The case for autoimmunity in glaucoma.

Although the majority of patients with glaucoma have elevated intraocular pressure as the presumed etiology for their resultant neuropathy, it is well known that approximately 25% of patients with glaucoma have intraocular pressure within the normal range for their race. These patients may have conditions that facilitate non-pressure related stress to the retina and optic nerve that might directly contribute to their glaucomatous neuropathy and include chronic or intermittent ischemia (i.e atherosclerosis, heart disease, vasospasm, migraine, sleep apnea), altered scleral/optic nerve head morphology that predisposes to glaucomatous stress (i.e myopia); genetic mutations that predispose to glaucoma damage at normal IOP (OPA-1,optineurin, myocilin) and evidence of aberrant immunity that suggests that their glaucoma might be a form of an autoimmune neuropathy (i.e. presumed autoimmune glaucoma). This review provides a critical assessment of the potential role for autoimmunity as an initiating or exacerbating etiology in some patients with glaucoma.

Regulation of gap junction coupling in bovine ciliary epithelium.

Aqueous humor is formed by fluid transfer from the ciliary stroma sequentially across the pigmented ciliary epithelial (PE) cells, gap junctions, and nonpigmented ciliary epithelial (NPE) cells. Which connexins (Cx) contribute to PE-NPE gap junctional formation appears species specific. We tested whether small interfering RNA (siRNA) against Cx43 (siCx43) affects bovine PE-NPE communication and whether cAMP affects communication. Native bovine ciliary epithelial cells were studied by dual-cell patch clamping, Lucifer Yellow (LY) transfer, quantitative polymerase chain reaction with reverse transcription (qRT-PCR), and Western immunoblot. qRT-PCR revealed at least 100-fold greater expression for Cx43 than Cx40. siCx43 knocked down target mRNA expression by 55 +/- 7% after 24 h, compared with nontargeting control siRNA (NTC1) transfection. After 48 h, siCx43 reduced Cx43 protein expression and LY transfer. The ratio of fluorescence intensity (R(f)) in recipient to donor cell was 0.47 +/- 0.09 (n = 11) 10 min after whole cell patch formation in couplets transfected with NTC1. siCx43 decreased R(f) by approximately 60% to 0.20 +/- 0.07 (n = 13, P < 0.02). Dibutyryl-cAMP (500 microM) also reduced LY dye transfer by approximately 60%, reducing R(f) from 0.41 +/- 0.05 (n = 15) to 0.17 +/- 0.05 (n = 20) after 10 min. Junctional currents were lowered by approximately 50% (n = 6) after 10-min perfusion with 500 microM dibutyryl-cAMP (n = 6); thereafter, heptanol abolished the currents (n = 5). Preincubation with the PKA inhibitor H-89 (2 microM) prevented cAMP-triggered current reduction (n = 6). We conclude that 1) Cx43, but not Cx40, is a major functional component of bovine PE-NPE gap junctions; and 2) under certain conditions, cAMP may act through PKA to inhibit bovine PE-NPE gap junctional communication.

Induced autoimmunity to heat shock proteins elicits glaucomatous loss of retinal ganglion cell neurons via activated T-cell-derived fas-ligand.

Glaucomatous optic neuropathy causes blindness through the degeneration of retinal ganglion cells (RGCs) and their axons, which comprise the optic nerve. Glaucoma traditionally is associated with elevated intraocular pressure, but often occurs or may progress with intraocular pressure in the normal range. Like other diseases of the CNS, a subset of glaucoma has been proposed to involve an autoimmune component to help explain the loss of RGCs in the absence of elevated intraocular pressure. One hypothesis involves heat shock proteins (HSPs), because increased serum levels of HSP autoantibodies are prominent in some glaucoma patients with normal pressures. In the first direct support of this hypothesis, we found that HSP27 and HSP60 immunization in the Lewis rat induced RGC degeneration and axon loss 1-4 months later in vivo in a pattern with similarities to human glaucoma, including topographic specificity of cell loss. Infiltration of increased numbers of T-cells in the retina occurred much earlier, 14-21 d after HSP immunization, and appeared to be transient. In vitro studies found that T-cells activated by HSP immunization induced RGC apoptosis via the release of the inflammatory cytokine FasL, whereas HSP immunization induced activation of microglia cells and upregulation of the FasL receptor in RGCs. In summary, our results suggest that RGC degeneration in glaucoma for selected individuals likely involves failed immunoregulation of the T-cell-RGC axis and is thus a disturbance of both proapoptotic and protective pathways.

Immunoregulation of retinal ganglion cell fate in glaucoma.

Glaucomatous neurodegeneration has been associated with the activation of multiple pathogenic mechanisms that can result in RGC death and axonal degeneration. Growing evidence obtained from clinical and experimental studies over the last decade also strongly suggests the involvement of the immune system in the neurodegenerative process of glaucoma. The roles of the immune system in glaucoma have been described as either neuroprotective or neurodestructive. It has been proposed that a critical balance between beneficial protective immunity and harmful sequelae of autoimmune neurodegenerative injury determines the ultimate fate of RGCs in response to various stressors in patients with glaucoma. Here, we review the key role for immunoregulation in cell fate decisions regarding RGC survival in response to glaucomatous tissue stress. Furthermore, we review the mechanisms by which autoimmunity to specific antigens such as heat shock proteins may result in RGC demise in some patients with glaucoma. In these patients, we hypothesized that one form of glaucoma may be an autoimmune optic neuropathy in which an individual's immune system facilitates a somatic or axonal degeneration of RGCs by the very system which normally serves to protect it against stress.

The effects of moxifloxacin ophthalmic solution 0.5% or gatifloxacin ophthalmic solution 0.3% treatment on corneal wound healing in pigmented rabbits following anterior keratectomy.

PURPOSE: These studies examined corneal healing rates, Type-IV collagen and zonula occludens membrane-associated protein (ZO-1) expression, as well as aqueous PGE(2) and IL-1 beta concentrations in pigmented rabbits treated with either moxifloxacin 0.5%, gatifloxacin 0.3% or BSS following anterior keratectomy. METHODS: Anterior keratectomy surgery was followed by topical administration with commercial ophthalmic formulations of either moxifloxacin or gatifloxacin or BSS (TID for 96 h). Images of the fluorescein-stained healing corneas were analyzed for wound area. At 48 or 96 h following surgery, aqueous humor samples were collected and analyzed for the inflammatory mediators PGE(2) and IL-1 beta using an ELISA. The corneas were subsequently evaluated using both scanning and transmission electron microscopy. In a second parallel study, corneas were evaluated at both 48 and 96 h for Type-IV collagen and ZO-1 expression using immunohistochemistry. RESULTS: Fluorescein-stained corneal images at 96 h postsurgery demonstrated that 90% +/- 8% re-epithelialization for moxifloxacin, 81% +/- 14% for gatifloxacin, and 88 +/- 6% for BSS((R)) (P > 0.05). PGE(2 )levels in the aqueous humor of fluoroquinolone treated eyes were reduced at 48 h compared to BSS treated eyes. IL-1 beta was undetectable in all samples. No differences in Type-IV collagen or ZO-1 expression were observed between any treatment groups. There were no differences between groups in histological appearance or in ultrastructural healing processes. CONCLUSIONS: These studies demonstrated that the commercial ophthalmic formulations of moxifloxacin and gatifloxacin were similar to each other in their effects on the levels of aqueous humor PGE(2) and rates of corneal wound re-epithelialization.

Glaucoma.

Glaucoma is a chronic neurodegenerative disease of the optic nerve, in which apoptosis of retinal ganglion cells (RGCs) and progressive loss of optic nerve axons result in structural and functional deficits in glaucoma patients. This neurodegenerative disease is indeed a leading cause of blindness in the world. The glaucomatous neurodegenerative environment has been associated with the activation of multiple pathogenic mechanisms for RGC death and axon degeneration. Growing evidence obtained from clinical and experimental studies over the last decade also strongly suggests the involvement of the immune system in this neurodegenerative process. Paradoxically, the roles of the immune system in glaucoma have been described as either neuroprotective or neurodestructive. A balance between beneficial immunity and harmful autoimmune neurodegeneration may ultimately determine the fate of RGCs in response to various stressors in glaucomatous eyes. Based on clinical data in humans, it has been proposed that one form of glaucoma may be an autoimmune neuropathy, in which an individual's immune response facilitates a somatic and/or axonal degeneration of RGCs by the very system which normally serves to protect it against tissue stress.

Aqueous humor sCD44 concentration and visual field loss in primary open-angle glaucoma.

PURPOSE: To correlate aqueous humor soluble CD44 (sCD44) concentration, visual field loss, and glaucoma risk factors in primary open-angle glaucoma (POAG) patients. METHODS: Aqueous samples were obtained by paracentesis from normal and glaucoma patients who were undergoing elective surgery and analyzed for sCD44 concentration by enzyme-linked immunosorbent assay. RESULTS: In normal aqueous (n=124) the sCD44 concentration was 5.88+/-0.27 ng/mL, whereas in POAG aqueous (n=90) the sCD44 concentration was 12.76+/-0.66 ng/mL, a 2.2-fold increase (P<0.000001). In POAG patients with prior successful filtration surgery (n=13), the sCD44 concentration was decreased by 43% to 7.32+/-1.44 (P=0.001) in comparison with POAG patients without filtration surgery; however, the sCD44 concentration in the prior successful filtration subgroup with no medications and normal intraocular pressure was 12.62+/-3.81 (P=0.05) compared with normal. The sCD44 concentration of normal pressure glaucoma patients was 9.19+/-1.75 ng/mL, a 1.6-fold increase compared with normal (P=0.02). Race and intraocular pressure pulse amplitude were significant POAG risk factors in this cohort of patients. In both normal and POAG patients with mild and moderate visual field loss, sCD44 concentration was greater in African Americans than in whites (P=0.04). CONCLUSIONS: sCD44 concentration in the aqueous of POAG patients correlated with the severity of visual field loss in all stages in white patients and in mild to moderate stages in African American patients. sCD44 concentration in aqueous is a possible protein biomarker of visual field loss in POAG.

Corneal wound healing in New Zealand White Rabbits following anterior keratectomy and treatment with moxifloxacin ophthalmic solution 0.5% or gatifloxacin ophthalmic solution 0.3%.

PURPOSE: These studies examined corneal reepithelialization rates and type IV collagen expression in rabbits treated with either moxifloxacin HCl ophthalmic solution 0.5% as base or gatifloxacin 0.3% ophthalmic solution following anterior keratectomy. METHODS: Animals (n = 6 per group) underwent surgery to create an 8-mm anterior keratectomy in the right eye. Rabbits were subsequently dosed with 1 drop, 3 times per day for 4 days with either moxifloxacin, gatifloxacin, or a commercially available irrigating solution. Fluorescein images were collected daily for the duration of the study. Approximately 96 h following surgery, the eyes were processed and evaluated for the presence of type IV collagen using immunohistochemical techniques. In two similar parallel studies, epithelial tissues were collected after the 48-h slit-lamp examination for a quantitative comparison of type IV collagen using either Western blot or quantitative polymerase chain reaction (Q-RT-PCR) techniques. RESULTS: Analysis of fluorescein images demonstrated that there were no significant differences in reepithelialization rates between the groups at any time point. At 96 h, 87%+/- 8% reepithelialization for moxifloxacin-treated eyes was observed compared with 77%+/- 10% for gatifloxacin-treated eyes and 85%+/-14% for BSS-treated eyes. The wound healing rates for the parallel studies demonstrated similar levels of reepithelialization for all groups. No discernable differences in type IV collagen expression were observed between treatment groups in the animals. The Q-RT-PCR analysis yielded no significant quantifiable difference in type IV collagen expression between any of the treatment groups. Expression values for alpha1 type IV collagen relative to the 18 S ribosomal RNA control were 0.0306+/-0.005 for BSS, 0.0251+/-0.002 for moxifloxacin, and 0.0254+/-0.006 for gatifloxacin. CONCLUSIONS: These studies indicate that there are no significant differences in corneal reepithelialization rates and type IV collagen expression between moxifloxacin ophthalmic solution 0.5%, gatifloxacin ophthalmic solution 0.3%, and the commercially available irrigating solution in this anterior keratectomy model.

Serum autoantibodies to alpha-fodrin are present in glaucoma patients from Germany and the United States.

PURPOSE: Glaucoma is characterized by a progressive loss of retinal ganglion cells that results in a characteristic optic neuropathy associated with visual field loss. In previous studies, changes in the antibody profiles have been shown in the sera of patients with glaucoma, and these findings suggest a role for autoimmune involvement in the pathogenesis of glaucoma in some patients. The purpose of this study was to compare the antibody profiles against optic nerve antigens in patients with glaucoma in two different study populations from Germany and the United States. METHODS: One hundred twenty patients were included in the study, 60 from Germany and 60 from the United States: a control group (CTRL, n = 20), a group of patients with primary open-angle glaucoma (POAG, n = 20), and one group of patients with normal-pressure glaucoma (NPG, n = 20) from each country. Western blot analyses against bovine optic nerve antigens were used to detect the IgG antibody patterns present in the patients' sera. The complex antibody profiles were analyzed by multivariate statistical techniques. RESULTS: Complex IgG autoantibody repertoires were present in all patients with glaucoma as well as healthy subjects from both the German and the United States study population. A large similarity between all antibody profiles in both study populations was demonstrated in the number and frequency of both up- and downregulation of antibody reactivities in patients with glaucoma of both national cohorts. The multivariate analysis of discriminance found a significant difference between the glaucoma groups and healthy subjects against optic nerve antigens. As in previous studies, the NPG group revealed the highest variance from the control group (P < 0.01). Furthermore, a newly described antibody biomarker in both study populations was identified as alpha-fodrin. Western blot results revealed that there was an increased frequency and enhanced immunoreactivity to alpha-fodrin (120 kDa) in the sera of patients with NPG. The presence of alpha-fodrin autoantibodies were confirmed by ELISA, in which a highly elevated anti-alpha-fodrin titer in patients with NPG was found to be significantly greater than in the control subjects (P < 0.01) or age-matched patients with POAG (P < 0.04). CONCLUSIONS: Complex IgG antibody patterns against optic nerve antigens can be reproducibly identified in the serum of study populations from the United States and Germany. In both cohorts, patients with glaucoma have characteristic differences in serum autoantibody repertoires from those in control subjects. A newly described autoantibody to alpha-fodrin found in other neurodegenerative diseases such as Alzheimer's, further implicate a role for autoimmunity and the neurodegenerative processes in glaucoma. The high correspondence of the autoantibody patterns found in the study populations from different continents provides further evidence that serum autoantibody patterns may be useful biomarkers for glaucoma detection or for determining prognosis in future studies by means of pattern-matching algorithms.

Rubeosis and anterior segment ischemia associated with systemic cryoglobulinemia.

PURPOSE: To report two cases of iris neovascularization associated with systemic cryoglobulinemia. DESIGN: Retrospective case report. METHODS: Patient chart review and review of literature. RESULTS: Two patients with iris neovascularization in the absence of retinal ischemia were subsequently found to have systemic cryoglobulinemia. Successful treatment of one patient's underlying lymphoma led to stabilization and resolution of neovascularization. CONCLUSIONS: Systemic cryoglobulinemia may be associated with anterior segment ischemia and neovascularization, and should be considered in the differential diagnosis of iris neovascularization in the absence of apparent retinal ischemia.

Administration of Menadione, Vitamin K3, Ameliorates Off-Target Effects on Corneal Epithelial Wound Healing Due to Receptor Tyrosine Kinase Inhibition.

PURPOSE: The antiangiogenic receptor tyrosine kinase inhibitor (RTKi), 3-[(4-bromo-2,6-difluorophenyl)methoxy]-5-[[[[4-(1-pyrrolidinyl) butyl] amino] carbonyl]amino]-4-isothiazolecarboxamide hydrochloride, targets VEGFR2 (half maximal inhibitory concentration [IC50] = 11 nM); however, off-target inhibition of epidermal growth factor receptor (EGFR) occurs at higher concentrations. (IC50 = 5.8 µM). This study was designed to determine the effect of topical RTKi treatment on EGF-mediated corneal epithelial wound healing and to develop new strategies to minimize off-target EGFR inhibition. METHODS: In vitro corneal epithelial wound healing was measured in response to EGF using a transformed human cell line (hTCEpi cells). In vivo corneal wound healing was assessed using a murine model. In these complementary assays, wound healing was measured in the presence of varying RTKi concentrations. Immunoblot analysis was used to examine EGFR and VEGFR2 phosphorylation and the kinetics of EGFR degradation. An Alamar Blue assay measured VEGFR2-mediated cell biology. RESULTS: Receptor tyrosine kinase inhibitor exposure caused dose-dependent inhibition of EGFR-mediated corneal epithelial wound healing in vitro and in vivo. Nanomolar concentrations of menadione, a vitamin K3 analog, when coadministered with the RTKi, slowed EGFR degradation and ameliorated the inhibitory effects on epithelial wound healing both in vitro and in vivo. Menadione did not alter the RTKi's IC50 against VEGFR2 phosphorylation or its inhibition of VEGF-induced retinal endothelial cell proliferation. CONCLUSIONS: An antiangiogenic RTKi exhibited off-target effects on the corneal epithelium that can be minimized by menadione without deleteriously affecting its on-target VEGFR2 blockade. These data indicate that menadione has potential as a topical supplement for individuals suffering from perturbations in corneal epithelial homeostasis, especially as an untoward side effect of kinase inhibitors.

Rapid Identification of Novel Inhibitors of the Human Aquaporin-1 Water Channel.

Aquaporins (AQPs) are a family of membrane proteins that function as channels facilitating water transport in response to osmotic gradients. These play critical roles in several normal physiological and pathological states and are targets for drug discovery. Selective inhibition of the AQP1 water channel may provide a new approach for the treatment of several disorders including ocular hypertension/glaucoma, congestive heart failure, brain swelling associated with a stroke, corneal and macular edema, pulmonary edema, and otic disorders such as hearing loss and vertigo. We developed a high-throughput assay to screen a library of compounds as potential AQP1 modulators by monitoring the fluorescence dequenching of entrapped calcein in a confluent layer of AQP1-overexpressing CHO cells that were exposed to a hypotonic shock. Promising candidates were tested in a Xenopus oocyte-swelling assay, which confirmed the identification of two lead classes of compounds belonging to aromatic sulfonamides and dihydrobenzofurans with IC50 s in the low micromolar range. These selected compounds directly inhibited water transport in AQP1-enriched stripped erythrocyte ghosts and in proteoliposomes reconstituted with purified AQP1. Validation of these lead compounds, by the three independent assays, establishes a set of attractive AQP1 blockers for developing novel, small-molecule functional modulators of human AQP1.

Noninvasive measurement of rodent intraocular pressure with a rebound tonometer.

PURPOSE: The present study evaluated the applicability of a rebound tonometer in measuring intraocular pressure (IOP) in rats and mice. METHODS: The accuracy of the TonoLab rebound tonometer was determined in cannulated mouse and rat eyes. IOP was manipulated by changing reservoir height, and tonometer pressure readings were recorded by an independent observer. IOP values were recorded in conscious Wistar rats and in four different strains of mice. The effects of anesthesia on IOP were evaluated in two different strains of mice. RESULTS: The IOP readings generated by the rebound tonometer correlated very well with the actual pressure in the eye. In rats, this linear correlation had a slope of 0.96 +/- 0.05 (mean +/- SEM, n = 4) and a Y-intercept of -2.1 +/- 1.2. In mice, the slope was 0.99 +/- 0.05 (n = 3), and the Y-intercept was 0.8 +/- 1.4. Using this method, the resting IOP of conscious male Wistar rats was observed to be 18.4 +/- 0.1 mm Hg (n = 132). In mice, strain differences in IOP were detected. Baseline IOP values in Balb/c, C57-BL/6, CBA, and 11- to 12-month-old DBA/2J mice were 10.6 +/- 0.6, 13.3 +/- 0.3, 16.4 +/- 0.3, and 19.3 +/- 0.4 mm Hg (n = 12), respectively. In separated studies, anesthesia lowered IOP from 14.3 +/- 0.9 to 9.2 +/- 0.5 mm Hg (n = 8) in C57-BL/6 mice, and from 16.6 +/- 0.4 to 9.4 +/- 0.6 mm Hg (n = 10) in CBA mice. CONCLUSIONS: The rebound tonometer was easy to use and accurately measured IOP in rats and mice. This technique, together with advances in genetic and other biological studies in rodents, will be valuable in the further understanding of the etiology and pathology of glaucoma.

Neurobiology of glaucomatous optic neuropathy: diverse cellular events in neurodegeneration and neuroprotection.

Although glaucomatous optic nerve degeneration is a leading cause of worldwide blindness, neither the precise cellular mechanisms underlying neurodegeneration in glaucoma, nor effective strategies for neuroprotection are yet clear. This review focuses on diverse cellular events associated with glaucomatous neurodegeneration whose balance is critical for determination of ultimate cell fate. An improved understanding of the site of primary injury to optic nerve, the mediator pathways of apoptotic cell death and intrinsic protection mechanisms in retinal ganglion cells, the role of glial activation on the survival and death of retinal ganglion cell bodies and their axons, and the protective and destructive consequences of immune system involvement can facilitate development of effective neuroprotective strategies in glaucoma.

Immunohistochemical assessment of the glial mitogen-activated protein kinase activation in glaucoma.

PURPOSE: To determine whether retinal glial cells exhibit an activated phenotype in glaucomatous human eyes and whether the mitogen-activated protein kinases (MAPKs) are associated with glial activation in glaucoma. METHODS: Activated phenotypes of retinal macroglia (astrocytes and Müller cells) and microglia were identified by morphologic assessment and immunostaining for the cell markers glial fibrillary acidic protein (GFAP) and HLA-DR, respectively, in 30 eyes obtained from glaucomatous donor eyes in comparison with normal control eyes from 20 age-matched donors. Cellular localization of the activated forms of MAPKs, including extracellular signal-regulated kinases (ERK), c-Jun amino(N)-terminal kinase (JNK), and p38, were studied in the retina of these eyes by immunoperoxidase staining and double immunofluorescence labeling with phosphorylation site-specific antibodies. RESULTS: Retinal astrocytes and Müller cells exhibited a hypertrophic morphology and increased immunostaining for GFAP in the glaucomatous retina. Although an increase was detectable in the number and size of cells positive for HLA-DR immunostaining in the glaucomatous retina compared with the control retina, microglial activation was not as prominent or widespread as the macroglial activation detected in the same eyes. The intensity of immunostaining and the number of immunostained cells for the activated MAPKs were greater in retina sections from glaucomatous eyes than in control eyes, being most prominent for phospho-ERK. Double immunofluorescence labeling demonstrated that the increased retinal immunostaining for phospho-ERK was predominantly, but not exclusively, localized to glial cells, whereas, the immunostaining for phospho-JNK or phospho-p38 was mainly associated with nonglial cells. CONCLUSIONS: These findings provide evidence that retinal glial cells undergo activation in the glaucomatous human retina. A prominent and persistent activation of ERK in activated glial cells suggests that this signaling pathway is probably associated with the induction and/or maintenance of the activated glial phenotype in glaucoma. Because MAPKs are involved in determination of ultimate cell fate, their differential activity in neuronal and activated glial cells in the glaucomatous retina may be associated, in part, with the differential susceptibility of these cell types to glaucomatous injury.

Interleukin-10 receptor signaling through STAT-3 regulates the apoptosis of retinal ganglion cells in response to stress.

PURPOSE: Interleukin (IL)-10 has recently been shown to promote survival of neurons and glia. The purpose of this report is to investigate whether IL-10 has any role in protecting retinal ganglion cells (RGCs) from death under conditions in which growth factors are removed, or in which oxidative stress is present. Signal transduction pathways that activate IL-10 signaling in RGCs were studied in both stress conditions. METHODS: Effects of various interleukins on the viability of the RGC cell line was determined, and apoptotic cells were quantified. Immunoblot analysis was preformed to identify the IL-10 receptor (IL-10R) and phosphorylated or nonphosphorylated Akt and STAT-3 proteins in RGC extracts. Immunohistochemistry was performed on the rat retinal sections to identify native IL-10R. RESULTS: Apoptosis of RGCs in the absence of growth factors with or without dexamethasone (1 microM) occurred in 68.5% +/- 3.4% and 53.4% +/- 2.6% of cells, respectively, after 96 hours. Addition of IL-10 at a concentration of 50 ng/mL significantly reduced the apoptotic population of RGCs to 28.2% +/- 2.3% in the absence of growth factors with dexamethasone and to 31% +/- 2.7% in the absence of growth factors alone. RGCs as well as native retina expressed functional IL-10R as determined by immunoblot analysis and by the ability of IL-10 to phosphorylate Stat-3. However, IL-10 failed to phosphorylate Akt in these cells. CONCLUSIONS: IL-10 caused a 59% and 42% reduction in the apoptotic population of serum-deprived cells with and without dexamethasone treatment, respectively. These observations establish that activation of IL-10R promotes survival of RGCs and this survival-promoting activity is due to IL-10 signaling through the Stat-3 pathway, which inhibits the cell death and not through the Akt cell survival pathway.

Detection of the free acid of bimatoprost in aqueous humor samples from human eyes treated with bimatoprost before cataract surgery.

PURPOSE: To determine whether bimatoprost is hydrolyzed to its free acid after topical application in humans in vivo. DESIGN: Prospective, masked, and vehicle controlled. PARTICIPANTS: Thirty-one eyes of 31 patients with cataracts. METHODS: Beginning 7 days before scheduled cataract surgery, one eye of each patient was treated with bimatoprost 0.03% or vehicle once daily, with the last drop administered 2 to 12 hours before anterior chamber paracentesis before cataract surgery. In a masked fashion, aqueous humor specimens were assayed for bimatoprost and its free acid by high-pressure liquid chromatography and mass spectrometry. MAIN OUTCOME MEASURE: Detection of the free acid of bimatoprost in aqueous humor. RESULTS: Aqueous humor concentrations of the free acid of bimatoprost were 22.0+/-7.0 nmol/l (mean +/- standard error of the mean, n = 12) and 7.0+/-4.6 nmol/l (n = 8) at 2 and 12 hours, respectively, and below the limit of detection after vehicle (n = 10). Concentrations of bimatoprost (amide) were 5.7+/-1.4 and 1.1+/-0.4 nmol/l at 2 and 12 hours, respectively, and undetectable after vehicle. CONCLUSION: After topical application of bimatoprost in humans, a sufficient concentration of its free acid, a potent FPprostanoid receptor agonist, is found in the aqueous humor to account for its ability to reduce intraocular pressure.

Hypoxia-inducible factor 1alpha in the glaucomatous retina and optic nerve head.

OBJECTIVE: To examine tissue hypoxia in the retina and optic nerve head of glaucomatous eyes by the assessment of a transcription factor, hypoxia-inducible factor 1alpha (HIF-1alpha), which is tightly regulated by the cellular oxygen concentration. METHODS: Using immunohistochemical analysis, the cellular localization of HIF-1alpha was studied in the retina and optic nerve head of 28 human donor eyes with glaucoma compared with 20 control eyes from healthy donors matched for several characteristics. The relationship between the retinal regions that exhibited immunostaining for HIF-1alpha and functional damage was examined using visual field data. RESULTS: There was an increase in the immunostaining for HIF-1alpha in the retina and optic nerve head of glaucomatous donor eyes compared with the control eyes. In addition, the retinal location of the increased immunostaining for HIF-1alpha in some of the glaucomatous eyes was closely concordant with the location of visual field defects recorded in these eyes. CONCLUSIONS: Because the regions of HIF-1alpha induction represent the areas of decreased oxygen delivery and hypoxic stress, information obtained from this study provides direct evidence that tissue hypoxia is present in the retina and optic nerve head of glaucomatous eyes, and hypoxic signaling is a likely component of the pathogenic mechanisms of glaucomatous neurodegeneration. Clinical Relevance These findings support the presence of tissue hypoxia in the retina and optic nerve head of glaucomatous patients.

Gold standard medical therapy for glaucoma: defining the criteria identifying measures for an evidence-based analysis.

BACKGROUND: Over the past decade, several new medical therapies have become available for the treatment of primary open-angle glaucoma (POAG). A systematic evidence-based approach for identifying an optimal therapeutic agent is lacking. OBJECTIVES: The aims of this review were to critically evaluate published treatment recommendations for POAG and, based on a systematic review of the literature, to develop criteria that would define a "gold standard" medical therapy that reflects new treatment advances and established therapeutic goals. METHODS: A MEDLINE search spanning the years 1966 to 2002 and using the search terms gold standard, drug of choice, agent of choice, benchmark, ophthalmology, eye, and glaucoma was conducted and the results reviewed by a panel of 15 experts in the field of glaucoma. Published treatment recommendations for POAG were discussed. Criteria, anchored to medical evidence, for distinguishing a standard of medical therapy for POAG were defined. RESULTS: The terms connoting a gold standard therapy were found in only 258 of approximately 368,000 ophthalmology-related citations and 53 of almost 23,000 glaucoma citations, validating the need to define therapeutic standards. The lack of recommendations for the use of new classes of ocular hypotensive agents was acknowledged. Criteria identified to evaluate intraocular pressure (IOP)-lowering agents as gold standards included the following: efficacy in reducing IOP consistently over a 24-hour period to a level that will preserve the visual field and protect the optic nerve without inducing tachyphylaxis and tolerance, paucity of local and systemic adverse effects, promotion of patient compliance, and applicability in diverse patient populations. CONCLUSIONS: These criteria should be employed as measures for evidence-based analyses to evaluate available and future IOP-lowering medical therapies for POAG. The conceptual framework presented may be applicable to other therapeutic areas.

Role of tumor necrosis factor receptor-1 in the death of retinal ganglion cells following optic nerve crush injury in mice.

To assess the specific role of tumor necrosis factor (TNF) death receptor signaling in the induction of retinal ganglion cell (RGC) death, optic nerves of mice deficient for TNF receptor-1 (TNF-R1-/-) and control mice (C57BL/6J) were unilaterally subjected to crush injury. Counts of RGCs and their axons 6 weeks after the injury demonstrated that their loss was significantly less in TNF-R1-/- mice compared to controls. The most prominent decrease in neuronal loss detected in TNF-R1-/- mice was beyond the initial 2-week period after the injury. This time period was correlated with the period of glial activation and increased glial immunolabeling for TNF-alpha in these eyes. No further protection against neuronal loss was detectable in TNF-R1-/- mice treated with D-JNKI1, a specific inhibitor of c-Jun N-terminal protein kinase (JNK). However, anti-JNK treatment of control animals provided a significant protection against neuronal loss during the same secondary degeneration period. Phospho-JNK immunolabeling of RGCs in control mice subjected to optic nerve crush significantly decreased following their treatment with D-JNKI1, and anti-JNK treatment protected RGCs from degeneration in these animals, similar to the lack of TNF-R1. These findings provide evidence that TNF death receptor signaling is involved in the secondary degeneration of RGCs following optic nerve injury, and is associated with JNK signaling. Since secondarily degenerating neurons are viable targets for neuroprotection, inhibition of TNF death receptor signaling may be an effective strategy to protect RGCs in several neurodegenerative injuries.