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Purpose Vascular endothelial growth factor (VEGF) and its receptor VEGFR2 are promising therapeutic targets for wet age-related macular degeneration (AMD). As a topically applicable option, we developed the peptide KAI to selectively interfere with VEGFR2 trafficking to the cell surface where it receives VEGF. This study sought to determine the efficacy of KAI in the mouse model of choroidal neovascularization (CNV). Methods The specificity of KAI was tested by surface plasmon resonance. The drug delivery was analyzed by cryosection and the ELISA after treatment of KAI eyedrop to the mouse eyes. For the laser-induced CNV model, mice with laser-induced ruptures in Bruch's membrane received daily treatment of KAI eyedrop or control peptide. The other groups of mice received intravitreal injection of anti-VEGF or IgG control. After two weeks, CNV was quantified and compared. Results First, we showed the specificity and high affinity of KAI to VEGFR2. Next, biodistribution revealed successful delivery of KAI eyedrop to the back of the mouse eyes. KAI significantly reduced the disease progression in laser-induced CNV. The comparison with current therapy suggests that KAI eyedrop is as effective as current therapy to prevent CNV in wet AMD. Moreover, the genetic deletion of a kinesin KIF13B, which mediates VEGFR2 trafficking to the cell surface, confirmed the pivotal role of KIF13B in disease progression of wet AMD and neovascularization from choroidal vessels. Conclusions Taken together, pharmacologic inhibition and genetic deletion complementarily suggest the therapeutic possibility of targeting VEGFR2 trafficking to inhibit pathological angiogenesis in wet AMD.

Purpose Patients receiving chemotherapy may experience ocular discomfort and dry eye–like symptoms; the latter may be neuropathic in nature. This study assessed corneal and somatic hypersensitivity in male rats treated with paclitaxel and whether it was relieved by nicotinamide riboside (NR). Methods Corneal sensitivity to tactile and chemical stimulation, basal tear production, and sensitivity of the hindpaw to tactile and cool stimuli were assessed before and after paclitaxel in the absence and presence of sustained treatment with 500 mg/kg per os NR. Corneal nerve density and hindpaw intraepidermal nerve fiber (IENF) density were also examined. Results Paclitaxel-treated rats developed corneal hypersensitivity to tactile stimuli, enhanced sensitivity to capsaicin but not hyperosmolar saline, and increased basal tear production. Corneal nerve density visualized with anti–β-tubulin or calcitonin gene-related peptide (CGRP) was unaffected. Paclitaxel induced tactile and cool hypersensitivity of the hindpaw and a loss of nonpeptidergic hindpaw IENFs visualized with anti-protein gene product (PGP) 9.5 and CGRP. NR reversed tactile hypersensitivity of the cornea without suppressing tear production or chemosensitivity; it did not alter corneal afferent density. NR also reversed tactile and cool hypersensitivity of the hindpaw without reversing the loss of hindpaw IENFs. Conclusions These findings suggest that paclitaxel may be a good translational model for chemotherapy-induced ocular discomfort and that NR may be useful for its relief. The ability of NR to relieve somatic tactile hypersensitivity independent of changes in sensory nerve innervation suggests that reversal of terminal arbor degeneration is not critical to the actions of NR.

Purpose To evaluate differences by sex in the neuroretina of rats with chronic glaucoma over 24 weeks of follow-up, and to assess by sex the influence on neurodegeneration of different methods of inducing ocular hypertension. Methods Forty-six Long-Evans rats-18 males and 28 females-with induced chronic glaucoma were analyzed. Glaucoma was achieved via 2 models: repeatedly sclerosing the episcleral veins (9 male/14 female) or by injecting poly(lactic-co-glycolic acid) microspheres measuring 20 to 10 µm (Ms20/10) into the anterior chamber (9 male/14 female). The IOP was measured weekly by tonometer; neuroretinal function was recorded by dark/light-adapted electroretinography at baseline and weeks 12 and 24; and structure was analyzed by optical coherence tomography using the retina posterior pole, retinal nerve fiber layer and ganglion cell layer protocols at baseline and weeks 8, 12, 18, and 24. Results Males showed statistically significant (P < 0.05) higher IOP in both chronic glaucoma models, and greater differences were found in the episcleral model at earlier stages. Males with episclerally induced glaucoma showed a statistically higher increase in retinal thickness in optical coherence tomography recordings than females and also when comparing Ms20/10 at 12 weeks. Males showed a higher percentage of retinal nerve fiber layer thickness loss in both models. Ganglion cell layer thickness loss was only detected in the Ms20/10 model. Males exhibited worse dark/light-adapted functionality in chronic glaucoma models, which worsened in the episcleral sclerosis model at 12 weeks, than females. Conclusions Female rats with chronic glaucoma experienced lower IOP and structural loss and better neuroretinal functionality than males. Sex and the ocular hypertension-inducing method influenced neuroretinal degeneration.

Purpose Phenylephrine has been shown to affect intraocular pressure (IOP) but the mechanism of action is poorly understood. However, its action as a vasoconstrictor suggests possible effects on episcleral venous pressure (EVP). In this study, we evaluated the effect of phenylephrine on EVP and IOP in healthy subjects. Methods Forty eyes of 20 subjects were included. Each subject received 3 drops of phenylephrine 2.5% in one eye at 1-minute intervals. The fellow eye served as control. Blood pressure, heart rate, and IOP and EVP of both eyes were measured at baseline, 15 minutes, and 60 minutes after instillation of phenylephrine. IOP was measured by pneumatonometry. EVP was assessed by using a computer-controlled episcleral venomanometer. Changes in IOP, EVP, blood pressure, and heart rate at 15 and 60 minutes were analyzed by paired t-tests. Results IOP increased 15 minutes after instillation of phenylephrine in both treated (P = 0.001) and control eyes (P = 0.01) and returned to baseline at 60 minutes. The change in IOP at 15 minutes was not significantly different between the 2 groups. EVP in treated eyes was unchanged at 15 minutes (P = 0.8) but decreased significantly at 60 minutes (P < 0.001). In control eyes, there was no change in EVP at any time (P > 0.6). There were no significant changes from baseline in systolic and diastolic blood pressure and heart rate after instillation of phenylephrine. Conclusions IOP elevation associated with topical phenylephrine is not caused by an increase in EVP in healthy subjects. Instead, EVP decreases with phenylephrine, but the mechanism remains to be determined.

Purpose Dysregulation of the alternative complement pathway is a major pathogenic mechanism in age-related macular degeneration. We investigated whether locally synthesized complement components contribute to AMD by profiling complement expression in postmortem eyes with and without AMD. Methods AMD severity grade 1 to 4 was determined by analysis of postmortem acquired fundus images and hematoxylin and eosin stained histological sections. TaqMan (donor eyes n = 39) and RNAscope/in situ hybridization (n = 10) were performed to detect complement mRNA. Meso scale discovery assay and Western blot (n = 31) were used to measure complement protein levels. Results The levels of complement mRNA and protein expression were approximately 15- to 100-fold (P < 0.0001-0.001) higher in macular retinal pigment epithelium (RPE)/choroid tissue than in neural retina, regardless of AMD grade status. Complement mRNA and protein levels were modestly elevated in vitreous and the macular neural retina in eyes with geographic atrophy (GA), but not in eyes with early or intermediate AMD, compared to normal eyes. Alternative and classical pathway complement mRNAs (C3, CFB, CFH, CFI, C1QA) identified by RNAscope were conspicuous in areas of atrophy; in those areas C3 mRNA was observed in a subset of IBA1+ microglia or macrophages. Conclusions We verified that RPE/choroid contains most ocular complement; thus RPE/choroid rather than the neural retina or vitreous is likely to be the key site for complement inhibition to treat GA or earlier stage of the disease. Outer retinal local production of complement mRNAs along with evidence of increased complement activation is a feature of GA.

Purpose The purpose of this study was to determine whether the ability of the rat retina to control its pH is affected by diabetes. Methods Double-barreled H+-selective microelectrodes were used to measure extracellular [H+] in the dark-adapted retina of intact control and diabetic Long-Evans rats 1 to 6 months after intraperitoneal injection of vehicle or streptozotocin, respectively. Two manipulations-increasing of blood glucose and intravenous injection of the carbonic anhydrase blocker dorzolamide (DZM)-were used to examine their effects on retinal pH regulation. Results An increase of retinal acidity was correlated with the diabetes-related increase in blood glucose, but only between 1 and 3 months of diabetes, not earlier or later. Adding intravenous glucose had no noticeable effect on the retinal acidity of control animals. In contrast, similar injections of glucose in diabetic rats significantly increased the acidity of the retina. Again, the largest increase of retinal acidity due to artificially elevated blood glucose was observed at 1 to 3 months of diabetes. Suppression of carbonic anhydrase by DZM dramatically increased the retinal acidity in both control and diabetic retinas to a similar degree. However, in controls, the strongest effect of DZM was recorded within 10 minutes after the injection, but in diabetics, the effect tended to increase with time and after 2 hours could be two to three times larger than at the beginning. Conclusions During development of diabetes in rats, the control over retinal pH is partly compromised so that conditions that perturb retinal pH lead to larger and/or more sustained changes than in control animals.

Purpose Clinical manifestations of photoreceptor degeneration include gradual thinning of the outer nuclear layer (ONL) and progressive reduction of electroretinogram (ERG) amplitudes and vision loss. Although preclinical evaluations of treatment strategies greatly depend on rodent models, the courses of these changes in mice remain unclear. We thus sought to investigate the temporal correlations in changes of spatial vision, ERG response, and ONL thickness in mice with progressive photoreceptor degeneration. Methods Adult wild-type (WT) mice and mice carrying rhodopsin deficiency (Rho-/-), a frequently used mouse model of human retinitis pigmentosa, were selected for investigation. Mouse spatial vision, including visual acuity (VA) and contrast sensitivity (CS), was determined using optomotor response (OMR) assays; ONL thickness was quantified by spectral-domain optical coherence tomography (SD-OCT), and ERG was performed to evaluate retinal functions. The mice were killed when they were 14 weeks old, and the cone photoreceptors in retinal sections were counted. Results Spatial vision, ONL thickness, and ERG amplitudes remained stable in WT mice at all examined time points. While 6-week-old Rho-/- mice had VA, CS, as well as ERG responses similar to those of WT mice, progressive reductions in the spatial vision and retinal functions were recorded thereafter. Most tested 12-week-old Rho-/- mice had no visual-evoked OMR and ERG responses. Moreover, CS, but not VA, displayed a linear decline that was closely associated with ONL thinning, reduction of ERG amplitudes, and loss of cones. Conclusions We presented a comprehensive study of the relation between the changes of spatial vision, retinal function, and ONL thickness in postnatal week (PW)6 to PW12 Rho-/- mice. CS is a more sensitive indicator of spatial vision compared to VA, although both are required as separate parameters for monitoring the visual changes in retina undergoing photoreceptor degeneration.

PURPOSE. Recent evidence suggests that the smaller retinal vessels are significantly involved in the regulation of retinal blood flow and that this regulation may differ among the macular area and the retinal periphery. An alternative to studying blood flow regulation in smaller retinal vessels that are difficult to resolve is to assess the metabolic consequences of changes in the microcirculation using oximetry. METHODS. In 20 normal persons aged (mean ± SD, range) 30.1 ± 3.8 (24-37) years, the oxygen saturation and diameter of retinal arterioles and venules to the macular area and the retinal periphery were studied before and during an increase in the arterial blood pressure induced by isometric exercise. RESULTS. The isometric exercise increased the mean arterial blood pressure by (mean ± SEM) 10.0 ± 1.1 mm Hg but induced no significant changes in the diameter of the arterioles (P = 0.83). The isometric exercise had no significant effect on the oxygen saturation in the arterioles supplying the macular area and the retinal periphery (P > 0.42 for both comparisons). However, there was a significant increase in the oxygen saturation in venules draining the retinal periphery to reduce the oxygen extraction from (mean ± SEM) 36.0% ± 2.3% to 30.6% ± 2.1% (P = 0.002) but no significant change in the preexisting low oxygen extraction in the macular area that changed from (mean ± SEM) 18.2% ± 3.0% to 16.2% ± 1.9% (P = 0.37). CONCLUSIONS. Minor changes in the arterial blood pressure can induce changes in retinal rheology with significant regional variation. The finding may help explain regional variations in manifestations of retinal vascular disease such as hyperpermeability in the macular area and capillary occlusion in the retinal periphery.

Purpose Details of the posterior eye water dynamics are unclear. Aquaporin-4 (AQP4), a water channel, plays an important role in water dynamics in the central nervous system and is also present in the ocular tissue. The purpose of this study was to reveal the role of AQP4 in the water dynamics of the posterior eye using in vivo JJ vicinal coupling proton exchange (JJVCPE) magnetic resonance imaging (MRI) of AQP4 knockout (KO) mice and their wild-type littermates (controls). Methods JJVCPE MRI of the eye was performed on five AQP4 KO mice and seven control mice. We assessed the normalized signal intensities of a region of interest (ROI) set in the vitreous body after H217O administration. The results of the two groups were compared using a two-tailed Mann-Whitney U test. Results A statistical analysis revealed that the normalized ROI signal intensities at the steady state were significantly lower (P = 0.010

Purpose: Glia and their communication via connexin 43 (Cx43) gap junctions are known to mediate neurovascular coupling, a process driven by metabolic demand. However, it is unclear whether Cx43 mediated glial communication intermediates classical autoregulation. Here we used viral transfection and a glial fibrillary acidic protein (GFAP) promoter to downregulate glial Cx43 to evaluate its role in retinal vascular autoregulation to ocular perfusion pressure (OPP) reduction. Methods: Adult rats were intravitreally injected with the viral active construct or a control. Three weeks after the injection, eyes were imaged using confocal scanning laser ophthalmoscopy before and during a period of OPP decrease induced by blood draw to lower blood pressure or by manometric IOP elevation. Vessel diameter responses to the OPP decrease were compared between Cx43-downregulated and control-injected eyes. The extent of Cx43 downregulation was evaluated by Western blot and immunohistochemistry. Results: In control eyes, the OPP decrease induced dilatation of arterioles, but not venules. In Cx43-downregulated eyes, Cx43 expression in whole retina was decreased by approximately 40%. In these eyes, the resting diameter of the venules increased significantly, but there was no effect on arterioles. In Cx43-downregulated eyes, vasoreactivity evoked by blood pressure lowering was significantly compromised in both arterioles (P = 0.005) and venules (P = 0.001). Cx43 downregulation did not affect the arteriole responses to IOP elevation, whereas the responses of the venules showed a significantly greater decrease in diameter (P < 0.001). Conclusions: The downregulation of retinal Cx43 in GFAP-expressing cells compromises vasoreactivity of both arterioles and venules in response to an OPP decrease achieved via blood pressure lowering or IOP elevation. The results also suggest that Cx43-mediated glial communication actively regulates resting venular diameter.

Purpose Tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie2) activation in Schlemm's canal (SC) endothelium is required for the maintenance of IOP, making the angiopoietin/Tie2 pathway a target for new and potentially disease modifying glaucoma therapies. The goal of the present study was to examine the effects of a Tie2 activator, AKB-9778, on IOP and outflow function. Methods AKB-9778 effects on IOP was evaluated in humans, rabbits, and mice. Localization studies of vascular endothelial protein tyrosine phosphatase (VE-PTP), the target of AKB-9778 and a negative regulator of Tie2, were performed in human and mouse eyes. Mechanistic studies were carried out in mice, monitoring AKB-9778 effects on outflow facility, Tie2 phosphorylation, and filtration area of SC. Results AKB-9778 lowered IOP in patients treated subcutaneously for diabetic eye disease. In addition to efficacious, dose-dependent IOP lowering in rabbit eyes, topical ocular AKB-9778 increased Tie2 activation in SC endothelium, reduced IOP, and increased outflow facility in mouse eyes. VE-PTP was localized to SC endothelial cells in human and mouse eyes. Mechanistically, AKB-9778 increased the filtration area of SC for aqueous humor efflux in both wild type and in Tie2+/- mice. Conclusions This is the first report of IOP lowering in humans with a Tie2 activator and functional demonstration of its action in remodeling SC to increase outflow facility and lower IOP in fully developed mice. Based on these studies, a phase II clinical trial is in progress to advance topical ocular AKB-9778 as a first in class, Tie2 activator for treatment for ocular hypertension and glaucoma.

Purpose The role of adrenergic innervation in the regulation of lacrimal gland (LG) ductal fluid secretion is unknown. The Aim of the present study was to investigate the effect of adrenergic stimulation on fluid secretion in isolated LG duct segments and to study the underlying intracellular mechanisms. Methods Fluid secretion of isolated mouse LG ducts was measured using video-microscopy. Effect of various adrenergic agonists (norepinephrine, phenylephrine, and isoproterenol) on fluid secretion as well as inhibitory effects of specific antagonists on adrenergic agonist-stimulated secretory response were analyzed. Changes in intracellular Ca2+ level [Ca2+i] were investigated with microfluorometry. Results Both norepinephrine and phenylephrine initiated a rapid and robust fluid secretory response, whereas isoproterenol did not cause any secretion. Phenylephrine-induced secretion was completely blocked by α1D-adrenergic receptor blocker BMY-7378. The endothelial nitric oxide synthase (eNOS) inhibitor L-NAME or guanylyl cyclase inhibitor ODQ reduced but not completely abolished the phenylephrine-induced fluid secretion, whereas co-administration of Ca2+-chelator BAPTA-AM resulted in a complete blockade. Phenylephrine stimulation induced a small, but statistically significant elevation in [\(Ca_i^{2 + }\)]. Conclusions Our results prove the direct role of α1-adrenergic stimulation on LG ductal fluid secretion. Lack of isoproterenol-induced fluid secretory response suggests the absence of β-receptor mediated pathway in mouse LG ducts. Complete blockade of phenylephrine-induced fluid secretion by BMY-7378 and predominant inhibition of the secretory response either by L-NAME or ODQ suggest that α-adrenergic agonists use the NO/cGMP pathway through α1D receptor. Ca2+ signaling independent from NO/cGMP pathway may also play an at least partial role in α-adrenergic induced ductal fluid secretion.

Purpose We determined whether δ-opioid receptor agonist (SNC-121) regulates acetylation homeostasis via controlling histone deacetylases (HDACs) activity and expression in optic nerve head (ONH) astrocytes. Methods ONH astrocytes were treated with SNC-121 (1 µM) for 24 hours. The HDAC activity was measured using HDAC-specific fluorophore-conjugated synthetic substrates, Boc-Lys(Ac)-AMC and (Boc-Lys(Tfa)-AMC). Protein and mRNA expression of each HDAC was determined by Western blotting and quantitative real-time PCR. IOP in rats was elevated by injecting 2.0 M hypertonic saline into the limbal veins. Results Delta opioid receptor agonist, SNC-121 (1 µM), treatment increased acetylation of histone H3, H2B, and H4 by 128 ± 3%, 45 ± 1%, and 68 ± 2%, respectively. The addition of Garcinol, a histone-acetyltransferase inhibitor, fully blocked SNC-121–induced histone H3 acetylation. SNC-121 reduced the activities of class I and IIb HDACs activities significantly (17 ± 3%) and this decrease in HDACs activities was fully blocked by a selective δ-opioid receptors antagonist, naltrindole. SNC-121 also decrease the mRNA expression of HDAC-3 and HDAC-6 by 19% and 18%, respectively. Furthermore, protein expression of HDAC 1, 2, 3, and 6 was significantly (P < 0.05) decreased by SNC-121 treatment. SNC-121 treatment also reduced lipopolysaccharide-induced TNF-α production from ONH astrocytes and glial fibrillary acidic protein immunostaining in the optic nerve of ocular hypertensive animals. Conclusions We provided evidence that δ-opioid receptor agonist activation increased histone acetylation, decrease HDACs class I and class IIb activities, mRNA, and protein expression, lipopolysaccharide-induced TNF-α production in ONH astrocytes. Our data also demonstrate that SNC-121 treatment decrease glial fibrillary acidic protein immunostaining in the optic nerves of animals with ocular hypertension.

Purpose Polymorphisms at the caveolin-1/2 locus are associated with glaucoma and IOP risk and deletion of caveolin-1 (Cav1) in mice elevates IOP and reduces outflow facility. However, the specific location/cell type responsible for Cav1-dependent regulation of IOP is unclear. We hypothesized that endothelial Cav1 in the conventional outflow (CO) pathway regulate IOP via endothelial nitric oxide synthase (eNOS) signaling. Methods We created a mouse with targeted deletion of Cav1 in endothelial cells (Cav1ΔEC) and evaluated IOP, outflow facility, outflow pathway distal vascular morphology, eNOS phosphorylation, and tyrosine nitration of iridocorneal angle tissues by Western blotting. Results Endothelial deletion of Cav1 resulted in significantly elevated IOP versus wild-type mice but not a concomitant decrease in outflow facility. Endothelial Cav1 deficiency did not alter the trabecular meshwork or Schlemm's canal morphology, suggesting that the effects observed were not due to developmental deformities. Endothelial Cav1 deletion resulted in eNOS hyperactivity, modestly increased protein nitration, and significant enlargement of the drainage vessels distal to Schlemm's canal. L-Nitro-arginine methyl ester treatment reduced outflow in Cav1ΔEC but not wild-type mice and had no effect on the size of drainage vessels. Endothelin-1 treatment decrease the outflow and drainage vessel size in both wild-type and Cav1ΔEC mice. Conclusions Our results suggest that hyperactive eNOS signaling in the CO pathway of both Cav1ΔEC and global Cav1 knockout mice results in chronic dilation of distal CO vessels and protein nitration, but that Cav1 expression in the trabecular meshwork is sufficient to rescue CO defects reported in global Cav1 knockout mice.

Purpose N-methyl-N-nitrosourea (MNU) is an alkylating toxicant with potent mutagenic ability. This study was designed to induce apoptosis in lens epithelial cells (LECs) and corneal endothelial cells (CECs) via MNU administration. We sought to build ocular disease models of cataract and corneal endothelial decompensation. Methods MNU was delivered into the intraperitoneal cavities of neonatal rats and the anterior chambers of adult rabbits. The MNU-treated animals were then subjected to a series of functional and morphological analyses at various time points. Results MNU treatment induced pervasive apoptosis of LECs and CECs. These effects were dose and time dependent. Mature cataracts were found in neonatal rats 3 weeks after MNU treatment. Histological analysis revealed that MNU toxicity induced swelling, vacuolation, and liquefaction in lens fibers of MNU-treated rats. Pentacam examination showed that the average density of rat lens increased significantly after MNU administration. Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) analysis showed pervasive apoptotic staining in the lenses of MNU-treated rats. In rabbit eyes, intracameral treatment with MNU induced corneal edema and significantly increased central corneal thickness, which peaked at P14. Morphological and immunohistochemical analysis showed that CECs were effectively ablated in the MNU-treated rabbits. The expression of 8-OHdG increased significantly in the cornea of MNU-treated rabbits, compared with vehicle-treated controls. Conclusions MNU is sufficient to induce ocular cell apoptosis in animal models. These models of MNU-induced cataract and corneal endothelial decompensation represent valuable tools for efforts to develop relevant therapies.

Purpose To examine ion transport across the mouse retinal pigment epithelium (RPE), measured by the short-circuit current (ISC) and transepithelial resistance (TER). Methods Sheets of RPE from mice (C57BL6/J) with retina, choroid, and sclera attached were mounted in Ussing chambers (0.031-cm2 aperture) and Krebs solution. The ISC and TER were recorded with voltage clamps. Receptors implicated in ion transport were blocked or stimulated by ligands applied to both sides. Results The mean initial ISC was -12.0 ± 3.9 µA/cm2 (basolateral negative), and mean TER was 67.1 ± 8.0 ohm·cm2. RPE preparations remained stable for 3 hours, with ISC decreasing by 0.078 ± 0,033 µA/cm2/hr. Adenosine triphosphate (100 µM) increased ISC by 2.22 ± 0.41 µA/cm2 (P = 0.003). Epinephrine (100 µM) increased ISC by 1.14 ± 0.19 µA/cm2 (P = 0.011). Bumetanide (100 µM) reduced ISC by 1.72 ± 0.73 µA/cm2 (P = 0.027). Ouabain (1 mM) induced a biphasic response: an ISC increase from -7.9 ± 2.4 to -15.49 ± 2.12 µA/cm2 and then a decrease to -3.7 ± 2.2 µA/cm2. Ouabain increased TER by 15.3 ± 4.8 ohm·cm2. These compounds were added sequentially. Apical [K+]o at zero mM transiently increased ISC by 3.36 ± 1.06 µA/cm2. Ba++ decreased ISC from -10.4 ± 3.1 to -6.6 ± 1.8 µA/cm2 (P = 0.01). Ba++ reversed the K+-free response, with Isc decreasing further from -5.65 ± 1.24 to -3.37 ± 0.79 µA/cm2 (P = 0.029). Conclusions The ISC and TER can be recorded from the mouse RPE for 3 hours. Adrenergic and purinergic receptors affect murine RPE ion transport. Sodium-potassium adenosine triphosphatase plays a role in net ion transport across mouse RPE, and Na-K-2Cl cotransporter activity partly accounts for transepithelial ion transport. Mimicking light-induced changes, low subretinal [K+]o increases ion transport transiently, dependent on K+ channels.

Purpose: Bestrophinopathies are a group of untreatable inherited retinal dystrophies caused by mutations in the retinal pigment epithelium (RPE) Cl− channel bestrophin 1. We tested whether sodium phenylbutyrate (4PBA) could rescue the function of mutant bestrophin 1 associated with autosomal dominant and recessive disease. We then sought analogues of 4PBA with increased potency and determined the mode of action for 4PBA and a lead compound 2-naphthoxyacetic acid (2-NOAA). Lastly, we tested if 4PBA and 2-NOAA could functionally rescue bestrophin 1 function in RPE generated from induced pluripotent stem cells (iPSC-RPEs) derived from patients with a dominant or recessive bestrophinopathy.Methods: Global and plasma membrane expression was determined by Western blot and immunofluorescent microscopy, respectively. The effect of 4PBA and 2-NOAA on transcription was measured by quantitative RT-PCR and the rate of protein turnover by cycloheximide chase and Western blot. Channel function was measured by whole-cell patch clamp.Results: 4PBA and 2-NOAA can rescue the global and membrane expression of mutant bestrophin 1 associated with autosomal dominant disease (Best vitelliform macular dystrophy [BVMD]) and autosome recessive bestrophinopathy (ARB), and these small molecules have different modes of action. Both 4PBA and 2-NOAA significantly increased the channel function of mutant BVMD and ARB bestrophin 1 in HEK293T and iPSC-RPE cells derived from patients with BVMD and ARB. For 4PBA, the increased mutant channel function in BVMD and ARB iPSC-RPE was equal to that of wild-type iPSC-RPE bestrophin 1.Conclusions: The restoration of bestrophin 1 function in patient-derived RPE confirms the US Food and Drug Administration–approved drug 4PBA as a promising therapeutic treatment for bestrophinopathies.

Purpose Rodent and primate models are commonly used in glaucoma research; however, both have their limitations. The tree shrew (Tupaia belangeri) is an emerging animal model for glaucoma research owing in part to having a human-like optic nerve head anatomy, specifically a collagenous load-bearing lamina. However, the anterior segment anatomy and function have not been extensively studied in the tree shrew. Thus, the purpose of this study was to provide the first detailed examination of the anterior segment anatomy and aqueous outflow facility in the tree shrew. Methods Aqueous outflow dynamics were measured in five ostensibly normal eyes from three tree shrews using the iPerfusion system over a range of pressures. Gross histological assessment and immunohistochemistry were performed to characterize anterior segment anatomy and to localize several key molecules related to aqueous outflow. Results Anterior segment anatomy in tree shrews is similar to humans, demonstrating a scleral spur, a multilayered trabecular meshwork and a circular Schlemm's canal with a single lumen. Average outflow facility was 0.193 µL/min/mm Hg (95% confidence interval, 0.153–0.244), and was stable over time. Outflow facility was more similar between contralateral eyes (approximately 5% average difference) than between eyes of different animals. No significant dependence of outflow facility on time or pressure was detected (pressure–flow nonlinearity parameter of 0.01 (95% % confidence interval, −0.29 to 0.31 CI µL/min/mm Hg). Conclusions These studies lend support to the usefulness of the tree shrew as a novel animal model in anterior segment glaucoma and pharmacology research. The tree shrew's cost, load-bearing collagenous lamina cribrosa, and lack of washout or anterior chamber deepening provides a distinct experimental and anatomic advantage over the current rodent and nonhuman primate models used for translational research.

Purpose Dry eye–induced chronic ocular pain is also called ocular neuropathic pain. However, details of the pathogenic mechanism remain unknown. The purpose of this study was to elucidate the pathogenic mechanism of dry eye–induced chronic pain in the anterior eye area and develop a pathophysiology-based therapeutic strategy. Methods We used a rat dry eye model with lacrimal gland excision (LGE) to elucidate the pathogenic mechanism of ocular neuropathic pain. Corneal epithelial damage, hypersensitivity, and hyperalgesia were evaluated on the LGE side and compared with the sham surgery side. We analyzed neuronal activity, microglial and astrocytic activity, α2δ–1 subunit expression, and inhibitory interneurons in the trigeminal nucleus. We also evaluated the therapeutic effects of ophthalmic treatment and chronic pregabalin administration on dry eye–induced ocular neuropathic pain. Results Dry eye caused hypersensitivity and hyperalgesia on the LGE side. In the trigeminal nucleus of the LGE side, neuronal hyperactivation, transient activation of microglia, persistent activation of astrocytes, α2δ–1 subunit upregulation, and reduced numbers of inhibitory interneurons were observed. Ophthalmic treatment alone did not improve hyperalgesia. In contrast, continuous treatment with pregabalin effectively ameliorated hypersensitivity and hyperalgesia and normalized neural activity, α2δ–1 subunit upregulation, and astrocyte activation. Conclusions These results suggest that dry eye–induced hypersensitivity and hyperalgesia are caused by central sensitization in the trigeminal nucleus with upregulation of the α2δ–1 subunit. Here, we showed that pregabalin is effective for treating dry eye–induced ocular neuropathic pain even after chronic pain has been established.

Purpose The role of cystic fibrosis transmembrane conductance regulator (CFTR) in lacrimal gland (LG) function has only recently received some attention, mainly from our group. In the present study, we investigated the potential changes of LG pathology, tear secretion, ocular surface integrity, and fluid secretion in isolated LG ducts from CFTR knockout (KO) mice. Methods Tear production and ocular surface integrity were investigated in anesthetized wild-type (WT) and KO mice using cotton threads and fluorescein staining, respectively. Immunofluorescence was used to localize CFTR protein in the LGs. Ductal fluid secretions evoked by forskolin (10 μM); cell-permeable cAMP analogue (8-bromo cAMP, 100 μM); or carbachol (100 μM) were measured in isolated LG ducts using video-microscopy. Intracellular Ca2+ homeostasis underlying carbachol stimulation was investigated with microfluorometry. Results Significant decrease in tear secretion and impaired ocular surface integrity were observed in KO mice. Immunofluorescence demonstrated the predominant presence of CFTR protein in the apical membranes of the duct cells from WT mice. Continuous fluid secretion was evoked by forskolin and 8-bromo cAMP in LG ducts from WT mice, while no secretory response was observed in ducts from KO mice. Carbachol caused similar secretory responses in ducts from WT and KO animals without significant differences in cytosolic Ca2+ signaling. Conclusions Our results suggest the important role of CFTR in LG ductal secretion and in the maintenance of ocular surface integrity, suggesting that CFTR may be a promising target of novel therapeutic approaches in the treatment of dry eye.

Purpose The development of myopia in guinea pigs can be inhibited by attenuating scleral hypoxia by increasing choroidal blood perfusion (ChBP). In this study, we reduced ChBP through surgical and pharmacological methods to determine the effect on myopia development. We also determined whether ChBP was reduced by quinpirole, a drug that enhances form-deprivation myopia (FDM). Methods ChBP was reduced in the right eyes of guinea pigs via transection of the temporal ciliary arteries or daily injections of phenylephrine into the inferior peribulbar space for one week during normal ocular growth. Other guinea pigs were subjected to two weeks of monocular FDM—with facemasks, along with daily injections of quinpirole, a dopamine D2 receptor agonist, to enhance the FDM. Changes in refraction, axial length, ChBP, and choroidal thickness (ChT) were measured in both treated and fellow eyes of the treatment and control groups. Scleral hypoxia labeling with pimonidazole adducts and α-smooth muscle actin (α-SMA) protein were also measured. Results Surgical and pharmacological reduction of ChBP induced myopia development in the treated eyes. These treatments rendered the scleral hypoxia and increased scleral α-SMA expression. Furthermore, quinpirole injections, which increased the magnitude of myopia, augmented the FDM-associated reductions in ChBP and ChT and increased the levels of scleral hypoxia and α-SMA protein. Conclusions Decreased ChBP in guinea pigs leads to scleral hypoxia and scleral myofibroblast transdifferentiation with increased α-SMA expression, ultimately resulting in myopia development. In future clinical trials, ChBP reduction can serve as a potential biomarker for early detection of myopia development.

Purpose Endogenous and exogenous stressors, including nutritional challenges, may alter circadian rhythms in the cornea. This study aimed to determine the effects of high fructose intake (HFI) on circadian homeostasis in murine cornea. Methods Corneas of male C57BL/6J mice subjected to 10 days of HFI (15% fructose in drinking water) were collected at 3-hour intervals over a 24-hour circadian cycle. Total extracted RNA was subjected to high-throughput RNA sequencing. Rhythmic transcriptional data were analyzed to determine the phase, rhythmicity, unique signature, metabolic pathways, and cell signaling pathways of transcripts with temporally coordinated expression. Corneas of HFI mice were collected for whole-mounted techniques after immunofluorescent staining to quantify mitotic cell number in the epithelium and trafficking of neutrophils and γδ-T cells to the limbal region over a circadian cycle. Results HFI significantly reprogrammed the circadian transcriptomic profiles of the normal cornea and reorganized unique temporal and clustering enrichment pathways, but did not affect core-clock machinery. HFI altered the distribution pattern and number of corneal epithelial mitotic cells and enhanced recruitment of neutrophils and γδ-T cell immune cells to the limbus across a circadian cycle. Cell cycle, immune function, metabolic processes, and neuronal-related transcription and associated pathways were altered in the corneas of HFI mice. Conclusions HFI significantly reprograms diurnal oscillations in the cornea based on temporal and spatial distributions of epithelial mitosis, immune cell trafficking, and cell signaling pathways. Our findings reveal novel molecular targets for treating pathologic alterations in the cornea after HFI.

Background: Myopia has emerged as a major health issue in the world, due to the sight-threatening pathologies associated with high myopia. Even though previous studies have implicated that myopia is determined by a complex interaction of environmental and genetic factors, the pathogenesis of this disease remains unclear. Methods: Here we utilized plasma from patients with myopia, myopic animal models including the adenomatous polyposis coli (APC) gene mutation mouse model, and the form deprivation induced mouse model of myopia. Findings: Using these systems, we demonstrated that the over-activation of canonical Wnt pathway is associated with myopia. Moreover, inhibition of canonical Wnt signaling in mouse models markedly reduced axial elongation, resulting in myopia regression. Interpretation: Together, these results suggest that dysregulation of canonical Wnt signaling is a characteristic of myopia and suggest that targeting Wnt signaling pathways has potential as a therapeutic strategy for myopia. Funding Statement: This study was supported by the Natural Science Foundation of Fujian (No. 2019J05007, ZL); National Basic Research Program of China (Project 973) (No.2011CB50460); National Key Program of China (No. 2018YFA0107304); and National Natural Science Foundation of China (No.81870627). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: Approval was received from the Human Ethics committee of Xiamen Eye Center Affiliated to Xiamen University, in accordance with the Declaration of Helsinki. Informed consents were obtained from all subjects after explanation of the nature and possible consequences of the study. Mice were used, treated, and cared for in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Guidelines of the Animal Experimental Committee of Xiamen University.

Purpose Patients that medicate with antidepressants commonly report dryness of eyes. The cause is often attributed to the anticholinergic properties of the drugs. However, regulation of tear production includes a substantial reflex-evoked component and is regulated via distinct centers in the brain. Further, the anticholinergic component varies greatly among antidepressants with different mechanisms of action. In the current study it was wondered if acute administration of antidepressants can disturb production of tears by affecting the afferent and/or central pathway. Methods Tear production was examined in vivo in anesthetized rats in the presence or absence of the tricyclic antidepressant (TCA) clomipramine or the selective serotonin reuptake inhibitor (SSRI) escitalopram. The reflex-evoked production of tears was measured by challenging the surface of the eye with menthol (0.1 mM) and cholinergic regulation was examined by intravenous injection with the nonselective muscarinic agonist methacholine (1-5 µg/kg). Results Acute administration of clomipramine significantly attenuated both reflex-evoked and methacholine-induced tear production. However, escitalopram only attenuated reflex-evoked tear production, while methacholine-induced production of tears remained unaffected. Conclusions This study shows that antidepressants with different mechanisms of action can impair tear production by attenuating reflex-evoked signaling. Further, antimuscarinic actions are verified as a likely cause of lacrimal gland hyposecretion in regard to clomipramine but not escitalopram. Future studies on antidepressants with different selectivity profiles and mechanisms of action are required to further elucidate the mechanisms by which antidepressants affect tear production.

Purpose: We assess the effect of intravitreal anti-VEGF injections on tonographic outflow facility. Methods: Patients with age-related macular degeneration who had received unilateral intravitreal anti-VEGF injections were recruited into two groups, those with ≤10 and those with ≥20 total anti-VEGF injections. Intraocular pressure and tonographic outflow facility of injected and uninjected fellow eyes were measured and compared between groups. Risk factors for development of reduced outflow facility also were assessed. Results: Outflow facility was 12% lower in the injected eyes of patients who received ≥20 anti-VEGF injections, compared to contralateral uninjected eyes (P = 0.02). In contrast, there was no facility reduction for patients with ≤10 anti-VEGF injections (P = 0.4). In patients with ocular hypertension in the uninjected eye (IOP > 21 mm Hg, n = 5), the outflow facility of injected eyes was on average 46% lower (P = 0.01) than in the uninjected fellow eyes. This was significantly greater than the difference observed in patients with IOP ≤ 21 mm Hg in the uninjected eye (P = 2 × 10−4). In patients with ocular hypertension in the injected eye (n = 6) the differences in facility and IOP between contralateral eyes were significantly greater than in patients with IOP ≤ 21 mm Hg in the injected eye (P = 2 × 10−4 and P = 7 × 10−4, respectively). Conclusions: Chronic anti-VEGF injections significantly reduce outflow facility in patients with AMD. The greatest facility reduction is observed in patients with baseline ocular hypertension. Ophthalmologists who administer anti-VEGF injections should be aware of these findings and monitor patients closely for changes in IOP or evidence of glaucoma, especially in those with pre-existing ocular hypertension.

Purpose The purpose of this study was to test the hypothesis that retinal glial cells modify basal vessel diameter and pressure-initiated vascular regulation in rat retina. Methods In rats, L-2-aminoadipic acid (LAA, 10 nM) was intravitreally injected to inhibit glial cell activity. Twenty-four hours following injection, retinal glial intracellular calcium (Ca2+) was labeled with the fluorescent calcium indicator Fluo-4/AM (F4, 1 mM). At 110 minutes after injection, intraocular pressure (IOP) was elevated from 20 to 50 mm Hg. Prior to and during IOP elevation, Ca2+ and retinal vessel diameter were assessed using a spectral-domain optical coherence tomography/confocal scanning laser ophthalmoscope. Dynamic changes in Ca2+ and diameter from IOP elevation were quantified. The response in LAA-treated eyes was compared with vehicle treated control eyes. Results L-2-Aminoadipic acid treatment significantly reduced F4-positive cells in the retina (LAA, 16 ± 20 vs. control, 55 ± 37 cells/mm2; P = 0.02). Twenty-four hours following LAA treatment, basal venous diameter was increased from 38.9 ± 3.9 to 51.8 ± 6.4 μm (P < 0.0001, n = 20), whereas arterial diameter was unchanged (from 30.3 ± 3.5 to 30.7 ± 2.8 μm; P = 0.64). In response to IOP elevation, LAA-treated eyes showed a smaller increase in glial cell Ca2+ around both arteries and veins in comparison with control (P < 0.001 for both). There was also significantly greater IOP-induced vasoconstriction in both vessel types (P = 0.05 and P = 0.02, respectively; n = 6 each). Conclusions The results suggest that glial cells can modulate basal retinal venous diameter and contribute to pressure-initiated vascular responses.

Purpose Benzalkonium chloride (BAK) is the most commonly used eye drop preservative. Benzalkonium chloride has been associated with toxic effects such as "dry eye" and trabecular meshwork degeneration, but the underlying biochemical mechanism of ocular toxicity by BAK is unclear. In this study, we propose a mechanistic basis for BAK's adverse effects. Method Mitochondrial O2 consumption rates of human corneal epithelial primary cells (HCEP), osteosarcoma cybrid cells carrying healthy (control) or Leber hereditary optic neuropathy (LHON) mutant mtDNA [11778(G>A)], were measured before and after acute treatment with BAK. Mitochondrial adenosine triphosphate (ATP) synthesis and cell viability were also measured in the BAK-treated control: LHON mutant and human-derived trabecular meshwork cells (HTM3). Results Benzalkonium chloride inhibited mitochondrial ATP (IC50, 5.3 μM) and O2 consumption (IC50, 10.9 μM) in a concentration-dependent manner, by directly targeting mitochondrial complex I. At its pharmaceutical concentrations (107-667 μM), BAK inhibited mitochondrial function >90%. In addition, BAK elicited concentration-dependent cytotoxicity to cybrid cells (IC50, 22.8 μM) and induced apoptosis in HTM3 cells at similar concentrations. Furthermore, we show that BAK directly inhibits mitochondrial O2 consumption in HCEP cells (IC50, 3.8 μM) at 50-fold lower concentrations than used in eye drops, and that cells bearing mitochondrial blindness (LHON) mutations are further sensitized to BAK's mitotoxic effect. Conclusions Benzalkonium chloride inhibits mitochondria of human corneal epithelial cells and cells bearing LHON mutations at pharmacologically relevant concentrations, and we suggest this is the basis of BAK's ocular toxicity. Prescribing BAK-containing eye drops should be avoided in patients with mitochondrial deficiency, including LHON patients, LHON carriers, and possibly primary open-angle glaucoma patients.

204 p.-40 fig.-7 tab., La preeclampsia es una patología específica del embarazo que se caracteriza por la aparición de novo de hipertensión y proteinuria a partir de la semana 20 de gestación (Rana y cols. 2013). Es una de las principales causas de morbilidad y mortalidad tanto materna como fetal y perinatal en los países desarrollados. Según las recomendaciones de la Organización Mundial de la Salud (OMS), la preeclampsia se diagnostica cuando se presenta una presión arterial (PA) elevada (≥140/90 mmHg) acompañada por una proteinuria considerable (≥300 mg/24 h) a partir de la segunda mitad del embarazo. Además de estos síntomas, la preeclampsia suele ir acompañada de disfunción endotelial, crecimiento fetal retardado, aparición de edemas, etc. Por todo ello, la preeclampsia es considerada un trastorno complejo y multisistémico (Pennington y cols. 2012). Debido a esto, y ante el incremento de evidencias que ponen en duda la idoneidad de la proteinuria y el crecimiento fetal retardado para el diagnóstico correcto de la preeclampsia, el American College of Obstetricians and Gynecologists ha propuesto una serie criterios para un mejor diagnóstico, los cuales son recogidos en la Tabla 1 (The American College of Obstetricians and Gynecologists y cols. 2013). Generalmente, la preeclampsia se clasifica en función del grado de hipertensión, proteinuria o disfunción orgánica materna, en estadio leve o grave, aunque también existe una clasificación en función del momento de la gestación en el que aparecen las manifestaciones clínicas (von Dadelszen y cols. 2003) (Tabla 2). La progresión de la patología desde los estadios más leves a otros más graves puede ser rápida, inesperada y, a veces, fulminante. Las formas más lesivas de la enfermedad, como pueden ser HELLP (del inglés, Hemolysis, Elevated Liver enzymes, Low Platelet count) y eclampsia, se asocian con una mayor tasa de mortalidad materna y fetal, tanto en la gestación como en el periodo neonatal (Pennington y cols. 2012; Sibai y Coppage 2004; Sibai y cols. 2005; Barnhart 2015; Dusse y cols. 2015)(Tabla 2). Se han descrito diferentes factores de riesgo relacionados con la aparición de la preeclampsia. La obesidad, la hipertensión crónica y la diabetes son algunos de estos factores. También se incluyen las primigrávidas, el embarazo adolescente o a edad avanzada, la exposición limitada a esperma, los estados de hiperplacentación (como en el caso de embarazo gemelar), así como alteraciones inmunológicas (lupus eritematoso). Además, debido a la variabilidad en la incidencia entre los diversos grupos (es mayor en mujeres afro-americanas y asiáticas que en mujeres caucásicas), y a que la preeclampsia materna se asocia, en un 70%, a preeclampsia en las hijas; se ha sugerido la implicación de factores genéticos en el desarrollo de esta patología (Nilsson y cols. 2004; Sibai y cols. 2005; Rao y cols. 2006; Eiland y cols. 2012; Emanuel y Butt 2015; Ali y Khalil 2015). A pesar de los numerosos estudios realizados sobre los diferentes factores de riesgo y sobre el desarrollo de la enfermedad, su patogenia no ha sido aún dilucidada. Lo que está comúnmente aceptado es que la placenta es el órgano imprescindible en la preeclampsia, ya que juega un papel esencial tanto en el establecimiento como en el desarrollo de la patología (De Maria y See 1966; Piering y cols. 1993). Esto queda patente ante la rápida mejoría de los síntomas maternos que tienen lugar tras un aborto o el alumbramiento, pero los síntomas persisten mientras no sea extraída la placenta (Piering y cols. 1993). Por otro ado, el papel del feto ha sido descartado, ya que se ha observado preeclampsia en embarazos extrauterinos y en embarazos molares, en los cuales no hay un embrión viable implantado, pero si se desarrolla la placenta (Chun y cols. 1964; Roberts y Escudero 2012)., Hasta la fecha, el único tratamiento efectivo para el tratamiento de la preeclampsia ha sido la finalización del embarazo, ya sea por una interrupción voluntaria o a través de la inducción del parto y expulsión de la placenta. Aunque el parto pre-término reduce los efectos lesivos de la preeclampsia sobre el bebé, el nacimiento prematuro puede producir otra serie de complicaciones secundarias, tales como problemas respiratorios, visuales, etc., derivadas de una gestación interrumpida. Estos problemas pueden llegar a poner en un grave riesgo la vida del bebé o, incluso, derivar en patologías que lo acompañarán toda su vida (Lopez-Rodriguez y cols. 2017; Leung y cols. 2018; Guyton y Hall 2008b). Todo esto pone de relieve la necesidad de tratamientos efectivos que permitan tratar la enfermedad sin poner fin al embarazo., Por otro lado, la prevención primaria de la preeclampsia en aquellas mujeres consideradas “de riesgo” es un tema de debate y objeto de investigación activa. Entre los tratamientos utilizados se incluyen suplementos de calcio y vitamina D, administración de magnesio o zinc, aspirina a bajas dosis o la administración de suplementos de vitaminas C y E, y otros antioxidantes (Poston y cols. 2006; Sasan y cols. 2009; Bujold y cols. 2010; Hofmeyr y cols. 2014). Sin embargo, ninguno de estos tratamientos ha demostrados ser lo suficientemente efectivo en la prevención del desarrollo de la preeclampsia (Eiland y cols. 2012). En los últimos años, se están llevando a cabo diversas líneas de investigación para intentar elucidar los mecanismos implicados en el establecimiento y el desarrollo de la enfermedad, con el fin de poder desarrollar nuevos tratamientos efectivos, así como un mejor y precoz diagnóstico. Recientemente, se ha propuesto el empleo de moléculas solubles presentes en el plasma de las mujeres con preeclampsia como potentes biomarcadores de diagnóstico y pronóstico de la enfermedad. En esta línea, se han descrito incrementos de factores pro-angiogénicos, disminución de factores anti-angiogénicos, o una desregulación de los estrógenos como posibles biomarcadores. Entre los factores pro-angiogénicos destacan la forma soluble del receptor de membrana endoglina (sEng), así como la forma soluble del receptor de membrana Flt-1 (sFlt-1), que están presentes en altas concentraciones en el plasma de estas pacientes, y cuya elevación de sus niveles se asocia estrechamente con el desarrollo de la enfermedad. La elevación de la concentración en plasma de sFlt-1 se observa a partir de la semana 5-6 de gestación, mientras que sEng aumenta entre el mes 2 y 3 de gestación. Es decir, que estos marcadores aparecen elevados semanas antes de la aparición de las manifestaciones clínicas, lo que los convierte en marcadores tempranos. Además, niveles elevados sEng y sFlt-1 se han correlacionado con una mayor gravedad y peor pronóstico de la patología (Levine y cols. 2006; López-Novoa 2007; Dechend y Luft 2008; Farina y cols. 2008; Grill y cols. 2009; Hod y cols. 2015; Troisi y cols. 2008; Schrey-Petersen y Stepan 2017; Berkane y cols. 2017; Sahai y cols. 2017)., A pesar de la manifiesta relación de estos factores solubles con la preeclampsia, sus orígenes y sus mecanismos de liberación no han sido completamente dilucidados. Tal como se ha mencionado anteriormente, en la actualidad se considera que la placenta puede ser el órgano central de la preeclampsia (De Maria y See 1966; Roberts y Escudero 2012). En concreto, se señala la posible alteración en la remodelación de las arterias espirales (Browne y Veall 1953; Brosens 1964). Generalmente, la causa de los defectos en la remodelación arterial se asocia con una alteración de la invasión de los trofoblastos endovasculares (Kaufmann y cols. 2003; Pijnenborg y cols. 2006). Esto hace que no se produzca el reemplazo de células del endotelio por los trofoblastos, y como consecuencia, los vasos se mantienen en un estado de baja capacitancia (Cnossen y cols. 2008; Meler y cols. 2010). Además, se ha observado que el calibre de las arterias uterinas de las mujeres con preeclampsia son mucho menores que en mujeres con embarazo normal, siendo más similares a los valores de las mujeres no gestantes (Brosens y cols. 1972). Todo esto genera hipoperfusión placentaria, y por lo tanto una restricción en el aporte de nutrientes y oxígeno. Esta hipoperfusión puede originar otros cambios patológicos en el resto de la placenta, tales como depósitos de fibrina entre la capa de sincitiotrofoblastos, daños en la vasculatura intrínseca de la placenta, reducción de la superficie de contacto de las vellosidades coriónicas y liberación de restos celulares a la circulación sistémica, procedentes de la necrosis trofoblástica (Fisher 2015). Todas estas alteraciones contribuirían a la generación del daño placentario, que culminaría el desarrollo de la patología., Este trabajo ha sido realizado con cargo a los siguientes proyectos de investigación: SAF2013-43421-R (2014-2018) ; ISCIII-CB06/07/0038(2006-2017). Unidad 707 del CIBERER ; SAF2013-45784-R (2014-2017); RETIC RD12/0021/0032. Red de Investigación Renal (REDinREN), Instituto de Salud Carlos III, Ministerio de Economía, Industria y Competitividad. (2015-2016). Fundación Renal Iñigo Alvarez de Toledo (FRIAT)

Purpose The concentration of protons in the aqueous humor (AH) of the vertebrate eye is maintained close to blood pH; however, pathologic conditions and surgery may shift it by orders of magnitude. We investigated whether and how changes in extra- and intracellular pH affect the physiology and function of trabecular meshwork (TM) cells that regulate AH outflow. Methods Electrophysiology, in conjunction with pharmacology, gene knockdown, and optical recording, was used to track the pH dependence of transmembrane currents and mechanotransduction in primary and immortalized human TM cells. Results Extracellular acidification depolarized the resting membrane potential by inhibiting an outward K+-mediated current, whereas alkalinization hyperpolarized the cells and augmented the outward conductance. Intracellular acidification with sodium bicarbonate hyperpolarized TM cells, whereas removal of intracellular protons with ammonium chloride depolarized the membrane potential. The effects of extra- and intracellular acid and alkaline loading were abolished by quinine, a pan-selective inhibitor of two-pore domain potassium (K2P) channels, and suppressed by shRNA-mediated downregulation of the mechanosensitive K2P channel TREK-1. Extracellular acidosis suppressed, whereas alkalosis facilitated, the amplitude of the pressure-evoked TREK-1-mediated outward current. Conclusions These results demonstrate that TM mechanotransduction mediated by TREK-1 channels is profoundly sensitive to extra- and intracellular pH shifts. Intracellular acidification might modulate aqueous outflow and IOP by stimulating TREK-1 channels.

Purpose Hyperglycemia, a hallmark of diabetes mellitus, is associated with retinal inflammation and impairment of endothelium-dependent nitric oxide (NO)–mediated dilation of retinal arterioles. However, molecular mechanisms involved in this diminished endothelial vasodilator function remain unclear. We examined whether inflammatory stress-activated kinases, c-Jun N-terminal kinase (JNK) and p38, contribute to retinal arteriolar dysfunction during exposure to acute and chronic hyperglycemia. Methods Retinal arterioles were isolated from streptozocin-induced diabetic pigs (2 weeks; chronic hyperglycemia, 471 ± 23 mg/dL) or age-matched control pigs (euglycemia, 79 ± 5 mg/dL), and then cannulated and pressurized for vasoreactivity study. For acute hyperglycemia study, vessels from nondiabetic pigs were exposed intraluminally to high glucose (25 mM ≈ 450 mg/dL) for 2 hours, and normal glucose (5 mM ≈ 90 mg/dL) served as the control. Results Endothelium-dependent vasodilation to bradykinin was reduced in a similar manner after exposure to acute or chronic hyperglycemia. Administration of NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) nearly abolished vasodilations either in control (euglycemia and normal glucose) or hyperglycemic (acute and chronic) vessels. Treatment of either acute or chronic hyperglycemic vessels with JNK inhibitor SP600125 or JNK-interacting protein-1 (JIP1) inhibitor BI-78D3, but not p38 inhibitor SB203580, preserved bradykinin-induced dilation in an L-NAME–sensitive manner. By contrast, endothelium-independent vasodilation to sodium nitroprusside was unaffected by acute or chronic hyperglycemia. Conclusions Activation of JIP1/JNK signaling in retinal arterioles during exposure to acute or chronic hyperglycemia leads to selective impairment of endothelium-dependent NO-mediated dilation. Therapeutic targeting of the vascular JNK pathway may improve retinal endothelial vasodilator function during early diabetes.

Purpose Inflammation associated with blood-retinal barrier (BRB) breakdown is a common feature of several retinal diseases. Therefore, the development of novel nonsteroidal anti-inflammatory approaches may provide important therapeutic options. Previous studies demonstrated that inhibition of dipeptidyl peptidase-IV, the enzyme responsible for the degradation of glucagon-like peptide-1 (GLP-1), led to insulin-independent prevention of diabetes-induced increases in BRB permeability, suggesting that incretin-based drugs may have beneficial pleiotropic effects in the retina. In the current study, the barrier protective and anti-inflammatory properties of exendin-4 (Ex-4), an analog of GLP-1, after ischemia-reperfusion (IR) injury were examined. Methods Ischemia-reperfusion injury was induced in rat retinas by increasing the intraocular pressure for 45 minutes followed by 48 hours of reperfusion. Rats were treated with Ex-4 prior to and following IR. Blood-retinal barrier permeability was assessed by Evans blue dye leakage. Retinal inflammatory gene expression and leukocytic infiltration were measured by qRT-PCR and immunofluorescence, respectively. A microglial cell line was used to determine the effects of Ex-4 on lipopolysaccharide (LPS)-induced inflammatory response. Results Exendin-4 dramatically reduced the BRB permeability induced by IR injury, which was associated with suppression of inflammatory gene expression. Moreover, in vitro studies showed that Ex-4 also reduced the inflammatory response to LPS and inhibited NF-κB activation. Conclusions The present work suggests that Ex-4 can prevent IR injury-induced BRB breakdown and inflammation through inhibition of inflammatory cytokine production by activated microglia and may provide a novel option for therapeutic intervention in diseases involving retinal inflammation.

Purpose The nitric oxide (NO)-cyclic guanosine-3',5'-monophosphate (cGMP) pathway regulates aqueous humor outflow and therefore, intraocular pressure. We investigated the pharmacologic effects of the soluble guanylate cyclase (sGC) stimulator IWP-953 on primary human trabecular meshwork (HTM) cells and conventional outflow facility in mouse eyes. Methods Cyclic GMP levels were determined in vitro in HEK-293 cells and four HTM cell strains (HTM120/HTM123: predominantly myofibroblast-like phenotype, HTM130/HTM141: predominantly endothelial-like phenotype), and in HTM cell culture supernatants. Conventional outflow facility was measured following intracameral injection of IWP-953 or DETA-NO using a computerized pressure-controlled perfusion system in enucleated mouse eyes ex vivo. Results IWP-953 markedly stimulated cGMP production in HEK-293 cells in the presence and absence of DETA-NO (half maximal effective concentrations: 17 nM, 9.5 μM). Similarly, IWP-953 stimulated cGMP production in myofibroblast-like HTM120 and HTM123 cells, an effect that was greatly amplified by the presence of DETA-NO. In contrast, IWP-953 stimulation of cGMP production in endothelial-like HTM130 and HTM141 cells was observed, but was markedly less prominent than in HTM120 and HTM123 cells. Notably, cGMP was found in all HTM culture supernatants, following IWP-953/DETA-NO stimulation. In paired enucleated mouse eyes, IWP-953 at 10, 30, 60, and 100 μM concentration-dependently increased outflow facility. This effect (89.5%) was maximal at 100 μM (P = 0.002) and in magnitude comparable to DETA-NO at 100 μM (97.5% increase, P = 0.030). Conclusions These data indicate that IWP-953, via modulation of the sGC-cGMP pathway, increases aqueous outflow facility in mouse eyes, suggesting therapeutic potential for sGC stimulators as novel ocular hypotensive drugs.