Glaucoma is associated with ocular hypertension, and lowering intraocular pressure is the primary objective of current therapies. Recent studies have established a key role for Schlemm's canal endothelium in this pressure increase and have shown that it has a unique, lymphatic-like hybrid phenotype characterized by expression of the lymphatic transcription factor PROX1. However, the functional importance of this hybrid phenotype in the adult canal remains unclear, as long-term studies have been limited by systemic requirements for lymphatic gene expression and a lack of Schlemm's canal-specific animal models. Here, we designed and validated a51 strategy using 4OH-tamoxifen-loaded nanocarriers to generate targeted, Schlemm's canal-52 specific Prox1 knockout mice that specifically lacked lymphatic characteristics in the canal53 endothelium. Within 4 weeks, intraocular pressure was significantly elevated, and ocular hypertension was maintained for at least 24 weeks. Unlike lymphatic vessels, which degenerate following Prox1 deletion, Schlemm's canal persisted but reverted to a less functional vein-like phenotype with no change in size or morphology. Together, these findings demonstrate the utility of nanocarrier-mediated tamoxifen delivery and establish the importance of the Schlemm's canal lymphatic-like phenotype in intraocular pressure regulation, providing targets for future glaucoma therapies and a mouse model of adult-onset ocular hypertension.

Anterior segment diseases, including glaucoma and uveitis, affect millions of patients worldwide. Nanocarriers hold transformative potential for treating these conditions, yet corneal epithelium impermeability necessitates intraocular injection. Given the discomfort and infection risk, an injectable hydrogel‐based depot offers a promising strategy for sustained delivery. However, because the aqueous humor is a large, fluid‐filled environment, achieving spatially confined gelation remains a key challenge, as injected materials rapidly diffuse. Herein, a composite hydrogel (C‐gel) is presented that enables localized in situ gelation and sustained nanocarrier release within the anterior chamber. This is achieved by embedding poly(ethylene glycol)‐ b ‐poly(propylene sulfide) (PEG‐ b ‐PPS) filomicelles (FMs) within a crosslinked multi‐arm PEG hydrogel. The FM structure facilitated the spatial confinement of DBCO‐ and azide‐PEG crosslinking reactions, promoting efficient gel formation—the first use of FM morphology for enhanced localized gelation. As a result, 90% of the injected polymer is retained within the crosslinked matrix. Embedded FMs undergo oxidation‐induced cylinder‐to‐sphere transitions, facilitating gradual release of micellar nanocarriers. The mechanical properties and release kinetics of C‐gels can be specified by adjusting the formulation parameters. Sustained release of dye‐loaded nanocarriers, used as a fluorescent model cargo, persisted for over a month under anterior chamber–mimicking conditions, underscoring the C‐gel's potential as a long‐acting depot for ocular drug delivery.

The development of sophisticated computational tools, combined with advanced ultrastructural imaging techniques, offers unprecedented opportunities to investigate aqueous humor outflow through the conventional pathway, the site of pathology responsible for ocular hypertension in glaucoma. Recently, a series of studies1-7 have used computational methods to study conventional outflow function. Regrettably, these studies1-7 appear to contain fundamental errors that lead to predictions that are inconsistent with established outflow physiology and raise concerns regarding methodology and apparent misrepresentations of published work. As a result, this body of work draws potentially misleading and erroneous conclusions about aqueous humor dynamics. It is therefore important to recognize and discuss these works to correct the archival record and avoid misdirecting future research.

Zweck: Diskussion der Ergebnisse von klinischen Studien, die zur FDA-Zulassung von Anti-Komplement-Therapien der geografischen Atrophie (GA) geführt haben, sowie der Perspektiven auf funktionale Daten aus klinischen GA-Studien und der Lehren aus der FDA-Zulassung, die für die künftige Grundlagen- und klinische Forschung zur AMD richtungsweisend sein können. Design: Übersicht über die ausgewählte Literatur mit Analyse und Perspektive. Methoden: Wir haben eine gezielte Überprüfung veröffentlichter Daten aus klinischen Studien zu Pegcetacoplan und Avacincaptad zur Behandlung von GA durchgeführt sowie der wissenschaftlichen Literatur zum Verlauf der GA, zur Genetik und zur Grundlagenforschung des Komplementsystems bei AMD. Ergebnisse: Die Zulassung von Pegcetacoplan und Avacincaptad basierte auf dem anatomischen Endpunkt einer Verringerung der GA-Ausbreitungsrate im Zeitverlauf. Funktionale Daten aus 2 klinischen Phase-3-Studien für jedes Medikament zeigten jedoch, dass sich für die Patienten in den Behandlungsgruppen kein visueller Vorteil ergab. Eine Betrachtung der Genetik der AMD und der grundlegenden wissenschaftlichen Erkenntnisse zur Rolle des Komplementsystems bei der AMD liefert nur mäßige Belege für die gezielte Verwendung des Komplementsystems als Behandlungsmethode gegen die GA-Ausbreitung. Daher werden alternative molekulare Ziele für die GA-Behandlung diskutiert. Gründe für die Diskrepanz zwischen den anatomischen und funktionalen Ergebnissen in den klinischen Studien zu Anti-Komplement-Therapien werden erörtert; diese geben einen Einblick in den möglichen Aufbau künftiger klinischer Studien zur GA. Schlussfolgerung: Während es sich bei Avacincaptad und Pegcetacoplan um die ersten verfügbaren, von der FDA zugelassenen Behandlungsformen der GA handelt, zeigte sich in den Ergebnissen der klinischen Studien nach 1 bzw. 2 Jahren keine funktionale Verbesserung, was die Frage aufwirft, ob die Medikamente tatsächlich einen «klinisch relevanten Outcome» liefern. Um die Chancen auf wirksamere Therapien in der Zukunft zu verbessern, i) liefern wir die wissenschaftlichen Grundlagen für die Verfolgung nicht komplementbezogener Ziele, ii) betonen wir die Bedeutung laufender klinischer Forschung, die die anatomischen Merkmale der GA genauer mit funktionellen Ergebnissen verknüpft, und iii) machen Vorschläge für klinische Endpunkte künftiger klinischer Studien zur GA.

Children typically prefer to attend to social stimuli (e.g. faces, smiles) over non-social stimuli (e.g. natural scene, household objects). This preference for social stimuli is believed to be an essential building block for later social skills and healthy social development. Preference for social stimuli are typically measured using either passive viewing or instrumental choice paradigms, but not both. Since these paradigms likely tap into different mechanisms, the current study addresses this gap by administering both of these paradigms on an overlapping sample. In this study, we use a preferential looking task and an instrumental choice task to measure preference for social stimuli in 3–9 year old typically developing children. Children spent longer looking at social stimuli in the preferential looking task but did not show a similar preference for social rewards on the instrumental choice task. Task performance in these two paradigms were not correlated. Social skills were found to be positively related to the preference for social rewards on the choice task. This study points to putatively different mechanisms underlying the preference for social stimuli, and highlights the importance of choice of paradigms in measuring this construct.

Minimally invasive glaucoma surgeries (MIGS) offer an effective way to lower intraocular pressure without inducing extensive trauma to the anterior segment. In order to predict their efficacy, an analytical model of the conventional aqueous humor outflow pathway is developed using a resistor network. The model describes outflow through the normal eye and allows for the effects of geometric changes in the outflow pathway as IOP changes. By selectively removing these resistors, the model can be used to examine and predict the outcomes of several surgical procedures currently used to treat glaucoma. Treatments examined include traditional trabeculectomy, several ab interno methods for trabeculotomy and trabeculectomy, as well as recently developed trabecular stents that bypass the trabecular meshwork and dilate Schlemm canal. The model's predictions for the efficacy of these procedures generally matched well with the efficacy determined in experimental studies, although it tended to somewhat overestimate the efficacy of these procedures. Matching the model to experimental data indicated that a partial trabeculotomy substantially increases flow to collector channels within that region and approximately 1.5 clock hours past the ends of the trabeculotomized region. Similarly, trabecular bypass stents substantially increase flow to collector channels up to 1.5 clock hours past the open ends of the stent. The resistor model we have developed can be used to predict the efficacy of a variety of MIGS procedures. Circumferential flow in Schlemm canal extends the efficacy of MIGS, but this effect is limited to a few clock hours.

Purpose To validate the ability of visible-light optical coherence tomography (vis-OCT) in imaging the full Schlemm's canal (SC) and its surrounding limbal vascular network in mice in vivo through a compound circumlimbal scan. Methods We developed an anterior segment vis-OCT system and a compound circumlimbal scanning method, which montages eight rotated raster scans. We calibrated the circumlimbal scan geometry using a three-dimensional printed phantom eyeball before imaging wild-type C57BL/6J mice. We measured SC size by segmenting SC cross sections from vis-OCT B-scan images and imaged the limbal microvascular network using vis-OCT angiography (vis-OCTA). To introduce changes in SC size, we used a manometer to adjust the intraocular pressure (IOP) to different levels. To create additional optical scattering contrast to enhance SC imaging, we surgically increased the episcleral venous pressure (EVP) and caused blood reflux into SC. Results Using the compound circumlimbal scan, our anterior segment vis-OCT noninvasively imaged the full SC and limbal microvascular network in mouse for the first time. We observed an average 123% increase in SC volume when we decreased the IOP by 10 mm Hg from the baseline IOP of 7 to 10 mm Hg and an average 72% decrease in SC volume when the IOP level was elevated by 10 mm Hg from the baseline IOP. We also observed location-dependent SC size responses to IOP changes. Blood reflux caused by increased EVP enabled vis-OCTA to directly visualize SC, which matched well with the segmented SC. Conclusions Vis-OCT and vis-OCTA can accurately image the entire SC and limbal microvascular network in vivo using the compound circumlimbal scan. Vis-OCT is also able to quantitatively measure SC responses to changing IOP levels.

Increased stiffness of the Schlemm's canal (SC) endothelium in the aqueous humor outflow pathways has been associated with elevated intraocular pressure (IOP) in glaucoma. Novel treatments that relax this endothelium, such as actin depolymerizers and rho kinase inhibitors, are in development. Unfortunately, these treatments have undesirable off-target effects and a lower than desired potency. To address these issues, a targeted PEG-b-PPS micelle loaded with actin depolymerizer latrunculin A (tLatA-MC) is developed. Targeting of SC cells is achieved by modifying the micelle surface with a high affinity peptide that binds the VEGFR3/FLT4 receptor, a lymphatic lineage marker found to be highly expressed by SC cells relative to other ocular cells. During in vitro optimization, increasing the peptide surface density increased micellar uptake in SC cells while unexpectedly decreasing uptake by human umbilical vein endothelial cells (HUVEC). The functional efficacy of tLatA-MC, as measured by decreased SC cell stiffness compared to non-targeted micelles (ntLatA-MC) or targeted blank micelles (tBL-MC), is verified using atomic force microscopy. tLatA-MC reduced IOP in an in vivo mouse model by 30-50%. The results validate the use of a cell-softening nanotherapy to selectively modulate stiffness of SC cells for therapeutic reduction of IOP and treatment of glaucoma.

In development, wound healing, and pathology, cell biomechanical properties are increasingly recognized as being of central importance. To measure these properties, experimental probes of various types have been developed, but how each probe reflects the properties of heterogeneous cell regions has remained obscure. To better understand differences attributable to the probe technology, as well as to define the relative sensitivity of each probe to different cellular structures, here we took a comprehensive approach. We studied two cell types-Schlemm's canal endothelial cells and mouse embryonic fibroblasts (MEFs)-using four different probe technologies: 1) atomic force microscopy (AFM) with sharp tip, 2) AFM with round tip, 3) optical magnetic twisting cytometry (OMTC), and 4) traction microscopy (TM). Perturbation of Schlemm's canal cells with dexamethasone treatment, α-actinin overexpression, or RhoA overexpression caused increases in traction reported by TM and stiffness reported by sharp-tip AFM as compared to corresponding controls. By contrast, under these same experimental conditions, stiffness reported by round-tip AFM and by OMTC indicated little change. Knockout (KO) of vimentin in MEFs caused a diminution of traction reported by TM, as well as stiffness reported by sharp-tip and round-tip AFM. However, stiffness reported by OMTC in vimentin-KO MEFs was greater than in wild type. Finite-element analysis demonstrated that this paradoxical OMTC result in vimentin-KO MEFs could be attributed to reduced cell thickness. Our results also suggest that vimentin contributes not only to intracellular network stiffness but also cortex stiffness. Taken together, this evidence suggests that AFM sharp tip and TM emphasize properties of the actin-rich shell of the cell, whereas round-tip AFM and OMTC emphasize those of the noncortical intracellular network.

The cause of the elevated outflow resistance and consequent ocular hypertension characteristic of glaucoma is unknown. To investigate possible causes for this flow resistance, we used atomic force microscopy (AFM) with 10-µm spherical tips to probe the stiffness of the inner wall of Schlemm's canal as a function of distance from the tissue surface in normal and glaucomatous postmortem human eyes, and 1-µm spherical AFM tips to probe the region immediately below the tissue surface. To localize flow resistance, perfusion and imaging methods were used to characterize the pressure drop in the immediate vicinity of the inner wall using giant vacuoles that form in Schlemm's canal cells as micropressure sensors. Tissue stiffness increased with increasing AFM indentation depth. Tissues from glaucomatous eyes were stiffer compared with normal eyes, with greatly increased stiffness residing within ∼1 µm of the inner-wall surface. Giant vacuole size and density were similar in normal and glaucomatous eyes despite lower flow rate through the latter due to their higher flow resistance. This implied that the elevated flow resistance found in the glaucomatous eyes was localized to the same region as the increased tissue stiffness. Our findings implicate pathological changes to biophysical characteristics of Schlemm's canal endothelia and/or their immediate underlying extracellular matrix as cause for ocular hypertension in glaucoma.

Changes in hydrostatic pressure, at levels as low as 10 mm Hg, have been reported in some studies to alter cell function in vitro; however, other studies have found no detectable changes using similar methodologies. We here investigate the hypothesis that the rate of depressurization, rather than elevated hydrostatic pressure itself, may be responsible for these reported changes. Hydrostatic pressure (100 mm Hg above atmospheric pressure) was applied to bovine aortic endothelial cells (BAECs) and PC12 neuronal cells using pressurized gas for periods ranging from 3 hours to 9 days, and then the system was either slowly (~30 minutes) or rapidly (~5 seconds) depressurized. Cell viability, apoptosis, proliferation, and F-actin distribution were then assayed. Our results did not show significant differences between rapidly and slowly depressurized cells that would explain differences previously reported in the literature. Moreover, we found no detectable effect of elevated hydrostatic pressure (with slow depressurization) on any measured variables. Our results do not confirm the findings of other groups that modest increases in hydrostatic pressure affect cell function, but we are not able to explain their findings.

Increased stiffness of Schlemm's canal endothelial cells (SC cells) is a major contributing factor to the increased pressure characteristic of primary open-angle glaucoma. New treatments for glaucoma are being developed using actin depolymerizers and rho kinase inhibitors to address this increased stiffness. However, these agents have off-target effects and are not as potent as had been hoped. We have developed a micellar nanocarrier assembled from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers capable of encapsulating latrunculin A (Lat A) with the goal of modulating SC cell stiffness. Lat A-loaded nanocarriers were similar in size and morphology to unloaded poly (ethylene glycol)-bl-poly(propylene sulfide) (PEG-bl-PPS) micelles, loaded Lat A at 62% encapsulation efficiency, and retained loaded Lat A for at least 22 days. The continued functional activity of Lat A following encapsulation within micelles was verified in murine macrophages, which are known to display decreased endocytosis in response to Lat A-dependent cytoskeletal disruption. Endocytic inhibition remained unchanged when comparing equal concentrations of micelle-loaded versus free form Lat A. Uptake of Lat A-loaded micelles by human SC cells was verified in vitro with no sign of cytotoxicity, and modulation of SC cell stiffness was measured by atomic force microscopy. Lat A-loaded micelles significantly decreased SC cell stiffness, which resulted in visible changes in cell morphology as observed by confocal microscopy. Our results demonstrate that PEG-bl-PPS micelles represent a tunable platform for the controlled intracellular delivery of latrunculin. These self-assembled polymeric nanobiomaterials may support the rational design and engineering of delivery systems for the treatment of glaucoma. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1771-1779, 2018.