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Secretogranin III (Scg3) is a diabetic retinopathy (DR)-restricted angiogenic factor identified in preclinical studies as a target for DR therapy. Previously, our group generated and characterized ML49.3, an anti-Scg3 monoclonal antibody (mAb) which we then converted into an EBP2 humanized antibody Fab fragment (hFab) with potential for clinical application. We also generated anti-Scg3 mT4 mAb and related EBP3 hFab. In this study, to identify the preferred hFab for DR therapy, we compared all four antibodies for binding, neutralizing and therapeutic activities in vitro and in vivo. Octet binding kinetics analyses revealed that ML49.3 mAb, EBP2 hFab, mT4 mAb and EBP3 hFab have Scg3-binding affinities of 35, 8.7, 0.859 and 0.116 nM, respectively. Both anti-Scg3 EBP2 and EBP3 hFabs significantly inhibited Scg3-induced proliferation and migration of human umbilical vein endothelial cells in vitro, and alleviated DR vascular leakage and choroidal neovascularization with high efficacy. Paired assays in DR mice revealed that intravitreally injected EBP3 hFab is 26.4% and 10.3% more effective than EBP2 hFab and aflibercept, respectively, for ameliorating DR leakage. In conclusion, this study confirms the markedly improved binding affinities of hFabs compared to mAbs and further identifies EBP3 hFab as the preferred antibody to develop for anti-Scg3 therapy.

Conventional angiogenic factors, such as vascular endothelial growth factor (VEGF), regulate both pathological and physiological angiogenesis indiscriminately, and their inhibitors may elicit adverse side effects. Secretogranin III (Scg3) was recently reported to be a diabetes-restricted VEGF-independent angiogenic factor, but the disease selectivity of Scg3 in retinopathy of prematurity (ROP), a retinal disease in preterm infants with concurrent pathological and physiological angiogenesis, was not defined. Here, using oxygen-induced retinopathy (OIR) mice, a surrogate model of ROP, we quantified an exclusive binding of Scg3 to diseased versus healthy developing neovessels that contrasted sharply with the ubiquitous binding of VEGF. Functional immunohistochemistry visualized Scg3 binding exclusively to disease-related disorganized retinal neovessels and neovascular tufts, whereas VEGF bound to both disorganized and well-organized neovessels. Homozygous deletion of the Scg3 gene showed undetectable effects on physiological retinal neovascularization but markedly reduced the severity of OIR-induced pathological angiogenesis. Furthermore, anti-Scg3 humanized antibody Fab (hFab) inhibited pathological angiogenesis with similar efficacy to anti-VEGF aflibercept. Aflibercept dose-dependently blocked physiological angiogenesis in neonatal retinas, whereas anti-Scg3 hFab was without adverse effects at any dose and supported a therapeutic window at least 10X wider than that of aflibercept. Therefore, Scg3 stringently regulates pathological but not physiological angiogenesis, and anti-Scg3 hFab satisfies essential criteria for development as a safe and effective disease-targeted anti-angiogenic therapy for ROP., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Aims: Our previous study showed that intravitreal delivery of self-complementary AAV2 (scAAV2)-mediated exoenzyme C3 transferase (C3) can attenuate retinal ischemia/reperfusion (I/R) injury. The current study investigated the neuroprotective effects of lentivirus (LV)-mediated C3 transgene expression on rat retinal I/R injury., Main Methods: The LV encoding C3 and green fluorescent protein (GFP) together (LV-C3-GFP) or GFP only (LV-GFP) was intravitreally injected to SPRAGUE-DAWLEY rats. On day 5 post-intravitreal injection, eyes were evaluated by slit-lamp examination. The GFP expression on retina was confirmed by in vivo and ex vivo assessments. RhoA GTPase expression in retina was examined by western blot. Retinal I/R injury was generated by transiently increasing intraocular pressure (110 mmHg, 90 min). Eyes were then enucleated, and retinas processed for morphological analysis and TdT-dUTP terminal nick-end labeling (TUNEL) assay., Key Findings: No obvious inflammatory reactions or surgical complications were observed after intravitreal injection of LV vectors. There was a significant decrease of total RhoA GTPase level in the retina treated with LV-C3-GFP. Compared to the blank control group, LV-C3-GFP and LV-GFP did not affect the retinal thickness, cell density in ganglion cell layer (GCL), or numbers of apoptotic cells in retinal flat-mounts. In the LV-GFP-treated retinas, I/R decreased the retinal thickness and GCL cell density and increased apoptotic retinal cell numbers. LV-C3-GFP significantly protected against all these degenerative effects of I/R., Significance: This study indicated that LV-mediated C3 transgene expression exhibits neuroprotective effects on the retinal I/R injury and holds potential as a novel neuroprotective approach targeting certain retinopathies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Retinal ganglion cell (RGC) death causes irreversible blindness in adult mammals. Death of RGC occurs in diseases including glaucoma or injuries to the optic nerve (ON). To investigate mechanisms involved in RGC degeneration, we evaluated the phosphoproteomic changes in the retina induced by ON injury. Intraorbital optic nerve crush (ONC) was performed in adult C57BL/6J mice. Retinas were collected at 0, 6, and 12 h following ONC. Retinal proteins labeled with CyDye-C2 were subject to 2D-PAGE, followed by phosphoprotein staining and in-gel/cross-gel image analysis. Proteins with significant changes in phosphorylation (ratios ≥1.2) in retinas of the injured eyes compared to the control eyes were spot-picked, tryptic digested, and peptide fragments were analyzed by MALDI-TOF (MS) and TOF/TOF (tandem MS/MS). Intraorbital ONC increased phosphorylation of many retinal proteins. Among them, 29 significantly phosphorylated proteins were identified. PANTHER analysis showed that these proteins are associated with a variety of protein classes, cellular components, biological processes and signaling pathways. One of the identified proteins, phosphoprotein enriched in astrocytes 15 (PEA15), was further validated by western blotting and immunofluorescence staining. Functions of PEA15 were determined in cultured astrocytes. PEA15 knockdown reduced astrocyte phagocytic activity but promoted cell migration. Long term PEA15 knockdown also decreased astrocyte ATP level. This study provides new insights into mechanisms of RGC degeneration after ON injury, as well as central nervous system (CNS) neurodegeneration, since the retina is an extension of the CNS. These new insights will lead to novel therapeutic targets for retinal and CNS neurodegeneration., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Glaucoma is one of the leading causes of vision impairment worldwide. In order to further understand the molecular pathobiology of this disease and to develop better therapies, clinically relevant animal models are necessary. In recent years, both the rat and mouse have become popular models in glaucoma research. Key reasons are: many important biological similarities shared among rodent eyes and the human eye; development of improved methods to induce glaucoma and to evaluate glaucomatous damage; availability of genetic tools in the mouse; as well as the relatively low cost of rodent studies. Commonly studied rat and mouse glaucoma models include intraocular pressure (IOP)-dependent and pressure-independent models. The pressure-dependent models address the most important risk factor of elevated IOP, whereas the pressure-independent models assess "normal tension" glaucoma and other "non-IOP" related factors associated with glaucomatous damage. The current article provides descriptions of these models, their characterizations, specific techniques to induce glaucoma, mechanisms of injury, advantages, and limitations., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Primary open-angle glaucoma (POAG) is a leading cause of irreversible blindness, and individuals with ocular hypertension are at risk to develop POAG. Currently, the only modifiable risk factor for glaucoma progression is lowering of intraocular pressure (IOP). A novel mechanism for lowering IOP involves activation of the type B natriuretic peptide receptor (NPR-B), the naturally occurring agonist of which is C-type natriuretic peptide (CNP). Being a cyclic peptide of 22 amino acids, CNP does not readily penetrate the cornea and its ocular hypotensive effect requires intraocular injection. TAK-639 is a synthetic, cornea-permeable, 9-amino acid CNP analog has been studied for the treatment of ocular hypertension and POAG. We assessed TAK-639 in a receptor binding profile and the effects of TAK-639 on NPR-B-mediated cyclic GMP production in cultured transformed human trabecular meshwork (TM) cells (GTM-3). We also evaluated the effects of topical ocular administration of TAK-639 on mouse IOP and aqueous humor dynamics. Among 89 non-natriuretic peptide receptors, transporters, and channels evaluated, TAK-639 at 10 μM displaced ligand binding by more than 50% to only two receptors: the type 2 angiotensin receptor (IC 50  = 8.2 μM) and the cholecystokinin A receptor (IC 50  = 25.8 μM). In vitro, TAK-639 selectively activates NPR-B (EC 50  = 61 ± 11 nM; GTM-3 cells) relative to NPR-A (EC 50  = 2179 ± 670 nM; 293T cells). In vivo, TAK-639 lowered mouse IOP by three mechanisms: increase in aqueous humor outflow facility (C), reduction in the aqueous humor formation rate (Fin), and reduction in episcleral venous pressure (Pe). The maximum mean IOP decreases from baseline were -12.1%, -21.0%, and -36.1% for 0.1%, 0.3%, and 0.6% doses of TAK-639, respectively. Maximum IOP-lowering effect was seen at 2 h, and the duration of action was >6 h. With TAK-639 0.6%, at 2 h post-dose, aqueous outflow facility (C) increased by 155.8%, Fin decreased by 41.0%, the uveoscleral outflow rate (Fu) decreased by 52.6%, and Pe decreased by 31.5% (all p < 0.05). No ocular adverse effects were observed. TAK-639 is an efficacious IOP-lowering agent, with a unique combination of mechanisms of action on both aqueous formation and aqueous outflow facility. Further study of this mechanism of treatment may optimize pharmacologic outcomes and provide disease management in patients with POAG and ocular hypertension., (Copyright © 2019. Published by Elsevier Ltd.)

Cataract is an eye disease with clouding of the eye lens leading to disrupted vision, which often develops slowly and causes blurriness of the eyesight. Although the restoration of the vision in people with cataract is conducted through surgery, the costs and risks remain an issue. Botanical drugs have been evaluated for their potential efficacies in reducing cataract formation decades ago and major active phytoconstituents were isolated from the plant extracts. The aim of this review is to find effective phytoconstituents in cataract treatments in vitro, ex vivo, and in vivo. A literature search was synthesized from the databases of Pubmed, Science Direct, Google Scholar, Web of Science, and Scopus using different combinations of keywords. Selection of all manuscripts were based on inclusion and exclusion criteria together with analysis of publication year, plant species, isolated phytoconstituents, and evaluated cataract activities. Scientists have focused their attention not only for anti-cataract activity in vitro, but also in ex vivo and in vivo from the review of active phytoconstituents in medicinal plants. In our present review, we identified 58 active phytoconstituents with strong anti-cataract effects at in vitro and ex vivo with lack of in vivo studies. Considering the benefits of anti-cataract activities require critical evaluation, more in vivo and clinical trials need to be conducted to increase our understanding on the possible mechanisms of action and the therapeutic effects.

Purpose: We evaluated the effects of lentivirus-mediated exoenzyme C3 transferase (C3) expression on cultured primary human trabecular meshwork (HTM) cells in vitro, and on rat intraocular pressure (IOP)., Methods: HTM cells were cultured and treated with lentivirus vectors expressing either green fluorescent protein (GFP) only (LV-GFP) or GFP and C3 together (LV-C3-GFP). Changes in cell morphology and actin stress fibers were assessed. The vectors were also injected into the anterior chamber of rats, and GFP expression was visualized by a Micron III Retinal Imaging Microscope in vivo and a fluorescence microscope ex vivo. Changes in rat IOP were monitored by using a rebound tonometer and the eyes were evaluated by slit lamp., Results: LV-mediated C3 expression induced morphologic changes in HTM cells. The cells became retracted and rounded. GFP expression in the anterior chamber angle of rats was observed in vivo from 8 days to 48 days after injection of LV-C3-GFP or LV-GFP. IOP was significantly decreased in the LV-C3-GFP group starting 3 days post injection, and lasting for at least 40 days, when compared to either the contralateral control eyes (the LV-GFP group) or the ipsilateral baseline before injection (P < 0.05). No obvious inflammatory signs were observed in either the LV-C3-GFP or LV-GFP groups., Conclusions: LV-mediated C3 expression induced changes in morphology of cultured HTM cells. Intracameral injection of LV-C3-GFP lowered rat IOP for at least 40 days. No significant inflammatory reactions were observed in either the LV-C3-GFP or LV-GFP groups. This study supports the possible use of C3 gene therapy for the treatment of glaucoma.

As productivity of pharmaceutical research and development (R&D) for small-molecule drugs declines, the trend in drug discovery strategies is shifting towards biologics, which predominantly target secreted or cell surface proteins. Receptors and ligands are the most-valuable drug targets. In contrast to conventional approaches of discovering one ligand at a time, the emerging technology of ligandomics can systematically map disease-selective cellular ligands in the absence of molecular probes. Biologics targeting these ligands with disease selectivity have the advantages of high efficacy, minimal adverse effects, wide therapeutic indices, and low safety-related attrition rates. Therefore, ligandomics represents a paradigm shift to address the bottleneck of target discovery for biologics development., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Mice and rats are being increasingly used in glaucoma research and much useful data have been generated from them. One aspect of using these animals for this purpose involves assessment of aqueous humor dynamics. Several techniques have been described in the literature for the determination of one or more of these parameters in rodents, in both living animals and eyes perfused ex vivo. Here, we describe the practical details for a technique for the determination of all principal parameters of aqueous humor dynamics (intraocular pressure (IOP), aqueous humor formation rate (Fin), uveoscleral outflow rate (Fu), aqueous outflow facility (C), and episcleral venous pressure (Pe)) in the living rat and mouse eye, in a single experimental session.

Oxidative injuries, such as those related to reactive oxygen species (ROS), have been implicated in various retinal and optic nerve disorders. Many ROS detection methods have been developed. Although widely utilized, many of these methods are useful only in post mortem tissues, or require relatively expensive equipment, or involve intraocular injection. In the present study, we demonstrated and characterized a chemiluminescent probe L-012 as a noninvasive, in vivo ROS detection agent in the mouse retina. Using optic nerve crush (ONC) and retinal ischemia/reperfusion (I/R) as injury models, we show that L-012 produced intensive luminescent signals specifically in the injured eyes. Histological examination showed that L-012 administration was safe to the retina. Additionally, compounds that reduce tissue superoxide levels, apocynin and TEMPOL, decreased injury-induced L-012 chemiluminescence. The decrease in L-012 signals correlated with their protective effects against retinal I/R-induced morphological and functional changes in the retina. Together, these data demonstrate the feasibility of a fast, simple, reproducible, and non-invasive detection method to monitor in vivo ROS in the retina. Furthermore, the results also show that reduction of ROS is a potential therapeutic approach for protection from these retinal injuries., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The human body harbors within it an intricate and delicate balance between oxidants and antioxidants. Any disruption in this checks-and-balances system can lead to harmful consequences in various organs and tissues, such as the eye. This review focuses on the effects of oxidative stress and the role of a particular antioxidant system-the Keap1-Nrf2-ARE pathway-on ocular diseases, specifically age-related macular degeneration, cataracts, diabetic retinopathy, and glaucoma. Together, they are the major causes of blindness in the world.

Oxidative stress-induced retinal pigment epithelial (RPE) cell damage is an important factor in the pathogenesis of age-related macular degeneration (AMD). Previous studies have shown that RTA 408, a synthetic triterpenoid compound, potently activates Nrf2. This study aimed to investigate the protective effects of RTA 408 in cultured RPE cells during oxidative stress and to determine the effects of RTA 408 on Nrf2 and its downstream target genes. Primary human RPE cells were pretreated with RTA 408 and then incubated in 200μM H2O2 for 6h. Cell viability was measured with the WST-8 assay. Apoptosis was quantitatively measured by annexin V/propidium iodide (PI) double staining and Hoechst 33342 fluorescent staining. Reduced (GSH) and oxidized glutathione (GSSG) were measured using colorimetric assays. Nrf2 activation and its downstream effects on phase II enzymes were examined by Western blot. Treatment of RPE cells with nanomolar ranges (10 and 100nM) of RTA 408 markedly attenuated H2O2-induced viability loss and apoptosis. RTA 408 pretreatment significantly protected cells from oxidative stress-induced GSH loss, GSSG formation and decreased ROS production. RTA 408 activated Nrf2 and increased the expression of its downstream genes, such as HO-1, NQO1, SOD2, catalase, Grx1, and Trx1. Consequently, the enzyme activities of NQO1, Grx1, and Trx1 were fully protected by RTA 408 pretreatment under oxidative stress. Moreover, knockdown of Nrf2 by siRNA significantly reduced the cytoprotective effects of RTA 408. In conclusion, our data suggest that RTA 408 protect primary human RPE cells from oxidative stress-induced damage by activating Nrf2 and its downstream genes., (Published by Elsevier B.V.)

Glaucoma is a major cause of blindness in China and the world. Currently, all therapeutic means in treating open-angle glaucoma are limited to control the progression of optic neuropathy by lowering intraocular pressure (IOP). Clinically available medicines lower IOP by either enhancing the uveoscleral pathway or inhibiting aqueous humor production. Since the primary cause of IOP elevation in POAG is elevated outflow resistance in the trabecular outflow pathway, current medicines are not able to correct the underlying pathogenesis and pathophysiology of the disease. In this review article, we discuss a series of new therapeutic targets and therapeutic approaches that are designed to directly modify the pathological changes related to the reduction in trabecular outflow in glaucoma patients. Some of these targets and approaches may produce a significant breakthrough in the treatment of this devastating disease. (Chin J Ophthalmol, 2016, 52: 471-475).

Background: The c-Jun N-terminal kinase (JNK) signaling pathway plays an important role in neuronal pathophysiology. Using JNK inhibitors, we examined involvement of the JNK pathway in cultured rat retinal ganglion cell (RGC) death and in mouse retinal ischemia/reperfusion (I/R) injury of the visual axis. The in vitro effects of JNK inhibitors were evaluated in cultured adult rat retinal cells enriched in RGCs. Retinal I/R was induced in C57BL/6J mice through elevation of intraocular pressure to 120 mmHg for 60 min followed by reperfusion. SP600125 was administered intraperitoneally once daily for 28 days. Phosphorylation of JNK and c-Jun in the retina was examined by immunoblotting and immunohistochemistry. The thickness of retinal layers and cell numbers in the ganglion cell layer (GCL) were examined using H&E stained retinal cross sections and spectral domain optical coherence tomography (SD-OCT). Retinal function was measured by scotopic flash electroretinography (ERG). Volumetric measurement of the superior colliculus (SC) as well as VGLUT2 and PSD95 expression were studied., Results: JNK inhibitors SP600125 and TAT-JNK-III, dose-dependently and significantly (p < 0.05) protected against glutamate excitotoxicity and trophic factor withdrawal induced RGC death in culture. In the I/R model, phosphorylation of JNK (pJNK) in the retina was significantly (p < 0.05) increased after injury. I/R injury significantly (p < 0.05) decreased the thickness of retinal layers, including the whole retina, inner plexiform layer, and inner nuclear layer and cell numbers in the GCL. Administration of SP600125 for 28 days protected against all these degenerative morphological changes (p < 0.05). In addition, SP600125 significantly (p < 0.05) protected against I/R-induced reduction in scotopic ERG b-wave amplitude at 3, 7, 14, 21 and 28 days after injury. SP600125 also protected against the I/R-induced losses in volume and levels of synaptic markers in the SC. Moreover, the protective effects of SP600125 in the retina and SC were also detected even with only 7 days (Days 1-7 after I/R) of SP600125 treatment., Conclusions: Our results demonstrate the important role the JNK pathway plays in retinal degeneration in both in vitro and in vivo models and suggest that JNK inhibitors may be a useful therapeutic strategy for neuroprotection of RGCs in the retina.

Glaucoma is a leading cause of blindness, which is treatable but currently incurable. Numerous animal models therefore have both been and continue to be utilized in the study of numerous aspects of this condition. One important facet associated with the use of such models is the ability to accurately and reproducibly measure (by cannulation) or estimate (by tonometry) intraocular pressure (IOP). At this juncture there are several different approaches to IOP measurement in different experimental animal species, and the list continues to grow. We feel therefore that a review of this subject matter is timely and should prove useful to others who wish to perform similar measurements. The general principles underlying various types of tonometric and non-tonometric techniques for non-continuous determination of IOP are considered. There follows discussion of specific details as to how these techniques are applied to experimental animal species involved in the research of this disease. Specific comments regarding anesthesia, circadian rhythm, and animal handling are also included, especially in the case of rodents. Brief consideration is also given to possible future developments., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Rodents are increasingly being used as glaucoma models to study ocular hypertension, optic neuropathy, and retinopathy. A number of different techniques are used to elevate intraocular pressure in rodent eyes by artificially obstructing the aqueous outflow pathway. Another successful technique to induce ocular hypertension is to transduce the trabecular meshwork of rodent eyes with viral vectors expressing glaucoma associated transgenes to provide more relevant models of glaucomatous damage to the trabecular meshwork. This technique has been used to validate newly discovered glaucoma pathogenesis pathways as well as to develop rodent models of primary open angle glaucoma. Ocular hypertension has successfully been induced by adenovirus 5 mediated delivery of mutant MYOC, bioactivated TGFβ2, SFRP1, DKK1, GREM1, and CD44. Advantages of this approach are: selective tropism for the trabecular meshwork, the ability to use numerous mouse strains, and the relatively rapid onset of IOP elevation. Disadvantages include mild-to-moderate ocular inflammation induced by the Ad5 vector and sometimes transient transgene expression. Current efforts are focused at discovering less immunogenic viral vectors that have tropism for the trabecular meshwork and drive sufficient transgene expression to induce ocular hypertension. This viral vector approach allows rapid proof of concept studies to study glaucomatous damage to the trabecular meshwork without the expensive and time-consuming generation of transgenic mouse lines., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Osteogenesis imperfecta (OI) is a genetically heterogeneous group of disorders, characterized by abnormal bone fragility, blue sclera, deafness, joint laxity, and soft-tissue dysplasia. The purpose of this study was to elucidate the genetic or molecular basis for OI type IA in a Chinese family. We evaluated the members of a family, in which six individuals are affected with increased bone fragility and blue sclera. Results of exome sequencing revealed a novel 1-bp deletion (c.2329delG, p.A777fs) in exon 33 of the COL1A1 gene in two affected individuals, but not in a control family member without OI. The variation co-segregated with the disease in all the OI patients but not in the unaffected family members. The mutation caused a frameshift alteration after codon 777, leading to premature termination of the COL1A1 protein. Thus, our findings identified a novel frameshift deletion c.2329delG (p.A777fs) in the COL1A1 gene, which is associated with OI type IA in a Chinese family.

Purpose: We evaluated differences in aqueous humor dynamics (AHD) among several mouse strains within younger and older age groups., Methods: Albino (A/J, BALB/cJ) and pigmented (C3H/HeJ, C57-BL/6J) mice (young [2½-4½ months] and aged [10-12 months]) were studied. Intraocular pressure (IOP) was measured. In cannulated eyes, episcleral venous pressure (Pe) was assessed (blood reflux). Other AHD parameters (outflow facility [C], aqueous humor formation rate [Fin]) were assessed (constant flow infusion). Uveoscleral outflow rate (Fu) was obtained by calculation (Fu(calc)) using the modified Goldmann equation, and in additional eyes (for comparison), by FITC-dextran perfusion (Fu(FITC-dex))., Results: Intraocular pressure was higher in pigmented strains, but did not exhibit age-dependence, except in the C57-BL/6J strain. Fu(calc) decreased with age in BALB/cJ (↓83.3%), C3H/HeJ (↓78.0%), and C57-BL/6J (↓85.0%) strains. In the A/J strain, Fu(calc) decreased with age (↓70.0%), but not significantly. Fin decreased with age in the C3H/HeJ (↓53.6%) strain. In C57-BL/6J and A/J strains, Fin decreased with age, but not significantly. C in the BALB/cJ strain increased with age (↑62.5%). In C3H/HeJ and C57-BL/6J strains, C increased with age, but not significantly. Episcleral venous pressure ranged from 6.0 to 6.6 mm Hg (albino strains) to 8.5 to 8.9 mm Hg (pigmented strains). Pe was not age dependent, but was higher in pigmented animals., Conclusions: In mouse, Fu and Fin diminish with age. C tends to increase as animals progress to middle life. There are strain differences in Fu, IOP, C, Fin, and Pe. The current findings provide an important foundation for comparisons among different strains in different study reports.

Background: Axonal injury of the optic nerve (ON) is involved in various ocular diseases, such as glaucoma and traumatic optic neuropathy, which leads to apoptotic death of retinal ganglion cells (RGCs) and loss of vision. Caspases have been implicated in RGC pathogenesis. However, the role of caspase-7, a functionally unique caspase, in ON injury and RGC apoptosis has not been reported previously. The purpose of this study is to evaluate the role of caspase-7 in ON injury-induced RGC apoptosis., Results: C57BL/6 (wildtype, WT) and caspase-7 knockout (Casp7(-/-)) mice were used. We show that ON crush activated caspase-7 and calpain-1, an upstream activator of caspase-7, in mouse RGCs, as well as hydrolysis of kinectin and co-chaperone P23, specific substrates of caspase-7. ON crush caused a progressive loss of RGCs to 28 days after injury. Knockout of caspase-7 partially and significantly protected against the ON injury-induced RGC loss; RGC density at 28 days post ON crush in Casp7(-/-) mice was approximately twice of that in WT ON injured retinas. Consistent with changes in RGC counts, spectral-domain optical coherence tomography analysis revealed that ON crush significantly reduced the in vivo thickness of the ganglion cell complex layer (including ganglion cell layer, nerve fiber layer, and inner plexiform layer) in the retina. The ON crush-induced thinning of retinal layer was significantly ameliorated in Casp7(-/-) mice when compared to WT mice. Moreover, electroretinography analysis demonstrated a decline in the positive component of scotopic threshold response amplitude in ON crushed eyes of the WT mice, whereas this RGC functional response was significantly higher in Casp7(-/-) mice at 28 days post injury., Conclusion: Altogether, our findings indicate that caspase-7 plays a critical role in ON injury-induced RGC death, and inhibition of caspase-7 activity may be a novel therapeutic strategy for glaucoma and other neurodegenerative diseases of the retina.

Social media are found to facilitate social information exchange among lesbian, gay, and bisexual (LGB) individuals who are subjected to social stigma. This study tested the protective role of LGB-tailored social media uses and gratifications in promoting LGB group membership, which we hypothesized to reduce LGB stigma and enhance mental health among LGB individuals in Hong Kong. Based on a sample of 233 Chinese LGB individuals in Hong Kong, structural equation modeling showed evidence for our hypotheses, χ(df=62)(2)= 88.20, GFI = 0.95, CFI = 0.98, NNFI = 0.98, SRMR = 0.07, RMSEA = 0.04. Community surveillance, identity expression, and emotional support on social media may promote mental health by instilling a sense of group membership and reducing stigma. Social media may build camaraderie and bolster resilience among LGB individuals that may otherwise be difficult in conservative regions.

Neuritin 1 (Nrn1) is an extracellular glycophosphatidylinositol-linked protein that stimulates axonal plasticity, dendritic arborization and synapse maturation in the central nervous system (CNS). The purpose of this study was to evaluate the neuroprotective and axogenic properties of Nrn1 on axotomized retinal ganglion cells (RGCs) in vitro and on the in vivo optic nerve crush (ONC) mouse model. Axotomized cultured RGCs treated with recombinant hNRN1 significantly increased survival of RGCs by 21% (n=6-7, P<0.01) and neurite outgrowth in RGCs by 141% compared to controls (n=15, P<0.05). RGC transduction with AAV2-CAG-hNRN1 prior to ONC promoted RGC survival (450%, n=3-7, P<0.05) and significantly preserved RGC function by 70% until 28 days post crush (dpc) (n=6, P<0.05) compared with the control AAV2-CAG-green fluorescent protein transduction group. Significantly elevated levels of RGC marker, RNA binding protein with multiple splicing (Rbpms; 73%, n=5-8, P<0.001) and growth cone marker, growth-associated protein 43 (Gap43; 36%, n=3, P<0.01) were observed 28 dpc in the retinas of the treatment group compared with the control group. Significant increase in Gap43 (100%, n=5-6, P<0.05) expression was observed within the optic nerves of the AAV2-hNRN1 group compared to controls. In conclusion, Nrn1 exhibited neuroprotective, regenerative effects and preserved RGC function on axotomized RGCs in vitro and after axonal injury in vivo. Nrn1 is a potential therapeutic target for CNS neurodegenerative diseases.

Purpose: To investigate the role of C/EBP homologous protein (CHOP), a proapoptotic protein, and the unfolded protein response (UPR) marker that is involved in endoplasmic reticulum (ER) stress-mediated apoptosis in mouse retinal ganglion cell (RGC) death following ischemia/reperfusion (I/R) injury., Methods: Retinal I/R injury was induced in adult C57BL/6J wild-type (WT) and CHOP knockout (Chop(-/-)) mice by raising IOP to 120 mm Hg for 60 minutes. Expression of CHOP and other UPR markers was studied by Western blot and immunohistochemistry. Retinal ganglion cell counts were performed in retinal flat mounts stained with an RGC marker. Retinal ganglion cell function was evaluated by scotopic threshold response (STR) electroretinography., Results: In WT mice, retinal CHOP was upregulated by 30% in I/R-injured eyes compared to uninjured eyes 3 days after injury (P < 0.05). Immunohistochemistry confirmed CHOP upregulation specifically in RGCs. CHOP knockout did not affect baseline RGC density or STR amplitude. Ischemia/reperfusion injury decreased RGC densities and STR amplitudes in both WT and Chop(-/-) mice. However, survival of RGCs in I/R-injured Chop(-/-) mouse was 48% higher (P < 0.05) than that in I/R-injured WT mouse 3 days after I/R injury. Similarly, RGC density was significantly higher in Chop(-/-) eyes at 7, 14, and 28 days after I/R injury. Scotopic threshold response amplitudes of Chop(-/-) mice were significantly higher at 3 and 7 days after I/R than those of WT mice., Conclusions: Absence of CHOP partially protects against RGC loss and reduction in retinal function after I/R injury, indicating that CHOP and, thus, ER stress play an important role in RGC apoptosis in retinal I/R injury., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: Transforming growth factor-β2 (TGF-β2) has been implicated in the pathogenesis of primary open-angle glaucoma through extracellular matrix (ECM) alteration among various mechanisms. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that regulates ECM within the trabecular meshwork (TM), and is highly upregulated by TGF-β2. We hypothesized that, in vivo, SPARC is a critical regulatory node in TGF-β2-mediated ocular hypertension., Methods: Empty (Ad.empty) or TGF-β2-containing adenovirus (Ad.TGF-β2) was injected intravitreally into C57BL6-SV129 WT and SPARC-null mice. An initial study was performed to identify a stable period for IOP measurement under isoflurane. The IOP was measured before injection and every other day for two weeks using rebound tonometry. Additional mice were euthanized at peak IOP for immunohistochemistry., Results: The IOP was stable under isoflurane during minutes 5 to 8. The IOP was significantly elevated in Ad.TGF-β2-injected (n = 8) versus Ad.empty-injected WT (n = 8) mice and contralateral uninjected eyes during days 4 to 11 (P < 0.03). The IOPs were not significantly elevated in Ad.TGF-β2-injected versus Ad.empty-injected SPARC-null mice. However, on day 8, the IOP of Ad.TGF-β2-injected SPARC-null eyes was elevated compared to that of contralateral uninjected eyes (P = 0.0385). Immunohistochemistry demonstrated that TGF-β2 stimulated increases in collagen IV, fibronectin, plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), and SPARC in WT mice, but only PAI-1 and CTGF in SPARC-null mice (P < 0.05)., Conclusions: SPARC is essential to the regulation of TGF-β2-mediated ocular hypertension. Deletion of SPARC significantly attenuates the effects of TGF-β2 by restricting collagen IV and fibronectin expression. These data provide further evidence that SPARC may have an important role in IOP regulation and possibly glaucoma pathogenesis., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

A series of 2,3,6-pyrazine Rho Kinase inhibitors were optimized for in vivo activity for topical ocular dosing. Modifications of the 2-(piperazin-1-yl)pyrazine derivatives produced compounds with improved solubility and physicochemical properties. Modifications of the 6-pyrazine substituent led to improvements in in vitro potency. Compound 9 had the best in vitro and in vivo potency of EC50=260 nM with a 30% reduction of IOP in a non-human primate model at a dose of 0.33%., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: CD44 plays major roles in multiple physiologic processes. The ectodomain concentration of the CD44 receptor, soluble CD44 (sCD44), is significantly increased in the aqueous humor of primary open-angle glaucoma (POAG). The purpose of this study was to determine if adenoviral constructs of CD44 and isolated 32-kDa sCD44 change intraocular pressure (IOP) in vivo and aqueous outflow resistance in vitro., Methods: Adenoviral constructs of human standard CD44 (Ad-CD44S), soluble CD44 (Ad-sCD44), and empty viral cDNA were injected into the vitreous of BALB/cJ mice, followed by serial IOP measurements. Overexpression of CD44S and sCD44 was verified in vitro by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Anterior segments of porcine eyes were perfused with the isolated sCD44. sCD44-treated human trabecular meshwork (TM) cells and microdissected porcine TM were examined by confocal microscopy and Optiprep density gradient with western blot analysis to determine changes in lipid raft components., Results: Intravitreous injection of adenoviral constructs with either Ad-CD44S or Ad-sCD44 vectors caused prolonged ocular hypertension in mice. Eight days after vector injection, Ad-CD44S significantly elevated IOP to 28.3±1.2 mmHg (mean±SEM, n=8; p<0.001); Ad-sCD44 increased IOP to 18.5±2.6 mmHg (n=8; p<0.01), whereas the IOP of uninjected eyes was 12.7±0.2 mmHg (n=16). The IOP elevation lasted more than 50 days. Topical administration of a γ-secretase inhibitor normalized Ad-sCD44-induced elevated IOP. sCD44 levels were significantly elevated in the aqueous humor of Ad-CD44S and Ad-sCD44 eyes versus contralateral uninjected eyes (p<0.01). Anterior segment perfusion of isolated 32-kDa sCD44 significantly decreased aqueous outflow rates. Co-administration of isolated sCD44 and CD44 neutralizing antibody or of γ-secretase inhibitor significantly enhanced flow rates. sCD44-treated human TM cells displayed cross-linked actin network formation. Optiprep density gradient and western blot analysis of human TM cells treated with sCD44 showed decreased annexin 2 expression and increased phosphorylated annexin 2 and caveolin 1 expression., Conclusions: Our data suggest that sCD44 increases outflow resistance in vivo and in vitro. Viral overexpression of both CD44S and sCD44 is sufficient to cause ocular hypertension. Infusion of sCD44 in porcine anterior segment eyes significantly decreased flow rates. Notably, sCD44 enhanced cross-linked actin network formation. The elevated sCD44 levels seen in POAG aqueous humor may play an important causative role in POAG pathogenesis.

The p38 mitogen-activated protein kinase (MAPK) isoforms are phosphorylated by a variety of stress stimuli in neurodegenerative disease and act as upstream activators of myriad pathogenic processes. Thus, p38 MAPK inhibitors are of growing interest as possible therapeutic interventions. Axonal dysfunction is an early component of most neurodegenerative disorders, including the most prevalent optic neuropathy, glaucoma. Sensitivity to intraocular pressure at an early stage disrupts anterograde transport along retinal ganglion cell (RGC) axons to projection targets in the brain with subsequent degeneration of the axons themselves; RGC body loss is much later. Here we show that elevated ocular pressure in rats increases p38 MAPK activation in retina, especially in RGC bodies. Topical eye-drop application of a potent and selective inhibitor of the p38 MAPK catalytic domain (Ro3206145) prevented both the degradation of anterograde transport to the brain and degeneration of axons in the optic nerve. Ro3206145 reduced in the retina phosphorylation of tau and heat-shock protein 27, both down-stream targets of p38 MAPK activation implicated in glaucoma, as well as expression of two inflammatory responses. We also observed increased p38 MAPK activation in mouse models. Thus, inhibition of p38 MAPK signaling in the retina may represent a therapeutic target for preventing early pathogenesis in optic neuropathies., (© 2013.)

Glaucoma, a disease of the optic nerve and retina, causes blindness in millions of people worldwide. Currently available therapies for this disease only attempt to reduce intraocular pressure, the major risk factor, without addressing the associated optic neuropathy and retinopathy. Development of glaucoma neuroprotective treatment is therefore a pressing unmet medical need. Unfortunately, many challenges hinder this effort, including an incomplete understanding of the mechanism of pathogenesis, leading to uncertain therapeutic targets and confounded by not yet validated preclinical models. Most importantly, with slow disease progression and a less than ideal endpoint measurement method, clinical trials are necessarily large, lengthy, expensive and, to many, prohibitive. No easy solution is available to overcome these challenges. Increased commitment to basic mechanistic research is an essential foundation for dealing with this problem. Innovations in clinical trials with novel surrogate endpoints, nontraditional study designs and the use of surrogate diseases might shorten the study time, reduce the patient sample size and consequently lower the budgetary hurdle for the development of new therapies.

The normal gene expression profiles of the tissues in the eye are a valuable resource for considering genes likely to be involved with disease processes. We profiled gene expression in ten ocular tissues from human donor eyes using Affymetrix Human Exon 1.0 ST arrays. Ten different tissues were obtained from six different individuals and RNA was pooled. The tissues included: retina, optic nerve head (ONH), optic nerve (ON), ciliary body (CB), trabecular meshwork (TM), sclera, lens, cornea, choroid/retinal pigment epithelium (RPE) and iris. Expression values were compared with publically available Expressed Sequence Tag (EST) and RNA-sequencing resources. Known tissue-specific genes were examined and they demonstrated correspondence of expression with the representative ocular tissues. The estimated gene and exon level abundances are available online at the Ocular Tissue Database., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Glaucoma and age-related macular degeneration are ocular diseases targeted clinically by anecortave acetate (AA). AA and its deacetylated metabolite, anecortave desacetate (AdesA), are intraocular pressure (IOP)-lowering and angiostatic cortisenes devoid of glucocorticoid activity but with an unknown mechanism of action. We used a methotrexate-anchored yeast three-hybrid (Y3H) technology to search for binding targets for AA in human trabecular meshwork (TM) cells, the target cell type that controls IOP, a major risk factor in glaucoma. Y3H hits were filtered by competitive Y3H screens and coimmunoprecipitation experiments and verified by surface plasmon resonance analysis to yield a single target, phosphodiesterase 6-delta (PDE6D). PDE6D is a prenyl-binding protein with additional function outside the PDE6 phototransduction system. Overexpression of PDE6D in mouse eyes caused elevated IOP, and this elevation was reversed by topical ocular application of either AA or AdesA. The identification of PDE6D as the molecular binding partner of AA provides insight into the role of this drug candidate in treating glaucoma.

Purpose: We previously discovered elevated levels of secreted frizzled-related protein 1 (sFRP1), the Wnt signaling pathway inhibitor, in the glaucomatous trabecular meshwork (GTM), and found that key canonical Wnt signaling pathway genes are expressed in the trabecular meshwork (TM). The purpose of our study was to determine whether a functional canonical Wnt signaling pathway exists in the human TM (HTM)., Methods: Western immunoblotting and/or immunofluorescent microscopy were used to study β-catenin translocation as well as the actin cytoskeleton in transformed and primary HTM cells. A TCF/LEF luciferase assay was used to study functional canonical Wnt signaling, which was confirmed further by WNT3a-induced expression of a pathway target gene, AXIN2, via quantitative PCR. Intravitreal injection of an Ad5 adenovirus expressing Dickkopf-related protein-1 (DKK1) was used to study the in vivo effect of canonical Wnt signaling on IOP in mice., Results: WNT3a induced β-catenin translocation in the HTM, which was blocked by co-treatment with sFRP1. Similarly, WNT3a enhanced luciferase levels in TCF/LEF luciferase assays, which also were blocked by sFRP1. Furthermore, AXIN2 expression was elevated significantly by WNT3a. However, neither WNT3a nor sFRP1 affected actin cytoskeleton organization, which theoretically could be regulated by noncanonical Wnt signaling in HTM cells. Exogenous DKK1, a specific inhibitor for the canonical Wnt signaling pathway, or sFRP1 elevated mouse IOP to equivalent levels., Conclusions: There is a canonical Wnt signaling pathway in the TM, and this canonical Wnt pathway, but not the noncanonical Wnt signaling pathway, regulates IOP.

Elevated intraocular pressure (IOP) is a causative risk factor for the development and progression of glaucoma. Glaucomatous mutations in myocilin (MYOC) damage the trabecular meshwork and elevate IOP in humans and in mice. Animal models of glaucoma are important to discover and better understand molecular pathogenic pathways and to test new glaucoma therapeutics. Although a number of different animal models of glaucoma have been developed and characterized, there are no true models of human primary open angle glaucoma (POAG). The overall goal of this work is to develop the first inducible mouse model of POAG using a human POAG relevant transgene (i.e. mutant MYOC) expression in mouse eyes to elevate IOP and cause pressure-induced damage to the optic nerve. Four mouse strains (A/J, BALB/cJ, C57BL/6J, and C3H/HeJ) were used in this study. Ad5.MYOC.Y437H (5 × 10(7) pfu) was injected intravitreally into one eye, with the uninjected contralateral eye serving as the control eye. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Optic nerve damage was determined by scoring PPD stained optic nerve cross sections. Retinal ganglion cell and superior colliculus damage was assessed by Nissl stain cell counts. Intravitreal administration of viral vector Ad5.MYOC.Y437H caused a prolonged, reproducible, and statistically significant IOP elevation in BALB/cJ, A/J, and C57BL/6J mice. IOPs increased to approximately 25 mm Hg for 8 weeks (p < 0.0001). In contrast, the C3H/HeJ mouse strain was resistant to Ad5.MYOC.Y437H induced IOP elevation for the 8-week time period. IOPs were stable (12-15 mm Hg) in the uninjected control eyes. We also determined whether there were any strain differences in pressure-induced optic nerve damage. Even though IOP was similarly elevated in three of the strains tested (BALB/cJ, C57BL/6J, and A/J) only the A/J strain had considerable and significant optic nerve damage at the end of 8 weeks with optic nerve damage score of 2.64 ± 0.19 (n = 18, p < 0.001) in the injected eye. There was no statistical difference in retinal ganglion cell death or superior colliculus damage at the 8-week time point in any of the strains tested. These results demonstrate strain dependent responses to Ad5.MYOC.Y437H-induced ocular hypertension and pressure-induced optic nerve damage., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose: To characterize the microbead-induced ocular hypertension (OHT) mouse model and investigate its potential use for preclinical screening and evaluation of ocular hypotensive agents, we tested the model's responses to major antiglaucoma drugs., Methods: Adult C57BL/6J mice were induced to develop OHT unilaterally by intracameral injection of microbeads. The effects of the most commonly used ocular hypotensive drugs, including timolol, brimonidine, brinzolamide, pilocarpine, and latanoprost, on IOP and glaucomatous neural damage were evaluated. Degeneration of retinal ganglion cells (RGCs) and optic nerve axons were quantitatively assessed using immunofluorescence labeling and histochemistry. Thickness of the ganglion cell complex (GCC) was also assessed with spectral-domain optical coherence tomography (SD-OCT)., Results: A microbead-induced OHT model promptly responded to drugs, such as timolol, brimonidine, and brinzolamide, that lower IOP through suppressing aqueous humor production and showed improved RGC and axon survival as compared to vehicle controls. Accordingly, SD-OCT detected significantly less reduction of GCC thickness in mice treated with all three aqueous production suppressants as compared to the vehicle contol-treated group. In contrast, drugs that increase aqueous outflow, such as pilocarpine and latanoprost, failed to decrease IOP in the microbead-induced OHT mice., Conclusions: Microbead-induced OHT mice carry dysfunctional aqueous outflow facility and therefore offer a unique model that allows selective screening of aqueous production suppressant antiglaucoma drugs or for studying the mechanisms regulating aqueous humor production. Our data set the stage for using GCC thickness assessed by SD-OCT as an imaging biomarker for noninvasive tracking of neuronal benefits of glaucoma therapy in this model.

Glaucoma is a neurodegenerative disease characterized by the apoptotic death of retinal ganglion cells (RGCs). The primary insult to RGCs in glaucoma is thought to occur to their axons as they exit the eye in the optic nerve head. However, pathological signaling pathways that exert central roles in triggering RGC death following axonal injury remain unidentified. It is likely that the first changes to occur following axonal injury are signal relay events that transduce the injury signal from the axon to the cell body. Here we focus on the c-Jun N-terminal kinase (JNK1-3) family, a signaling pathway implicated in axonal injury signaling and neurodegenerative apoptosis, and likely to function as a central node in axonal injury-induced RGC death. We show that JNK signaling is activated immediately after axonal injury in RGC axons at the site of injury. Following its early activation, sustained JNK signaling is observed in axonally-injured RGCs in the form of JUN phosphorylation and upregulation. Using mice lacking specific Jnk isoforms, we show that Jnk2 and Jnk3 are the isoforms activated in injured axons. Combined deficiency of Jnk2 and Jnk3 provides robust long-term protection against axonal injury-induced RGC death and prevents downregulation of the RGC marker, BRN3B, and phosphorylation of JUN. Finally, using Jun deficient mice, we show that JUN-dependent pathways are important for axonal injury-induced RGC death. Together these data demonstrate that JNK signaling is the major early pathway triggering RGC death after axonal injury and may directly link axon injury to transcriptional activity that controls RGC death., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: To correlate retinal ganglion cell (RGC) loss and optic nerve (ON) damage with the duration of acute glaucoma attacks in a rat experimental model and to determine whether the c-Jun N-terminal kinase (JNK) inhibitor SP600125 protects against such attacks., Methods: To model an acute glaucoma attack, rat intraocular pressure (IOP) was elevated by a controllable compression method using pulleys and specific weights. Intraocular pressure was measured with a TonoLab® rebound tonometer. Time-dependent ocular hypertension-induced damage was evaluated by ON morphology, retina morphology (both retina layer thickness in cross-sections and RGC counts in Dextran tetramethylrhodamine crystals [DTMR] labeled flatmounts), and scotopic flash electroretinography (ERG). A c-Jun N-terminal kinase (JNK) inhibitor, SP600125 (0, 1.5, 5, or 15 mg/kg), was administered by intraperitoneal injection immediately before and after induction of ocular hypertension, then once daily for seven days. Retinal cross-sections were measured to determine the thickness of various retinal layers and the cell density in the ganglion cell layer (GCL). Retinal flatmounts immunolabeled with anti-rat Brn-3a primary antibody were used to quantify RGC numbers., Results: Elevated rat IOP induced by corneal limbus compression correlated with the different weights. Elevation to 45 mmHg for up to 7 h did not significantly affect the thicknesses of the outer nuclear layer, outer plexiform layer, or inner nuclear layer. Amplitudes of A- and B-waves were not affected. However, elevation to 45 mmHg for up to 7 h decreased the inner retinal thickness and caused ON damage. Most importantly, IOP elevation induced a time-dependent RGC loss. Cell density in the GCL decreased to 70%, 62%, and 49% of that of the control after 5 h, 6 h, and 7 h, respectively, of pressure increases. In retinal flatmount studies, labeled RGCs were reduced 56±4% (mean±SEM) versus the control (p<0.001) after 7 h of ocular hypertension. SP600125 dose-dependently protected against ocular hypertension-induced RGC loss. The difference in RGC density between the vehicle and SP600125-treated (15 mg/kg) groups was statistically significant (p<0.001)., Conclusions: The correlation of inner retinal morphological changes with the duration of the application of 45 mmHg IOP was demonstrated. Treatment with SP600125 significantly protected RGC survival against this insult. Inhibitors of JNK may be an interesting pharmacological class for treating glaucoma.

Purpose: To characterize a technique that concurrently assesses all aqueous humor hydrodynamic parameters in mouse eyes., Methods: Mouse outflow facility (C) was determined by multiple flow-rate infusion and episcleral venous pressure (Pe) measured by manometry. The animals were then euthanatized, eliminating aqueous formation rate (Fin) and Pe. C was determined again (C(dead)) while uveoscleral outflow (Fu(dead)) and Fin were deduced. To assess whether Fu(dead) would remain the same as Fu(live), the animals were perfused with FITC-dextran and Fu determined. The effects of IOP-lowering drugs on the parameters of aqueous hydrodynamics were also evaluated., Results: Under the conditions tested, Fu(live) (0.012 ± 0.003 μL/min) was not different from Fu(dead) (0.015 ± 0.003 μL/min; P = 0.472). In anesthetized mice, IOP = 11.4 ± 0.2 mm Hg (mean ± SEM, n = 8), C = 0.018 ± 0.0006 μL/min/mm Hg, Pe = 5.4 ± 0.2 mm Hg, Fin = 0.14 ± 0.0007 μL/min, and Fu = 0.029 ± 0.005 μL/min. C(dead) was not different from C (P = 0.317). Latanoprost reduced IOP by increasing C by 0.009 ± 0.0003 μL/min/mm Hg (P < 0.001), without affecting Fin or Fu. Betaxolol reduced Fin by 0.075 ± 0.021 μL/min (P = 0.009). Brimonidine increased C by 0.005 ± 0.0005 μL/min/mm Hg (P < 0.001) and Fu by 0.013 ± 0.003 μL/min (P = 0.007)., Conclusions: In this study, a unique technique was developed to concurrently assess IOP, C, Pe, Fin, and Fu in the mouse eye. This experimental approach should be useful to evaluate effects of pharmacologic agents or genetic manipulations on aqueous humor dynamics in mice and other animal models.

Purpose: To characterize the effects of circadian rhythm, feeding time, age, general anesthesia, and ocular hypotensive compounds on intraocular pressure (IOP) of the Tibetan monkey (Macaca thibetana)., Methods: Tibetan monkeys were trained for IOP measurement with the TonoVet® rebound tonometer without sedation or anesthesia. Their circadian IOP fluctuation was monitored every 3 h. Effects of changing the feeding time, general anesthesia, age (2-3 year-old versus 8-15 year-old animals), and various pharmacological agents, such as travoprost, timolol, naphazoline and spiradoline, on IOP were also evaluated., Results: After behavioral training, conscious Tibetan monkeys were receptive to IOP measurement. The lowest and highest IOP values in a circadian cycle were recorded at 3:00 AM (19.8±0.4 mmHg, mean±SEM, n=12) and noon (29.3±0.9 mmHg), respectively. Changing the feeding time from 11:30 AM to 12:30 PM lowered the noon IOP to 25.1±1.2 mmHg. General anesthesia lowered IOP in these monkeys, while IOP of young and mature animals were similar. Three hours after topical ocular administration, travoprost reduced IOP by 5.2±0.6 mmHg (n=6, p<0.001), and timolol reduced IOP by 2.8±0.7 mmHg (p<0.05). Naphazoline and spiradoline lowered IOP by 4.8 mmHg and 2.5 mmHg (both p<0.001), respectively, 2 h after drug administration., Conclusions: The circadian IOP fluctuation in conscious Tibetan monkeys and their responses to travoprost, timolol, and other experimental conditions are similar to other primates. These monkeys appear to be a suitable model for glaucoma research.

PURPOSE. To evaluate the potential neuroprotective effects of C-type natriuretic peptide (CNP) on rat retinal ganglion cells (RGCs). METHODS. Cultured adult rat retinal cells were treated with vehicle, CNP, or atrial natriuretic peptide (ANP), followed by cytotoxic insults (glutamate, TNFalpha, or withdrawal of trophic factor). RGC survival was analyzed by counting Thy-1-positive cells in each well. For in vivo evaluation, N-methyl-d-aspartate (NMDA) with or without CNP was injected intravitreally into rat eyes. At various time points after injection, retinal cross-sections were analyzed for thickness changes in the retinal layers, and retinal flat mounts were assessed by counting cresyl violet-labeled or TUNEL-positive cells. Expressions of natriuretic peptide receptor-B (NPRB) and apoptosis-related genes in retina, including Bcl-xL, BAX, and micro-calpain, were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS. At 50 and 500 nM, CNP, but not ANP, significantly (P < 0.05) protected against glutamate-insult and trophic factor withdrawal-induced RGC death in vitro. Neither peptide significantly affected TNFalpha-induced cytotoxicity. Intravitreal injection of NMDA (20 nanomoles) significantly (P < 0.05) decreased the thickness of the inner plexiform layer (IPL), induced cell loss, increased the number of TUNEL-positive cells in the RGC layer, and upregulated the expression of Bcl-xL, BAX, and micro-calpain. All these effects were significantly (P < 0.05) alleviated by concomitant injection of CNP (4.5 nmol, 10 microg). The neuroprotective effects of CNP were maintained up to 14 days after CNP injection. CONCLUSIONS. CNP protects rat RGCs against the apoptotic damage induced by insults such as excitatory amino acid, both in vitro and in vivo.

Purpose. Glaucoma is a leading cause worldwide of blindness and visual impairment. Transforming growth factor-beta2 (TGFbeta2) has been implicated in the pathogenesis of primary open-angle glaucoma (POAG) based on elevated levels in glaucomatous aqueous humor and its ability to induce extracellular matrix (ECM) remodeling in the trabecular meshwork (TM). The goal of this study was to generate a rodent model of POAG using viral gene transfer of human TGFbeta2. Methods. Latent (hTGFbeta2(WT)) or active (C226S, C228S; hTGFbeta2(226/228)) TGFbeta2-encoding cDNA was cloned into the pac.Ad5.CMV.K-N.pA shuttle vector for generation of replication-deficient adenovirus. Empty adenovirus (Ad5.CMV.K-N.pA) was used as a control. Adenoviral expression of active and total TGFbeta2 was assayed in vitro by the transduction of Chinese hamster ovary and trabecular meshwork cells. BALB/cJ mice or Wistar rats were injected either intracamerally or intravitreally with the adenovectors and assessed for changes in intraocular pressure (IOP) using the rebound tonometer. At peak IOP, aqueous outflow facility and total TGFbeta2 levels in aqueous humor were measured. Mouse eye morphology was assessed by hematoxylin and eosin staining. Results. Adenoviral gene transfer of hTGFbeta2(226/228), but not hTGFbeta2(WT), to the rodent eye elevated IOP in rat (43%, P < 0.001) and mouse (110%, P < 0.001) and reduced aqueous humor outflow facility in the mouse. The TGFbeta2-induced ocular hypertension correlated with anterior segment TGFbeta2 expression levels (P < 0.0001). Conclusions. The adenoviral TGFbeta2 rodent model displays the glaucoma risk factors of elevated IOP and decreased aqueous outflow facility and may potentially serve as a model for studying glaucoma.

Purpose: To evaluate the usefulness of the TonoVet rebound tonometer in measuring intraocular pressure (IOP) of monkeys., Methods: The accuracy of the TonoVet rebound tonometer was determined in cannulated eyes of anesthetized rhesus monkeys where IOP was controlled by adjusting the height of a connected perfusate reservoir. To assess the applicability of the equipment through in vivo studies, the diurnal fluctuation of IOP and effects of IOP-lowering compounds were evaluated in monkeys., Results: IOP readings generated by the TonoVet tonometer correlated very well with the actual pressure in the cannulated monkey eye. The linear correlation had a slope of 0.922+/-0.014 (mean+/-SEM, n=4), a y-intercept of 3.04+/-0.61, and a correlation coefficient of r(2)=0.97. Using this method, diurnal IOP fluctuation of the rhesus monkey was demonstrated. The tonometer was also able to detect IOP changes induced by pharmacologically active compounds. A single topical ocular instillation (15 microg) of the rho kinase inhibitor, H1152, produced a 5-6 mmHg reduction (p<0.001) in IOP, lasting at least 4 h. In addition, topical administration of Travatan, a prostaglandin agonist, induced a small transient IOP increase (1.1 mmHg versus vehicle control; p=0.26) at 2 h after treatment followed by a pressure reduction at 23 h (-2.4 mmHg; p<0.05). Multiple daily dosing with the drug produced a persistent IOP-lowering effect. Three consecutive days of Travatan treatment produced ocular hypotension of -2.0 to -2.2 mmHg (p<0.05) the following day., Conclusions: The rebound tonometer was easy to use and accurately measured IOP in the rhesus monkey eye.

Purpose: The aims of the study were to characterize the signal transduction responses to platelet-activating factor (PAF) and to monitor the downstream effects of PAF on the production of proinflammatory cytokines in human conjunctival epithelial cells (HCECs)., Methods: The generation of inositol phosphates ([(3)H]IPs) from [(3)H]phosphoinositide (PI) hydrolysis and the mobilization of intracellular calcium ([Ca(2+)](i)) were evaluated using ion exchange chromatography and Fura-2 fluorescence techniques, respectively. The production of the cytokines (interleukin-6 [IL-6], interleukin-8 [IL-8], and granulocyte macrophage colony-stimulating factor [GM-CSF]) from PAF-stimulated HCECs was quantified using specific ELISA assays. Specific PAF antagonists were used to study the pharmacological aspects of PAF actions in HCECs., Results: PAF (100 nM) maximally stimulated PI turnover in HCECs by 2.3+/-0.02 fold (n=21) above basal levels and with a potency (EC(50)) of 5.9+/-1.7 nM (n=4). PAF or its stabilized analog, methyl carbamyl (mc)PAF (EC(50)=0.8 nM), rapidly mobilized [Ca(2+)](i), which peaked within 30-60 s and remained elevated for 3 min. PAF (10 nM-1 microM) stimulated the release of the proinflammatory cytokines, IL-6, IL-8, and GM-CSF, 1.4-3.5 fold above basal levels. The effects of PAF (100 nM) on PI turnover and [Ca(2+)](i) were potently antagonized by the PAF antagonists, 1-o-hexadecyl-2-o-acetyl-sn-glycero-3-phospho (N,N,N-trimethyl) hexanolamine (IC(50)=0.69 microM; K(i)=38 nM), methyl 2-(phenylthio)ethyl-1,4-dihydro-2,4,6-trimethyl-pyridine-3,5-dicsrboxylate (PCA-42481; IC(50)=0.89 microM; K(i)=50 nM), rac-3-(N-octadecylcarbomoyl)-2-methoxy) propyl-(2-thiazolioethyl) phosphate (CV-3988; IC(50)=13 microM; K(i)=771 nM), and (+/-)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one HCl (SM-10661; IC(50)=14 microM; K(i)=789 nM [n=3 for each antagonist]). PAF-induced production of IL-6, IL-8, and GM-CSF from HCECs was also blocked by these PAF antagonists (IC(50)=4.6- 8.6 microM)., Conclusions: HCECs respond to PAF by generating IPs, mobilizing [Ca(2+)](i), and then secreting cytokines into the extracellular medium. These results suggest that HCECs may be key target cells for the PAF released from conjunctival mast cells following ocular allergic reactions. Therefore, HCECs in culture represent suitable in vitro models for the investigation of the role of PAF in human ocular allergic and inflammatory diseases and for the discovery of therapeutically useful PAF antagonists.

Purpose: To search for and validate potential molecular pathogenic mechanisms in the trabecular meshwork (TM) responsible for the elevated intraocular pressure (IOP) associated with glaucoma., Methods: Gene chip arrays were used to identify differential gene expression in glaucomatous TM tissues. Serum amyloid A (SAA) upregulation was subsequently confirmed with quantitative PCR (QPCR) and ELISA. The effect of SAA on gene expression of cultured human TM cells was tested with gene chip arrays and verified with ELISA, and its effect on IOP was evaluated in the human ocular perfusion organ culture., Results: Microarray analysis showed that the expression of SAA2 was increased in TM tissues from donors with glaucoma. This finding was subsequently confirmed by QPCR. The SAA mRNA levels were increased in glaucoma TM tissues by more than 5-fold (P < 0.05) and in cultured TM cells derived from donors with glaucoma by 25-fold (P < 0.05) compared with controls. SAA protein levels in the TM of glaucoma patients were also significantly (P < 0.05) elevated by 2.9-fold. Treatment of cultured human TM cells with recombinant SAA affected gene expression, including a 22-fold up-regulation of interleukin-8 (P < 0.001). SAA increased IOP by approximately 40% (P < 0.05) in the human ocular perfusion organ culture without any observable changes in the morphology of the tissues involved in aqueous outflow., Conclusions: These findings indicate that SAA, which is an acute-phase apolipoprotein that plays important roles in infection, inflammation, and tissue repair, may contribute to the pathogenic changes to the TM in glaucoma.

The ability to measure intraocular pressure in rodents is very important for the advancement of glaucoma research. This review article describes various currently used methods, such as, microcannulation, servo-null micropipette, Tonopen tonometer, rebound tonometer, Goldmann applanation tonometer, Schiötz indentation tonometer, pneumatonometer, and optic interferometry tonometer. Their principles of operation, advantages and limitations, as well as a calibration method are also discussed.

Elevated intraocular pressure (IOP) is the principal risk factor for glaucoma and results from excessive impedance of the fluid outflow from the eye. This abnormality likely originates from outflow pathway tissues such as the trabecular meshwork (TM), but the associated molecular etiology is poorly understood. We discovered what we believe to be a novel role for secreted frizzled-related protein-1 (sFRP-1), an antagonist of Wnt signaling, in regulating IOP. sFRP1 was overexpressed in human glaucomatous TM cells. Genes involved in the Wnt signaling pathway were expressed in cultured TM cells and human TM tissues. Addition of recombinant sFRP-1 to ex vivo perfusion-cultured human eyes decreased outflow facility, concomitant with reduced levels of beta-catenin, the Wnt signaling mediator, in the TM. Intravitreal injection of an adenoviral vector encoding sFRP1 in mice produced a titer-dependent increase in IOP. Five days after vector injection, IOP increased 2 fold, which was significantly reduced by topical ocular administration of an inhibitor of a downstream suppressor of Wnt signaling. Thus, these data indicate that increased expression of sFRP1 in the TM appears to be responsible for elevated IOP in glaucoma and restoring Wnt signaling in the TM may be a novel disease intervention strategy for treating glaucoma.

Purpose: Gene transduction using adenoviral vectors is an important research tool. To assess and optimize this technique for glaucoma research, we characterized green fluorescent protein (GFP) expression in the mouse eye after intraocular injection of adenoviral vector encoding GFP (Ad5.CMV-GFP) and evaluated the effect of anti-CD40L antibody administration on GFP expression., Methods: Mice were injected with Ad5.CMV-GFP intracamerally (IC) or intravitreally (IVT) with or without anti-CD40L antibody treatment. GFP expression was assessed by in vivo fluorescence intensity with a standardized grading scale. Location of expression was analyzed histologically by fluorescence microscopy., Results: Intraocular injection of Ad5.CMV-GFP induced titer-dependent expression of GFP in the anterior segment. In vivo fluorescence was detectable but low after IC injection. After IVT injection, fluorescence in the eye peaked at days 4-7 with a fluorescence grade of 3.0 +/-0.0 (mean +/-SEM, n=6; injection with 1x10(8) pfu vector). After day 7, GFP expression declined significantly. Treatment with anti-CD40L antibody increased fluorescence intensity after IC injection, and prolonged GFP expression in the IVT group. At day 43, fluorescence grades of the IVT group were 2.8 +/-0.7 (with anti-CD40L) and 1.2 +/-0.6 (without antibody). Three-Way ANOVA confirmed that GFP expression was significantly higher in the anti-CD40L than the no antibody group (p=0.013), significantly higher in the IVT than the IC group (p=0.003), and significantly higher in the high viral titer (1x10(8) pfu) than the low titer (1x10(7) pfu) group (p=0.010). Fluorescence microscopic examination of cross-sections of eyes indicated GFP expression in the trabecular meshwork (TM), corneal endothelium, and sporadically iris, ciliary body and lens epithelium., Conclusions: Intraocular injection of Ad5.CMV-GFP induced GFP expression in the mouse anterior segment, including the TM. Expression was more prominent after IVT injection than IC injection. Anti-CD40L antibody treatment increased both intensity and duration of GFP expression. These findings provide important and practical means to improve duration and efficiency of adenovirus-mediated transgene expression in the eye.

Background: Nitric oxide synthase 2 (NOS2) contributes to neural death in some settings, but its role in glaucoma remains controversial. NOS2 is implicated in retinal ganglion cell degeneration in a rat glaucoma model in which intraocular pressure (IOP) is experimentally elevated by blood vessel cauterization, but not in a rat glaucoma model where IOP was elevated by injection of hypertonic saline. To test the importance of NOS2 for an inherited glaucoma, in this study we both genetically and pharmacologically decreased NOS2 activity in the DBA/2J mouse glaucoma model., Methods: The expression of Nos2 in the optic nerve head was analyzed at both the RNA and protein levels at different stages of disease pathogenesis. To test the involvement of Nos2 in glaucomatous neurodegeneration, a null allele of Nos2 was backcrossed into DBA/2J mice and the incidence and severity of glaucoma was assessed in mice of each Nos2 genotype. Additionally, DBA/2J mice were treated with the NOS2 inhibitor aminoguanidine and the disease compared to untreated mice., Results: Optic nerve head Nos2 RNA levels varied and increased during moderate but decreased at early and severe stages of disease. Despite the presence of a few NOS2 positive cells in the optic nerve head, NOS2 protein was not substantially increased during the glaucoma. Genetic deficiency of Nos2 or aminoguanidine treatment did not alter the IOP profile of DBA/2J mice. Additionally, neither Nos2 deficiency nor aminoguanidine had any detectable affect on the glaucomatous optic nerve damage., Conclusion: Glaucomatous neurodegeneration in DBA/2J mice does not require NOS2 activity. Further experiments involving various models are needed to assess the general importance of Nos2 in glaucoma.

Animal models are useful to elucidate the etiology and pathology of glaucoma and to develop novel and more effective therapies for the disease. Because of the substantial similarities between the rodent and primate eyes, and the advances of relevant study techniques, rat and mouse models of glaucoma have recently become popular as research tools. This review surveys research techniques used in the measurement of rodent intraocular pressure, and also the evaluation of pertinent morphologic, biochemical, and functional changes in the retina, optic nerve head, and optic nerve. This review further describes in detail the individual rodent models, some of which serve as surrogate models and do not entail ocular hypertension, whereas others involve transient or chronic increases of intraocular pressure. The technical considerations and theoretical concerns of these models, their advantages, and limitations, are also discussed.

Purpose: To determine the expression profile of neurotrophins and their receptors in cultured primary rat retinal ganglion cells (RGCs) and the transformed RGC-5 cells., Methods: Confocal microscopic immunocytochemistry with double fluorescent labeling with thy-1 as a marker for RGCs was used to demonstrate expression of neurotrophins and their receptors. An enzyme-linked immunosorbent assay (ELISA) was used to detect secretion of neurotrophins by RGC-5 cells., Results: Primary RGCs and RGC-5 cells expressed brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT3), neurotrophin-4 (NT4), and receptors TrkA, p75, with low levels of TrkB. However, minimal if any, expression of TrkC was observed in these cells. RGC-5 cells also secreted NT3 (1311+/-21 pg/ml), BDNF (92+/-9 pg/ml), NGF (86+/-7 pg/ml), and NT4 (21+/-1 pg/ml) into the cultured media., Conclusions: These results demonstrated that neurotrophins and TrkA, p75 with low levels of TrkB receptors are expressed by RGCs. Specific neurotrophins acting through TrkA, TrkB, and p75 receptors within the RGCs may be involved in the survival and apoptosis of the RGCs in various retinopathies, such as glaucoma.

Glaucoma is a leading cause of worldwide irreversible visual impairment and blindness and is a clinically and genetically heterogenous group of optic neuropathies. Specific mutations in the myocilin (MYOC) gene cause primary open angle glaucoma (POAG) with varying age-of-onset and degree of severity. We show a mutation-dependent, gain-of-function association between human myocilin and the peroxisomal targeting signal type 1 receptor (PTS1R). There was correlation between the glaucoma phenotype and the specific MYOC mutations, with the more severe early-onset POAG mutations having a higher degree of association with PTS1R. Expression of human myocilin glaucomatous mutations in mouse eyes causes elevated intraocular pressure, which is a major phenotype of MYOC glaucoma. This is the first demonstration of a disease resulting from mutation-induced exposure of a cryptic signaling site that causes mislocalization of mutant protein to peroxisomes and the first disease-gene-based animal model of human POAG.

Purpose: The primary causative factor of primary open-angle glaucoma (POAG) is elevated intraocular pressure (IOP) due to increased aqueous humor (AH) outflow resistance, which is associated with morphologic and biochemical changes in the trabecular meshwork (TM). Patients with glaucoma have elevated levels of transforming growth factor (TGF)-beta2 in their AH, and TGF-beta has been shown to increase TM extracellular matrix (ECM) production. The bone morphogenetic protein (BMP) signaling pathway modifies TGF-beta signaling in several different tissues, and a prior study demonstrated that TM cells and tissues express members of the BMP gene family. The purpose of this study was to determine whether BMPs can alter TGF-beta2 signaling in the TM and whether there are defects in BMP signaling in glaucoma., Methods: ELISA, Western immunoblot analysis, and immunohistochemistry were used to evaluate the expression of BMP proteins in TM cells and tissues. ELISA was used to determine the effects of TGF-beta2 and BMPs on TM fibronectin (FN) secretion. Gene expression was determined by gene microarrays and quantitative (q)PCR. Perfusion-cultured human anterior segments were used to study the effects of altered BMP signaling on IOP., Results: The human TM synthesized and secreted BMP-4 as well as expressed BMP receptor subtypes BMPRI and BMPRII. TM cells responded to exogenous BMP-4 by phosphorylating Smad signaling proteins. Cultured human TM cells treated with TGF-beta2 significantly increased FN levels, and BMP-4 blocked this FN induction. The expression of BMP family genes in normal and glaucomatous TM cells was profiled and significant elevation of mRNA and protein levels of the BMP antagonist gremlin were found in glaucomatous TM cells. In addition, Gremlin was present in human aqueous humor and in the perfusate medium of perfusion-cultured human eyes. Gremlin blocked the negative effect of BMP-4 on TGF-beta-induction of FN. Recombinant Gremlin added to the medium of ex vivo perfusion-cultured human eye anterior segments caused the glaucoma phenotype of elevated IOP., Conclusions: These results are consistent with the hypothesis that, in POAG, elevated expression of Gremlin by TM cells inhibits BMP-4 antagonism of TGF-beta2 and leads to increased ECM deposition and elevated IOP.