The Factors Affecting the Stability of IOP Homeostasis.

Purpose: Shear-induced nitric oxide (NO) production by Schlemm's canal (SC) endothelial cells provides a fast, IOP-sensitive feedback signal that normally contributes to IOP homeostasis. Our goal was to analyze the response of this homeostatic system under constant flow perfusion (as occurs in vivo) vs. constant pressure perfusion (as typical for laboratory perfusions). Methods: A mathematical model of aqueous humor dynamics, including shear-mediated NO signaling, was formulated and analyzed for stability. The model includes Goldmann's equation, accounting for proximal and distal outflow resistance, and describes how elevated IOP causes narrowing of SC lumen that increases the shear stress on SC cells. Elevated shear stress stimulates NO production, which acts to reduce outflow resistance and relax trabecular meshwork cells to decrease trabecular meshwork stiffness, affecting the SC luminal caliber. Results: During constant flow perfusion, the outflow system is typically stable, returning to baseline IOP after a perturbation. In contrast, during constant pressure perfusion, the outflow system can become unstable and exhibit a time-dependent change in outflow resistance that diverges from baseline. Conclusions: The stability of shear mediated IOP homeostasis is predicted to differ critically between constant flow vs. constant pressure perfusion. Because outflow facility is typically measured at a constant pressure in the laboratory, this instability may contribute to the characteristic time-dependent increase in outflow facility, known as washout, observed in many nonhuman species. Studies of IOP homeostasis should consider how the outflow system may respond differently under constant pressure vs. constant flow perfusion.

Pressure Clamping During Ocular Perfusions Drives Nitric Oxide-Mediated Washout.

Purpose: The aim of this study was to test the hypothesis that nitric oxide (NO) mediates a pressure-dependent, negative feedback loop that maintains conventional outflow homeostasis and thus IOP. If true, holding pressure during ocular perfusions will result in uncontrolled production of NO, hyper-relaxation of the trabecular meshwork, and washout. Methods: Paired porcine eyes were perfused at constant pressure of 15 mm Hg. After 1 hour acclimatization, one eye was exchanged with N5-[imino(nitroamino)methyl]-L-ornithine, methyl ester, monohydrochloride (L-NAME) (50 µm) and the contralateral eye with DBG, and perfused for 3 hours. In a separate group, one eye was exchanged with DETA-NO (100 nM) and the other with DBG and perfused for 30 minutes. Changes in conventional outflow tissue function and morphology were monitored. Results: Control eyes exhibited a washout rate of 15% (P = 0.0026), whereas eyes perfused with L-NAME showed a 10% decrease in outflow facility from baseline over 3 hours (P < 0.01); with nitrite levels in effluent positively correlating with time and facility. Compared with L-NAME-treated eyes, significant morphological changes in control eyes included increased distal vessel size, number of giant vacuoles, and juxtacanalicular tissue separation from the angular aqueous plexi (P < 0.05). For 30-minute perfusions, control eyes showed a washout rate of 11% (P = 0.075), whereas DETA-NO-treated eyes showed an increased washout rate of 33% from baseline (P < 0.005). Compared with control eyes, significant morphological changes in DETA-NO-treated eyes also included increased distal vessel size, number of giant vacuoles and juxtacanalicular tissue separation (P < 0.05). Conclusions: Uncontrolled NO production is responsible for washout during perfusions of nonhuman eyes where pressure is clamped.

Model of inverse bleb growth explains giant vacuole dynamics during cell mechanoadaptation.

Cells can withstand hostile environmental conditions manifest as large mechanical forces such as pressure gradients and/or shear stresses by dynamically changing their shape. Such conditions are realized in the Schlemm's canal of the eye where endothelial cells that cover the inner vessel wall are subjected to the hydrodynamic pressure gradients exerted by the aqueous humor outflow. These cells form fluid-filled dynamic outpouchings of their basal membrane called giant vacuoles. The inverses of giant vacuoles are reminiscent of cellular blebs, extracellular cytoplasmic protrusions triggered by local temporary disruption of the contractile actomyosin cortex. Inverse blebbing has also been first observed experimentally during sprouting angiogenesis, but its underlying physical mechanisms are poorly understood. Here, we hypothesize that giant vacuole formation can be described as inverse blebbing and formulate a biophysical model of this process. Our model elucidates how cell membrane mechanical properties affect the morphology and dynamics of giant vacuoles and predicts coarsening akin to Ostwald ripening between multiple invaginating vacuoles. Our results are in qualitative agreement with observations from the formation of giant vacuoles during perfusion experiments. Our model not only elucidates the biophysical mechanisms driving inverse blebbing and giant vacuole dynamics, but also identifies universal features of the cellular response to pressure loads that are relevant to many experimental contexts.

Segmental outflow dynamics in the trabecular meshwork of living mice.

Aqueous humour does not drain uniformly through the trabecular meshwork (TM), but rather follows non-uniform or "segmental" routes. In this study, we examined whether segmental outflow patterns in the TM change over time in living mice and whether such changes are affected by age. Segmental outflow patterns were labelled by constant-pressure infusion of fluorescent tracer microparticles into the anterior chamber of anesthetised C57BL/6J mice at 3 or 8 months of age. Two different tracer colours were infused at separate time points with an interval of Δt = 0, 2, 7 or 14 days. In a separate experiment, one tracer was infused in vivo while the second tracer was infused ex vivo after 2 days. The spatial relationship between the two tracer patterns was analysed using the Pearson's correlation coefficient, r. In 3-month-old mice, there was a time-dependent decay in r, which was near unity at Δt = 0 and near zero at Δt = 14 days. In 8-month-old mice, r remained elevated for 14 days. Segmental outflow patterns measured in young mice ex vivo were not significantly different from those measured in vivo after accounting for the expected changes over 2 days. Therefore, segmental outflow patterns are not static in the TM but redistribute over time, achieving near complete loss of correlation by 2 weeks in young healthy mice. There is an age-related decline in the rate at which segmental outflow patterns redistribute in the TM. Further research is needed to understand the dynamic factors controlling segmental outflow.

Measurement of postmortem outflow facility using iPerfusion.

The key risk factor for glaucoma is elevation of intraocular pressure (IOP) and alleviating it is the only effective therapeutic approach to inhibit further vision loss. IOP is regulated by the flow of aqueous humour across resistive tissues, and a reduction in outflow facility, is responsible for the IOP elevation in glaucoma. Measurement of outflow facility is therefore important when investigating the pathophysiology of glaucoma and testing candidate treatments for lowering IOP. Due to similar anatomy and response to pharmacological treatments, mouse eyes are a common model of human aqueous humour dynamics. The ex vivo preparation, in which an enucleated mouse eye is mounted in a temperature controlled bath and cannulated, has been well characterised and is widely used. The postmortem in situ model, in which the eyes are perfused within the cadaver, has received relatively little attention. In this study, we investigate the postmortem in situ model using the iPerfusion system, with a particular focus on i) the presence or absence of pressure-independent flow, ii) the effect of evaporation on measured flow rates and iii) the magnitude and pressure dependence of outflow facility and how these properties are affected by postmortem changes. Measurements immediately after cannulation and following multi-pressure facility measurement demonstrated negligible pressure-independent flow in postmortem eyes, in contrast to assumptions made in previous studies. Using a humidity chamber, we investigated whether the humidity of the surrounding air would influence measured flow rates. We found that at room levels of humidity, evaporation of saline droplets on the eye resulted in artefactual flow rates with a magnitude comparable to outflow, which were eliminated by a high relative humidity (>85%) environment. Average postmortem outflow facility was ∼4 nl/min/mmHg, similar to values observed ex vivo, irrespective of whether a postmortem delay was introduced prior to cannulation. The intra-animal variability of measured outflow facility values was also reduced relative to previous ex vivo data. The pressure-dependence of outflow facility was reduced in the postmortem relative to ex vivo model, and practically eliminated when eyes were cannulated >40 min after euthanisation. Overall, our results indicate that the moderately increased technical complexity associated with postmortem perfusion provides reduced variability and reduced pressure-dependence in outflow facility, when experimental conditions are properly controlled.

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

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.

The role of Piezo1 in conventional aqueous humor outflow dynamics.

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.

Smarce1 and Tensin 4 Are Putative Modulators of Corneoscleral Stiffness.

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.

Deletion of the gene encoding prostamide/prostaglandin F synthase reveals an important role in regulating intraocular pressure.

Prostamide/prostaglandin F synthase (PM/PGFS) is an enzyme with very narrow substrate specificity and is dedicated to the biosynthesis of prostamide F2α and prostaglandin F2α (PGF2α.). The importance of this enzyme, relative to the aldo-keto reductase (AKR) series, in providing functional tissue prostamide F2α levels was determined by creating a line of PM/PGFS gene deleted mice. Deletion of the gene encoding PM/PGFS (Fam213b / Prxl2b) was accomplished by a two exon disruption. Prostamide F2α levels in wild type (WT) and PM/PGFS knock-out (KO) mice were determined by LC/MS/MS. Deletion of Fam213b (Prxl2b) had no observed effect on behavior, appetite, or fertility. In contrast, tonometrically measured intraocular pressure was significantly elevated by approximately 4 mmHg in PM/PGFS KO mice compared to littermate WT mice. Outflow facility was measured in enucleated mouse eyes using the iPerfusion system. No effect on pressure dependent outflow facility occurred, which is consistent with the effects of prostamide F2α and PGF2α increasing outflow through the unconventional pathway. The elevation of intraocular pressure caused by deletion of the gene encoding the PM/PGFS enzyme likely results from a diversion of the endoperoxide precursor pathway to provide increased levels of those prostanoids known to raise intraocular pressure, namely prostaglandin D2 (PGD2) and thromboxane A2 (TxA2). It follows that PM/PGFS may serve an important regulatory role in the eye by providing PGF2α and prostamide F2α to constrain the influence of those prostanoids that raise intraocular pressure.

The ocular pulse decreases aqueous humor outflow resistance by stimulating nitric oxide production.

Intraocular pressure (IOP) is not static, but rather oscillates by 2-3 mmHg because of cardiac pulsations in ocular blood volume known as the ocular pulse. The ocular pulse induces pulsatile shear stress in Schlemm's canal (SC). We hypothesize that the ocular pulse modulates outflow facility by stimulating shear-induced nitric oxide (NO) production by SC cells. We confirmed that living mice exhibit an ocular pulse with a peak-to-peak (pk-pk) amplitude of 0.5 mmHg under anesthesia. Using iPerfusion, we measured outflow facility (flow/pressure) during alternating periods of steady or pulsatile IOP in both eyes of 16 cadaveric C57BL/6J mice (13-14 weeks). Eyes were retained in situ, with an applied mean pressure of 8 mmHg and 1.0 mmHg pk-pk pressure amplitude at 10 Hz to mimic the murine heart rate. One eye of each cadaver was perfused with 100 µM L-NAME to inhibit NO synthase, whereas the contralateral eye was perfused with vehicle. During the pulsatile period in the vehicle-treated eye, outflow facility increased by 16 [12, 20] % (P < 0.001) relative to the facility measured during the preceding and subsequent steady periods. This effect was partly inhibited by L-NAME, where pressure pulsations increased outflow facility by 8% [4, 12] (P < 0.001). Thus, the ocular pulse causes an immediate increase in outflow facility in mice, with roughly one-half of the facility increase attributable to NO production. These studies reveal a dynamic component to outflow function that responds instantly to the ocular pulse and may be important for outflow regulation and IOP homeostasis.

siRNA targeting Schlemm's canal endothelial tight junctions enhances outflow facility and reduces IOP in a steroid-induced OHT rodent model.

Systemic or localized application of glucocorticoids (GCs) can lead to iatrogenic ocular hypertension, which is a leading cause of secondary open-angle glaucoma and visual impairment. Previous work has shown that dexamethasone increases zonula occludens-1 (ZO-1) protein expression in trabecular meshwork (TM) cells, and that an antisense oligonucleotide inhibitor of ZO-1 can abolish the dexamethasone-induced increase in trans-endothelial flow resistance in cultured Schlemm's canal (SC) endothelial and TM cells. We have previously shown that intracameral inoculation of small interfering RNA (siRNA) targeting SC endothelial cell tight junction components, ZO-1 and tricellulin, increases aqueous humor outflow facility ex vivo in normotensive mice by reversibly opening SC endothelial paracellular pores. In this study, we show that targeted siRNA downregulation of these SC endothelial tight junctions reduces intraocular pressure (IOP) in vivo, with a concomitant increase in conventional outflow facility in a well-characterized chronic steroid-induced mouse model of ocular hypertension, thus representing a potential focused clinical application for this therapy in a sight-threatening scenario.

Infusion Mechanisms in Brain White Matter and Their Dependence on Microstructure: An Experimental Study of Hydraulic Permeability.

OBJECTIVE: Hydraulic permeability is a topic of deep interest in biological materials because of its important role in a range of drug delivery-based therapies. The strong dependence of permeability on the geometry and topology of pore structure and the lack of detailed knowledge of these parameters in the case of brain tissue makes the study more challenging. Although theoretical models have been developed for hydraulic permeability, there is limited consensus on the validity of existing experimental evidence to complement these models. In the present study, we measure the permeability of white matter (WM) of fresh ovine brain tissue considering the localised heterogeneities in the medium using an infusion-based experimental set up, iPerfusion. We measure the flow across different parts of the WM in response to applied pressures for a sample of specific dimensions and calculate the permeability from directly measured parameters. Furthermore, we directly probe the effect of anisotropy of the tissue on permeability by considering the directionality of tissue on the obtained values. Additionally, we investigate whether WM hydraulic permeability changes with post-mortem time. To our knowledge, this is the first report of experimental measurements of the localised WM permeability, also demonstrating the effect of axon directionality on permeability. This work provides a significant contribution to the successful development of intra-tumoural infusion-based technologies, such as convection-enhanced delivery (CED), which are based on the delivery of drugs directly by injection under positive pressure into the brain.

The role of EP2 receptors in mediating the ultra-long-lasting intraocular pressure reduction by JV-GL1.

BACKGROUND: A single application of JV-GL1 substantially lowers non-human primate intraocular pressure (IOP) for about a week, independent of dose. This highly protracted effect does not correlate with its ocular biodisposition or correlate with the once-daily dosing regimen for other prostanoid EP2 receptor agonists such as trapenepag or omidenepag. The underlying pharmacological mechanism for the multiday extended activity of JV-GL1 is highly intriguing. The present studies were intended to determine EP2 receptor involvement in mediating the long-term ocular hypotensive activity of JV-GL1 by using mice genetically deficient in EP2 receptors. METHODS: The protracted IOP reduction produced by JV-GL1 was investigated in C57BL/6J and EP2 receptor knock-out mice (B6.129-Ptger2tm1Brey /J; EP2KO). Both ocular normotensive and steroid-induced ocular hypertensive (SI-OHT) mice were studied. IOP was measured tonometrically under general anaesthesia. Aqueous humour outflow facility was measured ex vivo using iPerfusion in normotensive C57BL/6J mouse eyes perfused with 100 nM de-esterified JV-GL1 and in SI-OHT C57BL/6J mouse eyes that had received topical JV-GL1 (0.01%) 3 days prior. RESULTS: Both the initial 1-day and the protracted multiday effects of JV-GL1 in the SI-OHT model for glaucoma were abolished by deletion of the gene encoding the EP2 receptor. Thus, JV-GL1 did not lower IOP in SI-OHT EP2KO mice, but in littermate SI-OHT EP2WT control mice, JV-GL1 statistically significantly lowered IOP for 4-6 days. CONCLUSIONS: Both the 1-day and the long-term effects of JV-GL1 on IOP are entirely EP2 receptor dependent.

The vital role for nitric oxide in intraocular pressure homeostasis.

Catalyzed by endothelial nitric oxide (NO) synthase (eNOS) activity, NO is a gaseous signaling molecule maintaining endothelial and cardiovascular homeostasis. Principally, NO regulates the contractility of vascular smooth muscle cells and permeability of endothelial cells in response to either biochemical or biomechanical cues. In the conventional outflow pathway of the eye, the smooth muscle-like trabecular meshwork (TM) cells and Schlemm's canal (SC) endothelium control aqueous humor outflow resistance, and therefore intraocular pressure (IOP). The mechanisms by which outflow resistance is regulated are complicated, but NO appears to be a key player as enhancement or inhibition of NO signaling dramatically affects outflow function; and polymorphisms in NOS3, the gene that encodes eNOS modifies the relation between various environmental exposures and glaucoma. Based upon a comprehensive review of past foundational studies, we present a model whereby NO controls a feedback signaling loop in the conventional outflow pathway that is sensitive to changes in IOP and its oscillations. Thus, upon IOP elevation, the outflow pathway tissues distend, and the SC lumen narrows resulting in increased SC endothelial shear stress and stretch. In response, SC cells upregulate the production of NO, relaxing neighboring TM cells and increasing permeability of SC's inner wall. These IOP-dependent changes in the outflow pathway tissues reduce the resistance to aqueous humor drainage and lower IOP, which, in turn, diminishes the biomechanical signaling on SC. Similar to cardiovascular pathogenesis, dysregulation of the eNOS/NO system leads to dysfunctional outflow regulation and ocular hypertension, eventually resulting in primary open-angle glaucoma.

Aqueous Humor Outflow Requires Active Cellular Metabolism in Mice.

Purpose: Conventional wisdom posits that aqueous humor leaves the eye by passive bulk flow without involving energy-dependent processes. However, recent studies have shown that active processes, such as cell contractility, contribute to outflow regulation. Here, we examine whether inhibiting cellular metabolism affects outflow facility in mice. Methods: We measured outflow facility in paired enucleated eyes from C57BL/6J mice using iPerfusion. We had three Experimental Sets: ES1, perfused at 35°C versus 22°C; ES2, perfused with metabolic inhibitors versus vehicle at 35°C; and ES3, perfused at 35°C versus 22°C in the presence of metabolic inhibitors. Inhibitors targeted glycolysis and oxidative phosphorylation (2-deoxy-D-glucose, 3PO and sodium azide). We also measured adenosine triphosphate (ATP) levels in separate murine anterior segments treated like ES1 and ES2. Results: Reducing temperature decreased facility by 63% [38%, 78%] (mean [95% confidence interval (CI)], n = 10 pairs; P = 0.002) in ES1 after correcting for changes in viscosity. Metabolic inhibitors reduced facility by 21% [9%, 31%] (n = 9, P = 0.006) in ES2. In the presence of inhibitors, temperature reduction decreased facility by 44% [29%, 56%] (n = 8, P < 0.001) in ES3. Metabolic inhibitors reduced anterior segment adenosine triphosphate (ATP) levels by 90% [83%, 97%] (n = 5, P<<0.001), but reducing temperature did not affect ATP. Conclusions: Inhibiting cellular metabolism decreases outflow facility within minutes. This implies that outflow is not entirely passive, but depends partly on energy-dependent cellular processes, at least in mice. This study also suggests that there is a yet unidentified mechanism, which is strongly temperature-dependent but metabolism-independent, that is necessary for nearly half of normal outflow function in mice.

Age and Menopause Effects on Ocular Compliance and Aqueous Outflow.

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.

Shear Stress in Schlemm's Canal as a Sensor of Intraocular Pressure.

Elevated intraocular pressure (IOP) narrows Schlemm's canal (SC), theoretically increasing luminal shear stress. Using engineered adenoviruses containing a functional fragment of the shear-responsive endothelial nitric oxide synthase (eNOS) promoter, we tested effects of shear stress and elevated flow rate on reporter expression in vitro and ex vivo. Cultured human umbilical vein endothelial cells (HUVECs) and SC cells were transduced with adenovirus containing eNOS promoter driving secreted alkaline phosphatase (SEAP) or green fluorescent protein (GFP) and subjected to shear stress. In parallel, human anterior segments were perfused under controlled flow. After delivering adenoviruses to the SC lumen by retroperfusion, the flow rate in one anterior segment of pair was increased to double pressure. In response to high shear stress, HUVECs and SC cells expressed more SEAP and GFP than control. Similarly, human anterior segments perfused at higher flow rates released significantly more nitrites and SEAP into perfusion effluent, and SC cells expressed increased GFP near collector channel ostia compared to control. These data establish that engineered adenoviruses have the capacity to quantify and localize shear stress experienced by endothelial cells. This is the first in situ demonstration of shear-mediated SC mechanobiology as a key IOP-sensing mechanism necessary for IOP homeostasis.

The ß4-Subunit of the Large-Conductance Potassium Ion Channel KCa1.1 Regulates Outflow Facility in Mice.

Purpose: The large-conductance calcium-activated potassium channel KCa1.1 (BKCa, maxi-K) influences aqueous humor outflow facility, but the contribution of auxiliary ß-subunits to KCa1.1 activity in the outflow pathway is unknown. Methods: Using quantitative polymerase chain reaction, we measured expression of ß-subunit genes in anterior segments of C57BL/6J mice (Kcnmb1-4) and in cultured human trabecular meshwork (TM) and Schlemm's canal (SC) cells (KCNMB1-4). We also measured expression of Kcnma1/KCNMA1 that encodes the pore-forming α-subunit. Using confocal immunofluorescence, we visualized the distribution of ß4 in the conventional outflow pathway of mice. Using iPerfusion, we measured outflow facility in enucleated mouse eyes in response to 100 or 500 nM iberiotoxin (IbTX; N = 9) or 100 nM martentoxin (MarTX; N = 12). MarTX selectively blocks ß4-containing KCa1.1 channels, whereas IbTX blocks KCa1.1 channels that lack ß4. Results: Kcnmb4 was the most highly expressed ß-subunit in mouse conventional outflow tissues, expressed at a level comparable to Kcnma1. ß4 was present within the juxtacanalicular TM, appearing to label cellular processes connecting to SC cells. Accordingly, KCNMB4 was the most highly expressed ß-subunit in human TM cells, and the sole ß-subunit in human SC cells. To dissect functional contribution, MarTX decreased outflow facility by 35% (27%, 42%; mean, 95% confidence interval) relative to vehicle-treated contralateral eyes, whereas IbTX reduced outflow facility by 16% (6%, 25%). Conclusions: The ß4-subunit regulates KCa1.1 activity in the conventional outflow pathway, significantly influencing outflow function. Targeting ß4-containing KCa1.1 channels may be a promising approach to lower intraocular pressure to treat glaucoma.

Measurement of Ocular Compliance Using iPerfusion.

The pressure-volume relationship of the eye is determined by the biomechanical properties of the corneoscleral shell and is classically characterised by Friedenwald's coefficient of ocular rigidity or, alternatively, by the ocular compliance (OC), defined as dV/dP. OC is important in any situation where the volume (V) or pressure (P) of the eye is perturbed, as occurs during several physiological and pathological processes. However, accurately measuring OC is challenging, particularly in rodents. We measured OC in 24 untreated enucleated eyes from 12 C57BL/6 mice using the iPerfusion system to apply controlled pressure steps, whilst measuring the time-varying flow rate into the eye. Pressure and flow data were analysed by a "Discrete Volume" (integrating the flow trace) and "Step Response" method (fitting an analytical solution to the pressure trace). OC evaluated at 13 mmHg was similar between the two methods (Step Response, 41 [37, 46] vs. Discrete Volume, 42 [37, 48] nl/mmHg; mean [95% CI]), although the Step Response Method yielded tighter confidence bounds on individual eyes. OC was tightly correlated between contralateral eyes (R 2 = 0.75, p = 0.0003). Following treatment with the cross-linking agent genipin, OC decreased by 40 [33, 47]% (p = 0.0001; N = 6, Step Response Method). Measuring OC provides a powerful tool to assess corneoscleral biomechanics in mice and other species.

Ultrastructural variability of the juxtacanalicular tissue along the inner wall of Schlemm's canal.

Purpose: Increased resistance of aqueous humor drainage from the eye through Schlemm's canal (SC) is the basis for elevated intraocular pressure in glaucoma. Experimental evidence suggests that the bulk of outflow resistance lies in the vicinity of the inner wall endothelial lining of SC and the adjacent juxtacanalicular tissue (JCT). However, there is little understanding of how this resistance is generated, and a detailed understanding of the structure-function relationship of the outflow pathway has not been established yet. In the present study, regional variations in the ultrastructure of the JCT and the inner wall of SC were investigated in three dimensions. Methods: With the use of serial block face scanning electron microscopy (SBF-SEM), the volume occupied by the electron lucent spaces of the JCT compared to that occupied by the cellular and extracellular matrix was investigated and quantified. The distribution of giant vacuoles (GVs) and pores in the inner wall endothelium of SC was further examined. Results: With increasing distance from the inner wall of SC, the volume of the electron lucent spaces increased above 30%. In contrast, the volume of these spaces in immediate contact with the inner wall endothelium was minimal (<10%). Circumferential variability in the type and distribution of GVs was observed, and the percentage of GVs with pores varied between 3% and 27%. Conclusions: These studies provide a detailed quantitative analysis of the ultrastructure of JCT and the distribution of GVs along the circumference of SC in three dimensions, supporting the non-uniform or segmental aqueous outflow.