Taiki Kokubun

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.

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.

Relationship of Skin Autofluorescence to Severity of Retinopathy in Type 2 Diabetes

Abstract Purpose: To evaluate the relationship between skin autofluorescence (SAF), which reflects the accumulation of advanced glycation end products (AGEs), and the severity of diabetic retinopathy (DR) in patients with type 2 diabetes. Methods: Sixty-seven eyes of 67 patients with type 2 diabetes were enrolled. Sixty-seven age-matched non-diabetic subjects served as controls. Diabetic patients were classified by the severity of their DR: no DR (NDR), non-proliferative DR (NPDR), and proliferative DR (PDR). SAF was measured with an autofluorescence reader. Results: SAF in the diabetes patients was significantly higher than in the controls (median 2.5 (interquartile range 2.3–2.7) and 1.8 (1.6–2.3) arbitrary unit (AU), respectively, p < 0.001). There was a statistically significant increase in SAF along with the increasing severity of DR (from NDR to NPDR: p = 0.034; NPDR to PDR: p < 0.01). Logistic regression analysis revealed that SAF (OR, 17.2; p < 0.05) was an independent factor indicating the presence of PDR. Conclusions: SAF has an independent relationship with PDR in patients with type 2 diabetes. SAF measurement with an autofluorescence reader is a non-invasive way to assess the risk of DR. SAF may, therefore, be a surrogate marker candidate for the non-invasive evaluation of DR.

The traditional kampo medicine tokishakuyakusan increases ocular blood flow in healthy subjects.

The aim of this study was to examine the effects of oral administration of kampo medical formulas on ocular blood flow (OBF). A crossover protocol was used to randomly administer five grams of yokukansan, tokishakuyakusan (TSS), keishibukuryogan, or hachimijiogan to 13 healthy blinded subjects (mean age: 37.3 ± 12.3 years). The mean blur rate, a quantitative OBF index obtained with laser speckle flowgraphy, was measured at the optic nerve head before and 30 minutes after administration. Blood pressure (BP) and intraocular pressure (IOP) were also recorded. No significant changes were observed in mean BP or IOP after the administration of any of the kampo medical formulas. There was a significant increase in OBF 30 minutes after administration of TSS (100% to 103.6 ± 6.9%, P < 0.01). Next, TSS was administered to 19 healthy subjects (mean age: 32.0 ± 11.0 years) and OBF was measured before and 15, 30, 45, and 60 minutes after administration. Plain water was used as a control. OBF increased significantly after TSS administration compared to control (P < 0.01) and also increased from 30 to 60 minutes after administration compared to baseline (P < 0.05). These results suggest that TSS can increase OBF without affecting BP or IOP in healthy subjects.

Characteristic Profiles of Inflammatory Cytokines in the Aqueous Humor of Glaucomatous Eyes

ABSTRACT Purpose: To analyze cytokine profiles of the aqueous humor of eyes with primary open-angle glaucoma (POAG), neovascular glaucoma (NVG), and cataract (as controls). Methods: A multiplex bead assay was used to measure concentrations of 27 cytokines in aqueous humor samples from 54 eyes. Results: Detection rates were as follows: IL-7: NVG higher than POAG; IL-10: POAG lower than cataract or NVG; and GM-CSF: cataract higher than POAG or NVG. Concentrations were as follows: IL-8, IP-10, MCP-1, and MIP-1β: POAG and NVG higher than cataract; IL-9: POAG lower than NVG; IL-12: POAG lower than cataract or NVG; and VEGF: NVG higher than cataract or POAG and POAG lower than cataract. Further analysis showed that IL-8, IP-10, MCP-1, and MIP-1β were correlated with intraocular pressure and age. Conclusions: The detection rates and levels of various cytokines had different patterns in POAG and NVG patients, suggesting distinctive alterations in the microenvironment in different types of glaucoma.

Estimation of Jones matrix, birefringence and entropy using Cloude-Pottier decomposition in polarization-sensitive optical coherence tomography

Estimation of polarimetric parameters has been a fundamental issue to assess biological tissues that have form birefringence or polarization scrambling in polarization-sensitive optical coherence tomography (PS-OCT). We present a mathematical framework to provide a maximum likelihood estimation of the target covariance matrix and its incoherent target decomposition to estimate a Jones matrix of a dominant scattering mechanism, called Cloude-Pottier decomposition, thereby deriving the phase retardation and the optic axis of the sample. In addition, we introduce entropy that shows the randomness of the polarization property. Underestimation of the entropy at a low sampling number is mitigated by asymptotic quasi maximum likelihood estimator. A bias of the entropy from random noises is corrected to show only the polarization property inherent in the sample. The theory is validated with experimental measurements of a glass plate and waveplates, and applied to the imaging of a healthy human eye anterior segment as an image filter.

Quantification of the filtering bleb's structure with anterior segment optical coherence tomography.

The aim of the study is to determine the relationship between post‐trabeculectomy bleb structure evaluated using anterior segment optical coherence tomography (AS‐OCT) and postoperative intraocular pressure (IOP).

Metabolomic changes in the mouse retina after optic nerve injury

In glaucoma, although axonal injury drives retinal ganglion cell (RGC) death, little is known about the underlying pathomechanisms. To provide new mechanistic insights and identify new biomarkers, we combined latest non-targeting metabolomics analyses to profile altered metabolites in the mouse whole retina 2, 4, and 7 days after optic nerve crush (NC). Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry and liquid chromatography Fourier transform mass spectrometry covering wide spectrum of metabolites in combination highlighted 30 metabolites that changed its concentration after NC. The analysis displayed similar changes for purine nucleotide and glutathione as reported previously in another animal model of axonal injury and detected multiple metabolites that increased after the injury. After studying the specificity of the identified metabolites to RGCs in histological sections using imaging mass spectrometry, two metabolites, i.e., L-acetylcarnitine and phosphatidylcholine were increased not only preceding the peak of RGC death in the whole retina but also at the RGC layer (2.3-fold and 1.2-fold, respectively). These phospholipids propose novel mechanisms of RGC death and may serve as early biomarkers of axonal injury. The combinatory metabolomics analyses promise to illuminate pathomechanisms, reveal biomarkers, and allow the discovery of new therapeutic targets of glaucoma.

Anterior-Segment Optical Coherence Tomography for Predicting Postoperative Outcomes After Trabeculectomy

ABSTRACT Purpose: To determine whether anterior-segment optical coherence tomography (AS-OCT) can be used to predict post-trabeculectomy bleb outcomes. Materials and methods: We divided 58 eyes of 47 trabeculectomy patients into success or failure groups based on their status at 12 months after surgery. We then compared various AS-OCT measurement parameters between the two groups at 1 and 2 weeks and 1, 3, 6, and 12 months. We also analyzed the early post-trabeculectomy bleb parameters with multiple logistic regression, stepwise multiple regression, and the receiver operating characteristic (ROC) curve, to evaluate the power of these parameters to predict long-term outcomes. Results: Intraocular pressure 3 or more months after trabeculectomy was significantly lower in the success group than the failure group (all: P < 0.0016). Cleft volume was significantly higher 6 or more months after trabeculectomy in the success group than the failure group (P = 0.0027 and <0.0016). Reflectivity of the bleb wall was significantly higher in the failure group than the success group at 2 weeks and all later time points (all: P < 0.0016). Reflectivity of the bleb wall at 2 weeks after trabeculectomy was a risk factor for failure, with an odds ratio (OR) for failure of 2.48 (95% confidence interval, 1.31–4.68, increasing per 10 AU). The area under the ROC curve for reflectivity of the bleb wall at 2 weeks after trabeculectomy was 0.775 when the cutoff value was set at 122.8, with sensitivity, specificity, and OR of 78.3%, 80.0%, and 14.4, respectively. A stepwise multiple regression analysis showed that reflectivity of the bleb wall at 2 weeks was an independent factor indicating postoperative bleb survival period (β = −0.39, P = 0.007). Conclusions: Reflectivity of the bleb wall, measured by AS-OCT, may be an early post-trabeculectomy predictor of bleb outcome.

Association between mitochondrial DNA damage and ocular blood flow in patients with glaucoma

Background/Aims We determined the relationship between tissue mean blur rate (MT) and mitochondrial dysfunction, represented by the mitochondrial/nuclear DNA (mtDNA/nDNA) ratio. We also investigated the usefulness of these biomarkers. Methods We assessed ocular blood flow in 123 eyes of 123 patients with open-angle glaucoma (OAG) and 37 control eyes of 37 healthy subjects by measuring MT in the optic nerve head with laser speckle flowgraphy. We measured mtDNA and nDNA with PCR, calculated the mtDNA/nDNA ratio and compared this ratio with MT using Spearman’s rank test. We used multiple regression analysis to further investigate the association between MT and glaucoma in the most severe group. Results The control and the patients with glaucoma had significant differences in the mtDNA/nDNA ratio, circumpapillary retinal nerve fibre layer thickness and MT. There was no significant relationship between the mtDNA/nDNA ratio and MT in patients with OAG overall or the female patients with OAG, but there was a significant relationship between the mtDNA/nDNA ratio and MT, temporal-MT and superior-MT in male patients with severe OAG (r=−0.46, p=0.03; r=−0.51, p=0.02; r=−0.61, p<0.01, respectively). Furthermore, we found that the mtDNA/nDNA ratio was an independent contributor to temporal-MT and superior-MT in these patients (p<0.01 and p=0.03, respectively). Conclusion We found that there was a significant relationship between the mtDNA/nDNA ratio and MT in male patients with severe OAG, suggesting that the mtDNA/nDNA ratio may be a new biomarker in glaucoma and may help research on the vulnerability of these patients to mitochondrial dysfunction.