma. Here, we examined how menopause and age impact visual function and retinal structure in an experimental model of glaucoma. Young (3–4 months) and aged (9–10 months) female Brown Norway rats were divided into pre- and post-menopausal cohorts by surgically inducing menopause via ovariectomy (OVX). After six weeks, ocular hypertension (OHT) was induced unilaterally for a period of eight weeks. Four cohorts were successfully followed to eight weeks: young sham (n = 8), young OVX (n = 9), aged sham (n = 10), and aged OVX (n = 11) animals. Intraocular pressure (IOP) was monitored weekly in all groups. Prior to inducing OHT (baseline) and at four and eight weeks after inducing OHT, we assessed visual acuity via the optomotor response (OMR) and retinal structure using optical coherence tomography (OCT). OHT decreased the OMR in all cohorts. We found that spatial frequency thresholds decreased by 54% in OVX animals after OHT compared to sham animals after OHT, regardless of age (p < 0.001). We also found thinning of the retinal nerve fiber layer (RNFL) and loss of total retinal thickness after induction of OHT. Aged animals had more thinning of the RNFL and loss of total retinal thickness compared to young animals (p < 0.001). Overall, OHT caused significant changes in visual function and retinal structure. Observing that OVX in young and aged animals further decreased spatial frequency thresholds after OHT suggests that an estrogen deficiency may intensify visual impairment after OHT.

Purpose Glaucoma is the second leading cause of blindness worldwide. Recent work suggests that estrogen and the timing of menopause play a role in modulating the risk of developing glaucoma. Menopause is known to cause modest changes in intraocular pressure; yet, whether this change is mediated through the outflow pathway remains unknown. Menopause also affects tissue biomechanical properties throughout the body; however, the impact of menopause on ocular biomechanical properties is not well characterized. Methods Here, we simultaneously assessed the impact of menopause on aqueous outflow facility and ocular compliance, as a measure of corneoscleral shell biomechanics. We used young (3–4 months old) and middle-aged (9–10 months old) Brown Norway rats. Menopause was induced by ovariectomy (OVX), and control animals underwent sham surgery, resulting in the following groups: young sham (n = 5), young OVX (n = 6), middle-aged sham (n = 5), and middle-aged OVX (n = 5). Eight weeks postoperatively, we measured outflow facility and ocular compliance. Results Menopause resulted in a 34% decrease in outflow facility and a 19% increase in ocular compliance (P = 0.011) in OVX animals compared with sham controls (P = 0.019). Conclusions These observations reveal that menopause affects several key physiological factors known to be associated with glaucoma, suggesting that menopause may contribute to an increased risk of glaucoma in women.

Diabetic retinopathy (DR) is diagnosed clinically by directly viewing retinal vascular changes during ophthal-moscopy or through fundus photographs. However, electroretinography (ERG) studies in humans and rodents have revealed that retinal dysfunction is demonstrable prior to the development of visible vascular defects. Specifically, delays in dark-adapted ERG oscillatory potential (OP) implicit times in response to dim-flash stimuli (<21.8 log cd $ s/m2) occur prior to clinically recognized DR. Animal studies suggest that retinal dopamine deficiency underlies these early functional deficits. In this study, we randomized individuals with diabetes, without clinically detectable retinopathy, to treatment with either low-or high-dose Sinemet (levodopa plus carbidopa) for 2 weeks and compared their ERG findings with those of control subjects (no diabetes). We assessed dim-flash–stimulated OP delays using a novel handheld ERG system (RETeval) at baseline and 2 and 4 weeks. RETeval recordings identified significant OP implicit time delays in individuals with diabetes without retinopathy compared with age-matched control subjects (P < 0.001). After 2 weeks of Sinemet treatment, OP implicit times were restored to control values, and these improvements persisted even after a 2-week washout. We conclude that detection of dim-flash OP delays could provide early detection of DR and that Sinemet treatment may reverse retinal dysfunction.

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.

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.

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.