Toru Nakazawa

Reproducibility of retinal circulation measurements obtained using laser speckle flowgraphy-NAVI in patients with glaucoma

Background: Laser speckle flowgraphy (LSFG) enables noninvasive quantification of the retinal circulation in glaucoma patients. In this study, we tested the intrasession reproducibility of LSFG-NAVI, a modified LSFG technique. Methods: Sixty-five eyes from 33 subjects (male (M):female (F) = 17:16) with a mean age of 49.4 ± 11.2 years were examined in this study. Two parameters indicating reproducibility – the coefficient of variation (COV) and the intraclass correlation coefficient (ICC) – were analyzed three times on the same day that mean blur rate (MBR) was measured using LSFG-NAVI. The sites analyzed were the retinal artery and vein, the optic disk, and the choroid. Following classification according to the Glaucoma Hemifield Test (GHT; SITA-Standard 30-2 program), the COV and ICC were examined in patients with (GHT+; 38 eyes, M:F = 20:18, average age 48.9 ± 12.8 years) and without (GHT−; 27 eyes, M:F = 13:14, average age 50.1 ± 8.7 years) abnormal glaucomatous visual fields. Results: For all subjects, the intrasession reproducibility of MBR in the optic disk (COV: 3.4 ± 2.0; ICC: 0.95) and choroid (COV: 4.7 ± 3.4; ICC: 0.98) was excellent. The reproducibility for the retinal vein (COV: 8.4 ± 5.6, ICC: 0.90) and retinal artery (COV: 10.9 ± 9.9, ICC: 0.9) was moderate. MBRs in the optic disk had good reproducibility in both the GHT+ group (COV: 3.8 ± 2.0; ICC: 0.97) and the GHT− group (COV: 2.9 ± 2.1; ICC: 0.95). Local assessment of the optic disk in normal or glaucoma patients showed that the COVs of the quadrant optic disk areas were best in the temporal area of MBR (3.4%, 4.2%, respectively). Conclusion: LSFG-NAVI showed favorable reproducibility in evaluation of retinal circulation of glaucoma patients, particularly in the optic disk and choroid.

Critical role of Nrf2 in oxidative stress-induced retinal ganglion cell death

NF‐E2 related factor 2 (Nrf2) is a key transcription factor that plays a pivotal role in endogenous protection against oxidative stress. However, the role of Nrf2 in visual disorders remains unclear. It has been reported that oxidative stress is thought of as one of the causes of glaucoma. Here, we investigate whether the function of Nrf2 in oxidative stress‐induced retinal ganglion cell (RGC) death. This study used adult male Nrf2 deficient mice (Nrf2 KO) and age‐ and sex‐matched wild‐type (WT) mice. We dissociated and purified N‐4‐[4‐didecylaminostryryl]‐N‐methyl‐pyridinium iodide‐labeled RGCs with fluorescence‐activated cell sorting, and tried to detect the Nrf2 and Keap1 genes. In the absence of nerve crush (NC), the number of RGCs in Nrf2 KO mice was almost same as that in WT mice. 1‐(2‐cyano‐3‐, 12‐dioxooleana‐1, 9 (11)‐dien‐28‐oyl) imidazole (CDDO‐Im), an Nrf2 activator, prevented NC‐induced loss of RGCs in WT mice. Seven days after NC, without treatment, the number of RGCs in Nrf2 KO mice was significantly lower than in WT mice. In addition, after CDDO‐Im treatment, quantitative RT‐PCR showed increased expression of antioxidant and phase II detoxifying enzymes. These results suggest that up‐regulation of Nrf2 signaling after CDDO‐Im treatment may be a novel therapeutic strategy for the protection of RGCs, especially in glaucoma.

Waveform Analysis of Ocular Blood Flow and the Early Detection of Normal Tension Glaucoma

PURPOSE To investigate waveform changes in blood flow (BF) in the optic nerve head (ONH) and to evaluate their usefulness in identifying normal tension glaucoma (NTG). METHODS Sixty-one eyes of 61 patients with NTG and 21 eyes of age-matched healthy control subjects were included in this study. The NTG eyes were divided into the following three groups based on the progression of their visual field defects: mild (mean deviation [MD] greater than -6.0 decibels [dB]), moderate (MD between -6.0 and -12.0 dB), and severe (MD less than -12.0 dB). The ONH BF analysis was performed with laser speckle flowgraphy (LSFG) and included waveform variables such as skew, acceleration time index (ATI), and blowout time. RESULTS In the ONH, LSFG skew variables were significantly lower in the NTG eyes than in the control eyes (P < 0.001), and ATI was significantly higher (P < 0.01), despite similar systemic characteristics in the four groups. The differences were most marked in the mild NTG group. Multiple linear regression analysis showed that MD, average thickness of the circumpapillary retinal nerve fiber layer, and pulse rate were predictive factors for both skew and ATI. A receiver operating characteristic (ROC) curve analysis also revealed that skew (area under the ROC curve, 0.89) and ATI (area under the ROC curve, 0.80) had the greatest power to differentiate normal eyes from eyes with mild NTG. CONCLUSIONS These results suggest that LSFG measurements of waveform changes in ONH BF can differentiate healthy eyes from eyes with NTG, particularly those with mild NTG.

Significant correlations between optic nerve head microcirculation and visual field defects and nerve fiber layer loss in glaucoma patients with myopic glaucomatous disk

Background Eyes with glaucoma are characterized by optic neuropathy with visual field defects in the areas corresponding to the optic disk damage. The exact cause for the glaucomatous optic neuropathy has not been determined. Myopia has been shown to be a risk factor for glaucoma. The purpose of this study was to determine whether a significant correlation existed between the microcirculation of the optic disk and the visual field defects and the retinal nerve fiber layer thickness (RNFLT) in glaucoma patients with myopic optic disks. Methods Sixty eyes of 60 patients with myopic disks were studied; 36 eyes with glaucoma (men:women = 19:17) and 24 eyes with no ocular diseases (men:women = 14:10). The mean deviation (MD) determined by the Humphrey field analyzer, and the peripapillary RNFLT determined by the Stratus-OCT were compared between the two groups. The ocular circulation was determined by laser speckle flowgraphy (LSFG), and the mean blur rate (MBR) was compared between the two groups. The correlations between the RNFLT and MBR of the corresponding areas of the optic disk and between MD and MBR of the optic disk in the glaucoma group were determined by simple regression analyses. Results The average MBR for the entire optic disk was significantly lower in the glaucoma group than that in the control group. The differences of the MBR for the tissue in the superior, inferior, and temporal quadrants of the optic disk between the two groups were significant. The MBR for the entire optic disk was significantly correlated with the MD (r = 0.58, P = 0.0002) and the average RNFLT (r = 0.53, P = 0.0008). The tissue MBR of the optic disk was significantly correlated with the RNFLT in the superior, inferior, and temporal quadrants. Conclusions Our study suggests that there is a causal relationship between the thinner RNFLT that led to the MD and reduction in the microcirculation in the optic nerve head.

Tumor Necrosis Factor-α Mediates Photoreceptor Death in a Rodent Model of Retinal Detachment

PURPOSE Photoreceptor degeneration is a major cause of visual loss in various retinal diseases, including retinal detachment (RD) and neovascular AMD, but the underlying mechanisms remain elusive. In this study, the role of TNFα in RD-induced photoreceptor degeneration was investigated. METHODS RD was induced by subretinal injection of hyaluronic acid. Photoreceptor degeneration was assessed by counting the number of apoptotic cells with TdT-dUTP terminal nick-end labeling (TUNEL) 3 days after RD and measurement of the outer nuclear layer (ONL) thickness 7 days after RD. As the target of anti-inflammatory treatment, the expression of TNFα, with or without dexamethasone (DEX) was examined in rats by real-time PCR. To understand the role of TNFα in photoreceptor degeneration, RD was induced in mice deficient in TNFα or its receptors (TNFR1, TNFR2, and TNFR1 and -2), or in wild-type (WT) mice by using a functionally blocking antibody to TNFα. CD11b(+) cells in the outer plexiform layer (OPL) and subretinal space were counted by immunohistochemistry (IHC). RESULTS Treatment with DEX (P = 0.001) significantly suppressed RD-induced photoreceptor degeneration and the expression of TNFα. RD-induced photoreceptor degeneration was significantly suppressed with specific blockade of TNFα (P = 0.032), in mice deficient for TNFα (P < 0.001), TNFR2 (P = 0.001), or TNFR1 and -2 (P < 0.001). However, lack of TNFR1 did not protect against RD-induced photoreceptor degeneration (P = 0.060). Müller cell activation was unchanged in WT and TNFα(-/-) mice. Recruitment of CD11b(+) monocytes was significantly lower in the TNFα(-/-) mice compared to WT mice (P = 0.002). CONCLUSIONS TNFα plays a critical role in RD-induced photoreceptor degeneration. This pathway may become an important target in the prevention of RD-induced photoreceptor degeneration.

Critical role of calpain in axonal damage-induced retinal ganglion cell death

Calpain, an intracellular cysteine protease, has been widely reported to be involved in neuronal cell death. The purpose of this study is to investigate the role of calpain activation in axonal damage‐induced retinal ganglion cell (RGC) death. Twelve‐week‐old male calpstatin (an endogenous calpain inhibitor) knockout mice (CAST KO) and wild‐type (WT) mice were used in this study. Axonal damage was induced by optic nerve crush (NC) or tubulin destruction induced by leaving a gelatin sponge soaked with vinblastine (VB), a microtubule disassembly chemical, around the optic nerve. Calpain activation was assessed by immunoblot analysis, which indirectly quantified the cleaved α‐fodrin, a substrate of calpain. RGCs were retrogradely labeled by injecting a fluorescent tracer, Fluoro‐Gold (FG), and the retinas were harvested and flat‐mounted retinas prepared. The densities of FG‐labeled RGCs harvested from the WT and CAST KO groups were assessed and compared. Additionally, a calpain inhibitor (SNJ‐1945, 100 mg/kg/day) was administered orally, and the density of surviving RGCs was compared with that of the vehicle control group. The mean density of surviving RGCs in the CAST KO group was significantly lower than that observed in the WT group, both in NC and in VB. The mean density of surviving RGCs in the SNJ‐1945‐treated group was significantly higher than that of the control group. The calpain inhibitor SNJ‐1945 has a neuroprotective effect against axonal damage‐induced RGC death. This pathway may be an important therapeutic target for preventing this axonal damage‐induced RGC death, including glaucoma and diabetic optic neuropathy and other CNS diseases that share a common etiology.

Visual Outcome After Intravitreal Triamcinolone Acetonide Depends On Optical Coherence Tomographic Patterns In Patients With Diffuse Diabetic Macular Edema

Purpose: To evaluate the effectiveness of intravitreal triamcinolone acetonide (IVTA) on the reduction of diffuse diabetic macular edema with different optical coherence tomographic patterns. Methods: One hundred and thirty-five eyes with diffuse diabetic macular edema without any treatment that had received a single dose (4 mg in 0.1 mL) of IVTA were retrospectively examined. Each preoperative macular optical coherence tomographic image was classified according to its appearance as follows: sponge-like diffuse retinal thickening, cystoid macular edema (CME), and serous retinal detachment (SRD). Central macular thickness with optical coherence tomographic images and visual acuity with a logarithm of the minimum angle of resolution chart were assessed at 3 months postoperatively. Results: Of 135 eligible eyes, 49 eyes were identified as having only sponge-like diffuse retinal thickening, 45 eyes with CME, and 26 eyes with SRD. Of those 135 eyes, 15 eyes exhibited the combination of all types of diffuse diabetic macular edema, defined as FULL. After IVTA, central macular thickness was reduced to 31.0 ± 15.9% in the sponge-like diffuse retinal thickening, 40.7 ± 14.2% in the CME, 23.4 ± 15.0% in the SRD, and 25.8 ± 14.8% in the FULL group (P < 0.001; one-factor analysis of variance), while improvement in logarithm of the minimum angle of resolution visual acuity was −0.26 ± 0.21 in the sponge-like diffuse retinal thickening, −0.32 ± 0.20 in the CME, −0.17 ± 0.20 in the SRD, and −0.14 ± 0.22 in the FULL group (P = 0.018; one-factor analysis of variance). Conclusion: The effectiveness of IVTA on diffuse diabetic macular edema was dependent on the optical coherence tomographic pattern, and IVTA was found to be more effective in patients with CME, while IVTA was less effective in those with SRD.

Heat Shock Protein 70 (HSP70) Is Critical for the Photoreceptor Stress Response after Retinal Detachment via Modulating Anti-Apoptotic Akt Kinase

Photoreceptor apoptosis is a major cause of vision loss in many ocular diseases. Significant progress has been made to elucidate the molecular pathways involved in this process, yet little is known about proteins counteracting these apoptotic pathways. It is established that heat shock proteins (HSPs) function as molecular helper proteins (chaperones) by preventing protein aggregation and facilitating refolding of dysfunctional proteins, critical to the survival of all organisms. Here, we investigated the role of HSP70 on photoreceptor survival after experimental retinal detachment (RD) in mice and rats. We found that HSP70 was up-regulated after RD and associated with phosphorylated Akt, thereby preventing its dephosphorylation and further activation of cell death pathways. Administration of quercetin, which inhibits HSP70 and suppresses Akt phosphorylation significantly increased photoreceptor apoptosis. Similarly, RD-induced photoreceptor apoptosis was augmented in mice carrying hypomorphic mutations of the genes encoding HSP70. On the other hand, administration of geranylgeranylacetone, which induces an increase in HSP70 significantly decreased photoreceptor apoptosis after RD through prolonged activation of Akt pathway. Thus, HSP70 may be a favorable potential target to increase photoreceptor cell survival after RD.

Metabolic stress response implicated in diabetic retinopathy: The role of calpain, and the therapeutic impact of calpain inhibitor

To describe how a high fat diet (HFD) and hyperglycemia initiate a sequence of calpain activation and oxidative stress associated with neuro-degenerative changes in diabetic retinopathy (DR), hyperglycemia was induced with streptozotocin in mice lacking the gene for calpastatin (CAST KO), and in mice lacking the gene for the transcription factor NF-E2 related factor 2 (Nrf2 KO). All animals were fed a HFD. Retinal ganglion cell (RGC) density was estimated by labeling with fluorogold and immunohistochemistry. A potent calpain inhibitor, SNJ-1945, was administered daily until the animals were sacrificed. In vitro, oxidative stress-induced RGC loss was evaluated in a high glucose culture medium with and without SNJ-1945. Retinal mRNA of calpain-1 and calpain-2 was measured by quantitative RT-PCR. Pre-apoptotic substrates of cleaved α-fodrin and synaptophysin protein were quantified by immunoblot analysis. Axonal damage was examined in transverse sections of the optic nerve. A HFD and hyperglycemia significantly increased RGC and axonal degeneration 3 weeks into the experiment. Levels of cleaved α-fodrin were increased. In the CAST KO mice, the neurotoxicity was augmented significantly. Gene manipulation of CAST and orally administered SNJ-1945 successfully modified calpain levels in the retina and prevented RGC death. In vitro, a high-glucose culture of retinal cells without antioxidants showed more RGC death than that with antioxidant treatment. The expression of synaptophysin was significantly suppressed by SNJ-1945 treatment. These results suggest that calpain plays a crucial role in metabolic-induced RGC degeneration caused by hyperglycemia and oxidative stress. Antioxidant and calpain inhibition offers important opportunities for future neuroprotective treatment against RGC death in various metabolic stress-induced diseases including DR.

Association between optic nerve blood flow and objective examinations in glaucoma patients with generalized enlargement disc type.

Background The purpose of this study was to investigate the correlations between microcirculation in the optic disc, average peripapillary retinal nerve fiber layer thickness cupping parameters, and visual field defects in glaucoma patients with the generalized enlargement disc type. Methods A total of 38 eyes from 38 glaucoma patients with the generalized enlargement disc type were included. The microcirculation of the optic nerve head was examined with laser speckle flow graphy, and the mean blur rate in all areas, in vessel area, and in tissue area were calculated using the laser speckle flow graphy analyzer software. Average peripapillary retinal nerve fiber layer thickness was measured using Stratus optical coherence tomography, and cupping parameters were accessed using the Heidelberg retina tomograph. The mean deviation in the Humphrey field analyzer (30-2 SITA standard) was analyzed. The correlation between these parameters was evaluated using the Spearman rank correlation coefficient. Results The correlation coefficient of mean blur rate in all optic disc area to the average peripapillary retinal nerve fiber layer thickness, vertical C/D, and mean deviation were r = 0.7546 (P < 0.0001), r = −0.6208 (P < 0.0001), and r = 0.6010 (P = 0.0001), respectively. The mean blur rate in tissue area of the optic disc showed r = 0.7305 (P < 0.0001), r = −0.6438 (P < 0.0001), and r = 0.6338 (P < 0.0001). Conclusion We found that the mean blur rate in the optic disc was significantly correlated with the average peripapillary retinal nerve fiber layer thickness, vertical C/D, and mean deviation in patients with the generalized enlargement disc type of glaucoma. In particular, the mean blur rate in tissue area was more highly correlated than the vessel area with other results of examination in glaucoma patients with the generalized enlargement disc type.