SignificanceGlaucoma is the leading cause of irreversible blindness worldwide. The primary and only modifiable risk factor for the development of glaucoma is elevated intraocular pressure (IOP), and lowering IOP effectively slows glaucomatous disease progression. Unfortunately, the majority of available treatments do not target, or intentionally bypass, the diseased and stiffened glaucomatous outflow tissues responsible for IOP elevation. We recently established that conventional outflow tissue stiffness reflects tissue function. Therefore, detection of outflow tissue stiffness using noncontact, noninvasive optical coherence tomography, as we here demonstrate in an animal model of glaucoma, represents a valuable tool for assessing outflow tissue functional status. Such technology has the potential to monitor recently approved treatments targeting the outflow tissues, and to inform glaucoma surgery decisions.

Aqueous humor (AH) is a dynamic intraocular fluid that supports the vitality of tissues that regulate intraocular pressure. We recently discovered that extracellular nanovesicles called exosomes are a major constituent of AH. Exosomes function in extracellular communication and contain proteins and small RNA. Our goal was to characterize the physical properties of AH exosomes and their exosomal RNA (esRNA) content. We isolated exosomes from human AH collected during cataract surgery from five patients using serial ultracentrifugation. We measured the size and concentration of AH exosomes in solution using nanoparticle tracking analysis. We found a single population of vesicles having a mean size of 121±11nm in the unprocessed AH. Data show that centrifugation does not significantly affect the mean particle size (121±11nm versus 124±21nm), but does impact the final number of exosomes in solution (87% loss from the unprocessed AH; n=5). We extracted esRNA from the pooled human AH samples using miRCURY RNA isolation kit from Exiqon. The quality of extracted esRNA was evaluated using Agilent Bioanalyzer 2100 and was used to generate a sequencing library for small RNA sequencing with Illumina MiSeq sequencer. More than 10 different miRNAs were identified; abundant species included miR-486-5p, miR-204, and miR-184. We found that the majority of extracellular vesicles in the AH were in the exosome size range, suggesting that miRNAs housed within exosomes may function in communication between AH inflow and outflow tissues.

Visual impairment due to glaucoma currently impacts 70 million people worldwide. While disease progression can be slowed or stopped with effective lowering of intraocular pressure, current medical treatments are often inadequate. Fortunately, three new classes of therapeutics that target the diseased conventional outflow tissue responsible for ocular hypertension are in the final stages of human testing. The rho kinase inhibitors have proven particularly efficacious and additive to current therapies. Unfortunately, non-contact technology that monitors the health of outflow tissue and its response to conventional outflow therapy is not available clinically. Using optical coherence tomographic (OCT) imaging and novel segmentation software, we present the first demonstration of drug effects on conventional outflow tissues in living eyes. Topical netarsudil (formerly AR-13324), a rho kinase/ norepinephrine transporter inhibitor, affected both proximal (trabecular meshwork and Schlemm's Canal) and distal portions (intrascleral vessels) of the mouse conventional outflow tract. Hence, increased perfusion of outflow tissues was reliably resolved by OCT as widening of the trabecular meshwork and significant increases in cross-sectional area of Schlemm's canal following netarsudil treatment. These changes occurred in conjunction with increased outflow facility, increased speckle variance intensity of outflow vessels, increased tracer deposition in conventional outflow tissues and decreased intraocular pressure. This is the first report using live imaging to show real-time drug effects on conventional outflow tissues and specifically the mechanism of action of netarsudil in mouse eyes. Advancements here pave the way for development of a clinic-friendly OCT platform for monitoring glaucoma therapy.

Both exfoliation glaucoma (XFG) and primary open-angle glaucoma (POAG) have been linked to decreased conventional outflow of aqueous humor (AH). To better understand the molecular changes in the AH content under such conditions, we analyzed the miRNA profiles of AH samples from patients with POAG and XFG compared to non-glaucoma controls. Individual AH samples (n = 76) were collected from POAG and XFG patients and age-matched controls during surgical procedure. After RNA extraction, the miRNA profiles were individually determined in 12 POAG, 12 XFG and 11 control samples. We identified 205, 295 and 195 miRNAs in the POAG, XFG and control samples, respectively. Our differential expression analysis identified three miRNAs (miR-125b-5p, miR-302d-3p and miR-451a) significantly different between POAG and controls, five miRNAs (miR-122-5p, miR-3144-3p, miR-320a, miR-320e and miR-630) between XFG and controls and one miRNA (miR-302d-3p) between POAG and XFG. While none of these miRNAs have been previously linked to glaucoma, miR-122-5p may target three glaucoma-associated genes: OPTN, TMCO1 and TGF-ß1. Pathway analysis revealed that these miRNAs are involved in potential glaucoma pathways, including focal adhesion, tight junctions, and TGF-ß signaling. Comparison of the miRNA profile in AH to unrelated human serum (n = 12) exposed potential relationships between these two fluids, although they were not significantly correlated. In summary, we have successfully profiled the miRNA expression without amplification in individual human AH samples and identified several POAG or XFG-associated miRNAs. These miRNAs may play a role in pathways previously implicated in glaucoma and act as biomarkers for disease pathogenesis.

It has been suggested that common mechanisms may underlie the pathogenesis of primary open-angle glaucoma (POAG) and steroid-induced glaucoma (SIG). The biomechanical properties (stiffness) of the trabecular meshwork (TM) have been shown to differ between POAG patients and unaffected individuals. While features such as ocular hypertension and increased outflow resistance in POAG and SIG have been replicated in mouse models, whether changes of TM stiffness contributes to altered IOP homeostasis remains unknown. We found that outer TM was stiffer than the inner TM and, there was a significant positive correlation between outflow resistance and TM stiffness in mice where conditions are well controlled. This suggests that TM stiffness is intimately involved in establishing outflow resistance, motivating further studies to investigate factors underlying TM biomechanical property regulation. Such factors may play a role in the pathophysiology of ocular hypertension. Additionally, this finding may imply that manipulating TM may be a promising approach to restore normal outflow dynamics in glaucoma. Further, novel technologies are being developed to measure ocular tissue stiffness in situ. Thus, the changes of TM stiffness might be a surrogate marker to help in diagnosing altered conventional outflow pathway function if those technologies could be adapted to TM.