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Background: The ciliary muscle is known to play a part in presbyopia, but the mechanism has not received a comprehensive review, which this study aims to achieve. We examined relevant articles published from 1975 through 2022 that explored various properties of the muscle and related tissues in humans and rhesus monkeys. These properties include geometry, elasticity, rigidity, and composition, and were studied using a range of imaging technologies, computer models, and surgical methods. We identified a notable age-related displacement of the ciliary muscle apex that is characterized by anterior and medial shifts, and hypothesized to be primarily attributed to the accrual of connective tissue and tension exerted by the thickening lens. Other factors could also contribute to the movement, particularly the "inward bowing" of the sclera. Another noteworthy observation is that while the ciliary muscle experiences increasing constraint with advancing age due to adjacent anatomical structures, its contractile capacity remains unaltered, alongside the sustained constancy in both the concentration of muscarinic receptors and their binding affinity. Overall, more studies on human ciliary muscle are needed, as it ages differently from that of monkeys' ciliary muscle. These studies should explore other perspectives, including those regarding changes in the physical properties of the tissue and its relationship with other connected tissues., Methodology: This literature review utilized a systematic methodology to identify and analyze pertinent studies of the presbyopia and ciliary muscles. The approach encompassed a thorough examination of available literature across different academic databases, such as PubMed, Embase, and Cochrane Library., Results: Many studies have identified age-related thickening in the ciliary muscle and its potential causes, including the heightened deposition of connective tissues and traction exerted by the thickening lens. Notably, these inquiries culminated in the formulation of a geometric theory positing that the morphology of the ciliary muscle and its spatial relationship with adjacent structures exert pivotal influence over the tension exerted on zonular fibers., Conclusion: The decline in the accommodative response of the muscle is prevalent in advanced age, with reduced mobility likely attributable to the increased stiffness of the Bruch's Membrane-Choroid Complex (BMCC), where the tendons of the ciliary muscle insert, as well as the thickening and stiffening of the lens. Importantly, the ciliary muscle forces do not change with age., Competing Interests: The authors declare that they have no competing interests., (© 2024 Zuo et al.)

To elucidate the possible biological roles of fatty acid-binding protein 5 (FABP5) in the intraocular environment, the cells from which FABP5 originates were determined by using four different intraocular tissue-derived cell types including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cell lines, and the effects of FABP ligand 6, a specific inhibitor for FABP5 and FABP7 were analyzed by RNA sequencing and seahorse cellular metabolic measurements. Among these four different cell types, qPCR analysis showed that FABP5 was most prominently expressed in HNPCE cells, in which no mRNA expression of FABP7 was detected. In RNA sequencing analysis, 166 markedly up-regulated and 198 markedly down-regulated differentially expressed genes (DEGs) were detected between non-treated cells and cells treated with FABP ligand 6. IPA analysis of these DEGs suggested that FABP5 may be involved in essential roles required for cell development, cell survival and cell homeostasis. In support of this possibility, both mitochondrial and glycolytic functions of HNPCE cells, in which mRNA expression of FABP5, but not that of FABP7, was detected, were shown by using a Seahorse XFe96 Bioanalyzer to be dramatically suppressed by FABP ligand 6-induced inhibition of the activity of FABP5. Furthermore, in IPA upstream analysis, various unfolded protein response (UPR)-related factors were identified as upstream and causal network master regulators. Analysis by qPCR analysis showed significant upregulation of the mRNA expression of most of UPR-related factors and aquaporin1 (AQP1). The findings in this study suggest that HNPCE is one of intraocular cells producing FABP5 and may be involved in the maintenance of UPR and AQP1-related functions of HNPCE.

The ciliary muscle constitutes a crucial element in refractive regulation. Investigating the pathophysiological mechanisms within the ciliary muscle during excessive contraction holds significance in treating ciliary muscle dysfunction. A guinea pig model of excessive contraction of the ciliary muscle induced by drops pilocarpine was employed, alongside the primary ciliary muscle cells was employed in in vitro experiments. The results of the ophthalmic examination showed that pilocarpine did not significantly change refraction and axial length during the experiment, but had adverse effects on the regulatory power of the ciliary muscle. The current data reveal notable alterations in the expression profiles of hypoxia inducible factor 1 (HIF-1α), ATP2A2, P53, α-SMA, Caspase-3, and BAX within the ciliary muscle of animals subjected to pilocarpine exposure, alongside corresponding changes observed in cultured cells treated with pilocarpine. Augmented levels of ROS were detected in both tissue specimens and cells, culminating in a significant increase in cell apoptosis in in vivo and in vitro experiments. Further examination revealed that pilocarpine induced an increase in intracellular Ca 2+ levels and disrupted MMP, as evidenced by mitochondrial swelling and diminished cristae density compared to control conditions, concomitant with a noteworthy decline in antioxidant enzyme activity. However, subsequent blockade of Ca 2+ channels in cells resulted in downregulation of HIF-1α, ATP2A2, P53, α-SMA, Caspase-3, and BAX expression, alongside ameliorated mitochondrial function and morphology. The inhibition of Ca 2+ channels presents a viable approach to mitigate ciliary cells damage and sustain proper ciliary muscle function by curtailing the mitochondrial damage induced by excessive contractions., (© 2024 Federation of American Societies for Experimental Biology.)

Purpose: This study aims to reveal the immunopathogenesis of the high-risk corneal transplantation using a comparative proteomic approach., Methods: The immunological properties of ocular tissues (including corneal grafts, aqueous humour, and iris-ciliary body) were analysed using a high-risk rabbit corneal transplantation model employing a comparative proteomic approach., Results: The corneal grafts revealed a dramatic increase in the immune response both at the early (postoperative day 7) and rejection stages, along with the appearance of transplantation stress-induced cellular senescence in the early stage. The aqueous humour (AH) displayed persistent pathological alterations, indicated by the significant enrichment of complement and coagulation cascades pathway in the early stage and interleukin (IL)-17 signalling pathway in the rejection stage. More surprisingly, the pronounced elevation of immune response was also observed in the iris-ciliary body (I-CB) tissues at the early and rejection stages. The enriched immune-related pathways were associated with antigen processing and presentation, complement and coagulation cascades, and IL-17 signalling pathway. Furthermore, proteomic analysis revealed that the implantation of Cyclosporine A drug delivery system (CsA-DDS) into the anterior chamber obviously mitigated corneal transplantation rejection by inhibiting immunoreaction both in the corneal grafts and I-CB tissues., Conclusion: The results highlighted the involvement of intraocular immunity both in the grafts and I-CB tissues during corneal transplantation rejection, further suggesting the anterior chamber as an optimal drug-delivery site for its treatment., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aqueous humor (AQH) is a transparent fluid with characteristics similar to those of the interstitial fluid, which fills the eyeball posterior and anterior chambers and circulates in them from the sites of production to those of drainage. The AQH volume and pressure homeostasis is essential for the trophism of the ocular avascular tissues and their normal structure and function. Different AQH outflow pathways exist, including a main pathway, quite well defined anatomically and referred to as the conventional pathway, and some accessory pathways, more recently described and still not fully morphofunctionally understood, generically referred to as unconventional pathways. The conventional pathway is based on the existence of a series of conduits starting with the trabecular meshwork and Schlemm's Canal and continuing with a system of intrascleral and episcleral venules, which are tributaries to veins of the anterior segment of the eyeball. The unconventional pathways are mainly represented by the uveoscleral pathway, in which AQH flows through clefts, interstitial conduits located in the ciliary body and sclera, and then merges into the aforementioned intrascleral and episcleral venules. A further unconventional pathway, the lymphatic pathway, has been supported by the demonstration of lymphatic microvessels in the limbal sclera and, possibly, in the uvea (ciliary body, choroid) as well as by the ocular glymphatic channels, present in the neural retina and optic nerve. It follows that AQH may be drained from the eyeball through blood vessels (TM-SC pathway, US pathway) or lymphatic vessels (lymphatic pathway), and the different pathways may integrate or compensate for each other, optimizing the AQH drainage. The present review aims to define the state-of-the-art concerning the structural organization and the functional anatomy of all the AQH outflow pathways. Particular attention is paid to examining the regulatory mechanisms active in each of them. The new data on the anatomy and physiology of AQH outflow pathways is the key to understanding the pathophysiology of AQH outflow disorders and could open the way for novel approaches to their treatment., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Changes in the anterior segment of the eye due to type 2 diabetes mellitus (T2DM) are not well-characterized, in part due to the lack of a reliable animal model. This study evaluated changes in the anterior segment, including crystalline lens health, corneal endothelial cell density, aqueous humor metabolites, and ciliary body vasculature, in a rat model of T2DM compared with human eyes. Male Sprague-Dawley rats were fed a high-fat diet (45% fat) or normal diet, and rats fed the high-fat diet were injected with streptozotocin intraperitoneally to generate a model of T2DM. Cataract formation and corneal endothelial cell density were assessed using microscopic analysis. Diabetes-related rat aqueous humor alterations were assessed using metabolomics screening. Transmission electron microscopy was used to assess qualitative ultrastructural changes ciliary process microvessels at the site of aqueous formation in the eyes of diabetic rats and humans. Eyes from the diabetic rats demonstrated cataracts, lower corneal endothelial cell densities, altered aqueous metabolites, and ciliary body ultrastructural changes, including vascular endothelial cell activation, pericyte degeneration, perivascular edema, and basement membrane reduplication. These findings recapitulated diabetic changes in human eyes. These results support the use of this model for studying ocular manifestations of T2DM and support a hypothesis postulating blood-aqueous barrier breakdown and vascular leakage at the ciliary body as a mechanism for diabetic anterior segment pathology., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 American Society for Investigative Pathology. All rights reserved.)

The carbonic anhydrase 2 (Car2) gene encodes the primary isoenzyme responsible for aqueous humor (AH) production and plays a major role in the regulation of intraocular pressure (IOP). The CRISPR-Cas9 system, based on the ShH10 adenovirus-associated virus, can efficiently disrupt the Car2 gene in the ciliary body. With a single intravitreal injection, Car2 knockout can significantly and sustainably reduce IOP in both normal mice and glaucoma models by inhibiting AH production. Furthermore, it effectively delays and even halts glaucomatous damage induced by prolonged high IOP in a chronic ocular hypertension model, surpassing the efficacy of clinically available carbonic anhydrase inhibitors such as brinzolamide. The clinical application of CRISPR-Cas9 based disruption of Car2 is an attractive therapeutic strategy that could bring additional benefits to patients with glaucoma., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Glaucoma is a blinding disease. Reduction of intraocular pressure (IOP) is the mainstay of treatment, but current drugs show side effects or become progressively ineffective, highlighting the need for novel compounds. We have synthesized a family of perhydro-1,4-oxazepine derivatives of digoxin, the selective inhibitor of Na,K-ATPase. The cyclobutyl derivative (DcB) displays strong selectivity for the human α2 isoform and potently reduces IOP in rabbits. These observations appeared consistent with a hypothesis that in ciliary epithelium DcB inhibits the α2 isoform of Na,K-ATPase, which is expressed strongly in nonpigmented cells, reducing aqueous humor (AH) inflow. This paper extends assessment of efficacy and mechanism of action of DcB using an ocular hypertensive nonhuman primate model (OHT-NHP) ( Macaca fascicularis ). In OHT-NHP, DcB potently lowers IOP, in both acute (24 h) and extended (7-10 days) settings, accompanied by increased aqueous humor flow rate (AFR). By contrast, ocular normotensive animals (ONT-NHP) are poorly responsive to DcB, if at all. The mechanism of action of DcB has been analyzed using isolated porcine ciliary epithelium and perfused enucleated eyes to study AH inflow and AH outflow facility, respectively. 1 ) DcB significantly stimulates AH inflow although prior addition of 8-Br-cAMP, which raises AH inflow, precludes additional effects of DcB. 2 ) DcB significantly increases AH outflow facility via the trabecular meshwork (TM). Taken together, the data indicate that the original hypothesis on the mechanism of action must be revised. In the OHT-NHP, and presumably other species, DcB lowers IOP by increasing AH outflow facility rather than by decreasing AH inflow. NEW & NOTEWORTHY When applied topically, a cyclobutyl derivative of digoxin (DcB) potently reduces intraocular pressure in an ocular hypertensive nonhuman primate model ( Macaca fascicularis ), associated with increased aqueous humor (AH) flow rate (AFR). The mechanism of action of DcB involves increased AH outflow facility as detected in enucleated perfused porcine eyes and, in parallel, increased (AH) inflow as detected in isolated porcine ciliary epithelium. DcB might have potential as a drug for the treatment of open-angle human glaucoma.

Currently, researchers have mainly focused on the role of the tissues of the posterior segment of the eyes in the development of myopia. However, the ciliary body, an anterior ocular tissue that contracts to initiate the process of accommodation, may also play an important role in the progression of myopia due to the increased demand for near work. In the present study, we established a lens-induced myopia (LIM) animal model in guinea pigs and investigated the molecular changes in the ciliary body associated with the development of myopia based on RNA sequencing. As a result, 871 differentially expressed (DE) mRNAs and 19 DE lncRNAs were identified in the ciliary body between the LIM group and the normal control group. In addition, the lncRNA-mRNA co-expression analysis was performed to explore the target genes of lncRNAs, which were mainly enriched in the Rap1 signaling pathway, cytokine-cytokine receptor interaction, and complement and coagulation cascades pathways based on the functional enrichment analysis. Among the target genes of lncRNAs, three hub genes, including Ctnnb1, Pik3r1, and Itgb1, were found to be involved in the Rap1 signaling pathway. Interestingly, two crucial genes, Grk1 and Pde6a, which are mainly expressed in retinal photoreceptors, were enriched in visual perception in the ciliary body in functional analysis and were verified to be expressed in the ciliary body. These findings indicate the molecular pathogenetic role of the ciliary body in myopia and provide new insights into the underlying mechanism of myopia development. Further studies are needed to explore the specific contributions of these identified lncRNAs and mRNAs to the development of myopia., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Inflammation is closely associated with the pathogenesis of various ocular diseases. Uveitis is a condition characterized by the inflammation of the uvea and ocular tissues that causes extreme pain, decreases visual acuity, and may eventually lead to blindness. The pharmacological functions of morroniside, isolated from Cornus officinalis , are multifarious. Morroniside exerts various therapeutic effects, e.g., it ameliorates inflammation. However, the specific anti-inflammatory effect of morroniside on lipopolysaccharide-induced uveitis has not been reported widely. In this study, we investigated the anti-inflammatory effect of morroniside on uveitis in mice., Methods: An endotoxin-induced uveitis (EIU) mouse model was constructed and treated with morroniside. The inflammatory response was observed using slit lamp microscopy, and histopathological changes were observed by hematoxylin-eosin staining. The cell count in the aqueous humor was measured using a hemocytometer. The concentrations of TNF- α , IL-6, and IL-1 β in the ciliary body and retina were measured using ELISA kits. The expression of iNOS and Arg-1 in the ciliary body and retina was measured by immunofluorescence costaining, and western blotting was performed to measure the protein expression of JAK2, p-JAK2, STAT3, and p-STAT3 in the ciliary body and retina., Results: Morroniside effectively ameliorated the inflammatory response in EIU mice. Furthermore, morroniside significantly reduced the concentrations of IL-1 β , IL-6, and TNF- α in the ciliary body and retina. Morroniside treatment significantly reduced the expression of iNOS in the ciliary body and retinal tissues. It also significantly inhibited p-JAK2 and p-STAT3 expression and promoted Arg-1 expression. In addition, morroniside boosted the effect of JAK inhibitors on the above indices., Conclusions: Collectively, these findings suggest that morroniside may protect against LPS-induced inflammation in uveitis by promoting M2 polarization through the inhibition of the JAK/STAT pathway., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Wenjie Li et al.)

Secretion of melatonin, a natural hormone whose receptors are present in the ciliary epithelium, displays diurnal variation in the aqueous humor (AH), potentially contributing to the regulation of intraocular pressure. This study aimed to determine the effects of melatonin on AH secretion in porcine ciliary epithelium. The addition of 100 µM melatonin to both sides of the epithelium significantly increased the short-circuit current (Isc) by ~40%. Stromal administration alone had no effect on the Isc, but aqueous application triggered a 40% increase in Isc, similar to that of bilateral application without additive effect. Pre-treatment with niflumic acid abolished melatonin-induced Isc stimulation. More importantly, melatonin stimulated the fluid secretion across the intact ciliary epithelium by ~80% and elicited a sustained increase (~50-60%) in gap junctional permeability between pigmented ciliary epithelial (PE) cells and non-pigmented ciliary epithelial (NPE) cells. The expression of MT 3 receptor was found to be >10-fold higher than that of MT 1 and MT 2 in porcine ciliary epithelium. Aqueous pre-treatment with MT 1 /MT 2 antagonist luzindole failed to inhibit the melatonin-induced Isc response, while MT 3 antagonist prazosin pre-treatment abolished the Isc stimulation. We conclude that melatonin facilitates Cl - and fluid movement from PE to NPE cells, thereby stimulating AH secretion via NPE-cell MT 3 receptors.

The eye is an immune-privileged site, with both vasculature and lymphatics absent from the central light path. Unique adaptations have made it possible for immune cells to be recruited to this region of the eye in response to ocular injuries and pathogenic insults. The induction of such immune responses is typically activated by tissue resident immune cells, considered the sentinels of the immune system. We discovered that, despite the absence of an embedded vasculature, the embryonic lens becomes populated by resident immune cells. The paths by which they travel to the lens during development were not known. However, our previous studies show that in response to corneal wounding immune cells travel to the lens from the vascular-rich ciliary body across the zonules that link these two tissues. We now examined whether the zonule fibers provide a path for immune cells to the embryonic lens, and the zonule-associated matrix molecules that could promote immune cell migration. The vitreous also was examined as a potential source of lens resident immune cells. This matrix-rich site in the posterior of the eye harbors hyalocytes, an immune cell type with macrophage-like properties. We found that both the zonules and the vitreous of the embryonic eye contained fibrillin-2-based networks and that migration-promoting matrix proteins like fibronectin and tenascin-C were linked to these fibrils. Immune cells were seen emerging from the ciliary body, migrating along the ciliary zonules to the lens, and invading through the lens capsule at its equator. This is just adjacent to where immune cells take up residence in the embryonic lens. In contrast, the immune cells of the vitreous were not detected in the region of the lens. These results strongly suggest that the ciliary zonules are a primary path of immune cell delivery to the developing lens.

Bestrophin-2 (BEST2) is a member of the bestrophin family of calcium-activated anion channels that has a critical role in ocular physiology 1-4 . Here we uncover a directional permeability of BEST2 to glutamate that heavily favours glutamate exit, identify glutamine synthetase (GS) as a binding partner of BEST2 in the ciliary body of the eye, and solve the structure of the BEST2-GS complex. BEST2 reduces cytosolic GS activity by tethering GS to the cell membrane. GS extends the ion conducting pathway of BEST2 through its central cavity and inhibits BEST2 channel function in the absence of intracellular glutamate, but sensitizes BEST2 to intracellular glutamate, which promotes the opening of BEST2 and thus relieves the inhibitory effect of GS. We demonstrate the physiological role of BEST2 in conducting chloride and glutamate and the influence of GS in non-pigmented ciliary epithelial cells. Together, our results reveal a novel mechanism of glutamate release through BEST2-GS., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: The lymphatic structure of the eye is still under debate. It is mainly assumed that the retina is primarily drained by prelymphatics and not by lymphatics per se. We aimed to identify lymphatics in the rat retina., Methods: Eyes from ten Wistar rats were paraffin-embedded and the lymphatic marker podoplanin (D2-40) was investigated., Results: We identified in the rat retina a blunt-end network of lymphatic endothelial vessels. It consisted of circumferential vessels within the outer and, respectively, inner plexiform layers, connected by radial dichotomous vessels. Moreover, D2-40 expression was found within the choroid, ciliary body, and extraocular muscles., Conclusions: This in situ evidence is strongly supported by the recent in vitro demonstration of the expression of lymphatic markers in retinal endothelial cells. Further studies of comparative histology should use specific lymphatic markers to test whether other species besides rats have proper retinal lymphatics., Competing Interests: Disclosure statement The authors have no conflict of interests to declare., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

The ciliary body critically contributes to the ocular physiology with multiple responsibilities in the production of aqueous humor, vision accommodation and intraocular immunity. Comparatively little work, however, has revealed the single-cell molecular taxonomy of the human ciliary body required for studying these functionalities. In this study, we report a comprehensive atlas of the cellular and molecular components of human ciliary body as well as their interactions using single-cell RNA sequencing (scRNAseq). Cluster analysis of the transcriptome of 14,563 individual ciliary cells from the eyes of 3 human donors identified 14 distinct cell types, including the ciliary epithelium, smooth muscle, vascular endothelial cell, immune cell and other stromal cell populations. Cell-type discriminative gene markers were also revealed. Unique gene expression patterns essential for ciliary epithelium-mediated aqueous humor inflow and ciliary smooth muscle contractility were identified. Importantly, we discovered the transitional states that probably contribute to the transition of ciliary macrophage into retina microglia and verified no lymphatics in the ciliary body. Moreover, the utilization of CellPhoneDB allowed us to systemically infer cell-cell interactions among diverse ciliary cells including those that potentially participate in the pathogenesis of glaucoma and uveitis. Altogether, these new findings provide insights into the regulation of intraocular pressure, accommodation reflex and immune homeostasis under physiological and pathological conditions., (© 2022. The Author(s).)

Purpose: We develop a mathematical model that predicts aqueous humor (AH) production rate by the ciliary processes and aqueous composition in the posterior chamber (PC), with the aim of estimating how the aqueous production rate depends on the controlling parameters and how it can be manipulated., Methods: We propose a compartmental mathematical model that considers the stromal region, ciliary epithelium, and PC. All domains contain an aqueous solution with different chemical species. We impose the concentration of all species on the stromal side and exploit the various ion channels present on the cell membrane to compute the water flux produced by osmosis, the solute concentrations in the AH and the transepithelial potential difference., Results: With a feasible set of parameters, the model predictions of water flux from the stroma to the PC and of the solute concentrations in the AH are in good agreement with measurements. Key parameters which impact the aqueous production rate are identified. A relevant role is predicted to be played by cell membrane permeability to \(\text{K}^+\) and \(\text{Cl}^-\), by the level of transport due to the Na+-H+ exchanger and to the co-transporter of Na+/K+/2Cl-; and by carbonic anhydrase., Conclusions: The mathematical model predicts the formation and composition of AH, based on the structure of the ciliary epithelium. The model provides insight into the physical processes underlying the functioning of drugs that are adopted to regulate the aqueous production. It also suggests ion channels and cell membrane properties that may be targeted to manipulate the aqueous production rate.

The anterior segment of the eye consists of the cornea, iris, ciliary body, crystalline lens, and aqueous humor outflow pathways. Together, these tissues are essential for the proper functioning of the eye. Disorders of vision have been ascribed to defects in all of them; some disorders, including glaucoma and cataract, are among the most prevalent causes of blindness in the world. To characterize the cell types that compose these tissues, we generated an anterior segment cell atlas of the human eye using high-throughput single-nucleus RNA sequencing (snRNAseq). We profiled 195,248 nuclei from nondiseased anterior segment tissues of six human donors, identifying >60 cell types. Many of these cell types were discrete, whereas others, especially in the lens and cornea, formed continua corresponding to known developmental transitions that persist in adulthood. Having profiled each tissue separately, we performed an integrated analysis of the entire anterior segment, revealing that some cell types are unique to a single structure, whereas others are shared across tissues. The integrated cell atlas was then used to investigate cell type-specific expression patterns of more than 900 human ocular disease genes identified through either Mendelian inheritance patterns or genome-wide association studies.

The iris controls the level of retinal illumination by controlling pupil diameter. It is a site of diverse ophthalmologic diseases and it is a potential source of cells for ocular auto-transplantation. The present study provides foundational data on the mouse iris based on single nucleus RNA sequencing. More specifically, this work has (1) defined all of the major cell types in the mouse iris and ciliary body, (2) led to the discovery of two types of iris stromal cells and two types of iris sphincter cells, (3) revealed the differences in cell type-specific transcriptomes in the resting vs. dilated states, and (4) identified and validated antibody and in situ hybridization probes that can be used to visualize the major iris cell types. By immunostaining for specific iris cell types, we have observed and quantified distortions in nuclear morphology associated with iris dilation and clarified the neural crest contribution to the iris by showing that Wnt1-Cre -expressing progenitors contribute to nearly all iris cell types, whereas Sox10-Cre -expressing progenitors contribute only to stromal cells. This work should be useful as a point of reference for investigations of iris development, disease, and pharmacology, for the isolation and propagation of defined iris cell types, and for iris cell engineering and transplantation., Competing Interests: JW, AR, JN No competing interests declared, (© 2021, Wang et al.)

This study investigated the effects of omidenepag (OMD), a novel selective EP2 receptor agonist, on human trabecular meshwork (HTM) cells, monkey Schlemm's canal endothelial (SCE) cells, and porcine ciliary muscle (CM) to clarify the mechanism of intraocular pressure (IOP) reduction involving conventional outflow pathway. In HTM and SCE cells, the effects of OMD on transforming growth factor-β2 (TGF-β2)-induced changes were examined. The expression of actin cytoskeleton and extracellular matrix (ECM) proteins, myosin light chain (MLC) phosphorylation in HTM cells were evaluated using real-time quantitative PCR, immunocytochemistry, and western blotting. The expression of barrier-related proteins, ZO-1 and β-catenin, and permeability of SCE cells were evaluated using immunocytochemistry and transendothelial electrical resistance. The CM contraction was determined by contractibility assay. OMD significantly inhibited expression of TGF-β2 induced mRNA, protein, and MLC-phosphorylation on cytoskeletal and ECM remodeling in the HTM dose dependently. In SCE cells, OMD suppressed TGF-β2-induced expression of the barrier-related proteins and decreased SCE monolayer permeability. OMD at 3 µM significantly inhibited CM contraction, however, the effect was not significant at lower concentrations. IOP lowering effect of OMD through conventional outflow pathway is exerted by increasing outflow facilities with the modulation of TM cell fibrosis and SCE cell permeability., (© 2021. The Author(s).)

Purpose : To investigate the effect of NKR-1 antagonists in an established UVR-B-induced cataract mouse model. Furthermore, to examine the expression of pro-inflammatory cytokines/chemokines in mouse eyes following unilateral UVR-B exposure. Methods : Mice received intraperitoneally injections of Fosaprepitant and Spantide I, before and after unilateral exposure to UVR-B. After day 3 and 7 post-exposure, ocular tissues were extracted for the detection of NKR-1 protein level by ELISA. Results : Pretreatment with Fosaprepitant decreases NKR-1 expression in exposed ocular tissues as well as in the unexposed lens epithelium compared to the saline group. Spantide I treatment showed a tendency of NKR-1 overexpression in ocular tissues. Conclusion : The clinically approved NKR-1 receptor antagonist Fosaprepitant decreases NKR-1 protein expression effectively not only in the exposed but also in the unexposed partner eye in a UVR-B irradiation mouse model. No effect was seen on the protein concentration of pro-inflammatory cytokines/chemokines in either eye.

Purpose: To find immunohistochemical markers that distinguish adenocarcinoma of the nonpigmented ciliary epithelium (NPCE) from metastatic carcinoma, especially metastatic renal cell carcinoma., Design: Retrospective case series., Methods: Three cases of adenocarcinoma of the NPCE were examined histologically with hematoxylin-eosin stain and immunohistochemical stains including vimentin, AE1/AE3, Cam 5.2, CK7, PAX2, PAX8, AMACR, and CAIX. We also reviewed previously reported cases of this tumor., Results: We found that the immunohistochemical profile of adenocarcinoma of the NPCE can overlap with renal cell carcinoma. Both tumors can express vimentin, cytokeratin AE1/AE3, Cam 5.2, PAX2, PAX8, and AMACR. One of the adenocarcinomas of the NPCE in our series also expressed CD10 and the renal cell carcinoma marker (RCC Ma). Carbonic anhydrase IX (CAIX) was not detected in any of the 3 tumors., Conclusions: Adenocarcinomas arising in phthisic eyes can be diagnostically challenging. We have found it particularly difficult to distinguish adenocarcinoma of the NPCE from metastatic carcinoma, especially metastatic clear cell renal cell carcinoma and papillary renal cell carcinoma. Because of the immunophenotypic overlap, most patients will require systemic workup including imaging of the kidneys to be certain of the diagnosis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: Proper aqueous humor (AH) dynamics is crucial for maintaining the intraocular pressure (IOP) in the eye. This study aims to investigate the function of Piezo2, a newly discovered mechanosensitive ion channel, in regulating AH dynamics., Methods: Immunohistochemistry (IHC) analysis and western blotting were performed to detect Piezo2 expression. The Cre-lox system was applied to create a conditional knockout model of Piezo2. IOP and aqueous humor outflow facility in live animals were recorded with a Tonometer and a syringe-pump system for up to 2 weeks., Results: We first detected Piezo2 with robust expression in the human trabecular meshwork (TM), Schlemm's canal (SC), the ciliary body's epithelium, and ciliary muscle. In addition, we found Piezo2 in human retinal ganglion cells (RGCs) and astrocytes in the optic nerve head (ONH). Through the Cre-lox system, Piezo2 can be successfully downregulated in mouse iridocorneal angle tissues. However, Piezo2 downregulation cannot significantly influence the IOP and outflow facility through the conventional pathway. Instead, we observed an effect of downregulated Piezo2 on decreasing the intercept in the flow rate versus pressure plot. According to the Goldmann equation, Piezo2 may function in regulating unconventional outflow, AH production, and episcleral venous pressure., Conclusions: These findings, for the first time, demonstrate that Piezo2 acts as an essential mechanosensor in maintaining the proper aqueous humor dynamics in the eye., (Copyright © 2021 Molecular Vision.)

Purpose: To evaluate the distribution of the PAX8 transcription factor protein in ocular tissues and to investigate if immunohistochemical stains for this biomarker are useful in the diagnosis of intraocular tumors., Design: Observational case series., Participants: Excision and cytologic analysis specimens of 6 ciliary body epithelial neoplasms, 2 iris epithelial neoplasms, 3 retinal pigment epithelial neoplasms, 3 intraocular medulloepitheliomas, 15 uveal melanomas, and 5 uveal melanocytomas., Methods: Hematoxylin-eosin and PAX8 immunohistochemical stains were performed on all specimens. In appropriate cases, bleached preparations and other immunohistochemical stains, including AE1/AE3 cytokeratin, Lin28A, and CD45, were performed., Main Outcome Measures: Distribution of PAX8 expression in normal and neoplastic tissue., Results: Strong nuclear PAX8 expression was observed in the normal corneal epithelium, iris sphincter pupillae muscle, iris pigment epithelium and dilator muscle complex, nonpigmented and pigmented epithelia of the ciliary body, lens epithelium, and a subset of retinal neurons. The normal retinal pigment epithelium and uveal melanocytes did not stain for PAX8. The ciliary body epithelial and neuroepithelial tumors (adenoma, adenocarcinoma, and medulloepithelioma) showed uniform strong nuclear PAX8 immunoreactivity. All melanocytic tumors (iris melanoma, ciliary-choroidal melanoma, and melanocytoma) and retinal pigment epithelial neoplasms showed negative results for PAX8. A subset of tumor-associated lymphocytes, most prominent in uveal melanoma, showed positive results for PAX8. The uniformity of the PAX8 staining was superior to the variable cytokeratin staining in the ciliary epithelial neoplasms and the variable Lin28A staining in malignant medulloepithelioma. The veracity of PAX8 staining was equally as robust on cytologic analysis and open-flap biopsy specimens of ciliary epithelial and iris epithelial neoplasms, melanocytoma, and melanoma., Conclusions: PAX8 has proven to be a very useful diagnostic marker in a select group of adult intraocular tumors, and we highly recommend its inclusion in diagnostic antibody panels of morphologically challenging intraocular neoplasms., (Copyright © 2020 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: Our purpose is to report a patient with primary unilateral ciliary body marginal zone lymphoma who initially presented with hemorrhagic hypopyon., Methods: Retrospective review of the clinical, imaging, and immunohistopathological features of the case was performed., Results: A 59-year-old man was referred with right anterior uveitis of unknown etiology which was unresponsive to systemic treatment. Slit-lamp biomicroscopy showed normotensive hemorrhagic hypopyon in that eye. Anterior segment ultrasound biomicroscopy revealed an iridociliary mass lesion. Because an anterior chamber paracentesis was noncontributory, a diagnostic cyclectomy was performed. Histopathological evaluation showed that the neoplastic cells were positive for CD20, lambda light chain, and BCL 2. BCL 6, CD10, CD5, SOX11, kappa, and Cyclin D1 stains were negative. The final diagnosis was extranodal marginal zone lymphoma of the ciliary body., Conclusions: Although rare, ciliary lymphoma may be a cause of intractable anterior uveitis. Repeat biopsies could be carried out when there is a high level of clinical suspicion.

Purpose: Fibrillin-1 and -2 are major components of tissue microfibrils that compose the ciliary zonule and cornea. While mutations in human fibrillin-1 lead to ectopia lentis, a major manifestation of Marfan syndrome (MFS), in mice fibrillin-2 can compensate for reduced/lack of fibrillin-1 and maintain the integrity of ocular structures. Here we examine the consequences of a heterozygous dominant-negative mutation in the Fbn1 gene in the ocular system of the mgΔ lpn mouse model for MFS., Methods: Eyes from mgΔ lpn and wild-type mice at 3 and 6 months of age were analyzed by histology. The ciliary zonule was analyzed by scanning electron microscopy (SEM) and immunofluorescence., Results: Mutant mice presented a significantly larger distance of the ciliary body to the lens at 3 and 6 months of age when compared to wild-type, and ectopia lentis. Immunofluorescence and SEM corroborated those findings in MFS mice, revealing a disorganized mesh of microfibrils on the floor of the ciliary body. Moreover, mutant mice also had a larger volume of the anterior chamber, possibly due to excess aqueous humor. Finally, losartan treatment had limited efficacy in improving ocular phenotypes., Conclusions: In contrast with null or hypomorphic mutations, expression of a dominant-negative form of fibrillin-1 leads to disruption of microfibrils in the zonule of mice. This in turn causes lens dislocation and enlargement of the anterior chamber. Therefore, heterozygous mgΔ lpn mice recapitulate the major ocular phenotypes of MFS and can be instrumental in understanding the development of the disease., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Purpose: Ciliary epithelium (CE) of adult mammalian eyes contains quiescent retinal progenitor/stem cells that generate neurospheres in vitro and differentiate into retinal neurons. This ability doesn't evolve efficiently probably because of regulatory mechanisms, such as microRNAs (miRNAs) that control pluripotent, progenitor, and differentiation genes. Here we investigate the presence of Let-7 miRNAs and its regulator and target, Lin28 and Hmga2, in CE cells from neurospheres, newborns, and adult tissues., Methods: Newborn and adult rats CE cells were dissected into pigmented and nonpigmented epithelium (PE and NPE). Newborn PE cells were cultured with growth factors to form neurospheres and we analyzed Let-7, Lin28a, and Hmga2 expression. During the neurospheres formation, we added chemically modified single-stranded oligonucleotides designed to bind and inhibit or mimic endogenous mature Let-7b and Let-7c. After seven days in culture, we analyzed neurospheres size, number and expression of Let-7, Lin28, and Hmga2., Results: Let-7 miRNAs were expressed at low rates in newborn CE cells with significant increase in adult tissues, with higher levels on NPE cells, that does not present the stem cells reprogramming ability. The Lin28a and Hmga2 protein and transcripts were more expressed in newborns than adults cells, opposed to Let-7. Neurospheres presented higher Lin28 and Hmga2 expression than newborn and adult, but similar Let-7 than newborns. Let-7b inhibitor upregulated Hmga2 expression, whereas Let-7c mimics upregulated Lin28 and downregulated Hmga2., Conclusions: This study shows the dynamic of Lin28-Let-7-Hmga regulatory axis in CE cells. These components may develop different roles during neurospheres formation and postnatal CE cells.

Anterior segment dysgenesis is often associated with cornea diseases, cataracts, and glaucoma. In the anterior segment, the ciliary body (CB) containing inner and outer ciliary epithelia (ICE and OCE) secretes aqueous humor that maintains intraocular pressure (IOP). However, CB development and function remain poorly understood. Here, this study shows that NOTCH signaling in the CB maintains the vitreous, IOP, and eye structures by regulating CB morphogenesis, aqueous humor secretion, and vitreous protein expression. Notch2 and Notch3 function via RBPJ in the CB to control ICE-OCE adhesion, CB morphogenesis, aqueous humor secretion, and protein expression, thus maintaining IOP and eye structures. Mechanistically, NOTCH signaling transcriptionally controls Nectin1 expression in the OCE to promote cell adhesion for driving CB morphogenesis and to directly stabilize Cx43 for controlling aqueous humor secretion. Finally, NOTCH signaling directly controls vitreous protein secretion in the ICE. Therefore, this study provides important insight into CB functions and involvement in eye diseases., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Transient potential receptor vanilloid 4 (TRPV4) is an ion channel responsible for sensing osmotic and mechanical signals, which in turn regulates calcium signaling across cell membranes. TRPV4 is widely expressed throughout the body, and plays an important role in normal physiological function, as well as different pathologies, however, its role in the eye is not well known. In the eye, TRPV4 is expressed in various tissues, such as the retina, corneal epithelium, ciliary body, and the lens. In this review, we provide an overview on TRPV4 structure, activation, mutations, and summarize the current knowledge of TRPV4 function and signaling mechanisms in various locations throughout the eye, as well as its role in ocular diseases, such as glaucoma and diabetic retinopathy. Based on the available data, we highlight the therapeutic potential of TRPV4 as well as the shortcomings of current research. Finally, we provide future perspectives on the implications of targeting TRPV4 to treat various ocular pathologies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Herein partially summarizes one scientist-clinician's wanderings through the jungles of primate aqueous humor outflow over the past ~45 years. Totally removing the iris has no effect on outflow facility or its response to pilocarpine, whereas disinserting the ciliary muscle (CM) from the scleral spur/trabecular meshwork (TM) completely abolishes pilocarpine's effect. Epinephrine increases facility in CM disinserted eyes. Cytochalasins and latrunculins increase outflow facility, subthreshold doses of cytochalasins and epinephrine given together increase facility, and phalloidin, which has no effect on facility, partially blocks the effect of both cytochalasins and epinephrine. H-7, ML7, Y27632 and nitric oxide - donating compounds all increase facility, consistent with a mechanosensitive TM/SC. Adenosine A1 agonists increase and angiotensin II decrease facility. OCT and optical imaging techniques now permit visualization and digital recording of the distal outflow pathways in real time. Prostaglandin (PG) F2α analogues increase the synthesis and release of matrix metalloproteinases by the CM cells, causing remodeling and thinning of the interbundle extracellular matrix (ECM), thereby increasing uveoscleral outflow and reducing IOP. Combination molecules (one molecule, two or more effects) and fixed combination products (two molecules in one bottle) simplify drug regimens for patients. Gene and stem cell therapies to enhance aqueous outflow have been successful in laboratory models and may fill an unmet need in terms of patient compliance, taking the patient out of the delivery system. Functional transfer of genes inhibiting the rho cascade or decoupling actin from myosin increase facility, while genes preferentially expressed in the glaucomatous TM decrease facility. In live NHP, reporter genes are expressed for 2+ years in the TM after a single intracameral injection, with no adverse reaction. However, except for one recent report, injection of facility-effective genes in monkey organ cultured anterior segments (MOCAS) have no effect in live NHP. While intracameral injection of an FIV. BOVPGFS-myc.GFP PGF synthase vector construct reproducibly induces an ~2 mmHg reduction in IOP, the effect is much less than that of topical PGF 2⍺ analogue eyedrops, and dissipates after 5 months. The turnoff mechanism has yet to be defeated, although proteasome inhibition enhances reporter gene expression in MOCAS. Intracanalicular injection might minimize off-target effects that activate turn-off mechanisms. An AD-P21 vector injected sub-tenon is effective in 'right-timing' wound healing after trabeculectomy in live laser-induced glaucomatous monkeys. In human (H)OCAS, depletion of TM cells by saponification eliminates the aqueous flow response to pressure elevation, which can be restored by either cultured TM cells or by IPSC-derived TM cells. There were many other steps along the way, but much was accomplished, biologically and therapeutically over the past half century of research and development focused on one very small but complex ocular apparatus. I am deeply grateful for this award, named for a giant in our field that none of us can live up to., (Copyright © 2020. Published by Elsevier Ltd.)

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade extracellular matrix (ECM) components such as collagen and have important roles in multiple biological processes, including development and tissue remodeling, both in health and disease. The activity of MMPs is influenced by the expression of MMPs and tissue inhibitors of metalloproteinase (TIMPs). In the eye, MMP-mediated ECM turnover in the juxtacanalicular region of the trabecular meshwork (TM) reduces outflow resistance in the conventional outflow pathway and helps maintain intraocular pressure (IOP) homeostasis. An imbalance in the MMP/TIMP ratio may be involved in the elevated IOP often associated with glaucoma. The prostaglandin analog/prostamide (PGA) class of topical ocular hypotensive medications used in glaucoma treatment reduces IOP by increasing outflow through both conventional and unconventional (uveoscleral) outflow pathways. Evidence from in vivo and in vitro studies using animal models and anterior segment explant and cell cultures indicates that the mechanism of IOP lowering by PGAs involves increased MMP expression in the TM and ciliary body, leading to tissue remodeling that enhances conventional and unconventional outflow. PGA effects on MMP expression are dependent on the identity and concentration of the PGA. An intracameral sustained-release PGA implant (Bimatoprost SR) in development for glaucoma treatment can reduce IOP for many months after expected intraocular drug bioavailability. We hypothesize that the higher concentrations of bimatoprost achieved in ocular outflow tissues with the implant produce greater MMP upregulation and more extensive, sustained MMP-mediated target tissue remodeling, providing an extended duration of effect.

Three-dimensional respiratory tissue models have been generated using, for example, human primary airway epithelial cells (hAEC) or respective cell lines. To investigate ciliopathies, such as primary ciliary dyskinesia, the presence of functional kinocilia in vitro is an essential prerequisite. Since access to hAEC of healthy donors is limited, we aimed to identify a respiratory epithelial cell line that is capable to display functional kinocilia on at least 60% of the apical surface. Thus, we cultured four different human respiratory cell lines with human primary airway fibroblasts under airlift conditions, characterized the morphology, and analyzed ciliary function. Only one of the tested cell lines showed beating kinocilia; however, <10% of the whole surface was covered and ciliary beating was undirected. Positive control tissue models using hAEC and fibroblasts displayed expected directed ciliary beating pattern around 11 Hz. Our data show that the available cell lines are not suitable for basic and applied research questions whenever functional kinocilia are required and that, rather, hAEC- or human induced pluripotent stem cell-derived tissue models need to be generated. Impact Statement To study ciliopathies or Bordetella pertussis infection in vitro , three-dimensional respiratory tissue models with functional kinocilia covering at least 60% of the model's surface are mandatory. We cultured four respiratory cell lines on a fibroblast-loaded biological scaffold and showed that none of them met this requirement. In contrast, primary airway cell-derived models sufficiently reflected the mucociliary phenotype. To further search for an alternative to primary respiratory cells, investigations on other cell lines should be conducted or even new cell lines have to be generated.

Purpose: Elevated IOP can cause the development of glaucoma. The circadian rhythm of IOP depends on the dynamics of the aqueous humor and is synchronized with the circadian rhythm pacemaker, that is, the suprachiasmatic nucleus. The suprachiasmatic nucleus resets peripheral clocks via sympathetic nerves or adrenal glucocorticoids. However, the detailed mechanisms underlying IOP rhythmicity remain unclear. The purpose of this study was to verify this regulatory pathway., Methods: Adrenalectomy and/or superior cervical ganglionectomy were performed in C57BL/6J mice. Their IOP rhythms were measured under light/dark cycle and constant dark conditions. Ocular administration of corticosterone or norepinephrine was also performed. Localization of adrenergic receptors, glucocorticoid receptors, and clock proteins Bmal1 and Per1 were analyzed using immunohistochemistry. Period2::luciferase rhythms in the cultured iris/ciliary bodies of adrenalectomized and/or superior cervical ganglionectomized mice were monitored to evaluate the effect of the procedures on the local clock. The IOP rhythm of retina and ciliary epithelium-specific Bmal1 knockout mice were measured to determine the significance of the local clock., Results: Adrenalectomy and superior cervical ganglionectomy disrupted IOP rhythms and the circadian clock in the iris/ciliary body cultures. Instillation of corticosterone and norepinephrine restored the IOP rhythm. β2-Adrenergic receptors, glucocorticoid receptors, and clock proteins were strongly expressed within the nonpigmented epithelia of the ciliary body. However, tissue-specific Bmal1 knock-out mice maintained their IOP rhythm., Conclusions: These findings suggest direct driving of the IOP rhythm by the suprachiasmatic nucleus, via the dual corticosterone and norepinephrine pathway, but not the ciliary clock, which may be useful for chronotherapy of glaucoma.

Glaucoma is a common cause of blindness, yet current therapeutic options are imperfect. Clinical trials have invariably shown that reduction in intraocular pressure (IOP) regardless of disease subtype prevents visual loss. Reducing ciliary body aqueous humor production can lower IOP, and the adeno-associated virus ShH10 serotype was identified as able to transduce mouse ciliary body epithelium following intravitreal injection. Using ShH10 to deliver a single vector CRISPR-Cas9 system disrupting Aquaporin 1 resulted in reduced IOP in treated eyes (10.4 ± 2.4 mmHg) compared with control (13.2 ± 2.0 mmHg) or non-injected eyes (13.1 ± 2.8 mmHg; p < 0.001; n = 12). Editing in the aquaporin 1 gene could be detected in ciliary body, and no off-target increases in corneal or retinal thickness were identified. In experimental mouse models of corticosteroid and microbead-induced ocular hypertension, IOP could be reduced to prevent ganglion cell loss (32 ± 4 /mm 2 ) compared with untreated eyes (25 ± 5/mm 2 ; p < 0.01). ShH10 could transduce human ciliary body from post-mortem donor eyes in ex vivo culture with indel formation detectable in the Aquaporin 1 locus. Clinical translation of this approach to patients with glaucoma may permit long-term reduction of IOP following a single injection., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Experiments in the late nineties showed an inverse relationship in the eye levels of melatonin and dopamine, thereby constituting an example of eye parameters that are prone to circadian variations. The underlying mechanisms are not known but these relevant molecules act via specific cell surface dopamine and melatonin receptors. This study investigated whether these receptors formed heteromers whose function impact on eye physiology. We performed biophysical assays to identify interactions in heterologous systems. Particular heteromer functionality was detected using Gi coupling, MAPK activation, and label-free assays. The expression of the heteroreceptor complexes was assessed using proximity ligation assays in cells producing the aqueous humor and human eye samples. Dopamine D 3 receptors (D 3 Rs) were identified in eye ciliary body epithelial cells. We discovered heteromers formed by D 3 R and either MT 1 (MT 1 R) or MT 2 (MT 2 R) melatonin receptors. Heteromerization led to the blockade of D 3 R-Gi coupling and regulation of signaling to the MAPK pathway. Heteromer expression was negatively correlated with intraocular hypertension., Conclusions: Heteromers likely mediate melatonin and dopamine actions in structures regulating intraocular pressure. Significant expression of D 3 R-MT 1 R and D 3 R-MT 1 R was associated with normotensive conditions, whereas expression diminished in a cell model of hypertension. A clear trend of expression reduction was observed in samples from glaucoma cases. The trend was marked but no statistical analysis was possible as the number of available eyes was 2.

Purpose: The avascular cornea, trabecular meshwork (TM), and lens obtain iron, an essential biometal, from the aqueous humor (AH). The mechanism by which this exchange is regulated, however, is unclear. Recently we reported that non-pigmented ciliary epithelial cells express ferroportin (Fpn) (Ashok, 2018b), an iron export protein modulated by hepcidin, the master regulator of iron homeostasis secreted mainly by the liver. Here, we explored whether ciliary epithelial and other cells in the anterior segment synthesize hepcidin, suggesting local regulation of iron exchange at this site., Methods: Human and bovine eyes were dissected to isolate the ciliary body (CB), corneal endothelial (CE), TM, lens epithelial (LE), and outer epithelial cell layer of the iris. Total mRNA and protein lysates were processed to evaluate the synthesis and expression of hepcidin, the iron regulatory peptide hormone, Fpn, the only known iron export protein, ceruloplasmin (Cp), a ferroxidase necessary for iron export, transferrin receptor (TfR), a major iron uptake protein, and ferritin, a major iron storage protein. A combination of techniques including reverse transcription polymerase chain reaction (RT-PCR) of total mRNA, Western blotting of protein lysates, and immunofluorescence of fixed tissue sections were used to accomplish these goals., Results: RT-PCR of isolated tissue samples revealed hepcidin-specific mRNA in the CB, TM, CE, and LE of the bovine eye. Western blotting of protein lysates from these tissues showed reactivity for hepcidin, Fpn, ferritin, and TfR. Western blotting and immunohistochemistry of similar tissues isolated from cadaveric human eyes showed expression of hepcidin, Fpn, and Cp in these samples. Notably, Fpn and Cp were expressed on the basolateral membrane of non-pigmented ciliary epithelial cells, facing the AH., Conclusions: Synthesis and expression of hepcidin and Fpn in the ciliary epithelium suggests local regulation of iron transport from choroidal plexus in the ciliary body to the AH across the blood-aqueous barrier. Expression of hepcidin and Fpn in CE, TM, and LE cells indicates additional regulation of iron exchange between the AH and cornea, TM, and lens, suggesting autonomous regulation of iron homeostasis in the anterior segment. Physiological and pathological implications of these observations are discussed., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: To investigate whether we can identify different patterns of inflammation in the aqueous humor of a uveal melanoma (UM)-containing eye, and whether these are related to prognosis., Methods: Ninety samples of aqueous humor from UM-containing eyes were analyzed using a high-throughput multiplex immunoassay that enables simultaneous analysis of 92 predefined protein biomarkers. Cytokine expression was compared to clinical and histopathological characteristics. Cluster analysis was performed, after which the clusters were compared with clinical and histopathological tumor characteristics., Results: Cluster analysis revealed three distinct clusters, with one cluster showing hardly any inflammatory cytokines, one showing intermediate levels, and one showing a high expression of inflammation-related biomarkers. Significant differences between the clusters were seen with regard to patient age (P = 0.008), tumor prominence (P = 0.001), ciliary body involvement (P < 0.001), American Joint Committee on Cancer (AJCC) stage (P < 0.001), monosomy of chromosome 3 (P = 0.03), and gain of chromosome 8q (P = 0.04), with the cluster with a highest cytokine expression having the worst prognostic markers. Especially apoptosis-related cytokines were differentially expressed., Conclusions: Analysis of cytokines in the aqueous humor shows distinct differences between aqueous humor samples and allocates these samples into three different prognostic tumor clusters. Especially large tumors with ciliary body involvement and monosomy 3 were associated with many cytokines, especially apoptosis-related cytokines. The presence of these cytokines in the aqueous humor may play a role in the lack of effective antitumor immune responses.

Purpose: Lenses have an intracellular hydrostatic pressure gradient to drive fluid from central fiber cells to surface epithelial cells. Pressure is regulated by a feedback control system that relies on transient receptor potential vanilloid (TRPV)1 and TRPV4 channels. The ciliary muscle transmits tension to the lens through the zonules of Zinn. Here, we have examined if ciliary muscle tension influenced the lens intracellular hydrostatic pressure gradient., Methods: We measured the ciliary body position and intracellular hydrostatic pressures in mouse lenses while pharmacologically causing relaxation or contraction of the ciliary muscle. We also used inhibitors of TRPV1 and TRPV4, in addition to phosphoinositide 3-kinase (PI3K) p110α knockout mice and immunostaining of phosphorylated protein kinase B (Akt), to determine how changes in ciliary muscle tension resulted in altered hydrostatic pressure., Results: Ciliary muscle relaxation increased the distance between the ciliary body and the lens and caused a decrease in intracellular hydrostatic pressure that was dependent on intact zonules and could be blocked by inhibition of TRPV4. Ciliary contraction moved the ciliary body toward the lens and caused an increase in intracellular hydrostatic pressure and Akt phosphorylation that required intact zonules and was blocked by either inhibition of TRPV1 or genetic deletion of the p110α catalytic subunit of PI3K., Conclusions: These results show that the hydrostatic pressure gradient within the lens was influenced by the tension exerted on the lens by the ciliary muscle through the zonules of Zinn. Modulation of the gradient of intracellular hydrostatic pressure in the lens could alter the water content, and the gradient of refractive index.

Ocular hypotony is an infrequent, yet potentially vision-threatening, entity. The list of differential causes is extensive, involving any condition that may compromise aqueous humor dynamics or the integrity of the globe and sometimes following medical treatments or procedures. Depending on the cause and the clinical impact, treatment options aim to correct the underlying pathology and to reestablish anatomical integrity, as well as visual function. We review the pathophysiology, clinical presentation, different causes, and associated therapeutic options of ocular hypotony., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Inwardly rectifying K + channel Kir7.1 is expressed in epithelia where it shares membrane localisation with the Na + /K + -pump. The ciliary body epithelium (CBE) of the eye is a determinant of intraocular pressure (IOP) through NaCl-driven fluid secretion of aqueous humour. In the present study we explored the presence Kir7.1 in this epithelium in the mouse and its possible functional role in the generation of IOP. Use heterozygous animals for total Kir7.1 knockout expressing β-galactosidase under the control of Kir7.1 promoter, identified the expression of Kir7.1 in non-pigmented epithelial cells of CBE. Using conditional, floxed knockout Kir7.1 mice as negative controls, we found Kir7.1 at the basolateral membrane of the same CBE cell layer. This was confirmed using a knockin mouse expressing the Kir7.1 protein tagged with a haemagglutinin epitope. Measurements using the conditional knockout mouse show only a minor effect of Kir7.1 inactivation on steady-state IOP. Transient increases in IOP in response to general anaesthetics, or to water injection, are absent or markedly curtailed in Kir7.1-deficient mice. These results suggest a role for Kir7.1 in IOP regulation through a possible modulation of aqueous humour production by the CBE non-pigmented epithelial cells. The location of Kir7.1 in the CBE, together with the effect of its removal on dynamic changes in IOP, point to a possible role of the channel as a leak pathway preventing cellular overload of K + during the secretion process. Kir7.1 could be used as a potential therapeutic target in pathological conditions leading to elevated intraocular pressure., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Photoreception is pivotal to our experience and perception of the natural world; hence the eye is of prime importance for most vertebrate animals to sense light. Central to visual health is mitochondrial homeostasis, and the selective autophagic turnover of mitochondria (mitophagy) is predicted to play a key role here. Despite studies that link aberrant mitophagy to ocular dysfunction, little is known about the prevalence of basal mitophagy, or its relationship to general autophagy, in the visual system. In this study, we utilize the mito -QC mouse and a closely related general macroautophagy reporter model to profile basal mitophagy and macroautophagy in the adult and developing eye. We report that ocular macroautophagy is widespread, but surprisingly mitophagy does not always follow the same pattern of occurrence. We observe low levels of mitophagy in the lens and ciliary body, in stark contrast to the high levels of general MAP1LC3-dependent macroautophagy in these regions. We uncover a striking reversal of this process in the adult retina, where mitophagy accounts for a larger degree of the macroautophagy taking place, specifically in the photoreceptor neurons of the outer nuclear layer. We also show the developmental regulation of autophagy in a variety of ocular tissues. In particular, mitophagy in the adult mouse retina is reversed in localization during the latter stages of development. Our work thus defines the landscape of mitochondrial homeostasis in the mammalian eye, and in doing so highlights the selective nature of autophagy in vivo and the specificity of the reporters used. Abbreviations: ATG: autophagy related; GFP: green fluorescent protein; LC3: microtubule associated protein 1 light chain 3; ONH: optic nerve head; ONL: outer nuclear layer; RPE: retinal pigment epithelium.

The receptor for growth hormone-releasing hormone (GHRH-R) has been shown to upregulate specifically in the ciliary and iris epithelial cells and infiltrating cells in the aqueous humor in a rat model of acute anterior uveitis. Treatment with GHRHR-R antagonist alleviates significantly these inflammatory responses. Herein we investigated whether the ciliary and iris epithelial cells can respond directly to lipopolysaccharide (LPS) without the influences of circulating leukocytes to produce inflammatory mediators through a GHRH-R mediated mechanism. In explant cultures of rat ciliary body and iris, LPS caused a substantial increase of GHRH-R in 24 h. Immunohistochemistry showed a localization of TLR4, the receptor for LPS, and an elevated expression of IL-6 and IL-1β in ciliary and iris epithelial cells after LPS treatment. LPS also elevated the level of IL-1β, IL-6, and iNOS and increased secretion of IL-1β and IL-6 from the explants. The GHRH-R antagonist, MIA-602, suppressed the elevated expression of IL-1β and IL-6, and reduced the release of IL-6. Such effects were not seen for the GHRHR agonist, MR-409. When co-cultured with leukocytes, expression of GHRH-R in the ocular explants was further enhanced during LPS treatment. Our results demonstrate a direct action of LPS on ciliary and iris epithelial cells to produce pro-inflammatory factors through a GHRH-R mediated mechanism, and suggest a role of these epithelial cells, in addition to the resident antigen presenting cells, in immune surveillance of the eye. Infiltrating leukocytes may enhance these inflammatory responses by regulating GHRH-R in ciliary and iris epithelial cells, in addition to their functions of synthesizing proinflammatory cytokines., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Sequence variants in LOXL1 coding for the secreted enzyme lysyl oxidase homolog 1 (LOXL1) associate with pseudoexfoliation (PEX) syndrome, a condition that is characterized by the deposition of extracellular fibrillar PEX material in the anterior eye and other parts of the body. Since the specific role of LOXL1 in the pathogenesis of PEX is unclear, and an increase in its expression was reported for early stages of PEX syndrome, we generated and studied transgenic mice with ocular overexpression of its mouse ortholog Loxl1. The chicken βB1-crystallin promoter was used to overexpress Loxl1 in the lenses of βB1-crystallin-Loxl1 transgenic mice. Transgenic lenses contained high levels of the protein LOXL1 and its mRNA, which were both not detectable in lenses of wildtype littermates. In wildtype mice, immunoreactivity for LOXL1 was mainly seen extracellularly in region of the ciliary zonules. βB1-crystallin-Loxl1 littermates showed an additional diffuse immunostaining in lens fibers and capsule, and in the inner limiting membrane and retina indicating secretion of soluble LOXL1 from transgenic lenses. In addition, lens fibers of transgenic animals contained multiple distinct spots of very intense LOXL1 immunoreactivity. By transmission electron microscopy, those spots correlated with electron-dense round or oval bodies of 20-50 nm in diameter which were localized in the rough endoplasmic reticulum and not seen in wildtype lenses. Immunogold electron microscopy confirmed that the electron-dense bodies contained LOXL1 indicating aggregation of insoluble LOXL1. Similar structures were seen in the extracellular lens capsule suggesting their secretion from lens fibers. Otherwise, no changes were seen between the eyes of βB1-crystallin-Loxl1 mice and their wildtype littermates, neither by light microscopy and funduscopy of whole eyes, nor by scanning and quantitative transmission electron microscopy of ciliary epithelium and zonules. At one month of age, intraocular pressure was significantly higher in transgenic mice than in wildtype littermates. No differences in IOP were seen though at 2-5 months of age. We conclude that LOXL1 has a strong tendency to aggregate in the rER when expressed in vivo at high amounts. A similar scenario, involving intracellular aggregation of LOXL1 and secretion of LOXL1 aggregates into the extracellular space, may be involved in the early pathogenetic events in eyes of PEX patients., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This chapter provides an overview of the early developmental origins of six ocular tissues: the cornea, lens, ciliary body, iris, neural retina, and retina pigment epithelium. Many of these tissue types are concurrently specified and undergo a complex set of morphogenetic movements that facilitate their structural interconnection. Within the context of vertebrate eye organogenesis, we also discuss the genetic hierarchies of transcription factors and signaling pathways that regulate growth, patterning, cell type specification and differentiation., (© 2019 Elsevier Inc. All rights reserved.)

The aim of this study was to investigate the effects of pilocarpine-induced ciliary body constant contraction for a long time period on the elastic modulus and collagen in corneal and scleral tissues in the early developmental stage. Twelve one-month-old New Zealand white rabbits were randomly monocularly treated with pilocarpine to cause ciliary body constant contraction. After 1- and 2-months, the sclera tissues and the eyeballs were obtained to assess the cornea and three regions of the sclera-anterior, equatorial and posterior. The corneal tissues and the three regions of the scleral tissues were cut into strips for elastic modulus measurement using an Instron 5544, the size distribution of collagen fibrils was examined using electron microscopy, the samples were homogenized, and the concentration of hydroxyproline was measured to determine the collagen content. For corneal tissues, long-term pilocarpine-induced ciliary body constant contraction did not affect the elastic modulus and collagen. For scleral tissues, long-term pilocarpine-induced ciliary body constant contraction affected the elastic modulus, diameter of collagen fibrils and collagen content in the equatorial and posterior sclera, however, the anterior sclera were unaffected. These results suggested that pilocarpine-induced ciliary body contraction could affect the scleral structure and lead to deformation of the sclera and eyeball, thereby affecting visual functions function in the process of ocular emmetropization., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Defects in autophagy and the resulting deposition of protein aggregates have been implicated in aging and neurodegenerative diseases. While gene targeting in the mouse has facilitated the characterization of these processes in different types of neurons, potential roles of autophagy and accumulation of protein substrates in neuroepithelial cells have remained elusive. Here we report that Atg7 f/f Tyr-Cre mice, in which autophagy-related 7 (Atg7) is conditionally deleted under the control of the tyrosinase promoter, are a model for accumulations of the autophagy adapter and substrate sequestosome-1/p62 in both neuronal and neuroepithelial cells. In the brain of Atg7 f/f Tyr-Cre but not of fully autophagy competent control mice, p62 aggregates were present in sporadic neurons in the cortex and other brain regions as well in epithelial cells of the choroid plexus and the ependyma. Western blot analysis confirmed a dramatic increase of p62 abundance and formation of high-molecular weight species of p62 in the brain of Atg7 f/f Tyr-Cre mice relative to Atg7 f/f controls. Immuno-electron microscopy showed that p62 formed filamentous aggregates in neurons and ependymal cells. p62 aggregates were also highly abundant in the ciliary body in the eye. Atg7 f/f Tyr-Cre mice reached an age of more than 2 years although neurological defects manifesting in abnormal hindlimb clasping reflexes were evident in old mice. These results show that p62 filaments form in response to impaired autophagy in vivo and suggest that Atg7 f/f Tyr-Cre mice are a model useful to study the long-term effects of autophagy deficiency on the homeostasis of different neuroectoderm-derived cells.

Absence of the developing lens results in severe eye defects, including substantial reductions in eye size. How the lens controls eye expansion and the underlying signalling pathways are very poorly defined. We identified RDH10 , a gene crucial for retinoic acid synthesis during embryogenesis, as a key factor downregulated in the peripheral retina (presumptive ciliary body region) of lens-removed embryonic chicken eyes prior to overt reductions in eye size. This is associated with a significant decrease in retinoic acid synthesis by lens-removed eyes. Restoring retinoic acid signalling in lens-removed eyes by implanting beads soaked in retinoic acid or retinal, but not vitamin A, rescued eye size. Conversely, blocking retinoic acid synthesis decreased eye size in lens-containing eyes. Production of collagen II and collagen IX, which are major vitreal proteins, is also regulated by the lens and retinoic acid signalling. These data mechanistically link the known roles of both the lens and retinoic acid in normal eye development, and support a model whereby retinoic acid production by the peripheral retina acts downstream of the lens to support vitreous production and eye expansion., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Iron is an essential biometal in the aqueous humor, the principal source of nutrients for the avascular cornea and the lens. Here, we explored whether the ciliary body (CB), the source of aqueous humor, transports iron, and if the prion protein (PrP C ) facilitates this process as in the outer retina. Using a combination of human, bovine, and mouse eyes as models, we report the expression of iron export proteins ferroportin and ceruloplasmin, and major iron uptake and storage proteins transferrin, transferrin receptor, and ferritin in the ciliary epithelium, indicating active exchange of iron at this site. Ferroportin and transferrin receptor are also expressed in the corneal endothelium. However, the relative expression of iron export and uptake proteins suggests export from the ciliary epithelium and import by corneal endothelium. In addition, abundant expression of PrP C , a ferrireductase that facilitates iron transport, is noted in pigmented and non-pigmented epithelium of the CB, posterior pigmented epithelium of the iris, corneal endothelium and epithelium, and lens epithelium. Notably, majority of PrP C in the ciliary epithelium is cleaved at the β-site as in retinal pigment epithelial cells, suggesting a role in iron transport. Most of the PrP C in the cornea, however, is full-length, and susceptible to aggregation by intracerebrally inoculated PrP-scrapie, an infectious conformation of PrP C responsible for human and animal prion disorders. Soluble PrP C is present in the aqueous and vitreous humor, a provocative observation with significant implications. Together, these observations suggest independent cycling of iron in the anterior segment, and a prominent role of PrP C in this process. Aggregation of PrP C in the cornea of PrP-scrapie-infected animals raises the alarming possibility of transmission of animal prions through corneal abrasions., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Non-infectious uveitis (NIU) is the first cause of blindness that can be cured if optimal anti-inflammatory therapy can be achieved. Systemic anti-TNF (Tumor Necrosis Factor) agents have been recently approved for NIU but no local delivery of anti-TNF is available. For sustained production of secreted therapeutic proteins into the eye, non-viral gene therapy using plasmid electrotransfer in the ciliary muscle has been proposed. In this paper, we report the development steps of pEYS606, a clinical-grade plasmid DNA, devoid of antiobiotic selection gene, encoding a fusion protein consisting of the extracellular domain of the soluble p55 TNF-α receptor linked to the human IgG1 Fc domain (hTNFR-Is/hIgG1 or Protein 6), with high affinity for human TNF-α, for non-viral gene transfer into the ocular ciliary muscle. Electrotransfer of pEYS606 in the ciliary muscle significantly reduced ocular inflammation in two well-established rat models of uveitis, the endotoxin-induced uveitis (EIU) and the experimental autoimmune uveitis (EAU). In addition, in EAU, a significant protection of photoreceptors was demonstrated after pEYS606 treatment. The improved pharmacokinetic profile of intraocularly-secreted protein as compared to direct intravitreous injection of recombinant protein allowed to demonstrate Protein 6 efficacy at very low concentrations. Based on these results, a phase I/II clinical trial is conducted [ClinicalTrials.gov Identifier: NCT03308045]., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Purpose: Gap junctions provide a conduit between the intracellular fluids of the pigmented (PE) and non-pigmented (NPE) ciliary epithelial cells, and are therefore critical in the secretion of the aqueous humor (AH). However, opinions differ concerning the connexin (Cx) composition of the gap junctions. Therefore, we aimed to characterize the expression of Cx in the porcine ciliary epithelium (CE), a favorable model for humans; and determine the contribution of the highest expressed Cx to AH secretion., Methods: Freshly-harvested porcine CE cells were used. The mRNA and protein expressions of gap junctions were assessed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (WB), respectively. The relative gene expressions of various Cx were determined by quantitative PCR. The gap junction permeability of isolated PE-NPE cell couplets was evaluated by Lucifer Yellow dye transfer., Results: Using RT-PCR and WB, Cx43, Cx45, Cx47, Cx50, and Cx60 were present in porcine CE, with Cx43 being the most abundant isoform, having over 200-fold higher expression than other Cx. Cx43 was primarily localized in the PE-NPE interface and the basolateral membranes of PE cells. Knockdown of Cx43 by siRNA significantly reduced gene and protein expressions, resulting in reduction of transcellular fluid flow by 90%., Conclusions: Cx43 was found to be the major component of gap junctions in porcine CE. Consistent with results from a bovine model, our results support the important role of Cx43 in mediating AH secretion. This finding may shed light on the development of a novel ocular hypotensive agent.

Choroid plexus tumors and ciliary body medulloepithelioma are predominantly pediatric neoplasms. Progress in understanding the pathogenesis of these tumors has been hindered by their rarity and lack of models that faithfully recapitulate the disease. Here, we find that endogenous Myc proto-oncogene protein is down-regulated in the forebrain neuroepithelium, whose neural plate border domains give rise to the anterior choroid plexus and ciliary body. To uncover the consequences of persistent Myc expression, MYC expression was forced in multipotent neural precursors (nestin-Cre:Myc), which produced fully penetrant models of choroid plexus carcinoma and ciliary body medulloepithelioma. Nestin-mediated MYC expression in the epithelial cells of choroid plexus leads to the regionalized formation of choroid plexus carcinoma in the posterior domain of the lateral ventricle choroid plexus and the fourth ventricle choroid plexus that is accompanied by loss of multiple cilia, up-regulation of protein biosynthetic machinery, and hydrocephalus. Parallel MYC expression in the ciliary body leads also to up-regulation of protein biosynthetic machinery. Additionally, Myc expression in human choroid plexus tumors increases with aggressiveness of disease. Collectively, our findings expose a select vulnerability of the neuroepithelial lineage to postnatal tumorigenesis and provide a new mouse model for investigating the pathogenesis of these rare pediatric neoplasms., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)