NADPH oxidase-induced oxidative stress in the eyes of hypertensive rats.

Purpose: Increased reactive oxygen species (ROS) released by NADPH oxidase and inflammation are associated with arterial hypertension and eye diseases associated with high blood pressure, including glaucoma, retinopathies (e.g., age-related macular degeneration), and choroidopathies affecting ocular function; however, the mechanisms underlying these adverse outcomes remain undefined. The present study was designed to highlight the importance of oxidative stress in severe hypertension-related eye damage. Methods: Male Wistar rats (n = 7, unless otherwise specified for specific experiments) were administered an oral dose of 30 mg of Nω-nitro-L-arginine methyl ester (L-NAME) per kilogram of bodyweight and day for 3 weeks; chronic administration with L-NAME is a validated experimental approach resulting in severe hypertension secondary to nitric oxide (NO) depletion and subsequent vasoconstriction in the systemic circulation. Upon treatment completion, histomorphometric studies, NADPH oxidase activity, and ROS production were measured in eyecup homogenates and paraffin-embedded sections from control and L-NAME-treated animals. In addition, immunohistofluorescence, western blotting, and real-time PCR (RT-qPCR) analyses were performed in the eye and the retina to evaluate the expression of i) NADPH oxidase main isoforms (NOX1, NOX2, and NOX4) and subunits (p22phox and p47phox); ii) glial fibrillary acidic protein (GFAP), as a marker of microglial activation in the retina; iii) antioxidant enzymes; and iv) endothelial constitutive (eNOS) and inflammation inducible (iNOS) nitric oxide synthase isoforms, and nitrotyrosine as a versatile biomarker of oxidative stress. Results: Increased activity of NADPH oxidase and superoxide anion production, accompanied by transcriptional upregulation of this enzyme isoforms, was found in the retina and choroid of the hypertensive rats in comparison with the untreated controls. Histomorphometric analyses revealed a significant reduction in the thickness of the ganglion cell layer and the outer retinal layers in the hypertensive animals, which also showed a positive strong signal of GFAP in the retinal outer segment and plexiform layers. In addition, L-NAME-treated animals presented with upregulation of nitric oxide synthase (including inducible and endothelial isoforms) and abnormally elevated nitrotyrosine levels. Experiments on protein and mRNA expression of antioxidant enzymes revealed depletion of superoxide dismutase and glutathione peroxidase in the eyes of the hypertensive animals; however, glutathione reductase was significantly higher than in the normotensive controls. Conclusions: The present study demonstrated structural changes in the retinas of the L-NAME-treated hypertensive animals and strengthens the importance of NADPH oxidase as a major ROS-generating enzyme system in the oxidative and inflammatory processes surrounding hypertensive eye diseases. These observations might contribute to unveiling pathogenic mechanisms responsible for developing ocular disturbances in the context of severe hypertension.

Knockout of tissue transglutaminase ameliorates TGFß2-induced ocular hypertension: A novel therapeutic target for glaucoma?

Glaucoma is a vision threatening optic neuropathy that affects millions of people worldwide. In primary open angle, increased intraocular pressure (IOP) is the main risk factor for the development of this disease. Studies investigating the causes and mechanisms of increased IOP show fibrotic changes in the trabecular meshwork (TM) that are different from those of age-matched controls. Tissue transglutaminase (TGM2), an extracellular matrix (ECM) crosslinking enzyme, covalently crosslinks ECM proteins and causes excessive ECM protein deposition in the TM that could cause increased IOP. Previous literature reports increased expression of TGM2 in glaucomatous eyes compared to controls. We recently have shown that overexpression of TGM2 causes increased ECM crosslinking in the TM, increases IOP, and decreases aqueous humor (AH) outflow facility in mouse eyes. Therefore, we wanted to study the effect of TGM2 knockout (KO) on IOP in TGM2 floxed mice. Ad5.Cre transduction caused partial KO of TGM2, which decreased TGM2 expression in the TM region of mouse eyes. TGM2 KO significantly decreased IOP by itself and also in TGFß2 induced ocular hypertensive mice. TGM2 KO also restores the outflow facility in TGFß2 transduced eyes. Overall, TGM2 KO rescued the TGFß2-induced ocular hypertensive phenotype. Thus, TGM2 may offer potential as a new therapeutic target for glaucoma.

RhoA GTPase-induced ocular hypertension in a rodent model is associated with increased fibrogenic activity in the trabecular meshwork.

Ocular hypertension arising from increased resistance to aqueous humor (AH) outflow through the trabecular meshwork is a primary risk factor for open-angle glaucoma, a leading cause of blindness. Ongoing efforts have found little about the molecular and cellular bases of increased resistance to AH outflow through the trabecular meshwork in ocular hypertension patients. To test the hypothesis that dysregulated Rho GTPase signaling and a resulting fibrotic activity within the trabecular meshwork may result in ocular hypertension, we investigated the effects of expressing a constitutively active RhoA GTPase (RhoAV14) in the AH outflow pathway in Sprague-Dawley rats by using lentiviral vector-based gene delivery. Rats expressing RhoAV14 in the iridocorneal angle exhibited a significantly elevated intraocular pressure. Elevated intraocular pressure in the RhoAV14-expressing rats was associated with fibrotic trabecular meshwork and increased levels of F-actin, phosphorylated myosin light chain, α-smooth muscle actin, collagen-1A, and total collagen in the trabecular AH outflow pathway. Most of these changes were ameliorated by topical application of Rho kinase inhibitor. Human autopsy eyes from patients with glaucoma exhibited significant increases in levels of collagen-1A and total collagen in the trabecular AH outflow pathway. Collectively, these observations indicate that increased fibrogenic activity because of dysregulated RhoA GTPase activity in the trabecular AH outflow pathway increases intraocular pressure in a Rho kinase-dependent manner.

Topical trans-resveratrol ameliorates steroid-induced anterior and posterior segment changes in rats.

Steroid-induced hypertension and glaucoma is associated with increased extracellular meshwork (ECM) deposition in trabecular meshwork (TM). Previous studies have shown that single drop application of trans-resveratrol lowers IOP in steroid-induced ocular hypertensive (SIOH) rats. This IOP lowering is attributed to activation of adenosine A1 receptors, which may lead to increased matrix metalloproteinase (MMP)-2 activity. This study evaluated the effect of repeated topical application of trans-resveratrol for 21 days in SIOH animals on IOP, changes in MMP-2 level in aqueous humor, trabecular meshwork and retinal morphology and retinal redox status. We observed that treatment with trans-resveratrol results in significant and sustained IOP reduction in SIOH rats. This IOP reduction is associated with significantly higher aqueous humor total MMP-2 level; significantly reduced TM thickness and increased number of TM cells. Treatment with trans-resveratrol also significantly increased ganglion cell layer (GCL) thickness, the linear cell density in the GCL and inner retina thickness; and significantly reduced retinal oxidative stress compared to the SIOH vehicle-treated group. In conclusion, repeated dose topical application of trans-resveratrol produces sustained IOP lowering effect, which is associated with increased level of aqueous humor MMP-2, normalization of TM and retinal morphology and restoration of retinal redox status.

Digoxin derivatives with enhanced selectivity for the α2 isoform of Na,K-ATPase: effects on intraocular pressure in rabbits.

In the ciliary epithelium of the eye, the pigmented cells express the α1ß1 isoform of Na,K-ATPase, whereas the non-pigmented cells express mainly the α2ß3 isoform of Na,K-ATPase. In principle, a Na,K-ATPase inhibitor with selectivity for α2 could effectively reduce intraocular pressure with only minimal local and systemic toxicity. Such an inhibitor could be applied topically provided it was sufficiently permeable via the cornea. Previous experiments with recombinant human α1ß1, α2ß1, and α3ß1 isoforms showed that the classical cardiac glycoside, digoxin, is partially α2-selective and also that the trisdigitoxose moiety is responsible for isoform selectivity. This led to a prediction that modification of the third digitoxose might increase α2 selectivity. A series of perhydro-1,4-oxazepine derivatives of digoxin have been synthesized by periodate oxidation and reductive amination using a variety of R-NH2 substituents. Several derivatives show enhanced selectivity for α2 over α1, close to 8-fold in the best case. Effects of topically applied cardiac glycosides on intraocular pressure in rabbits have been assessed by their ability to either prevent or reverse acute intraocular pressure increases induced by 4-aminopyridine or a selective agonist of the A3 adenosine receptor. Two relatively α2-selective digoxin derivatives efficiently normalize the ocular hypertension, by comparison with digoxin, digoxigenin, or ouabain. This observation is consistent with a major role of α2 in aqueous humor production and suggests that, potentially, α2-selective digoxin derivatives could be of interest as novel drugs for control of intraocular pressure.

VEGF-A is necessary and sufficient for retinal neuroprotection in models of experimental glaucoma.

Vascular endothelial growth factor A (VEGF-A) is a validated therapeutic target in several angiogenic- and vascular permeability-related pathological conditions, including certain cancers and potentially blinding diseases, such as age-related macular degeneration and diabetic retinopathy. We and others have shown that VEGF-A also plays an important role in neuronal development and neuroprotection, including in the neural retina. Antagonism of VEGF-A function might therefore present a risk to neuronal survival as a significant adverse effect. Herein, we demonstrate that VEGF-A acts directly on retinal ganglion cells (RGCs) to promote survival. VEGF receptor-2 signaling via the phosphoinositide-3-kinase/Akt pathway was required for the survival response in isolated RGCs. These results were confirmed in animal models of staurosporine-induced RGC death and experimental hypertensive glaucoma. Importantly, we observed that VEGF-A blockade significantly exacerbated neuronal cell death in the hypertensive glaucoma model. Our findings highlight the need to better define the risks associated with use of VEGF-A antagonists in the ocular setting.

Lamotrigine monotherapy does not provide protection against the loss of optic nerve axons in a rat model of ocular hypertension.

Sodium channel blocking agents such as lamotrigine are potent agents for neuroprotection in several animal models of neurodegenerative and neuroinflammatory disease. We therefore explored whether lamotrigine therapy was neuroprotective in a rat model of ocular hypertension characterized by axonal injury and selective loss of retinal ganglion cells. Twenty-seven male Wistar rats were injected subcutaneously twice daily with either lamotrigine (14 mg/kg/day) or vehicle. Two weeks after the first injection, experimental ocular hypertension was induced in one eye by 532 nm trabecular laser treatment. Intraocular pressure (IOP) was monitored by rebound tonometry and four weeks after the elevation of IOP the loss of optic nerve axons was quantified relative to eyes without either IOP elevation or lamotrigine exposure. In other animals with ocular hypertension, the optic nerves were examined by immunohistochemistry for the expression of the inducible form of nitric oxide synthase (iNOS) at 7 and 28 days. Four weeks after initiation of IOP elevation, no significant difference in axonal loss was observed between rats treated with lamotrigine (30.8% ± 10.5%) or vehicle (17.8% ± 5.7%) (P = 0.19, T-test). There was no significant difference in mean IOP, peak IOP and integral IOP exposure. Furthermore, optic nerve axon counts per unit integral IOP exposure were similar in both groups (P = 0.44). The optic nerves were not positive for the expression of iNOS. In conclusion, this study provides no evidence that lamotrigine is neuroprotective for RGC axons after four weeks of experimental ocular hypertension in the rat, in a model where axonal degeneration occurs in the absence of iNOS expression.

Involvement of carbonic anhydrases in the ocular hypotensive effect of melatonin analogue 5-MCA-NAT.

We have previously demonstrated that melatonin and its analogue, 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT), reduce intraocular pressure (IOP) in New Zealand rabbits. More recently, we have shown that 5-MCA-NAT can also regulate ciliary adrenoceptor gene expression. Like adrenoceptors, carbonic anhydrase (CA) enzymes are involved in aqueous humour secretion by the ocular ciliary epithelium. Moreover, CA enzymes have been reported to be regulated by melatonin. Hence, the aim of this study was to investigate whether the hypotensive effect of 5-MCA-NAT is also because of a regulation of CA genes and enzymes. Time course of 5-MCA-NAT effect on rabbit IOP was followed for 7 hr every day for up to 144 hr (6 days). 5-MCA-NAT reduced IOP, maximally by 51.30 ± 2.41% (at 3 hr), and the hypotensive effect was maintained for up to 96 hr with a single application. IOP studies with 5-MCA-NAT plus Trusopt(®) and immunohistochemical analysis confirmed that CA are molecular targets of 5-MCA-NAT. In addition, real-time quantitative PCR (qPCR) and immunocytochemical assays were performed to determine changes in CA2 (CAII) and CA12 (CAXII) expression in cultured rabbit nonpigmented ciliary epithelial cells (NPE) treated with 5-MCA-NAT. NPE cells showed a prominent decrease in both CA, at the mRNA and protein levels. These data confirm that the long-term hypotensive effect of 5-MCA-NAT is also due, to a down-regulation of CA2 (CAII) and CA12 (CAXII) expression.

Honeycomb Epithelial Edema Associated With Rho Kinase Inhibition: A Case Series and Review of the Literature.

ABSTRACT: The Rho kinase inhibitor netarsudil is a recently approved therapeutic option for the management of increased intraocular pressure in the United States. Although phase 3 clinical trials noted corneal changes related to the medication-namely, nonvisually-significant corneal verticillata-descriptions of a unique form of cystic epithelial edema began to surface as netarsudil (and its sister drug ripasudil, approved in Japan) gained widespread use. This series adds 3 new cases and reviews the current literature on this unique side effect.

Benzothiophene containing Rho kinase inhibitors: Efficacy in an animal model of glaucoma.

We identified a new benzothiophene containing Rho kinase inhibitor scaffold in an ultra high-throughput enzymatic activity screen. SAR studies, driven by a novel label-free cellular impedance assay, were used to derive 39, which substantially reduced intraocular pressure in a monkey model of glaucoma-associated ocular hypertension.

Mitochondrial cytochrome c oxidase expression in the central nervous system is elevated at sites of pressure gradient elevation but not absolute pressure increase.

The paucity of suitable experimental models has made it difficult to isolate the pathogenic role of mitochondria in central nervous system diseases associated with absolute pressure elevation and increased pressure gradients. Experimental models of traumatic brain injury (TBI) and hydrocephalus have been useful for examining the mitochondrial response following pressure increase in the central nervous system; however, the presence of multiple pathogenic factors acting on the brain in these previous studies has made it difficult to determine whether the induced changes were a result of mechanical damage, intracranial pressure elevation, or other pathogenic factors. By direct monitoring and control of pressures in the intraocular, intracranial, and vascular compartments, we use the pig optic nerve, a typical central white matter tract, to compare the temporal sequence of cytochrome c oxidase (CcO) levels between regions of absolute pressure elevation and pressure gradient increase. We demonstrate that a rise in pressure gradient without traumatic injury up-regulates CcO levels across the site of the gradient, in a manner similar to what has been previously reported for hydrocephalus. We also demonstrate that CcO changes do not occur following an absolute pressure rise. These findings taken together with our recent reports suggest that mitochondria initiate an early compensatory response to axonal damage following pressure gradient increase. Extrapolation of our results also suggests that decreased CcO levels in TBI may be secondary to mechanical damage. This study emphasises the importance of pressure gradients in regulating mitochondrial function in the central nervous system.

Carbonic anhydrase inhibition and the management of glaucoma: a literature and patent review 2013-2019.

Introduction: Glaucoma affects more than 70 million people worldwide. One of the major therapeutic options for its management is based on the inhibition of the metalloenzyme carbonic anhydrases (CAs, EC 4.2.1.1). CA inhibitors (CAIs) diminish ocular hypertension in glaucomatous patients by reducing the rate of bicarbonate formation and thus, the secretion of the aqueous humor. Areas covered: This review is intended to cover the major contributions in terms of patent literature reports for the treatment of ophthalmic diseases by means of CAIs in a time frame spanning from 2013 to date. Expert opinion: The patent literature is dominated by innovative pharmaceutical formulations including a CAI alone or in combination with other therapeutic agents. Very few novelties within drug discovery are currently present and they mainly account for new CAI moieties and classical CAIs merged into scaffolds bearing additional chemical functionalities beneficial for the pharmacological treatment of the disease. It is reasonable to expect that in the near future the so-called 'old drugs' will achieve pharmacological performances in the management of ocular hypertension beyond any expectations and thus open a new era of drug repurposing merely based on material science advancements.

Differential roles of phosphatidylinositol 3-kinase/akt pathway in retinal ganglion cell survival in rats with or without acute ocular hypertension.

Intraocular pressure (IOP) elevation has often been used as an experimental model to study mechanisms underlying retinal ganglion cell (RGC) death associated with ocular ischemic injury and glaucoma. The aim of the present study, using both in vitro and in vivo approaches, was to investigate the role of phosphatidylinositol 3-kinase (PI3K)/akt pathway in RGC viability in normal rats and rats following transient IOP elevation. For in vivo studies, pathway inhibitors were administered intravitreally on days 3, 9, and 15 post-2-h IOP elevation at 110 mm Hg. Toward the end of the 3-week examination period, the fluorescent dye Fluorogold was used to retrogradely label surviving RGCs. In order to examine the role of macrophages that were recruited into the eye following the pathway inhibition, clodronate liposomes were used to deplete phagocytic cells in the eye. PI3K/akt pathway activity and location in the retina were examined using Western blot and immunohistochemistry, respectively. Here we showed that PI3K/akt inhibitors 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and KY12420 at low concentrations (2 microM or 20 microM) did not influence RGC survival but caused RGC loss at high concentration (200 muM) in retinal explants derived from intact rats. In contrast, both LY294002 and KY12420 at 20 microM led to RGC loss in retinal explants derived from IOP-elevated eyes. A detrimental action of phagocytic cells on RGC survival was also seen in these retinas. In vivo results confirmed the detrimental actions of PI3K/akt inhibition and macrophages on RGC survival in IOP-elevated, but not intact eyes even with high concentration of LY294002. Low level of PI3K/akt activity was detected in the ganglion cell layer (GCL) in intact retina. Acute IOP elevation activated PI3K/akt pathway in the inner nuclear layer and GCL including RGCs. This study thus demonstrates that PI3K/akt pathway mediates RGC survival after IOP elevation but not under normal condition.

Roles of PI3K and JAK pathways in viability of retinal ganglion cells after acute elevation of intraocular pressure in rats with different autoimmune backgrounds.

BACKGROUND: We recently showed that whereas inhibition of PI3K/akt or JAK/STAT pathway promoted retinal ganglion cell (RGC) survival after optic nerve (ON) injury in Fischer 344 (F344) rats, the same inhibition resulted in aggravated RGC loss after acute intraocular pressure (IOP) elevation in Sprague Dawley (SPD) rats. In addition, the responses of macrophages to ON injury and acute IOP elevation were different between F344 and Lewis rats, i.e., different autoimmune profiles. Using an acute IOP elevation paradigm in this study, we investigated 1) whether autoimmune background influences PI3K/akt and JAK/STAT functions by examining the effect of PI3K/akt and JAK/STAT pathway inhibition on RGC survival in F344 and Lewis rats, and 2) whether differential actions of macrophages occur in PI3K/akt and JAK/STAT pathways-dependent modulation of RGC survival. IOP elevation was performed at 110 mmHg for 2 hours. PI3K/akt and JAK/STAT pathway inhibitors were applied intravitreally to block their respective pathway signaling transduction. Because macrophage invasion was seen in the eye after the pathway inhibition, to examine the role of these pathways independent of macrophages, macrophages in the retina were removed by intravitreal application of clodronate liposomes. Viable RGCs were retrogradely labelled by FluoroGold 40 hours before animal sacrifice. RESULTS: Similar to what was previously observed, significantly more RGCs were lost in Lewis than F344 rats 3 weeks after acute IOP elevation. As in SPD rats, inhibition of the PI3K/akt or JAK/STAT pathway increased the loss of RGCs in both F344 and Lewis rats. Removal of macrophages in the eye by clodronate liposomes reduced RGC loss due to pathway inhibition in both strains. CONCLUSION: This study demonstrates that following acute IOP elevation 1) PI3K/akt and JAK/STAT pathways mediate RGC survival in both F344 and Lewis rats, 2) autoimmune responses do not influence the functions of these two pathways, and 3) PI3K/akt and JAK/STAT pathway inhibition-dependent activation of macrophages is detrimental to RGCs.

The relationship between plasma MMP-9 and TIMP-2 levels and intraocular pressure elevation in diabetic patients after intravitreal triamcinolone injection.

PURPOSE: To find out the relationship between steroid-induced intraocular pressure (IOP) rise and the plasma levels of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of MMP-2 (TIMP-2) in diabetic patients who underwent intravitreal triamcinolone acetonide (IVTA) injection for the treatment of diabetic macular edema. SUBJECTS AND METHODS: A total of 34 patients with diabetic macular edema who were treated with IVTA and 17 healthy subjects who served as control group for plasma MMP and TIMP levels were participated. Before IVTA treatment, patients and control subjects underwent complete ophthalmologic examination, including best-corrected visual acuity, slit-lamp biomicroscopy, IOP measurement with Goldmann applanation tonometry, and indirect ophthalmoscopy; and peripheral blood samples were collected from each study participants. Plasma levels of MMP-9, TIMP-2, and HbA1c levels were measured. Patients were seen 1, 6, 12, and 24 weeks after treatment and then every 6 months for up to 1 year for probable IOP rise. Patients were divided into 2 groups as having nonproliferative or proliferative diabetic retinopathy. These 2 groups were further classified according to their IOP levels as normal or elevated IOP (>21 mm Hg). RESULTS: The mean age of diabetic group of patients (n=34) and healthy control subjects (n=17) were 57.6+/-10.2 years (range: 22 to 70 y) and 53.1+/-10.3 years (range: 29 to 68 y), respectively. Seventeen (50%) diabetic patients had developed elevated IOP after a mean 2.2 months after IVTA injection. MMP-9 and TIMP-2 levels were found to be significantly higher in the diabetic groups with and without elevated IOP when compared with control group (P<0.001). MMP-9/TIMP-2 did not change significantly among the groups. Logistic regression analysis showed that only higher plasma TIMP-2 levels increase the risk of IOP elevation after IVTA injection in proliferative diabetic retinopathy (odds ratio=1.06, P<0.05). No significant relationship was found between IOP rise and HbA1c levels. CONCLUSIONS: The high levels of TIMP in diabetic patients might have a role on steroid-induced IOP rise. The key pathogenetic events that up-regulate TIMP levels should be investigated in steroid IOP rise in diabetics.

Jnk2 deficiency increases the rate of glaucomatous neurodegeneration in ocular hypertensive DBA/2J mice.

The cJun N-terminal kinases (JNKs; JNK1, JNK2, and JNK3) promote degenerative processes after neuronal injury and in disease. JNK2 and JNK3 have been shown to promote retinal ganglion cell (RGC) death after optic nerve injury. In their absence, long-term survival of RGC somas is significantly increased after mechanical optic nerve injury. In glaucoma, because optic nerve damage is thought to be a major cause of RGC death, JNKs are an important potential target for therapeutic intervention. To assess the role of JNK2 and JNK3 in an ocular hypertensive model of glaucoma, null alleles of Jnk2 and Jnk3 were backcrossed into the DBA/2J (D2) mouse. JNK activation occurred in RGCs following increased intraocular pressure in D2 mice. However, deficiency of both Jnk2 and Jnk3 together did not lessen optic nerve damage or RGC death. These results differentiate the molecular pathways controlling cell death in ocular hypertensive glaucoma compared with mechanical optic nerve injury. It is further shown that JUN, a pro-death component of the JNK pathway in RGCs, can be activated in glaucoma in the absence of JNK2 and JNK3. This implicates JNK1 in glaucomatous RGC death. Unexpectedly, at younger ages, Jnk2-deficient mice were more likely to develop features of glaucomatous neurodegeneration than D2 mice expressing Jnk2. This appears to be due to a neuroprotective effect of JNK2 and not due to a change in intraocular pressure. The Jnk2-deficient context also unmasked a lesser role for Jnk3 in glaucoma. Jnk2 and Jnk3 double knockout mice had a modestly increased risk of neurodegeneration compared with mice only deficient in Jnk2. Overall, these findings are consistent with pleiotropic effects of JNK isoforms in glaucoma and suggest caution is warranted when using JNK inhibitors to treat chronic neurodegenerative conditions.

Netarsudil ophthalmic solution 0.02% for the treatment of patients with open-angle glaucoma or ocular hypertension.

Once-daily (p.m.) netarsudil ophthalmic solution 0.02% (Rhopressa) is approved in the United States for lowering elevated intraocular pressure (IOP) in patients with open-angle glaucoma or ocular hypertension. Netarsudil, a Rho kinase (ROCK) inhibitor that lowers IOP primarily by increasing trabecular outflow, produces statistically and clinically significant reductions in mean IOP from baseline, with comparable effects on nocturnal and diurnal IOP. In three phase III trials of patients with elevated IOP, the ocular hypotensive efficacy of once-daily netarsudil 0.02% met the criteria for noninferiority to twice-daily timolol 0.5% at all time points over 3 months in patients with baseline IOP less than 25 mmHg. The most frequent adverse event (AE) was generally mild conjunctival hyperemia, the severity of which did not increase with continued dosing. Netarsudil was associated with minimal treatment-related serious or systemic AEs, likely due to the lack of systemic exposure. This report summarizes the available preclinical and clinical data on netarsudil.

Evaluation of inducible nitric oxide synthase in glaucomatous optic neuropathy and pressure-induced optic nerve damage.

PURPOSE: To determine whether inducible nitric oxide synthase (NOS-2) is involved in glaucomatous optic neuropathy. METHODS: Chronic elevation of rat intraocular pressure (IOP) leading to optic nerve damage was induced by episcleral injection of hypertonic saline, which caused sclerosis and blockade of aqueous humor outflow pathways. Expression of NOS-2 in the retina and optic nerve head (ONH) was evaluated by immunohistochemistry, gene array analysis, and quantitative PCR (Q-PCR). Immunohistochemistry was also used to assess the NOS-2 level in the ONH from primary open-angle glaucoma (POAG) and nonglaucomatous human eyes. Finally, an NOS-2 inhibitor, aminoguanidine, administered orally in the drinking water, was tested for its effect on optic nerve injury in rats with ocular hypertension. RESULTS: Chronically elevated IOP in the rat produced optic nerve damage that correlated with pressure change (r(2) = 0.77), but did not increase NOS-2 immunoreactivity in the optic nerve, ONH, or ganglion cell layer. Retinal and ONH NOS-2 mRNA levels did not correlate with either IOP level or severity of optic nerve injury. Similarly, there was no difference in NOS-2 immunoreactivity in the optic nerve or ONH between POAG and nonglaucomatous eyes. Furthermore, aminoguanidine treatment did not affect the development of pressure-induced optic neuropathy in the rat. CONCLUSIONS: As demonstrated by several independent methods, glaucomatous optic neuropathy was not associated with a significant change in the expression of NOS-2 in the retina, ONH, or optic nerve.

Aqueous Humor Outflow Physiology in NOS3 Knockout Mice.

PURPOSE: To investigate the role of endothelial nitric oxide synthase (eNOS) on conventional outflow function using NOS3 knockout (KO) mice. METHODS: Intraocular pressure was measured in both NOS3 KO and wild type (WT) by rebound tonometry. Outflow facility was measured by perfusing enucleated mouse eyes at multiple pressure steps. A subset of eyes was sectioned and stained for histology. Mock aqueous humor ± the nitric oxide (NO) donors nitroprusside dihydrate (SNP) or S-Nitroso-N-Acetyl-D,L-Penicillamine (SNAP) was perfused into enucleated eyes. SNP and SNAP was administered topically at 0, 1, 2, and 3 hours while the contralateral eyes served as vehicle controls. Intraocular pressure was measured in both eyes before and after the last drug treatment. RESULTS: Intraocular pressure was higher in KO mice (18.2 ± 0.7 mm Hg vs. 13.9 ± 0.5 mm Hg, mean ± SEM, n = 30, P < 0.05), and pressure-dependent conventional drainage was significantly lower (0.0058 ± 0.0005 µL/min/mm Hg, mean ± SEM, n = 21) compared with WT mice (0.0082 ± 0.0013 µL/min/mm Hg, n = 23, P < 0.05). No obvious morphological differences in iridiocorneal angle tissues were observed in hematoxylin and eosin (H&E)-stained sections. SNP and SNAP significantly increased pressure-dependent drainage in KO animals (n = 12, P < 0.05). In WT mice, SNP and SNAP caused a significant increase in pressure dependent drainage (n = 12, P < 0.05) to a similar degree as in KO mice. Topical application of SNP significantly reduced IOP in WT and KO mice (n = 12, P < 0.05), but SNAP did not change IOP significantly (n = 19). CONCLUSIONS: NOS3 KO mice have elevated IOP, which is likely the result of reduced pressure-dependent drainage. These findings are consistent with human data showing polymorphisms in the NOS3 gene associate with ocular hypertension and the development of glaucoma.

Glutamate-induced glutamine synthetase expression in retinal Muller cells after short-term ocular hypertension in the rat.

PURPOSE: To determine the effect of intraocular pressure (IOP) elevation on glutamate-induced expression of glutamine synthetase (GS) in retinal Müller cells of rat eyes. METHODS: Six groups of three rats each were studied. Group I was a normal control group. In Group II, one eye received an intravitreal glutamate injection (75 nmoles) while the contralateral eye served as control. In Groups III and IV, IOP was raised in one eye by episcleral vein cauterization. Moderately elevated IOP was maintained for 1 day in Group III or 1 week in Group IV (35 +/- 1.9-45 +/- 5.2 mm Hg). An additional two groups of rats received bilateral intravitreal glutamate injections (75 nmoles) immediately (Group V), or 6 days (Group VI), after induction of IOP elevation in one eye. One day after glutamate injection the rats in all groups were killed, and the eyes enucleated and fixed. Retinas from left and right eyes of each animal were embedded together in LR White resin (Ted Pella, Redding, CA). Sections were processed for GS immunolabeling with antibodies to GS by two-stage immunogold labeling with silver enhancement. Images of labeled retinas from the two eyes were captured under identical light microscopic conditions and the GS immunoreactivity in Müller cells was compared between the left and right retinas in the same section by image analysis. An additional five rats were included in Group II and the retinas were analyzed by Western blot analysis to confirm immunohistochemical findings. RESULTS: Similar to the finding in the control group (Group I), the GS immunoreactivity of the left and right eyes of Group III and IV remained unchanged even though the right eyes in the two groups had elevation of IOP lasting for 24 hours and 1 week, respectively. However, GS levels were significantly increased by 40 +/- 5.7% in normotensive eyes 24 hours after intravitreal injection of glutamate (Group II). The rise in GS immunoreactivity was abolished in eyes with acute IOP elevation (Group V). In contrast, when the eyes were exposed to high IOP for 1 week (Group VI), the glutamate-induced increase in GS immunoreactivity was restored. CONCLUSIONS: Elevated levels of vitreal glutamate can increase the expression of GS in retinal Müller cells. This increase was blocked if IOP was acutely elevated for 24 hours but was restored if IOP remained elevated for 1 week. This finding suggests that moderate elevation of IOP causes only short-term functional changes of glutamate metabolism (amidation) by retinal Müller cells. However, it is not known to what extent endogenous extracellular glutamate can regulate GS expression in normal eyes or in eyes with glaucoma.