Publications by authors named "Hajime Shinoda"

71 Publications

Amplification-free RNA detection with CRISPR-Cas13.

Commun Biol 2021 04 19;4(1):476. Epub 2021 Apr 19.

Molecular Physiology Laboratory, Cluster for Pioneering Research, RIKEN, Saitama, Japan.

CRISPR-based nucleic-acid detection is an emerging technology for molecular diagnostics. However, these methods generally require several hours and could cause amplification errors, due to the pre-amplification of target nucleic acids to enhance the detection sensitivity. Here, we developed a platform that allows "CRISPR-based amplification-free digital RNA detection (SATORI)", by combining CRISPR-Cas13-based RNA detection and microchamber-array technologies. SATORI detected single-stranded RNA targets with maximal sensitivity of ~10 fM in <5 min, with high specificity. Furthermore, the simultaneous use of multiple different guide RNAs enhanced the sensitivity, thereby enabling the detection of the SARS-CoV-2 N-gene RNA at ~5 fM levels. Therefore, we hope SATORI will serve as a powerful class of accurate and rapid diagnostics.
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http://dx.doi.org/10.1038/s42003-021-02001-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055673PMC
April 2021

Association between axial length and choroidal thickness in early age-related macular degeneration.

PLoS One 2020 9;15(10):e0240357. Epub 2020 Oct 9.

Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.

The clinical course of age-related macular degeneration (AMD) is related to choroidal conditions, and can be determined by the evaluation of the central choroidal thickness (CCT). The aim of this study was to determine the association between the axial length (AL) and choroidal thickness in AMD by measuring these parameters in patients with and without AMD. Seventy eyes of 70 patients (34 men and 36 women; age, 64-88 years; mean age, 77.0 ± 6.5 years) who underwent cataract surgery from February 2015 to March 2020 at the Department of Ophthalmology, Keio University School of Medicine were retrospectively analyzed. The AMD group (29 patients, 29 eyes) included eyes with early AMD, whereas the control group (41 patients, 41 eyes) included those without ocular diseases other than cataract. Optical coherence tomography images were used to measure the CCT and the choroidal vessel diameter (CVD). The IOL Master was used to measure the AL. The results revealed that mean CCT was greater in the AMD group (238.3 ± 108.3 μm) compared with the age-matched control group (187.2 ± 66.8 μm) (p = 0.03). The CCT was negatively correlated with AL in the overall sample (r = -0.42, p = 0.001), the AMD group (r = -0.42, p = 0.02), and the control group (r = -0.42, p = 0.006). Note that all eyes with CCT > 350 μm were included in the AMD group. CCT and CVD were positively correlated in the overall sample (r = 0.76, p < 0.001) as well as in the individual groups (AMD: r = 0.82, p < 0.001; control: r = 0.76, p = 0.004). Given that CCT is an important parameter for predicting the prognosis of subfoveal diseases, routine evaluation of AL may be valuable for a better understanding of the pathogenesis of AMD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240357PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546466PMC
December 2020

Risk of newly developing visual field defect and neurodegeneration after pars plana vitrectomy for idiopathic epiretinal membrane.

Br J Ophthalmol 2020 Oct 5. Epub 2020 Oct 5.

Department of Ophthalmology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan

Background/aims: Pars plana vitrectomy (PPV) is widely performed in patients with idiopathic epiretinal membrane (iERM) to improve vision. Postoperative visual field defects (VFDs) have been previously reported. However, whether they occur when using the most recent PPV system, and the frequency of VFDs as measured by standard automated perimetry, remain poorly documented and were examined in this study.

Methods: Data of 30 eyes (30 patients; mean age, 66.1 years; 15 men) who underwent PPV for iERM during February 2016-June 2019 and had preoperative and postoperative visual field measurements using standard automated perimetry (Humphrey visual field analyser 30-2 program) were retrospectively analysed. Eyes with diseases other than iERM, including moderate-to-severe cataract or preoperative VFDs were excluded.

Results: VFD, defined by the Anderson and Patella's criteria, was found in 73.3% of the eyes 1 month after PPV. After age adjustment, internal limiting membrane (ILM) peeling was identified as a risk factor for postoperative VFD (p=0.035; 95% CI 1.173 to 92.8). Postoperative VFD was frequently observed nasally (86.4%, p=0.002), and on optical coherence tomography measurements, ganglion cell layer (GCL) thinning was found temporal to the fovea (p=0.008). Thinning of the superior and inferior retinal nerve fibre layers and of the GCL temporal to the fovea were significant in eyes after ILM peeling (all p<0.05).

Conclusion: ILM peeling may cause inner retinal degeneration and lead to the development of VFDs after PPV, which should be further examined.
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http://dx.doi.org/10.1136/bjophthalmol-2020-317478DOI Listing
October 2020

Hyperreflective Material in Optical Coherence Tomography Images of Eyes with Myopic Choroidal Neovascularization May Affect the Visual Outcome.

J Clin Med 2020 Jul 27;9(8). Epub 2020 Jul 27.

Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

The visual outcome of myopic choroidal neovascularization (CNV) after anti-vascular endothelial growth factor (anti-VEGF) therapy varies among individuals. We retrospectively analyzed the data of 24 eyes (24 patients) with treatment-naïve myopic CNV who underwent anti-VEGF monotherapy following a pro-re-nata regimen at the Division of Medical Retina Clinic, Department of Ophthalmology, Keio University Hospital between May 2014 and December 2017. The mean age was 70.6 ± 2.1 years, and 16 (66.7%) patients were female. Overall, the mean best-corrected visual acuity (BCVA) improved ( = 0.034), and the mean height of the hyperreflective material (HRM), involving the CNV lesion recorded by optical coherence tomography, decreased ( < 0.01) 12 months after the initial treatment. Fifteen eyes (62.5%) achieved a BCVA of better than 0.10 in LogMAR at 12 months; they had a better BCVA ( = 0.015) and lower HRM intensity ( = 0.033) at baseline than the others. Remarkably, the BCVA improved ( < 0.05) and the HRM height ( < 0.01) decreased only in eyes with a final BCVA better than 0.10 as early as 1 month after the initial treatment, which was still present at 12 months. The HRM height and intensity, not only the BCVA, would be valuable in evaluating the prognosis of myopic CNV after anti-VEGF therapy, although further study is required.
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http://dx.doi.org/10.3390/jcm9082394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7466026PMC
July 2020

Ocular and Systemic Effects of Antioxidative Supplement Use in Young and Healthy Adults: Real-World Cross-Sectional Data.

Antioxidants (Basel) 2020 Jun 3;9(6). Epub 2020 Jun 3.

Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

Randomized controlled studies have shown that antioxidative supplements are effective in suppressing the progression of age-related macular degeneration and visual display terminal syndrome. However, effects of their general use in the real-world and by young and healthy individuals have not been well documented. We analyzed 27 participants who were under 35 years of age and had no diagnosed diseases. Mean functional visual acuity (FVA) score and visual maintenance ratio, which represent quick recognition of a target, both measured using FVA system, were better (both < 0.01) in subjects who had had regular antioxidative supplement intake for more than 2 months (11 participants) compared with those who had not. Systemic data, i.e., total cholesterol, hemoglobin A1c (HbA1c), and high-sensitivity C-reactive protein (hs-CRP) levels, which correspond to chronic low-grade inflammation, were lower (all < 0.05) in the former. Overall, hs-CRP levels had a correlation with total cholesterol ( < 0.05) and a trend of correlation with HbA1c ( = 0.054) levels. Thus, current real-world data showed that young, healthy participants who had a regular intake of antioxidative supplements had better visual acuity and systemic levels of metabolic and low-grade inflammation markers. This study will help promote future research into the effects of general antioxidative supplement use.
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http://dx.doi.org/10.3390/antiox9060487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7346213PMC
June 2020

The Area and Number of Intraretinal Cystoid Spaces Predict the Visual Outcome after Ranibizumab Monotherapy in Diabetic Macular Edema.

J Clin Med 2020 May 8;9(5). Epub 2020 May 8.

Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

Visual outcomes in diabetic macular edema (DME) after anti-vascular endothelial growth factor therapy vary across individuals. We retrospectively reviewed the clinical records for 46 treatment-naive eyes of 46 patients with DME who underwent intravitreal ranibizumab (IVR) monotherapy with a pro re nata regimen for 12 months. Overall, mean best-corrected visual acuity (BCVA) improved. Multivariate analyses adjusted for age and baseline BCVA showed that the area ratio, compared with the retinal area, and the number of intraretinal cystoid spaces evaluated on OCT (optical coherence tomography) images at baseline positively correlated with LogMAR BCVA and the extents of ellipsoid zone and external limiting membrane disruption at 12 months, and negatively correlated with central retinal thickness at the time of edema resolution. Therefore, a high area ratio and large number of intraretinal cystoid spaces resulted in a disorganized outer retinal structure at 12 months, a thin and atrophic retina after edema resolution, and a worse visual outcome. The area ratio and number of intraretinal cystoid spaces on initial OCT images were predictors of the visual outcome after IVR therapy in DME irrespective of baseline age and BCVA. The factors were related to retinal neurodegenerative changes in DME and could help in obtaining proper informed consent before treatment.
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http://dx.doi.org/10.3390/jcm9051391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290842PMC
May 2020

Macular Pigment Optical Density and Photoreceptor Outer Segment Length as Predisease Biomarkers for Age-Related Macular Degeneration.

J Clin Med 2020 May 5;9(5). Epub 2020 May 5.

Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

To explore predisease biomarkers, which may help screen for the risk of age-related macular degeneration (AMD) at very early stages, macular pigment optical density (MPOD) and photoreceptor outer segment (PROS) length were analyzed. Thirty late AMD fellow eyes, which are at high risk and represent the predisease condition of AMD, were evaluated and compared with 30 age-matched control eyes without retinal diseases; there was no early AMD involvement in the AMD fellow eyes. MPOD was measured using MPS2 (M.E. Technica Co. Ltd., Tokyo, Japan), and PROS length was measured based on optical coherence tomography images. MPOD levels and PROS length in the AMD fellow eyes were significantly lower and shorter, respectively, than in control eyes. MPOD and PROS length were positively correlated in control eyes (R = 0.386; = 0.035) but not in AMD fellow eyes. Twenty (67%) AMD fellow eyes met the criteria of MPOD < 0.65 and/or PROS length < 35 μm, while only five (17%) control eyes did. After adjusting for age and sex, AMD fellow eyes more frequently satisfied the definition ( < 0.001; 95% confidence interval, 3.50-60.4; odds ratio, 14.6). The combination of MPOD and PROS length may be a useful biomarker for screening predisease AMD patients, although further studies are required in this regard.
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http://dx.doi.org/10.3390/jcm9051347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290696PMC
May 2020

Correlation between Macular Pigment Optical Density and Neural Thickness and Volume of the Retina.

Nutrients 2020 Mar 25;12(4). Epub 2020 Mar 25.

Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

Macular pigment (MP), which is composed of lutein/zeaxanthin/mezo-zeaxanthin, is concentrated in the central part of the retina, the macula. It protects the macula by absorbing short-wavelength light and suppressing oxidative stress. To evaluate whether MP levels are related to retinal neural protection and resulting health, we analyzed the association between the MP optical density (MPOD), and the macular thickness and volumes. Forty-three eyes of 43 healthy adult volunteers (21 men and 22 women; age: 22-48 (average 31.4 ± 1.1) years) were analyzed. Highly myopic eyes (<-6 diopters) were excluded. MPOD was measured using MPS2®, and the neural retinal thickness and volume were measured using optical coherence tomography. The mean MPOD was 0.589 ± 0.024, and it positively correlated with the central retinal thickness ( = 0.017, R = 0.360) and retinal volume of the fovea (1-mm diameter around the fovea; = 0.029, R = 0.332), parafovea (1-3-mm diameter; = 0.002, R = 0.458), and macula (6-mm diameter; = 0.003, R = 0.447). In the macular area (diameter: 6 mm), MPOD was correlated with the retinal neural volume of the ganglion cell layer ( = 0.037, R = 0.320), inner plexiform layer ( = 0.029, R = 0.333), and outer nuclear layer ( = 0.020, R = 0.353). Thus, MPOD may help in estimating neural health. Further studies should determine the impact of MP levels on neuroprotection.
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http://dx.doi.org/10.3390/nu12040888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7230595PMC
March 2020

Effect of axial length and age on the visual outcome of patients with idiopathic epiretinal membrane after pars plana vitrectomy.

Sci Rep 2019 12 13;9(1):19056. Epub 2019 Dec 13.

Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.

We evaluated predictive factors for visual outcomes in patients with idiopathic epiretinal membrane (iERM) after pars plana vitrectomy (PPV). Clinical records for 114 eyes (114 patients, mean age: 70.6 years) with iERM treated by PPV between March 2012 and March 2018 were retrospectively reviewed. Overall, the mean postoperative best-corrected visual acuity (BCVA) and central retinal thickness measured by optical coherence tomography improved as early as 1 month after surgery, and further improved until 3 months (P < 0.01). Multiple linear regression analyses adjusted for the preoperative BCVA showed that older age (B, 0.010; 95% confidence interval, 0.003 to 0.016; P = 0.003) and a shorter axial length (AL; B, -0.059; 95% confidence interval, -0.099 to -0.019; P = 0.005) predicted worse postoperative BCVA. The Mann-Whitney U test showed that the postoperative BCVA was worse in eyes with AL < 23.6 mm than in eyes with AL ≥ 23.6 mm (P = 0.037), and in patients aged ≥69 years than in patients aged <69 years (P = 0.024). The findings may help in evaluating surgical indications for each patient to obtain satisfactory outcomes, irrespective of the preoperative BCVA.
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http://dx.doi.org/10.1038/s41598-019-55544-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911110PMC
December 2019

Dynamic changes in neural retinal images during the development of a lamellar macular hole: A case report.

Medicine (Baltimore) 2019 Dec;98(49):e18297

Department of Ophthalmology, Keio University, School of Medicine, Shinjukuku, Tokyo, Japan.

Rationale: Lamellar macular hole (LMH) comprises a partial-thickness defect of the macula, the central part of the neural retina. Classification of LMHs into degenerative and tractional types has been proposed based on macular morphology observed on optical coherence tomography (OCT) imaging. Although LMHs are assumed to develop from aborted full-thickness macular holes, the clinical course of LMH development is not fully understood.

Patient Concerns: A 67-year-old man noticed slight changes in his central vision, and exhibited gradual change in macular morphology.

Diagnosis: The patient was first diagnosed with macular hole, which closed spontaneously. He then developed an LMH.

Interventions: The patient was placed under observation.

Outcomes: The patient first exhibited a macular hole, which resolved spontaneously. However, the tractional force at the surface of the macula became more severe, thereby causing retinoschisis-like appearance, which is the characteristic finding of a tractional LMH. The same eye then developed a lamellar hole-associated epiretinal proliferation (LHEP)-like structure, which is often observed in a degenerative LMH; importantly, the retinoschisis-like appearance persisted. Finally, the macula formed a typical degenerative LMH, characterized by intraretinal cavitation with persistent LHEP and disappearance of the retinoschisis-like structure.

Lessons: The present report shows dynamic changes of neural retinal morphology during development of an LMH: a tractional LMH developed initially; it then transformed into a degenerative LMH, which comprises a neurodegenerative disease. The findings in this report may help to understand the pathogenesis of LMHs and to elucidate the neurodegenerative disease process in various fields.
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http://dx.doi.org/10.1097/MD.0000000000018297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919539PMC
December 2019

Spatial-sweep steady-state pattern electroretinography can detect subtle differences in visual function among healthy adults.

Sci Rep 2019 12 2;9(1):18119. Epub 2019 Dec 2.

Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.

We aimed to establish a highly sensitive method for measuring visual function using spatial-sweep steady-state pattern electroretinography (swpPERG). Overall, 35 eyes of 35 healthy adults (18 men; mean age, 32.3 years) were examined using swpPERG, and the data were recorded using spatial-patterned and contrast-reversed stimuli of size 1 (thickest) to 6. Data were converted into frequency-domain using a Fourier transform and expressed by signal-to-noise ratio (SNR). The number of participants who showed SNR ≥ 1 was significantly lesser at stimulus sizes 5 and 6 compared with those at greater stimulus sizes. Among the data with SNR ≥ 1, SNRs were negatively correlated with age at stimulus size 5 (r = -0.500, P = 0.029), and positively correlated with macular volume evaluated by optical coherence tomography (OCT) within a 6-mm circle diameter from the fovea of the retinal nerve fibre layer at size 4 (r = 0.409, P = 0.025) and of the ganglion cell layer at size 5 (r = 0.567, P = 0.011). We found that SNRs of swpPERG, recorded using the EvokeDx system, were correlated with age and macular morphology in participants without diagnosed eye diseases. The system detected subtle differences in retinal function, which may help in early disease diagnosis and visual evaluation in neuroprotective interventions in the future.
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http://dx.doi.org/10.1038/s41598-019-54606-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889279PMC
December 2019

Predicting recurrences of macular edema due to branch retinal vein occlusion during anti-vascular endothelial growth factor therapy.

Graefes Arch Clin Exp Ophthalmol 2020 Jan 15;258(1):49-56. Epub 2019 Nov 15.

Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.

Purpose: To determine the predictive factors for recurrent macular edema due to branch retinal vein occlusion (BRVO) during intravitreal ranibizumab (IVR) monotherapy.

Methods: Clinical records were retrospectively reviewed for 65 patients (mean age 66.5 years, 65 eyes) who were diagnosed with macular edema due to BRVO and treated with IVR monotherapy for 12 months at the Medical Retina Division, Department of Ophthalmology, Keio University Hospital between October 2013 and August 2017. Best-corrected visual acuity (BCVA), fundus findings, and sectional optical coherence tomography (OCT) images were analyzed.

Results: Overall BCVA and central retinal thickness (CRT) improved (all p < 0.01). BCVA at 12 months was significantly worse in patients with recurrent macular edema (40 eyes [61.5%]) (p < 0.01) than in those without, while CRT decreased and was comparable in both groups at 12 months. Logistic regression analyses showed association of recurrence with disorganization of the retinal inner layer (DRIL) temporal to the fovea at baseline (odds ratio = 7.74; 95% confidence interval 1.62-37.08, p = 0.01), after adjusting for age, gender, and initial CRT.

Conclusion: Recurrent macular edema due to BRVO affects visual outcome and is associated with initial DRIL temporal to the fovea, evaluated using OCT sectional images before treatments. DRIL may facilitate determination of follow-up schedules in clinical practice.
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http://dx.doi.org/10.1007/s00417-019-04495-9DOI Listing
January 2020

Acid-Tolerant Reversibly Switchable Green Fluorescent Protein for Super-resolution Imaging under Acidic Conditions.

Cell Chem Biol 2019 10 15;26(10):1469-1479.e6. Epub 2019 Aug 15.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan; The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047, Japan. Electronic address:

Reversibly switchable fluorescent proteins (RSFPs) are crucial tags for super-resolution observation of protein localization and dynamics inside living cells. However, due to the high fluorescence pK (∼5-6) of most RSFPs, their usage in acidic conditions (pH 4.5-6.0) has been limited. Here, we investigated a new photochromic mechanism in Gamillus, a recently developed green fluorescent protein with acid tolerance. Gamillus exhibits negative switching with especially high contrast in acidic conditions, and its off switching is caused by trans-to-cis isomerization of the chromophore hydroxyphenyl ring that accompanies protonation. Through a combination of rational design and saturation mutagenesis, we developed two variants with enhanced switching contrasts and off-switching speeds, designated rsGamillus-S and rsGamillus-F, respectively. The fluorescence intensity, off-switching speed, and switching contrast of the rsGamillus variants are only slightly affected by changes in pH between 4.5 and 7.5. Exploiting these properties, we succeeded in high-contrast super-resolution imaging of cellular architectures in acidic conditions.
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http://dx.doi.org/10.1016/j.chembiol.2019.07.012DOI Listing
October 2019

Dynamic changes in choroidal conditions during anti-vascular endothelial growth factor therapy in polypoidal choroidal vasculopathy.

Sci Rep 2019 08 6;9(1):11389. Epub 2019 Aug 6.

Laboratory of Retinal Cell Biology, Keio University, School of Medicine, Tokyo, Japan.

We defined the relationships between initial choroidal conditions and their dynamics and exudative changes during anti-vascular endothelial growth factor (anti-VEGF) therapy in polypoidal choroidal vasculopathy (PCV). One hundred treatment-naïve eyes of 100 patients with PCV treated for 24 months at Keio University Hospital with intravitreal ranibizumab or aflibercept monotherapy (three injections and PRN thereafter) were retrospectively analyzed. Wet macula risk after three induction injections, which affected visual prognosis, was predicted by initial pachyvessels in the choroid (foveal greatest vertical choroidal vessel diameter [CVD] ≥180 μm) and pachychoroid (central choroidal thickness [CCT] ≥220 μm) recorded by optical coherence tomography. The risk for recurrent exudative change was greater in the pachyvessel groups irrespective of presence or absence of pachychoroid. Mean CVD and CCT decreased with anti-VEGF therapy when achieving a dry macula, suggesting that exudative changes are regulated by VEGF. Mean CVD and CCT at remission were greater in patients with initial pachyvessels and pachychoroid than in those without; the basal levels of CVD and CCT most likely represent VEGF-unrelated conditions. CVD increase preceded CCT increase and recurrent exudative changes, suggesting that the VEGF-related CVD increase may regulate CCT and exudative change; and that CVD may be a biomarker of exudative change.
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http://dx.doi.org/10.1038/s41598-019-47738-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684594PMC
August 2019

QD laser eyewear as a visual field aid in a visual field defect model.

Sci Rep 2019 01 30;9(1):1010. Epub 2019 Jan 30.

Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.

Visual field defects interfere with free actions and influence the quality of life of patients with retinitis pigmentosa; the prevalence of this disease is increasing in aging societies. Patients with progressive disease may require visual aids; however, no such devices are currently available. We utilized a retinal projection eyewear system, QD laser eyewear, which includes a projector inside the spectacle frame, to draw the image taken by a connected portable camera with a wide field lens. The images are projected onto the retina using a Maxwellian view optical system, which is not influenced by refractive error or the amount of incident light. Goldmann perimetry and figure recognition tests with the QD laser eyewear showed increased visual field areas and angles, and shortened the time for recognition of the number of figures in a sheet, in a limited visual field model that we developed by using a pin-hole system to simulate the tunnel vision of retinitis pigmentosa in 19 healthy adults. The device supported the quality of vision. Additionally, the visual field defect model used in healthy adults was useful for validating the device in the development stage of the study, to clarify both advantages and future goals for improving the device.
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http://dx.doi.org/10.1038/s41598-018-37744-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6353865PMC
January 2019

Retinal inflammation diagnosed as an idiopathic macular hole with multiple recurrences and spontaneous closures: A case report.

Medicine (Baltimore) 2019 Jan;98(4):e14230

Department of Ophthalmology, Keio University, School of Medicine, 35 Shinanomachi, Shinjukuku.

Rationale: An idiopathic macular hole that causes substantial reduction in central visual acuity is believed to involve no obvious underlying diseases; thus, it is suspected to form due to the presence of idiopathic tractional forces at the vitreoretinal interface. Importantly, it is effectively treated with pars plana vitrectomy (PPV), which removes the mechanical forces. However, while it is exceedingly rare, a macular hole can develop in eyes after PPV; fresh or postoperative macular holes can close spontaneously without surgical removal of traction. Thus, another mechanism might be involved, although it remains obscure.

Patients Concerns: A 67-year-old woman experienced 4 episodes of distorted and/or blurred vision.

Diagnosis: She was diagnosed with recurrent macular hole formation.

Interventions: For each episode, she either underwent surgery or was placed under observation.

Outcomes: The macular hole was twice closed with PPV and twice without. The 2nd PPV procedure, which was performed at the time of 2nd recurrence, confirmed the absence of the epiretinal membrane and internal limiting membrane that cause tractional forces at the vitreoretinal interface in the macular area. At the time of the 3rd recurrence, fluorescein angiographies (FAs) revealed the presence of mild and diffuse inflammation throughout the peripheral retina, although there were no other findings indicative of ocular inflammation during the general eye examination conducted for every episode of macular hole formation. After the initiation of topical steroid treatment, inflammation (as recorded on FA) was reduced, and the macular hole subsequently closed. Development and resolution of perifoveal cystoid change and retinal protrusion were observed in every episode in optical coherence tomography (OCT) images. A bridging element in an OCT image was observed during the 4th closure of the macular hole.

Lessons: Dynamic changes in FA and OCT images unraveled the pathogenesis of a macular hole that was originally diagnosed as idiopathic; mild inflammation was involved. The FA is typically not used for the diagnosis and management of macular hole formation; however, its use in this case helped determine a new mechanism in an otherwise idiopathic disease.
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http://dx.doi.org/10.1097/MD.0000000000014230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358394PMC
January 2019

Fluorescent Proteins for Investigating Biological Events in Acidic Environments.

Int J Mol Sci 2018 May 23;19(6). Epub 2018 May 23.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan.

The interior lumen of acidic organelles (e.g., endosomes, secretory granules, lysosomes and plant vacuoles) is an important platform for modification, transport and degradation of biomolecules as well as signal transduction, which remains challenging to investigate using conventional fluorescent proteins (FPs). Due to the highly acidic luminal environment (pH ~ 4.5⁻6.0), most FPs and related sensors are apt to lose their fluorescence. To address the need to image in acidic environments, several research groups have developed acid-tolerant FPs in a wide color range. Furthermore, the engineering of pH insensitive sensors, and their concomitant use with pH sensitive sensors for the purpose of pH-calibration has enabled characterization of the role of luminal ions. In this short review, we summarize the recent development of acid-tolerant FPs and related functional sensors and discuss the future prospects for this field.
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http://dx.doi.org/10.3390/ijms19061548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032295PMC
May 2018

Optic neuropathy causing vertical unilateral hemianopsia after pars plana vitrectomy for a macular hole: A case report.

Medicine (Baltimore) 2018 Apr;97(17):e0321

Laboratory of Retinal Cell Biology.

Introduction: Recent progress in medical technology has resulted in improved surgical outcomes of pars plana vitrectomy (PPV); with microincision systems, the incidence of procedure-related complications during surgery has been reduced. However, unpredictable visual field defects after PPV remain an unresolved issue. A few reports have shown that damage to the retinal neurofibers owing to dry-up during air/fluid exchange or retinal neurotoxicity of the dye used to visualize the internal limiting membrane (ILM), as well as unintentional removal of retinal neurofibers during ILM peeling, are responsible for such visual field disorders. In this report, we present a case of extensive visual field defect due to optic neuropathy exhibiting vertical hemianopsia after PPV.

Case Summary: A 50-year-old woman underwent PPV and cataract surgery for a macular hole and mild cataract under retrobulbar anesthesia with 3.5 mL of xylocaine. At the time of opening an infusion cannula for PPV, the intraocular lens was herniating, with an acute increase in pressure from the posterior eyeball; thus, intraocular pressure configuration level had to be decreased from the default level, whereas the other procedures including 20% SF6 injection were performed without any modification. The macular hole was closed postoperatively. However, the patient experienced nasal hemianopsia, which turned out to be optic neuropathy, as assessed via electric physiological examinations. The pattern of the visual field defect was not typical for glaucoma or anterior ischemic optic neuropathy. Her optic nerve head was pale at the temporal side soon after the surgery, and her blood pressure was low, suggesting that there may have been a congestion of the optic nerve feeder vessels because of the relatively high pressure in the orbit. The space occupancy with xylocaine and extensively stretched and plumped out eye ball with infusion during PPV may have pressed the surrounding tissue of the optic nerve and the feeder vessels.

Conclusion: PPV is safe for most patients; however, individual variations in local and/or systemic conditions may cause complications. Future studies to optimize the surgical condition for each individual patient may be warranted.
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http://dx.doi.org/10.1097/MD.0000000000010321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5944518PMC
April 2018

Benefits of aflibercept treatment for age-related macular degeneration patients with good best-corrected visual acuity at baseline.

Sci Rep 2018 01 8;8(1):58. Epub 2018 Jan 8.

Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.

Currently, age-related macular degeneration (AMD) is treated while patients exhibit good best-corrected visual acuity (BCVA). However, previous clinical trials only include patients with poor BCVA. We prospectively analyzed the benefits of intravitreal aflibercept (IVA) treatment for AMD patients exhibiting good BCVA at baseline. Twenty-nine treatment-naive AMD patients (29 eyes) with BCVA better than 0.6 (74 letters in ETDRS chart) were treated with IVA once a month for 3 months and every 2 months thereafter with no additional treatments. Improvement in mean BCVA, measured using the conventional Landolt C chart, contrast VA chart, and functional VA (FVA) system, and reductions in mean central retinal thickness (CRT), central choroidal thickness, macular volume (MV), and choroidal area on optical coherence tomography images were observed at 6 and 12 months. Improvements in contrast VA and FVA scores, in contrast to conventional BCVA, correlated with MV reduction; no VA scores correlated with a reduced CRT. The MV correlated with choroidal area after IVA. No severe adverse events occurred. IVA improved visual function, retinal condition, and quality of life evaluated by Visual Function Questionnaire, and was beneficial in these patients. The contrast VA and FVA scores and MVs, which detect subtle changes, helped demonstrate the benefits.
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http://dx.doi.org/10.1038/s41598-017-18255-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758719PMC
January 2018

Acid-Tolerant Monomeric GFP from Olindias formosa.

Cell Chem Biol 2018 03 28;25(3):330-338.e7. Epub 2017 Dec 28.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan; The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki 567-0047, Japan. Electronic address:

The fluorescent protein (FP) color palette has greatly contributed to the visualization of molecular and cellular processes. However, most FPs lose fluorescence at a pH lower than their neutral pK (∼6), and this has hampered their application in acidic organelles (pH ∼4.5-6.0). Currently, several cyan- and red-colored acid-tolerant FPs are available; however, there are few reports of acid-tolerant green FPs (GFPs) that are practically applicable to bioimaging. Here, we developed the acid-tolerant monomeric GFP "Gamillus" from the jellyfish Olindias formosa, with excellent brightness, maturation speed, and photostability. Results from X-ray crystallography and point mutagenesis suggest that across a broad pH range the acid tolerance is attributed to stabilization of deprotonation in the chromophore phenyl ring by forming a unique trans configuration. We demonstrate that Gamillus can serve as a molecular tag suitable for imaging in acidic organelles through autophagy-mediated molecular tracking to lysosomes.
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http://dx.doi.org/10.1016/j.chembiol.2017.12.005DOI Listing
March 2018

Absolute and estimated values of macular pigment optical density in young and aged Asian participants with or without age-related macular degeneration.

BMC Ophthalmol 2017 Aug 29;17(1):161. Epub 2017 Aug 29.

Department of Ophthalmology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.

Background: Lutein and zeaxanthin are suggested micronutrient supplements to prevent the progression of age-related macular degeneration (AMD), a leading cause of blindness worldwide. To monitor the levels of lutein/zeaxanthin in the macula, macular pigment optical density (MPOD) is measured. A commercially available device (MPSII®, Elektron Technology, Switzerland), using technology based on heterochromatic flicker photometry, can measure both absolute and estimated values of MPOD. However, whether the estimated value is applicable to Asian individuals and/or AMD patients remains to be determined.

Methods: The absolute and estimated values of MPOD were measured using the MPSII® device in 77 participants with a best-corrected visual acuity (BCVA) > 0.099 (logMAR score).

Results: The studied eyes included 17 young (20-29 years) healthy, 26 aged (>50 years) healthy, 18 aged and AMD-fellow, and 16 aged AMD eyes. The mean BCVA among the groups were not significantly different. Both absolute and estimated values were measurable in all eyes of young healthy group. However, absolute values were measurable in only 57.7%, 66.7%, and 43.8%, of the aged healthy, AMD-fellow, and AMD groups, respectively, and 56.7% of the eyes included in the 3 aged groups. In contrast, the estimated value was measurable in 84.6%, 88.9% and 93.8% of the groups, respectively, and 88.3% of eyes in the pooled aged group. The estimated value was correlated with absolute value in individuals from all groups by Spearman's correlation coefficient analyses (young healthy: R = 0.885, P = 0.0001; aged healthy: R = 0.765, P = 0.001; AMD-fellow: R = 0.851, P = 0.0001; and AMD: R = 0.860, P = 0.013). Using the estimated value, significantly lower MPOD values were found in aged AMD-related eyes, which included both AMD-fellow and AMD eyes, compared with aged healthy eyes by Student's t-test (P = 0.02).

Conclusions: Absolute, in contrast to estimated, value was measurable in a limited number of aged participants; however, it was correlated with estimated value both in young and aged Asian populations with or without AMD. These results may inform future clinical studies investigating the measurement of MPOD in understanding the role of macular pigments in the pathogenesis of AMD.
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http://dx.doi.org/10.1186/s12886-017-0557-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576241PMC
August 2017

Maculopapular rash after intravitreal injection of an antivascular endothelial growth factor, aflibercept, for treating age-related macular degeneration: A case report.

Medicine (Baltimore) 2017 May;96(21):e6965

Laboratory of Retinal Cell Biology Department of Ophthalmology Department of Pathology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.

Rationale: Aflibercept, an anti-vascular endothelial growth factor (VEGF) drug, is used for treatment of colon cancer as well as retinal diseases, including wet age-related macular degeneration (AMD). It is injected into the vitreous cavity of eyes for treatment of AMD. Although vascular suppression-including cardiovascular events-and local infection related to the injection procedure are well-known potential adverse events, pathological immune responses after intravitreal aflibercept (IVA) injection have not been described.

Patient Concerns: A 60-year-old Japanese man diagnosed with polypoidal choroidal vasculopathy (PCV), a subtype of wet AMD, was treated by anti-VEGF injection. Ten hours after the last IVA injection, he presented with systemic erythema with itching.

Diagnoses: On the basis of the palpable erythema and papules observed on the trunk and extremities, along with redness of the pharynx, the patient was diagnosed with maculopapular-type drug eruption. The findings of biopsy of erythematous skin on the back revealed lymphocyte infiltration and telangiectasia in the upper dermis, thus confirming the diagnosis.

Interventions: The patient was administered 30 mg prednisolone to resolve the immunoreaction.

Outcomes: With this treatment, the eruption turned brown, and the pharyngeal lesion and itching were resolved, and the maculopapular rash after intravitreal IVA was resolved.

Lessons: This case illustrates the importance of medical staff being aware of aflibercept-a widely used anti-VEGF drug in various fields, including retinal diseases-as a potential cause of drug allergy.
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http://dx.doi.org/10.1097/MD.0000000000006965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457872PMC
May 2017

The 5-HT Receptor Antagonist Ondansetron Attenuates Pancreatic Injury in Cerulein-Induced Acute Pancreatitis Model.

Inflammation 2017 Aug;40(4):1409-1415

Laboratory of Laboratory Animal Science, Azabu University, School of Veterinary Medicine, 1-17-71 Fuchinobe, Chuou-ku, Sagamihara, Kanagawa, 252-5201, Japan.

The 5-hydroxytryptamine-3 receptor (5-HTR) antagonist ondansetron has been clinically approved as an anti-emetic agent. Recent findings indicate that ondansetron has anti-inflammatory properties. The aim of the present study was to assess the therapeutic action of ondansetron in cerulein-induced acute pancreatitis model. Male-BALB/c mice were used in the present study. Acute pancreatitis was induced by an hourly injection of cerulein. Ondansetron was administered subcutaneously at a dose of 3 mg/kg. The messenger RNA (mRNA) expression of 5-HT R in pancreatic tissue was assessed with RT-PCR. Plasma amylase, lipase, and interleukin (IL)-6 levels were evaluated. Pancreatic injury was histopathologically graded, and myeloperoxidase (MPO)-positive cells were counted. 5-HTR mRNA was expressed in the pancreas. In acute pancreatitis model mice, amylase, lipase, and IL-6 levels were significantly increased in the blood. With ondansetron treatment, these levels were significantly decreased. Histopathological evaluation revealed that ondansetron attenuated the inflammatory damage in acute pancreatitis. The number of infiltrated neutrophils stained by MPO was decreased by ondansetron treatment. In summary, the 5-HTR antagonist ondansetron attenuated pancreatic injury through its anti-inflammatory action. These findings suggest that ondansetron may potentially be of use for therapy of acute pancreatitis.
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http://dx.doi.org/10.1007/s10753-017-0584-7DOI Listing
August 2017

Predictive factors of better outcomes by monotherapy of an antivascular endothelial growth factor drug, ranibizumab, for diabetic macular edema in clinical practice.

Medicine (Baltimore) 2017 Apr;96(16):e6459

aDepartment of Ophthalmology bLaboratory of Retinal Cell Biology, Keio University, School of Medicine, Tokyo, Japan.

Intravitreal ranibizumab (IVR) has been approved for treating diabetic macular edema (DME), and is used in daily clinical practice. However, the treatment efficacies of IVR monotherapy in real-world clinical settings are not well known.The medical records of 56 eyes from 38 patients who received their first IVR for DME between April 2014 and March 2015, and were retreated with IVR monotherapy as needed with no rescue treatment, such as laser photocoagulation, were retrospectively reviewed. The clinical course, best-corrected visual acuity (BCVA), and fundus findings at baseline, before the initial IVR injection, and at 12 months, were evaluated.Twenty-five eyes from 25 patients (16 men; mean age 68.7 ± 9.8 years) who received IVR in the first eye, or unilaterally, without any other treatments during follow-up were included. After 12 months, mean central retinal thickness (CRT), which includes edema, was reduced (P = .003), although mean BCVA remained unchanged. There was a negative correlation between individual changes in BCVA (r = -0.57; P = .003) and CRT (r = -0.60; P = .002) at 12 months compared with baseline values. BCVA changes were greater in individuals with a history of pan-retinal photocoagulation at baseline (P = .026). After adjusting for age and sex, CRT improvement >100 μm at 12 months was associated with a greater CRT at baseline (OR 0.87 per 10 μm [95% CI 0.72-0.97]; P = .018) according to logistic regression analyses; however, better BCVA and CRT at 12 months were associated with a better BCVA (r = 0.77; P < .001) and lower CRT (r = 0.41; P = .039) at baseline, respectively, according to linear regression analyses.IVR monotherapy suppressed DME, and the effects varied according to baseline conditions. Eyes that had poorer BCVA or greater CRT, or a history of pan-retinal photocoagulation at baseline, demonstrated greater improvement with IVR monotherapy. In contrast, to achieve better outcome values, DME eyes should be treated before the BCVA and CRT deteriorate. These findings advance our understanding of the optimal use of IVR for DME in daily clinical practice, although further study is warranted.
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http://dx.doi.org/10.1097/MD.0000000000006459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406051PMC
April 2017

Five colour variants of bright luminescent protein for real-time multicolour bioimaging.

Nat Commun 2016 12 14;7:13718. Epub 2016 Dec 14.

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan.

Luminescence imaging has gained attention as a promising bio-imaging modality in situations where fluorescence imaging cannot be applied. However, wider application to multicolour and dynamic imaging is limited by the lack of bright luminescent proteins with emissions across the visible spectrum. Here we report five new spectral variants of the bright luminescent protein, enhanced Nano-lantern (eNL), made by concatenation of the brightest luciferase, NanoLuc, with various colour hues of fluorescent proteins. eNLs allow five-colour live-cell imaging, as well as detection of single protein complexes and even single molecules. We also develop an eNL-based Ca indicator with a 500% signal change, which can image spontaneous Ca dynamics in cardiomyocyte and neural cell models. These eNL probes facilitate not only multicolour imaging in living cells but also sensitive imaging of a wide repertoire of proteins, even at very low expression levels.
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http://dx.doi.org/10.1038/ncomms13718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5171807PMC
December 2016

Dysregulation of a potassium channel, THIK-1, targeted by caspase-8 accelerates cell shrinkage.

Biochim Biophys Acta 2016 11 23;1863(11):2766-2783. Epub 2016 Aug 23.

Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.

Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the physiological role of CASP8 is not fully understood. Here, we identified a two-pore domain potassium channel, tandem-pore domain halothane-inhibited K channel 1 (THIK-1), as a novel CASP8 substrate. The intracellular region of THIK-1 was cleaved by CASP8 in apoptotic cells. Overexpression of THIK-1, but not its mutant lacking the CASP8-target sequence in the intracellular portion, accelerated cell shrinkage in response to apoptotic stimuli. In contrast, knockdown of endogenous THIK-1 by RNA interference resulted in delayed shrinkage and potassium efflux. Furthermore, a truncated THIK-1 mutant lacking the intracellular region, which mimics the form cleaved by CASP8, led to a decrease of cell volume of cultured cells without apoptotic stimulation and excessively promoted irregular development of Xenopus embryos. Taken together, these results indicate that THIK-1 is involved in the acceleration of cell shrinkage. Thus, we have demonstrated a novel physiological role of CASP8: creating a cascade that advances the cell to the next stage in the apoptotic process.
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http://dx.doi.org/10.1016/j.bbamcr.2016.08.010DOI Listing
November 2016

Reply.

Am J Ophthalmol 2016 09 15;169:295-296. Epub 2016 Jul 15.

Tokyo, Japan.

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http://dx.doi.org/10.1016/j.ajo.2016.06.018DOI Listing
September 2016

Non-responsiveness to intravitreal aflibercept treatment in neovascular age-related macular degeneration: implications of serous pigment epithelial detachment.

Sci Rep 2016 07 11;6:29619. Epub 2016 Jul 11.

Laboratory of Retinal Cell Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

The prognosis of neovascular age-related macular degeneration (AMD) has been improved by anti-vascular endothelial growth factor treatments, including intravitreal aflibercept (IVA) treatment. However, many patients remain incurable. In this study, we retrospectively evaluated non-responsiveness to IVA monotherapy at 12 months in 133 eyes of 133 AMD patients. Sixty-two patients were initially treatment-naive, and 71 had received other treatments before IVA (the treatment-switched group). Mean best-corrected visual acuity (BCVA) was improved in the treatment-naive group but not in the treatment-switched group, although mean central retinal thickness (CRT) decreased in both groups. The respective percentages of non-responders as determined by worsened BCVA in the treatment-naive and treatment-switched groups were 8.1% and 15.5%, and via fundus findings, they were 12.9% and 8.5%. Multivariate analyses adjusted for age, gender, CRT, and greatest linear dimension showed that serous pigment epithelial detachment (PED) at baseline was associated with non-responsiveness in both groups as determined by BCVA and by fundus findings, and fibrovascular PED measurements indicated no response as determined by fundus findings in the treatment-switched group. The results reported herein may assist the formulation of appropriate treatment protocols for AMD patients.
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http://dx.doi.org/10.1038/srep29619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939600PMC
July 2016

Distinct Responsiveness to Intravitreal Ranibizumab Therapy in Polypoidal Choroidal Vasculopathy With Single or Multiple Polyps.

Am J Ophthalmol 2016 06 24;166:52-59. Epub 2016 Mar 24.

Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan. Electronic address:

Purpose: To understand the prognosis of polypoidal choroidal vasculopathy (PCV) by evaluating the responsiveness to intravitreal ranibizumab (IVR) monotherapy according to the presence of a single or multiple polyps.

Design: Retrospective case series.

Methods: We included 48 treatment-naïve eyes of 48 patients who received IVR monotherapy at the Medical Retina Division Clinic, Keio University Hospital between March 2009 and January 2013 and attended the clinic for at least 12 months. All patients received 3 monthly IVR injections followed by pro re nata injections and were divided into single polyp and multiple polyps groups according to indocyanine green angiography and optical coherence tomography (OCT) findings. The outcome measures included changes in best-corrected visual acuity (BCVA) and OCT findings over 2 years after initial IVR.

Results: At baseline, the multiple polyps group exhibited a poorer BCVA, larger greatest linear dimension, and higher prevalence of fibrovascular pigment epithelial detachment compared with the single polyp group. Over 2 years, the multiple polyps group showed no improvement in BCVA, although the central retinal thickness (CRT) decreased in both groups. The multiple polyps group exhibited a significantly greater CRT at 1 year and required more injections in the first year compared with the single polyp group; furthermore, it included a higher number of nonresponders judged either by BCVA or fundus findings at 1 year and fundus findings at 2 years.

Conclusions: We propose that the stratification of PCV lesions according to the presence of single or multiple polyps may be valuable to understand the prognosis.
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http://dx.doi.org/10.1016/j.ajo.2016.03.024DOI Listing
June 2016