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Purpose: To assess the combined estimate of retinal ganglion cell (RGC) count developed by Medeiros et al. as a tool for diagnosis of glaucoma in Japanese patients., Study Design: Cross-sectional study., Methods: Thirty-one eyes of 19 healthy controls and 106 eyes of 70 glaucoma patients underwent standard automated perimetry (SAP) and three types of spectral domain optical coherence tomography (SD-OCT) imaging using the Cirrus, RTVue, and 3D-OCT instruments. RGC counts derived from SAP and SD-OCT data were estimated using the Harwerth model (SAPrgc and OCTrgc, respectively), from which the combined RGC count estimates (CRGC) were calculated using the formula developed by Medeiros et al. Receiver operating characteristic curve (ROC) analyses were conducted for mean deviation (MD), retinal nerve fiber layer thickness (RNFLT), and CRGC., Results: The mean OCTrgc derived from the Cirrus, RTVue, and 3D-OCT instruments were 1150, 1245, and 1316 (× 1000 cells), respectively, for the control group and 463, 519, and 516 (× 1000 cells), respectively, for the patient group. SAPrgc of the controls' group was 1526 and the patients' group, 731 (× 1000 cells), and were consistently greater than OCTrgc in both groups (a generalized estimating equation model, p < 0.001). Partial area under the curve (pAUC) of MD was 0.178, and that of RNFLT and CRGC for the three OCT instruments were 0.185, 0.18, 0.189 and 0.196, 0.196, 0.197, respectively. CRGC had larger pAUC than MD, whereas there was no or marginal difference in pAUC between CRGC and cpRNFLT, irrespective of OCT device used or glaucoma severity., Conclusion: CRGC proved well suited to discriminate glaucoma patients from controls. However, its clinical utility did not seem to overwhelm isolated structural measures in the tested Japanese patients.

Purposes: To evaluate the effects of axial length and age on the circumpapillary retinal nerve fiber layer (cpRNFL) and the inner macular parameters measured using 3 spectral-domain optical coherence tomography (OCT) instruments., Methods: A total of 102 normal eyes (1 eye per subject) were imaged using Cirrus, RTVue, and 3D-OCT. The cpRNFL and the macular retinal nerve fiber layer (mRNFL), the ganglion cell layer+inner plexiform layer (GCLIPL), and the mRNFL+GCLIPL ganglion cell complex, GCC thicknesses were analyzed. The correlations between these values and the axial length or age were evaluated using a partial correlation analysis. These correlations were corrected using the axial length-related magnification effect., Results: All but the nasal quadrant cpRNFL thicknesses and GCC thicknesses obtained using the 3 OCT instruments were significantly correlated with age. The average cpRNFL thickness and GCC thickness measured using the Cirrus and RTVue, but not by the 3D-OCT, had a negative correlation with the axial length. The temporal quadrant cpRNFL thickness measured using the 3 instruments was positively correlated with the axial length. The magnification correction made the most correlations positive., Conclusions: The average cpRNFL and GCC thicknesses measured using these 3 instruments decreased with age. The axial length affected the cpRNFL and GCC thicknesses as measured using the Cirrus and RTVue; this effect likely depended on the fundus area of analyses.

Background/aims: Chiasmal compression affects the crossed nerve fibres originating from the nasal hemiretina, as opposed to the uncrossed fibres from the temporal hemiretina. The objectives were to evaluate circumpapillary retinal nerve fibre layer (cpRNFL) thickness by spectral-domain optical coherence tomography in eyes with band atrophy (BA) accompanying temporal hemianopia due to chiasmal damage and to estimate the distribution pattern of cpRNFL from the nasal hemiretina., Methods: This cross-sectional study included 53 eyes with optic neuropathy due to chiasmal lesions and 72 normal eyes. Visual field sensitivity (VFS) was evaluated by standard automated perimetry. Eyes with abnormalities in the nasal visual hemifield were excluded. The structure-function relationships (cpRNFL thickness and VFS in the temporal hemifield) were evaluated in eyes with BA. The base levels composed of only non-neuronal elements and cpRNFL from the temporal hemiretina were estimated in the average and 12 sector-cpRNFL thicknesses using regression analysis., Results: The base level in the average cpRNFL thickness was 71.2 µm in eyes with BA, which corresponded to 70% of average thickness of normal controls. However, the estimated base level of 12 sector-cpRNFL thicknesses represented the unique distribution pattern, in which base level-thickness localised at the 1 o'clock and 5 o'clock sectors was extensively reduced, with an even distribution of base levels at other sectors., Conclusions: The RNFL originating from the nasal hemiretina is estimated to enter into the optic disc predominantly at the 1 o'clock and 5 o'clock angles., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Aquaporin 9 (AQP9), an aquaglyceroporin, is not only permeable to water but also to non-charged solutes, such as lactate. Lactate can be an energy source for retinal neurons. This study aimed to evaluate the effect of the downregulation of AQP9 expression on the survival rates and reactive oxygen species accumulation in RGC-5 cells cultured in a medium containing lactate. The Live/Dead assay revealed that the cell death rate of RGC-5 cells transfected with the control siRNA (siControl) was 3.65%±0.75% in the 5-mM glucose medium. The death rate was significantly increased by five-fold in the no glucose and 10-mM d-lactate media but not in the 10-mM l-lactate medium. In comparison, the death rate of cells transfected with siRNA targeting AQP9 (siAQP9) was 8.07%±1.01% in the 5-mM glucose medium, which was significantly increased by two-fold in the other medium conditions, indicating that the downregulation of AQP9 expression eliminated the prosurvival effect of l-lactate. Few RGC-5 cells transfected with siControl showed dichlorofluorescein (DCF) fluorescence when cultured in 5-mM glucose and 10-mM l-lactate media. However, approximately 70% of those showed DCF fluorescence when cultured in the no glucose and 10-mM d-lactate media. The downregulation of AQP9 significantly increased the DCF fluorescence rate to 50.44%±6.13% in the 10-mM l-lactate medium, whereas, it did not increase the rate in the other medium conditions. These results demonstrate that AQP9 expression is required for l-lactate to maintain retinal neuronal survival., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Purpose: To compare the relationship between visual field sensitivity (VFS) and macular parameters measured using three spectral-domain optical coherence tomography (SD-OCT) instruments and to determine a base level (=floor effect) for macular parameters., Methods: We imaged 127 glaucomatous eyes (1 eye per subject) using three different OCT instruments, i.e., the Cirrus, RTVue and 3D OCT devices; 76 normal eyes were evaluated as controls using the same instruments. The thicknesses of the macular retinal nerve fiber layer (mRNFL), ganglion cell layer+inner plexiform layer (GCL/IPL), and mRNFL+GCL/IPL (GCC) were analyzed. The VFS of the area analyzed by OCT was expressed in decibels and the 1/Lambert scale. For each parameter, the structure-function relationship and the base level were evaluated by regression analysis. The strength of the correlations between the instruments was compared by the bootstrapping method., Results: All of the macular parameters evaluated exhibited statistically significant correlations with VFS. The average GCC measured by all three SD-OCT instruments and the average mRNFL thickness measured by the Cirrus and 3D OCT instruments had similar correlations with VFS. The average GCL/IPL thickness measured by the Cirrus OCT instrument was better correlated with VFS that was measured by the 3D OCT instrument (p = 0.031). The base level GCC thickness measured by all three instruments was approximately 65% of that of normal eyes. The base level mRNFL thickness measured with the Cirrus and OCT instruments was 52 and 48%, respectively, of that of normal eyes. The base level GCL/IPL thickness measured with the Cirrus and 3D instruments was 71 and 75%, respectively, of that of normal eyes., Conclusions: The three SD-OCT instruments evaluated showed similar structure-function relationships in terms of GCC and mRNFL measurements. The base levels of the macular parameters determined by the three instruments differed, due, at least partly, to the scanning area defined by each instrument.

Purpose: To assess the diagnostic performance of the macular parameters detected by spectral-domain optical coherence tomography (SD-OCT) in band atrophy (BA) eyes., Methods: Forty-nine BA eyes with permanent temporal hemianopia and 89 normal eyes were enrolled. Any patients who had nasal visual field loss were excluded. Each participant was imaged by three-dimensional (3D) OCT-2000, and 10 × 10 grids in the macula were automatically allocated. The thickness of the macular retinal nerve fiber layer (mRNFL), ganglion cell layer (GCL)+ (GCL+inner plexiform layer [IPL]), and GCL++ (RNFL+GCL+IPL) in both nasal and temporal hemiretina was calculated and compared between the BA and normal eyes. The areas under the receiver operating characteristic curves (AUCs) in these parameters were compared between the nasal hemiretina and the temporal hemiretina., Results: All the parameters in the BA eyes were significantly thinner than those in the normal eyes. The AUCs for the mRNFL, GCL+, and GCL++ thickness in the nasal hemiretina were 0.890, 0.988, and 0.981, respectively. The parameters in the nasal hemiretina showed significantly higher AUCs than those parameters in the temporal hemiretina. In the temporal hemiretina, the damaged grids in the mRNFL were located in the arcuate areas in each hemifield., Conclusions: The inner macular parameters in the nasal hemiretina exhibited high diagnostic abilities to detect BA. The GCL+ was more affected than mRNFL. The characteristic pattern of mRNFL and GCL+ thinning was implicated in the anatomical architecture regarding the nasotemporal overlap of the crossed and uncrossed fibers around the fovea. (www.umin.ac.jp/ctr number, UMIN000006900.)., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: Using spectral-domain optical coherence tomography (SD-OCT), we assessed the ability of cluster analyses, based on the grid-pattern of macular parameters, to detect glaucoma., Methods: We enrolled 75 normal eyes, 64 early glaucomatous eyes (EG), and 40 preperimetric glaucomatous eyes (PPG). Each participant was imaged using 3-dimensional optical coherence tomography (3D-OCT) to examine the macular retinal nerve fiber layer (mRNFL) and the thickness of the ganglion cell layer, together with the inner plexiform layer (GCL/IPL). Diagnostic criteria based on the clustering of abnormal grids from the mRNFL and GCL/IPL measurements were applied. The sensitivity and specificity of glaucoma detection were compared between the cluster criteria (CC) and the average thickness criteria (ATC) of total and hemiretinal sectors, and the cut-off criteria were determined using receiver operating characteristic (ROC) curve analyses from our normal controls., Results: The specificity values of CC and ATC from mRNFL measurements were 97% and 100%, respectively. The sensitivity of CC was 94% for EG and 68% for PPG. The sensitivity of ATC was 81% for EG and 38% for PPG. The specificity values of CC and ATC from GCL/IPL measurements were 96% and 100%, respectively. The sensitivity values of CC and ATC were 92% for EG and 63% for PPG. The sensitivity of ATC was 84% for EG and 25% for PPG. When compared to ATC and ROC-based cut-off criteria, CC showed a higher diagnostic capability., Conclusions: Judging abnormality based on a clustering of abnormal grids from macular OCT parameters may be a reliable approach for diagnosing early glaucoma. (http://www.umin.ac.jp/ctr/index/htm9 number, UMIN000006900.).

Purpose: To compare the ability of circumpapillary retinal nerve fiber layer (cpRNFL) thickness and macular parameters obtained by three spectral-domain optical coherence tomography (SD-OCT) instruments to detect highly myopic glaucoma., Methods: In this study, 84 glaucomatous eyes, 53 normal eyes with high myopia, and 86 normal eyes (not highly myopic) were enrolled. Each participant was imaged using Cirrus, RTVue, and 3D OCT to evaluate the average and quadrant cpRNFL thicknesses. The macular retinal nerve fiber layer (mRNFL), ganglion cell layer + inner plexiform layer (GCL/IPL), and mRNFL + GCL/IPL (GCC) thicknesses were analyzed. The areas under the receiver operating characteristic curves (AUCs) were compared between the instruments. In addition, the best parameters for the AUC were compared between the cpRNFL parameters and macular parameters in each instrument., Results: These analyses revealed similar AUCs for the average cpRNFL and GCC thicknesses between the instruments. RTVue displayed a significantly higher AUC in the nasal cpRNFL thickness than both Cirrus (P = 0.0004) and 3D OCT (P = 0.0006). 3D OCT showed higher AUCs than Cirrus in the mRNFL parameters. There were no significant differences between the best cpRNFL parameters and macular parameters in each instrument., Conclusions: The average cpRNFL and GCC thicknesses that were measured using these OCT instruments exhibited similar abilities for diagnosing highly myopic glaucoma, and RTVue exhibited better diagnostic abilities than Cirrus and 3D OCT for nasal cpRNFL. 3D OCT had better ability than Cirrus in the mRNFL. Both cpRNFL and GCC in each instrument were comparable, and their measurements offered good ability for diagnosing glaucoma with high myopia. (http://www.umin.ac.jp/ctr/index/htm9, UMIN000006900.).

Purpose: We compared the ability of circumpapillary retinal nerve fiber layer (cpRNFL) thickness and macular parameters obtained by three spectral-domain optical coherence tomography (SD-OCT) instruments to detect glaucoma., Methods: We enrolled 87 normal eyes and 145 glaucomatous eyes (75 early glaucomatous eyes (EGs), mean deviation > -6 dB). Each participant was imaged using Cirrus, RTVue, and 3D-OCT to evaluate the average and quadrant cpRNFL thicknesses. The macular retinal nerve fiber layer (mRNFL), ganglion cell layer plus inner plexiform layer (GCL/IPL), and mRNFL + GCL/IPL (ganglion cell complex [GCC]) thicknesses were analyzed. The areas under the receiver operating characteristic curves (AUCs) were compared among the instruments., Results: These instruments revealed similar AUCs for the average cpRNFL and GCC thicknesses in EGs, and total all-stage glaucomatous eyes (TGs). RTVue showed better performance in the nasal cpRNFL thickness than Cirrus and 3D-OCT, and better performance in the temporal cpRNFL thickness than 3D-OCT in TGs. RTVue had a higher AUC for the superior GCC thickness compared to Cirrus and 3D-OCT in EGs, and TGs. Cirrus had higher AUCs for GCL/IPL parameters in TGs, and lower AUCs for the mRNFL parameters in EGs and TGs compared to 3D-OCT., Conclusions: The average cpRNFL and GCC thicknesses measured using these OCT instruments exhibited similar abilities in the diagnosis of glaucoma, and RTVue exhibited better diagnostic abilities than Cirrus and 3D-OCT for nasal cpRNFL, and superior GCC thicknesses. The diagnostic performance of Cirrus and 3D-OCT was different for GCL/IPL and mRNFL parameters. (http://www.umin.ac.jp/ctr number, UMIN000006900.)

Aquaporin 9 (AQP9), an aquaglyceroporin belonging to the AQP water channel family, is permeable not only to water but also to noncharged solutes such as lactate. In neurons, lactate presumably acts as an energy substrate and as a source of NADH (the reduced form of nicotinamide adenine dinucleotide), a scavenger of reactive oxygen species (ROS). We previously reported that retinal ganglion cells (RGCs) express AQP9 and that elevated intraocular pressure reduces AQP9 expression and increases death of neurons in the retinal ganglion cell layer of rodents. In the present study, we investigated the association of AQP9 expression with serum deprivation-induced death of RGC-5 cells and with death of neurons in the rat retinal ganglion cell layer after optic nerve transection (ONT). The effect of AQP9 RNA interference on serum deprivation-induced apoptosis, ROS accumulation, and the NAD(+)/NADH ratio in RGC-5 cells was examined. Both serum deprivation and ONT significantly reduced AQP9 protein expression in RGCs and increased the rate of RGC death. Retinal AQP9 gene expression also declined after ONT. Down-regulation of AQP9 significantly increased apoptosis, ROS accumulation, and the NAD(+)/NADH ratio in the RGC-5 cells. These findings suggest that AQP9 loss adversely affects survival of RGCs, at least partly because of decreased transport of lactate as a substrate for energy and/or ROS scavenger., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: To compare the relationships of parapapillary retinal nerve fibre layer (RNFL) thickness among three spectral-domain optical coherence tomography (SD-OCT) instruments with visual field sensitivity (VFS)., Methods: One hundred and thirteen eyes of 113 Japanese subjects with glaucoma (mean deviation in standard automated perimetry = -8.9 ± 6.7 dB) were imaged by Cirrus, RTVue and 3D OCT. The average hemi-superior and hemi-inferior RNFL thicknesses were obtained. In addition, the structure-function relationship using two retinotopic maps that consisted of six or nine sectors was also evaluated. Decibel (dB) scale and 1/Lamert (1/L) were used to express VFS. RNFL thickness correlation with corresponding VFS in visual field sectors was compared among the three instruments. Background RNFL thickness (non-neuronal tissue) for each instrument was estimated using a linear regression model at 1/L = 0., Results: The correlation between the average hemi-superior and hemi-inferior RNFL thickness in the three SD-OCT instruments with corresponding VFS was similar. In the analysis of retinotopic maps, 3D OCT showed a higher correlation in superotemporal sectors than RTVue, whereas RTVue had a stronger relationship in the nasal sector than Cirrus. The background RNFL thickness extrapolated was approximately 60% that of normal eyes in all the instruments., Conclusions: The three SD-OCT showed similar overall correspondence with VFS with a partially discordant retinotopic relationship. RNFL measured with any SD-OCT devices likely contains non-neuronal tissue, comprising 60% of its thickness., (© 2013 The Authors. Acta Ophthalmologica © 2013 Acta Ophthalmologica Scandinavica Foundation.)

Background: Unilateral injury of the optic tract leads to asymmetrical optic atrophy in both eyes derived from the crossing of the nerve fibers at the chiasm. This report demonstrates unique imaging appearances of optic atrophy due to this uncommon condition detected by spectral-domain optical coherence tomography (SD-OCT)., Methods: Cirrus and RTVue measurements were performed in four cases of optic tract syndrome. Circumpapillary retinal nerve fiber layer (cpRNFL) thickness was obtained from both instruments and ganglion cell complex (GCC) integrity was obtained from RTVue. The presumable reduction rates of quadrant cpRNFL thickness were calculated from the published normative database and compared between eyes with temporal hemianopia and those with nasal hemianopia., Results: Both devices showed significant reduction of cpRNFL thickness, but did not have statistical difference in the reduction rates at temporal or nasal quadrant cpRNFL between contralateral and ipsilateral eyes to the lesion. Color-coded maps helped to visualize the unique pattern of cpRNFL and GCC thinning., Conclusions: SD-OCT can be used as a diagnostic tool for the optic tract syndrome.

Background: To assess the agreement and diagnostic performance between retinal nerve fiber layer (RNFL) thickness measurements obtained using the Cirrus (Carl Zeiss Meditec) and RTVue (Optovue Inc.) devices for detection of band atrophy (BA) in patients with permanent temporal hemianopia., Methods: In this retrospective study, 26 eyes with BA and 64 control eyes were enrolled. The Cirrus optic disc cube protocol and the RTVue optic nerve head map protocol were used. The Cirrus measurements were extracted and regrouped to be topographically matched with the RTVue measurements. Concordance correlation and 95 % limits of agreement were assessed. Areas under the receiver operating characteristic curves (AUC) and the Spearman's correlation coefficient between average Humphrey total deviation in the temporal hemifield and average RNFL thickness were calculated., Results: RTVue measured consistently thicker values than Cirrus in controls, whereas in eyes with BA, nasal segment measurements from the RTVue were thinner than those obtained using the Cirrus. Each quadrant showed moderate to close agreement in controls, whereas in eyes with BA, the nasal and temporal quadrants exhibited poor agreement. The RTVue measurements demonstrated significantly higher AUCs for nasal segments just above (0.95) and below (0.96) the horizontal meridian than Cirrus measurements (0.80 and 0.66, respectively) and a significant correlation with visual field loss (r(s) = 0.46, P = 0.02 for RTVue vs. r(s) = 0.26, P = 0.22 for Cirrus)., Conclusions: The RTVue RNFL thickness measurements in nasal sectors showed better diagnostic performance in detecting BA and higher correlations with temporal hemianopia than the Cirrus measurements.

Axonal injury and degeneration are pivotal pathological events in diseases of the nervous system. In the past decade, it has been recognized that the process of axonal degeneration is distinct from somal degeneration and that axoprotective strategies may be distinct from those that protect the soma. Preserving the cell body via neuroprotection cannot improve function if the axon is damaged, because the soma is still disconnected from its target. Therefore, understanding the mechanisms of axonal degeneration is critical for developing new therapeutic interventions for axonal disease treatment. We combined in vivo imaging with a multilaser confocal scanning laser ophthalmoscope and in vivo axotomy with a diode-pumped solid-state laser to assess the time course of Wallerian and retrograde degeneration of unmyelinated retinal ganglion cell axons in living rats for 4 weeks after intraretinal axotomy. Laser injury resulted in reproducible axon loss both distal and proximal to the site of injury. Longitudinal polarization-sensitive imaging of axons demonstrated that Wallerian and retrograde degeneration occurred synchronously. Neurofilament immunostaining of retinal whole-mounts confirmed axonal loss and demonstrated sparing of adjacent axons to the axotomy site. In vivo fluorescent imaging of axonal transport and photobleaching of labeled axons demonstrated that the laser axotomy model did not affect adjacent axon function. These results are consistent with a shared mechanism for Wallerian and retrograde degeneration., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: To evaluate the effects of ocular rotation on parapapillary retinal nerve fiber layer (RNFL) thickness measured by spectral-domain optical coherence tomography (SD-OCT)., Patients and Methods: Eighty-eight normal and 205 glaucomatous eyes were studied. RNFL thickness was measured by 3D OCT. Ocular rotation angle was measured from a fundus image obtained by a non-mydriatic fundus camera equipped with 3D OCT. The average, hemi-superior, and hemi-inferior RNFL thicknesses as well as those in the 4 quadrants and the 16-segmented superotemporal (ST-1) and inferotemporal (IT-4) sectors were compared both before and after correcting for ocular rotation. Receiver operating characteristic curves and the areas under the curve (AUC) for the RNFL thicknesses were calculated on the basis of the data from glaucomatous and control eyes. The relationships between RNFL thickness and retinal sensitivity in the corresponding visual field were analyzed using a Humphrey field analyzer., Results: Correction for ocular rotation did not affect the AUCs of the hemi-superior and hemi-inferior RNFL thicknesses. RNFL thicknesses in all of the quadrants and in ST-1 and IT-4 were significantly changed by correcting for ocular rotation. The correlations between all RNFL sectors and retinal sensitivity were not changed by correcting for the ocular rotation angle., Conclusions: Ocular rotation compensation affected RNFL thickness measurement with 3D OCT. However, the effect was clinically negligible in the diagnosis of glaucoma.

Backgrounds/aims: To evaluate the agreement of parapapillary retinal nerve fibre layer (RNFL) thickness among three spectral-domain optical coherence tomography (OCT) instruments., Methods: Two hundred and three glaucomatous eyes and 88 normal eyes were imaged by Cirrus, RTVue and 3D OCT. The average and the four quadrant RNFL thicknesses were evaluated. Agreement among RNFL measurements was evaluated using Bland-Altman analysis and linear regression analysis. The percentage of each quadrant in the average RNFL thickness value was compared among the three instruments., Results: Cirrus showed significantly smaller thickness values than RTVue (difference=8.8 μm, p<0.0001) and 3D OCT (difference=8.1 μm, p<0.0001). Although RNFL measurements among the instruments were highly correlated, the Bland-Altman analysis revealed proportional biases for most of the pair-wise agreements. Additionally, 3D OCT showed strong proportional biases with RTVue and 3D OCT. RTVue had a smaller occupied proportion of nasal quadrants (30.2%) and a larger proportion of inferior quadrants (32.4%) compared with Cirrus and 3D OCT., Conclusions: RNFL measurements among the instruments were well correlated but had different values for thickness. The measurement circle of RTVue might be more superior-temporally located compared with the other instruments. Differences in the measurement protocols might be affected by the disagreements. These instruments should not be used interchangeably.

Optic neuropathy is the leading cause of irreversible blindness, and a paradigm for central nervous system axonal disease. The primary event is damage to retinal ganglion cell axons, with subsequent death of the cell body by apoptosis. Trials of neuroprotection for these and other neuronal diseases have mostly failed, primarily because mechanisms of neuroprotection in animals do not necessarily translate to humans. We developed a methodology for imaging an intracellular transduction pathway that signals neuronal death in the living animal. Using longitudinal confocal scanning multilaser ophthalmoscopy, we identified the production of superoxide within retrograde-labelled rat retinal ganglion cells after optic nerve transection. Superoxide was visualized by real-time imaging of its reaction product with intravitreally administered hydroethidine and confirmed by differential spectroscopy of the specific product 2-hydroxyethidium. Retinal ganglion cell superoxide increased within 24 h after axotomy, peaking at 4 days, and was not observed in contralateral untransected eyes. The superoxide signal preceded phosphatidylserine externalization, indicating that superoxide generation was an early event and preceded apoptosis. Intravitreal pegylated superoxide dismutase blocked superoxide generation after axotomy and delayed retinal ganglion cell death. Together, these results are consistent with superoxide being an upstream signal for retinal ganglion cell apoptosis after optic nerve injury. Early detection of axonal injury with superoxide could serve as a predictive biomarker for patients with optic neuropathy.

Corroles are tetrapyrrolic macrocycles that have come under increased attention because of their unique capabilities for oxidation catalysis, reduction catalysis, and biomedical applications. Corrole-metal complexes (metallocorroles) can decompose certain reactive oxygen species (ROS), similar to metalloporphyrins. We investigated whether Fe-, Mn-, and Ga-corroles have neuroprotective effects on neurons and correlated this with superoxide scavenging activity in vitro and in vivo. Apoptosis was induced in retinal ganglion cell-5 neuronal precursor cells by serum deprivation. Cell death was measured with sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene-sulfonic acid hydrate and calcein-AM/propidium iodide assays. Fe- and Mn-corroles, but not the non-redox-active Ga-corrole used as control, reduced RGC-5 cell death after serum deprivation. Serum deprivation caused increased levels of intracellular superoxide, detected by an increase in the fluorescence intensity of 2-hydroxyethidium, and this was blocked by Fe- and Mn-corroles, but not Ga-corrole. In vivo real-time confocal imaging of retinas after optic nerve transection assessed the superoxide production within individual rat retinal ganglion cells. Fe- and Mn-corroles, but not Ga-corrole, scavenged neuronal superoxide in vivo. Given that the neuroprotective activity of metallocorroles correlated with superoxide scavenging activity, Fe- and Mn-corroles could be candidate drugs for delaying neuronal death after axonal injury in optic neuropathies, such as glaucoma.

Purpose. Optic nerve injury causes loss of retinal ganglion cells (RGCs) and their axons. The reduction in RGC counts over time in axonal injury is well studied, but the correlation with the timing of anterograde and retrograde axonal degeneration is less clear. The authors longitudinally imaged RGC axons stained with a chloromethyl derivative of fluorescein diacetate (CMFDA) in live rats after optic nerve injury. Methods. Optic nerves were transected. Three days later CMFDA was intravitreously injected. Confocal scanning laser ophthalmoscopy was performed daily, and mean fluorescence intensity and the number of CMFDA bundles were calculated. RGC soma survival was studied after retrograde fluorescence labeling. Retinal nerve fiber layer (RNFL) thickness was evaluated histologically. Results. CMFDA-positive RGC axon bundles could be imaged in vivo. Axons lost 68% +/- 29% of their fluorescence by 7 days after transection compared with 25% +/- 21% in nontransected eyes. The number of labeled axon bundles decreased by 61% +/- 28% at 7 days after transection compared with 26% +/- 9% in nontransected eyes. The number of retrograde-labeled RGCs detected in vivo declined by 53% at 7 days and by 76% at 14 days after transection. RGC soma and CMFDA axon counts decreased most rapidly between 5 and 7 days after transection. Histologic examination demonstrated a reduction in RNFL thickness 7 days after transection. Conclusions. Intravitreal CMFDA can be used to longitudinally monitor RGC axons within the RNFL in vivo. Imaging the disappearance of retrograde-labeled RGC somas and axons indicates that axonal and somal degeneration occur in parallel after axotomy.

Background: To investigate whether tafluprost, which is a prostaglandin-related compound and an anti-glaucoma drug, has a direct anti-apoptotic effect in cultured retinal ganglion cells (RGCs) and rat RGCs in retinas with optic nerve crush (ONC)., Methods: RGC-5 cells were induced to undergo apoptosis by a serum deprivation and by exogenous glutamate. The level of cell death with or without tafluprost was monitored by an XTT assay and by immunocytochemistry with activated caspase-3. Changes in intracellular calcium ([Ca(2+)]i) levels were measured with fluo-4 fluorescence. Rat RGCs were degenerated by ONC. After topical instillation of tafluprost for 7 and 14 days, the numbers of retrograde-labeled RGCs were counted. Retinal flatmounts were subjected to terminal dUTP nick end labeling (TUNEL) staining to detect apoptotic cells., Results: Tafluprost dose-dependently promoted RGC-5 cell viability with an optimum concentration of 3 microM (p = 0.006). Tafluprost significantly reduced caspase-3-positive cells and suppressed [Ca(+2)]i evoked by exogenous glutamate. The cGMP-dependent protein kinase inhibitor and KT-5823 partially blocked the rescue effect of tafluprost (p = 0.002). The survival rate of RGCs significantly increased in eyes treated with tafluprost (p = 0.01), and the prevalence of TUNEL-positive cells was significantly decreased 14 days after ONC (p < 0.001)., Conclusions: These data suggest that tafluprost has an anti-apoptotic effect in RGCs.

We investigated whether latanoprost has a direct anti-apoptotic effect in retinal ganglion cell (RGC) line and RGCs in the rat. RGC-5 cells were induced to undergo apoptosis by serum deprivation and exogenous glutamate. The level of cell death with or without latanoprost acid was monitored by an XTT assay and by immunocytochemistry with activated caspase-3. Changes in the level of intracellular calcium ([Ca(2+)]i) were measured with fluo-4 fluorescence. The XTT assay revealed that latanoprost acid increased RGC-5 cell viability. Latanoprost acid significantly reduced caspase-3 positive cells and suppressed [Ca(2+)]i evoked by glutamate. U0126, a mitogen-activated protein/extracellular signal-regulated kinase 1 and 2 inhibitor, partially blocked the rescue effect of latnanoprost acid (p=0.013). In vivo, rat RGCs were degenerated by optic nerve crush. After topical instillation of latanoprost for 7days, RGCs labeled with fluorogold were significantly. Retinal flatmounts were subjected to terminal dUTP nick end labeling (TUNEL) staining to detect apoptotic cells. TUNEL-positive cells were significantly decreased in eyes with topically instilled latanoprost (p=0.015). These data suggest that latanoprost has an neuroprotective ability in RGCs.

Objective: To establish the structure-function relationship between peripapillary retinal nerve fiber layer (RNFL) thickness and visual field (VF) test points in standard automated perimetry., Methods: We included 213 eyes with open-angle glaucoma and VF loss in this cross-sectional study. Correlations between individual VF sensitivity at 52 test points and peripapillary RNFL thickness divided into 16 sectors were calculated. The RNFL thickness was measured by Stratus optical coherence tomography. A new VF cluster map corresponding to RNFL sectors was generated by grouping the VF test points with the highest relation to each RNFL sector., Results: The VF sensitivity at each test point was significantly correlated with the sectoral RNFL thickness. The highest coefficient of determination (R(2)) for a superotemporal RNFL sector and VF sensitivity at an inferotemporal test point (9 degrees temporal and 15 degrees inferior from the center) in standard automated perimetry was 0.500 (P < .001). Clustered VF test points most highly related to the RNFL sectors were asymmetrically located between the upper and lower hemifields. A newly developed map revealed significant structure-function relationships., Conclusions: We describe an association between VF sensitivity at test points and sectoral RNFL thickness. Nine clustered VF test points corresponding to 9 RNFL regions were demonstrated from the structure-function relationships.

Purpose: To investigate if nipradilol has an anti-apoptotic effect in serum-deprived RGC-5 cells and in the streptozotocin-induced diabetic rat retina., Methods: Apoptosis was quantified by activated caspase-3 immunohistochemistry or terminal dUTP nick end-labeling assay., Results: Nipradilol dose-dependently suppressed apoptosis in a protein kinase A- and G-dependent manner and counteracted glutamate-induced calcium entry in the RGC-5 cells and reduced apoptotic cells in the retinal ganglion cell layer of 4- and 12-week diabetic retinas compared to controls when instilled for 5 days. Removal of the nitric oxide moiety from nipradilol blocked these effects., Conclusions: Nipradilol protects RGCs from apoptosis induced by serum-deprivation in vitro and by diabetes in vivo. The NO-related signaling pathway mediates the anti-apoptotic ability of nipradilol.

Background: Optic tract syndrome (OTS) is characterized by incongruous homonymous hemianopia and a perpendicular pattern of bilateral optic atrophy due to the optic tract lesion. However, loss of retinal nerve fiber layer thickness (RNFLT) associated with OTS has not been quantitatively assessed., Case: A 20-year-old woman with blunt head trauma showed normal visual acuity, color vision, ocular motility, and intraocular pressure. Because of a relative afferent pupillary defect in her left eye and left-sided homonymous hemianopia, we suspected right-sided optic tract damage, although magnetic resonance imaging detected no intracranial lesion., Observations: Using optical coherence tomography (OCT), the RNFLT of this case was measured at 31 months after the trauma and compared with age-matched normal controls (n = 41). Nasal, temporal, superior, and inferior quadrant RNFLT was reduced by 22%, 21%, 5%, and 46% in the right eye and 76%, 64%, 25%, and 27% in the left eye, respectively. The reduction was > 3 x the standard deviation of the normal mean values in the nasal and temporal quadrants of the left eye and in the inferior quadrant of the right eye., Conclusions: OCT can determine the RNFLT reduction corresponding to the characteristic patterns of optic atrophy of OTS., ((c) Japanese Ophthalmological Society 2005.)

Objectives: To analyze retinal nerve fiber layer (RNFL) thickness in eyes with band atrophy by use of optical coherence tomography (OCT) and to evaluate the ability of OCT to detect this characteristic pattern of RNFL loss., Design: Cross-sectional, retrospective study., Participants: Thirty-four eyes of 18 patients with bitemporal hemianopia caused by optic chiasm compression by chiasmal tumors were studied. All eyes were divided into 3 groups according to visual field loss grading after Goldmann perimetry., Interventions: Retinal nerve fiber layer thickness measurements with OCT., Main Outcome Measures: Retinal nerve fiber layer thickness around the optic disc was measured by OCT (3.4-mm diameter circle). Calculation of the changes in OCT parameters, including the horizontal (nasal + temporal quadrant RNFL thickness) and vertical values (superior + inferior quadrant RNFL thickness) was based on data from 160 normal eyes. Comparison between the 3 visual field grading groups was done with the analysis of variance test. The receiver operating characteristic (ROC) curve for the horizontal and vertical value were calculated, and the areas under the curve (AUC) were compared., Results: Retinal nerve fiber layer thickness in eyes with band atrophy decreased in all OCT parameters. The reduction rate in average and temporal RNFL thickness and horizontal value was correlated with visual field grading. The AUC of horizontal value was 0.970+/-0.011, which was significantly different from AUC of vertical value (0.903+/-0.022)., Conclusions: The degree of RNFL thickness reduction correlated with that of visual field defects. Optical coherence tomography was able to identify the characteristic pattern of RNFL loss in these eyes.

Purpose: Diabetes mellitus (DM) is a risk factor for open-angle glaucoma, although the mechanistic interrelationship of the two is debatable. The purpose of this study is to test whether DM augments neural apoptosis in rat retina with chronically elevated intraocular pressure (IOP)., Methods: Sprague-Dawley rats were made diabetic by intraperitoneal injection of streptozotocin (STZ). At one month after STZ injection, three episcleral veins in one eye were cauterized to elevate IOP. Rats without STZ injection were treated likewise as diabetic controls. At 2 weeks, 1 month, and 2 months after cauterization, the retina was dissected, flat-mounted, and subjected to terminal dUTP nick end labeling (TUNEL) staining. TUNEL positive cells per unit area of the whole retina were measured., Results: DM did not affect base line IOP or augment IOP elevation due to episcleral vein cauterization. TUNEL positive cells, which primarily consisted of the neurons and glial cells in the inner retina including retinal ganglion cell (RGC), were counted consistently eight times more in the diabetic retina without IOP elevation than diabetic controls (n = 9, p < 0.001). The cauterization significantly elevated IOP up to 28.9 mmHg (p < 0.001), which was reduced over time, and substantially induced apoptosis in a IOP-dependent fashion (p < 0.001). Ocular hypertensive retinas with DM had significantly more TUNEL positive cells than those without DM despite of the similar time course of IOP changes (p < 0.001)., Conclusions: DM has an additive effect on apoptosis induction by chronic elevation of IOP. Diabetes may act as a risk factor of open-angle glaucoma by increasing susceptibility of retinal cells including retinal ganglion cells to apoptosis triggered by additional stresses such as elevated IOP.

Purpose: To evaluate the relationship between visual field and retinal nerve fiber layer (RNFL) thickness measured by optical coherent tomography (OCT) and to assess the diagnostic ability of OCT to distinguish between early glaucomatous or glaucoma-suspect eyes from normal eyes., Design: Retrospective, non-randomized, cross-sectional study., Methods: A total of 160 eyes of 120 normal Japanese adults, 23 eyes of 16 patients with ocular hypertension, 38 eyes of 35 glaucoma-suspect patients, and 237 glaucomatous eyes of 140 glaucoma patients were enrolled in the study. The glaucoma group included 89 early glaucomatous eyes. Thickness of the RNFL around the optic disk was determined with three 3.4-mm diameter circle OCT scans. Average and segmental RNFL thickness values were compared among all groups. The correlation between mean deviation and RNFL thickness in glaucomatous eyes was also analyzed. Receiver operating characteristic (ROC) curve area was calculated to discriminate normal eyes from early glaucomatous or glaucoma-suspect eyes., Results: A significant relationship existed between the mean deviation and RNFL thickness in all parameters excluding the 3-o'clock area. The average RNFL thickness had the strongest correlation in all parameters (r = -0.729, P <.001). Retinal nerve fiber layer thickness at the 7-o'clock inferotemporal segment had the widest areas under the ROC curves in all parameters for early glaucomatous eyes (0.873)., Conclusions: Measurement of RNFL thickness by OCT is useful in detecting early RNFL damage. Furthermore, OCT measurements of RNFL thickness may provide clinically relevant information in monitoring glaucomatous changes.

Purpose: An autoantibody against aquaporin-4 (AQP4 Ab) is highly specific for neuromyelitis optica spectrum disorder and plays a pathogenic role in this disease. The purpose of this study was to investigate the impact of AQP4 Ab on inner retinal structure, function, and the structure−function relationships in eyes with optic neuritis. Methods: Thirty five eyes from 25 cases who had received visual function tests and RTVue optical coherence tomography (OCT) measurement at least six months after the latest episode of optic neuritis were enrolled. Patients with multiple sclerosis were excluded. AQP4 Ab was measured using a cell-based assay. Visual acuity, mean deviation (MD) of the Humphrey visual field SITA standard 30–2 tests, retinal nerve fiber layer (RNFL), ganglion cell complex (GCC) thicknesses, and other clinical variables were compared between the AQP4 Ab-positive and -negative groups. Parameters associated with visual functions were evaluated by generalized estimating equation (GEE) models. Results: The AQP4 Ab-positive group (20 eyes from 12 cases) had a higher proportion of bilateral involvement and longer duration of follow-up than the AQP4 Ab-negative group (15 eyes from 13 cases). Linear mixed effect models revealed worse MD and visual acuity in AQP4 Ab-positive eyes than those in AQP4 Ab-negative eyes after adjusting for within-patient inter-eye dependence, whereas there were no differences in RNFL and GCC thickness between the two groups. In seropositive eyes, GEE regression analyses revealed that depending on age and the number of recurrences of ON episodes, OCT parameters correlated strongly with MD and more weakly with visual acuity. Conclusions: Reductions in RNFL and GCC thickness were proportional to the visual field defect in eyes with AQP4 Ab but not in eyes without AQP4 Ab. The presence of AQP4 Ab probably plays a critical role in retinal ganglion cell loss in optic neuritis. [ABSTRACT FROM AUTHOR]

The impairment of mitochondrial function is an important pathogenic factor in glaucoma and other optic neuropathies in which retinal ganglion cell (RGC) death is the fundamental pathology. Syntaphilin was recently discovered as a docking protein that affects mitochondrial mobility. However, no reports have investigated the involvement of syntaphilin in the visual system. We investigated the expression of syntaphilin in the rat retina, optic nerve and brain. The expression of syntaphilin exhibited varying patterns in the visual system. Syntaphilin was expressed in retinal ganglion cells in the retina, in the cell bodies of neurons in the superior colliculus and was abundant in the astrocytes of rat optic nerves (similar to the findings that syntaphilin is expressed in human optic nerves). After optic nerve transection, which caused RGC death and axonal degeneration, quantitative real-time RT-PCR was used to assess changes in gene expression in the rat retina and optic nerve. Syntaphilin gene and protein expression in the optic nerve was downregulated 3 and 7 days after optic nerve transection. Our study suggests that syntaphilin expression in astrocytes at the optic nerve might be involved in axonal injury. [ABSTRACT FROM AUTHOR]

Purpose: To establish a rat glaucoma model with chronic intraocular pressure (IOP) elevation induced by microbeads suspended in sodium sulfate-sodium hyaluronate. Methods: Chronic elevation of IOP was induced unilaterally by injecting polystyrene microbeads, suspended in 4 % sodium sulfate and 3 % sodium hyaluronate, into the anterior chamber. The microbead suspension was injected through either the clear corneal (CC) or sclerocorneal (SC) tunnel. IOP changes were monitored up to 8 weeks after injection. The loss of retinal ganglion cells (RGCs) was assessed using fluorogold retrograde labeling of RGCs. RGC axons were evaluated by immunohistochemistry and immunoblotting. Results: The resulting IOP elevation was maintained up to 3 weeks after the intracameral injection of microbeads through the CC route and up to 4 weeks after injection through the SC route. The density of RGCs was significantly reduced at 4 weeks after injection, with the SC route leading to more RGC loss than the CC route ( p = 0.037). The neurofilament immunoreactivity and protein levels in the optic nerve were also significantly reduced at 4 weeks after injection. Some eyes in the SC route cohort received re-injection of the microbead suspension at 4 weeks after the initial injection, which led to an elevated IOP more than 8 weeks after the initial injection, and eventually a 27.5 % loss of RGC density compared with the control eyes. Conclusion: The intracameral injection of microbeads suspended in hyaluronate effectively produced chronic IOP elevation and subsequent RGC degeneration in rat eyes. The sclerocorneal tunnel approach yielded a longer period of IOP elevation than the clear corneal approach. Our modified microbead injection offers a reliable high-pressure glaucoma model. [ABSTRACT FROM AUTHOR]

Background: Unilateral injury of the optic tract leads to asymmetrical optic atrophy in both eyes derived from the crossing of the nerve fibers at the chiasm. This report demonstrates unique imaging appearances of optic atrophy due to this uncommon condition detected by spectral-domain optical coherence tomography (SD-OCT). Methods: Cirrus and RTVue measurements were performed in four cases of optic tract syndrome. Circumpapillary retinal nerve fiber layer (cpRNFL) thickness was obtained from both instruments and ganglion cell complex (GCC) integrity was obtained from RTVue. The presumable reduction rates of quadrant cpRNFL thickness were calculated from the published normative database and compared between eyes with temporal hemianopia and those with nasal hemianopia. Results: Both devices showed significant reduction of cpRNFL thickness, but did not have statistical difference in the reduction rates at temporal or nasal quadrant cpRNFL between contralateral and ipsilateral eyes to the lesion. Color-coded maps helped to visualize the unique pattern of cpRNFL and GCC thinning. Conclusions: SD-OCT can be used as a diagnostic tool for the optic tract syndrome. [ABSTRACT FROM AUTHOR]

Optic neuropathy is the leading cause of irreversible blindness, and a paradigm for central nervous system axonal disease. The primary event is damage to retinal ganglion cell axons, with subsequent death of the cell body by apoptosis. Trials of neuroprotection for these and other neuronal diseases have mostly failed, primarily because mechanisms of neuroprotection in animals do not necessarily translate to humans. We developed a methodology for imaging an intracellular transduction pathway that signals neuronal death in the living animal. Using longitudinal confocal scanning multilaser ophthalmoscopy, we identified the production of superoxide within retrograde-labelled rat retinal ganglion cells after optic nerve transection. Superoxide was visualized by real-time imaging of its reaction product with intravitreally administered hydroethidine and confirmed by differential spectroscopy of the specific product 2-hydroxyethidium. Retinal ganglion cell superoxide increased within 24 h after axotomy, peaking at 4 days, and was not observed in contralateral untransected eyes. The superoxide signal preceded phosphatidylserine externalization, indicating that superoxide generation was an early event and preceded apoptosis. Intravitreal pegylated superoxide dismutase blocked superoxide generation after axotomy and delayed retinal ganglion cell death. Together, these results are consistent with superoxide being an upstream signal for retinal ganglion cell apoptosis after optic nerve injury. Early detection of axonal injury with superoxide could serve as a predictive biomarker for patients with optic neuropathy. [ABSTRACT FROM PUBLISHER]

To investigate whether tafluprost, which is a prostaglandin-related compound and an anti-glaucoma drug, has a direct anti-apoptotic effect in cultured retinal ganglion cells (RGCs) and rat RGCs in retinas with optic nerve crush (ONC). RGC-5 cells were induced to undergo apoptosis by a serum deprivation and by exogenous glutamate. The level of cell death with or without tafluprost was monitored by an XTT assay and by immunocytochemistry with activated caspase-3. Changes in intracellular calcium ([Ca2+]i) levels were measured with fluo-4 fluorescence. Rat RGCs were degenerated by ONC. After topical instillation of tafluprost for 7 and 14 days, the numbers of retrograde-labeled RGCs were counted. Retinal flatmounts were subjected to terminal dUTP nick end labeling (TUNEL) staining to detect apoptotic cells. Tafluprost dose-dependently promoted RGC-5 cell viability with an optimum concentration of 3 μM ( p = 0.006). Tafluprost significantly reduced caspase-3-positive cells and suppressed [Ca+2]i evoked by exogenous glutamate. The cGMP-dependent protein kinase inhibitor and KT-5823 partially blocked the rescue effect of tafluprost ( p = 0.002). The survival rate of RGCs significantly increased in eyes treated with tafluprost ( p = 0.01), and the prevalence of TUNEL-positive cells was significantly decreased 14 days after ONC ( p < 0.001). These data suggest that tafluprost has an anti-apoptotic effect in RGCs. [ABSTRACT FROM AUTHOR]

The Akt serine/threonine kinase mediates pro-survival signalings in retina and was reported to be activated in a response to some retinal and optic nerve injuries. Human and experimental glaucoma induce apoptosis of retinal ganglion cells (RGCs). The purpose of this study is to test whether episcleral vein cauterization (EVC) to chronically elevate intraocular pressures (IOPs) in rats increase apoptosis of RGCs and affect activation of Akt and its upstream insulin-like growth factor (IGF)-1 receptor/Insulin receptor. Three episcleral veins in left eyes of Sprague–Dawley rats were cauterized to elevate IOPs. Up to 6 months, IOPs were monitored and the retina was dissected at several time points. The numbers of terminal dUTP nick end labeling (TUNEL)-positive cells and those of RGCs labeled with fluorogold were counted in flat-mounted retina. Immunohistochemistry and immunoblotting were performed to identify cells expressing phosphorylated Akt and to quantify the phospho- to total ratios of Akt and IGF-1 receptor/insulin receptor. EVC significantly elevated IOPs up to 2 months, increased TUNEL-positive cells in an IOP-dependent fashion, and reduced 34.5% of RGCs at 6 months (P<0.001) compared with contralateral retinas. Phosphorylated Akt was specifically expressed in RGCs until 1 month after cauterization. Akt (P=0.036) and IGF-1 receptor/Insulin receptor (P=0.003) were transiently phosphorylated at 3 days. Intrinsic activation of the IGF-1 receptor/Insulin receptor to Akt pathway may occur in RGCs in retina with EVC. [Copyright &y& Elsevier]