Satoru Tsuda

Development of Azo‐Based Fluorescent Probes to Detect Different Levels of Hypoxia

Let it shine: New hypoxia-sensitive fluorescent probes were developed; they consist of a rhodamine moiety with an azo group directly conjugated to the fluorophore. Because of an ultrafast conformational change around the NN bond, the compounds are nonfluorescent under normoxia. However, under hypoxia, the azo group is reduced, and a strongly fluorescent rhodamine derivative is released.

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.

Profiles of extracellular miRNAs in the aqueous humor of glaucoma patients assessed with a microarray system.

Aqueous humor (AH) is one of the body fluids in the eye, which is known to be related with various ocular diseases, but the complete RNAs characteristic of the AH in patients is not yet known. The aim of this study was, with a microarray analysis, to reveal the disease-related extracellular miRNAs profiles in individual patients AH. 100 μl of AH was collected by anterior chamber paracentesis from 10 glaucoma, 5 cataract, and 5 epiretinal membrane patients. The extracted total RNAs were shorter than 200 nt, and their amount was 5.27 ± 0.41 ng in average. Among 530.5 ± 44.6 miRNA types detected in each sample with a microarray detectable 2019 types of matured miRNAs, 172 miRNAs were detected in all 10 glaucoma or control patients. From the glaucoma group, 11 significantly up-regulated and 18 significantly down-regulated miRNAs (P < 0.05 for both) were found to have areas under the curve better than 0.74 in a receiver operating characteristic analysis. They also formed a cluster composed only of glaucoma patients in a hierarchal cluster analysis. AH had a possibility of becoming a source of miRNA that can serve as a biomarker and a therapeutic target.

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.

Pulse-Waveform Analysis of Normal Population using Laser Speckle Flowgraphy

Abstract Purpose: Laser speckle flowgraphy (LSFG), a new, non-invasive method of measuring the mean blur rate (MBR) of ocular blood flow, allows for the analysis of the pulse waveform of a heartbeat as it changes dynamically. Here, we investigated the relationship between the pulse waveform and clinical parameters, particularly age. Materials and methods: Sixty eyes of 60 healthy subjects without diabetes were enrolled from among patients undergoing annual health examinations. LSFG, and its analysis software, were used to determine pulse waveform parameters including MBR, skew, blowout score (BOS), blowout time (BOT), rising rate and falling rate in the optic nerve head (ONH), both specifically in the tissue area and in the ONH overall. Fifteen clinical parameters were also recorded, including age and blood pressure, as well as triglyceride and creatinine levels. Results: Skew, BOT and falling rate had a strong correlation (|r| > 0.60) with age, but not with the other clinical parameters. This correlation with age was stronger in the tissue area (BOT: p < 0.0001, r = − 0.68; skew: p < 0.0001, r = 0.65; falling rate: p < 0.0001, r = 0.61) than in the ONH overall (BOT: p < 0.0001, r = −0.67; skew: p < 0.0001, r = 0.60; falling rate: p < 0.0001, r = 0.59). Stepwise multiple regression analysis revealed that tissue area falling rate was an independent factor indicating age, and conversely that age was an independent factor indicating tissue area falling rate. Conclusions: The significant correlation of LSFG-measured tissue area falling rate with age suggests that it may be a new candidate biomarker for age-dependent microcirculation.

The Influence of Posture Change on Ocular Blood Flow in Normal Subjects, Measured by Laser Speckle Flowgraphy

Abstract Purpose: To investigate, using laser speckle flowgraphy (LSFG), the autoregulation of ocular blood flow (BF) in response to posture change. Methods: This study comprised 20 healthy volunteers (mean age 30.0 ± 8.5). The mean blur rate (MBR) of the ocular circulation in the subjects was assessed in both a sitting and a supine position every 2 min over the course of 10 min. Baseline measurements of the MBR at the optic nerve head (ONH) and the choroid were taken in a sitting position. Increases in the MBR ratio in a supine position were calculated with reference to this baseline. Intraocular pressure (IOP), systemic blood pressure and heart rate in the brachial artery were also recorded. Results: In the ONH, the MBR ratio increased significantly over the baseline after 2 min (104.8 ± 5.0%, p = 0.001) and 4 min (104.4 ± 5.6%, p = 0.005), in a supine position, but decreased to the initial level after only 6 min. In the choroid, on the other hand, while the MBR ratio also increased significantly after 2 min in a supine position (113.7 ± 8.1%, p < 0.001), it kept this significant increase over the time course of 10 min. After 10 min in a supine position, IOP increased significantly (p < 0.001), systolic blood pressure decreased significantly (p < 0.001), but diastolic blood pressure did not change significantly compared to the baseline. (p = 0.07) Conclusions: ONH and choroidal circulation have significantly different hemodynamics in response to posture change in healthy volunteers. This finding suggests that LSFG enables us to assess the autoregulation of BF in the ONH.

RNA Sequence Reveals Mouse Retinal Transcriptome Changes Early after Axonal Injury

Glaucoma is an ocular disease characterized by progressive retinal ganglion cell (RGC) death caused by axonal injury. However, the underlying mechanisms involved in RGC death remain unclear. In this study, we investigated changes in the transcriptome profile following axonal injury in mice (C57BL/6) with RNA sequencing (RNA-seq) technology. The experiment group underwent an optic nerve crush (ONC) procedure to induce axonal injury in the right eye, and the control group underwent a sham procedure. Two days later, we extracted the retinas and performed RNA-seq and a pathway analysis. We identified 177 differentially expressed genes with RNA-seq, notably the endoplasmic reticulum (ER) stress-related genes Atf3, Atf4, Atf5, Chac1, Chop, Egr1 and Trb3, which were significantly upregulated. The pathway analysis revealed that ATF4 was the most significant upstream regulator. The antioxidative response-related genes Hmox1 and Srxn1, as well as the immune response-related genes C1qa, C1qb and C1qc, were also significantly upregulated. To our knowledge, this is the first reported RNA-seq investigation of the retinal transcriptome and molecular pathways in the early stages after axonal injury. Our results indicated that ER stress plays a key role under these conditions. Furthermore, the antioxidative defense and immune responses occurred concurrently in the early stages after axonal injury. We believe that our study will lead to a better understanding of and insight into the molecular mechanisms underlying RGC death after axonal injury.

Effect of Topical Tafluprost on Optic Nerve Head Blood Flow in Patients With Myopic Disc Type

Purpose:To investigate the effect of topical tafluprost on optic disc blood flow in patients with myopic disc. Materials and Methods:Forty-eight eyes in 24 patients with a myopic disc type (oval shaped) optic disc tilted to the temporal, with a crescent peripapillary atrophy were included in this study. Twenty-eight eyes were diagnosed as normal tension glaucoma and 20 eyes were in normal subjects. None had any treatment for glaucoma. Average age was 45.3±11.9 years. One eye was treated with topical tafluprost and the fellow eye served as the control. Ocular blood flow was measured by laser speckle flowgraphy (LSFG-NAVI) at 30, 60, 90, and 120 minutes after tafluprost administration, and the mean blur rate (MBR) on the optic disc was analyzed. Blood pressure and intraocular pressure (IOP) were recorded. Results:In all subjects, topical tafluprost (a) significantly reduced IOP versus baseline from 60 minutes after treatment (baseline: 15.2±3.4 mm Hg, 60 min: 13.3±3.2 mm Hg, P=0.001, 90 min: 13.3±3.6 mm Hg, P=0.002, 120 min: 13.7±3.4 mm Hg, P=0.007); and (b) significantly increased the MBR versus baseline (60 min: +4.3±6.6%, P=0.008, 90 min: +5.0±4.9%, P<0.001, 120 min: +6.7±7.0%, P<0.001). Conclusions:Topical tafluprost increased MBR in the optic nerve head and significantly reduced IOP, effects that may represent beneficial treatment for glaucoma patients with a myopic disc type.

Characteristics of uveitic glaucoma and evaluation of its surgical treatment.

Purpose To investigate the characteristics of uveitic glaucoma (UG) and evaluate surgical treatments. Methods This study examined a retrospective, nonrandomized comparative interventional case series of 105 UG patients (141 eyes) followed between April 1, 2001 and July 30, 2014 at the outpatient clinic of Tohoku University Hospital. The study group included 47 patients (47 eyes) who underwent glaucoma surgery: trabeculectomy, trabeculotomy, and trabectome surgery. The analysis used Kaplan–Meier life tables, with surgical failure defined as intraocular pressure ≧21 mmHg or the need for additional glaucoma surgery. Results UG patients represented 9.73% of our database of glaucoma patients. The mean follow-up period was 40.32±32.53 months. Seventy-one patients had granulomatous uveitis (67.62%) and 34 had nongranulomatous uveitis (32.38%). The causes of uveitis included sarcoidosis (n=25), Behçet’s disease (n=11), Vogt–Koyanagi–Harada disease (n=9), Posner–Schlossman syndrome (n=12), herpes simplex virus infectious uveitis (n=7), acute anterior uveitis (n=5), intermediate uveitis (n=4), scleritis (n=4), inflammatory bowel disease (n=4), varicella zoster virus uveitis (n=2), and others (n=6). An additional 16 patients were diagnosed with idiopathic UG. Surgical success rates were 82.86% for trabeculectomy, 62.50% for trabeculotomy, and 75.00% for trabectome. Significant risk factors for surgical failure included male sex (P=0.02), age less than 45 years (P=0.0009), nongranulomatous uveitis (P=0.04), and postoperative inflammation (P=0.01). Conclusion Young male patients with nongranulomatous uveitis had a significant risk of surgical failure. Moreover, prolonged postoperative inflammation created a susceptibility to surgical failure, indicating the importance of postoperative inflammation reduction.

Fiber-based polarization-sensitive OCT for birefringence imaging of the anterior eye segment

We demonstrate a prototype system of polarization-sensitive optical coherence tomography (PS-OCT) designed for clinical studies of the anterior eye segment imaging. The system can measure Jones matrices of the sample with depth-multiplexing of two orthogonal incident polarizations and polarization-sensitive detection. An optical clock is generated using a quadrature modulator and a logical circuit to double the clock frequency. Systematic artifacts in measured Jones matrices are theoretically analyzed and numerically compensated using signals at the surface of the sample. Local retardation images of filtering blebs after trabeculectomy show improved visualization of subconjunctival tissue, sclera, and scar tissue of the bleb wall in the anterior eye segment.