Factors associated with intrachoroidal cavitation and sinkhole formation in eyes with glaucomatous visual-field defects.

PURPOSE: To investigate factors associated with intrachoroidal cavitation (ICC) and sinkhole formation in eyes with glaucomatous visual-field defects. METHODS: This retrospective, cross-sectional study enrolled a total of 2808 eyes of 1482 patients who were diagnosed/treated for glaucoma and underwent swept-source optical coherence tomography (OCT). We first determined the prevalence of ICCs and sinkholes and their locations. Next, we selected one eye from each patient and compared the clinical characteristics of eyes with and without ICCs. Finally, in eyes with ICCs, we compared the clinical characteristics of eyes with and without sinkholes. Blood flow (BF), represented by laser speckle flowgraphy-measured tissue-area mean blur rate (MBR), was measured in the temporal optic nerve head (ONH), temporal peripapillary chorioretinal atrophy (PPA) zone, and in the ICC zone. ICC area and angle were analyzed in OCT en-face images. Mean deviation and total deviation in the central area (TD-central) were measured with Humphrey visual-field testing. RESULTS: A total of 86 eyes (3.1%) had ICCs and 52 eyes (1.9%) had sinkholes. ICC eyes had a lower spherical equivalent and longer axial length (AL) than non-ICC eyes (P < 0.05). Patients with eyes with sinkholes were more elderly and had worse best-corrected visual acuity, worse TD-central, a larger ICC, and lower tissue-area MBR in the temporal ONH, temporal PPA zone, and ICC zone (P < 0.05). CONCLUSION: In eyes with glaucoma, AL elongation might be linked to ICC formation. Sinkhole formation might be associated with ICC enlargement, impaired ocular BF, and impaired retinal structure and function involving the central area.

The association between intraocular pressure dynamics during dark-room prone testing and intraocular pressure over a relatively long-term follow-up period in primary open-glaucoma patients.

PURPOSE: To investigate the relationship between the dynamics of intraocular pressure (IOP) during dark-room prone testing (DRPT) and IOP over a relatively long-term follow-up period. METHODS: This retrospective study enrolled 84 eyes of 51 primary open-angle glaucoma patients who underwent DRPT for whom at least three IOP measurements made using Goldmann applanation tonometry were available over a maximum follow-up period of two years. We excluded eyes with a history of intraocular surgery or laser treatment and those with changes in topical anti-glaucoma medication during the follow-up period. In DRPT, IOP was measured in the sitting position, and after 60 min in the prone position in a dark room, IOP was measured again. In this study, IOP fluctuation refers to the standard deviation (SD) of IOP, and IOP max indicates the maximum value of IOP during the follow-up. The relationship between these parameters was analyzed with a linear mixed-effects model, adjusting for clinical parameters including age, gender, and axial length. RESULTS: IOP increased after DRPT with a mean of 6.13 ± 3.55 mmHg. IOP max was significantly associated with IOP after DRPT (ß = 0.38; p < 0.001). IOP fluctuation was significantly associated with IOP change in DRPT (ß = 0.29; p = 0.007). CONCLUSION: Our findings suggest that short-term and relatively long-term IOP dynamics are associated. Long-term IOP dynamics can be predicted by DRPT to some extent.

The relationship between equol production status and normal tension glaucoma.

PURPOSE: Equol is metabolized by intestinal bacteria from soy isoflavones and is chemically similar to estrogen. Dietary habits, such as consumption of soy products, influence equol production. A relationship between glaucoma and estrogen has been identified; here, we investigated the relationship between equol production status and glaucoma in Japan. METHODS: We recruited 68 normal-tension glaucoma (NTG) patients (male to female ratio 26:42, average age 63.0 ± 7.6 years) and 31 controls (male to female ratio 13:18, average age 66.0 ± 6.3 years) from our hospital. All women included were postmenopausal. Urinary equol concentration was quantified with the ELISA method. MD was calculated based on the Humphrey visual field. The association between MD and equol was analyzed with Spearman's rank correlation coefficient. The Mann-Whitney U test was used to compare the equol-producing (> 1 µM) and non-producing (< 1 µM) subjects. We also investigated the association between equol and glaucoma with a logistic regression analysis. RESULTS: There was a significant association between equol and MD (r = 0.36, P < 0.01) in the NTG patients. Glaucoma, represented by MD, was significantly milder in the equol-producing subjects than the non-equol producing subjects (P = 0.03). A multivariate analysis revealed the independent contributions of equol, cpRNFLT, and IOP to MD (P = 0.03, P = 0.04, and P < 0.01, respectively). CONCLUSION: Our results suggest that equol, acting through estrogen receptor-mediated neuroprotective effects, might be involved in suppressing the progression of NTG. This result also adds to evidence that glaucoma may be influenced by lifestyle.

The GPR84 molecule is a mediator of a subpopulation of retinal microglia that promote TNF/IL-1α expression via the rho-ROCK pathway after optic nerve injury.

Resident microglia are important to maintain homeostasis in the central nervous system, which includes the retina. The retinal microglia become activated in numerous pathological conditions, but the molecular signatures of these changes are poorly understood. Here, using an approach based on FACS and RNA-seq, we show that microglial gene expression patterns gradually change during RGC degeneration induced by optic nerve injury. Most importantly, we found that the microglial cells strongly expressed Tnf and Il1α, both of which are known to induce neurotoxic reactive astrocytes, and were characterized by Gpr84high -expressing cells in a particular subpopulation. Moreover, ripasudil, a Rho kinase inhibitor, significantly blunted Gpr84 expression and cytokine induction in vitro and in vivo. Finally, GPR84-deficient mice prevented RGC loss in optic nerve-injured retina. These results reveal that Rho kinase-mediated GPR84 alteration strongly contribute to microglial activation and promote neurotoxicity, suggesting that Rho-ROCK and GPR84 signaling may be potential therapeutic targets to prevent the neurotoxic microglial phenotype induced by optic nerve damage, such as occurs in traumatic optic neuropathy and glaucoma.

Predictive potential of optical coherence tomography parameters for the prognosis of decreased visual acuity after trabeculectomy in open-angle glaucoma patients with good vision.

BACKGROUND: Trabeculectomy (trab) is the most effective surgical procedure for lowering IOP and preventing glaucoma progression. However, decline in best-corrected visual acuity (BCVA) is one of the most serious postoperative complications of trab. Here, we investigated methods to predict decreased BCVA after trab in glaucoma patients with good preoperative BCVA. METHODS: This study included 35 eyes of 35 open-angle glaucoma (OAG) patients (male / female: 21 / 14, age: 64.0 ± 9.7 years old, preoperative intraocular pressure: 15.9 ± 5.4 mmHg, mean deviation: -18.1 ± 5.6 dB) with preoperative BCVA of 0.7 or better who underwent trab and were observed for more than 12 months. As a preoperative analysis, we measured temporal quadrant circumpapillary retinal nerve fiber layer thickness (cpRNFLT) and ganglion cell complex thickness in a central strip between the disc and fovea (csGCCT), an area that corresponds to the location of the papillomacular bundle (PMB) in swept-source optical coherence tomography (OCT). We defined BCVA decline as a loss of more than 3 lines of BCVA after 12 months. Measurement parameters were compared between the BCVA-decline group and the non-BCVA-decline group. RESULTS: BCVA decline was detected in 11 cases (31.4%) 12 months after trab. There was a statistically significant difference in axial length (P = 0.049). A single logistic analysis showed that the BCVA-decline group had significantly lower cpRNFLT than the non-BCVA-decline group (27.7 ± 8.0 µm vs. 45.1 ± 5.3 µm, P < 0.001, cut-off value: 33.4 µm), as well as lower csGCCT (72.4 ± 7.7 µm vs. 87.5 ± 5.1 µm, P = 0.002, cut-off value: 82.3 µm). Multivariable logistic analysis showed that the BCVA-decline group had significantly lower temporal quadrant cpRNFLT (P < 0.001) and lower middle csGCCT (P < 0.001) compared to the non-BCVA-decline group. CONCLUSIONS: Lower temporal quadrant cpRNFLT and middle csGCCT, OCT scan areas that correspond to the location of the PMB, might be biomarkers that predict BCVA decline after trab in OAG patients with good vision.

Clinical characteristics of glaucoma patients with various risk factors.

BACKGROUND: Glaucoma is multifactorial, but the interrelationship between risk factors and structural changes remains unclear. Here, we adjusted for confounding factors in glaucoma patients with differing risk factors, and compared differences in structure and susceptible areas in the optic disc and macula. METHODS: In 458 eyes with glaucoma, we determined confounding factors for intraocular pressure (IOP), central corneal thickness (CCT), axial length (AL), LSFG-measured ocular blood flow (OBF), which was assessed with laser speckle flowgraphy-measured mean blur rate in the tissue area (MT) of the optic nerve head, biological antioxidant potential (BAP), and systemic abnormalities in diastolic blood pressure (dBP). To compensate for measurement bias, we also analyzed corrected IOP (cIOP; corrected for CCT) and corrected MT (cMT; corrected for age, weighted retinal ganglion cell count, and AL). Then, we determined the distribution of these parameters in low-, middle-, and high-value subgroups and compared them with the Kruskal-Wallis test. Pairwise comparisons used the Steel-Dwass test. RESULTS: The high-cIOP subgroup had significantly worse mean deviation (MD), temporal, superior, and inferior loss of circumpapillary retinal nerve fiber layer thickness (cpRNFLT), and large cupping. The low-CCT subgroup had temporal cpRNFLT loss; the high-CCT subgroup had low cup volume. The high-AL subgroup had macular ganglion cell complex thickness (GCCT) loss; the low-AL subgroup had temporal cpRNFLT loss. The high-systemic-dBP subgroup had worse MD, total, superior, and inferior cpRNFLT loss and macular GCCT loss. The low-BAP subgroup had more male patients, higher dBP, and cpRNFLT loss in the 10 o'clock area. The high-OBF subgroup had higher total, superior and temporal cpRNFLT and macular GCCT. CONCLUSIONS: Structural changes and local susceptibility to glaucomatous damage show unique variations in patients with different risk factors, which might suggest that specific risk factors induce specific types of pathogenesis and corresponding glaucoma phenotypes. Our study may open new avenues for the development of precision medicine for glaucoma.

Time-Course Changes in Optic Nerve Head Blood Flow and Retinal Nerve Fiber Layer Thickness in Eyes with Open-angle Glaucoma.

PURPOSE: To determine whether decreased optic nerve head (ONH) blood flow (BF) precedes or follows decreased circumpapillary retinal nerve fiber layer thickness (cpRNFLT) in eyes with open-angle glaucoma (OAG). DESIGN: Retrospective, longitudinal study. PARTICIPANTS: This study followed up 350 eyes of 225 OAG patients for at least 2 years and collected data from each patient from at least 5 examinations obtained with laser speckle flowgraphy (LSFG) and OCT. METHODS: In the superior, temporal, and inferior ONH quadrants, tissue area mean blur rate (MT), representing ONH tissue BF, was measured with LSFG, whereas cpRNFLT was measured with OCT. A multivariate linear mixed-effects model was used to identify potential predictors of faster MT decrease, adjusting for possible confounding factors. Based on these results, each quadrant of each patient was assigned a risk point if the quadrant was the superior or temporal, if patient age was older than the median (61 years), and if patient pulse rate was higher than median (74 beats per minute). The quadrants were then compared with a mixed-effects Cox model for MT and cpRNFLT changes, defined as a difference between the baseline value and the values from the latest 2 consecutive follow-up visits of more than 1.96 × the corresponding coefficient of variation. MAIN OUTCOME MEASURES: Ophthalmic and systemic variables and MT and cpRNFLT in the superior, temporal, and inferior quadrants. RESULTS: The multivariate model showed that MT decrease was faster in older patients with higher pulse rate and slower in inferior quadrants (P < 0.05). Quadrants with 0 risk points showed primary cpRNFLT decrease (P = 0.048), 1-risk point quadrants showed simultaneous cpRNFLT and MT decrease (P = 0.260), and 2-risk point and 3-risk point quadrants showed primary MT decrease (P < 0.001). CONCLUSIONS: Older patients with higher pulse rate are at greater risk of a primary reduction in ONH tissue BF, that is, preceding cpRNFLT decrease, in the superior and temporal quadrants.

CHOP deletion and anti-neuroinflammation treatment with hesperidin synergistically attenuate NMDA retinal injury in mice.

Glaucoma is a leading cause of blindness worldwide and is characterized by degeneration associated with the death of retinal ganglion cells (RGCs). It is believed that glaucoma is a group of heterogeneous diseases with multifactorial pathomechanisms. Here, we investigate whether anti-inflammation treatment with an ER stress blockade can selectively promote neuroprotection against NMDA injury in the RGCs. Retinal excitotoxicity was induced with an intravitreal NMDA injection. Microglial activation and neuroinflammation were evaluated with Iba1 immunostaining and cytokine gene expression. A stable HT22 cell line transfected with an NF-kB reporter was used to assess NF-kB activity after hesperidin treatment. CHOP-deficient mice were used as a model of ER stress blockade. Retinal cell death was evaluated with a TUNEL assay. As results, in the NMDA injury group, Iba1-positive microglia increased 6 h after NMDA injection. Also at 6 h, pro-inflammatory cytokines and chemokine increased, including TNFα, IL-1b, IL-6 and MCP-1. In addition, the MCP-1 promoter-driven EGFP signal, which we previously identified as a stress signal in injured RGCs, also increased; hesperidin treatment suppressed this inflammatory response and reduced stressed RGCs. In CHOP-deficient mice that received an NMDA injection, the gene expression of pro-inflammatory cytokines, chemokines, markers of active microglia, and inflammatory regulators was greater than in WT mice. In WT mice, hesperidin treatment partially prevented retinal cell death after NMDA injury; this neuroprotective effect was enhanced in CHOP-deficient mice. These findings demonstrate that ER stress blockade is not enough by itself to prevent RGC loss due to neuroinflammation in the retina, but it has a synergistic neuroprotective effect after NMDA injury when combined with an anti-inflammatory treatment based on hesperidin.

A pyruvate dehydrogenase kinase inhibitor prevents retinal cell death and improves energy metabolism in rat retinas after ischemia/reperfusion injury.

We aimed to assess the neuroprotective effect of a pyruvate dehydrogenase kinase (PDK) inhibitor, Nov3r after ischemia/reperfusion (IR) injury in rats. IR injury was induced by applying 150 mmHg of intraocular pressure for 50 min. Nov3r was orally administered (100 mg/kg) 3 h before and 24 h after IR injury. TUNEL-positive cells increased and immunoreactive RBPMS-positive cells decreased in the rat retinas after IR injury. Administration of Nov3r significantly ameliorated the increase in TUNEL-positive cells and prevented the RBPMS-positive cell decrease. Similarly, the number of IR-induced Iba1-positive microglial cells was significantly reduced with Nov3r treatment. Among metabolic parameters, IR damage induced the elevation of lactate and pyruvate, and the reduction of ATP. Oral administration of Nov3r ameliorated these changes. Our data suggest that the Nov3r had a retinal neuroprotective effect in IR injury in rats. This finding suggests that the regulation of pyruvate dehydrogenase (PDH) activity has potential therapeutic value by enabling metabolic reprograming in diseases associated with ischemic retinal damage, such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, ischemic optic neuropathy and glaucoma.

Genetic characteristics of retinitis pigmentosa in 1204 Japanese patients.

BACKGROUND: The genetic profile of retinitis pigmentosa (RP) in East Asian populations has not been well characterised. Therefore, we conducted a large-scale sequencing study to investigate the genes and variants causing RP in a Japanese population. METHODS: A total of 1209 Japanese patients diagnosed with typical RP were enrolled. We performed deep resequencing of 83 known causative genes of RP using next-generation sequencing. We defined pathogenic variants as those that were putatively deleterious or registered as pathogenic in the Human Gene Mutation Database or ClinVar database and had a minor allele frequency in any ethnic population of ≤0.5% for recessive genes or ≤0.01% for dominant genes as determined using population-based databases. RESULTS: We successfully sequenced 1204 patients with RP and determined 200 pathogenic variants in 38 genes as the cause of RP in 356 patients (29.6%). Variants in six genes (EYS, USH2A, RP1L1, RHO, RP1 and RPGR) caused RP in 65.4% (233/356) of those patients. Among autosomal recessive genes, two known founder variants in EYS [p.(Ser1653fs) and p.(Tyr2935*)] and four East Asian-specific variants [p.(Gly2752Arg) in USH2A, p.(Arg658*) in RP1L1, p.(Gly2186Glu) in EYS and p.(Ile535Asn) in PDE6B] and p.(Cys934Trp) in USH2A were found in ≥10 patients. Among autosomal dominant genes, four pathogenic variants [p.(Pro347Leu) in RHO, p.(Arg872fs) in RP1, p.(Arg41Trp) in CRX and p.(Gly381fs) in PRPF31] were found in ≥4 patients, while these variants were unreported or extremely rare in both East Asian and non-East Asian population-based databases. CONCLUSIONS: East Asian-specific variants in causative genes were the major causes of RP in the Japanese population.

Radial peripapillary capillary density in superior segmental optic hypoplasia measured with OCT angiography.

BACKGROUND: To investigate the diagnostic power of radial peripapillary capillary (RPC) density, measured with optical coherence tomography angiography (OCT-A), in patients with superior segmental optic hypoplasia (SSOH). METHODS: Forty subjects with SSOH and 40 age- and axial length-matched control subjects were retrospectively registered for this study. SSOH was defined as intraocular pressure less than 21 mmHg with the presence of two of the following: superior rim thinning, superior entrance of the central retinal artery, scleral halo, and pale optic disc; as well as non-progressive visual field loss. RPC density was measured with swept-source OCT-A (Triton, Topcon) overall, in the quadrants, and in the 12 clock-wise sectors. Changes in RPC density were also compared in SSOH patients and age-matched patients with mild- or moderate-stage of glaucoma. RPC density was compared in pairs of groups with Welch's t-test. Diagnostic power was assessed with the area under the receiver operating characteristics curve (AUC). RESULTS: Overall cpRNFLT was significantly different in the normal (106.7 ± 9.5 µm) and SSOH (77.2 ± 13.7 µm, p <  0.001) subjects. RPC density overall and in the superior, nasal, and inferior quadrants was significantly lower in the SSOH group (all, p <  0.001), but not in the temporal (p = 0.756) quadrant. The diagnostic power of RPC density was highest in the superior quadrant (AUC = 0.928) and the 1 o'clock sector (0.896). Comparing the SSOH and glaucoma patients showed that there were no significant differences in RPC density either overall (p = 0.391) or in the superior quadrant (p = 0.268), while RPC density was significantly higher in the inferior (p = 0.005) and temporal quadrants (p <  0.001) and lower in the nasal quadrant (p = 0.029). CONCLUSIONS: Low RPC density was found in the three non-temporal quadrants of the optic nerve head in SSOH patients, in comparison to normal subjects. Regionally, RPC density in SSOH was lower in the nasal quadrant and higher in the inferior and temporal quadrants in comparison to glaucoma patients. Measuring RPC density with OCT-A may help the diagnosis of SSOH and may improve the management of glaucoma.

The neuroprotective effect of latanoprost acts via klotho-mediated suppression of calpain activation after optic nerve transection.

Latanoprost was first developed for use in glaucoma therapy as an ocular hypotensive agent targeting the prostaglandin F2α (FP) receptor. Subsequently, latanoprost showed a neuroprotective effect, an additional pharmacological action. However, although it is well-known that latanoprost exerts an ocular hypotensive effect via the FP receptor, it is not known whether this is also true of its neuroprotective effect. Klotho was firstly identified as the gene linked to the suppression of aging phenotype: the defect of klotho gene in mice results aging phenotype such as hypokinesis, arteriosclerosis, and short lifespan. After that, the function of klotho was also reported to maintain calcium homeostasis and to exert a neuroprotective effect in various models of neurodegenerative disease. However, the function of klotho in eyes including retina is still poorly understood. Here, we show that klotho is a key factor underlying the neuroprotective effect of latanoprost during post-axotomy retinal ganglion cell (RGC) degeneration. Importantly, a quantitative RT-PCR gene expression analysis of klotho in sorted rat retinal cells revealed that the highest expression level of klotho in the retina was in the RGCs. Latanoprost acid, the biologically active form of latanoprost, inhibits post-traumatic calpain activation and concomitantly facilitates the expression and shedding of klotho in axotomized RGCs. This expression profile is a good match with the localization, not of the FP receptor, but of organic anion transporting polypeptide 2B1, known as a prostaglandin transporter, in the ocular tissue. Furthermore, an organic anion transporting polypeptide 2B1 inhibitor suppressed latanoprost acid-mediated klotho shedding ex vivo, whereas an FP receptor antagonist did not. The klotho fragments shed from the RGCs reduced the intracellular level of reactive oxygen species, and a specific klotho inhibitor accelerated and increased RGC death after axotomy. We conclude that the shed klotho fragments might contribute to the attenuation of axonal injury-induced calpain activation and oxidative stress, thereby protecting RGCs from post-traumatic neuronal degeneration.

Relationship between laser speckle flowgraphy and optical coherence tomography angiography measurements of ocular microcirculation.

PURPOSE: The purpose of this study was to investigate the relationship between laser speckle flowgraphy (LSFG) and optical coherence tomography angiography (OCTA) measurements of ocular microcirculation in normal and open-angle glaucoma (OAG) subjects. METHODS: This study included 18 eyes of 18 OAG patients and ten eyes of ten age-matched healthy controls. LSFG was used to measure mean blur rate (MBR) in the optic nerve head (ONH) vessel area (MV) and tissue area (MT). OCTA was used to measure a new parameter, peripapillary relative intensity (PRI), in the superficial retina, superficial choroid, and deep choroid. Statistical associations were then determined. RESULTS: MV, MT, superficial-retinal PRI, and superficial-choroidal PRI were lower in the OAG subjects than the controls (P = 0.02, P < 0.001, P = 0.02 and P = 0.008, respectively). Superficial-retinal PRI was correlated with MV and MT (R = 0.68, P < 0.001 and R = 0.63, P < 0.001, respectively). Superficial-choroidal PRI was also correlated with MV and MT (R = 0.45, P = 0.02 and R = 0.57, P = 0.002, respectively). Multiple regression analysis revealed that MV and MT independently contributed to superficial-retinal PRI (P = 0.008 and P = 0.04, respectively), while only MT contributed to superficial-choroidal PRI (P = 0.03). CONCLUSIONS: Our finding that OCTA-measured PRI was related to LSFG-measured MBR was reasonable, considering the vascular anatomy of the eye. Thus, PRI, like MBR, may be a promising biomarker of ocular microcirculation that can reveal the presence of ocular diseases such as OAG.

Phase Difference-Enhanced Magnetic Resonance (MR) Imaging (PADRE) Technique for the Detection of Age-Related Microstructural Changes in Optic Radiation: Comparison with Diffusion Tensor Imaging (DTI).

BACKGROUND The optic radiation (OR) is a white-matter bundle connecting the lateral geniculate body and the visual cortex. Phase difference-enhanced imaging (PADRE) is a new MRI technique that is able to achieve precise delineation of the OR. The aim of this study was to investigate the effect of age on the volume and signal intensity of the OR using PADRE, and to establish a volumetric reference of the OR from a healthy population, compared with diffusion tensor imaging (DTI). MATERIAL AND METHODS Thirty-nine healthy volunteers underwent MR imaging with PADRE and DTI sequences on a 3.0-T scanner. For the volumetric analysis with PADRE, the OR corresponding to the external sagittal stratum was manually traced, while an automated thresholding method was used for the DTI-based volumetric analysis of the OR. RESULTS The mean right and left OR volumes measured from the PADRE images were 1469.0±242.4 mm³ and 1372.6±310.2 mm³, respectively. Although OR volume showed no significant correlation with age, the normalized OR signal intensity showed a linear correlation with increasing age (r²=0.50-0.53; P<0.01). The OR signal intensity on PADRE and DTI-related quantitative parameters for the OR showed significant correlations (r²=0.46-0.49; P<0.01). CONCLUSIONS The PADRE technique revealed exceptional preservation of OR volume, even in later life. Moreover, PADRE was able to detect age-related changes in signal intensity of the OR and may contribute to future analyses of pathological neurodegeneration in patients with glaucoma and multiple sclerosis.

The association between systemic oxidative stress and ocular blood flow in patients with normal-tension glaucoma.

PURPOSE: To evaluate the association between ocular blood flow and biomarkers of systemic oxidative stress, as well as the potential of these biomarkers to assess normal-tension glaucoma (NTG). METHODS: This study included 73 eyes of 73 patients with NTG. We assessed ocular blood flow by measuring mean blur rate (MBR) in the optic nerve head using laser speckle flowgraphy, both overall and separately in the vessel and tissue areas. We also measured urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and skin autofluorescence (SAF), and lastly, determined correlations between these measurements and with other clinical parameters. RESULTS: SAF was correlated with age, circumpapillary retinal nerve fiber layer thickness (cpRNFLT), mean deviation (MD), and overall MBR (P = 0.003, P = 0.013, P = 0.015 and P = 0.006, respectively). SAF and 8-OHdG were both correlated with tissue-area MBR (P = 0.006 and P = 0.010, respectively). Visual acuity, cpRNFLT, mean deviation and tissue-area MBR had a significant tendency to change with NTG severity (P = 0.014, P < 0.001, P < 0.001 and P = 0.006, respectively). Multiple regression analysis revealed that cpRNFLT and 8-OHdG were independent contributing factors to MD (P < 0.001 and P = 0.040, respectively), and that cpRNFLT and 8-OHdG were independent contributing factors to tissue-area MBR (P = 0.005 and P = 0.028, respectively). CONCLUSIONS: We found a close relationship between cpRNFLT, MD, tissue MBR, SAF and 8-OHdG, suggesting that systemic oxidative stress is associated with decreased ocular blood flow and may be involved in the pathogenesis of NTG.

Estimation of the Disc Damage Likelihood Scale in primary open-angle glaucoma: the Glaucoma Stereo Analysis Study.

PURPOSE: The Glaucoma Stereo Analysis Study (GSAS), a cross-sectional multicenter collaborative study, used a stereo fundus camera (nonmyd WX) to assess various morphological parameters of the optic nerve head (ONH) in glaucoma patients. We examined the associations between the Disc Damage Likelihood Scale (DDLS), a grading system for estimating glaucomatous ONH damage, and each parameter. METHODS: The study included 187 eyes of 187 patients with primary open-angle glaucoma or normal-tension glaucoma. ONH morphological parameters including the DDLS stage were calculated with prototype analysis software. Three independent graders classified each optic disc appearance into four different types: focal ischemic, myopic glaucomatous, senile sclerotic, and generalized enlargement. The correlations between the DDLS and patient characteristics or each ONH parameter were analyzed with Spearman's rank correlation coefficient. RESULTS: The DDLS was correlated positively with baseline intraocular pressure and visual field pattern standard deviation, and negatively with visual field mean deviation. The DDLS was strongly correlated with vertical cup-to-disc ratio and horizontal cup-to-disc ratio positively, and with minimum rim-disc ratio negatively. The mean DDLS stage in the myopic glaucomatous type tended to be higher than the scores in other types. CONCLUSION: The DDLS obtained through three-dimensional ONH analysis correlates well with the severity of glaucomatous ONH and visual field damage.

The role of calpain in an in vivo model of oxidative stress-induced retinal ganglion cell damage.

PURPOSE: In this study, we set out to establish an in vivo animal model of oxidative stress in the retinal ganglion cells (RGCs) and determine whether there is a link between oxidative stress in the RGCs and the activation of calpain, a major part of the apoptotic pathway. MATERIALS AND METHODS: Oxidative stress was induced in the RGCs of C57BL/6 mice by the intravitreal administration of 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH, 30mM, 2µl). Control eyes were injected with 2µl of vehicle. Surviving Fluorogold (FG)-labeled RGCs were then counted in retinal flat mounts. Double staining with CellROX and Annexin V was performed to investigate the co-localization of free radical generation and apoptosis. An immunoblot assay was used both to indirectly evaluate calpain activation in the AAPH-treated eyes by confirming α-fodrin cleavage, and also to evaluate the effect of SNJ-1945 (a specific calpain inhibitor: 4% w/v, 100mg/kg, intraperitoneal administration) in these eyes. RESULTS: Intravitreal administration of AAPH led to a significant decrease in FG-labeled RGCs 7days after treatment (control: 3806.7±575.2RGCs/mm(2), AAPH: 3156.1±371.2RGCs/mm(2), P<0.01). CellROX and Annexin V signals were co-localized in the FG-labeled RGCs 24h after AAPH injection. An immunoblot assay revealed a cleaved α-fodrin band that increased significantly 24h after AAPH administration. Intraperitoneally administered SNJ-1945 prevented the cleavage of α-fodrin and had a neuroprotective effect against AAPH-induced RGC death (AAPH: 3354.0±226.9RGCs/mm(2), AAPH+SNJ-1945: 3717.1±614.6RGCs/mm(2), P<0.01). CONCLUSION: AAPH administration was an effective model of oxidative stress in the RGCs, showing that oxidative stress directly activated the calpain pathway and induced RGC death. Furthermore, inhibition of the calpain pathway protected the RGCs after AAPH administration.

Artemin augments survival and axon regeneration in axotomized retinal ganglion cells.

Artemin, a recently discovered member of the glial cell line-derived neurotrophic factor (GDNF) family, has neurotrophic effects on damaged neurons, including sympathetic neurons, dopamine neurons, and spiral ganglion neurons both in vivo and in vitro. However, its effects on retinal cells and its intracellular signaling remain relatively unexplored. During development, expression of GFRα3, a specific receptor for artemin, is strong in the immature retina and gradually decreases during maturation, suggesting a possible role in the formation of retinal connections. Optic nerve damage in mature rats causes levels of GFRα3 mRNA to increase tenfold in the retina within 3 days. GFRα3 mRNA levels continue to rise within the first week and then decline. Artemin, a specific ligand for GFRα3, has a neuroprotective effect on axotomized retinal ganglion cells (RGCs) in vivo and in vitro via activation of the extracellular signal-related kinase- and phosphoinositide 3-kinase-Akt signaling pathways. Artemin also has a substantial effect on axon regeneration in RGCs both in vivo and in vitro, whereas other GDNF family members do not. Therefore, artemin/GFRα3, but not other GDNF family members, may be of value for optic nerve regeneration in mature mammals.

Critical neuroprotective roles of heme oxygenase-1 induction against axonal injury-induced retinal ganglion cell death.

Although axonal damage induces significant retinal ganglion cell (RGC) death, small numbers of RGCs are able to survive up to 7 days after optic nerve crush (NC) injury. To develop new treatments, we set out to identify patterns of change in the gene expression of axonal damage-resistant RGCs. To compensate for the low density of RGCs in the retina, we performed retrograde labeling of these cells with 4Di-10ASP in adult mice and 7 days after NC purified the RGCs with fluorescence-activated cell sorting. Gene expression in the cells was determined with a microarray, and the expression of Ho-1 was determined with quantitative PCR (qPCR). Changes in protein expression were assessed with immunohistochemistry and immunoblotting. Additionally, the density of Fluoro-gold-labeled RGCs was counted in retinas from mice pretreated with CoPP, a potent HO-1 inducer. The microarray and qPCR analyses showed increased expression of Ho-1 in the post-NC RGCs. Immunohistochemistry also showed that HO-1-positive cells were present in the ganglion cell layer (GCL), and cell counting showed that the proportion of HO-1-positive cells in the GCL rose significantly after NC. Seven days after NC, the number of RGCs in the CoPP-treated mice was significantly higher than in the control mice. Combined pretreatment with SnPP, an HO-1 inhibitor, suppressed the neuroprotective effect of CoPP. These results reflect changes in HO-1 activity to RGCs that are a key part of RGC survival. Upregulation of HO-1 signaling may therefore be a novel therapeutic strategy for glaucoma.

Correlation between structure/function and optic disc microcirculation in myopic glaucoma, measured with laser speckle flowgraphy.

BACKGROUND: It is difficult to identify glaucoma in myopic eyes because the configuration of the optic disc varies; yet it is important clinically. Here, we used laser speckle flowgraphy (LSFG) to measure mean blur rate (MBR), representing optic disc microcirculation, and assessed its ability to identify glaucoma in eyes with myopic optic discs. METHODS: 129 eyes (normal disc: 21 eyes; myopic disc: 108 eyes) were enrolled. The eyes were classified as normal or mildly, moderately, or severely glaucomatous with standard automated perimetry (SAP). We determined the relationship between optic nerve head (ONH) MBR, measured with LSFG, mean deviation (MD), measured with SAP, and circumpapillary retinal nerve fiber layer thickness (cpRNFLT), measured with optical coherence tomography (OCT). RESULTS: ONH MBR and cpRNFLT decreased significantly with the severity of glaucoma. MBR was significantly correlated with cpRNFLT and MD (r =0.65 and r =0.63, respectively). A multiple regression analysis revealed that MBR and cpRNFLT were independent factors indicating glaucoma severity. A logistic regression analysis revealed that MBR and cpRNFLT were also independent factors indicating the presence of glaucoma. In a receiver operating characteristic (ROC) analysis, MBR and cpRNFLT could both differentiate between normal and glaucomatous eyes (MBR area under the ROC curve: 0.86, with a cut-off score of 24.0 AU). CONCLUSION: These results suggest that in addition to cpRNFLT, non-invasive and objective LSFG measurements of MBR may enable the identification of glaucoma and the classification of its severity in eyes with myopic optic discs.