Details of a Researcher - Kurihara, Toshihide

Papers - Kurihara, Toshihide

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Dose-dependent myopia-suppressing effect of 4-phenlybutyric acid eye drops in a mouse myopia model under masked condition

Ikeda SI, Sone K, Takai Y, Miyano T, Negishi K, Tsubota K, Kurihara T†

BMC Ophthalmol 25 ( 1 ) 383 2025.07

Last author, Corresponding author, Accepted

Background: Myopia, characterized by excessive axial elongation of the eyeball, has become a significant global public health issue, particularly in Asia. Severe myopia increases the risk of ocular complications, including retinal degeneration and glaucoma. Recent studies have identified the role of scleral extracellular matrix remodeling and endoplasmic reticulum (ER) stress in myopia pathogenesis. Our previous study identified scleral ER stress as a critical factor in myopia development. This study aimed to investigate the therapeutic potential of 4-phenylbutyric acid (4-PBA), a chemical chaperone, in mitigating scleral ER stress and its effects on myopia progression in a mouse myopia model under masked condition. Methods: A lens-induced myopia (LIM) mouse model was utilized to evaluate the effects of 4-PBA administered as eye drops. Mice received varying concentrations of 4-PBA or phosphate-buffered saline (PBS) as a control. Changes in refractive error and axial length were measured to assess myopia progression. In addition, further experiments were also conducted under masked conditions, where the experimenter was unaware of the treatment was being applied, for the three conditions of 2%, 0.5% 4-PBA eye drops or vehicle treatment of LIM mice. Results: Administration of 4-PBA at concentrations of 0.5% and higher significantly suppressed the myopic shift in refraction and axial elongation associated with myopia compared to mice in the PBS control group. In a blinded, masked experiment, 2% and 0.5% 4-PBA eye drops also suppressed the progression of myopia in a dose-dependent manner. Conclusions: This study demonstrated that 4-PBA effectively mitigated myopia progression in a mouse model by targeting scleral ER stress. The dose-dependent suppression of myopic shifts and axial elongation under masked experimental condtion highlights the potential of 4-PBA as a therapeutic agent for managing myopia. These findings pave the way for further research and potential clinical applications in myopia treatment.

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Antibiotics-Induced Gut Microbiome Dysbiosis Affects Susceptibility to Minus Lens-Induced Myopia in Mice

Ikeda SI, Lee D, Chen J, Fukuda S, Negishi K, Tsubota K†, Kurihara T†

Invest Ophthal Vis Sci. (Association for Research in Vision and Ophthalmology (ARVO)) 66 ( 6 ) 76 2025.06

Last author, Corresponding author, Accepted, ISSN 1552-5783

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Evaluation of Anti-VEGFR2 Specific Photoimmunotherapy for Targeted Regression of Neovascularization in an AMD Model

Osada H, Nishimura T, Mitsunaga M, Saruta M, Tsubota K, Negishi K, Kurihara T, Ban N†

Invest Ophthalmol Vis Sci. (Association for Research in Vision and Ophthalmology (ARVO)) 66 ( 6 ) 70 2025.06

Accepted, ISSN 1552-5783

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Thicker peripapillary choroid may be associated with behavioral factors in Tokyo's children.

Hashimoto A*, Yotsukura E*, Ogawa M, Mori K, Hanyuda A, Tsubota K, Kurihara T, Negishi K†, Torii H†

Sci Rep. 15 ( 1 ) 17868 - 17868 2025.05

Accepted

The study goals were measurement of the choroidal thickness (CT) and investigation of related factors among children. Third-grade students at an elementary school in Tokyo participated in this study. A total of 119 schoolchildren underwent measurements of the axial length (AL), non-cycloplegic objective refraction (SE), and CT. The CT was divided into nine regions: the subfoveal area with a diameter of 1 mm (C0), a 5-mm-diameter circle-C0 (C1), and a 9-mm-diameter circle (C2); each was further divided into four quadrants. The SE, AL, and C0 CT were, respectively, - 1.57 ± 1.56 diopters, 23.39 ± 0.80 mm, and 297.73 ± 57.88 μm. Multiple regression analysis showed that the time spent using a computer was associated significantly with the CT in the C0 and the inferior C2 (C0: β = -0.728, P = 0.028; inferior C2: β = -0.527, P = 0.048), and the reading distance was associated significantly with the CT in the superior and nasal C2 (superior, β = 1.664, P = 0.007; nasal, β = 1.128, P = 0.015). In addition, time spent using smartphones, tablets, or handheld gaming devices was associated significantly with the CT in the superior and inferior C2 (superior C2: β = 0.259, P = 0.018; inferior C2: β = 0.262, P = 0.013). In conclusion, reading distance and time spent on digital devices may be associated with choroid thickness in children.

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Intensive outdoor activity for 1 week increases choroidal thickness in Japanese schoolchildren: a prospective observational study

Ogawa M, Torii H†, Yotsukura E, Mori K, Hanyuda A, Matsumura J, Fukuoka K, Negishi K, Kurihara T†, Tsubota K†

BMC Ophthalmol. 25 ( 1 ) 300 2025.05

Corresponding author, Accepted

Background: Outdoor activity is important to prevent myopia progression among schoolchildren, and maintaining the choroidal thickness is important to retard myopia progression. However, no research has reported the effects of short-term intensive outdoor activities on the choroidal thickness. This institution-based prospective observational study aims to assess the impact of outdoor activity for 1 week during a camp program to study ocular parameters including the choroidal thickness. Methods: The intensive outdoor activity program included an average of 6.15 ± 2.98 h of daily time spent outdoors during the camp on subsequent days for 1 week at a low-altitude mountain camp. Twenty-four children participated in this program. Results: The main outcome was the change in the choroidal thickness compared with baseline. The data were measured at the beginning and end of the program over the course of 1 week and the changes analyzed. The mean age of the participants (50% female) was 11.5 ± 0.5 (standard deviation) years, and the mean changes were as follows: the refractive error became more positive, the axial length decreased, and the choroidal thickness (µm) increased, respectively, by 0.21 ± 1.35 diopters (P = 0.742), -0.01 ± 0.02 mm (P = 0.241), and 30.7 ± 20.3 μm (P < 0.001). Conclusion: The results suggested that intensive outdoor activity for only 1 week increased the choroidal thickness in Japanese schoolchildren.

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Intraocular Vasculature Formation Precedes Extraocular Vasculature Penetration of Mouse Eyes

Imanishi S., Tomita Y., Negishi K., Tsubota K., Kurihara T†

SSRN 2025.05

Last author, Corresponding author

Assessing public awareness of myopia after the COVID-19 pandemic: an infodemiology study

Chen J, Lee D., Ikeda SI, Zhang Y., Negishi K., Tsubota K., Kurihara T†

BMJ Health Care Inform. 32 ( 1 ) e101156 2025.05

Last author, Corresponding author, Accepted

Objectives Since the COVID-19 pandemic started, an increasing number of individuals use online resources to obtain health-related information, including myopia management. We aimed to obtain the search interest of global public health on myopia and prevention/treatment methods before and after the outbreak of COVID-19. Methods Google Trends was used to assess the global search interest in myopia and related terms. Data spanning from 2019 to 2023 were collected and segmented into three periods: prepandemic (2019), pandemic year (2020) and postpandemic (2021-2023). Each average search volume index was used to examine public awareness with the Kruskal-Wallis test. Results Among commonly used search keywords, Myopia is preferred over more colloquial terms (Nearsighted and Shortsighted). During the pandemic, searches for Myopia remained stable, while postpandemic, interest in Myopia surged (31.54%, p<0.0001). Myopia prevention/treatment awareness from 2019 to 2023 indicated notable shifts. In 2020, searches for Contact lenses and Spectacles decreased (-14.09% and -21.97%, respectively, both p<0.0001). These trends persisted postpandemic, with declining searches for Orthokeratology (-41.38%, p<0.01). Public interest for Light therapy (43.00%, p<0.0001) and Atropine (27.42%, p<0.0001) increased. Discussion The research highlights significant increases in global online awareness of myopia during the COVID-19 pandemic. The extended period of remote work during the pandemic may alter lifestyle habits and affect public perception of treatment options. Those findings could offer valuable perspectives on global interest patterns related to myopia, which supports subsequent myopia studies and is further useful to develop relevant public health policies.

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Unilateral Common Carotid Artery Occlusion in Adult Mice with Streptozotocin Comorbidity Leads to Early Retinal Inflammation

Gettinger K*, Lee D*, Negishi K, Kurihara T†

Int J Mol Sci. 26 ( 9 ) 4385 2025.05

Last author, Corresponding author, Accepted, ISSN 16616596

Diabetic retinopathy (DR) is a leading cause of visual impairment. To better understand the pathology, clinically relevant experimental models are needed. Widely used DR models (especially streptozotocin (STZ)-induced) require extended timeframes to reach DR phenotype endpoints and lack ischemic phenotypes, which are in contrast to the human condition. Unilateral common carotid artery occlusion (UCCAO) could provide a retinal ischemic insult. We explored the pathologic synergistic effects of UCCAO in STZ mice. STZ (90 mg/kg) was injected intraperitoneally into adult C57BL/6 mice for three days. Four weeks later, right UCCAO was performed. One week after UCCAO, retinal samples were stained with isolectin B4 to analyze cellular and vascular changes. Retinal samples were obtained one day and one week after UCCAO and quantitative PCR (qPCR) were performed to observe inflammatory and ischemic responses. Only the STZ UCCAO group showed increased inflammatory cells. STZ UCCAO retina demonstrated a significant difference in capillary and large vessel size compared to other groups. At one day and one week, there was a change in mRNA expressions in inflammatory genes Ccl2, Ccl12, Bnip3, Pdk1, Hsp25, and Vegfa in the STZ UCCAO group compared to other groups. Our model can serve as an accelerated DR model for studying inflammatory vascular changes.

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Violet Light Is Abundant Outdoors but Deficient Indoors in Modern Lifestyle in Tokyo

Kondo S*, Jiang X*, Torii H, Mori K, Negishi K, Kurihara T†, Tsubota K†

Int J Enviro Res Public Health. (MDPI AG) 22 ( 3 ) 444 - 444 2025.03

Corresponding author, Accepted, ISSN 16617827

This study examines the role of violet light (VL) in preventing myopia progression, addressing a critical need in urban environments where VL exposure is limited. Recent research suggests that VL, within the 360–400 nm wavelength range, may reduce myopia risk. To investigate, we conducted spectroscopic measurements in various settings across Tokyo, quantifying VL irradiance in natural sunlight. The results showed high VL levels outdoors, averaging 583 μW/cm2 on sunny days and 271 μW/cm2 on cloudy days, leading to a weighted annual average of approximately 310 μW/cm2. In contrast, indoor environments lacked VL due to UV-blocking materials in windows, glasses, and lighting. This deficiency may contribute to the rising incidence of myopia, particularly in urban areas with reduced outdoor exposure. Our findings highlight the need for innovative solutions to mitigate VL deficiency indoors, such as optimizing architectural designs and artificial lighting to better incorporate VL. This study provides foundational insights for future interventions aimed at reducing myopia risk through improved indoor light environments.

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第128回日本眼科学会総会評議員会指名講演II 視覚とニューロサイエンス光生物学を基盤とした眼疾患病態生理の理解と治療開発

栗原俊英

日眼会誌 129 ( 3 ) 354 - 378 2025.03

Lead author, Last author, Corresponding author, Accepted

Axial Length Shortened in Approximately 20% of Patients Wearing Extended Depth-of-Focus Contact Lenses

Hazra D*, Yotsukura E*, Oyama S*, Shigeno Y, Mori K, Takeuchi R, Nakajima Y, Hanyuda A, Ogawa M, Kurihara T, Torii H†, Negishi K†

J Clin Med. 14 ( 5 ) 1750 2025.03

Accepted

Background/Objectives: Preceding studies have reported the efficacy of multifocal contact lenses (MFCLs) in slowing myopia progression. Recently, a novel type of MFCL, i.e., extended depth-of-focus (EDOF) contact lens (CL), was designed. Here, we retrospectively investigated myopia progression associated with EDOF CL wear. Methods: Twenty-four consecutive myopic children (24 eyes; mean age, 13.9 years) received EDOF CLs to control myopia progression and participated in the study. We measured the axial length (AL), spherical equivalent (SE), and choroidal thickness (CT) at baseline and after 1 year of lens wear and compared the changes. Results: The mean baseline AL, SE, and CT were, respectively, 26.31 mm, −6.38 diopter (D), and 235 μm, and at 1 year 26.40 mm (p = 0.03), −6.61 D (p = 0.05), and 244 μm (p = 0.18). The AL decreased in 20.8% of cases (≧−0.05 mm/year), whereas 20.8% and 58.4% of cases had stabilization of the AL or an increased AL (≧+0.05 mm/year), respectively. The patients with a decreased AL engaged in a mean outdoor activity time of 200.6 min/day, the patients with an increased AL (≧+0.05 mm/year) engaged in a mean outdoor activity time of 126.7 min/day. The change in the AL was correlated significantly with the change in the CT (β = −0.46, p < 0.05), and 80% of patients with a shortened AL had increased CT (≧+20 μm/year). Conclusions: Our data showed that the AL stabilized or decreased in over 40% of myopic patients wearing EDOF CLs.

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Tropism of the AAV6.2 Vector in the Murine Retina

Suzuki R*, Katada Y*, Fujii M, Serizawa N, Negishi K, Kurihara T†

Int J Mol Sci. 26 ( 4 ) 1580 2025.02

Last author, Corresponding author, Accepted, ISSN 16616596

Retinitis pigmentosa (RP) is a progressive inherited retinal dystrophy (IRD) that primarily affects rod photoreceptor cells, leading to the degeneration of photoreceptors and the gradual loss of vision. While RP is one of the most studied IRDs, other neurodegenerative diseases affecting the retina and optic nerve, such as glaucoma, also involve common mechanisms of cellular stress and degeneration. Current therapeutic approaches under investigation include gene therapy, retina prosthesis, and neuroprotection. Among these approaches, gene therapy has shown promise, though challenges related to viral vector tropism and transduction efficiency persist. The adeno-associated virus (AAV) vector is commonly employed for gene delivery, but novel serotypes and engineered variants are being explored to improve specificity and efficacy. This study evaluates the gene transfer efficiency of the AAV6.2 vector following intravitreal injection into the murine retina. Male C57BL/6 mice (9 weeks old) were intravitreally injected with 1 µL of AAV2-CMV-EGFP, AAV6-CMV-EGFP, or AAV6.2-CMV-EGFP at a titer of 3.2 × 1012 vg/mL per eye. Retinal transduction was assessed using in vivo fluorescence imaging, flat-mount imaging, and immunohistochemistry. EGFP expression in retinal ganglion cells, Müller cells, amacrine cells, and bipolar cells was quantitatively analyzed. All three AAV serotypes effectively transduced retinal ganglion cells, but AAV6.2 exhibited enhanced transduction in Müller cells and other neuronal retinal cells, including bipolar and amacrine cells. AAV6.2 demonstrated more localized expression around retinal blood vessels compared to the diffuse expression observed with AAV2. Immunohistochemical analysis revealed that AAV6.2 had significantly higher transduction efficiency in Müller cells (p < 0.001) compared to AAV2 and AAV6. AAV6.2 shows superior transduction efficiency in Müller cells, positioning it as a promising vector for gene therapies targeting retinal degenerative diseases such as RP. Its ability to effectively transduce Müller cells suggests potential applications in neuroprotection and gene replacement therapies.

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幼少期の強膜穿孔創から58年経過して外因性細菌性眼内炎を発症した1例

上村綾音, 伴紀充, 國見洋光, 富田洋平, 栗原俊英, 篠田肇, 根岸一乃

眼科臨床紀要 17 ( 10 ) 749 - 752 2024.12

Accepted

Acquisition of high-sensitivity human aqueous humor Raman spectra

Shinojima A, Sawada M, Kuroda M,Lee D, Kinoshita T, Negishi K, Kurihara T†

Res Sq. 2024.10

Last author, Corresponding author

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Deep Spectral Library of Mice Retina for Myopia Research: Proteomics Dataset generated by SWATH and DIA-NN

Sze YH, Tse DYY, Zuo B, Li KK, Zhao Q, Jiang X, Kurihara T, Tsubota K, Lam TC†

Sci Data. 11 ( 1 ) 1115 - 1115 2024.10

Accepted

The retina plays a crucial role in processing and decoding visual information, both in normal development and during myopia progression. Recent advancements have introduced a library-independent approach for data-independent acquisition (DIA) analyses. This study demonstrates deep proteome identification and quantification in individual mice retinas during myopia development, with an average of 6,263 ± 86 unique protein groups. We anticipate that the use of a predicted retinal-specific spectral library combined with the robust quantification achieved within this dataset will contribute to a better understanding of the proteome complexity. Furthermore, a comprehensive mice retinal-specific spectral library was generated, encompassing a total identification of 9,401 protein groups, 70,041 peptides, 95,339 precursors, and 761,868 transitions acquired using SWATH-MS acquisition on a ZenoTOF 7600 mass spectrometer. This dataset surpasses the spectral library generated through high-pH reversed-phase fractionation by data-dependent acquisition (DDA). The data is available via ProteomeXchange with the identifier PXD046983. It will also serve as an indispensable reference for investigations in myopia research and other retinal or neurological diseases.

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Myopic shift in female mice after ovariectomy

Zhang Y, Mori K, Jeong H, Chen J, Liang Y, Negishi K, Tsubota K†, Kurihara T†

Sci Rep. 14 ( 1 ) 22946 - 22946 2024.10

Last author, Corresponding author, Accepted

Myopia is a global public health concern, with a higher prevalence in women than that in male. As the relationship between sex hormone and myopia remains unclear, we aimed to reveal the relationship between sex hormone and myopia by removing the ovaries of mice and measuring changes in ocular parameters related to myopia. Lens-induced myopia (LIM) surgeries were performed on 3-week-old male mice and age-matched female mice to evaluate the effects of sex on myopia development, which indicated a high degree of myopia and rapid progression of axial elongation in male mice. Bilateral ovariectomy (OVX) performed on 4-week-old female mice induced myopic refraction status in ovariectomized mice. Although axial length elongation was larger in ovariectomized mice than that in sham control mice, the result was insignificant. To further reveal the relationship between female sex hormones and myopia, LIM combined with OVX was performed, which revealed a magnified myopic refraction status in ovariectomized mice. Nevertheless, elongation of the anterior chamber depth in the - 30 D lens-treated eyes significantly increased in LIM + OVX mice compared with the frame-treated eyes in LIM + OVX mice and - 30 D lens-treated eyes in LIM + Sham surgery mice. Sex hormones play a role in regulating myopia development in female mice.

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Recent Insights into Roles of Hypoxia-Inducible Factors in Retinal Diseases

Lee D, Tomita Y, Miwa Y, Kunimi H, Nakai A, Shoda C, Negishi K, Kurihara T†

Int J Mol Sci. 25 ( 18 ) 10140 2024.09

Last author, Corresponding author, Accepted, ISSN 16616596

Hypoxia-inducible factors (HIFs) are transcriptional factors that function as strong regulators of oxygen homeostasis and cellular metabolisms. The maintenance of cellular oxygen levels is critical as either insufficient or excessive oxygen affects development and physiologic and pathologic conditions. In the eye, retinas have a high metabolic demand for oxygen. Retinal ischemia can cause visual impairment in various sight-threating disorders including age-related macular degeneration, diabetic retinopathy, and some types of glaucoma. Therefore, understanding the potential roles of HIFs in the retina is highly important for managing disease development and progression. This review focuses on the physiologic and pathologic roles of HIFs as regulators of oxygen homeostasis and cellular metabolism in the retina, drawing on recent evidence. Our summary will promote comprehensive approaches to targeting HIFs for therapeutic purposes in retinal diseases.

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Fundus Image Analysis of Retinitis Pigmentosa Using Artificial Intelligence

Ubukata S*, Masayoshi K*, Katada Y†, Yang L, Ozawa N, Ibuki M, Negishi K, Kurihara T†

Res Sq. 2024.09

Last author, Corresponding author

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Establishment of an in vitro choroid complex system for vascular response screening

Jeong H, Lee D, Negishi K, Tsubota K†, Kurihara T†

Sci Rep. 14 ( 1 ) 16129 2024.07

Corresponding author, Accepted

The choroid, a vascularized tissue situated between the retina and the sclera, plays a crucial role in maintaining ocular homeostasis. Despite its significance, research on choroidal abnormalities and the establishment of effective in vitro models have been limited. In this study, we developed an in vitro choroid model through the co-culture of human induced pluripotent stem cells (hiPSC)-derived endothelial cells (ECs) and mouse choroidal fibroblasts (msCFs) with hiPSC-derived retinal pigment epithelial (RPE) cells via a permeable membrane. This model, inclusive of ECs, CFs, and RPE cells, exhibited similarities with in vivo choroidal vessels, as confirmed through immunohistochemistry of extracellular matrix markers and vascular-related markers, as well as choroid angiogenesis sprouting assay analysis. The effectiveness of our in vitro model was demonstrated in assessing vascular changes induced by drugs targeting vasoregulation. Our model offers a valuable tool for gaining insights into the pathological mechanisms underlying choroid development and the progression of choroidal vascular diseases.

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Long-term Outcomes of Anti-Vascular Endothelial Growth Factor Drug Therapies for Neovascular Age-related Macular Degeneration Based on its Subtypes

Kato S, Uchida A†, Nagai N, Suzuki M, Minami S, Shinojima A, Ban N, Horiuchi N, Fujioka S,Kurihara T, Shinoda H, Ozawa Y, Negishi K

J Jpn Ophthalmol Soc. 128 ( 6 ) 473 - 486 2024.06

Accepted

Choroidal thinning in myopia is associated with axial elongation and severity of myopic maculopathy

Midorikawa M*, Mori K*, Torii H, Tomita Y, Zhang Y, Tsubota K, Kurihara T†, Negishi K

Res Sq. (Research Square Platform LLC) 14 ( 1 ) 17600 2024.05

Corresponding author

Abstract High myopia can lead to pathologic myopia and visual impairment, whereas its causes are unclear. We retrospectively researched high myopia cases from patient records to investigate the association between axial elongation and myopic maculopathy. Sixty-four eyes were examined in patients who visited the department between July 2017 and June 2018, had an axial length of 26 mm or more, underwent fundus photography, and had their axial length measured twice or more. The average axial length was 28.29±1.69 mm (mean±standard deviation). The average age was 58.3 ± 14.4 years old. Myopic maculopathy was categorized as mild (grades 0 and 1) and severe (grades 2, 3, and 4). The severe group had longer axial lengths than the mild group (p<0.05). Moreover, the severe group exhibited thinner choroidal thickness than the mild group (p<0.05). When subjects were grouped by axial elongation over median value within a year, the elongation group showed thinner central choroidal thickness than the non-elongation group (142.1±91.9 vs. 82.9±69.8 ㎛, p<0.05). In conclusion, in patients with high myopia, the severity of maculopathy correlated with choroidal thickness and axial length. Thinner choroidal thickness was associated with axial elongation based on the baseline axial length.

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Myopia Is an Ischemic Eye Condition: A Review from the Perspective of Choroidal Blood Flow

Baksh J*, Lee D*, Mori K, Zhang Y, Torii H, Jeong H, Hou J, Negishi K, Tsubota K†, Kurihara T†

J Clin Med. (MDPI AG) 13 ( 10 ) 2777 - 2777 2024.05

Last author, Corresponding author, Accepted

Myopia is a common refractive error that affects a large proportion of the population. Recent studies have revealed that alterations in choroidal thickness (ChT) and choroidal blood flow (ChBF) play important roles in the progression of myopia. Reduced ChBF could affect scleral cellular matrix remodeling, which leads to axial elongation and further myopia progression. As ChT and ChBF could be used as potential biomarkers for the progression of myopia, several recent myopia treatments have targeted alterations in ChT and ChBF. Our review provides a comprehensive overview of the recent literature review on the relationship between ChBF and myopia. We also highlight the importance of ChT and ChBF in the progression of myopia and the potential of ChT as an important biomarker for myopia progression. This summary has significant implications for the development of novel strategies for preventing and treating myopia.

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Retinal ischemic diseases and promising therapeutic molecular targets

Lee D*, Tomita Y*, Negishi K, Kurihara T†

Histol Histopathol. 40 ( 1 ) 18756 - 20 2024.05

Last author, Corresponding author, Accepted, ISSN 02133911

Retinal ischemia is a fundamental pathologic condition associated with retinal vascular occlusion, glaucoma, diabetic retinopathy, age-related macular degeneration, and other eye diseases. Extensive inflammation, redox imbalance, apoptosis, and abnormal vascular formation in retinal ischemia could lead to visual impairments. Developing or finding effective treatments is urgently needed to protect the eye against retinal ischemia and related damage. To address the demand, we have searched for promising therapeutic molecular targets in the eye (e.g., hypoxia-inducible factor [HIF], peroxisome proliferator-activated receptor-alpha [PPARα], and nicotinamide adenine dinucleotide [NAD+]), and found that modulations of each molecular target might protect the eye against retinal ischemic damage in terms of complex pathologic mechanisms. In the current article, we review and update the therapeutic evidence of modulation of HIF, PPARα, or NAD+ and discuss future directions for developing promising drugs based on these molecular targets. This summary urges research to obtain more solid evidence of each molecular target in retinal ischemic diseases.

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Drusen in AMD from the Perspective of Cholesterol Metabolism and Hypoxic Response

Ban N, Shinojima A, Negishi K, Kurihara T*

J Clin Med. (Journal of Clinical Medicine) 13 ( 9 ) 2608 2024.04

Last author, Corresponding author, Accepted

Drusen are one of the most characteristic pathologies of precursor lesion of age-related macular degeneration (AMD). Drusen comprise a yellowish white substance that accumulates typically under the retinal pigment epithelium (RPE), and their constituents are lipids, complement, amyloid, crystallin, and others. In the past, many researchers have focused on drusen and tried to elucidate the pathophysiology of AMD because they believed that disease progression from early AMD to advanced AMD might be based on drusen or drusen might cause AMD. In fact, it is well established that drusen are the hallmark of precursor lesion of AMD and a major risk factor for AMD progression mainly based on their size and number. However, the existence of advanced AMD without drusen has long been recognized. For example, polypoidal choroidal vasculopathy (PCV), which comprises the majority of AMD cases in Asians, often lacks drusen. Thus, there is the possibility that drusen might be no more than a biomarker of AMD and not a cause of AMD. Now is the time to reconsider the relationship between AMD and drusen. In this review, we focus on early AMD pathogenesis based on basic research from the perspective of cholesterol metabolism and hypoxic response in the retina, and we discuss the role of drusen.

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Halofuginone prevents outer retinal degeneration in a mouse model of light-induced retinopathy

Miwa Y*, Lee D*, Shoda C, Jeong H, Negishi K, Kurihara T†

PLOS ONE (Public Library of Science (PLoS)) 19 ( 3 ) e0300045 2024.03

Last author, Corresponding author, Accepted

Photoreceptor cell death can cause progressive and irreversible visual impairments. Still, effective therapies on retinal neuroprotection are not available. Hypoxia-inducible factors (HIFs) are transcriptional factors which strongly regulate angiogenesis, erythropoiesis, intracellular metabolism, and programed cell death under a hypoxic or an abnormal metabolic oxidative stress condition. Therefore, we aimed to unravel that inhibition of HIFs could prevent disease progression in photoreceptor cell death, as recent studies showed that HIFs might be pathologic factors in retinal diseases. Adult male balb/cAJcl (8 weeks old; BALB/c) were used to investigate preventive effects of a novel HIF inhibitor halofuginone (HF) on a murine model of light-induced retinopathy. After intraperitoneal injections of phosphate-buffered saline (PBS) or HF (0.4 mg/kg in PBS) for 5 days, male BALB/c mice were subjected to a dark-adaption to being exposed to a white LED light source at an intensity of 3,000 lux for 1 hour in order to induce light-induced retinal damage. After extensive light exposure, retinal damage was evaluated using electroretinography (ERG), optical coherence tomography (OCT), and TUNEL assay. Light-induced retinal dysfunction was suppressed by HF administration. The amplitudes of scotopic a-wave and b-wave as well as that of photopic b-wave were preserved in the HF-administered retina. Outer retinal thinning after extensive light exposure was suppressed by HF administration. Based on the TUNEL assay, cell death in the outer retina was seen after light exposure. However, its cell death was not detected in the HF-administered retina. Halofuginone was found to exert preventive effects on light-induced outer retinal cell death.

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Scleral remodeling during myopia development in mice eyes: a potential role of thrombospondin-1

Chen J*, Ikeda SI*, Yang Y, Zhang Y, Ma Z, Liang Y, Negishi K, Tsubota K†, Kurihara T†

Mol Med. (Molecular Medicine) 30 ( 1 ) 25 2024.02

Last author, Corresponding author, Accepted, ISSN 10761551

Background: Scleral extracellular matrix (ECM) remodeling plays a crucial role in the development of myopia, particularly in ocular axial elongation. Thrombospondin-1 (THBS1), also known as TSP-1, is a significant cellular protein involved in matrix remodeling in various tissues. However, the specific role of THBS1 in myopia development remains unclear. Method: We employed the HumanNet database to predict genes related to myopic sclera remodeling, followed by screening and visualization of the predicted genes using bioinformatics tools. To investigate the potential target gene Thbs1, we utilized lens-induced myopia models in male C57BL/6J mice and performed Western blot analysis to detect the expression level of scleral THBS1 during myopia development. Additionally, we evaluated the effects of scleral THBS1 knockdown on myopia development through AAV sub-Tenon’s injection. The refractive status and axial length were measured using a refractometer and SD-OCT system. Results: During lens-induced myopia, THBS1 protein expression in the sclera was downregulated, particularly in the early stages of myopia induction. Moreover, the mice in the THBS1 knockdown group exhibited alterations in myopia development in both refraction and axial length changed compared to the control group. Western blotting analysis confirmed the effectiveness of AAV-mediated knockdown, demonstrating a decrease in COLA1 expression and an increase in MMP9 levels in the sclera. Conclusion: Our findings indicate that sclera THBS1 levels decreased during myopia development and subsequent THBS1 knockdown showed a decrease in scleral COLA1 expression. Taken together, these results suggest that THBS1 plays a role in maintaining the homeostasis of scleral extracellular matrix, and the reduction of THBS1 may promote the remodeling process and then affect ocular axial elongation during myopia progression.

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Slowing of Greater Axial Length Elongation Stemming from the COVID-19 Pandemic with Increasing Time Outdoors: The Tokyo Myopia Study

Yotsukura E, Torii H†, Mori K, Ogawa M, Hanyuda A, Negishi K, Kurihara T†, Tsubota K†

Ophthalmol Sci. (Elsevier BV) 4 ( 5 ) 100491 - 100491 2024.02

Corresponding author, Accepted, ISSN 2666-9145

Purpose: To investigate the changes in axial length (AL) elongation and other ocular parameters before and during the coronavirus disease 2019 pandemic. Design: A longitudinal school-based study. Participants: Public elementary schoolchildren in Tokyo (grades 1–6; age, 6–12 years) participated in this study from 2018 to 2021. Methods: All participants underwent eye examinations and provided written consent to measurements of the noncycloplegic refraction and ocular biometry including AL, among others. The students’ parents also completed a questionnaire about the students’ lifestyles. We included the right eye in our analysis and compared the changes in the ocular parameters among the periods using a linear mixed-effects model for repeated measures and examined the univariate and step-wise multiple regression analyses to evaluate the associations between myopia and other covariates. Main Outcome Measures: Changes in AL elongation and other ocular parameters from 2018 to 2019 (prepandemic), that of 2019 to 2020 (immediately after the pandemic onset), and that of 2020 to 2021 (during the pandemic). Results: A total of 578 students before the pandemic period, 432 immediately after the pandemic onset, and 457 during the pandemic period were evaluated. The changes in the ALs and spherical equivalents (SEs) a year before, immediately after onset, and during the pandemic were 0.31 mm/−0.20 diopter, 0.38 mm/−0.27 diopter, and 0.28 mm/−0.47 diopter, respectively (ALs, P < 0.001; SEs, P = 0.014). The results of the questionnaire showed that time spent outdoors daily had changed during the 3 years to 79, 63, and 77 minutes/day, respectively (P < 0.001). Time spent using smartphones or tablets increased year by year to 41, 52, and 62 minutes/day (P < 0.001). The greatest AL elongation occurred during the period when the shortest amount of time was spent outdoors during the 3 years. Conclusions: These results suggested that the school closures and decreasing time spent outdoors might have caused greater AL elongation among schoolchildren in Tokyo; however, it is possible that, although the time spent in near work still increased, the return to the time spent outdoors to the prepandemic levels may have affected the slowing of AL elongation after lockdown. Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

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Deep Learning Segmentation of Non-perfusion Area from Color Fundus Images and AI-generated Fluorescein Angiography

Masayoshi K*, Katada Y*, Ozawa N, Ibuki M, Negishi K, Kurihara T†

Res Sq. (Research Square Platform LLC) 14 ( 1 ) https://doi.org/10.21203/rs.3.rs-3871406/v1 2024.01

Last author, Corresponding author

Abstract

The non-perfusion area (NPA) of the retina is an important indicator in the visual prognosis of patients with retinal vein occlusion (RVO). However, the current evaluation method of NPA, fluorescein angiography (FA), is invasive and burdensome. In this study, we examined the use of deep learning models for detecting NPA in color fundus images, bypassing the need for FA, and we also investigated the utility of synthetic FA generated from color fundus images. The models were evaluated using the Dice score and Monte Carlo dropout uncertainty. We retrospectively collected 403 sets of color fundus and FA images from 319 RVO patients. We trained three deep learning models on FA, color fundus images, and synthetic FA. As a result, though the FA model achieved the highest score, the other two models also performed comparably. We found no statistical significance in median Dice scores between the models. However, the color fundus model showed significantly higher uncertainty than the other models (p < 0.05). In conclusion, deep learning models can detect NPAs from color fundus images with reasonable accuracy, though with somewhat less prediction stability. Synthetic FA stabilizes the prediction and reduces misleading uncertainty estimates by enhancing image quality.

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Modulation of Hypoxia-Inducible Factors and Vascular Endothelial Growth Factor Expressions by Superfood Camu-Camu (Myrciaria dubia) Treatment in ARPE-19 and Fetal Human RPE Cells

Nakai A, Lee D, Shoda C, Negishi K, Nakashizuka H, Yamagami S, Kurihara T†

J Ophthalmol. (Hindawi Limited) 2023 6617981 2023.12

Last author, Corresponding author, Accepted, ISSN 2090-004X

Background. Anti-vascular endothelial growth factor (anti-VEGF) therapy via intravitreal injection is an effective treatment for patients with abnormal ocular neovascularization, such as age-related macular degeneration (AMD) and diabetic macular edema (DME). However, prolonged and frequent anti-VEGF treatment is associated with a risk of local and systemic adverse events, including geographic atrophy, cerebrovascular disease, and death. Furthermore, some patients do not adequately respond to anti-VEGF therapy. Hypoxia-inducible factor (HIF) is a transcription factor that controls the expression of hypoxia-responsive genes involved in angiogenesis, inflammation, and metabolism. The HIF/VEGF pathway plays an important role in neovascularization, and the inhibition of HIF activation could be an effective biomolecular target for neovascular diseases. The demand for disease prevention or treatment using functional foods such as superfoods has increased in recent years. Few reports to date have focused on the antineovascular effects of superfoods in the retinal pigment epithelium (RPE). In light of the growing demand for functional foods, we aimed to find novel HIF inhibitors from superfoods worked in RPE cells, which could be an adjuvant for anti-VEGF therapy. Methods. Seven superfoods were examined to identify novel HIF inhibitor candidates using luciferase assay screening. We used the human RPE cell line ARPE-19 and fetal human RPE (fhRPE) to investigate the biomolecular actions of novel HIF inhibitors using quantitative PCR and western blotting. Results. Under CoCl2-induced pseudohypoxic condition and 1% oxygen hypoxic incubation, camu-camu (Myrciaria dubia) showed HIF inhibitory effects determined by luciferase assays. Camu-camu downregulated HIF-1α and VEGFA mRNA expressions in a concentration-dependent manner. Camu-camu also inhibited HIF-1α protein expressions, and its inhibitory effect was greater than that of vitamin C, which is present at high levels in camu-camu. Conclusion. The camu-camu extract suppressed the activation of HIF and VEGF in RPE cells. This could assist anti-VEGF therapy in patients with abnormal ocular neovascularization.

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Inhibition of hypoxia-inducible factors suppresses subretinal fibrosis

Shoda C*, Lee D*, Miwa Y, Yamagami S, Nakashizuka H, Nimura K, Okamoto K, Kawagishi H, Negishi K, Kurihara T†

FASEB J. (Cold Spring Harbor Laboratory) 38 ( 13 ) 571193 2023.12

Last author, Corresponding author, ISSN 08926638

Age-related macular degeneration (AMD) is a common cause of vision loss. The aggressive form of AMD is associated with ocular neovascularization and subretinal fibrosis, representing a responsive outcome against neovascularization mediated by epithelial-mesenchymal transition of retinal pigment epithelium cells. A failure of the current treatment (anti-vascular endothelial growth factor therapy) has also been attributed to the progression of subretinal fibrosis. Hypoxia-inducible factors (HIFs) increase gene expressions to promote fibrosis and neovascularization. HIFs act as a central pathway in the pathogenesis of AMD. HIF inhibitors may suppress ocular neovascularization. Nonetheless, further investigation is required to unravel the aspects of subretinal fibrosis. In this study, we used RPE-specific HIFs or von Hippel-Lindau (VHL, a regulator of HIFs) conditional knockout (cKO) mice, along with pharmacological HIF inhibitors, to demonstrate the suppression of subretinal fibrosis. Fibrosis was suppressed by treatments of HIF inhibitors, and similar suppressive effects were detected in RPE-specific Hif1a/Hif2a- and Hif1a-cKO mice. Promotive effects were observed in RPE-specific Vhl-cKO mice, where fibrosis-mediated pathologic processes were evident. Marine products extracts and their component taurine suppressed fibrosis as HIF inhibitors. Our study shows critical roles of HIFs in the progression of fibrosis, linking them to the potential development of therapeutics for AMD.

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Tight junction component protein claudin-1 deficiency in retinal pigment epithelium leads to early and intermediate age-related macular degeneration phenotypes in mice.

Nakai A, Lee D, Zhang Y, Shoda C, Imanishi S, Jeong H, Ikeda SI, Negishi K, Nakashizuka H, Yamagami S, Kubo A, Kurihara T†

bioRxiv. (Cold Spring Harbor Laboratory) 570525 2023.12

Last author, Corresponding author

The early and intermediate age-related macular degeneration (AMD) is characterized by the presence of drusen and pigmentary abnormalities in the retinal pigment epithelial (RPE) cells which form the outer blood retinal barrier (oBRB). Fluid leakage through the disrupted oBRB from the choroid to the neural retina has been implicated in the pathogenesis of AMD, however; the molecular mechanisms still remain unclear. The family of four transmembrane proteins, claudins are known to form tight junctions (TJs) in the oBRB. Nonetheless, there are few reports showing how they function in the oBRB in vivo. We found that claudin-1 is dominantly expressed in TJs of the mouse RPE. To investigate the role of claudin-1 in the RPE, we generated RPE-specific Cldn1 conditional knockout mice (Best1-Cre+/- Cldn1flox/flox mice: Cldn1 cKO mice). Deficiency of Cldn1 led to age-related lipid deposits such as subretinal drusenoid deposits (SDD), increased lipid droplets in the RPE, basal lamellar deposits (BlamD) and membranous debris in the Bruch's membrane. In addition, pigmentary abnormalities such as RPE hypertrophy, multilayered-RPE cells, and ectopic pigment granules outside the RPE were observed in Cldn1 cKO mice. Our study provides new insights into the possible association of the TJ protein claudin-1 with lipid metabolism and cellular ageing in the RPE contributing to the early onset of AMD.

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Topical application of bunazosin hydrochloride suppresses myopia progression with an increase in choroidal blood perfusion

Jeong H, Lee D, Jiang X, Negishi K, Tsubota K†, Kurihara T†

Invest Ophthalmol Vis Sci. (Investigative ophthalmology & visual science) 64 ( 14 ) 15 2023.11

Last author, Corresponding author, Accepted, ISSN 01460404

Purpose: The incidence of myopia has rapidly increased in recent decades, making it a growing public health concern worldwide. Interventions to suppress the progression of myopia are needed; one suggested strategy is the prevention of choroidal thinning, which can improve choroidal blood perfusion (ChBP). Bunazosin hydrochloride (BH) is an alpha1-adrenergic blocker and commercialized glaucoma eye drop that increases in blood circulation in the eye. In this study, we evaluated the efficacy of BH in suppressing the progression of myopia in a lens-induced murine model. Methods: Lens-induced myopia was induced in 3-week-old C57BL/6 J mice with -30 diopter (D) lenses for three weeks. Refractive error, axial length, and choroidal thickness were evaluated at three and six weeks of age using an infrared photorefractor and a spectral domain optical coherence tomography (OCT) system. Moreover, ChBP and scleral thickness were evaluated using swept-source OCT and histological analysis. Results: Compared with the controls, the administration of BH eye drops suppressed the myopic shift of refractive error (mean difference ± standard error in the eye with -30 D lens, -13.65 ± 5.69 D vs. 2.55 ± 4.30 D; P < 0.001), axial elongation (0.226 ± 0.013 mm vs. 0.183 ± 0.023 mm; P < 0.05), choroidal thinning (-2.01 ± 1.80 µm vs. 1.88 ± 1.27 µm; P < 0.001), and scleral thinning (11.41 ± 3.91 µm vs. 19.72 ± 4.01 µm; P < 0.01) with myopia progression and increased ChBP (52.0% ± 4.1% vs. 59.5% ± 6.3%; P < 0.05). The suppressive effect of BH eye drops was dose-dependent and higher than that of other glaucoma eye drops and alpha1 blockers. Conclusions: These results demonstrate the potential of BH eye drops in the treatment of myopia and support further investigation of their efficacy in humans. Further studies are needed to determine the mechanism of action and long-term safety of this treatment.

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Establishment of a novel ER-stress induced myopia model in mice

Kang L*, Ikeda SI*, Yang Y, Jeong H, Chen J, Zhang Y, Negishi K, Tsubota K†, Kurihara T†

Eye Vis. (Eye and Vision) 10 ( 1 ) 44 2023.11

Last author, Corresponding author, Accepted

Background: Recent studies have indicated a strong correlation between endoplasmic reticulum (ER) stress and myopia and that eyedrops containing the ER stress inducer tunicamycin (Tm) can induce myopic changes in C57BL/6 J mice. Therefore, this study aimed to create a new myopia model using Tm eyedrops and to explore the mechanism of ER stress-mediated myopia development. Methods: Three-week-old C57BL/6 J mice were treated with different concentrations (0, 25, 50, and 100 μg/mL) and/or number of applications (zero, one, three, and seven) of Tm eyedrops. Refraction and axial length (AL) were measured before and one week after Tm treatment. Scleral collagen alterations were evaluated under polarised light after picrosirius red staining. ER stress-related indicators, such as the expression of collagen I and cleaved collagen were detected using Western blotting. Results: Compared with the control group, mice administered eyedrops with 50 μg/mL Tm only once showed the greatest myopic shifts in refraction and AL elongation and reduced scleral expression of collagen I. Picrosirius red staining showed a lower percentage of bundled collagen in the Tm group. Expression of ER-stress indicators increased in the Tm groups. Furthermore, optimised administration of Tm induced matrix metalloproteinase-2 (MMP2) expression in the sclera, which plays a major role in collagen degradation. Conclusions: We have demonstrated that ER stress in the sclera is involved in myopia progression. Tm eyedrops induced myopic changes, loosening of the scleral collagen and decreased expression of collagen I. This process may be associated with ER stress in the sclera, which upregulates the expression of MMP2 leading to collagen degradation.

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Bisphenol A exposure triggers endoplasmic reticulum stress pathway leading to ocular axial elongation in mice

Chen J*, Ikeda SI*, Kang L, Negishi K, Tsubota K†, Kurihara T†

Front Med. (Frontiers in Medicine) 10 1255121 2023.08

Last author, Corresponding author, Accepted

Background: Ocular axial elongation is one of the features of myopia progression. Endoplasmic reticulum (ER) stress-associated scleral remodeling plays an important role in ocular axial elongation. Bisphenol A (BPA) is one of the most common environmental pollutants and is known to affect various human organs through ER stress. However, whether BPA exerts an effect on scleral remodeling remains unknown. The purpose of this study was to determine the effect of BPA on the development of myopia and scleral ER stress. Methods: BPA was administered by intraperitoneal injection. 4-PBA was administered as an endoplasmic reticulum stress inhibitor by eye drops. Refraction and axial length were measured by refractometer and SD-OCT system. Western blot was performed to detect the expression level of ER stress-related proteins. Results: BPA-administered mice exhibit axial elongation and myopic refractive shift with endoplasmic reticulum stress in the sclera. BPA administration activated scleral PERK and ATF6 pathways, and 4-PBA eye drops attenuated ER stress response and suppressed myopia progression. Conclusion: BPA controlled axial elongation during myopia development in a mouse model by inducing scleral ER stress and activation of the PERK/ATF6 pathway. 4-PBA eye drops as ER stress inhibitor suppressed BPA-induced myopia development.

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“Fruiting Liquid” of Mushroom-Forming Fungi, A Novel Source of Bioactive Compounds – Fruiting-Body Inducer and HIF and Axl Inhibitors

Wu J, Uchida K, Yoshikawa A, Hashimoto M, Kondo M, Nihei K, Ishii M, Choi JH, Miwa Y, Shoda C, Lee D, Nakai A, Kurihara T, D’Alessandro-Gabazza CN, Toda M, Yasuma T, Gabazza EC, Hirai H, Kawagishi H†

J Agric Food Chem. (American Chemical Society (ACS)) 71 ( 36 ) 13338 - 13345 2023.08

Accepted, ISSN 0021-8561

In general, mushroom-forming fungi secrete liquid on the surface of mycelia just before fruiting-body formation. However, no researchers in mushroom science have paid attention to the liquid until now. We formulated a hypothesis that the liquid plays an important role(s) in the formation of the fruiting body and produces various bioactive compounds and named it the “fruiting liquid (FL)”. Four novel compounds (1-4) were isolated from FL of Hypholoma lateritium and Hericium erinaceus. The structures of 1-4 except for their stereochemistry were determined by interpretation of MS and NMR data. The absolute configurations of compounds 1-4 were determined by quantum chemical calculation of the ECD spectrum, by single-crystal X-ray diffraction analyses, or by chemical syntheses. Compounds 1, 3, and 4 induced fruiting body formation of Flammulina velutipes. Compound 4 inhibited the activity of hypoxia-inducible factor, and compounds 2-4 suppressed receptor tyrosine kinase (Axl) expression.

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Highly-sensitive visual restoration and protection via ectopic expression of chimeric rhodopsin in mice

Katada Y, Yoshida K, Serizawa N, Lee D, Kobayashi K, Negishi K, Okano H, Kandori H, Tsubota K, Kurihara T†

iScience. (Elsevier BV) 26 ( 10 ) 107716 2023.08

Last author, Corresponding author, Accepted, ISSN 2589-0042

Photoreception requires amplification by mammalian rhodopsin through G protein activation, which requires a visual cycle. To achieve this in retinal gene therapy, we incorporated human rhodopsin cytoplasmic loops into Gloeobacter rhodopsin, thereby generating Gloeobacter and human chimeric rhodopsin (GHCR). In a murine model of inherited retinal degeneration, we induced retinal GHCR expression by intravitreal injection of a recombinant adeno-associated virus vector. Retinal explant and visual thalamus electrophysiological recordings, behavioral tests, and histological analysis showed that GHCR restored dim-environment vision and prevented the progression of retinal degeneration. Thus, GHCR may be a potent clinical tool for the treatment of retinal disorders.

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Identification of potential therapeutic targets for myopic choroidal neovascularization via discovery-driven data mining

Chen J, Ikeda SI, Negishi K, Tsubota K†, Kurihara T†

Curr Eye Res. (Current Eye Research) 48 ( 12 ) 1160 - 1169 2023.08

Last author, Corresponding author, Accepted, ISSN 02713683

Purpose: Myopic choroidal neovascularization (mCNV) is a prevalent cause of vision loss. However, the development of effective therapeutic targets for mCNV has been hindered by the paucity of suitable animal models. Therefore, the aim of this study is to identify potential genes and pathways associated with mCNV and to unearth prospective therapeutic targets that can be utilized to devise efficacious treatments. Methods: Text data mining was used to identify genes linked to choroid, neovascularization, and myopia. g: Profiler was utilized to analyze the biological processes of gene ontology and the Reactome pathways. Protein interaction network analysis was performed using strings and visualized in Cytoscape. MCODE and cytoHubba were used for further screening. Results: Discovery-driven text data mining identified 55 potential genes related to choroid, neovascularization, and myopia. Gene enrichment analysis revealed 11 biological processes and seven Reactome pathways. A protein-protein interaction network with 47 nodes was constructed and analyzed using centrality ranking. Key clusters were identified through algorithm tools. Finally, 14 genes (IL6, FGF2, MMP9, IL10, TNF, MMP2, HGF, MMP3, IGF1, CCL2, CTNNB1, BDNF, NGF, and EDN1), in addition to VEGFA, were evaluated as targets with potential as future therapeutics. Conclusions: This study provides new potential therapeutic targets for mCNV, including IL6, FGF2, MMP9, IL10, TNF, MMP2, HGF, MMP3, IGF1, CCL2, CTNNB1, BDNF, NGF, and EDN1, which correspond to seven potential enriched pathways. These findings provide a basis for further research and offer new possibilities for developing therapeutic interventions for this condition.

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Long chain acyl-CoA synthetase 6 facilitates the local distribution of di-docosahexaenoic acid- and ultra-long-chain-PUFA-containing phospholipids in the retina to support normal visual function in mice

Kuroha S, Katada Y, Isobe Y, Uchino H, Shishikura K, Nirasawa T, Tsubota K, Negishi K, Kurihara T†, Arita M†

FASEB J. (FASEB Journal) 37 ( 9 ) e23151 2023.08

Corresponding author, Accepted, ISSN 08926638

Docosahexaenoic acid (DHA) and ultra-long-chain polyunsaturated fatty acids (ULC–PUFAs) are uniquely enriched in membrane phospholipids of retinal photoreceptors. Several studies have shown that di-DHA- and ULC–PUFA-containing phospholipids in photoreceptors have an important role in maintaining normal visual function; however, the molecular mechanisms underlying the synthesis and enrichment of these unique lipids in the retina, and their specific roles in retinal function remain unclear. Long-chain acyl-coenzyme A (CoA) synthetase 6 (ACSL6) preferentially converts DHA into DHA–CoA, which is a substrate during DHA-containing lipid biosynthesis. Here, we report that Acsl6 mRNA is expressed in the inner segment of photoreceptor cells and the retinal pigment epithelial cells, and genetic deletion of ACSL6 resulted in the selective depletion of di-DHA– and ULC–PUFA-containing phospholipids, but not mono-DHA-containing phospholipids in the retina. MALDI mass spectrometry imaging (MALDI–MSI) revealed the selective distribution of di-DHA– and ULC–PUFA-containing phospholipids in the photoreceptor outer segment (OS). Electroretinogram of Acsl6−/− mice exhibited photoreceptor cell-derived visual impairment, whereas the expression levels and localization of opsin proteins were unchanged. Acsl6−/− mice exhibited an age-dependent progressive decrease of the thickness of the outer nuclear layers, whereas the inner nuclear layers and OSs were normal. These results demonstrate that ACSL6 facilitates the local enrichment of di-DHA– and ULC–PUFA-containing phospholipids in the retina, which supports normal visual function and retinal homeostasis.

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Associations between fatty acid intake and diabetic retinopathy in a Japanese population

Sasaki M†, Yuki K, Hanyuda A, Yamagishi K†, Motomura K, Kurihara T, Tomita Y, Mori K, Ozawa N, Ozawa Y, Sawada N, Negishi K, Tsubota K, Tsugane S, Iso H

Sci Rep. (Springer Science and Business Media LLC) 13 ( 1 ) 12903 2023.08

Accepted

Abstract

Residents of Chikusei City, aged 40–74 years, underwent systemic and ophthalmological screening, and participants with diabetes were included in this analysis. Dietary intake was assessed using a food frequency questionnaire and calculated as a percentage of the total energy. The presence of diabetic retinopathy (DR) was defined as Early Treatment Diabetic Retinopathy Study levels ≥ 20 in either eye. The association between dietary fatty acid intake and DR has been examined in a cross-sectional study. Among the 647 diabetic participants, 100 had DR. The mean total fat and saturated fatty acid (SFA) intakes were 22.0% and 7.3% of the total energy intake, respectively. After adjusting for potential confounders, the highest quartiles of total fat and SFA intake were positively associated with the presence of DR compared with the lowest quartiles (odds ratios (95% confidence intervals), 2.61 (1.07–6.39), p for trend = 0.025, and 2.40 (1.12–5.17), p for trend = 0.013, respectively). No significant associations were found between DR prevalence and monounsaturated or unsaturated fatty acid intake. These results suggest that a high intake of fat and SFA may affect the development of DR, even in individuals whose total fat intake is generally much lower than that of Westerners.

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System for Transmitting Laser and its Safety Evaluation for Measuring Metabolites in Aqueous Humor

Toshihide Kurihara, Ari Shinojima, Taku Kinoshita, Saori Nishizaki, Yoshinori Shirakawa, Sho Kimura, Deokho Lee, Yuki Hidaka, Yasuyo Nishi, Yukari Tsuneyoshi, Hidemasa Torii, Kazuo Tsubota, Kazuno Negishi

J Jpn Ophthalmol Soc. 127 ( 7 ) 672 - 679 2023.07

Lead author, Corresponding author, Accepted

Nicotinamide mononucleotide, a potential future treatment in ocular diseases

Lee D, Tomita Y, Shinojima A, Ban N, Yamaguchi S, Nishioka K, Negishi K, Yoshino J, Kurihara T†

Graefes Arch Clin Exp Ophthalmol. (Graefe's Archive for Clinical and Experimental Ophthalmology) 262 ( 3 ) 689 - 700 2023.06

Last author, Corresponding author, Accepted, ISSN 0721832X

Purpose: The burden of ocular diseases has been gradually increasing worldwide. Various factors are suggested for the development and progression of ocular diseases, such as ocular inflammation, oxidative stress, and complex metabolic dysregulation. Thus, managing ocular diseases requires the modulation of pathologic signaling pathways through many mechanisms. Nicotinamide mononucleotide (NMN) is a bioactive molecule naturally found in life forms. NMN is a direct precursor of the important molecule nicotinamide adenine dinucleotide (NAD+), an essential co-enzyme required for enormous cellular functions in most life forms. While the recent experimental evidence of NMN treatment in various metabolic diseases has been well-reviewed, NMN treatment in ocular diseases has not been comprehensively summarized yet. In this regard, we aimed to focus on the therapeutic roles of NMN treatment in various ocular diseases with recent advances. Methods: How we came to our current opinion with a recent summary was described based on our own recent reports as well as a search of the related literature. Results: We found that NMN treatment might be available for the prevention of and protection from various experimental ocular diseases, as NMN treatment modulated ocular inflammation, oxidative stress, and complex metabolic dysregulation in murine models for eye diseases such as ischemic retinopathy, corneal defect, glaucoma, and age-related macular degeneration. Conclusion: Our current review suggests and discusses new modes of actions of NMN for the prevention of and protection from various ocular diseases and can urge future research to obtain more solid evidence on a potential future NMN treatment in ocular diseases at the preclinical stages.

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The orientation of choroidal macrophage polarization significantly influences the development of myopia in murine models

Hou J*, Ikeda SI*, Mori K, Jeong H, Torii H, Negishi K, Tsubota K†, Kurihara T†

bioRxiv. (Cold Spring Harbor Laboratory) 544445 2023.06

Last author, Corresponding author

Abstract

Myopia is a primary contributor to visual impairment and has emerged as a global public health concern. Evidence indicates that one of the main structural features of myopia is the corresponding decrease in choroidal thickness, and choroidal macrophages play an important role in maintaining the choroidal thickness. Nevertheless, the effect of choroidal macrophages on myopia remains unclear. Here, we discovered that the continuous intraperitoneal injection of clodronate liposomes depleted choroidal macrophages and leads to myopia, which confirmed that the presence of choroidal macrophages plays an important role in myopia development.

Subsequently, based on the phenotypic characteristics of macrophages, experiments were designed to study the effects of different polarization directions of macrophages on myopia development. We found that lipopolysaccharides (LPS) injection can induce the polarization of choroidal M1 macrophages and result in myopia. Conversely, IL-4 or IL-13 injection causes choroidal M2 macrophage polarization and suppresses the progression of myopia. Additionally, we demonstrated that the opposite effects of M1 and M2 macrophages on myopia development may be related to their impacts on inflammation and oxidative stress response. These findings establish that choroidal macrophages are critically important in the development of myopia and provide new strategies for the development of myopic therapies.

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Encephalopsin (OPN3) is required for normal refractive development and the GO/GROW response to induced myopia

Linne C, Mon KY, D’Souza S, Jeong H, Jiang X, Brown DM, Zhang K, Vemaraju S, Tsubota K, Kurihara T, Pardue MT, Lang RA†

Mol Vis. (Molecular vision) 29 39 - 57 2023.05

Accepted

Purpose: Myopia, or nearsightedness, is the most common form of refractive error and is increasing in prevalence. While significant efforts have been made to identify genetic variants that predispose individuals to myopia, these variants are believed to account for only a small portion of the myopia prevalence, leading to a feedback theory of emmetropization, which depends on the active perception of environmental visual cues. Consequently, there has been renewed interest in studying myopia in the context of light perception, beginning with the opsin family of G-protein coupled receptors (GPCRs). Refractive phenotypes have been characterized in every opsin signaling pathway studied, leaving only Opsin 3 (OPN3), the most widely expressed and blue-light sensing noncanonical opsin, to be investigated for function in the eye and refraction. Methods: Opn3 expression was assessed in various ocular tissues using an Opn3eGFP reporter. Weekly refractive development in Opn3 retinal and germline mutants from 3 to 9 weeks of age was measured using an infrared photorefractor and spectral domain optical coherence tomography (SD-OCT). Susceptibility to lens-induced myopia was then assessed using skull-mounted goggles with a -30 diopter experimental and a 0 diopter control lens. Mouse eye biometry was similarly tracked from 3 to 6 weeks. A myopia gene expression signature was assessed 24 h after lens induction for germline mutants to further assess myopia-induced changes. Results: Opn3 was found to be expressed in a subset of retinal ganglion cells and a limited number of choroidal cells. Based on an assessment of Opn3 mutants, the OPN3 germline, but not retina conditional Opn3 knockout, exhibits a refractive myopia phenotype, which manifests in decreased lens thickness, shallower aqueous compartment depth, and shorter axial length, atypical of traditional axial myopias. Despite the short axial length, Opn3 null eyes demonstrate normal axial elongation in response to myopia induction and mild changes in choroidal thinning and myopic shift, suggesting that susceptibility to lens-induced myopia is largely unchanged. Additionally, the Opn3 null retinal gene expression signature in response to induced myopia after 24 h is distinct, with opposing Ctgf, Cx43, and Egr1 polarity compared to controls. Conclusions: The data suggest that an OPN3 expression domain outside the retina can control lens shape and thus the refractive performance of the eye. Prior to this study, the role of Opn3 in the eye had not been investigated. This work adds OPN3 to the list of opsin family GPCRs that are implicated in emmetropization and myopia. Further, the work to exclude retinal OPN3 as the contributing domain in this refractive phenotype is unique and suggests a distinct mechanism when compared to other opsins.

Myopia Is suppressed by digested lactoferrin or holo-lactoferrin administration

Liang Y*, Ikeda SI*, Chen J, Zhang Y, Negishi K, Tsubota K†, Kurihara T†

Int J Mol Sci. (International Journal of Molecular Sciences) 24 ( 6 ) 5815 2023.03

Last author, Corresponding author, Accepted, ISSN 16616596

Myopia is becoming a leading cause of vision impairment. An effective intervention is needed. Lactoferrin (LF) is a protein that has been reported to inhibit myopia progression when taken orally. This study looked at the effects of different forms of LF, such as native LF and digested LF, on myopia in mice. Mice were given different forms of LF from 3 weeks of age, and myopia was induced with minus lenses from 4 weeks of age. Results showed that mice given digested LF or holo-LF had a less elongated axial length and thinned choroid, compared to those given native-LF. Gene expression analysis also showed that the groups given native-LF and its derivatives had lower levels of certain cytokines and growth factors associated with myopia. These results suggest that myopia can be more effectively suppressed by digested LF or holo-LF than native-LF.

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Contralateral effect in progression and recovery of lens-induced myopia in mice

Ma Z*, Jeong H*, Yang Y, Jiang X, Ikeda SI, Negishi K, Kurihara T†, Tsubota K†

Ophthal Physiol Opt. (Ophthalmic and Physiological Optics) 00 ( 3 ) 1 - 8 2023.03

Corresponding author, Accepted, ISSN 02755408

Purpose: Apart from genetic factors, recent animal studies on myopia have focused on localised mechanisms. In this study, we aimed to examine the contralateral effects of monocular experimental myopia and recovery, which cannot be explained by a mere local mechanism. Methods: One eye of 3-week-old C57BL/6 male mice was fitted with a −30 dioptre (D) lens. The mice were distributed into two groups based on different conditions in the contralateral eye: either no lens (NLC) (n = 10) or a Plano lens on the contralateral eye (PLC) group (n = 6). Mice receiving no treatment on either eye were set as a control group (n = 6). Lenses were removed after 3 weeks of myopia induction. All mice were allowed to recover for 1 week in the same environment. Refractive status, axial length (AL) and choroidal thickness were measured before myopia induction, after 1 and 3 weeks of lens wear and after 1 week of recovery. Results: One week after removing the lenses, complete recovery was observed in the eyes that wore the −30 D lenses. In both the PLC and NLC groups, the refractive status showed a myopic shift after lens removal. Additionally, the choroid was significantly thinned in these eyes. The −30 D wearing eye showed a significant increase in AL after 3 weeks of lens wear. While the AL of the −30 D wearing eye ceased to grow after the lens was removed, the AL in the PLC and NLC contralateral eyes increased, and the binocular ALs gradually converged. Conclusions: Recovery of lens-induced myopia was observed in mouse models. In the fellow eyes, the effects, including thinning of the choroid and changes in refractive status, were triggered by contralateral visual cues.

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Molecular and cellular regulations in the development of the choroidal circulation

Imanishi S, Tomita Y, Negishi K, Tsubota K†, Kurihara T†

Int J Mol Sci. (International journal of molecular sciences) 24 ( 6 ) 5371 2023.03

Last author, Corresponding author, Accepted, ISSN 16616596

Disorders in the development and regulation of blood vessels are involved in various ocular disorders, such as persistent hyperplastic primary vitreous, familial exudative vitreoretinopathy, and choroidal dystrophy. Thus, the appropriate regulation of vascular development is essential for healthy ocular functions. However, regulation of the developing choroidal circulation system has not been well studied compared with vascular regulation in the vitreous and the retina. The choroid is a vascular-rich and uniquely structured tissue supplying oxygen and nutrients to the retina, and hypoplasia and the degeneration of the choroid are involved in many ocular disorders. Therefore, understanding the developing choroidal circulation system expands our knowledge of ocular development and supports our understanding of ocular disorders. In this review, we examine studies on regulating the developing choroidal circulation system at the cellular and molecular levels and discuss the relevance to human diseases.

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Ginkgo biloba extracts improve choroidal circulation leading to suppression of myopia in mice

Hou J*, Mori K*, Ikeda SI, Jeong H, Torii H, Negishi K, Kurihara T†, Tsubota K†

Sci Rep. (Scientific Reports) 13 ( 1 ) 3772 2023.03

Corresponding author, Accepted

Myopia is becoming more common across the world, necessitating the development of preventive methods. We investigated the activity of early growth response 1 (EGR-1) protein and discovered that Ginkgo biloba extracts (GBEs) activated EGR-1 in vitro. In vivo, C57BL/6 J mice were fed either normal or 0.0667% GBEs (200 mg/kg) mixed chow (n = 6 each), and myopia was induced with − 30 diopter (D) lenses from 3 to 6 weeks of age. Refraction and axial length were measured by an infrared photorefractor and an SD-OCT system, respectively. In lens-induced myopia mice, oral GBEs significantly improved refractive errors (− 9.92 ± 1.53 D vs. − 1.67 ± 3.51 D, p < 0.001) and axial elongation (0.22 ± 0.02 mm vs. 0.19 ± 0.02 mm, p < 0.05). To confirm the mechanism of GBEs in preventing myopia progression, the 3-week-old mice were divided into normally fed with either myopic-induced or non-myopic-induced groups and GBEs fed with either myopic-induced or non-myopic-induced groups (n = 10 each). Choroidal blood perfusion was measured with optical coherence tomography angiography (OCTA). In both non-myopic induced groups, compared to normal chow, oral GBEs significantly improved choroidal blood perfusion (8.48 ± 15.75%Area vs. 21.74 ± 10.54%Area, p < 0.05) and expression of Egr-1 and endothelial nitric oxide synthase (eNOS) in the choroid. In both myopic-induced groups, compared to normal chow, oral GBEs also improved choroidal blood perfusion (− 9.82 ± 9.47%Area vs. 2.29 ± 11.84%Area, p < 0.05) and was positively correlated with the change in choroidal thickness. These findings suggest that GBEs may inhibit the progression of myopia by improving choroidal blood perfusion.

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Suppressive effects of violet light transmission on myopia progression in a mouse model of lens-induced myopia

Jeong H, Kurihara T†, Jiang X, Kondo S, Ueno Y, Hayashi Y, Lee D, Ikeda SI, Mori K, Torii H, Negishi K, Tsubota K†

Exp Eye Res. (Elsevier BV) 228 109414 2023.02

Corresponding author, Accepted, ISSN 0014-4835

The prevalence of myopia has been steadily increasing for several decades, and this condition can cause extensive medical and economic issues in society. Exposure to violet light (VL), a short wavelength (360–400 nm) of visible light from sunlight, has been suggested as an effective preventive and suppressive treatments for the development and progression of myopia. However, the clinical application of VL remains unclear. In this study, we aimed to investigate the preventive and suppressive effects of VL on myopia progression. Various transmittances of VL (40%, 70%, and 100%) were tested in C57BL/6J mice with lens-induced myopia (LIM). Changes in the refractive error, axial length, and choroid thickness during the 3-week LIM were measured. The myopic shift in refractive error and difference in axial length between the 0 and -30 diopter lens was lessened in a transmission-dependent manner. Choroidal thinning, which was observed in myopic conditions, was suppressed by VL exposure and affected by its transmission. The results suggest that myopia progression can be managed using VL transmittance. Therefore, these factors should be considered for the prevention and treatment of myopia.

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Pemafibrate prevents choroidal neovascularization in a mouse model of neovascular age-related macular degeneration

Lee D, Nakai A, Miwa Y, Negishi K, Tomita Y, Kurihara T†

PeerJ. (PeerJ) 11 e14611 2023.01

Last author, Corresponding author, Accepted

Background. Pathological choroidal neovascularization (CNV) is one of the major causes of visual impairment in neovascular age-related macular degeneration (AMD). CNV has been suppressed by using anti-vascular endothelial growth factor (VEGF) antibodies. However, some clinical cases have demonstrated the failure of anti-VEGF therapies. Furthermore, anti-VEGF agents might induce the development of ocular atrophy. Recently, peroxisome proliferator-activated receptor alpha (PPARα) activation using pemafibrate treatment was suggested as one of the promising therapeutic targets in the prevention of ocular ischemia. However, the preventive role of pemafibrate remains unclear in CNV. We aimed to examine the preventive role of pemafibrate on laser-induced pathological CNV. Methods. Adult male C57BL/6 mice were orally supplied pemafibrate (0.5 mg/kg) for four days, followed by laser irradiation. Then, pemafibrate was consecutively given to mice with the same condition. CNV was visualized with isolectin-IB4. The eye (retina and/or retinal pigment epithelium [RPE]-choroid), liver, and serum were used for biomolecular analyses. Results. We found that pemafibrate administration suppressed CNV volumes. Pemafibrate administration activated PPARα downstream genes in the liver and eye (especially, RPE-choroid). Furthermore, pemafibrate administration elevated serum fibroblast growth factor 21 levels and reduced serum levels of triglycerides. Conclusions. Our data suggest a promising pemafibrate therapy for suppressing CNV in AMD.

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Association between fatty acid intakes and age-related macular degeneration in a Japanese population: JPHC-NEXT Eye Study

Yasukawa T*, Sasaki M*†, Motomura K, Yuki K, Kurihara T, Tomita Y, Mori K, Ozawa N, Ozawa Y, Yamagishi K†, Hanyuda A, Sawada N, Tsubota K, Tsugane S, Iso H

Transl Vis Sci Technol. (Translational Vision Science and Technology) 12 ( 1 ) 3 2023.01

Accepted

Purpose: To determine the associations between fatty acid intakes and the prevalence of age-related macular degeneration (AMD) under a population-based cross-sectional study. Methods: Residents of Chikusei City aged ≥40 years underwent systemic and eye screening. AMD was graded according to a modified version of the Age-Related Eye Disease Study classification. Dietary intake was assessed using a food frequency questionnaire and was adjusted for total energy intake. Results: Altogether, 10,788 eyes of 5394 participants, 2116 men (mean [standard devia-tion (SD)] age, 62.4 [9.4] years) and 3278 women (60.6 [9.5] years), were included. The mean daily total fat intakes were 52.8 g and 59.0 g in men and women, respectively. After adjustments for potential confounders, saturated fatty acid (SFA) intake was inversely associated with the prevalence of any AMD in men (for each energy-adjusted 1-SD increase: odds ratio [OR], 0.86; 95% confidence interval [CI], 0.74–1.00). Significant trends were found for decreasing odds ratios of AMD with increasing SFA, monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) intake (P for trend = 0.02, 0.04, and 0.04, respectively). In women, only a significant association was observed between the second quartile of linolenic acid intake and the prevalence of any AMD (OR, 0.78; 95% CI, 0.62–0.99). Conclusions: We found an inverse association of SFA intake and a weak inverse association of MUFA and PUFA intakes with the prevalence of any AMD in a Japanese popula-tion. Translational Relevance: Adequate fatty acid intake may be necessary to prevent or decelerate AMD.

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Deep-learning-based AI for evaluating estimated nonperfusion areas requiring further examination in ultra-widefield fundus images

Inoda S, Takahashi H†, Yamagata H, Hisadome Y, Kondo Y, Tampo H, Sakamoto S, Katada Y, Kurihara T, Kawashima H, Yanagi Y

Sci Rep. (Springer Science and Business Media LLC) 12 ( 1 ) 21826 2022.12

Accepted

Abstract

We herein propose a PraNet-based deep-learning model for estimating the size of non-perfusion area (NPA) in pseudo-color fundus photos from an ultra-wide-field (UWF) image. We trained the model with focal loss and weighted binary cross-entropy loss to deal with the class-imbalanced dataset, and optimized hyperparameters in order to minimize validation loss. As expected, the resultant PraNet-based deep-learning model outperformed previously published methods. For verification, we used UWF fundus images with NPA and used Bland–Altman plots to compare estimated NPA with the ground truth in FA, which demonstrated that bias between the eNPA and ground truth was smaller than 10% of the confidence limits zone and that the number of outliers was less than 10% of observed paired images. The accuracy of the model was also tested on an external dataset from another institution, which confirmed the generalization of the model. For validation, we employed a contingency table for ROC analysis to judge the sensitivity and specificity of the estimated-NPA (eNPA). The results demonstrated that the sensitivity and specificity ranged from 83.3–87.0% and 79.3–85.7%, respectively. In conclusion, we developed an AI model capable of estimating NPA size from only an UWF image without angiography using PraNet-based deep learning. This is a potentially useful tool in monitoring eyes with ischemic retinal diseases.

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Axl, Immune Checkpoint Molecules and HIF Inhibitors from the Culture Broth of Lepista luscina

Kotajima M*, Choi JH*, Kondo M, D’Alessandro-Gabazza CN, Toda M, Yasuma T, Gabazza EC, Miwa Y, Shoda C, Lee D, Nakai A, Kurihara T, Wu J, Hirai H, Kawagishi H†

Molecules. (MDPI AG) 27 ( 24 ) 8925 - 8925 2022.12

Accepted

Two compounds 1 and 2 were isolated from the culture broth of Lepista luscina. This is the first time that compound 1 was isolated from a natural source. The structure of compound 1 was identified via 1D and 2D NMR and HRESIMS data. Compounds 1 and 2 along with 8-nitrotryptanthrin (4) were evaluated for their biological activities using the A549 lung cancer cell line. As a result, 1 and 2 inhibited the expression of Axl and immune checkpoint molecules. In addition, compounds 1, 2 and 4 were tested for HIF inhibitory activity. Compound 2 demonstrated statistically significant HIF inhibitory effects on NIH3T3 cells and 1 and 2 against ARPE19 cells.

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Relationship of choroidal thickness and axial length with posterior vitreous detachment in patients with high myopia

Hanyuda A*, Torii H*, Hayashi K, Uchida A, Mori K, Yotsukura E, Ogawa M, Negishi K, Kurihara T†, Tsubota T†

Sci Rep. (Springer Science and Business Media LLC) 12 ( 1 ) 4093 2022.12

Accepted

<title>Abstract</title>Although accumulating evidence suggests a higher prevalence of posterior vitreous detachment (PVD) in highly myopic eyes, the relation between ocular biometric features and PVD stages in such eyes remains unclear. Therefore, we enrolled 170 patients with high myopia (axial length ≥ 26.0 mm) to investigate the status of PVD regarding subfoveal choroidal thickness and axial length. Utilising swept-source optical coherence tomography, we classified the PVD status into five stages. The distribution of PVD grades increased as the choroidal thickness decreased and axial length increased (P < 0.01). On adjusting for age and sex, decreased choroidal thickness and increased axial length were associated with more advanced PVD stages: odds ratios with the highest vs. lowest groups were 0.31 (95% confidence interval [CI] 0.09–1.01; Ptrend = 0.009) for choroidal thickness and 5.16 (95% CI 1.34–19.80; Ptrend = 0.002) for axial length. The inverse association between choroidal thickness and PVD status seemed stronger in women than in men (Pinteraction = 0.05). In conclusion, we firstly observed a significant trend of decreased choroidal thickness, along with increased axial length, with increased grade of PVD, particularly among women with highly myopic eyes, suggesting that advanced morphological myopic changes contribute to PVD in middle-aged adults.

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Relation between Dry Eye and Myopia Based on Tear Film Breakup Time, Higher Order Aberration, Choroidal Thickness, and Axial Length

Hazra D*, Yotsukura E*, Torii H*†, Mori K, Maruyama T, Ogawa M, Hanyuda A, Tsubota K, Kurihara T†, Negishi K†

Sci Rep. (Scientific Reports) 12 ( 1 ) 10891 2022.12

Accepted

The purpose of this study was to investigate the association between dry eye disease (DED) and myopia by evaluating higher order aberrations (HOAs) and choroidal thickness (CT). We recruited 72 myopic children with DED symptoms (mean age 12.8 years), measured the tear film breakup time (TBUT), corneal/intraocular/total ocular HOAs, CT, and axial length (AL), administered lifestyle questionnaires, and evaluated the relationships among TBUT, HOAs, CT, and AL. The TBUT was correlated significantly with the corneal HOAs and intraocular HOAs but not with the total ocular HOAs. Multiple regression analyses showed that the AL was associated significantly with the TBUT (β = − 0.067, P = 0.004), the intraocular HOAs, and total ocular HOAs but not with the corneal HOAs. The CT was associated significantly with the TBUT and AL (β = 9.15 and − 7.85, respectively; P < 0.001 and = 0.01, respectively). Our data suggested the association between DED and myopia might be independent of the HOAs. We showed that the TBUT was associated with the CT, which is related to the AL. Because the parasympathetic nervous system affects the lacrimal glands and CT, the parasympathetic nervous system might be a common upstream factor in the association between DED and myopia.

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Nicotinamide Mononucleotide Protects against Retinal Dysfunction in a Murine Model of Carotid Artery Occlusion

Lee D, Tomita Y, Miwa Y, Jeong H, Shinojima A, Ban N, Yamaguchi S, Nishioka K, Negishi K, Yoshino J, Kurihara T†

Intl J Mol Sci. (MDPI AG) 23 ( 23 ) 14711 2022.11

Last author, Corresponding author, Accepted, ISSN 16616596

Cardiovascular abnormality-mediated retinal ischemia causes severe visual impairment. Retinal ischemia is involved in enormous pathological processes including oxidative stress, reactive gliosis, and retinal functional deficits. Thus, maintaining retinal function by modulating those pathological processes may prevent or protect against vision loss. Over the decades, nicotinamide mononucleotide (NMN), a crucial nicotinamide adenine dinucleotide (NAD+) intermediate, has been nominated as a promising therapeutic target in retinal diseases. Nonetheless, a protective effect of NMN has not been examined in cardiovascular diseases-induced retinal ischemia. In our study, we aimed to investigate its promising effect of NMN in the ischemic retina of a murine model of carotid artery occlusion. After surgical unilateral common carotid artery occlusion (UCCAO) in adult male C57BL/6 mice, NMN (500 mg/kg/day) was intraperitoneally injected to mice every day until the end of experiments. Electroretinography and biomolecular assays were utilized to measure ocular functional and further molecular alterations in the retina. We found that UCCAO-induced retinal dysfunction was suppressed, pathological gliosis was reduced, retinal NAD+ levels were preserved, and the expression of an antioxidant molecule (nuclear factor erythroid-2-related factor 2; Nrf2) was upregulated by consecutive administration of NMN. Our present outcomes first suggest a promising NMN therapy for the suppression of cardiovascular diseases-mediated retinal ischemic dysfunction.

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Pemafibrate, a potent selective peroxisome proliferator-activated receptor α modulator, a promising novel treatment for ischemic retinopathy?

Lee D, Tomita Y, Negishi K, Kurihara T†

Neural Regen Res. (Medknow) 18 ( 7 ) 1495 - 1496 2022.11

Last author, Corresponding author, Accepted, ISSN 1673-5374

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Therapeutic roles of PPARα activation in ocular ischemic diseases

Lee D*, Tomita Y*, Negishi K, Kurihara T†

Histol Histopathol. (Histology and histopathology) 38 ( 4 ) 18542 - 401 2022.10

Last author, Corresponding author, Accepted, ISSN 02133911

Ocular ischemia is one of the leading causes of blindness. It is related to various ocular diseases and disorders, including age-related macular degeneration, diabetic retinopathy, glaucoma, and corneal injury. Ocular ischemia occurs due to an abnormal supply of oxygen and nutrients to the eye, resulting in ocular metabolic dysfunction. These changes can be linked with pathologic conditions in the eye, such as inflammation, neovascularization, and cell death, ultimately leading to vision loss. The current treatment care for ocular ischemia is limited. Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor protein functioning in regulating lipid metabolism, fatty acid oxidation, and glucose homeostasis. Recently, PPARα activation has been suggested as a useful therapeutic target in treating ocular ischemia. However, its applications have not been well summarized. In this review, we cover an overview of the therapeutic roles of PPARα activation in various ocular ischemic conditions with recent experimental evidence and further provide clinical implications of its therapeutic applications. Our review will enable more approaches to comprehensively understand the therapeutic roles of PPARα activation for preventing ocular ischemic diseases.

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Short-Term Exposure to Violet Light Emitted from Eyeglass Frames in Myopic Children: A Randomized Pilot Clinical Trial

Torii H*†, Mori K*, Takashi Okano T, Kondo S, Yang HY, Yotsukura E, Hanyuda A, Ogawa M, Negishi K, Kurihara T†, Tsubota K†

J Clin Med. (Journal of Clinical Medicine) 11 ( 20 ) 6000 2022.10

Corresponding author, Accepted

Violet light (VL), 360–400 nm wavelength, is contained in the sunlight and is an effective element for myopia suppression. This study is to investigate the safety and efficacy of novel eyeglasses that emit VL from the frames. This is a double-masked, randomized, pilot clinical trial conducted in a clinic in Japan. Forty-three children with myopia were enrolled. Participants were randomly assigned to two groups, wearing VL-emitting eyeglass frames (VLf) that emitted VL of 310 μW/cm2 (VLf group, n = 22) or pseudo-placebo eyeglass frames with a minimal emission of VL (<10 μW/cm2) (control group, n = 21). The exposure time was 3 h per day. The primary outcomes were visual acuity, tear film break-up time, corneal endothelial cell density, and the slit-lamp/fundus examinations. The secondary outcome was the 6-month changes in the axial lengths and cycloplegic refractions. Forty-one (95%) participants were included; twenty-one in the VLf group and twenty in the control group. No significant differences were seen in any safety evaluation. Significant changes were seen in axial elongation, choroidal thickness, and cycloplegic refractions in the subgroup analysis of 8- to 10-year-old children (p < 0.05), but otherwise no significant differences were seen. The VLf showed short-term safety and effectiveness against myopia progression.

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Scleral PERK and ATF6 as targets of myopic axial elongation of mouse eyes

Ikeda SI, Kurihara T†, Jiang X, Miwa Y, Lee D, Serizawa N, Jeong H, Mori K, Katada Y, Kunimi H, Ozawa N, Shoda C, Ibuki M, Negishi K, Torii H, Tsubota K†

Nat Commun. (Springer Science and Business Media LLC) 13 ( 1 ) 5859 2022.10

Corresponding author, Accepted

Abstract

Axial length is the primary determinant of eye size, and it is elongated in myopia. However, the underlying mechanism of the onset and progression of axial elongation remain unclear. Here, we show that endoplasmic reticulum (ER) stress in sclera is an essential regulator of axial elongation in myopia development through activation of both PERK and ATF6 axis followed by scleral collagen remodeling. Mice with lens-induced myopia (LIM) showed ER stress in sclera. Pharmacological interventions for ER stress could induce or inhibit myopia progression. LIM activated all IRE1, PERK and ATF6 axis, and pharmacological inhibition of both PERK and ATF6 suppressed myopia progression, which was confirmed by knocking down above two genes via CRISPR/Cas9 system. LIM dramatically changed the expression of scleral collagen genes responsible for ER stress. Furthermore, collagen fiber thinning and expression of dysregulated collagens in LIM were ameliorated by 4-PBA administration. We demonstrate that scleral ER stress and PERK/ATF6 pathway controls axial elongation during the myopia development in vivo model and 4-PBA eye drop is promising drug for myopia suppression/treatment.

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Degeneration of retinal ganglion cells in hypoxic responses: hypoxia-inducible factor inhibition, a new therapeutic insight

Lee D, Kunimi H, Negishi K, Kurihara T†

Neural Regen Res. (Neural Regeneration Research) 17 ( 10 ) 2230 - 2231 2022.10

Accepted, ISSN 16735374

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Nicotinamide Mononucleotide Prevents Retinal Dysfunction in a Mouse Model of Retinal Ischemia/Reperfusion Injury

Lee D, Tomita Y, Miwa Y, Shinojima A, Ban N, Yamaguchi S, Nishioka K, Negishi K, Yoshino J, Kurihara T†

Int J Mol Sci. (International Journal of Molecular Sciences) 23 ( 19 ) 11228 2022.09

Last author, Corresponding author, Accepted, ISSN 16616596

Retinal ischemia/reperfusion (I/R) injury can cause severe vision impairment. Retinal I/R injury is associated with pathological increases in reactive oxygen species and inflammation, resulting in retinal neuronal cell death. To date, effective therapies have not been developed. Nicotinamide mononucleotide (NMN), a key nicotinamide adenine dinucleotide (NAD+) intermediate, has been shown to exert neuroprotection for retinal diseases. However, it remains unclear whether NMN can prevent retinal I/R injury. Thus, we aimed to determine whether NMN therapy is useful for retinal I/R injury-induced retinal degeneration. One day after NMN intraperitoneal (IP) injection, adult mice were subjected to retinal I/R injury. Then, the mice were injected with NMN once every day for three days. Electroretinography and immunohistochemistry were used to measure retinal functional alterations and retinal inflammation, respectively. The protective effect of NMN administration was further examined using a retinal cell line, 661W, under CoCl2-induced oxidative stress conditions. NMN IP injection significantly suppressed retinal functional damage, as well as inflammation. NMN treatment showed protective effects against oxidative stress-induced cell death. The antioxidant pathway (Nrf2 and Hmox-1) was activated by NMN treatment. In conclusion, NMN could be a promising preventive neuroprotective drug for ischemic retinopathy.

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Vascular endothelial growth factor from retinal pigment epithelium is essential in choriocapillaris and axial length maintenance

Zhang Y*, Jeong H*, Mori K, Ikeda SI, Shoda C, Miwa Y, Nakai A, Chen J, Ma Z, Jiang X, Torii H, Kubota Y, Negishi K, Kurihara T†, Tsubota K†

PNAS Nexus (Oxford University Press (OUP)) 1 ( 4 ) pgac166 2022.08

Corresponding author, Accepted

Abstract

Myopia, which prevalence is rapidly increasing, causes visual impairment; however, the onset mechanism of pathological axial length (AL) elongation remains unclear. A highly vascularized choroid between the retinal pigment epithelium (RPE) and sclera not only maintains physiological activities, but also contributes to ocular development and growth regulation. Vascular endothelial growth factor (VEGF) secreted from the RPE to the choroid is essential for retinal function and maintenance of the choriocapillaris. Herein, we demonstrated that the loss of VEGF secreted from the RPE caused abnormal choriocapillaris development and AL elongation, with features similar to those of the lens-induced myopia (LIM) mouse model, whereas VEGF overexpression by knocking-out von Hippel–Lindau (VHL) specific to the RPE expands the choriocapillaris and shortens the AL. Additionally, LDL Receptor Related Protein 2 (LRP2) deletion in the RPE downregulated VEGF expression and leads to pathological AL elongation. Furthermore, high myopia patients without choriocapillaris demonstrated longer ALs than did those with preserved choriocapillaris. These results suggest that physiological secretion of VEGF from the RPE is required for proper AL development by maintaining the choriocapillaris. The pinpoint application of VEGF to the choriocapillaris may become a potential intervention for the prevention and treatment of axial myopia progression.

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Automatic segmentation of non-perfusion area from fluorescein angiography using deep learning with uncertainty estimation

Masayoshi K*, Katada Y*, Ozawa N, Ibuki M, Negishi K, Kurihara T†

Inform Med Unlocked. (Elsevier BV) 32 101060 - 101060 2022.08

Last author, Corresponding author, Accepted, ISSN 2352-9148

The non-perfusion area (NPA) of the retina is an important indicator in the visual prognosis of patients with retinal vein occlusion. Therefore, automatic detection of NPA will help its management. Deep learning models for NPA segmentation in fluorescein angiography have been reported. However, typical deep learning models do not adequately address the uncertainty of the prediction, which may lead to missed lesions and difficulties in working with medical professionals. In this study, we developed deep segmentation models with uncertainty estimation using Monte Carlo dropout and compared the accuracy of prediction and reliability of uncertainty in different models (U-Net, PSPNet, and DeepLabv3+) and uncertainty measures (standard deviation and mutual information). The study included 403 Japanese fluorescein angiography images of retinal vein occlusion. The mean Dice scores were 65.6 ± 9.6%, 66.8 ± 12.3%, and 73.6 ± 9.4% for U-Net, PSPNet, and DeepLabv3+, respectively. The uncertainty scores were best for U-Net, which suggests that the model complexity may deteriorate the quality of uncertainty estimation. Over-looked lesions and inconsistent prediction led to high uncertainty values. The results indicated that the uncertainty estimation would help decrease the risk of missed lesions.

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Retinal degeneration induced in a mouse model of ischemia–reperfusion injury and its management by pemafibrate treatment

Lee D, Nakai A, Miwa Y, Tomita Y, Kunimi H, Chen J, Ikeda SI, Tsubota K, Negishi K, Kurihara T†

FASEB J. (FASEB Journal) 36 ( 9 ) e22497 2022.08

Last author, Corresponding author, Accepted, ISSN 08926638

Retinal ischemia–reperfusion (I/R) injury is a common cause of visual impairment. To date, no effective treatment is available for retinal I/R injury. In addition, the precise pathological mechanisms still need to be established. Recently, pemafibrate, a peroxisome proliferator-activated receptor α (PPARα) modulator, was shown to be a promising drug for retinal ischemia. However, the role of pemafibrate in preventing retinal I/R injury has not been documented. Here, we investigated how retinal degeneration occurs in a mouse model of retinal I/R injury by elevation of intraocular pressure and examined whether pemafibrate could be beneficial against retinal degeneration. Adult mice were orally administered pemafibrate (0.5 mg/kg/day) for 4 days, followed by retinal I/R injury. The mice were continuously administered pemafibrate once every day until the end of the experiments. Retinal functional changes were measured using electroretinography. Retina, liver, and serum samples were used for western blotting, quantitative PCR, immunohistochemistry, or enzyme linked immunosorbent assay. Retinal degeneration induced by retinal inflammation was prevented by pemafibrate administration. Pemafibrate administration increased the hepatic PPARα target gene expression and serum levels of fibroblast growth factor 21, a neuroprotective molecule in the eye. The expression of hypoxia-response and pro-and anti-apoptotic/inflammatory genes increased in the retina following retinal I/R injury; however, these changes were modulated by pemafibrate administration. In conclusion, pemafibrate is a promising preventive drug for ischemic retinopathies.

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Children in Tokyo Have a Long Sustained Axial Length from Age 3 Years: The Tokyo Myopia Study

Maruyama T*, Yotsukura E*, Torii H*†, Mori K, Inokuchi M, Tokumura M, Hazra D, Ogawa M, Hanyuda A, Tsubota K, Kurihara T†, Negishi K†

J Clin Med. (Journal of Clinical Medicine) 11 ( 15 ) 4413 2022.07

Corresponding author, Accepted

Background: myopia prevalence is high among Japanese schoolchildren, but the underlying causes are unclear. Objective: To examine the distributions of ocular biometry and refraction and their associations with lifestyle variables among Japanese schoolchildren. Methods: This cross-sectional school-based study included 2140 children aged 3–14 years in Tokyo, Japan, and evaluated the distributions under non-cycloplegic conditions and the associated environmental factors. Results: The prevalence of spherical equivalent (SE) ≤−0.75 diopter among preschoolers (aged 3–6 years), elementary school students (aged 6–11 years), and junior high school students (aged 12–14 years) was 49.7%, 72.4%, and 87.7%, respectively. Multiple linear regression analyses showed that the time spent using digital devices was associated positively with lens thickness (β = 0.010; p < 0.050) but not SE, axial length, or vitreous chamber depth. The time spent reading was associated negatively with lens thickness (β = −0.012; p < 0.050), SE (β = −0.152; p < 0.010), axial length (β = 0.110; p < 0.001), and vitreous chamber depth (β = 0.110; p < 0.001). Conclusions: The data indicated that almost half of preschoolers may be myopic. The association with the lens thickness differed depending on the type of near work performed.

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Lipidomic analysis revealed n‐3 polyunsaturated fatty acids suppressed choroidal thinning and myopia progression in mice

Mori K*, Kuroha S*, Hou J, Jeong H, Ogawa M, Ikeda SI, Kang JX, Negishi K, Torii H, Arita M†, Kurihara T†, Tsubota K

FASEB J. (Wiley) 36 ( 6 ) e22312 2022.06

Accepted, ISSN 08926638

Myopia is increasing worldwide and its preventable measure should urgently be pursued. N-3 polyunsaturated fatty acids (PUFAs) have been reported to have various effects such as vasodilative and anti-inflammatory, which myopia may be involved in. This study is to investigate the inhibitory effect of PUFAs on myopia progression. A lens-induced myopia (LIM) model was prepared using C57B L6/J 3-week-old mice, which were equipped with a -30 diopter lens to the right eye. Chows containing two different ratios of n-3/n-6 PUFA were administered to the mice, and myopic shifts were confirmed in choroidal thickness, refraction, and axial length in the n-3 PUFA-enriched chow group after 5 weeks. To exclude the possibility that the other ingredients in the chow may have taken the suppressive effect, fat-1 transgenic mice, which can produce n-3 PUFAs endogenously, demonstrated significant suppression of myopia. To identify what elements in n-3 PUFAs took effects on myopia suppression, enucleated eyes were used for targeted lipidomic analysis, and eicosapentaenoic acid (EPA) were characteristically distributed. Administration of EPA to the LIM model confirmed the inhibitory effect on choroidal thinning and myopia progression. Subsequently, to identify the elements and the metabolites of fatty acids effective on myopia suppression, targeted lipidomic analysis was performed and it demonstrated that metabolites of EPA were involved in myopia suppression, whereas prostaglandin E2 and 14,15-dihydrotestosterone were associated with progression of myopia. In conclusion, EPA and its metabolites are related to myopia suppression and inhibition of choroidal thinning.

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Multiple Factors Causing Myopia and the Possible Treatments: A Mini Review

Shinojima A, Negishi K, Tsubota K, Kurihara T†

Front Public Health. (Frontiers in Public Health) 10 897600 2022.05

The myopia epidemic has become a global public health problem. Although myopia is progressing worldwide, the recent coronavirus infections 2019 (COVID-19) outbreak has spurred myopia progression. The current evidence-based treatments for humans are atropine eye drops, optical treatment with defocus, use of orthokeratology, extending proximity working distance, pausing from near work every half hour and increased time outside the home. Studies on myopia using animal models have been conducted for more than 40 years. In recent years, new mechanisms of myopia suppression have been revealed from animal experiments such as inflammation control, intraocular pressure control, light control, and the activity of early growth response protein 1 control. This mini-review provides a summary of the scientific evidence currently available on the control of myopia, and the possible treatments mitigating myopia.

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Ocular Ischemic Syndrome and Its Related Experimental Models

Lee D*, Tomita Y*, Yang L, Negishi K, Kurihara T†

Int J Mol Sci. (MDPI AG) 23 ( 9 ) 5249 - 5249 2022.05

Accepted, ISSN 16616596

Ocular ischemic syndrome (OIS) is one of the severe ocular disorders occurring from stenosis or occlusion of the carotid arteries. As the ophthalmic artery is derived from the branch of the carotid artery, stenosis or occlusion of the carotid arteries could induce chronic ocular hypoperfusion, finally leading to the development of OIS. To date, the pathophysiology of OIS is still not clearly unraveled. To better explore the pathophysiology of OIS, several experimental models have been developed in rats and mice. Surgical occlusion or stenosis of common carotid arteries or internal carotid arteries was conducted bilaterally or unilaterally for model development. In this regard, final ischemic outcomes in the eye varied depending on the surgical procedure, even though similar findings on ocular hypoperfusion could be observed. In the current review, we provide an overview of the pathophysiology of OIS from various experimental models, as well as several clinical cases. Moreover, we cover the status of current therapies for OIS along with promising preclinical treatments with recent advances. Our review will enable more comprehensive therapeutic approaches to prevent the development and/or progression of OIS.

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Non-Perfusion Area Index for Prognostic Prediction in Diabetic Retinopathy

Ofuji Y*, Katada Y*, Tomita Y, Nagai N, Sonobe H, Watanabe K, Shinoda H, Ozawa Y, Negishi K, Tsubota K, Kurihara T†

Life. (MDPI AG) 12 ( 4 ) 542 - 542 2022.04

Accepted

Fundus fluorescent angiography is a standard examination in Japan that can directly visualize the circulatory failure in diabetic retinopathy but is not used in Western countries. In this study, we examine the relationship between the non-perfusion area in fundus fluorescent angiography and the progression of diabetic retinopathy. We evaluated 22 eyes between 22 patients who had their first fundus fluorescent angiography during a clinical episode at Keio University Hospital from January 2012 to May 2015, were diagnosed as having preproliferative diabetic retinopathy, and could be followed for at least three years. The non-perfusion area index (%) in nine segmented fundi in the initial fundus fluorescent angiography was calculated, and the progression to proliferative diabetic retinopathy over three years was evaluated. Three out of the 22 eyes (13.6%) developed proliferative diabetic retinopathy over three years. The non-perfusion area index for the initial fundus fluorescent angiography was significantly associated with progression to proliferative diabetic retinopathy. The non-perfusion area index in the posterior pole was most strongly correlated with the progression to proliferative diabetic retinopathy. Thus, the non-perfusion area index in the posterior pole among those with preproliferative diabetic retinopathy may predict the progression to proliferative diabetic retinopathy in the subsequent three years.

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HIF Inhibition Therapy in Ocular Diseases

Lee D*, Miwa Y*, Kunimi H, Ibuki M, Shoda C, Nakai A, Kurihara T†

Keio J Med. (Keio Journal of Medicine) 71 ( 1 ) 1 - 12 2022

Accepted, ISSN 00229717

The uncontrolled growth of blood vessels is a major pathological factor in human eye diseases that can result in blindness. This effect is termed ocular neovascularization and is seen in diabetic retinopathy, age-related macular degeneration, glaucoma and retinopathy of prematurity. Current treatments for these diseases include laser photocoagulation, topical injection of corticosteroids, intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) agents and vitreoretinal surgery. Although strategies to inhibit VEGF have proved to be dramatically successful in some clinical studies, there remains the possibility of significant adverse effects regarding the blockade of crucial physiological roles of VEGF and the invasive nature of the treatments. Moreover, it is evident that other pro-angiogenic factors also play important roles in the development of these diseases, as seen in cases in which antiVEGF therapies have failed. Therefore, new types of effective treatments are required. In this review, we discuss a promising strategy for the treatment of ocular neovascular diseases, i.e., the inhibition of hypoxia-inducible factor (HIF), a master regulator of angiogenesis. We also summarize promising recently investigated HIF inhibitors as treatments for ocular diseases. This review will facilitate more comprehensive approaches to understanding the protective aspects of HIF inhibition in the prevention of ocular diseases. (DOI: 10.2302/kjm.2021-0004-IR; Keio J Med 71 (1): 1–12, March 2022).

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Retinal Diseases Regulated by Hypoxia—Basic and Clinical Perspectives: A Comprehensive Review

Shinojima A, Lee D, Tsubota K, Negishi K, Kurihara T†

J Clin Med. (Journal of Clinical Medicine) 10 ( 23 ) 5496 2021.12

Accepted

In recent years, the number of patients with age-related macular degeneration (AMD) is increasing worldwide along with increased life expectancy. Currently, the standard treatment for wet-AMD is intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs. The upstream of VEGF is hypoxia-inducible factor (HIF), a master regulator of hypoxia-responsive genes responsive to acute and chronic hypoxia. HIF activation induces various pathological pro-angiogenic gene expressions including VEGF under retinal hypoxia, ultimately leading to the development of ocular ischemic neovascular diseases. In this regard, HIF is considered as a promising therapeutic target in ocular ischemic diseases. In clinical ophthalmology, abnormal hypofluorescent areas have been detected in the late-phase of indocyanine green angiography, which are thought to be lipid deposits at the level of Bruch’s membrane to choriocapillaris in vitreoretinal diseases. These deposits may interfere with the oxygen and nutrients that should be supplied to the retinal pigment epithelium, and that HIF/VEGF is highly suspected to be expressed in the hypoxic retinal pigment epithelium, leading to neovascularization. In this review, we comprehensively summarize pathophysiology of AMD-related ocular diseases with the HIF/VEGF pathway from basic and clinic researches with recent findings.

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Association between ocular axial length and anthropometrics of Asian adults

Shinojima A, Kurihara T†, Mori K, Iwai Y, Hanyuda A, Negishi K, Torii H, Tsubota K†

BMC Res Notes. (BMC Research Notes) 14 ( 1 ) 328 - 328 2021.12

Accepted

OBJECTIVE: The purpose of this study is to analyze axial length, body height, hand length, and foot length to find new factors that predict myopia and to identify gender differences as one of the factors of high myopia. A cross-sectional study was conducted as a single observation. Body height, hand length, and foot length were measured according to standard anthropometric methods. Axial length, retinal thickness, and choroidal thickness were measured using the IOL Master 700 and the Heidelberg Spectralis-OCT. To account for body height differences among participants, foot length/body height and hand length/body height were analyzed using a mixed-effects model. RESULTS: A total of 80 eyes (men, n = 20, 40 eyes; women, n = 20, 40 eyes) were analyzed. The mean age was 33.5 years (range 21-59 years, SD: 9.6). For choroidal thickness, there was a significant association with axial length in men (p < 0.001) and a trend toward an association in women (p = 0.072). There was also a significant association between foot length/body height and axial length in men (p = 0.015), but not in women (p = 0.58). These results suggest that factors that determine body height and foot length may be related to axial length, although they vary by gender.

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PPARα Modulation-Based Therapy in Central Nervous System Diseases.

Lee D*, Tomita Y*, Allen W, Tsubota K, Negishi K, Kurihara T†

Life. (Life) 11 ( 11 ) 2021.11

Accepted

The burden of neurodegenerative diseases in the central nervous system (CNS) is increasing globally. There are various risk factors for the development and progression of CNS diseases, such as inflammatory responses and metabolic derangements. Thus, curing CNS diseases requires the modulation of damaging signaling pathways through a multitude of mechanisms. Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear hormone receptors (PPARα, PPARβ/δ, and PPARγ), and they work as master sensors and modulators of cellular metabolism. In this regard, PPARs have recently been suggested as promising therapeutic targets for suppressing the development of CNS diseases and their progressions. While the therapeutic role of PPARγ modulation in CNS diseases has been well reviewed, the role of PPARα modulation in these diseases has not been comprehensively summarized. The current review focuses on the therapeutic roles of PPARα modulation in CNS diseases, including those affecting the brain, spinal cord, and eye, with recent advances. Our review will enable more comprehensive therapeutic approaches to modulate PPARα for the prevention of and protection from various CNS diseases.

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Effect of Violet Light-Transmitting Eyeglasses on Axial Elongation in Myopic Children: A Randomized Controlled Trial

Mori K*, Torii H*, Hara Y, Hara M, Yotsukura E, Hanyuda A, Negishi K, Kurihara T†, Tsubota K†

J Clin Med. (Journal of Clinical Medicine) 10 ( 22 ) 5462 2021.11

Accepted

The fact that outdoor light environment is an important suppressive factor against myopia led us to invent violet light-transmitting eyeglasses (VL glasses) which can transmit violet light (VL), 360–400 nm in wavelength, for the suppression of myopia, and can meanwhile block harmful ultra-violet waves from sunlight. The current study is a double-blinded randomized clinical trial to investigate the myopia-suppressive effect of VL glasses compared to conventional eyeglasses (placebo glasses) that do not transmit VL. The subjects were children aged from 6 to 12 years old, the population in which myopia progression is generally accelerated, and the myopia suppressive effect was followed up for two years in a city in Japan. Periodical ophthalmic examinations, interviews, and measurements of reflection and axial length under mydriasis were performed at the initial visit (the baseline) and at 1, 6, 12, 18, and 24 months. The mean change in axial length in the VL glasses group was significantly smaller than in the placebo glasses group when time for near-work was less than 180 min and when the subjects were limited to those who had never used eyeglasses before this trial (p < 0.01); however, this change was not significant without subgrouping. The suppressive rate for axial elongation in the VL glasses group was 21.4% for two years.

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Updates on the Current Treatments for Diabetic Retinopathy and Possibility of Future Oral Therapy

Tomita Y*, Lee D*, Tsubota K, Negishi K, Kurihara T†

J Clin Med. (Journal of Clinical Medicine) 10 ( 20 ) 4666 2021.10

Accepted

Diabetic retinopathy (DR) is a complication of diabetes and one of the leading causes of vision loss worldwide. Despite extensive efforts to reduce visual impairment, the prevalence of DR is still increasing. The initial pathophysiology of DR includes damage to vascular endothelial cells and loss of pericytes. Ensuing hypoxic responses trigger the expression of vascular endothelial growth factor (VEGF) and other pro-angiogenic factors. At present, the most effective treatment for DR and diabetic macular edema (DME) is the control of blood glucose levels. More advanced cases require laser, anti-VEGF therapy, steroid, and vitrectomy. Pan-retinal photocoagulation for non-proliferative diabetic retinopathy (NPDR) is well established and has demonstrated promising outcomes for preventing the progressive stage of DR. Furthermore, the efficacy of laser therapies such as grid and subthreshold diode laser micropulse photocoagulation (SDM) for DME has been re-ported. Vitrectomy has been performed for vitreous hemorrhage and tractional retinal detachment for patients with PDR. In addition, anti-VEGF treatment has been widely used for DME, and re-cently its potential to prevent the progression of PDR has been remarked. Even with these treat-ments, many patients with DR lose their vision and suffer from potential side effects. Thus, we need alternative treatments to address these limitations. In recent years, the relationship between DR, lipid metabolism, and inflammation has been featured. Research in diabetic animal models points to peroxisome proliferator-activated receptor alpha (PPARα) activation in cellular metabolism and inflammation by oral fenofibrate and/or pemafibrate as a promising target for DR. In this paper, we review the status of existing therapies, summarize PPARα activation therapies for DR, and discuss their potentials as promising DR treatments.

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Shorter Axial Length Is a Risk Factor for Proliferative Vitreoretinopathy Grade C in Eyes Unmodified by Surgical Invasion.

Minami S, Uchida A, Nagai N, Shinoda H, Kurihara T, Ban N, Terasaki H, Takagi H, Tsubota K, Sakamoto T, Ozawa Y†

J Clin Med. (Journal of Clinical Medicine) 10 ( 17 ) 3944 2021.09

Accepted

PURPOSE: To investigate the risk factors for the development of proliferative vitreoretinopathy grade C (PVR-C), independent of prior surgical invasion. METHODS: Patients who underwent surgery for rhegmatogenous retinal detachment were prospectively registered with the Japan-Retinal Detachment Registry, organized by the Japanese Retina and Vitreous Society, between February 2016 and March 2017. Data obtained from general ophthalmic examinations performed before and at 1, 3, and 6 months after surgery were analyzed. RESULTS: We included 2013 eyes of 2013 patients (men, 1326 (65.9%); mean age, 55.2 ± 15.2 years) from amongst 3446 registered patients. Preoperative PVR-C was observed in 3.6% of patients. Propensity score matching revealed that a shorter axial length (AL) was a risk factor for preoperative PVR-C (OR (Odds Ratio), 0.81; 95% CI (Confidence Interval), 0.69 to 0.96; p = 0.015), which was a risk factor for surgical failure (OR, 4.22; 95% CI, 1.12 to 15.93; p = 0.034); the association was particularly significant for eyes with an AL < 25.0 mm (p = 0.016), while it was insignificant for eyes with an AL ≥ 25.0 mm. CONCLUSIONS: A shorter AL was related to the development of PVR-C before surgical invasion. Our results will help elucidate the fundamental pathogenesis of PVR and caution clinicians to meticulously examine eyes with a shorter AL to detect retinal detachment before PVR development.

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Pemafibrate Prevents Retinal Dysfunction in a Mouse Model of Unilateral Common Carotid Artery Occlusion

Lee D*, Tomita Y*, Jeong H, Miwa Y, Tsubota K, Negishi K, Kurihara T†

Int J Mol Sci. (MDPI AG) 22 ( 17 ) 9408 - 9408 2021.09

Accepted, ISSN 16616596

Cardiovascular diseases lead to retinal ischemia, one of the leading causes of blindness. Retinal ischemia triggers pathological retinal glial responses and functional deficits. Therefore, maintaining retinal neuronal activities and modulating pathological gliosis may prevent loss of vision. Previously, pemafibrate, a selective peroxisome proliferator-activated receptor alpha modulator, was nominated as a promising drug in retinal ischemia. However, a protective role of pemafibrate remains untouched in cardiovascular diseases-mediated retinal ischemia. Therefore, we aimed to unravel systemic and retinal alterations by treating pemafibrate in a new murine model of retinal ischemia caused by cardiovascular diseases. Adult C57BL/6 mice were orally administered pemafibrate (0.5 mg/kg) for 4 days, followed by unilateral common carotid artery occlusion (UCCAO). After UCCAO, pemafibrate was continuously supplied to mice until the end of experiments. Retinal function (a-and b-waves and the oscillatory potentials) was measured using electroretinography on day 5 and 12 after UCCAO. Moreover, the retina, liver, and serum were subjected to qPCR, immunohistochemistry, or ELISA analysis. We found that pemafibrate enhanced liver function, elevated serum levels of fibroblast growth factor 21 (FGF21), one of the neuroprotective molecules in the eye, and protected against UCCAO-induced retinal dysfunction, observed with modulation of retinal gliosis and preservation of oscillatory potentials. Our current data suggest a promising pemafibrate therapy for the suppression of retinal dysfunction in cardiovascular diseases.

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Glucose levels between the anterior chamber of the eye and blood are correlated based on blood glucose dynamics

Kurihara T*†, Lee D*, Shinojima A*, Kinoshita T, Nishizaki S, Arita Y, Hidaka Y, Nishi Y, Shirakawa Y, Kimura S, Tsuneyoshi Y, Torii H, Tsubota K, Negishi K†

PLOS ONE. (PLoS ONE) 16 ( 9 September ) e0256986 2021.09

Accepted

Glycemic control is essential to manage metabolic diseases such as diabetes. Frequent measurements of systemic glucose levels with prompt managements can prevent organ damages. The eye is a glucose highly demanding organ in our body, and the anterior chamber (AC) in the eye has been suggested for a noninvasive blood glucose monitoring site. However, calculating blood glucose levels from measuring glucose levels in AC has been difficult and unclear. In this study, we aimed to examine glucose levels from AC and find a correlation with blood glucose levels. A total of 30 patients with cataracts (men and women, study 1; 7 and 3, study 2; 9 and 11) who visited Keio University Hospital from 2015 to 2018 and agreed to participate in this study were recruited. Glucose levels from AC and the blood were examined by a UV-hexokinase or H2O2-electrode method before/during the cataract surgery. These values were analyzed with regression analyses depending on the groups (blood glucose-ascending and descending groups). In the blood glucose-descending group, glucose levels from AC were strongly correlated with blood glucose levels (a high R2 value, 0.8636). However, the relatively moderate correlation was seen in the blood glucose-ascending group (a low R2 value, 0.5228). Taken together, we showed different correlation ratios on glucose levels between AC and the blood, based on blood glucose dynamics. Stacking data regarding this issue would enable establishing noninvasive blood glucose monitoring from measuring glucose levels in AC more correctly, which will be helpful for proper and prompt managements for glucose-mediated complications.

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Inhibition of the HIF-1α/BNIP3 pathway has a retinal neuroprotective effect

Kunimi H*, Lee D*, Ibuki M, Katada Y, Negishi K, Tsubota K†, Kurihara T†

FASEB J. (FASEB Journal) 35 ( 8 ) e21829 2021.08

Accepted, ISSN 08926638

Retinal ischemia is a leading cause of irreversible blindness worldwide. Inner retinal dysfunction including loss of retinal ganglion cells is encountered in a number of retinal ischemic disorders. We previously reported administration of two different hypoxia-inducible factor (HIF) inhibitors exerted neuroprotective effects in a murine model of retinal ischemia/reperfusion (I/R) which mimics these disorders, as inner retinal degeneration could be involved in pathological HIF induction. However, this notion needs further investigation. Therefore, in this study, we attempted to use retina-specific Hif-1α conditional knockout (cKO) mice to uncover this notion more clearly under the same condition. Hif-1α cKO mice showed inner retinal neurodegeneration to a lesser extent than control mice. Hif-1α depletion in a murine 661W retinal cell line reduced cell death under pseudohypoxic and hypoxic conditions. Among hypoxia-related genes, the expression of BCL2 19 kDa protein-interacting protein 3 (Bnip3) was substantially upregulated in the inner retinal layer after retinal I/R. In this regard, we further examined Bnip3 depletion in retinal neurons in vitro and in vivo and found the similar neuroprotective effects. Our results support the notion that the HIF-1α/BNIP3 pathway may have a critical role in inner retinal neurodegeneration, which can be linked with the development of new promising therapeutics for inner retinal ischemic disorders.

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Retinal dysfunction induced in a mouse model of unilateral common carotid artery occlusion

Lee D*, Jeong H*, Miwa Y, Shinojima A, Katada Y, Tsubota K†, Kurihara T†

PeerJ. (PeerJ) 9 e11665 2021.06

Accepted

Background: Retinal ischemic stresses are associated with the pathogenesis of various retinal vascular diseases. To investigate pathological mechanisms of retinal ischemia, reproducible, robust and clinically significant experimental rodent models are highly needed. Previously, we established a stable murine model of chronic hypoperfusion retinal injuries by permanent unilateral common carotid artery occlusion (UCCAO) and demonstrated chronic pathological processes in the ischemic retina after the occlusion; however, retinal functional deficits and other acute retinal ischemic injuries by UCCAO still remain obscure. In this study, we attempted to examine retinal functional changes as well as acute retinal ischemic alterations such as retinal thinning, gliosis and cell death after UCCAO. Methods: Adult mice (male C57BL/6, 6-8 weeks old) were subjected to UCCAO in the right side, and retinal function was primarily measured using electroretinography for 14 days after the surgery. Furthermore, retinal thinning, gliosis and cell death were investigated using optical coherence tomography, immunohistochemistry and TUNEL assay, respectively. Results: Functional deficits in the unilateral right retina started to be seen 7 days after the occlusion. Specifically, the amplitude of b-wave dramatically decreased while that of a-wave was slightly affected. 14 days after the occlusion, the amplitudes of both waves and oscillatory potentials were significantly detected decreased in the unilateral right retina. Even though a change in retinal thickness was not dramatically observed among all the eyes, retinal gliosis and cell death in the unilateral right retina were substantially observed after UCCAO. Conclusions: Along with previous retinal ischemic results in this model, UCCAO can stimulate retinal ischemia leading to functional, morphological and molecular changes in the retina. This model can be useful for the investigation of pathological mechanisms for human ischemic retinopathies and furthermore can be utilized to test new drugs for various ischemic ocular diseases.

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Iris metastasis as the initial presentation of metastatic esophageal cancer diagnosed by fine needle aspiration biopsy: A case report

Ozawa H, Usui Y, Takano Y, Horiuchi N, Kuribayashi T, Kurihara T, Smith LEH, Tsubota K†, Tomita Y†

Medicine. (Medicine) 100 ( 22 ) e26232 2021.06

Accepted, ISSN 00257974

RATIONALE: Metastasis of neoplasms to the eye is quite uncommon. In this case report, we describe a patient where primary esophageal cancer was diagnosed by fine needle aspiration biopsy (FNAB) of an iris tumor. PATIENT CONCERNS: A 70-year-old male complained of redness and discomfort in the right eye. DIAGNOSIS AND INTERVENTIONS: The patient's right eye was diagnosed as idiopathic uveitis, and a topical steroid was administered. As vitreous opacities were observed even after topical therapy, oral prednisolone was administered. On slit-lamp examination of the right eye, an iris mass with neovascularization was seen in the anterior chamber. A metastatic tumor was suspected, and FNAB was performed. Histology revealed squamous cell carcinoma. Systemic workup revealed esophageal cancer with several metastases. Best-corrected visual acuity decreased to 20/400, and intraocular pressure was 40 mmHg in the right eye. Two iris tumors with neovascularization were present extending into the anterior chamber with posterior iris synechiae and 360 degree peripheral anterior synechiae. Intraocular pressure in the right eye was medically managed with hypotensive eye drops and oral acetazolamide. Iris metastases were treated with 40 Gray of radiation therapy and concurrent chemotherapy. OUTCOMES: The tumor regressed, but intraocular pressure was refractory to treatment because of 360 degree goniosynechial closure. The right eye lost light perception six months after treatment commenced, and the patient died 9 months after the onset of therapy due to multiple systemic metastases. LESSONS: This is a rare case of masquerade syndrome without systemic symptoms in which FNAB of an iris tumor led to a diagnosis of metastatic esophageal squamous cell carcinoma. Although the patient lost his sight due to uncontrollable ocular hypertension, systemic chemotherapy, and radiation therapy were initially effective in the treatment of the metastatic iris tumor. As the prognosis of patients with metastatic iris tumors is poor, it is important for ophthalmologists to consider such diagnoses and conduct systemic investigations when necessary.

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Violet light suppresses lens-induced myopia via neuropsin (OPN5) in mice

Jiang X*, Pardue MT*, Mori K, Ikeda SI, Torii H, D’Souza S, Lang RA, Kurihara T†, Tsubota K†

Proc Natl Acad Sci U S A. (Proceedings of the National Academy of Sciences) 118 ( 22 ) e2018840118 2021.06

Accepted, ISSN 00278424

Myopia has become a major public health concern, particularly across much of Asia. It has been shown in multiple studies that outdoor activity has a protective effect on myopia. Recent reports have shown that short-wavelength visible violet light is the component of sunlight that appears to play an important role in preventing myopia progression in mice, chicks, and humans. The mechanism underlying this effect has not been understood. Here, we show that violet light prevents lens defocus–induced myopia in mice. This violet light effect was dependent on both time of day and retinal expression of the violet light sensitive atypical opsin, neuropsin (OPN5). These findings identify <italic>Opn5</italic>-expressing retinal ganglion cells as crucial for emmetropization in mice and suggest a strategy for myopia prevention in humans.

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Assessment of Hypofluorescent Foci on Late-Phase Indocyanine Green Angiography in Central Serous Chorioretinopathy

Shinojima A*†, Ozawa Y*†, Uchida A, Nagai N, Shinoda H, Kurihara T, Suzuki M, Minami S, Negishi K, Tsubota K

J Clin Med. (Journal of Clinical Medicine) 10 ( 10 ) 2178 2021.05

Accepted

To assess the hypofluorescent foci (HFF) on late-phase indocyanine green angiography (ICGA) in central serous chorioretinopathy (CSC) using short-wavelength fundus autofluorescence (SW-FAF), near-infrared autofluorescence (NIR-AF), and fluorescein angiography (FA). The HFF area on late-phase ICGA for at least 20 min was compared with the area of abnormal foci on SW-FAF, NIR-AF, and FA. In 14 consecutive patients (12 men, including 1 with bilateral CSC; and 2 women with unilateral CSC), four kinds of images of 27 eyes were acquired. The mean age ± standard deviation (range) was 46 ± 9.2 years (31–69 years). The HFF on late-phase ICGA were found in 23 eyes (in all 15 CSC eyes and the contralateral 8 eyes). From the results of simple regression analysis, we obtained the following three formulas. The HFF area on ICGA = 1.058 × [abnormal SW-FAF area] + 0.135, the HFF area on ICGA = 1.001 × [abnormal NIR-AF area] + 0.015, and the HFF area on ICGA = 1.089 × [abnormal FA area] + 0.135. Compared to SW-FAF and FA, NIR-AF was found to be the easiest method to detect the HFF on late-phase ICGA, which may indicate melanin abnormalities, especially a decrease, in the retinal pigment epithelium.

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Efficacy and safety of 0.01% atropine for prevention of childhood myopia in a 2-year randomized placebo-controlled study

Hieda O, Hiraoka T, Fujikado T, Ishiko S, Hasebe S, Torii H, Takahashi H, Nakamura Y, Sotozono C, Oshika T, Morimoto T, Nishida K, Nishikawa N, Song YS, Tokutake T, Nishi Y, Shigeno Y, Kurihara T, Negishi K, Tsubota K, Ono M, Nakai T, Tan D, Tanaka S, Kinoshita S†, ATOM-J. Study Group

Jpn J Ophthalmol. (Japanese Journal of Ophthalmology) 65 ( 3 ) 315 - 325 2021.05

Accepted, ISSN 00215155

PURPOSE: Atropine eye drops prevent the progression of myopia, but their use has not been tested in the Japanese schoolchildren population. Here, we evaluate the efficacy and safety of 0.01% atropine eye drops for myopia control in Japanese children. STUDY DESIGN: Multicenter (7 university hospitals), randomized, double-masked, placebo-controlled trial. METHODS: Participants were 171 Japanese schoolchildren aged 6 to 12 years, with progressive myopia, spherical equivalence (SE) of -1.00 to -6.00 diopters (D), and astigmatism of ≤1.5 D. They were randomized to receive either 0.01% atropine (n=85) or placebo (n=86) eye drops once nightly OU for 24 months. Primary and secondary efficacy endpoints were changes in SE and axial length (AL), respectively, from baseline to month 24. RESULTS: Data from 168 subjects were analyzed. At month 24, compliance was similar in both groups (atropine: 83.3%; placebo: 85.7%). The least squares mean change in SE and AL from baseline were, respectively, -1.26 D (95% confidence interval [CI]: -1.35, -1.17) and 0.63 mm (0.59, 0.67) for atropine and -1.48 D (- 1.57, -1.39) and 0.77 mm (0.73, 0.81) for placebo. Inter-group differences were 0.22 D (95% CI: 0.09, 0.35; P < 0.001) for SE and - 0.14 mm (-0.20, -0.08; P < 0.001) for AL. Three patients experienced mild allergic conjunctivitis side effects, with no inter-group difference in incidence (atropine: 2.4%; 2/84 patients; placebo: 1.4%; 1/84 patients). CONCLUSION: With good compliance, 0.01% atropine is effective and safe for preventing the progression of childhood myopia.

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Fenofibrate Protects against Retinal Dysfunction in a Murine Model of Common Carotid Artery Occlusion-Induced Ocular Ischemia

Lee D*, Tomita Y*, Miwa Y, Jeong H, Mori K, Tsubota K†, Kurihara T†

Pharmaceuticals. (Pharmaceuticals) 14 ( 3 ) 223 2021.03

Accepted

Ocular ischemia is a common cause of blindness and plays a detrimental role in various diseases such as diabetic retinopathy, occlusion of central retinal arteries, and ocular ischemic syndrome. Abnormalities of neuronal activities in the eye occur under ocular ischemic conditions. Therefore, protecting their activities may prevent vision loss. Previously, peroxisome proliferator-activated receptor alpha (PPARα) agonists were suggested as promising drugs in ocular ischemia. However, the potential therapeutic roles of PPARα agonists in ocular ischemia are still unknown. Thus, we attempted to unravel systemic and ocular changes by treatment of fenofibrate, a well-known PPARα agonist, in a new murine model of ocular ischemia. Adult mice were orally administered fenofibrate (60 mg/kg) for 4 days once a day, followed by induction of ocular ischemia by unilateral common carotid artery occlusion (UCCAO). After UCCAO, fenofibrate was continuously supplied to mice once every 2 days during the experiment period. Electroretinography was performed to measure retinal functional changes. Furthermore, samples from the retina, liver, and blood were subjected to qPCR, Western blot, or ELISA analysis. We found that fenofibrate boosted liver function, increased serum levels of fibroblast growth factor 21 (FGF21), one of the neuroprotective molecules in the central nervous system, and protected against UCCAO-induced retinal dysfunction. Our current data suggest a promising fenofibrate therapy in ischemic retinopathies.

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Spatial Functional Characteristics of East Asian Patients with Occult Macular Dystrophy (Miyake disease); EAOMD Report No.2.

Yang L*, Joo K*, Tsunoda K, Kondo M, Fujinami-Yokokawa Y, Arno G, Pontikos N, Liu X, Nakamura N, Kurihara T, Tsubota K, Iwata T, Li H, Zou X, Wu S, Sun Z, Ahn, SJ, Kim MS, Mun YS, Park KH, Robson AG, Miyake Y, Woo SJ†, Sui R†, Fujinami K†

Am J Ophthalmol. (American Journal of Ophthalmology) 221 169 - 180 2021.01

Accepted, ISSN 00029394

PURPOSE: To describe the functional phenotypic features of East Asian patients with RP1L1-associated occult macular dystrophy (i.e., Miyake disease). DESIGN: An international multi-center retrospective cohort study. METHODS: Twenty-eight participants (53 eyes) with Miyake disease were enrolled at three centres: in Japan, China, and Korea. Ophthalmological examinations including spectral-domain optic coherence tomography (SD-OCT) and multifocal electroretinogram (mfERG) were performed. Patients were classified into three functional groups based on mfERG: Group 1, paracentral dysfunction with relatively preserved central/peripheral function; Group 2, homogeneous central dysfunction with preserved peripheral function; and Group 3, widespread dysfunction over the recorded area. Three functional phenotypes were compared in clinical parameters and SD-OCT morphological classification (severe phenotype, blurred/flat ellipsoid zone and absence of the interdigitation zone; mild phenotype, preserved ellipsoid zone). RESULTS: There were eight eyes in Group 1, 40 eyes in Group 2, and five eyes in Group 3. The patients in Group 1 showed significantly later onset (P=.005) and shorter disease duration (P=.002), compared with those in Group 2. All eight eyes in Group 1 showed the mild morphological phenotype, while 43/45 eyes in Groups 2 and 3 presented the severe phenotype, which identified a significant association between the functional grouping and the morphological classification (P<.001). CONCLUSIONS: A spectrum of functional phenotypes of Miyake disease was first documented with identifying three functional subtypes. Patients with paracentral dysfunction had the mildest phenotype, and those with homogeneous central or widespread dysfunction showed overlapping clinical findings with severe photoreceptor changes, suggesting various extents of visual impairment.

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Axial Length and Prevalence of Myopia among Schoolchildren in the Equatorial Region of Brazil

Yotsukura E*, Torii H*, Ozawa H*, Hida RY, Shiraishi T, Corso Teixeira I, Rautha YVBL, Moraes do, Nascimento CF, Mori K, Uchino M, Kurihara T†, Negishi K†, Tsubota K†

J Clin Med. (Journal of Clinical Medicine) 10 ( 1 ) 1 - 11 2021.01

Accepted

The prevalence of myopia is increasing globally, and the outdoor light environment is considered as a possible factor that can retard myopia. The aim of this study was to evaluate the prevalence of myopia and the light environment in Aracati, equatorial Brazil. We surveyed 421 children (421 right eyes; mean age, 10.6 years) and performed ocular examinations that included non-cycloplegic refraction and axial length (AL). Multiple regression analyses were performed to identify factors affecting myopia such as time spent outdoors and in near work. We measured illuminance and violet light irradiance in Aracati. The mean spherical equivalent (SE) and AL were -0.44 ± 1.38 diopters (D) and 22.98 ± 0.87 mm, respectively. The prevalence of myopia (SE ≤ -0.75 D) and high myopia (SE ≤ -6.0 D/AL ≥ 26.0 mm) was 20.4 and 1.4/0.48%, respectively. Multiple regression analyses showed that myopia was not associated with lifestyle factors. The average illuminance in Aracati was about 100,000 lux from morning to evening. The current results reflect the ALs and the prevalence of myopia among Brazilian schoolchildren. There is a possibility that the light environment in addition to other confounding factors including racial differences affects the ALs and refractive errors.

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Prediction of causative genes in inherited retinal disorder from fundus photography and autofluorescence imaging using deep learning techniques

Fujinami-Yokokawa Y, Ninomiya H, Liu X, Yang L, Pontikos N, Yoshitake K, Iwata T, Sato Y, Hashimoto T, Tsunoda K, Miyata H, Fujinami K†

Br J Ophthalmol. (BMJ) bjophthalmol-2020-318544 2021

Accepted, ISSN 00071161

<sec><title>Background/Aims</title>To investigate the utility of a data-driven deep learning approach in patients with inherited retinal disorder (IRD) and to predict the causative genes based on fundus photography and fundus autofluorescence (FAF) imaging.

</sec><sec><title>Methods</title>Clinical and genetic data from 1302 subjects from 729 genetically confirmed families with IRD registered with the Japan Eye Genetics Consortium were reviewed. Three categories of genetic diagnosis were selected, based on the high prevalence of their causative genes: Stargardt disease (<italic>ABCA4</italic>), retinitis pigmentosa (<italic>EYS</italic>) and occult macular dystrophy (<italic>RP1L1</italic>). Fundus photographs and FAF images were cropped in a standardised manner with a macro algorithm. Images for training/testing were selected using a randomised, fourfold cross-validation method. The application program interface was established to reach the learning accuracy of concordance (target: >80%) between the genetic diagnosis and the machine diagnosis (<italic>ABCA4</italic>, <italic>EYS</italic>, <italic>RP1L1</italic> and normal).

</sec><sec><title>Results</title>A total of 417 images from 156 Japanese subjects were examined, including 115 genetically confirmed patients caused by the three prevalent causative genes and 41 normal subjects. The mean overall test accuracy for fundus photographs and FAF images was 88.2% and 81.3%, respectively. The mean overall sensitivity/specificity values for fundus photographs and FAF images were 88.3%/97.4% and 81.8%/95.5%, respectively.

</sec><sec><title>Conclusion</title>A novel application of deep neural networks in the prediction of the causative IRD genes from fundus photographs and FAF, with a high prediction accuracy of over 80%, was highlighted. These achievements will extensively promote the quality of medical care by facilitating early diagnosis, especially by non-specialists, access to care, reducing the cost of referrals, and preventing unnecessary clinical and genetic testing.

</sec>

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Retinal Degeneration in a Murine Model of Retinal Ischemia by Unilateral Common Carotid Artery Occlusion

Lee D, Nakai A, Miwa Y, Tomita Y, Serizawa N, Katada Y, Hatanaka Y, Tsubota K, Negishi K, Kurihara T†

Biomed Res Int. (Hindawi Limited) 2021 7727648 2021

Accepted, ISSN 23146133

Retinal degeneration is a progressive retinal damage in ocular vascular diseases. There are several reasons for this, such as occlusion of arteries or veins, diabetic retinopathy, or hereditary retinal diseases. To study pathological mechanisms of retinal degeneration, it is required to develop experimentally reproducible and clinically relevant models. In our previous studies, we developed a murine model of retinal hypoperfusion by unilateral common carotid artery occlusion (UCCAO) which mimics the pathophysiology of ocular ischemic syndrome (OIS) in humans, and described broad pathological mechanisms in the retina after UCCAO. However, there still remain missing pieces of the ocular pathologic process by UCCAO. In this study, we examined those unfound mechanisms. UCCAO was performed on adult mice. Ocular dysfunctions, histological deficits, and inflammation were examined after UCCAO, compared with sham-operated mice. Evaluation values were analyzed by electrophysiological, histological, and molecular biological methods. Eyelid drooping was permanently seen after UCCAO. Induction time point of acute reversible cataract under anesthesia was shortened. Retinal/visual dysfunctions were detected 2-4 weeks after UCCAO. Specifically, scotopic b-wave was more affected than a-wave, with the dysfunction of photopic b-wave. Impaired oscillatory potentials and visual evoked potential were constantly observed. Pathological Müller gliosis/inflammation was featured with NeuN-positive cell loss in the ganglion cell layer. Axial length, intraocular pressure, pupillary light reflex, and retinal pigment epithelium/choroidal thickness were not changed by UCCAO. A murine model of retinal ischemia by UCCAO can be useful for studying a series of degenerative process in the ischemic retina.

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Rice Bran and Vitamin B6 Suppress Pathological Neovascularization in a Murine Model of Age-Related Macular Degeneration as Novel HIF Inhibitors

Ibuki M*, Lee D*, Shinojima A*, Miwa Y, Tsubota K†, Kurihara T†

Int J Mol Sci. (MDPI AG) 21 ( 23 ) 1 - 24 2020.12

Accepted, ISSN 16616596

Pathological neovascularization in the eye is a leading cause of blindness in all age groups from retinopathy of prematurity (ROP) in children to age-related macular degeneration (AMD) in the elderly. Inhibiting neovascularization via antivascular endothelial growth factor (VEGF) drugs has been used for the effective treatment. However, anti-VEGF therapies may cause development of chorioretinal atrophy as they affect a physiological amount of VEGF essential for retinal homeostasis. Furthermore, anti-VEGF therapies are still ineffective in some cases, especially in patients with AMD. Hypoxia-inducible factor (HIF) is a strong regulator of VEGF induction under hypoxic and other stress conditions. Our previous reports have indicated that HIF is associated with pathological retinal neovascularization in murine models of ROP and AMD, and HIF inhibition suppresses neovascularization by reducing an abnormal increase in VEGF expression. Along with this, we attempted to find novel effective HIF inhibitors from natural foods of our daily lives. Food ingredients were screened for prospective HIF inhibitors in ocular cell lines of 661W and ARPE-19, and a murine AMD model was utilized for examining suppressive effects of the ingredients on retinal neovascularization. As a result, rice bran and its component, vitamin B6 showed inhibitory effects on HIF activation and suppressed VEGF mRNA induction under a CoCl2-induced pseudo-hypoxic condition. Dietary supplement of these significantly suppressed retinal neovascularization in the AMD model. These data suggest that rice bran could have promising therapeutic values in the management of pathological ocular neovascularization.

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Relationship between nerve fiber layer defect and the presence of epiretinal membrane in a Japanese population: The JPHC-NEXT Eye Study.

Uchida A, Sasaki M†, Motomura K, Yuki K, Kurihara T, Tomita Y, Ozawa Y, Yamagishi K†, Kawasaki R, Hanyuda A, Sawada N, Tsubota K, Tsugane S, Iso H

Sci Rep. (Scientific Reports) 10 ( 1 ) 779 - 779 2020.12

Accepted

The study subjects were residents of Chikusei city, Japan, aged 40 years or older who attended annual health check-up programs and participated in the JPHC-NEXT Eye Study which performed non-mydriatic fundus photography of both eyes. The relationship of glaucomatous fundus changes such as optic disc cupping (cup to disc ratio ≥ 0.7) and retinal nerve fiber layer defect (NFLD) with the presence of epiretinal membrane (ERM) were examined cross-sectionally. A total of 1990 persons gave consent to participate in this study in 2013. The overall prevalence of ERM was 12.9%. Of these, 1755 had fundus photographs of sufficient quality and no history of intraocular surgery (mean age: 62.3 ± 10.0 years). After adjusting for age, sex and refractive error, NFLD was positively associated with the presence of ERM (odds ratio [OR]: 2.48; 95% confidence interval [CI]: 1.24, 4.96; P = 0.010), but optic disc cupping was not (OR: 1.33; CI: 0.71, 2.48; P = 0.37). The results did not necessarily suggest an association between glaucoma and ERM, but indicated an association between NFLD and ERM.

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Genetic Spectrum of EYS-associated Retinal Disease in a Large Japanese Cohort: Identification of Disease-associated Variants with Relatively High Allele Frequency.

Yang L*, Fujinami K*†, Ueno S, Kuniyoshi K, Hayashi T, Kondo M, Mizota A, Naoi N, Shinoda K, Kameya S, Fujinami-Yokokawa Y, Liu X, Arno G, Pontikos N, Kominami T, Terasaki H, Sakuramoto H, Katagiri S, Mizobuchi K, Nakamura N, Mawatari G, Kurihara T, Tsubota K, Miyake Y, Yoshitake K, Iwata T, Tsunoda K

Sci Rep. (Scientific Reports) 10 ( 1 ) 5497 - 5497 2020.12

Accepted

Biallelic variants in the EYS gene are a major cause of autosomal recessive inherited retinal disease (IRD), with a high prevalence in the Asian population. The purpose of this study was to identify pathogenic EYS variants, to determine the clinical/genetic spectrum of EYS-associated retinal disease (EYS-RD), and to discover disease-associated variants with relatively high allele frequency (1%-10%) in a nationwide Japanese cohort. Sixty-six affected subjects from 61 families with biallelic or multiple pathogenic/disease-associated EYS variants were ascertained by whole-exome sequencing. Three phenotype groups were identified in EYS-RD: retinitis pigmentosa (RP; 85.94%), cone-rod dystrophy (CORD; 10.94%), and Leber congenital amaurosis (LCA; 3.12%). Twenty-six pathogenic/disease-associated EYS variants were identified, including seven novel variants. The two most prevalent variants, p.(Gly843Glu) and p.(Thr2465Ser) were found in 26 and twelve families (42.6%, 19.7%), respectively, for which the allele frequency (AF) in the Japanese population was 2.2% and 3.0%, respectively. These results expand the phenotypic and genotypic spectrum of EYS-RD, accounting for a high proportion of EYS-RD both in autosomal recessive RP (23.4%) and autosomal recessive CORD (9.9%) in the Japanese population. The presence of EYS variants with relatively high AF highlights the importance of considering the pathogenicity of non-rare variants in relatively prevalent Mendelian disorders.

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Clinical and Genetic Characteristics of 18 Patients from 13 Japanese Families with CRX-associated retinal disorder: Identification of Genotype-phenotype Association.

Fujinami-Yokokawa Y, Fujinami K†, Kuniyoshi K, Hayashi T, Ueno S, Mizota A, Shinoda K, Arno G, Pontikos N, Yang L, Liu X, Sakuramoto H, Katagiri S, Mizobuchi K, Kominami T, Terasaki H, Nakamura N, Kameya S, Yoshitake K, Miyake Y, Kurihara T, Tsubota K, Miyata H, Iwata T, Tsunoda K

Sci Rep. (Scientific Reports) 10 ( 1 ) 9531 - 9531 2020.12

Accepted

Inherited retinal disorder (IRD) is a leading cause of blindness, and CRX is one of a number of genes reported to harbour autosomal dominant (AD) and recessive (AR) causative variants. Eighteen patients from 13 families with CRX-associated retinal disorder (CRX-RD) were identified from 730 Japanese families with IRD. Ophthalmological examinations and phenotype subgroup classification were performed. The median age of onset/latest examination was 45.0/62.5 years (range, 15-77/25-94). The median visual acuity in the right/left eye was 0.52/0.40 (range, -0.08-2.00/-0.18-1.70) logarithm of the minimum angle of resolution (LogMAR) units. There was one family with macular dystrophy, nine with cone-rod dystrophy (CORD), and three with retinitis pigmentosa. In silico analysis of CRX variants was conducted for genotype subgroup classification based on inheritance and the presence of truncating variants. Eight pathogenic CRX variants were identified, including three novel heterozygous variants (p.R43H, p.P145Lfs*42, and p.P197Afs*22). A trend of a genotype-phenotype association was revealed between the phenotype and genotype subgroups. A considerably high proportion of CRX-RD in ADCORD was determined in the Japanese cohort (39.1%), often showing the mild phenotype (CORD) with late-onset disease (sixth decade). Frequently found heterozygous missense variants located within the homeodomain underlie this mild phenotype. This large cohort study delineates the disease spectrum of CRX-RD in the Japanese population.

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Oral Bovine Milk Lactoferrin Administration Suppressed Myopia Development through Matrix Metalloproteinase 2 in a Mouse Model

Ikeda SI*, Kurihara T†, Toda M, Jiang X, Torii H, Tsubota K†

Nutrients. (MDPI AG) 12 ( 12 ) 1 - 9 2020.12

Accepted

Recent studies have reported an association between myopia development and local ocular inflammation. Lactoferrin (LF) is an iron-binding protein present in saliva, tears, and mother’s milk. Furthermore, sequestering iron by LF can cause its antibacterial property. Moreover, LF has an anti-inflammatory effect. We aimed to determine the suppressive effect of LF against the development and progress of myopia using a murine lens-induced myopia (LIM) model. We divided male C57BL/6J mice (3 weeks old) into two groups. While the experimental group was orally administered LF (1600 mg/kg/day, from 3-weeks-old to 7-weeks-old), a similar volume of Ringer’s solution was administered to the control group. We subjected the 4-week-old mice to −30 diopter lenses and no lenses on the right and left eyes, respectively. We measured the refraction and the axial length at baseline and 3 weeks after using a refractometer and a spectral domain optical coherence tomography (SD-OCT) system in both eyes. Furthermore, we determined the matrix metalloproteinase-2 (MMP-2) activity, and the amount of interleukin-6 (IL-6), MMP-2, and collagen 1A1 in the choroid or sclera. The eyes with a minus lens showed a refractive error shift and an axial length elongation in the control group, thus indicating the successful induction of myopia. However, there were no significant differences in the aforementioned parameters in the LF group. While LIM increased IL-6 expression and MMP-2 activity, it decreased collagen 1A1 content. However, orally administered LF reversed these effects. Thus, oral administration of LF suppressed lens-induced myopia development by modifying the extracellular matrix remodeling through the IL-6–MMP-2 axis in mice.

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Efficacy of the Newly Invented Eyelid Clamper in Ultra-Widefield Fundus Imaging

Ozawa N*, Mori K*, Katada Y, Tsubota K†, Kurihara T†

Life. (Life) 10 ( 12 ) 1 - 7 2020.12

Accepted

BACKGROUND: Ultra-widefield fundus imaging is widely used for obtaining wide angle images of the retina in one single image. Although it has a potential to obtain a wide area of retinal photographs, images are often obstructed by eyelashes or eye lids. In this study, we used a newly invented eyelid clamper, which can keep an eye open without touching conjunctiva or lid margin, to assess the efficacy in clinical use by comparing with conventional tape fixation. METHODS: Ultra-widefield fundus images were captured with an ultra-widefield imaging system in 19 patients who visited to the outpatient clinic of Department of Ophthalmology, Keio University Hospital with the eyelid clamper or a conventional tape fixation. The area of imaged retinas was outlined and quantified with pixels. After obtaining images, patients answered a questionnaire. RESULTS: The average number of pixels in total areas with the eyelid clamper or with tape fixation were 4.31 ± 0.35 and 4.32 ± 0.34 mega pixels, respectively, showing no significant difference between the groups (p = 0.889). The average face pain scale of the eyelid clamper was 1.13 on a scale of 0 to 5. The number of patients who did not feel any pain was nine (47.4%). CONCLUSIONS: The eyelid clamper can be applied in clinical setting and can better support obtaining sufficiently wide fundus images compared to a conventional tape fixation.

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Axial length shortening in a myopic child with anisometropic amblyopia after wearing violet light-transmitting eyeglasses for 2 years

Ofuji Y*, Torii H*, Yotsukura E, Mori K, Kurihara T, Negishi K, Tsubota K†

Am J Ophthalmol Case Rep. (Elsevier BV) 20 101002 2020.12

Accepted, ISSN 24519936

© 2020 The Authors Purpose: To report a case in which the axial length (AL) shortened and the choroid thickened due to the use of violet light-transmitting eyeglasses. Observations: A 4-year-old boy with high myopia was referred to Keio University Hospital. He was prescribed standard eyeglasses. Six months after the first visit, his best-corrected visual acuities were 1.2 and 0.4 in the right and left eyes, respectively, with the standard eyeglasses, and he was diagnosed with anisometropic amblyopia. The right eye then was patched for 6 hours daily during the daytime. Because of the availability of violet light-transmitting eyeglasses, we changed the eyeglasses and instructed his parents to have him engage in outdoor activities for over 2 hours daily to be exposed to sufficient violet light. As a result, the violet light entered his left eye and minimal violet light entered his right eye. The changes in the ALs, choroidal thicknesses, and cycloplegic objective refractions in the right and left eyes during 2 years of wearing violet light-transmitting eyeglasses were +0.85 and −0.20 mm, +4.9 and + 115.7 μm, and −1.02 and + 1.88 D, respectively. Conclusions and importance: We successfully described a case in which the myopia improved, the AL shortened, and the choroid thickened after using violet light-transmitting eyeglasses.

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Automatic screening for diabetic retinopathy in interracial fundus images using artificial intelligence

Katada Y*, Ozawa N*, Masayoshi K, Ofuji Y, Tsubota K†, Kurihara T†

Intell Based Med. (Elsevier BV) 3-4 100024 2020.12

Accepted, ISSN 2666-5212

The development of deep learning technology and high-performance computers in recent years has enabled significant improvement in the accuracy of image recognition, and efforts are currently being made toward the development of ophthalmic artificial intelligence (AI). In this study, deep learning was applied to fundus image analysis. The fundus images were automatically classified by their severity index and the recognition accuracies for Japanese and American data were compared. We trained a deep convolutional neural network and a support vector machine using a data set consisting of 35,126 open-source American clinical fundus images, which were graded for diabetic retinopathy by licensed ophthalmologists. The grading accuracy of the trained AI model was evaluated by comparison with its performance for 200 Japanese fundus images obtained at Keio University Hospital. The trained AI model exhibited a sensitivity of 81.5% and specificity of 71.9% for the American validation data set, and a sensitivity of 90.8% and specificity of 80.0% for the Japanese data set. This indicates that the proposed AI program developed using American image data can be applied to fundus photographs of Japanese subjects and thus represents an interracial screening model. It can be used for screening tests and applied to telemedicine systems.

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A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Lee D*, Miwa Y*, Jeong H, Ikeda SI, Katada Y, Tsubota K†, KuriharaT†

J Vis Exp. (Journal of Visualized Experiments) 2020 ( 165 ) 1 - 19 2020.11

Accepted, ISSN 1940087X

© 2020 JoVE Journal of Visualized Experiments. Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

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PPARα Agonist Oral Therapy in Diabetic Retinopathy

Tomita Y*, Lee D*, Tsubota K†, Kurihara T†

Biomedicines. (MDPI AG) 8 ( 10 ) 1 - 18 2020.10

Accepted

Diabetic retinopathy (DR) is an eye condition that develops after chronically poorly-managed diabetes, and is presently the main cause for blindness on a global scale. Current treatments for DR such as laser photocoagulation, topical injection of corticosteroids, intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents and vitreoretinal surgery are only applicable at the late stages of DR and there are possibilities of significant adverse effects. Moreover, the forms of treatment available for DR are highly invasive to the eyes. Safer and more effective pharmacological treatments are required for DR treatment, in particular at an early stage. In this review, we cover recently investigated promising oral pharmacotherapies, the methods of which are safer, easier to use, patient-friendly and pain-free, in clinical studies. We especially focus on peroxisome proliferator-activator receptor alpha (PPARα) agonists in which experimental evidence suggests PPARα activation may be closely related to the attenuation of vascular damages, including lipid-induced toxicity, inflammation, an excess of free radical generation, endothelial dysfunction and angiogenesis. Furthermore, oral administration of selective peroxisome proliferator-activated receptor alpha modulator (SPPARMα) agonists may induce hepatic fibroblast growth factor 21 expression, indirectly resulting in retinal protection in animal studies. Our review will enable more comprehensive approaches for understanding protective roles of PPARα for the prevention of DR development.

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

Sato M, Minami S, Nagai N, Suzuki M, Kurihara T, Shinojima A, Sonobe H, Akino K, Ban N, Watanabe K, Uchida A, Shinoda H, Tsubota K, Ozawa Y†

PLOS ONE. (PLoS ONE) 15 ( 10 ) e0240357 2020.10

Accepted

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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A Fairy Chemical Suppresses Retinal Angiogenesis as a HIF Inhibitor

Lee D*, Miwa Y*, Wu J, Shoda C, Jeong H, Kawagishi H, Tsubota K†, Kurihara T†

Biomolecules. (MDPI AG) 10 ( 10 ) 1 - 14 2020.10

Accepted

Neovascular retinal degeneration is a leading cause of blindness in advanced countries. Anti-vascular endothelial growth factor (VEGF) drugs have been used for neovascular retinal diseases; however, anti-VEGF drugs may cause the development of chorioretinal atrophy in chronic therapy as they affect the physiological amount of VEGF needed for retinal homeostasis. Hypoxia-inducible factor (HIF) is a transcription factor inducing VEGF expression under hypoxic and other stress conditions. Previously, we demonstrated that HIF was involved with pathological retinal angiogenesis in murine models of oxygen-induced retinopathy (OIR), and pharmacological HIF inhibition prevented retinal neovascularization by reducing an ectopic amount of VEGF. Along with this, we attempted to find novel effective HIF inhibitors. Compounds originally isolated from mushroom-forming fungi were screened for prospective HIF inhibitors utilizing cell lines of 3T3, ARPE-19 and 661W. A murine OIR model was used to examine the anti-angiogenic effects of the compounds. As a result, 2-azahypoxanthine (AHX) showed an inhibitory effect on HIF activation and suppressed Vegf mRNA upregulation under CoCl2-induced pseudo-hypoxic conditions. Oral administration of AHX significantly suppressed retinal neovascular tufts in the OIR model. These data suggest that AHX could be a promising anti-angiogenic agent in retinal neovascularization by inhibiting HIF activation.

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High-sensitivity vision restoration via ectopic expression of chimeric rhodopsin in mice

Katada Y, Yoshida K, Kobayashi K, Okano H, Kandori H, Tsubota K†, Kurihara T†

bioRxiv. (Cold Spring Harbor Laboratory) 2020.09

Last author, Corresponding author

<title>Abstract</title>Photoreception requires amplification by mammalian rhodopsin through G protein activation, which requires a visual cycle. To achieve this in retinal gene therapy, we incorporated human rhodopsin cytoplasmic loops into <italic>Gloeobacter</italic> rhodopsin, thereby generating <italic>Gloeobacter</italic> and human chimeric rhodopsin (GHCR). In a murine model of inherited retinal degeneration, we induced retinal GHCR expression by intravitreal injection of a recombinant adeno-associated virus vector. Retinal explant and visual thalamus electrophysiological recordings, behavioral tests, and histological analysis showed that GHCR restored dim-environment vision and prevented the progression of retinal degeneration. Thus, GHCR may be a potent clinical tool for the treatment of retinal disorders.

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RP2‐associated retinal disorder in a Japanese cohort: Report of novel variants and a literature review, identifying a genotype–phenotype association.

Fujinami K*†, Liu X*, Ueno S, Mizota A, Shinoda K, Kuniyoshi K, Fujinami-Yokokawa Y, Yang L, Arno G, Pontikos N, Kameya S, Kominami T, Terasaki H, Sakuramoto H, Nakamura N, Kurihara T, Tsubota K, Miyake Y, Yoshiake K, Iwata T, Tsunoda K

Am J Med Genet C Semin Med Genet. (Wiley) 184 ( 3 ) 675 - 693 2020.09

Accepted, ISSN 15524868

© 2020 The Authors. American Journal of Medical Genetics Part C: Seminars in Medical Genetics published by Wiley Periodicals LLC. The retinitis pigmentosa 2 (RP2) gene is one of the causative genes for X-linked inherited retinal disorder. We characterized the clinical/genetic features of four patients with RP2-associated retinal disorder (RP2-RD) from four Japanese families in a nationwide cohort. A systematic review of RP2-RD in the Japanese population was also performed. All four patients were clinically diagnosed with retinitis pigmentosa (RP). The mean age at examination was 36.5 (10–47) years, and the mean visual acuity in the right/left eye was 1.40 (0.52–2.0)/1.10 (0.52–1.7) in the logarithm of the minimum angle of resolution unit, respectively. Three patients showed extensive retinal atrophy with macular involvement, and one had central retinal atrophy. Four RP2 variants were identified, including two novel missense (p.Ser6Phe, p.Leu189Pro) and two previously reported truncating variants (p.Arg120Ter, p.Glu269CysfsTer3). The phenotypes of two patients with truncating variants were more severe than the phenotypes of two patients with missense variants. A systematic review revealed additional 11 variants, including three missense and eight deleterious (null) variants, and a statistically significant association between phenotype severity and genotype severity was revealed. The clinical and genetic spectrum of RP2-RD was illustrated in the Japanese population, identifying the characteristic features of a severe form of RP with early macular involvement.

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Pemafibrate Protects Against Retinal Dysfunction in a Murine Model of Diabetic Retinopathy.

Tomita Y*, Lee D*, Miwa Y, Jiang X, Ohta M, Tsubota K†, Kurihara T†

Int J Mol Sci. (International Journal of Molecular Sciences) 21 ( 17 ) 1 - 17 2020.09

Accepted, ISSN 16616596

© 2020, MDPI AG. All rights reserved. Diabetic retinopathy (DR) is one of the leading causes of blindness globally. Retinal neuronal abnormalities occur in the early stage in DR. Therefore, maintaining retinal neuronal activity in DR may prevent vision loss. Previously, pemafibrate, a novel selective peroxisome proliferator-activated receptor alpha modulator, was suggested as a promising drug in hypertriglyceridemia. However, the role of pemafibrate remains obscure in DR. Therefore, we aimed to unravel systemic and retinal changes by pemafibrate in diabetes. Adult mice were intraperitoneally injected with streptozotocin (STZ) to induce diabetes. After STZ injection, diet supplemented with pemafibrate was given to STZ-induced diabetic mice for 12 weeks. During the experiment period, body weight and blood glucose levels were examined. Electroretinography was performed to check the retinal neural function. After sacrifice, the retina, liver, and blood samples were subjected to molecular analyses. We found pemafibrate mildly improved blood glucose level as well as lipid metabolism, boosted liver function, increased serum fibroblast growth factor21 level, restored retinal functional deficits, and increased retinal synaptophysin protein expression in STZ-induced diabetic mice. Our present data suggest a promising pemafibrate therapy for the prevention of early DR by improving systemic metabolism and protecting retinal function.

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Clinical and genetic characteristics of Stargardt disease in a large Western China cohort: Report 1.

Liu X, Meng X, Yang L, Long Y, Fujinami-Yokokawa Y, Ren J, Kurihara T, Tsubota K, Tsunoda K, Fujinami K†, Li S†

Am J Med Genet C Semin Med Genet. (American Journal of Medical Genetics, Part C: Seminars in Medical Genetics) 184 ( 3 ) 694 - 707 2020.09

Accepted, ISSN 15524868

© 2020 The Authors. American Journal of Medical Genetics Part C: Seminars in Medical Genetics published by Wiley Periodicals LLC. Stargardt disease 1 (STGD1) is the most prevalent retinal dystrophy caused by pathogenic biallelic ABCA4 variants. Forty-two unrelated patients mostly originating from Western China were recruited. Comprehensive ophthalmological examinations, including visual acuity measurements (subjective function), fundus autofluorescence (retinal imaging), and full-field electroretinography (objective function), were performed. Next-generation sequencing (target/whole exome) and direct sequencing were conducted. Genotype grouping was performed based on the presence of deleterious variants. The median age of onset/age was 10.0 (5–52)/29.5 (12–72) years, and the median visual acuity in the right/left eye was 1.30 (0.15–2.28)/1.30 (0.15–2.28) in the logarithm of the minimum angle of resolution unit. Ten patients (10/38, 27.0%) showed confined macular dysfunction, and 27 (27/37, 73.7%) had generalized retinal dysfunction. Fifty-eight pathogenic/likely pathogenic ABCA4 variants, including 14 novel variants, were identified. Eight patients (8/35, 22.8%) harbored multiple deleterious variants, and 17 (17/35, 48.6%) had a single deleterious variant. Significant associations were revealed between subjective functional, retinal imaging, and objective functional groups, identifying a significant genotype–phenotype association. This study illustrates a large phenotypic/genotypic spectrum in a large well-characterized STGD1 cohort. A distinct genetic background of the Chinese population from the Caucasian population was identified; meanwhile, a genotype–phenotype association was similarly represented.

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Clinical and genetic characteristics of 10 Japanese patients with PROM1-associated retinal disorder: A report of the phenotype spectrum and a literature review in the Japanese population.

Fujinami K*†, Oishi A*, Yang L, Arno G, Pontikos N, Yoshitake K, Fujinami, Yokokawa Y, Liu X, Hayashi T, Katagiri S, Mizobuchi K, Mizota A, Shinoda K, Nakamura N, Kurihara T, Tsubota K, Miyake Y, Iwata T, Tsujikawa A, Tsunoda K

Am J Med Genet C Semin Med Genet. (American Journal of Medical Genetics, Part C: Seminars in Medical Genetics) 184 ( 3 ) 656 - 674 2020.09

Accepted, ISSN 15524868

Variants in the PROM1 gene are associated with cone (-rod) dystrophy, macular dystrophy, and other phenotypes. We describe the clinical and genetic characteristics of 10 patients from eight Japanese families with PROM1-associated retinal disorder (PROM1-RD) in a nationwide cohort. A literature review of PROM1-RD in the Japanese population was also performed. The median age at onset/examination of 10 patients was 31.0 (range, 10-45)/44.5 (22-73) years. All 10 patients showed atrophic macular changes. Seven patients (70.0%) had spared fovea to various degrees, approximately half of whom had maintained visual acuity. Generalized cone (-rod) dysfunction was demonstrated in all nine subjects with available electrophysiological data. Three PROM1 variants were identified in this study: one recurrent disease-causing variant (p.Arg373Cys), one novel putative disease-causing variant (p.Cys112Arg), and one novel variant of uncertain significance (VUS; p.Gly53Asp). Characteristic features of macular atrophy with generalized cone-dominated retinal dysfunction were shared among all 10 subjects with PROM1-RD, and the presence of foveal sparing was crucial in maintaining visual acuity. Together with the three previously reported variants [p.R373C, c.1551+1G>A (pathogenic), p.Asn580His (likely benign)] in the literature of Japanese patients, one prevalent missense variant (p.Arg373Cys, 6/9 families, 66.7%) detected in multiple studies was determined in the Japanese population, which was also frequently detected in the European population.

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Closure of macular hole secondary to ischemic hemi-central retinal vein occlusion by retinal photocoagulation and topical anti-inflammatory treatment.

Asato T, Nagai N, Suzuki M, Uchida A, Kurihara T, Ban N, Minami S, Shinoda H, Tsubota K, Ozawa Y†

Lasers Med Sci. (Lasers in Medical Science) 36 ( 2 ) 469 - 471 2020.08

Accepted, ISSN 02688921

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Starburst amacrine cells amplify optogenetic visual restoration through gap junctions.

Katada Y, Kunimi H, Kobayashi K, Okano H, Tanaka KF, Tsubota K†, Kurihara T†

bioRxiv. (Molecular Therapy - Methods and Clinical Development) 30 1 - 14 2020.08

Last author, Corresponding author

Ectopic induction of optogenetic actuators, such as channelrhodopsin, is a promising approach to restoring vision in the degenerating retina. However, the cell type-specific response of ectopic photoreception has not been well understood. There are limits to obtaining efficient gene expression in a specifically targeted cell population by a transgenic approach. In the present study, we established a murine model with high efficiency of gene induction to retinal ganglion cells (RGCs) and amacrine cells using an improved tetracycline transactivator-operator bipartite system (KENGE-tet system). To investigate the cell type-specific visual restorative effect, we expressed the channelrhodopsin gene into RGCs and amacrine cells using the KENGE-tet system. As a result, enhancement in the visual restorative effect was observed to RGCs and starburst amacrine cells. In conclusion, a photoresponse from amacrine cells may enhance the maintained response of RGCs and further increase or improve the visual restorative effect.

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

Mushiga Y, Minami S, Uchida A, Nagai N, Suzuki M, Kurihara T, Sonobe H, Ban N, Watanabe K, Shinoda H, Tsubota K, Ozawa Y†

J Clin Med. (Journal of Clinical Medicine) 9 ( 8 ) 1 - 10 2020.08

Accepted

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 (p = 0.034), and the mean height of the hyperreflective material (HRM), involving the CNV lesion recorded by optical coherence tomography, decreased (p < 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 (p = 0.015) and lower HRM intensity (p = 0.033) at baseline than the others. Remarkably, the BCVA improved (p < 0.05) and the HRM height (p < 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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Retinal microglia are critical for subretinal neovascular formation.

Usui-Ouchi A, Usui Y, Kurihara T, Aguilar E, Dorrell MI, Ideguchi Y, Sakimoto S, Bravo S, Friedlander M†

JCI Insight. (JCI Insight) 5 ( 12 ) 137317 2020.06

Accepted

Abnormal subretinal neovascularization is a characteristic of vision-threatening retinal diseases, including macular telangiectasia (MacTel) and retinal angiomatous proliferation (RAP). Subretinal neovascular tufts and photoreceptor dysfunction are observed in very-low-density lipoprotein receptor (Vldlr-/-) mutant mice. These changes mirror those observed in patients with MacTel and RAP, but the pathogenesis is largely unknown. In this study, we show that retinal microglia were closely associated with retinal neovascular tufts in Vldlr-/- mice and retinal tissue from patients with MacTel; ablation of microglia/macrophages dramatically prevented formation of retinal neovascular tufts and improved neuronal function, as assessed by electroretinography. Vldlr-/- mice with retinal pigmented epithelium-specific (RPE-specific) Vegfa had greatly reduced subretinal infiltration of microglia/macrophages, subsequently reducing neovascular tufts. These findings highlight the contribution of microglia/macrophages to the pathogenesis of neovascularization, provide valuable clues regarding potential causative cellular mechanisms for subretinal neovascularization in patients with MacTel and RAP and suggest that targeting microglia activation may be a therapeutic option in these diseases.

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

Nagai N, Suzuki M, Uchida A, Kurihara T, Ban N, Minami S, Shinoda H, Tsubota K, Ozawa Y†

J Clin Med. (Journal of Clinical Medicine) 9 ( 5 ) E1391 2020.05

Accepted

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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Clinical and Genetic Characteristics of 15 Affected Patients From 12 Japanese Families with GUCY2D-Associated Retinal Disorder.

Liu X*, Fujinami K*†, Kuniyoshi K, Kondo M, Ueno S, Hayashi T, Mochizuki K, Kameya S, Yang L, Fujinami-Yokokawa Y, Arno G, Pontikos N, Sakuramoto H, Kominami T, Terasaki H, Katagiri S, Mizobuchi K, Nakamura N, Yoshitake K, Miyake Y, Li S, Kurihara T, Tsubota K, Iwata T, Tsunoda K

Transl Vis Sci Technol. (Translational Vision Science and Technology) 9 ( 6 ) 2 - 2 2020.05

Accepted

Purpose: To determine the clinical and genetic characteristics of patients with GUCY2D-associated retinal disorder (GUCY2D-RD). Methods: Fifteen patients from 12 families with inherited retinal disorder (IRD) and harboring GUCY2D variants were ascertained from 730 Japanese families with IRD. Comprehensive ophthalmological examinations, including visual acuity (VA) measurement, retinal imaging, and electrophysiological assessment were performed to classify patients into three phenotype subgroups; macular dystrophy (MD), cone-rod dystrophy (CORD), and Leber congenital amaurosis (LCA). In silico analysis was performed for the detected variants, and the molecularly confirmed inheritance pattern was determined (autosomal dominant/recessive [AD/AR]). Results: The median age of onset/examination was 22.0/38.0 years (ranges, 0-55 and 1-73) with a median VA of 0.80/0.70 LogMAR units (ranges, 0.00-1.52 and 0.10-1.52) in the right/left eye, respectively. Macular atrophy was identified in seven patients (46.7%), and two had diffuse fundus disturbance (13.3%), and six had an essentially normal fundus (40.0%). There were 11 patients with generalized cone-rod dysfunction (78.6%), two with entire functional loss (14.3%), and one with confined macular dysfunction (7.1%). There were nine families with ADCORD, one with ARCORD, one with ADMD, and one with ARLCA. Ten GUCY2D variants were identified, including four novel variants (p.Val56GlyfsTer262, p.Met246Ile, p.Arg761Trp, p.Glu874Lys). Conclusions: This large cohort study delineates the disease spectrum of GUCY2D-RD. Diverse clinical presentations with various severities of ADCORD and the early-onset severe phenotype of ARLCA are illustrated. A relatively lower prevalence of GUCY2D-RD for ADCORD and ARLCA in the Japanese population was revealed. Translational Relevance: The obtained data help to monitor and counsel patients, especially in East Asia, as well as to design future therapeutic approaches.

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

Nagai N, Minami S, Suzuki M, Shinoda H, Kurihara T, Sonobe H, Watanabe K, Uchida A, Ban N, Tsubota K, Ozawa Y†

J Clin Med. (Journal of Clinical Medicine) 9 ( 5 ) E1347 2020.05

Accepted

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; p = 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 (p < 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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Hypoxia-Inducible Factor Inhibitors Derived from Marine Products Suppress a Murine Model of Neovascular Retinopathy.

Shoda C, Miwa Y, Nimura K, Okamoto K, Yamagami S, Tsubota K†, KuriharaT†

Nutrients. (Nutrients) 12 ( 4 ) 1055 2020.04

Accepted

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Neovascular retinal degenerative diseases are the leading causes of blindness in developed countries. Anti-vascular endothelial growth factor (VEGF) therapy is commonly used to treat these diseases currently. However, recent reports indicate that long term suppression of VEGF in the eye is associated with chorioretinal atrophy. Therefore, a physiological amount of VEGF is required for retinal homeostasis. Hypoxia-inducible factor (HIF) is a transcriptional factor upstream of VEGF. We previously reported that HIF regulated pathological angiogenesis in the retina of murine models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. Most of the known HIF inhibitors are anti-cancer agents which may have systemic adverse effects in for clinical use; thus, there is a need for safer and less invasive HIF inhibitors. In this study, we screened marine products, especially fish ingredients, and found that six species of fish had HIF inhibitory effects. Among them, administration of Decapterus tabl ingredients significantly suppressed retinal neovascular tufts by inhibiting HIF expression in a murine oxygen-induced retinopathy model. These results indicate that particular fish ingredients can act as anti-angiogenic agents in retinal neovascularization diseases.

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

Nagai N, Asato T, Minami S, Suzuki M, Shinoda H, Kurihara T, Sonobe H, Watanabe K, Uchida A, Ban N, Tsubota K, Ozawa Y†

Nutrients. (Nutrients) 12 ( 4 ) 1 - 10 2020.04

Accepted

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 (P = 0.017, R = 0.360) and retinal volume of the fovea (1-mm diameter around the fovea; P = 0.029, R = 0.332), parafovea (1-3-mm diameter; P = 0.002, R = 0.458), and macula (6-mm diameter; P = 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 (P = 0.037, R = 0.320), inner plexiform layer (P = 0.029, R = 0.333), and outer nuclear layer (P = 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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Lactoferrin Has a Therapeutic Effect via HIF Inhibition in a Murine Model of Choroidal Neovascularization.

Ibuki M, Shoda C, Miwa Y, Ishida A, Tsubota K†, Kurihara T†

Front Pharmacol. (Frontiers in Pharmacology) 11 174 - 174 2020.02

Accepted

Background: Lactoferrin, a type of glycoprotein, is contained in exocrine fluids such as tears, breast milk, sweat, and saliva, and is known to have anti-microbial, antioxidant, and anti-cancer effects. In the ophthalmological field, topical administration of lactoferrin has been reported to have a therapeutic effect in a murine dry eye model. Hypoxia-inducible factor (HIF) regulates various gene expressions under hypoxia, including vascular endothelial growth factor (VEGF), and is considered as an alternative target for neovascular ocular diseases such as age-related macular degeneration (AMD). We previously screened natural products and identified lactoferrin as a novel HIF inhibitor. In this study, we confirmed that lactoferrin has an HIF inhibitory effect and a therapeutic effect in a murine model of neovascular AMD. Methods: HIF inhibitory effects of lactoferrin were evaluated using a luciferase assay and western blotting in vitro. The quantified volume of choroidal neovascularization (CNV) induced by laser irradiation was compared with oral lactoferrin administration or conditional tissue specific Hif1a knockout mice. Results: Lactoferrin administration showed a significant HIF inhibitory effect in the retinal neuronal cells. Oral administration of lactoferrin or conditional Hif1a gene deletion significantly reduced CNV volume compared to controls. Conclusions: Lactoferrin has a therapeutic effect in a laser CNV model by suppressing the retinal HIF activity.

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

Suzuki M, Nagai N, Minami S, Kurihara T, Kamoshita M, Sonobe H, Watanabe K, Shinoda H, Tsubota K, Ozawa Y†

Graefes Arch Clin Exp Ophthalmol. (Graefe's Archive for Clinical and Experimental Ophthalmology) 258 ( 1 ) 49 - 56 2020.01

Accepted, ISSN 0721832X

© 2019, The Author(s). 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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Estimation of the minimum effective dose of dietary supplement crocetin for prevention of myopia progression in mice.

Mori K, Kurihara T†, Jiang X, Ikeda SI, Yotsukura E, Torii H, Tsubota K†

Nutrients. 12 ( 1 ) 180 2020.01

Corresponding author, Accepted

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

Akino K*, Nagai N*, Watanabe K, Ban N, Kurihara T, Uchida A, Shinoda H, Tsubota K, Ozawa Y†

Br J Ophtahlmol. (BMJ) 105 ( 12 ) 1683 - 1687 2020

Accepted, ISSN 00071161

<sec id="s1"><title>Background/Aims</title>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.

</sec><sec id="s2"><title>Methods</title>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.

</sec><sec id="s3"><title>Results</title>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).

</sec><sec id="s4"><title>Conclusion</title>ILM peeling may cause inner retinal degeneration and lead to the development of VFDs after PPV, which should be further examined.

</sec>

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Estimation of the minimum eﬀective dose of dietary supplement crocetin for prevention of myopia progression in mice

Mori K., Kurihara T., Jiang X., Ikeda S.I., Yotsukura E., Torii H., Tsubota K.

Nutrients (Nutrients) 12 ( 1 ) 2020

© 2020, MDPI AG. All rights reserved. The natural carotenoid crocetin has been reported to suppress phenotypes of an experimental myopia model in mice. We investigated the minimum effective dose to prevent myopia progression in a murine model. Three-week-old male mice (C57B6/J) were equipped with a −30 diopter (D) lens to induce myopia, and fed with normal chow, 0.0003%, or 0.001% of crocetin-containing chow. Changes in refractive errors and axial lengths (AL) were evaluated after three weeks. Pharmacokinetics of crocetin in the plasma and the eyeballs of mice was evaluated with specific high sensitivity quantitative analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the minimum effective dosage. A concentration of 0.001% of crocetin-containing chow showed a significant (p < 0.001) suppressive effect against both refractive and AL changes in the murine model. Meanwhile, there was no significant difference of AL change between the 0.0003% and the normal chow groups. The concentration of crocetin in the plasma and the eyeballs from mice fed with 0.001% crocetin-containing chow was significantly higher than control and 0.0003% crocetin-containing chow. In conclusion, we suggest 0.001% of crocetin-containing extract is the minimum effective dose showing a significant suppressive effect against both refractive and AL changes in the murine model.

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Long-term follow-up of a Chinese patient with KCNV2-retinopathy

Lie H*, Wang G*, Liu X*, Meng X, Long Y, Ren J, Yang L, Fujinami-Yokokawa Y, Kurihara T, Tsubota K, Fujinami K†, Li S†

Ophthalmic Genet. (Ophthalmic Genetics) 42 ( 2 ) 144 - 149 2020

Accepted, ISSN 13816810

Purpose: To characterize and monitor the clinical and electrophysiological features of a Chinese patient with KCNV2 retinopathy. Methods: A 17-year-old Chinese male with the diagnosis of cone dystrophy with supernormal rod response (CDSRR) was followed-up for 5 years, with full ophthalmological examinations, including decimal best corrected visual acuity (BCVA), fundus photography, fundus autofluorescence (FAF) imaging, spectral-domain optical coherence tomography (SD-OCT), and full-field electroretinogram (ERG). Genetic screening was performed to detect the sequence variations in the retinal dystrophy associated genes in the patient and his parents. Results: The patient demonstrated the characteristic full-field electroretinography (ERG) features of CDSRR, namely a profound enlargement of the dark-adapted ERG b-wave amplitude with increasing flash strength and a broadened a-wave trough; this case also had undetectable light-adapted ERGs. A BCVA of 0.15 was maintained over 5 years in both eyes; while progressive macular atrophy was identified. Molecular genetic analyses revealed two novel disease-causing KCNV2 variants in compound heterozygous state: c.1408 G > C (p.Gly470Arg) and c.1500 C > G (p.Tyr500Ter). Conclusions: This is the first long-term case study of an East Asian patient with molecularly confirmed CDSRR. The progressive atrophy with maintained VA demonstrated in this case will be valuable for increasing the understanding of the natural course of KCNV2 retinopathy and it will help in counselling patients with this disease.

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

Minami S, Nagai N, Suzuki M, Kurihara T, Sonobe H, Watanabe K, Shinoda H, Takagi H, Tsubota K, Ozawa Y†

Sci Rep. (Scientific Reports) 9 ( 1 ) 18119 2019.12

Accepted

© 2019, The Author(s). 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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QD laser eyewear as a visual field aid in a visual field defect model.

Iyama C, Shigeno Y, Hirano E, Kamoshita M, Nagai N, Suzuki M, Minami S, Kurihara T, Sonobe H, Watanabe K, Shinoda H, Tsubota K, Ozawa Y†

Sci Rep. (Scientific Reports) 9 ( 1 ) 1010 2019.12

Accepted

© 2019, The Author(s). 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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Pemafibrate Prevents Retinal Pathological Neovascularization by Increasing FGF21 Level in a Murine Oxygen-Induced Retinopathy Model.

Tomita Y*, Ozawa N*, Miwa Y, Ishida A, Ohta M, Tsubota K†, Kurihara T†

Int J Mol Sci. (International Journal of Molecular Sciences) 20 ( 23 ) 5878 2019.12

Accepted, ISSN 16616596

Large-scale clinical trials, such as the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) and the Action to Control Cardiovascular Risk in Diabetes (ACCORD) studies, have shown that the administration of fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARα) agonist, suppresses the progression of diabetic retinopathy. In this paper, we reveal a therapeutic effect of a selective PPARα modulator (SPPARMα), pemafibrate, against pathological angiogenesis in murine models of retinopathy. Oxygen-induced retinopathy (OIR) was induced in C57BL/6J mice by exposure to 85% oxygen from postnatal day eight (P8) for 72 h. Vehicle, pemafibrate or fenofibrate was administrated by oral gavage from P12 to P16 daily. Administration of pemafibrate, but not fenofibrate, significantly reduced pathological angiogenesis in OIR. After oral pemafibrate administration, expression levels of downstream PPARα targets such as acyl-CoA oxidase 1 (Acox1), fatty acid binding protein 4 (Fabp4), and fibroblast growth factor 21 (Fgf21) were significantly increased in the liver but not in the retina. A significant increase in plasma FGF21 and reduced retinal hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor A (Vegfa) were also observed after this treatment. In an in vitro HIF-luciferase assay, a long-acting FGF21 analogue, but not pemafibrate, suppressed HIF activity. These data indicate that SPPARMα pemafibrate administration may prevent retinal pathological neovascularization by upregulating FGF21 in the liver.

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Effects of Hyperoxia on the Refraction in Murine Neonatal and Adult Models.

Mori K, Kurihara T†, Jiang X, Ikeda SI, Ishida A, Torii H, Tsubota K†

Int J Mol Sci. (International Journal of Molecular Sciences) 20 ( 23 ) 6014 2019.12

Accepted, ISSN 16616596

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Whether hyperoxia affects the refraction in neonatal and adult mice is unknown. The mice exposed to 85% oxygen at postnatal 8 days (P8d) for 3 days and the mice exposed to normal air were assigned to the neonatal hyperoxia and normoxia groups, respectively. The refraction, the corneal curvature radius (CR) and the axial length (AL) were measured at P30d and P47d. Postnatal 6 weeks (P6w) adult mice were divided into the adult hyperoxia and normoxia groups. These parameters were measured before oxygen exposure, after 1 and 6 weeks, and every 7 weeks. The lens elasticity was measured at P7w and P26w by enucleation. The neonatal hyperoxia group showed a significantly larger myopic change than the neonatal normoxia group (P47d −6.56 ± 5.89 D, +4.11 ± 2.02 D, p < 0.001), whereas the changes in AL were not significantly different (P47d, 3.31 ± 0.04 mm, 3.31 ± 0.05 mm, p = 0.852). The adult hyperoxia group also showed a significantly larger myopic change (P12w, −7.20 ± 4.09 D, +7.52 ± 2.54 D, p < 0.001). The AL did not show significant difference (P12w, 3.44 ± 0.03 mm, 3.43 ± 0.01 mm, p = 0.545); however, the CR in the adult hyperoxia group was significantly smaller than the adult normoxia group (P12w, 1.44 ± 0.03 mm, 1.50 ± 0.03 mm, p = 0.003). In conclusion, hyperoxia was demonstrated to induce myopic shift both in neonatal and adult mice, which was attributed to the change in the CR rather than the AL. Elucidation of the mechanisms of hyperoxia and the application of this result to humans should be carried out in future studies.

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

Minami S, Shinoda H, Shigeno Y, Nagai N, Kurihara T, Watanabe K, Sonobe H, Takagi H, Tsubota K, Ozawa Y†

Sci Rep. (Scientific Reports) 9 ( 1 ) 19056 2019.12

Accepted

© 2019, The Author(s). 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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Dynamic changes in choroidal conditions during anti-vascular endothelial growth factor therapy in polypoidal choroidal vasculopathy.

Nagai N, Suzuki M, Minami S, Kurihara T, Kamoshita M, Sonobe H, Watanabe K, Uchida A, Shinoda H, Tsubota K, Ozawa Y†

Sci Rep. (Scientific Reports) 9 ( 1 ) 11389 2019.12

Accepted

© 2019, The Author(s). 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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Oral crocetin administration suppressed refractive shift and axial elongation in a murine model of lens-induced myopia.

Mori K, Kurihara T†, Miyauchi M, Ishida A, Jiang X, Ikeda SI, Torii H, Tsubota K†

Sci Rep. (Scientific Reports) 9 ( 1 ) 295 2019.12

Accepted

© 2019, The Author(s). Increased global incidence of myopia necessitates establishment of therapeutic approaches against its progression. To explore agents which may control myopia, we screened 207 types of natural compounds and chemical reagents based on an activity of a myopia suppressive factor, early growth response protein 1 (Egr-1) in vitro. Among the candidates, crocetin showed the highest and dose-dependent activation of Egr-1. For in vivo analysis, experimental myopia was induced in 3-week-old C57BL/6 J mice with −30 diopter (D) lenses for 3 weeks. Animals were fed with normal or mixed chow containing 0.003% (n = 19) and 0.03% (n = 7) of crocetin during myopia induction. Refraction and axial length were measured at 3-week-old and the 6-week-old with an infrared photorefractor and a SD-OCT system. Compared to controls (n = 14), crocetin administration showed a significant smaller change of refractive errors (−13.62 ± 8.14 vs +0.82 ± 5.81 D for 0.003%, p < 0.01, −2.00 ± 4.52 D for 0.03%, p < 0.01) and axial elongation (0.27 ± 0.03 vs 0.22 ± 0.04 mm for 0.003%, p < 0.01, 0.23 ± 0.05 mm for 0.03%, p < 0.05). These results suggest that a dietary factor crocetin may have a preventive effect against myopia progression.

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The role of sphingosine 1-phosphate receptors on retinal pigment epithelial cells barrier function and angiogenic effects.

Terao R, Honjo M, Totsuka K, Miwa Y, Kurihara T, Aihara M†

Prostaglandins Other Lipid Mediat. (Prostaglandins and Other Lipid Mediators) 145 106365 2019.12

Accepted, ISSN 10988823

© 2019 Sphingosine-1-phosphate (S1P) is a lysophospholipid mediator, promoting angiogenesis and inflammation via interactions with its receptors (S1P1–5), but the receptors and signaling pathways responsible for the progression of choroidal neovascularization (CNV) remain unknown. We investigated the roles of S1P/S1P receptors in RPE cells. ARPE-19 cells were treated with S1P dissolved in carrier proteins of albumin or apolipoprotein M (ApoM). The mRNA expression levels of interleukin-8 (IL-8), C-C motif chemokine ligand 2 (CCL2), and vascular endothelial growth factor (VEGF) were evaluated using quantitative real-time polymerase chain reaction. The protein level of hypoxia-inducible factor (HIF)-1α was assessed via enzyme-linked immunosorbent assay. HIF transcriptional activity was evaluated with a dual-reporter luciferase assay. Cellular barrier integrity was evaluated using transepithelial electrical resistance and the FITC-dextran permeability assay. The suppressive effect of an S1P antagonist on CNV progression was investigated with a laser-induced CNV model in mice. The increase in expression of IL-8, CCL2, and VEGF due to albumin-bound S1P was significantly mitigated by an S1P2 antagonist. The expression of HIF-1α significantly decreased with inhibition of S1P2 and S1P3. In addition, albumin-bound S1P disrupted the barrier integrity of retinal pigment epithelial cells via S1P2, whereas integrity was strengthened by ApoM-bound S1P. CNV lesions were significantly reduced in the mouse model with intravitreal injection of S1P2 antagonist. This study demonstrated that S1P significantly promotes angiogenesis, inflammation, and barrier integrity, which was attenuated by inhibition of S1P2 or S1P3, suggesting that regulation of S1P2 and S1P3 is a novel therapeutic target for CNV.

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組織型プラスミノーゲンアクチベーター（tPA；モンテプラーゼ）硝子体内投与による黄斑下血腫の治療経過

園部秀樹, 篠田肇, 鴨下衛, 渡邊一弘, 栗原俊英, 永井紀博, 坪田一男, 小沢洋子

あたらしい眼科 36 ( 11 ) 104 - 108 2019.11

Current Prevalence of Myopia and Association of Myopia With Environmental Factors Among Schoolchildren in Japan.

Yotsukura E*, Torii H*, Inokuchi M, Tokumura M, Uchino M, Nakamura K, Hyodo M, Mori K, Jiang X, Ikeda SI, Kondo S, Negishi K, Kurihara T, Tsubota K

JAMA Ophthalmol. (JAMA Ophthalmology) 137 ( 11 ) 1233 - 1239 2019.11

Accepted, ISSN 21686165

© 2019 American Medical Association. All rights reserved. Importance: Given the estimates of increasing prevalence of myopia, especially in Asia, it is important to determine the current prevalence of myopia among populations of schoolchildren in Japan. Objective: To investigate the current prevalence rate of myopia and the association between environmental factors and myopia in Japanese schoolchildren. Design, Setting, and Participants: This cross-sectional study assessed 1478 participants, including 726 elementary school students and 752 junior high school students, at 2 schools in Tokyo, Japan, who underwent eye examinations from April 1 to May 31, 2017, that included measurement of the refractive errors by autorefractometry with noncycloplegic refraction and ocular biometric factors. After excluding those who had been treated with atropine or orthokeratology (n = 11), had a history of eye disease (n = 2), had no parental consent (n = 41), and were absent (n = 8), 1416 schoolchildren were analyzed. Main Outcomes and Measures: The primary outcome was the prevalence of myopia and high myopia. Secondary outcomes were environmental factors that were associated with myopia. Results: A total of 1416 schoolchildren (mean [SD] age, 10.8 [2.7] years; 792 [55.9%] male) were studied. The prevalence rates of myopia (spherical equivalent ≤-0.5 diopters [D]) were 76.5% (95% CI, 73.4%-79.7%) among the elementary school students and 94.9% (95% CI, 93.3%-96.5%) among the junior high school students. The prevalence rates of high myopia (spherical equivalent ≤-6.0 D) were 4.0% (95% CI, 2.5%-5.4%) among the elementary school students and 11.3% (95% CI, 8.8%-13.7%) among the junior high school students. The prevalence rates of high myopia classified based on axial length of 26.0 mm or longer were 1.2% (95% CI, 0.4%-2.0%) among elementary school students and 15.2% (95% CI, 12.5%-17.8%) among junior high school students. Multiple regression analysis showed that higher-order aberrations and dry eye disease were associated with refractive error in elementary school students (spherical aberration: β = 6.152; 95% CI, 3.161-9.143; P <.001; dry eye disease: β = -0.626; 95% CI, -1.189 to -0.063; P =.03) and with axial length in junior high school students (spherical-like aberration: β = 26.546; 95% CI, 18.708-34.385; P <.001; dry eye disease: β = 0.354; 95% CI, 0.131-0.578; P <.002). Conclusions and Relevance: Although the use of noncycloplegic autorefraction with a cutoff of -0.50 D could lead to overestimation of results, these findings suggest that the current prevalence rates of myopia among elementary and junior high school students in Asia are high, especially if the results from these 2 schools are generalizable to all schoolchildren in Japan and Asia..

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High Myopia and Its Associated Factors in JPHC-NEXT Eye Study: A Cross-Sectional Observational Study.

Mori K, Kurihara T†, Uchino M, Torii H, Kawashima M, Sasaki M, Ozawa Y, Yamagishi K†, Iso H, Sawada N, Tsugane S, Yuki K, Tsubota K

J Clin Med. (Journal of Clinical Medicine) 8 ( 11 ) 1788 2019.11

Accepted

The increasing prevalence of high myopia has been noted. We investigated the epidemiological characteristics and the related factors of high myopia in a Japanese adult population. Japan Public Health Center-Based Prospective Study for the Next Generation (JPHC-NEXT) Eye Study was performed in Chikusei-city, a rural area in mid-east Japan, between 2013 and 2015. A cross-sectional observational analysis was conducted to investigate prevalence and related factors of high myopia. A total of 6101 participants aged ≥40 years without a history of ocular surgeries was included. High myopia was defined as a spherical equivalent refraction of ≤-6.00 diopters according to the American Academy of Ophthalmology. Potential high myopia-related factors included intraocular pressure (IOP), corneal structure, corneal endothelial cell density, age, height, body mass index, heart rate, blood pressure, biochemical profile, and current history of systemic and ocular disorders. The odds ratios of high myopia were estimated using the logistic regression models adjusted for the associated factors. The prevalence of high myopia was 3.8% in males and 5.9% in females with a significant difference. Age was inversely associated, IOP was positively associated, and none of other factors were associated with high myopia in both sexes. In conclusion, only age and IOP were associated with high myopia in this community-based sample.

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Therapeutic Effect of Garcinia cambogia Extract and Hydroxycitric Acid Inhibiting Hypoxia-Inducible Factor in a Murine Model of Age-Related Macular Degeneration.

Ibuki M, Shoda C, Miwa Y, Ishida A, Tsubota K†, Kurihara T†

Int J Mol Sci. (International Journal of Molecular Sciences) 20 ( 20 ) 5049 2019.10

Accepted, ISSN 16616596

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Background: Age-related macular degeneration (AMD) is the leading cause of blindness and can be classified into two types called atrophic AMD (dry AMD) and neovascular AMD (wet AMD). Dry AMD is characterized by cellular degeneration of the retinal pigment epithelium, choriocapillaris, and photoreceptors. Wet AMD is characterized by the invasion of abnormal vessels from the choroid. Although anti-vascular endothelial growth factor (VEGF) therapy has a potent therapeutic effect against the disease, there is a possibility of chorio-retinal atrophy and adverse systemic events due to long-term robust VEGF antagonism. We focused on hypoxia-inducible factor (HIF) regulation of VEGF transcription, and report the suppressive effects of HIF inhibition against ocular phenotypes in animal models. Many of the known HIF inhibitors are categorized as anti-cancer drugs, and their systemic side effects are cause for concern in clinical use. In this study, we explored food ingredients that have HIF inhibitory effects and verified their effects in an animal model of AMD. Methods: Food ingredients were screened using a luciferase assay. C57BL6/J mice were administered the Garcinia cambogia extract (Garcinia extract) and hydroxycitric acid (HCA). Choroidal neovascularization (CNV) was induced by laser irradiation. Results: Garcinia extract and HCA showed inhibitory effects on HIF in the luciferase assay. The laser CNV model mice showed significant reduction of CNV volume by administering Garcinia extract and HCA. Conclusions: Garcinia extract and HCA showed therapeutic effects in a murine AMD model.

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Ultra-Widefield Retinal Imaging for Analyzing the Association Between Types of Pathological Myopia and Posterior Staphyloma.

Mimura R*, Mori K*, Torii H, Nagai N, Suzuki M, Minami S, Ozawa Y, Kurihara T†, Tsubota K†

J Clin Med. (Journal of Clinical Medicine) 8 ( 10 ) 1505 2019.10

Accepted

High myopia may develop to pathologic myopia, which brings severe visual impairment; however, the etiology is not fully understood. We, therefore, investigated the relationship between the presence of posterior staphyloma and posterior ocular disorders by assessing the patients with high myopia. A retrospective study was performed for the patients, who have more than 26 mm of the axial length and of whom fundus photography was taken with an ultra-widefield retinal imaging system. The objectives were 138 cases encompassing 229 eyes. In 138 cases, 91 were bilateral and 47 were unilateral. The averages ± SD of axial length of bilateral and unilateral were 28.8 ± 2.2 mm, 27.3 ± 1.2 mm, respectively, showing statistically significant difference. The number of eyes with and without posterior staphyloma were 107 (46.7%) and 122 (53.3%), respectively. Retinal detachment and retinal breaks are more observed in cases without posterior staphyloma (p = 0.017). Myopic choroidal neovascularization (mCNV) (p = 0.002), chorioretinal atrophy (p < 0.001), retinoschisis (p < 0.001), and optic neuropathy (p = 0.038) are more often seen in cases with posterior staphyloma. In conclusion, the prevalence rates of myopic choroidal neovascularization, retinal choroidal atrophy, and optic neuropathy were significantly higher with posterior staphyloma. The rate of periocular disorders such as retinal detachment was significantly higher without posterior staphyloma. These results indicate associations between types of pathological myopia and presence or absence of posterior staphyloma analyzed by ultra-widefield retinal imaging.

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The long dystrophin gene product Dp427 modulates retinal function and vascular morphology in response to age and retinal ischemia.

Bucher F, Friedlander MSH, Aguilar E, Kurihara T, Krohne TU, Usui Y, Friedlander M†

Neruochem Int. (Neurochemistry International) 129 104489 2019.10

Accepted, ISSN 01970186

© 2019 Elsevier Ltd Mutations in dystrophin are the major cause of muscular dystrophies. Continuous muscular degeneration and late stage complications, including cardiomyopathy and respiratory insufficiency, dominate the clinical phenotype. Gene expression and regulation of the dystrophin gene outside of muscular tissue is far more complex. Multiple tissue-specific dystrophin gene products are widely expressed throughout the body, including the central nervous system and eye, predisposing affected patients to secondary complications in non-muscular tissues. In this study, we evaluated the impact of the full-length dystrophin gene product, Dp427, on retinal homeostasis and angiogenesis. Based on the clinical case of a Duchenne muscular dystrophy (DMD) patient who developed severe fibrovascular changes in the retina in response to hypoxic stress, we hypothesized that defects in Dp427 make the retina more susceptible to stresses such as ageing and ischemia. To further study this, a mouse strain lacking Dp427 expression (Mdx) was studied during retinal development, ageing and in the oxygen-induced retinopathy (OIR) model. While retinal vascular morphology was normal during development and ageing, retinal function measured by electroretinography (ERG) was slightly reduced in young adult Mdx mice and deteriorated with age. Mdx mice also had increased retinal neovascularization in response to OIR and more pronounced long-term deterioration in retinal function following OIR. Based on these results, we suggest that DMD patients with a mutation in Dp427 may experience disturbed retinal homeostasis with increasing age and therefore be prone to develop excessive retinal neovascular changes in response to hypoxic stress. DMD patients in late disease stages should, thus, be regularly examined to detect asymptomatic retinal abnormalities and prevent visual impairment.

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Clinical and Genetic Characteristics of East Asian Patients with Occult Macular Dystrophy (Miyake Disease): East Asia Occult Macular Dystrophy Studies Report Number 1.

Fujinami K*†, Yang L*, Joo K*, Tsunoda K, , Kameya S, Hanazono G, Fujinami-Yokokawa Y, Arno G, Kondo M, Nakamura N, Kurihara T, , Tsubota K, , Zou X, Li H, Park KH, Iwata T, Miyake Y, Woo SJ†, Sui R†

Ophthalmology. (Ophthalmology) 126 ( 10 ) 1432 - 1444 2019.10

Accepted, ISSN 01616420

PURPOSE: To describe the clinical and genetic characteristics of the cohort enrolled in the East Asian studies of occult macular dystrophy (OMD). DESIGN: International, multicenter, retrospective cohort studies. PARTICIPANTS: A total of 36 participants from 21 families with a clinical diagnosis of OMD and harboring pathogenic RP1L1 variants (i.e., Miyake disease) were enrolled from 3 centers in Japan, China, and South Korea. METHODS: A detailed history was obtained, and comprehensive ophthalmological examinations including spectral-domain OCT were performed. All detected sequence variants in the RP1L1 gene were reviewed, and in silico analysis was performed, including allele frequency analyses and pathogenicity predictions. MAIN OUTCOME MEASURES: Onset of disease, visual acuity (VA) converted to the logarithm of the minimum angle of resolution (logMAR), OCT findings, and effect of detected variants. RESULTS: Eleven families from Japan, 6 from South Korea, and 4 from China were recruited. There were 12 female and 24 male participants. The median age of onset was 25.5 years (range, 2-73), and the median age at the latest examination was 46.0 years (range, 11-86). The median VA (logMAR) was 0.65 (range, -0.08-1.22) in the right eye and 0.65 (-0.08-1.10) in the left eye. A significant correlation between onset of disease and VA was revealed. The Classical morphologic phenotype showing both blurred ellipsoid zone and absence of interdigitation zone of the photoreceptors was demonstrated in 30 patients (83.3%), and subtle photoreceptor architectural changes were demonstrated in 6 patients (16.6%). Eight pathogenic RP1L1 variants were identified, including 6 reported variants and 1 novel variant: p.R45W, p.T1194M/p.T1196I (complex), p.S1199C, p.G1200A, p.G1200D, p.V1201G, and p.S1198F, respectively. Two variants were recurrent: p.R45W (11 families, 52.4%) and p.S1199C (5 families, 23.8%). The pathogenic missense variants in 10 families (47.6%) were located within the previously reported unique motif, including 6 amino acids (1196-1201). CONCLUSIONS: There is a large spectrum of clinical findings in Miyake disease, including various onset of disease and VA, whereas the characteristic photoreceptor microstructures were shared in most cases. Two hot spots including amino acid numbers 45 and 1196-1201 in the RP1L1 gene were confirmed in the East Asian population.

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Pharmacological HIF inhibition prevents retinal neovascularization with improved visual function in a murine oxygen-induced retinopathy model

Miwa Y, Hoshino Y, Shoda C, Jiang X, Tsubota K†, Kurihara T†

Neurochem Int. (Neurochemistry International) 128 21 - 31 2019.09

Accepted, ISSN 01970186

© 2019 Elsevier Ltd Neovascular retinal diseases are the leading causes of blindness in advanced countries. To date, anti-VEGF (vascular endothelial growth factor) drugs are clinically effective and widely used for these diseases. However, recent animal and clinical studies reported that potent and long-term VEGF antagonism may induce chorioretinal atrophy. Thus, physiological amount of VEGF is required for the homeostasis in the retina. Hypoxia-inducible factors (HIFs) are transcription factors located upstream of VEGF. We hypothesized that ectopically stabilized HIFs induce pathological amount of VEGF involved with retinal neovascularization. Therefore, HIF inhibition could be an alternative therapeutic candidate targeting the pathological amount of VEGF while holding a physiological amount of VEGF. To test this hypothesis, topotecan and doxorubicin, HIF inhibitors with different mechanisms were administered to the murine oxygen-induced retinopathy (OIR) model. We found that both topotecan and doxorubicin significantly prevented pathological but not physiological neovascularization in OIR. Furthermore, impaired visual function observed in OIR can also be suppressed by administering topotecan. These data suggested that HIF inhibition may be effective for pathological angiogenesis and neurodegeneration of the retina.

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Ocular-Component-Specific miRNA Expression in a Murine Model of Lens-Induced Myopia.

Tanaka Y, Kurihara T†, Hagiwara Y, Ikeda SI, Mori K, Jiang X, Torii H, Tsubota K†

Int J Mol Sci. (International Journal of Molecular Sciences) 20 ( 15 ) 3629 2019.08

Accepted, ISSN 16616596

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. To identify tissues and molecules involved in refractive myopic shift and axial length elongation in a murine lens-induced myopia model, we performed a comprehensive analysis of microRNA (miRNA) expression. Three weeks after negative 30 diopter lens fixation on three-week-old C57BL/6J mice, total RNA was extracted from individual ocular components including cornea, iris, lens, retina, retinal pigment epithelium (RPE)/choroid, and sclera tissue. The miRNA expression analysis was pooled from three samples and carried out using Agilent Mouse miRNA Microarray (8 × 60 K) miRBase21.0. The expression ratio was calculated, and differentially expressed miRNAs were extracted, using GeneSpring GX 14.5. Myopic induction showed a significant myopic refractive change, axial elongation, and choroidal thinning. Through the comprehensive miRNA analysis, several upregulated miRNAs (56 in cornea tissue, 13 in iris tissue, 6 in lens tissue, 0 in retina tissue, 29 in RPE/choroid tissue, and 30 in sclera tissue) and downregulated miRNAs (7 in cornea tissue, 28 in iris tissue, 17 in lens tissue, 9 in retina tissue, 7 in RPE/choroid tissue, and 40 in sclera tissue) were observed. Overlapping expression changes in miRNAs were also found in different ocular components. Some of this miRNA dysregulation may be functionally involved in refractive myopia shift and axial length elongation.

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The Effect of Dietary Supplementation of Crocetin for Myopia Control in Children: A Randomized Clinical Trial

Mori K*, Torii H*, Fujimoto S*, Jiang X, Ikeda SI, Yotsukura E, Koh S, Kurihara T†, Nishida K†, Tsubota K†

J Clin Med. (Journal of Clinical Medicine) 8 ( 8 ) 1179 2019.08

Accepted

The prevalence of myopia has been increasing in recent years. The natural carotenoid crocetin has been reported to suppress experimental myopia in mice. We evaluated the effects of crocetin on myopia suppression in children. A multicenter randomized double-blind placebo-controlled clinical trial was performed with 69 participants aged 6 to 12 years, whose cycloplegic spherical equivalent refractions (SER) were between −1.5 and −4.5 diopter (D). The participants were randomized to receive either a placebo or crocetin and followed up for 24 weeks. Axial length (AL) elongation and changes in SER were evaluated for 24 weeks. Both written informed assent from the participants and written informed consent from legal guardians were obtained in this study because the selection criteria of this trial included children aged between 6 and 12 years old. This trial was approved by the institutional review boards. A mixed-effects model was used for analysis, using both eyes. Two participants dropped out and 67 children completed this trial. The change in SER in the placebo group, −0.41 ± 0.05 D (mean ± standard deviation), was significantly more myopic compared to that in the crocetin group, −0.33 ± 0.05 D (p = 0.049). The AL elongation in the placebo group, 0.21 ± 0.02 mm, was significantly bigger than that in the crocetin group, 0.18 ± 0.02 mm (p = 0.046). In conclusion, dietary crocetin may have a suppressive effect on myopia progression in children, but large-scale studies are required in order to confirm this effect.

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A Novel HIF Inhibitor Halofuginone Prevents Neurodegeneration in a Murine Model of Retinal Ischemia-Reperfusion

Kunimi H, Miwa Y, Inoue H, Tsubota K†, Kurihara T†

Int J Mol Sci. (International Journal of Molecular Sciences) 20 ( 13 ) 3171 2019.07

Accepted, ISSN 16616596

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Neurodegeneration caused with retinal ischemia or high intraocular pressure is irreversible in general. We have focused on the role of hypoxia-inducible factor (HIF) in retinal homeostasis and revealed that HIF inhibition may be effective against retinal neovascular and neurodegeneration. In this study, we performed in vitro screening of natural products and found halofuginone, which is a derivative of febrifugine extracted from hydrangea, as a novel HIF inhibitor. Administration of halofuginone showed a significant neuroprotective effect by inhibiting HIF-1α expression in a murine retinal ischemia-reperfusion model histologically and functionally. These results indicate that halofuginone can be a neuroprotective agent in ischemic retinal degenerative diseases.

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Three Cases of Developing Myopia in Newly Developed Dry Eye Disease after Hematopoietic Stem Cell Transplantation

伊藤賀一, 小川葉子, 清水映輔, 鈴木孝典, 西條裕美子, 内野美樹, 栗原俊英, 坪田一男

あたらしい眼科 ((株)メディカル葵出版) 36 ( 5 ) 684 - 688 2019.05

ISSN 0910-1810

移植片対宿主病(GVHD)によるドライアイ(DE)発症に伴い近視化を伴う3症例を経験したので報告する。症例1は32歳、男性。Fanconi貧血(先天性再生不良性貧血)に対し非血縁者骨髄移植を行った。移植前の自覚的視力検査による等価球面度数(SE)は右眼-2.75Dと左眼-3.00Dであった。移植後4ヵ月時GVHDによるDEを発症し、同時期のSEは右眼-2.75Dと左眼-4.50Dで左眼の近視化を認めた。移植後7ヵ月時のSEは右眼-3.00Dと左眼-4.625Dで時期の差があるが両眼の近視化を認めた。症例2は34歳、女性。急性リンパ性白血病に対し非血縁骨髄移植を行った。移植前の自覚的視力検査によるSEは右眼+3.375Dと左眼+0.50D、移植後13ヵ月時にGVHDによるDEを発症し、同時期のSEは右眼+2.50Dと左眼0.00Dと両眼の近視化を認めた。症例3は40歳、女性。急性骨髄性白血病に対し血縁末梢血幹細胞移植を行った。移植前の自覚的視力検査のSEは右眼-1.50Dと左眼0.00D。移植後9ヵ月時にDEを発症し、同時期のSEは右眼-1.50Dと左眼-0.625Dで左眼の近視化を認めた。他覚的検査では移植前後のSEの差が右眼1.125D、左眼1.75Dと両眼の近視化を認めた。3症例ともに白内障は認めず、眼底に異常は認められなかった。炎症性疾患であるGVHDによるDE発症時期に、近視化する3症例を認めた。GVHDと近視化との関連に関する因子について今後さらなる検討が必要である。(著者抄録)

Inducement and Evaluation of a Murine Model of Experimental Myopia.

Jiang X, Kurihara T†, Ikeda SI, Kunimi H, Mori K, Torii H, Tsubota K†

J Vis Exp. (Journal of Visualized Experiments) 2019 ( 143 ) e58822 2019.01

Accepted, ISSN 1940087X

© 2019 Journal of Visualized Experiments. Murine model of myopia can be a powerful tool for myopia research because of the comparatively easy genetic manipulation. One way to induce myopia in animals is to put clear minus lenses in front of eyes for weeks (lens-induced myopia, LIM). However, extant protocols for inducement and evaluation vary from laboratory to laboratory. Here, we described a highly practical and reproducible method to induce LIM in mice using newly designed eyeglasses. The method fixes the lens stably in front of the mouse eye while allows the lens to be taken off for cleaning or topical drug administration. The phenotype is robust and efficient, and the variance is small. The method described here can be applied to mice right after weaning which extends the possible duration for experiments. We also gave technical advises for achieving reproducible results in refraction and axial length measurements. We hope the step-by-step protocol described here and the detailed article can help researchers perform myopia experiments with myopia more smoothly and make the data comparable across laboratories.

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HIF inhibitor topotecan has a neuroprotective effect in a murine retinal ischemia-reperfusion model.

Kunimi H, Miwa Y, Katada Y, Tsubota K†, Kurihara T†

PeerJ. (PeerJ) 2019 ( 10 ) e7849 2019

Accepted

Copyright 2019 Kunimi et al. Purpose. The therapeutic approach for retinal ganglion cell (RGC) degeneration has not been fully established. Recently, it has been reported that hypoxia-inducible factor (HIF) may be involved with retinal neurodegeneration. In this study, we investigated neuroprotective effects of a HIF inhibitor against RGC degeneration induced in a murine model of retinal ischemia-reperfusion (I/R). Methods. Eight-weeks-old male C57/BL6J mice were treated with intraperitoneal injection of a HIF inhibitor topotecan (1.25 mg/kg) for 14 days followed by a retinal I/R procedure. Seven days after the I/R injury, the therapeutic effect was evaluated histologically and electrophysiologically. Results. The increase of HIF-1α expression and the decrease of retinal thickness and RGC number in I/R were significantly suppressed by administration of topotecan. Impaired visual function in I/R was improved by topotecan evaluated with elec-troretinogram and visual evoked potentials. Conclusions. Topotecan administration suppressed HIF-1a expression and improved RGC survival resulting in a functional protection against retinal I/R. These data indicated that the HIF inhibitor topotecan may have therapeutic potentials for RGC degeneration induced with retinal ischemia or high intraocular pressure.

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Evaluation of AAV-DJ vector for retinal gene therapy

Katada Y, Kobayashi K, Tsubota K†, Kurihara T†

PeerJ. (PeerJ) 2019 ( 1 ) e6317 2019

Accepted

Copyright 2019 Katada et al. Purpose. The most common virus vector used in gene therapy research for ophthalmologic diseases is the adeno-associated virus (AAV) vector, which has been used successfully in a number of preclinical and clinical studies. It is important to evaluate novel AAV vectors in animal models for application of clinical gene therapy. The AAV-DJ (type 2/type 8/type 9 chimera) was engineered from shuffling eight different wild-type native viruses. In this study, we investigated the efficiency of gene transfer by AAV-DJ injections into the retina. Methods. One microliter of AAV-2-CAGGS-EGFP or AAV-DJ-CAGGS-EGFP vector at a titer of 1.4 x 10e12 vg/ml was injected intravitreally or subretinally in each eye of C57BL/6 mice. We evaluated the transduction characteristics of AAV-2 and -DJ vectors using fluorescence microscopy and electroretinography. Results. The results confirmed that AAV-DJ could deeply transfer gene to photoreceptor layer with intravitreal injection and has an efficient gene transfer to various cell types especially the Mueller cells in the retina. Retinal function was not affected by AAV-DJ infection or ectopic EGFP expression. Conclusions. The AAV-DJ vector efficiently induces the reporter gene in both the inner and outer murine retina without functional toxicity. These data indicated that the AAV-DJ vector is a useful tool for the gene therapy research targeting retinal disorders.

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Effect of midazolam, medetomidine, and butorphanol tartrate combination anesthetic on electroretinograms of mice.

Miwa Y, Tsubota K†, Kurihara T†

Mol Vis. (Molecular Vision) 25 645 - 653 2019

Accepted

© 2019 Molecular Vision. Purpose: To evaluate electroretinogram (ERG) responses under anesthesia with midazolam, medetomidine, and butorphanol tartrate (MMB) combination compared with pentobarbital sodium and ketamine/xylazine (KX). Methods: Six-week-old male C57BL/6J mice were divided into MMB-, pentobarbital sodium-, and KX-administered groups. Following overnight dark adaptation, an ERG was performed. The parameters sensitivity (S), log maximum amplitude (Rmax), Rmax, and time delay to the onset (Td) of the ERG a-waves were computed based on the Lamb and Pugh model. The parameters light intensity at half maximum amplitude (K), Rmax, and n of the ERG b-waves were computed based on the Naka-Rushton equation. The amplitude and the implicit time of oscillatory potentials (OPs) were quantified. Results: The Td of the dark-adapted a-waves was statistically significantly larger under anesthesia with the MMB combination and pentobarbital sodium compared to KX. The K of the dark-adapted b-waves was statistically significantly larger under anesthesia with pentobarbital sodium compared to the MMB combination. The amplitude of the dark-adapted OPs was statistically significantly larger under anesthesia with the MMB combination compared to pentobarbital sodium. The implicit time of the dark-adapted OPs was statistically significantly smaller under anesthesia with the KX combination compared to pentobarbital sodium. Conclusions: The results suggested that ERG responses, especially in OPs, are greatly affected by the type of anesthetic. It is important to consider the sensitive responses influenced by the selection of anesthetics when ERG is performed.

Lactobacillus paracasei KW3110 Prevents Blue Light-Induced Inflammation and Degeneration in the Retina.

Morita Y*†, Miwa Y*, Jounai K, Fujiwara D, Kurihara T, Kanauchi O

Nutrients. (Nutrients) 10 ( 12 ) pii: E1991 2018.12

Accepted

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. Age-related macular degeneration and retinitis pigmentosa are leading causes of blindness and share a pathological feature, which is photoreceptor degeneration. To date, the lack of a potential treatment to prevent such diseases has raised great concern. Photoreceptor degeneration can be accelerated by excessive light exposure via an inflammatory response; therefore, anti-inflammatory agents would be candidates to prevent the progress of photoreceptor degeneration. We previously reported that a lactic acid bacterium, Lactobacillus paracasei KW3110 (L. paracasei KW3110), activated macrophages suppressing inflammation in mice and humans. Recently, we also showed that intake of L. paracasei KW3110 could mitigate visual display terminal (VDT) load-induced ocular disorders in humans. However, the biological mechanism of L. paracasei KW3110 to retain visual function remains unclear. In this study, we found that L. paracasei KW3110 activated M2 macrophages inducing anti-inflammatory cytokine interleukin-10 (IL-10) production in vitro using bone marrow-derived M2 macrophages. We also show that IL-10 gene expression was significantly increased in the intestinal immune tissues 6 h after oral administration of L. paracasei KW3110 in vivo. Furthermore, we demonstrated that intake of L. paracasei KW3110 suppressed inflammation and photoreceptor degeneration in a murine model of light-induced retinopathy. These results suggest that L. paracasei KW3110 may have a preventive effect against degrative retinal diseases.

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A highly efficient murine model of experimental myopia.

Jiang X, Kurihara T†, Kunimi H, Miyauchi M, Ikeda SI, Mori K, Tsubota K, Torii H, Tsubota K†

Sci Rep. (Scientific Reports) 8 ( 1 ) 2026 2018.12

Accepted

© 2018 The Author(s). Despite the global pandemic of myopia, the precise molecular mechanism of the onset of myopia remains largely unknown. This is partially because of the lack of efficient murine myopic models that allow genetic manipulation at low cost. Here we report a highly practical and reproducible lens-induced myopia model by specially designed frames and lenses for mice. A lens power dependent myopic induction in mice was shown until minus 30 diopter lenses. The phenotype was significantly stronger than form-deprivation myopia. We presented the protocol for precise evaluations of the state of myopia, including refraction, corneal curvature and axial length using up-to-date devices. We also found that myopic mouse eyes showed decreased visual acuity on optokinetic response examination. Finally, we confirmed the anti-myopic effect of 1% atropine using this model, which showed its potential in drug screening. The strong phenotype, stable evaluation and the potential for gene manipulation utilizing the presented method in mice will accelerate the translational research of myopia.

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

Minami S, Nagai N, Suzuki M, Kurihara T, Sonobe H, Kamoshita M, Uchida A, Shinoda H, Takagi H, Sonoda S, Sakamoto T, Tsubota K, Ozawa Y†

Sci Rep. (Scientific Reports) 8 ( 1 ) 58 2018.12

Accepted

© 2017 The Author(s). 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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Roles of hypoxia response in retinal development and pathophysiology

Kurihara T†

Keio J Med. (Keio Journal of Medicine) 67 ( 1 ) 1 - 9 2018

Accepted, ISSN 00229717

© 2017 by The Keio Journal of Medicine. The hypoxia response is a fundamental phenomenon mainly regulated by hypoxia-inducible factors (HIFs). For more than a decade, we have investigated and revealed the roles of the hypoxia response in the development, physiology, and pathophysiology of the retina by generating and utilizing cell-typespecific conditional knockout mice. To investigate the functions of genes related to the hypoxia response in cells composing the retina, we generated various mouse lines that lack HIFs and/or related genes specifically in retinal neurons, astrocytes, myeloid cells, or retinal pigment epithelium cells. We found that these genes in the different types of retinal cells contribute in various ways to the homeostasis of ocular vascular and visual function. We hypothesized that the activation of HIFs is likely involved in the development and progress of retinal diseases, and we subsequently confirmed the pathological roles of HIFs in animal models of neovascular and atrophic ocular diseases. Currently, anti-vascular endothelial growth factor (anti-VEGF) therapy is a first-line treatment widely used for neovascular retinal diseases. However, alternative or additional targets are now required because several recent large-scale clinical trials and animal studies, including our own research, have indicated that VEGF antagonism may induce retinal vascular and neuronal degeneration. We have identified and confirmed a microRNA as a candidate for an alternative target against neovascular retinal diseases, and we are now working to establish a novel HIF inhibitor for clinical use based on the disease mechanism that we identified.

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Progress and Control of Myopia by Light Environments.

Jiang X, Kurihara T†, Torii H, Tsubota K†

Eye Contact Lens. (Eye and Contact Lens) 44 ( 5 ) 273 - 278 2018

Accepted, ISSN 15422321

Copyright © 2018 Contact Lens Association of Ophthalmologists, Inc. Unauthorized reproduction of this article is prohibited. During the past 30 years, the prevalence rate of myopia has been increased dramatically. Myopia has become one of the leading causes of vision loss in some countries, whereas the mechanism of the main pathological change in myopia is still largely unknown. Although several studies showed genetic background influences the phenotype of myopia to some extent, the sudden increase of morbidity cannot be explained by genetics only. The change in lifestyle results in tremendous change in the light environment, which can be considered to play an important role in the onset and progression of myopia. The difference between indoor and outdoor light environments such as intensity and wavelength of modern electronic lighting equipment may be a cue for myopia control as environmental factors. In this review, we discuss the relationship between myopia and light environment focusing on the basic and clinical studies.

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Elevated Aqueous Cytokine Levels in Eyes With Ocular Surface Diseases.

Aketa N, Yamaguchi T†, Asato T, Yagi-Yaguchi Y, Suzuki T, Higa K, Kurihara T, Satake Y, Tsubota K, Shimazaki J

Am J Ophthalmol. (ELSEVIER SCIENCE INC) 184 42 - 51 2017.12

Accepted, ISSN 0002-9394

PURPOSE: To evaluate cytokine and protein levels in the aqueous humor (AqH) of eyes with ocular surface diseases.
DESIGN: Prospective consecutive case series.
METHODS: This study includes 14 patients (aged 62.4 +/- 13.7 years) with chronic-phase ocular surface diseases (4 with ocular cicatricial pemphigoid, 5 with chemical burns, 2 with a thermal burn, 2 with Stevens-Johnson syndrome, and 1 with exposure keratitis), 14 matched patients without ocular surface disease (controls with corneal scar), and 30 patients who underwent cataract surgery (healthy controls). AqH samples were collected at the beginning of surgery. AqH levels of cytokines (interleukin [IL]-1 alpha, IL-1 beta, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, monocyte chemotactic protein [MCP]-1, interferon [IFN]-alpha, IFN-gamma, macrophage inflammatory protein [MIP]-1 alpha, MIP-1 beta, P-selectin, E-selectin, soluble-intercellular adhesion molecule [s-ICAM]-1, tumor necrosis factor [TNF]-alpha, granulocyte-macrophage colony-stimulating factor [GMCSF], IFN-gamma-induced protein [IP]-10) were measured using multiplex beads immunoassays.
RESULTS: The levels of IL-6, IL-10, IL-17A, GM-CSF, E-selectin, P-selectin, and s-ICAM in AqH were significantly elevated in eyes with ocular surface diseases (in pg/mL: 1696 +/- 804, 4.0 +/- 1.0, 24.3 +/- 9.8, 26.0 +/- 18.3, 5150 +/- 1232, 13122 +/- 7219, and 7914 +/- 2813, respectively), compared to healthy controls (IL-6: 6.36 +/- 0.94, P = .001; IL-10: 1.68 +/- 0.04, P = .0006; IL-17A: 3.7 +/- 0.2, P = .008; GM-CSF: 2.7 +/- 0.3, P = .007; E-selectin: 2093 +/- 37, P = .0001; P-selectin: 3658 +/- 137, P = .0001; sICAM-1: 1397 +/- 119, P = .008). The levels of IL-6, IL-17A, E-selectin, and P-selectin in AqH were significantly higher in eyes with ocular surface diseases compared to those with corneal scar (IL-6: 44.1 +/- 15.0, P = .0077; IL-17A: 4.1 +/- 0.7, P = .034; E-selectin: 2439 +/- 302, P = .039; and P-selectin: 5673 +/- 1553, P = .017).
CONCLUSIONS: Multiple AqH cytokine levels were elevated in chronic ocular surface diseases. ((C) 2017 Elsevier Inc. All rights reserved.)

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Violet Light Transmission is Related to Myopia Progression in Adult High Myopia

Torii H, Ohnuma K, Kurihara T, Tsubota K, Negishi K†

Sci Rep. (NATURE PUBLISHING GROUP) 7 ( 1 ) 14523 2017.11

Accepted, ISSN 2045-2322

Myopia is increasing worldwide. Although the exact etiology of myopia is unknown, outdoor activity is one of the most important environmental factors for myopia control. We previously reported that violet light (VL, 360-400 nm wavelength), which is abundant in the outdoor environment, suppressed myopia progression for individuals under 20 years of age. However, whether VL is also effective for adult high myopia, which can be sight-threatening, has remained unknown. To investigate the influence of VL for adult myopia, we retrospectively compared the myopic progression and the axial length elongation over five years in adult high myopic patients over 25 years of age after two types (non-VL transmitting and VL transmitting) of phakic intraocular lens (pIOL) implantation. We found that high myopic patients with the non-VL transmitting pIOLs implanted are almost two times more myopic in the change of refraction and four times longer in the change of axial length, compared to those implanted with the VL transmitting pIOLs. This result indicated that the VL transmitting pIOL suppressed myopia progression and axial length elongation compared with the non-VL transmitting one. In conclusion, our study showed the VL possibly has an anti-myopia effect for human adults with high myopia.

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

Ozawa Y*†, Shigeno Y*, Nagai N, Suzuki M, Kurihara T, Minami S, Hirano E, Shinoda H, Kobayashi S, Tsubota K

BMC Ophthalmol. (BIOMED CENTRAL LTD) 17 ( 1 ) 161 2017.08

Accepted, ISSN 1471-2415

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 (R), 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 (R) 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-2 = 0.885, P = 0. 0001; aged healthy: R-2 = 0.765, P = 0.001; AMD-fellow: R-2 = 0.851, P = 0.0001; and AMD: R-2 = 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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Predictive factors of better outcomes by monotherapy of an antivascular endothelial growth factor drug, ranibizumab, for diabetic macular edema in clinical practice.

Sato S, Shinoda H, Nagai N, Suzuki M, Uchida A, Kurihara T, Kamoshita M, Tomita Y, Iyama C, Minami S, Yuki K, Tsubota K, Ozawa Y†

Medicine. (LIPPINCOTT WILLIAMS & WILKINS) 96 ( 16 ) e6459 2017.04

Accepted, ISSN 0025-7974

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=-.57; P=.003) and CRT (r=-.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 mu m at 12 months was associated with a greater CRT at baseline (OR 0.87 per 10 mu m [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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Violet Light Exposure Can Be a Preventive Strategy Against Myopia Progression.

Torii H, Kurihara T, Seko Y, Negishi K, Ohnuma K, Inaba T, Kawashima M, Jiang X, Kondo S, Miyauchi M, Miwa Y, Katada Y, Mori K, Kato K, Tsubota K, Goto H, Oda M, Hatori M, Tsubota K†

EBioMedicine. (ELSEVIER SCIENCE BV) 15 210 - 219 2017.02

Accepted, ISSN 2352-3964

Prevalence of myopia is increasing worldwide. Outdoor activity is one of the most important environmental factors for myopia control. Here we show that violet light (VL, 360-400 nm wavelength) suppresses myopia progression. First, we confirmed that VL suppressed the axial length (AL) elongation in the chick myopia model. Expression microarray analyses revealed that myopia suppressive gene EGR1 was upregulated by VL exposure. VL exposure induced significantly higher upregulation of EGR1 in chick chorioretinal tissues than blue light under the same conditions. Next, we conducted clinical research retrospectively to compare the AL elongation among myopic children who wore eyeglasses (VL blocked) and two types of contact lenses (partially VL blocked and VL transmitting). The data showed the VL transmitting contact lenses suppressed myopia progression most. These results suggest that VL is one of the important outdoor environmental factors for myopia control. Since VL is apt to be excluded from our modern society due to the excessive UV protection, VL exposure can be a preventive strategy against myopia progression. (C) 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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

Nagai, Norihiro, Suzuki, Misa, Uchida, Atsuro, Kurihara, Toshihide, Kamoshita, Mamoru, Minami, Sakiko, Shinoda, Hajime, Tsubota, Kazuo, Ozawa, Yoko

SCIENTIFIC REPORTS (NATURE PUBLISHING GROUP) 6 52 - 59 2016.07

Research paper (scientific journal), Accepted, ISSN 2045-2322

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

Suzuki, Misa, Nagai, Norihiro, Shinoda, Hajime, Uchida, Atsuro, Kurihara, Toshihide, Tomita, Yohei, Kamoshita, Mamoru, Iyama, Chigusa, Tsubota, Kazuo, Ozawa, Yoko

AMERICAN JOURNAL OF OPHTHALMOLOGY (ELSEVIER SCIENCE INC) 166 52 - 59 2016.06

Research paper (scientific journal), Accepted, ISSN 0002-9394

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-naive 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. (C) 2016 The Authors. Published by Elsevier Inc.

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Development and pathological changes of neurovascular unit regulated by hypoxia response in the retina.

Kurihara T†

Prog Brain Res. (ELSEVIER SCIENCE BV) 225 201 - 211 2016.04

Lead author, Last author, Corresponding author, Accepted, ISSN 0079-6123

Retina is a highly vascularized tissue with a high oxygen and metabolic demand receiving light located in the back of the eye. The development and the maintenance of the retinal vasculature are important to regulate the homeostasis in the tissue. a Subunits of hypoxia-inducible factor (HIF) are key molecules in hypoxia response inducing genes required for cell survival such as vascular endothelial growth factor under hypoxia. Neurons, glia, and vascular endothelium cells interdependently form neurovascular unit in the retina tightly regulated by hypoxia response via HIF expression. A corruption of the precise hypoxia response in the developmental or matured retinal tissue may lead congenital vascular anomalies or adult neovascular ocular diseases. To regulate hypoxia response through HIF activity would be an ideal therapeutic target for these vision-threatening eye diseases.

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Hypoxia-induced metabolic stress in retinal pigment epithelial cells is sufficient to induce photoreceptor degeneration

Kurihara, Toshihide, Westenskow, Peter D., Gantner, Marin L., Usui, Yoshihiko, Schultz, Andrew, Bravo, Stephen, Aguilar, Edith, Wittgrove, Carli, Friedlander, Mollie S. H., Paris, Liliana P., Chew, Emily, Siuzdak, Gary, Friedlander, Martin

ELIFE (ELIFE SCIENCES PUBLICATIONS LTD) 5 52 - 59 2016.03

Research paper (scientific journal), Accepted, ISSN 2050-084X

Photoreceptors are the most numerous and metabolically demanding cells in the retina. Their primary nutrient source is the choriocapillaris, and both the choriocapillaris and photoreceptors require trophic and functional support from retinal pigment epithelium (RPE) cells. Defects in RPE, photoreceptors, and the choriocapillaris are characteristic of age-related macular degeneration (AMD), a common vision-threatening disease. RPE dysfunction or death is a primary event in AMD, but the combination(s) of cellular stresses that affect the function and survival of RPE are incompletely understood. Here, using mouse models in which hypoxia can be genetically triggered in RPE, we show that hypoxia-induced metabolic stress alone leads to photoreceptor atrophy. Glucose and lipid metabolism are radically altered in hypoxic RPE cells; these changes impact nutrient availability for the sensory retina and promote progressive photoreceptor degeneration. Understanding the molecular pathways that control these responses may provide important clues about AMD pathogenesis and inform future therapies.

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iPSC-Derived Retinal Pigment Epithelium Allografts Do Not Elicit Detrimental Effects in Rats: A Follow-Up Study.

Westenskow PD, Bucher F, Bravo S, Kurihara T, Feitelberg D, Paris LP, Aguilar E, Lin JH, Friedlander M†

Stem Cells Int. (HINDAWI LTD) 2016 Article ID 8470263 2016.01

Accepted, ISSN 1687-966X

Phototransduction is accomplished in the retina by photoreceptor neurons and retinal pigment epithelium (RPE) cells. Photoreceptors rely heavily on the RPE, and death or dysfunction of RPE is characteristic of age-related macular degeneration (AMD), a very common neurodegenerative disease for which no cure exists. RPE replacement is a promising therapeutic intervention for AMD, and large numbers of RPE cells can be generated from pluripotent stem cells. However, questions persist regarding iPSC-derived RPE (iPS-RPE) viability, immunogenicity, and tumorigenesis potential. We showed previously that iPS-RPE prevent photoreceptor atrophy in dystrophic rats up until 24 weeks after implantation. In this follow-up study, we longitudinally monitored the same implanted iPS-RPE, in the same animals. We observed no gross abnormalities in the eyes, livers, spleens, brains, and blood in aging rats with iPSC-RPE grafts. iPS-RPE cells that integrated into the subretinal space outlived the photoreceptors and survived for as long as 21/2 years while nonintegrating RPE cells were ingested by host macrophages. Both populations could be distinguished using immunohistochemistry and electron microscopy. iPSC-RPE could be isolated from the grafts and maintained in culture; these cells also phagocytosed isolated photoreceptor outer segments. We conclude that iPS-RPE grafts remain viable and do not induce any obvious associated pathological changes.

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Global metabolomics reveals metabolic dysregulation in ischemic retinopathy

Paris, Liliana P., Johnson, Caroline H., Aguilar, Edith, Usui, Yoshihiko, Cho, Kevin, Hoang, Lihn T., Feitelberg, Daniel, Benton, H. Paul, Westenskow, Peter D., Kurihara, Toshihide, Trombley, Jennifer, Tsubota, Kinya, Ueda, Shunichiro, Wakabayashi, Yoshihiro, Patti, Gary J., Ivanisevic, Julijana, Siuzdak, Gary, Friedlander, Martin

METABOLOMICS (SPRINGER) 12 ( 1 ) 52 - 59 2016.01

Research paper (scientific journal), Accepted, ISSN 1573-3882

Proliferative diabetic retinopathy (PDR) is the most severe form of diabetic retinopathy and, along with diabetic macular edema, is responsible for the majority of blindness in adults below the age of 65. Therapeutic strategies for PDR are ineffective at curtailing disease progression in all cases; however a deeper understanding of the ocular metabolic landscape in PDR through metabolomic analysis may offer new therapeutic targets. Here, global and targeted mass spectrometry-based metabolomics were used to investigate metabolism. Initial analyses on vitreous humor from patients with PDR (n = 9) and non-diabetic controls (n = 11) revealed an increase of arginine and acylcarnitine metabolism in PDR. The oxygen-induced-retinopathy (OIR) mouse model, which exhibits comparable pathological manifestations to human PDR, revealed similar increases of arginine and other metabolites in the urea cycle, as well as downregulation of purine metabolism. We validated our findings by targeted multiple reaction monitoring and through the analysis of a second set of patient samples [PDR (n = 11) and non-diabetic controls (n = 20)]. These results confirmed a predominant and consistent increase in proline in both the OIR mouse model and vitreous samples from patients with PDR, suggesting that over activity in the arginine-to-proline pathway could be used as a therapeutic target in diabetic retinopathy.

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The Neuroprotective Effect of Rapamycin as a Modulator of the mTOR-NF-kappa B Axis during Retinal Inflammation

Okamoto, Tomohiro, Ozawa, Yoko, Kamoshita, Mamoru, Osada, Hideto, Toda, Eriko, Kurihara, Toshihide, Nagai, Norihiro, Umezawa, Kazuo, Tsubota, Kazuo

PLOS ONE (PUBLIC LIBRARY SCIENCE) 11 ( 1 ) 52 - 59 2016.01

Research paper (scientific journal), Accepted, ISSN 1932-6203

Purpose
The determination of the molecular mechanism underlying retinal pathogenesis and visual dysfunction during innate inflammation, and the treatment effect of rapamycin thereon.
Methods
The endotoxin-induced uveitis and retinitis mouse model was established by injecting lipopolysaccharide. The mice were subsequently treated with rapamycin, a mammalian target of rapamycin (mTOR) inhibitor. The rhodopsin mRNA and protein expression level in the retina and the photoreceptor outer segment (OS) length in immunohistochemical stainings were measured, and visual function was recorded by electroretinography. Inflammatory cytokines, their related molecules, mTOR, and LC3 levels were measured by real-time PCR and/or immunoblotting. Leukocyte adhesion during inflammation was analyzed using concanavalin A lectin.
Results
The post-transcriptional reduction in the visual pigment of rod photoreceptor cells, rhodopsin, OS shortening, and rod photoreceptor cell dysfunction during inflammation were suppressed by rapamycin. Activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B) and induction of inflammatory cytokines, such as interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), and the activation of the downstream signaling protein, signal transducer and activator of transcription 3 (STAT3), which reduces rhodopsin in the retina during inflammation, were attenuated by rapamycin. Increased leukocyte adhesion was also attenuated by rapamycin. Interestingly, although mTOR activation was observed after NF-kappa B activation, mTOR inhibition suppressed NF-kappa B activation at the early phase, indicating that the basal level of activated mTOR was sufficient to activate NF-kappa B in the retina. In addition, the inhibition of NF-kappa B suppressed mTOR activation, suggesting a positive feedback loop of mTOR and NF-kappa B during inflammation. The ratio of LC3II to LC3I, which reflects autophagy induction, was not changed by inflammation but was increased by rapamycin.
Conclusions
Our results propose the potential use of rapamycin as a neuroprotective therapy to suppress local activated mTOR levels, related inflammatory molecules, and the subsequent visual dysfunction during retinal inflammation.

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Effects of Constant Intake of Lutein-rich Spinach on Macular Pigment Optical Density: a Pilot Study

Ozawa Y†, Nagai N, Suzuki M, Kurihara T, Shinoda H, Watanabe M, Tsubota K

Nippon Ganka Gakkai zasshi 120 ( 1 ) 41 - 48 2016.01

Accepted, ISSN 0029-0203

Wide-Angle Viewing System versus Conventional Indirect Ophthalmoscopy for Scleral Buckling

Tomita, Yohei, Kurihara, Toshihide, Uchida, Atsuro, Nagai, Norihiro, Shinoda, Hajime, Tsubota, Kazuo, Ozawa, Yoko

SCIENTIFIC REPORTS (NATURE PUBLISHING GROUP) 5 275 - 281 2015.09

Research paper (scientific journal), Accepted, ISSN 2045-2322

Wide-angle viewing systems (WAVSs) were originally established for pars plana vitrectomy. However, their application to scleral buckling surgery was recently reported. In this study, we compared the outcomes of scleral buckling using a noncontact WAVS with that of scleral buckling using conventional indirect ophthalmoscopy for rhegmatogenous retinal detachment. The clinical records of 39 eyes (39 patients) with rhegmatogenous retinal detachment primarily treated between November 2012 and June 2014 at the Vitreo-Retina Surgical Division Clinic at the Department of Ophthalmology, Keio University Hospital were retrospectively reviewed. Scleral bucking was performed using WAVS with surgical placement of an endoilluminator in 16 eyes and indirect ophthalmoscopy in 23 eyes. The patients in these groups were consecutive over different intervals. The preoperative demographics, success rate of retinal reattachment, intraoperative findings, and postoperative complications were evaluated. There were no significant differences in pre- or postoperative conditions between groups, and similar surgical outcomes were achieved with the WAVS and conventional procedures. However, compared with the conventional procedure, the WAVS procedure resulted in fewer intraoperative corneal epithelial disorders (p = 0.049) and decreased the surgical duration of segmental buckling (p = 0.02); therefore, it may be suggested as an effective alternative procedure.

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Neurovascular crosstalk between interneurons and capillaries is required for vision

Usui, Voshihiko, Westenskow, Peter D., Kurihara, Toshihide, Aguilar, Edith, Sakimoto, Susumu, Paris, Liliana P., Wittgrove, Carli, Feitelberg, Daniel, Friedlander, Mollie S. H., Moreno, StaceyR., Dorrell, Michael I., Friedlander, Martin

JOURNAL OF CLINICAL INVESTIGATION (AMER SOC CLINICAL INVESTIGATION INC) 125 ( 6 ) 2335 - 2346 2015.06

Research paper (scientific journal), Accepted, ISSN 0021-9738

Functional interactions between neurons, vasculature, and glia within neurovascular units are critical for maintenance of the retina and other CNS tissues. For example, the architecture of the neurosensory retina is a highly organized structure with alternating layers of neurons and blood vessels that match the metabolic demand of neuronal activity with an appropriate supply of oxygen within perfused blood. Here, using murine genetic models and cell ablation strategies, we have demonstrated that a subset of retinal interneurons, the amacrine and horizontal cells, form neurovascular units with capillaries in 2 of the 3 retinal vascular plexuses. Moreover, we determined that these cells are required for generating and maintaining the intraretinal vasculature through precise regulation of hypoxia-inducible and proangiogenic factors, and that amacrine and horizontal cell dysfunction induces alterations to the intraretinal vasculature and substantial visual deficits. These findings demonstrate that specific retinal interneurons and the intraretinal vasculature are highly interdependent, and loss of either or both elicits profound effects on photoreceptor survival and function.

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Blue light-induced inflammatory marker expression in the retinal pigment epithelium-choroid of mice and the protective effect of a yellow intraocular lens material in vivo

Narimatsu, Toshio, Negishi, Kazuno, Miyake, Seiji, Hirasawa, Manabu, Osada, Hideto, Kurihara, Toshihide, Tsubota, Kazuo, Ozawa, Yoko

EXPERIMENTAL EYE RESEARCH 132 48 - 51 2015.03

Research paper (scientific journal), Accepted, ISSN 0014-4835

Oxidative stress in the retinal pigment epithelium (RPE) is a well-accepted pathogenic change in vision-threatening diseases such as age-related macular degeneration. One source of oxidative stress is excessive light exposure, which causes excessive activation of the visual cycle. Because short wavelength light (blue light) has more energy, it is reported to be more harmful to photoreceptor cells than the other wavelengths of light. However, the biological effect of blue light in the RPE of living animals and the protective effect of a yellow intraocular lens (IOL) material that blocks blue light is still obscure. Therefore, we compared the pathogenic effect in the RPE-choroid complexes of mice exposed to light in a box made of a clear or a yellow IOL material. We measured the level of reactive oxygen species (ROS) using 2', 7'-dichlorodihydrofluorescein diacetate, the mRNA levels of inflammatory cytokines and a macrophage marker by real-time polymerase chain reaction, and the protein level of monocyte chemotactic protein-1 (MCP-1) by ELISA. The ROS level after light exposure was suppressed in the RPE-choroids of light-exposed mice in the yellow IOL material box. In parallel, all the inflammatory cytokines that we measured and a macrophage marker were also suppressed in the RPE-choroids of light-exposed mice in the yellow IOL material box. Therefore, a yellow IOL material suppressed, and thus blue light exacerbated, the increase in the ROS level and inflammatory cytokine expression as well as macrophage recruitment in the RPE-choroid in vivo after light exposure.

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Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

Westenskow, Peter D., Kurihara, Toshihide, Bravo, Stephen, Feitelberg, Daniel, Sedillo, Zack A., Aguilar, Edith, Friedlander, Martin

JOVE-JOURNAL OF VISUALIZED EXPERIMENTS (JOURNAL OF VISUALIZED EXPERIMENTS) 23 ( 95 ) 2335 - 2346 2015.01

Research paper (scientific journal), Accepted, ISSN 1940-087X

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

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Neurons Limit Angiogenesis by Titrating VEGF in Retina

Okabe, Keisuke, Kobayashi, Sakiko, Yamada, Toru, Kurihara, Toshihide, Tai-Nagara, Ikue, Miyamoto, Takeshi, Mukouyama, Yoh-suke, Sato, Thomas N., Suda, Toshio, Ema, Masatsugu, Kubota, Yoshiaki

CELL (CELL PRESS) 159 ( 3 ) 584 - 596 2014.10

Research paper (scientific journal), Accepted, ISSN 0092-8674

Vascular and nervous systems, two major networks in mammalian bodies, show a high degree of anatomical parallelism and functional crosstalk. During development, neurons guide and attract blood vessels, and consequently this parallelism is established. Here, we identified a noncanonical neurovascular interaction in eye development and disease. VEGFR2, a critical endothelial receptor for VEGF, was more abundantly expressed in retinal neurons than in endothelial cells, including endothelial tip cells. Genetic deletion of VEGFR2 in neurons caused misdirected angiogenesis toward neurons, resulting in abnormally increased vascular density around neurons. Further genetic experiments revealed that this misdirected angiogenesis was attributable to an excessive amount of VEGF protein around neurons caused by insufficient engulfment of VEGF by VEGFR2-deficient neurons. Moreover, absence of neuronal VEGFR2 caused misdirected regenerative angiogenesis in ischemic retinopathy. Thus, this study revealed neurovascular crosstalk and unprecedented cellular regulation of VEGF: retinal neurons titrate VEGF to limit neuronal vascularization.

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Intraoperative and fluorescein angiographic findings of a secondary macular hole associated with age-related macular degeneration treated by pars plana vitrectomy

Okamoto, Tomohiro, Shinoda, Hajime, Kurihara, Toshihide, Nagai, Norihiro, Tsubota, Kazuo, Ozawa, Yoko

BMC OPHTHALMOLOGY (BIOMED CENTRAL LTD) 14 24561 - 24570 2014.09

Research paper (scientific journal), Accepted, ISSN 1471-2415

Background: Macular hole results from a tractional force at the vitreo-retinal interface which is developed by modification and subsequent degeneration of the posterior precortical vitreous and the internal limiting membrane (ILM). Meanwhile, the wet type of age-related macular degeneration (AMD) is caused by the submacular formation of choroidal neovascularization (CNV). Although exudative changes derived from CNV may cause epiretinal membrane (ERM) formation, which can also cause tractional force at the vitreo-retinal interface, there have been few reports of AMD-associated macular hole development in which the full thickness of the retinal tissue is completely torn by the tractional force. Moreover, intraoperative finding of macular hole associated with AMD with a possible involvement of subretinal lesion has not been described.
Case presentation: A 78-year-old man diagnosed with wet AMD with subretinal fluid and mild cataract received 8 treatments with intravitreal pegaptanib. After AMD remission, he developed a secondary macular hole in the same eye. He underwent a pars plana vitrectomy that successfully closed the macular hole. Intraoperatively, it was found that the patient's vitreous was formed and that the ERM and ILM were adherent, suggesting the involvement of a tractional force at the vitreo-retinal interface due to an inflammatory reaction related to AMD and/or intravitreally injected chemical compounds, resulting in macular hole development. Changes in the condition of his AMD and the RPE were observed on a fluorescein angiogram (FA) and an indocyanine green angiogram (IA) that preceded macular hole development, suggesting that subretinal changes may also have been involved in the pathogenesis.
Conclusion: These clinical data, including the intraoperative findings and the temporal changes in the angiograms, suggest that an inflammatory reaction at the vitreo-retinal interface and subretinal lesion related to AMD contribute to the macular hole development in AMD patients treated with intravitreal injection.

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Local acting Sticky-trap inhibits vascular endothelial growth factor dependent pathological angiogenesis in the eye

Michael, Iacovos P., Westenskow, Peter D., Hacibekiroglu, Sabiha, Greenwald, Alissa Cohen, Ballios, Brian G., Kurihara, Toshihide, Li, Zhijie, Warren, Carmen M., Zhang, Puzheng, Aguilar, Edith, Donaldson, Laura, Marchetti, Valentina, Baba, Takeshi, Hussein, Samer M., Sung, Hoon-Ki, Iruela-Arispe, M. Luisa, Rini, James M., van der Kooy, Derek, Friedlander, Martin, Nagy, Andras

EMBO MOLECULAR MEDICINE (WILEY) 6 ( 5 ) 604 - 623 2014.05

Research paper (scientific journal), Accepted, ISSN 1757-4676

Current therapeutic antiangiogenic biologics used for the treatment of pathological ocular angiogenesis could have serious side effects due to their interference with normal blood vessel physiology. Here, we report the generation of novel antivascular endothelial growth factor-A (VEGF) biologics, termed VEGF Sticky-traps, with unique properties that allow for local inhibition of angiogenesis without detectable systemic side effects. Using genetic and pharmacological approaches, we demonstrated that Sticky-traps could locally inhibit angiogenesis to at least the same extent as the original VEGF-trap that also gains whole-body access. Sticky-traps did not cause systemic effects, as shown by uncompromised wound healing and normal tracheal vessel density. Moreover, if injected intravitreally, recombinant Sticky-trap remained localized to various regions of the eye, such as the inner-limiting membrane and ciliary body, for prolonged time periods, without gaining access either to the photoreceptors/choriocapillaris area or the circulation. These unique pharmacological characteristics of Sticky-trap could allow for safe treatment of pathological angiogenesis in patients with diabetic retinopathy and retinopathy of pre-maturity.

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Utilizing Stem Cell-Derived RPE Cells as A Therapeutic Intervention for Age-Related Macular Degeneration

Westenskow, Peter D., Kurihara, Toshihide, Friedlander, Martin

RETINAL DEGENERATIVE DISEASES: MECHANISMS AND EXPERIMENTAL THERAPY (SPRINGER-VERLAG BERLIN) 801 323 - 329 2014

Research paper (scientific journal), Accepted, ISSN 0065-2598

Degeneration or dysfunction of the retinal pigment epithelium (RPE) can induce secondary photoreceptor atrophy and catastrophic vision loss in patients with agerelated macular degeneration (AMD). AMD is the leading cause of vision loss in the elderly in industrialized countries and no cure exists for the "dry" or atrophic form to date. However, recent pre-clinical data from several groups suggests that embryonic stem cell-derived RPE cell transplantation may prevent photoreceptor degeneration in animal models of RPE degeneration. Another approach may be to derive RPE cells from autologous induced pluripotent stem cells (iPSCs) reprogrammed from dermal tissue. However, the safety of this approach has been questioned on several levels. In this chapter we will summarize work reported by several groups, including our own, that clearly demonstrate that transplanted RPE cells can provide anatomical and functional photoreceptor rescue in animal models of retinal degeneration. We will also discuss some of the prevailing concerns and challenges associated with this technique.

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Hypoxia-Inducible Factor (HIF)/Vascular Endothelial Growth Factor (VEGF) Signaling in the Retina

Kurihara, Toshihide, Westenskow, Peter D., Friedlander, Martin

RETINAL DEGENERATIVE DISEASES: MECHANISMS AND EXPERIMENTAL THERAPY (SPRINGER-VERLAG BERLIN) 801 275 - 281 2014

Research paper (scientific journal), Accepted, ISSN 0065-2598

Over a span of two decades, it has become increasingly clear that vascular endothelial growth factor (VEGF) plays an important role in the pathogenesis of retinal diseases including age-related macular degeneration (AMD) and diabetic retinopathy (DR). Based on these observations, anti-VEGF therapies are being developed and approved for clinical use in the treatment of neovascular eye diseases. Hypoxia-inducible factors (HIFs) are transcriptional factors that are stabilized and activated under hypoxic conditions and induce expression of gene products, including VEGF, that are required for cell survival under hypoxia. Here we discuss recent findings from our lab and others that define roles of the HIF-VEGF axis in the retina.

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Ras pathway inhibition prevents neovascularization by repressing endothelial cell sprouting

Westenskow, Peter D., Kurihara, Toshihide, Aguilar, Edith, Scheppke, Elizabeth L., Moreno, Stacey K., Wittgrove, Carli, Marchetti, Valentina, Michael, Iacovos R., Anand, Sudarshan, Nagy, Andras, Cheresh, David, Friedlander, Martin

JOURNAL OF CLINICAL INVESTIGATION (AMER SOC CLINICAL INVESTIGATION INC) 123 ( 11 ) 4900 - 4908 2013.11

Research paper (scientific journal), Accepted, ISSN 0021-9738

Vascular networks develop from a growing vascular front that responds to VEGF and other guidance cues. Angiogenesis is required for normal tissue function, but, under conditions of stress, inappropriate vascularization can lead to disease. Therefore, inhibition of angiogenic sprouting may prevent neovascularization in patients with blinding neovascular eye diseases, including macular degeneration. VEGF antagonists have therapeutic benefits but also can elicit off-target effects. Here, we found that the Ras pathway, which functions downstream of a wide range of cytokines including VEGF, is active in the growing vascular front of developing and pathological vascular networks. The endogenous Ras inhibitor p120RasGAP was expressed predominately in quiescent VEGF-insensitive endothelial cells and was ectopically downregulated in multiple neovascular models. MicroRNA-132 negatively regulated p120RasGAP expression. Experimental delivery of alpha-miR-132 to developing mouse eyes disrupted tip cell Ras activity and prevented angiogenic sprouting. This strategy prevented ocular neovascularization in multiple rodent models even more potently than the VEGF antagonist, VEGF-trap. Targeting microRNA-132 as a therapeutic strategy may prove useful for treating multiple neovascular diseases of the eye and for preventing vision loss regardless of the neovascular stimulus.

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VEGF antagonism and age-related macular degeneration: too much of a good thing?

Kurihara T, Westenskow PD, Aguilar E, Friedlander M†

Exp Rev Ophthalmol. 8 ( 2 ) 103 - 105 2013.04

Lead author

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Targeted deletion of Vegfa in adult mice induces vision loss

Kurihara, Toshihide, Westenskow, Peter D., Bravo, Stephen, Aguilar, Edith, Friedlander, Martin

JOURNAL OF CLINICAL INVESTIGATION (AMER SOC CLINICAL INVESTIGATION INC) 122 ( 11 ) 4213 - 4217 2012.11

Research paper (scientific journal), Accepted, ISSN 0021-9738

Current therapies directed at controlling vascular abnormalities in cancers and neovascular eye diseases target VEGF and can slow the progression of these diseases. While the critical role of VEGF in development has been well described, the function of locally synthesized VEGF in the adult eye is incompletely understood. Here, we show that conditionally knocking out Vegfa in adult mouse retinal pigmented epithelial (RPE) cells, which regulate retinal homeostasis, rapidly leads to vision loss and ablation of the choriocapillaris, the major blood supply for the outer retina and photoreceptor cells. This deletion also caused rapid dysfunction of cone photoreceptors, the cells responsible for fine visual acuity and color vision. Furthermore, Vegfa deletion showed significant downregulation of multiple angiogenic genes in both physiological and pathological states, whereas the deletion of the upstream regulatory transcriptional factors HIFs did not affect the physiological expressions of angiogenic genes. These results suggest that endogenous VEGF provides critical trophic support necessary for retinal function. Targeting factors upstream of VEGF, such as HIFs, may be therapeutically advantageous compared with more potent and selective VEGF antagonists, which may have more off-target inhibitory trophic effects.

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Using Flow Cytometry to Compare the Dynamics of Photoreceptor Outer Segment Phagocytosis in iPS-Derived RPE Cells

Westenskow, Peter D., Moreno, Stacey K., Krohne, Tim U., Kurihara, Toshihide, Zhu, Saiyong, Zhang, Zhen-ning, Zhao, Tongbiao, Xu, Yang, Ding, Sheng, Friedlander, Martin

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 53 ( 10 ) 6282 - 6290 2012.09

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. Retinal pigment epithelium (RPE) autologous grafts can be readily derived from induced pluripotent stem (iPS) cells. It is critical to stringently characterize iPS-RPE using standardized and quantifiable methods to be confident that they are safe and adequate replacements for diseased RPE before utilizing them in clinical settings. One important and required function is that the iPS-RPE phagocytose photoreceptor outer segments (POS).
METHODS. We developed a flow cytometry-based assay to monitor binding and internalization of FITC labeled POS by ARPE-19, human fetal RPE (hfRPE), and two types of iPS-RPE. Expression and density of alpha(v)beta(5) integrin, CD36, and MerTK receptors, which are required for phagocytosis, were compared.
RESULTS. Trypsinization of treated RPE cells results in the release of bound POS. The number of freed POS, the percentage of cells that internalized POS, the brightness of the FITC signal from the cells, and the surface density of the phagocytosis receptors on single RPE cells were measured using flow cytometry. These assays reveal that receptor density is dynamic during differentiation and this can affect the binding and internalization dynamics of the RPE cells. Highly differentiated iPS-RPE phagocytose POS more efficiently than hfRPE.
CONCLUSIONS. Caution should be exercised to not use RPE grafts until demonstrating that they are fully functional. The density of the phagocytosis receptors is dynamic and may be used as a predictor for how well the iPS-RPE cells will function in vivo. The phagocytosis dynamics observed between iPS-RPE and primary RPE is very encouraging and adds to mounting evidence that iPS-RPE may be a viable replacement for dysfunctional or dying RPE in human patients. (Invest Ophthalmol Vis Sci. 2012;53:6282-6290) DOI:10.1167/iovs.12-9721

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Biological role of lutein in the light-induced retinal degeneration

Sasaki, Mariko, Yuki, Kenya, Kurihara, Toshihide, Miyake, Seiji, Noda, Kosuke, Kobayashi, Saori, Ishida, Susumu, Tsubota, Kazuo, Ozawa, Yoko

JOURNAL OF NUTRITIONAL BIOCHEMISTRY (ELSEVIER SCIENCE INC) 23 ( 5 ) 423 - 429 2012.05

Research paper (scientific journal), Accepted, ISSN 0955-2863

Lutein, a xanthophyll of a carotenoid, is anticipated as a therapeutic product to prevent human eye diseases. However, its biological mechanism is still unclear. Here, we show the molecular mechanism of lutein's effect to reduce photodamage of the retina. We analyzed the light-exposed retinas of Balb/c mice given lutein-supplemented or normal diet. Visual function was measured by electroretinogram, and histological changes were observed. Immunohistochemical and immunoblot analyses were performed to analyze molecular mechanism. The reactive oxygen species induced in the retina was evaluated by fluorescent probes. In the mice after light exposure, reduction of a-wave and b-wave amplitudes in electroretinogram, indicating visual impairment, and thinning of the photoreceptor cell layer owing to apoptosis were both attenuated by lutein diet. Interestingly, gamma-H2AX, a marker for double-strand breaks (DSBs) in DNA, was up-regulated in the photoreceptor cells after light exposure, but this increase was attenuated by lutein diet, suggesting that DSBs caused by photodamage contributed to the photoreceptor cell death and that this change was suppressed by lutein. Moreover, the expression of eyes absent (EYA), which promotes DNA repair and cell survival, was significantly up-regulated with lutein diet in the light-exposed retina. Therefore, lutein induced EYA for DNA repair, which could suppress DNA damage and photoreceptor cell apoptosis. Lutein reduced light-induced oxidative stress in the retina, which might contribute to promote DNA repair. The lutein-supplemented diet attenuated light-induced visual impairment by protecting the photoreceptor cells' DNA. (C) 2012 Elsevier Inc. All rights reserved.

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Renin-Angiotensin System Hyperactivation Can Induce Inflammation and Retinal Neural Dysfunction.

Kurihara T†, Ozawa Y, Ishida S, Okano H, Tsubota K

Int J Inflam. 2012 581695 2012.03

Lead author, Corresponding author, Accepted

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The formation of an angiogenic astrocyte template is regulated by the neuroretina in a HIF-1-dependent manner

Nakamura-Ishizu, Ayako, Kurihara, Toshihide, Okuno, Yuji, Ozawa, Yoko, Kishi, Kazuo, Goda, Nobuhito, Tsubota, Kazuo, Okano, Hideyuki, Suda, Toshio, Kubota, Yoshiaki

DEVELOPMENTAL BIOLOGY (ACADEMIC PRESS INC ELSEVIER SCIENCE) 363 ( 1 ) 106 - 114 2012.03

Research paper (scientific journal), Accepted, ISSN 0012-1606

The vascular and nervous systems display a high degree of cross-talk and depend on each other functionally. In the vascularization of the central nervous system, astrocytes have been thought to sense tissue oxygen levels in hypoxia-inducible factors (HIFs)-dependent manner and control the vascular growth into the hypoxic area by secreting VEGF. However, recent genetic evidences demonstrate that not only astrocyte HIFs but also astrocyte VEGF expression is dispensable for developmental angiogenesis of the retina. This study demonstrates that hypoxia-inducible factor 1 alpha subunit (HIF-1 alpha), a key transcription factor involved in cellular responses to hypoxia, is most abundantly expressed in the neuroretina, especially retinal progenitor cells (RPCs). A neuroretina-specific knockout of HIF-1 alpha (alpha Cre(+)Hif1 alpha(flox/flox)) showed impaired vascular development characterized by decreased tip cell filopodia and reduced vessel branching. The astrocyte network was hypoplastic in alpha Cre(+)Hif1 alpha(flox/flox) mice. Mechanistically, platelet-derived growth factor A (PDGF-A), a mitogen for astrocytes, was downregulated in the neuroretina of alpha Cre(+)Hif1 alpha(flox/flox) mice. Supplementing PDGF-A restored reduced astrocytic and vascular density in alpha Cre(+)Hif1 alpha(flox/flox) mice. Our data demonstrates that the neuroretina but not astrocytes acts as a primary oxygen sensor which ultimately controls the retinal vascular development by regulating an angiogenic astrocyte template. (C) 2011 Elsevier Inc. All rights reserved.

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Generation of Retinal Pigment Epithelial Cells from Small Molecules and OCT4 Reprogrammed Human Induced Pluripotent Stem Cells

Krohne, Tim U., Westenskow, Peter D., Kurihara, Toshihide, Friedlander, David F., Lehmann, Mandy, Dorsey, Alison L., Li, Wenlin, Zhu, Saiyong, Schultz, Andrew, Wang, Junhua, Siuzdak, Gary, Ding, Sheng, Friedlander, Martin

STEM CELLS TRANSLATIONAL MEDICINE (ALPHAMED PRESS) 1 ( 2 ) 96 - 109 2012.02

Research paper (scientific journal), Accepted, ISSN 2157-6564

Autologous retinal pigment epithelium (RPE) grafts derived from induced pluripotent stem cells (iPSCs) may be used to cure blinding diseases in which RPE dysfunction results in photoreceptor degeneration. Four-, two-, and one-factor-derived iPSCs (4F-, 2F-, and 1F-iPSC5, respectively) were differentiated into fully functional cuboidal pigmented cells in polarized monolayers that express RPE-specific markers. 1F-iPSC-RPE (1F-iPS-RPE) strongly resembles primary human fetal RPE (hfRPE) based on proteomic and untargeted metabolomic analyses, and using novel in vivo imaging technology coupled with electroretinography, we demonstrated that 1F-iPS-RPE mediate anatomical and functional rescue of photoreceptors after transplantation in an animal model of RPE-mediated retinal degeneration. 1F-iPS-RPE cells were injected subretinally as a suspension and formed a monolayer dispersed between host RPE cells. Furthermore, 1F-iPS-RPE do not simply provide trophic support to rescue photoreceptors as previously speculated but actually phagocytose photoreceptor outer segments in vivo and maintain visual cycling. Thus, 1F-iPS-RPE grafts may be superior to conventional iPS-RPE for clinical use because 1F-iPS-RPE closely resemble hfRPE, mediate anatomical and functional photoreceptor rescue in vivo, and are generated using a reduced number of potentially oncogenic reprogramming factors. STEM CELLS TRANSLATIONAL MEDICINE 2012;1:96-109

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Roles of AMP-Activated Protein Kinase in Diabetes-Induced Retinal Inflammation

Kubota, Shunsuke, Ozawa, Yoko, Kurihara, Toshihide, Sasaki, Mariko, Yuki, Kenya, Miyake, Seiji, Noda, Kousuke, Ishida, Susumu, Tsubota, Kazuo

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 52 ( 12 ) 9142 - 9148 2011.11

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. The purpose of the present study was to elucidate the roles of AMPK in the pathogenesis of diabetic retinopathy using the known AMPK activators resveratrol and AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) in a mouse model.
METHODS. C57BL/6 mice with streptozotocin-induced diabetes were treated with resveratrol orally at 50 mg/kg for 7 days or with AICAR intraperitoneally at 100 mg/kg 24 hours before death. Retinal protein levels of phosphorylated and total AMPK, phosphorylated nuclear factor (NF)-kappa B p65, intercellular adhesion molecule (ICAM)-1, and vascular endothelial growth factor (VEGF) were evaluated by Western blot analysis or enzyme-linked immunosorbent assay. Retinal activity of sirtuin (SIRT) 1 was measured by deacetylase fluorometric assay. Leukocyte adhesion to the retinal vasculature was examined with a concanavalin A lectin perfusion-labeling technique.
RESULTS. Induction of diabetes in mice led to retinal AMPK dephosphorylation, which was significantly reversed by either resveratrol or AICAR. Either resveratrol or AICAR significantly reversed SIRT1 deactivation and NF-kappa B phosphorylation, both of which were induced in the diabetic retina. Administration of resveratrol to diabetic mice significantly reduced diabetes-induced retinal leukocyte adhesion, together with retinal expression of ICAM-1 and VEGF.
CONCLUSIONS. The present findings reveal that diabetes-induced retinal inflammation stems from downregulation of the AMPK pathway, leading subsequently to SIRT1 deactivation and NF-kappa B activation. The data also suggest the potential use of the AMPK activator resveratrol as a therapeutic agent for diabetic retinopathy. (Invest Ophthalmol Vis Sci. 2011;52:9142-9148) DOI: 10.1167/iovs.11-8041

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Astrocyte pVHL and HIF-alpha isoforms are required for embryonic-to-adult vascular transition in the eye

Kurihara, Toshihide, Westenskow, Peter D., Krohne, Tim U., Aguilar, Edith, Johnson, Randall S., Friedlander, Martin

JOURNAL OF CELL BIOLOGY (ROCKEFELLER UNIV PRESS) 195 ( 4 ) 689 - 701 2011.11

Research paper (scientific journal), Accepted, ISSN 0021-9525

Successful transition from embryonic to adult circulation is critical for survival of mammalian organisms. This shift occurs in the central cardiovascular circulation and in the eye as oxygen tension increases. However, its regulation is not well understood. We have used combinatorial gene deletion and overexpression assays to assess the effect of astrocyte-targeted deletion of von Hippel-Lindau tumor suppressor (Vhl), hypoxia-inducible factor-alpha s (Hif-alpha s), and Vegf on the normal regression of the hyaloidal vessels, the fetal ocular circulation system. Astrocytic Vhl deletion induced accelerated hyaloidal regression and subsequent massive secondary outgrowth. Combinatorial gene deletion involving Vhl, Hif-alpha s, and Vegf genes revealed that HIF-2 alpha/vascular endothelial growth factor signaling induces secondary outgrowth in Vhl mutants. Conversely, HIF-1 alpha regulated macrophage migration inhibitory factor and promoted macrophage infiltration that accelerates hyaloidal vessel regression. The phenotype observed in Vhl mutants strongly resembles human persistent hyperplastic primary vitreous cases and may provide insights into vascular remodeling mechanisms in other systems.

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Retinal Ganglion Cell Loss in Superoxide Dismutase 1 Deficiency

Yuki, Kenya, Ozawa, Yoko, Yoshida, Tetsu, Kurihara, Toshihide, Hirasawa, Manabu, Ozeki, Naoki, Shiba, Daisuke, Noda, Kousuke, Ishida, Susumu, Tsubota, Kazuo

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 52 ( 7 ) 4143 - 4150 2011.06

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. To investigate the influence of deficiency in superoxide dismutase (SOD) 1, a major antioxidative enzyme, on retinal ganglion cells (RGCs).
METHODS. In the SOD1 total knockout (SOD1-deficient) mice, the level of superoxide anion was measured using dihydroethidium. The number of RGCs was counted in both the retinal sections and the flat-mount retinas after retrograde labeling. Thickness of nerve fiber layer (NFL) was measured in the sections, and the amount of neurofilament protein was measured by immunoblot analysis. Pattern electroretinogram (ERG), which reflects the function of retinal ganglion cells, dark-adapted ERG, and cone ERG were performed. The intraocular pressure (IOP) was measured with an induction-impact tonometer. The levels of SOD-1 and -2 were measured by ELISA, in the serum of 47 newly diagnosed consecutive normal tension glaucoma (NTG) patients and 44 consecutive control subjects.
RESULTS. The level of superoxide anion in the RGC layer was significantly higher in 24-week-old SOD1-deficient mice than in wild-type mice. The RGC number was significantly reduced in 24-week-old SOD1-deficient mice, although they were not in 8-week-old mice. The NFL thickness and neurofilament protein were reduced in 24-week-old SOD1-deficient mice. The amplitude of pattern ERG was significantly reduced, although dark-adapted and cone ERGs showed no impairment, in 24-week-old SOD1-deficient mice. The IOP level was not changed in the SOD1-deficient mice. The serum level of SOD1, but not SOD2, was significantly lower in the NTG patients than in the healthy controls.
CONCLUSIONS. SOD1 deficiency causes RGC vulnerability, which may be involved in the underlying condition of NTG. (Invest Ophthalmol Vis Sci. 2011;52:4143-4150) DOI:10.1167/iovs.106294

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Hydrogen and N-Acetyl-L-Cysteine Rescue Oxidative Stress-Induced Angiogenesis in a Mouse Corneal Alkali-Burn Model

Kubota, Miyuki, Shimmura, Shigeto, Kubota, Shunsuke, Miyashita, Hideyuki, Kato, Naoko, Noda, Kousuke, Ozawa, Yoko, Usui, Tomohiko, Ishida, Susumu, Umezawa, Kazuo, Kurihara, Toshihide, Tsubota, Kazuo

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 52 ( 1 ) 427 - 433 2011.01

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. To investigate the role of reactive oxygen species (ROS) as the prime initiators of the angiogenic response after alkali injury of the cornea and observe the effects of antioxidants in preventing angiogenesis.
METHODS. The corneal epithelia of SOD-1-deficient mice or wild-type (WT) mice were removed after application of 0.15 N NaOH to establish the animal model of alkali burn. ROS production was semiquantitatively measured by dihydroethidium (DHE) fluorescence. Angiogenesis was visualized by CD31 immunohistochemistry. The effects of the specific NF-kappa B inhibitor DHMEQ, the antioxidant N-acetyl-L-cysteine (NAC), and hydrogen (H(2)) solution were observed.
RESULTS. ROS production in the cornea was enhanced immediately after alkali injury, as shown by increased DHE fluorescence (P < 0.01). NF-kappa B activation and the upregulation of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) were significantly enhanced (P < 0.01), leading to a significantly larger area of angiogenesis. Angiogenesis in SOD-1(-/-) mice corneas were significantly higher in WT mice (P < 0.01), confirming the role of ROS. Pretreatment with the specific NF-kappa B inhibitor DHMEQ or the antioxidant NAC significantly reduced corneal angiogenesis by downregulating the NF-kappa B pathway (P < 0.01) in both WT and SOD-1(-/-) mice. Furthermore, we showed that irrigation of the cornea with hydrogen (H 2) solution significantly reduced angiogenesis after alkali-burn injury (P < 0.01).
CONCLUSIONS. Immediate antioxidant therapy with H(2)-enriched irrigation solution is a new potent treatment of angiogenesis in cornea to prevent blindness caused by alkali burn. (Invest Ophthalmol Vis Sci. 2011;52:427-433) DOI:10.1167/iovs.106167

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Neural Degeneration in the Retina of the Streptozotocin-Induced Type 1 Diabetes Model

Ozawa, Yoko, Kurihara, Toshihide, Sasaki, Mariko, Ban, Norimitsu, Yuki, Kenya, Kubota, Shunsuke, Tsubota, Kazuo

EXPERIMENTAL DIABETES RESEARCH (HINDAWI PUBLISHING CORPORATION) 2011 1433 - 1439 2011

Research paper (scientific journal), Accepted, ISSN 1687-5214

Diabetic retinopathy, a vision-threatening disease, has been regarded as a vascular disorder. However, impaired oscillatory potentials (OPs) in the electroretinogram (ERG) and visual dysfunction are recorded before severe vascular lesions appear. Here, we review the molecular mechanisms underlying the retinal neural degeneration observed in the streptozotocin-(STZ-) induced type 1 diabetes model. The renin-angiotensin system (RAS) and reactive oxygen species (ROS) both cause OP impairment and reduced levels of synaptophysin, a synaptic vesicle protein for neurotransmitter release, most likely through excessive protein degradation by the ubiquitin-proteasome system. ROS also decrease brain-derived neurotrophic factor (BDNF) and inner retinal neuronal cells. The influence of both RAS and ROS on synaptophysin suggests that RAS-ROS crosstalk occurs in the diabetic retina. Therefore, suppressors of RAS or ROS, such as angiotensin II type 1 receptor blockers or the antioxidant lutein, respectively, are potential candidates for neuroprotective and preventive therapies to improve the visual prognosis.

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Regulation of Posttranscriptional Modification as a Possible Therapeutic Approach for Retinal Neuroprotection

Ozawa, Yoko, Kurihara, Toshihide, Tsubota, Kazuo, Okano, Hideyuki

JOURNAL OF OPHTHALMOLOGY (HINDAWI LTD) 2011 663 - 665 2011

Research paper (scientific journal), Accepted, ISSN 2090-004X

Understanding pathogenesis at the molecular level is the first step toward developing new therapeutic approaches. Here, we review the molecular mechanisms of visual dysfunction in two common diseases, innate chorioretinal inflammation and diabetic retinopathy, and the role of the ubiquitin-proteasome system (UPS) in both processes. In innate chorioretinal inflammation, interleukin-6 family ligands induce STAT3 activation in photoreceptors, which causes UPS-mediated excessive degradation of the visual substance, rhodopsin. In diabetic retinopathy, angiotensin II type 1 receptor (AT1R) signaling activates ERK in the inner layers of the retina, causing UPS-mediated excessive degradation of the synaptic vesicle protein, synaptophysin. This latter effect may decrease synaptic activity, in turn adversely affecting neuronal survival. Both mechanisms involve increased UPS activity and the subsequent excessive degradation of a protein required for visual function. Finally, we review the therapeutic potential of regulating the UPS to protect tissue function, citing examples from clinical applications in other medical fields.

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Resveratrol Prevents Light-Induced Retinal Degeneration via Suppressing Activator Protein-1 Activation

Kubota, Shunsuke, Kurihara, Toshihide, Ebinuma, Mari, Kubota, Miyuki, Yuki, Kenya, Sasaki, Mariko, Noda, Kousuke, Ozawa, Yoko, Oike, Yuichi, Ishida, Susumu, Tsubota, Kazuo

AMERICAN JOURNAL OF PATHOLOGY (AMER SOC INVESTIGATIVE PATHOLOGY, INC) 177 ( 4 ) 1725 - 1731 2010.10

Research paper (scientific journal), Accepted, ISSN 0002-9440

Light damage to the retina accelerates retinal degeneration in human diseases and rodent models. Recently, the polyphenolic phytoalexin resveratrol has been shown to exert various bioactivities in addition to its classical antioxidant property. In the present study, we investigated the effect of resveratrol on light-induced retinal degeneration together with its underlying molecular mechanisms. BALB/c mice with light exposure (5000-lux white light for 3 hours) were orally pretreated with resveratrol at a dose of 50 mg/kg for 5 days. Retinal damage was evaluated by TdT-mediated dUTP nick-end labeling, outer nuclear layer morphometry, and electroretinography. Administration of resveratrol to mice with light exposure led to a significant suppression of light-induced pathological parameters, including TdT-mediated dUTP nick-end labeling-positive retinal cells, outer nuclear layer thinning, and electroretinography changes. To clarify the underlying molecular mechanisms, the nuclear translocation of activator protein-1 subunit c-fos was evaluated by enzyme-linked immunosorbent assay, and the retinal activity of sirtuin 1 was measured by deacetylase fluorometric assay. Retinal activator protein-1 activation, up-regulated following light exposure, was significantly reduced by application of resveratrol. In parallel, retinal sirtuin 1 activity, reduced in animals with light damage, was significantly augmented by resveratrol treatment. Our data suggest the potential use of resveratrol as a therapeutic agent to prevent retinal degeneration related to light damage. (Am J Pathol 2010, 1 77:1725-1731; DOI: 10.2353/ajpath.2010.100098)

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Astrocyte Hypoxic Response Is Essential for Pathological But Not Developmental Angiogenesis of the Retina

Weidemann, Alexander, Krohne, Tim U., Aguilar, Edith, Kurihara, Toshihide, Takeda, Norihiko, Dorrell, Michael I., Simon, M. Celeste, Haase, Volker H., Friedlander, Martin, Johnson, Randall S.

GLIA (WILEY-LISS) 58 ( 10 ) 1177 - 1185 2010.08

Research paper (scientific journal), Accepted, ISSN 0894-1491

Vascular/parenchymal crosstalk is increasingly recognized as important in the development and maintenance of healthy vascularized tissues. The retina is an excellent model in which to study the role of cell type-specific contributions to the process of blood vessel and neuronal growth. During retinal vascular development, glial cells such as astrocytes provide the template over which endothelial cells migrate to form the retinal vascular network, and hypoxia-regulated vascular endothelial growth factor (VEGF) has been demonstrated to play a critical role in this process as well as pathological neovascularization. To investigate the nature of cell-specific contributions to this process, we deleted VEGF and its upstream regulators, the hypoxia-inducible transcription factors HIF-1 alpha and HIF-2 alpha, and the negative regulator of HIF alpha, von Hippel Lindau protein (VHL), in astrocytes. We found that loss of hypoxic response and VEGF production in astrocytes does not impair normal development of retinal vasculature, indicating that astrocyte-derived VEGF is not essential for this process. In contrast, using a model of oxygen-induced ischemic retinopathy, we show that astrocyte-derived VEGF is essential for hypoxia-induced neovascularization. Thus, we demonstrate that astrocytes in the retina have highly divergent roles during developmental, physiological angiogenesis, and ischemia-driven, pathological neovascularization. (C) 2010 Wiley-Liss, Inc.

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von Hippel-Lindau protein regulates transition from the fetal to the adult circulatory system in retina

Kurihara, Toshihide, Kubota, Yoshiaki, Ozawa, Yoko, Takubo, Keiyo, Noda, Kousuke, Simon, M. Celeste, Johnson, Randall S., Suematsu, Makoto, Tsubota, Kazuo, Ishida, Susumu, Goda, Nobuhito, Suda, Toshio, Okano, Hideyuki

DEVELOPMENT (COMPANY OF BIOLOGISTS LTD) 137 ( 9 ) 1563 - 1571 2010.05

Research paper (scientific journal), Accepted, ISSN 0950-1991

In early neonates, the fetal circulatory system undergoes dramatic transition to the adult circulatory system. Normally, embryonic connecting vessels, such as the ductus arteriosus and the foramen ovale, close and regress. In the neonatal retina, hyaloid vessels maintaining blood flow in the embryonic retina regress, and retinal vessels take over to form the adult-type circulatory system. This process is regulated by a programmed cell death switch mediated by macrophages via Wnt and angiopoietin 2 pathways. In this study, we seek other mechanisms that regulate this process, and focus on the dramatic change in oxygen environment at the point of birth. The von Hippel-Lindau tumor suppressor protein (pVHL) is a substrate recognition component of an E3-ubiquitin ligase that rapidly destabilizes hypoxia-inducible factor alpha s (HIF-alpha s) under normoxic, but not hypoxic, conditions. To examine the role of oxygen-sensing mechanisms in retinal circulatory system transition, we generated retina-specific conditional-knockout mice for VHL (Vhl(alpha-CreKO) mice). These mice exhibit arrested transition from the fetal to the adult circulatory system, persistence of hyaloid vessels and poorly formed retinal vessels. These defects are suppressed by intraocular injection of FLT1-Fc protein [a vascular endothelial growth factor (VEGF) receptor-1 (FLT1)/Fc chimeric protein that can bind VEGF and inhibit its activity], or by inactivating the HIF-1 alpha gene. Our results suggest that not only macrophages but also tissue oxygen-sensing mechanisms regulate the transition from the fetal to the adult circulatory system in the retina.

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Neurodegenerative influence of oxidative stress in the retina of a murine model of diabetes.

Sasaki M, Ozawa Y†, Kurihara T, Kubota S, Yuki K, Noda K, Kobayashi S, Ishida S, Tsubota K

Diabetologia. (SPRINGER) 53 ( 5 ) 971 - 979 2010.05

Accepted, ISSN 0012-186X

Diabetic retinopathy is a progressive neurodegenerative disease, but the underlying mechanism is still obscure. Here, we focused on oxidative stress in the retina, and analysed its influence on retinal neurodegeneration, using an antioxidant, lutein.
C57BL/6 mice with streptozotocin-induced diabetes were constantly fed either a lutein-supplemented diet or a control diet from the onset of diabetes, and their metabolic data were recorded. In 1-month-diabetic mice, reactive oxygen species (ROS) in the retina were measured using dihydroethidium and visual function was evaluated by electroretinograms. Levels of activated extracellular signal-regulated kinase (ERK), synaptophysin and brain-derived neurotrophic factor (BDNF) were also measured by immunoblotting in the retina of 1-month-diabetic mice. In the retinal sections of 4-month-diabetic mice, histological changes, cleaved caspase-3 and TUNEL staining were analysed.
Lutein did not affect the metabolic status of the diabetic mice, but it prevented ROS generation in the retina and the visual impairment induced by diabetes. ERK activation, the subsequent synaptophysin reduction, and the BDNF depletion in the diabetic retina were all prevented by lutein. Later, in 4-month-diabetic mice, a decrease in the thickness of the inner plexiform and nuclear layers, and ganglion cell number, together with increase in cleaved caspase-3- and TUNEL-positive cells, were avoided in the retina of lutein-fed mice.
The results indicated that local oxidative stress that has a neurodegenerative influence in the diabetic retina is prevented by constant intake of a lutein-supplemented diet. The antioxidant, lutein may be a potential therapeutic approach to protect visual function in diabetes.

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Retinal phototoxicity in a novel murine model of intraocular lens implantation

Kurihara, Toshihide, Omoto, Masahiro, Noda, Kousuke, Ebinuma, Mari, Kubota, Shunsuke, Koizumi, Haruna, Yoshida, Satoru, Ozawa, Yoko, Shimmura, Shigeto, Ishida, Susumu, Tsubota, Kazuo

MOLECULAR VISION (MOLECULAR VISION) 15 ( 290 ) 323 - 329 2009.12

Research paper (scientific journal), Accepted, ISSN 1090-0535

Purpose: To establish a novel murine intraocular lens (IOL) implantation model to study the protective effects of colored-IOLs against retinal phototoxicity.
Methods: Two-millimeter diameter IOL buttons were created from IOLs for clinical use. Extra-capsular crystalline lens extraction and IOL implantation were performed in BALB/c mice using a technique similar to human cataract surgery. For light exposure experiments, mice were exposed to 5,000 LUX of white light for 24 h on the day after surgery. To investigate the protective effects of yellow IOL against light exposure, ERG measurements were conducted in vivo, followed by TdT-mediated dUTP Nick-End Labeling (TUNEL) and outer nuclear layer (ONL) thickness measurement of retinal tissue in yellow or clear IOL-implanted mice and control mice without surgery.
Results: IOLs were successfully implanted in all animals, and IOL buttons without haptics were well stabilized in the capsular bag. Murine eyes developed posterior capsule opacification (PCO) after IOL implantation by postoperative day 5 at the latest. In contrast to the clear IOL-implanted animals stimulated by light exposure, the yellow IOL-implanted animals had significantly reduced numbers of TUNEL-positive cells and retained thickness of the ONL. The ERG showed that yellow IOL implantation prevents a decrease of amplitude in both the a-wave and b-wave compared with clear IOL implantation.
Conclusions: We established a new animal model of IOL implantation and demonstrated the protective effects of colored-IOL against retinal phototoxicity after cataract surgery.

Bilateral acute functional disturbance in the retina following placental abruption

Kurihara, Toshihide, Ohde, Hisao, Inoue, Makoto, Ogata, Masaro, Shinoda, Kei, Tsubota, Kazuo

JAPANESE JOURNAL OF OPHTHALMOLOGY (SPRINGER TOKYO) 53 ( 6 ) 663 - 665 2009.11

Research paper (scientific journal), Accepted, ISSN 0021-5155

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Involvement of Hyaluronan and Its Receptor CD44 with Choroidal Neovascularization

Mochimaru, Hiroshi, Takahashi, Eri, Tsukamoto, Nobuo, Miyazaki, Junichiro, Yaguchi, Tomonori, Koto, Takashi, Kurihara, Toshihide, Noda, Kousuke, Ozawa, Yoko, Ishimoto, Takatsugu, Kawakami, Yutaka, Tanihara, Hidenobu, Saya, Hideyuki, Ishida, Susumu, Tsubota, Kazuo

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 50 ( 9 ) 4410 - 4415 2009.09

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. CD44 is a cell-surface adhesion molecule and receptor for hyaluronan (HA), one of the major extracellular matrix components. The purpose of the present study was to clarify a role of HA and CD44 in the development of choroidal neovascularization (CNV).
METHODS. Laser photocoagulation was used to induce CNV in C57BL/6 mice or CD44-deficient mice. The mRNA expression of CD44 and HA synthase (HAS)-2 in the retinal pigment epithelium (RPE)-choroid complex was evaluated by DNA microarray and real-time RT-PCR analyses 3 days after laser treatment. HA synthesis and CD44 expression were examined by immunohistochemistry 1 week after photocoagulation. Mice with laser-induced CNV were systemically administered the HA synthesis inhibitor 4-methylumbelliferone (MU) or an antiCD44-neutralizing antibody. The response of CNV was analyzed by volumetric measurements 1 week after photocoagulation. Macrophage infiltration into CNV lesions was evaluated by real-time RT-PCR for F4/80 3 days after laser-induced injury.
RESULTS. The induction of CNV led to a significant increase in expression of CD44 and HAS2 mRNA. HA and CD44 were immunopositive in the CNV lesions. Compared with vehicle treatment, the systemic application of MU significantly attenuated CNV volume in a dose-dependent fashion, together with macrophage infiltration into the lesions. Consistently, antibody-based blockade of CD44 resulted in a significant reduction of CNV volume, compared with the isotype control. In contrast, genetic ablation of CD44 significantly augmented CNV formation together with HA accumulation and macrophage infiltration, compared with wild-type mice.
CONCLUSIONS. These results indicate a significant role of HA and its receptor CD44 in the development of CNV. (Invest Ophthalmol Vis Sci. 2009;50:4410-4415) DOI: 10.1167/iovs.08-3044

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Prevention of Ocular Inflammation in Endotoxin-Induced Uveitis with Resveratrol by Inhibiting Oxidative Damage and Nuclear Factor-kappa B Activation

Kubota, Shunsuke, Kurihara, Toshihide, Mochimaru, Hiroshi, Satofuka, Shingo, Noda, Kousuke, Ozawa, Yoko, Oike, Yuichi, Ishida, Susumu, Tsubota, Kazuo

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 50 ( 7 ) 3512 - 3519 2009.07

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. Resveratrol is known as one of the antioxidant polyphenols contained in red wine and grape skin. The purpose of the present study was to investigate the role of resveratrol in ocular inflammation in endotoxin-induced uveitis (EIU).
METHODS. EIU was induced in male C57/B6 mice at the age of 6 weeks by a single intraperitoneal injection of lipopolysaccharide (LPS). Animals had received oral supplementation of resveratrol at the doses of 5, 50, 100, or 200 mg/kg for 5 days until LPS injection. Twenty-four hours after LPS administration, leukocyte adhesion to the retinal vasculature was examined with a concanavalin A lectin perfusion-labeling technique. Retinal and retinal pigment epithelium (RPE)-choroidal levels of intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nuclear translocation of nuclear factor (NF)-kappa B p65 were evaluated by enzyme-linked immunosorbent assay. Retinal and RPE-choroidal activities of silent information regulator two ortholog (SIRT) 1 were measured by deacetylase fluorometric assay.
RESULTS. Resveratrol pretreatment led to significant and dose-dependent suppression of leukocyte adhesion to retinal vessels of EIU mice compared with vehicle application. Protein levels of MCP-1 and ICAM-1 in the retina and the RPE-choroid of EIU animals were significantly reduced by resveratrol administration. Importantly, resveratrol-treated animals showed significant decline of retinal 8-OHdG generation and nuclear NF-kappa B P65 translocation, both of which were upregulated after EIU induction. RPE-choroidal SIRT1 activity, reduced in EIU animals, was significantly augmented by treatment with resveratrol.
CONCLUSIONS. Resveratrol prevented EIU-associated cellular and molecular inflammatory responses by inhibiting oxidative damage and redox-sensitive NF-kappa B activation. (Invest Ophthalmol Vis Sci. 2009; 50: 3512-3519) DOI: 10.1167/iovs.08-2666

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Neuroprotective Effect of an Antioxidant, Lutein, during Retinal Inflammation

Sasaki, Mariko, Ozawa, Yoko, Kurihara, Toshihide, Noda, Kousuke, Imamura, Yutaka, Kobayashi, Saori, Ishida, Susumu, Tsubota, Kazuo

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 50 ( 3 ) 1433 - 1439 2009.03

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. Lutein has been the focus of recent study as a possible therapeutic approach for retinal diseases, but the molecular mechanism of its neuroprotective effect remains to be elucidated. The aim of this study was to investigate, with the use of a mouse endotoxin-induced uveitis (EIU) model, the neuroprotective effects of lutein against retinal neural damage caused by inflammation.
METHODS. EIU was induced by intraperitoneal injection of lipopolysaccharide (LPS). Each animal was given a subcutaneous injection of lutein or vehicle three times: concurrently with and 3 hours before and after the LPS injection. Analysis was carried out 24 hours after EIU induction. Levels of rhodopsin protein and STAT3 activation were analyzed by immunoblotting. Lengths of the outer segments of the photoreceptor cells were measured. Dark-adapted full-field electroretinograms were recorded. Oxidative stress in the retina was analyzed by dihydroethidium and fluorescent probe. Expression of glial fibrillary acidic protein (GFAP) was shown immunohistochemically.
RESULTS. The EIU-induced decrease in rhodopsin expression followed by shortening of the outer segments and reduction in a-wave amplitude were prevented by lutein treatment. Levels of STAT3 activation, downstream of inflammatory cytokine signals, and reactive oxygen species (ROS), which are both upregulated during EIU, were reduced by lutein. Pathologic change of Muller glial cells, represented by GFAP expression, was also prevented by lutein.
CONCLUSIONS. The present data revealed that the antioxidant lutein was neuroprotective during EIU, suggesting a potential approach for suppressing retinal neural damage during inflammation. (Invest Ophthalmol Vis Sci. 2009; 50: 1433-1439) DOI: 10.1167/iovs.08-2493

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NEW RETRACTOR CAPABLE OF ASPIRATING FLUIDS DURING SCLERAL BUCKLING SURGERY

Kurihara, Toshihide, Suzuki, Kotaro, Noda, Kousuke, Kawamura, Ryosuke, Nagai, Norihiro, Shinoda, Hajime, Shinoda, Kei, Tsubota, Kazuo, Ishida, Susumu, Inoue, Makoto

RETINA-THE JOURNAL OF RETINAL AND VITREOUS DISEASES (LIPPINCOTT WILLIAMS & WILKINS) 29 ( 10 ) 1542 - 1544 2009.01

Research paper (scientific journal), Accepted, ISSN 0275-004X

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Roles of STAT3/SOCS3 pathway in regulating the visual function and ubiquitin-proteasome-dependent degradation of rhodopsin during retinal inflammation

Ozawa, Yoko, Nakao, Keiko, Kurihara, Toshihide, Shimazaki, Takuya, Shimmura, Shigeto, Ishida, Susumu, Yoshimura, Akihiko, Tsubota, Kazuo, Okano, Hideyuki

JOURNAL OF BIOLOGICAL CHEMISTRY (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC) 283 ( 36 ) 24561 - 24570 2008.09

Research paper (scientific journal), Accepted, ISSN 0021-9258

Inflammatory cytokines cause tissue dysfunction. We previously reported that retinal inflammation down-regulates rhodopsin expression and impairs visual function by an unknown mechanism. Here, we demonstrate that rhodopsin levels were preserved by suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of STAT3 activation. SOCS3 was expressed mainly in photoreceptor cells in the retina. In the SOCS3-deficient retinas, rhodopsin protein levels dropped sooner, and the reduction was more profound than in the wild type. Visual dysfunction, measured by electroretinogram, was prolonged in retina-specific SOCS3 conditional knockout mice. Visual dysfunction and decreased rhodopsin levels both correlated with increased STAT3 activation enhanced by SOCS3 deficiency. Interleukin 6, one of the inflammatory cytokines found during retinal inflammation, activated STAT3 and decreased rhodopsin protein in adult retinal explants. This was enhanced by inhibiting SOCS3 function in vitro, indicating that rhodopsin reduction was not a secondary effect in the mutant mice. Interestingly, in the inflamed SOCS3-deficient adult retina, rhodopsin decreased post-transcriptionally at least partly through ubiquitin-proteasome-dependent degradation accelerated by STAT3 activation and not transcriptionally as in the developing retina, on which we reported previously. A STAT3-dependent E3 ubiquitin ligase, Ubr1, was responsible for rhodopsin degradation and was up-regulated in the inflamed SOCS3-deficient retinas. These results indicate that in wild-type animals, a decrease in rhodopsin during inflammation is minimized by endogenous SOCS3. However, when STAT3 activation exceeds some threshold beyond the compensatory activity of endogenous SOCS3, rhodopsin levels decrease. These findings suggest SOCS3 as a potential therapeutic target molecule for protecting photoreceptor cell function during inflammation.

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Angiotensin II type 1 receptor signaling contributes to synaptophysin degradation and neuronal dysfunction in the diabetic retina

Kurihara, Toshihide, Ozawa, Yoko, Nagai, Norihiro, Shinoda, Kei, Noda, Kousuke, Imamura, Yutaka, Tsubota, Kazuo, Okano, Hideyuki, Oike, Yuichi, Ishida, Susumu

DIABETES 57 ( 8 ) 2191 - 2198 2008.08

Research paper (scientific journal), ISSN 0012-1797

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Retinal dysfunction and progressive retinal cell death in SOD1-deficient mice

Hashizume, Kouhei, Hirasawa, Manabu, Imamura, Yutaka, Noda, Setsuko, Shimizu, Takahiko, Shinoda, Kei, Kurihara, Toshihide, Noda, Kousuke, Ozawa, Yoko, Ishida, Susumu, Miyake, Yozo, Shirasawa, Takuji, Tsubota, Kazuo

AMERICAN JOURNAL OF PATHOLOGY (AMER SOC INVESTIGATIVE PATHOLOGY, INC) 172 ( 5 ) 1325 - 1331 2008.05

Research paper (scientific journal), Accepted, ISSN 0002-9440

The superoxide dismutase (SOD) family is a major antioxidant system, and deficiency of Cu,Zn-superoxide dismutase (SOD1) in Mice leads to many different phenotypes that resemble accelerated aging. The purpose of this study was to examine the morphology and physiology of the sensory retina in Sod1(-/-) mice. The amplitudes of the a- and b-waves of electroretinograms elicited by stimuli of different intensity were reduced in senescent Sod1(-/-) mice, and this reduction in amplitude was more pronounced with increasing age. Retinal morphometric analyses showed a reduced number of nuclei in both the inner nuclear cell layer and outer nuclear cell layer. Electron microscopy revealed swollen cells and degenerated mitochondria in the inner nuclear cell and outer nuclear cell layer of senescent Sod1(-/-) mice indicating necrotic cell death. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling revealed no significant differences in the number of apoptotic cells between Sod1(-/-) and wild-type mice, and activated caspase-3 could not be detected in the retina of Sod1(-/-) mice. In addition to the age-related macular degeneration-like phenotypes previously reported, Sod1(-/-) mice also present progressive retinal degeneration. Our results indicate that Sod1(-/-) mice may be a good model system in which to study the mechanism of reactive oxygen species-mediated retinal degeneration.

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Angiotensin II type 1 receptor signaling contributes to synaptophysin degradation and neuronal dysfunction in the diabetic retina

Kurihara, Toshihide, Ozawa, Yoko, Tsubota, Kazuo, Okano, Hideyuki, Ishida, Susumu

NEUROSCIENCE RESEARCH 61 S220 - S220 2008

Research paper (scientific journal), ISSN 0168-0102

Suppression of choroidal neovascularization by dendritic cell vaccination targeting VEGFR2

Mochimaru, Hiroshi, Nagai, Norihiro, Hasegawa, Go, Kudo-Saito, Chie, Yaguchi, Tomonori, Usui, Yoshihiko, Kurihara, Toshihide, Koto, Takashi, Satofuka, Shingo, Shinoda, Hajime, Ozawa, Yoko, Tsubota, Kazuo, Kawakami, Yutaka, Ishida, Susumu

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 48 ( 10 ) 4795 - 4801 2007.10

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. To investigate whether the induction of cellular immunity against vascular endothelial growth factor receptor ( VEGFR) 2 inhibits the development of choroidal neovascularization (CNV).
METHODS. H-2Db-restricted peptide corresponding to amino acids 400 to 408 of VEGFR2 was used as an epitope peptide. Dendritic cells (DCs) were harvested from bone marrow progenitors of C57BL/6 mice. Six-week-old C57BL/6 mice received subcutaneous injections of the epitope peptide-pulsed mature DCs three times at 6-day intervals. After the third immunization, laser photocoagulation was performed to induce CNV. One week after photocoagulation, mice were killed to harvest the choroid and splenocytes. CNV volume was evaluated by volumetric measurements. To confirm the specific immunogenicity of the epitope peptides in C57BL/6 mice, CD8 T cells isolated from harvested splenocytes were restimulated to measure interferon (IFN)-gamma and tumor necrosis factor (TNF)-gamma production through enzyme-linked immunospot assay and ELISA. To determine the T-cell subset responsible for the immunotherapy, mice were intraperitoneally injected with an anti-CD4 or anti-CD8 depletion antibody.
RESULTS. CNV volume was significantly lower in mice immunized with the VEGFR2 epitope peptide than in those not immunized or immunized with a control peptide gp70. Cytokine assays showed the peptide-specific production of IFN-gamma and TNF-alpha from the CD8 T cells in a dose-dependent manner. In vivo depletion of CD8, but not CD4, T cells significantly reversed the suppressive effect of the VEGFR2 peptide-pulsed DC vaccination on CNV to the level observed in nonimmunized or gp70-immunized animals.
CONCLUSIONS. These results indicate that the VEGFR2 peptide-specific induction of cellular immunity inhibits CNV through the cytotoxicity of CD8 T cells. Results of the present study suggested the possibility of DC vaccination targeting VEGFR2 as a novel therapeutic strategy for CNV.

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Eicosapentaenoic acid is anti-inflammatory in preventing choroidal neovascularization in mice

Koto, Takashi, Nagai, Norihiro, Mochimaru, Hiroshi, Kurihara, Toshihide, Izumi-Nagai, Kanako, Satofuka, Shingo, Shinoda, Hajime, Noda, Kousuke, Ozawa, Yoko, Inoue, Makoto, Tsubota, Kazuo, Oike, Yuichi, Ishida, Susumu

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 48 ( 9 ) 4328 - 4334 2007.09

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. To investigate the role of eicosapentaenoic acid (EPA), the major omega-3 polyunsaturated fatty acid (PUFA), in the development of choroidal neovascularization (CNV), together with underlying molecular mechanisms.
METHODS. Six-week-old C57BL/ 6 mice were fed with laboratory chow with 5% EPA or the omega-6 PUFA linoleic acid ( LA) for 4 weeks. Laser photocoagulation was performed to induce CNV, and the volume of CNV tissue was evaluated by volumetric measurements. The expression and production of intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1, vascular endothelial growth factor ( VEGF) and interleukin (IL)-6 in the retinal pigment epithelium (RPE)-choroid in vivo, and stimulated b-End3 endothelial cells and RAW264.7 macrophages in vitro were evaluated by RT-PCR and ELISA. Fatty acid composition in the serum and the RPE-choroid was analyzed by gas chromatography and high-performance liquid chromatography, respectively. Serum levels of C-reactive protein (CRP), IL-6, VEGF, MCP-1, and soluble ICAM-1 were examined by ELISA.
RESULTS. The CNV volume in EPA-fed animals was significantly suppressed compared with that in control mice, whereas the LA-rich diet did not affect CNV. The mRNA expression and protein levels of ICAM- 1, MCP-1, VEGF, and IL- 6 after CNV induction were significantly reduced in EPA-supplemented mice. In vitro, EPA application led to significant inhibition of mRNA and protein levels of ICAM- 1 and MCP-1 in endothelial cells and VEGF and IL- 6 in macrophages. EPA- fed mice exhibited significantly higher levels of EPA and lower levels of the omega-6 PUFA arachidonic acid in the serum and the RPE-choroid than control animals. EPA supplementation also led to significant reduction of serum levels of IL- 6 and CRP after CNV induction.
CONCLUSIONS. The present study demonstrates for the first time that an EPA- rich diet results in significant suppression of CNV and CNV-related inflammatory molecules in vivo and in vitro. These results suggest that frequent consumption of omega-3 PUFAs may prevent CNV and lower the risk of blindness due to age-related macular degeneration.

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Interleukin-6 receptor-mediated activation of signal transducer and activator of transcription-3 (STAT3) promotes choroidal neovascularization

Izumi-Nagai, Kanako, Nagai, Norihiro, Ozawa, Yoko, Mihara, Masahiko, Ohsugi, Yoshiyuki, Kurihara, Toshihide, Koto, Takashi, Satofuka, Shingo, Inoue, Makoto, Tsubota, Kazuo, Okano, Hideyuki, Oike, Yuichi, Ishida, Susumu

AMERICAN JOURNAL OF PATHOLOGY (AMER SOC INVESTIGATIVE PATHOLOGY, INC) 170 ( 6 ) 2149 - 2158 2007.06

Research paper (scientific journal), Accepted, ISSN 0002-9440

Interleukin (IL)-6, a potent proinflammatory cytokine, is suggested to be a risk factor for choroidal neovascularization (CNV) because of its increased levels in the serum of patients with age-related macular degeneration; however, the role of IL-6 in CNV has not been defined. The present study reveals the critical contribution of IL-6 signaling and its downstream STAT3 pathway to the murine model of laser-induced CNV. CNV induction by laser treatment stimulated IL-6 expression in the retinal pigment epithelium-choroid complex, and antibody-based blockade of M-6 receptor or genetic ablation of M-6 led to significant suppression of CNV. CNV generation was accompanied by STAT3 activation in choroidal endothelial cells and macrophages, and M-6 receptor blockade resulted in selectively inhibited phosphorylation of STAT3 but not extracellular signal-regulated kinase 1/2. Consistently, pharmacological blockade of STAT3 pathway also suppressed CNV. in addition, M-6 receptor neutralization led to significant inhibition of the in vivo and in vitro expression of inflammation-related molecules including monocyte chemotactic protein, intercellular adhesion molecule-1, and vascular endothelial growth factor, and of macrophage infiltration into CNV. These results indicate the significant involvement of H,6 receptor-mediated activation of STAT3 inflammatory pathway in CNV generation, suggesting the possibility of M-6 receptor blockade as a therapeutic strategy to suppress CNV associated with age-related macular degeneration.

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SOCS3 is required to temporally fine-tune photoreceptor cell differentiation

Ozawa, Yoko, Nakao, Keiko, Shimazaki, Takuya, Shimmura, Shigeto, Kurihara, Toshihide, Ishida, Susumu, Yoshimura, Akihiko, Tsubota, Kazuo, Okano, Hideyuki

DEVELOPMENTAL BIOLOGY (ACADEMIC PRESS INC ELSEVIER SCIENCE) 303 ( 2 ) 591 - 600 2007.03

Research paper (scientific journal), Accepted, ISSN 0012-1606

Suppressor of cytokine signaling 3 (SOCS3) is an intracellular, ligand-induced negative feedback modulator of STAT3 activation that acts during inflammation. Here, we dernonstrate that SOCS3 expression is important for normal retinal development in the perinatal period. STAT3 is highly activated in the late-embryonic retina, then downregulated at postnatal day 0 (PO), presumably by the depletion of upstream ligands. We found that SOCS3 was required after PO to shut down the residual STAT3 activation; this loss of activated STAT3 leads to Rhodopsin expression and rod photoreceptor cell differentiation. SOCS3 deficiency failed to terminate STAT3 activation, thereby delaying expression of Rhodopsin and its upstream transcription factor, crx. Development subsequently continued, but its course was temporally erratic, probably because of faulty compensation. Interestingly, SOCS3 protein expression was first detected postnatally, after STAT3 activation was mostly downregulated. It initially appeared in some of the presumptive photoreceptor cells and gradually spread. SOCS3 mRNA level was constant from the late-embryonic to early-postnatal period. Post-transcriptional inhibition of SOCS3 protein expression maintains a high STAT3 activation during late embryogenesis, and after PO, releasing from the inhibition promptly terminates STAT3 activation. Thus, SOCS3 can act as a temporal fine-tuner of STAT3 activation during photoreceptor cell differentiation. (c) 2006 Elsevier Inc. All rights reserved.

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Neuroprotective effects of angiotensin II type 1 receptor (AT1R) blocker, telmisartan, via modulating AT1R and AT2R signaling in retinal inflammation

Kurihara, Toshihide, Ozawa, Yoko, Shinoda, Kei, Nagai, Norihiro, Inoue, Makoto, Oike, Yuichi, Tsubota, Kazuo, Ishida, Susumu, Okano, Hideyuki

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE (ASSOC RESEARCH VISION OPHTHALMOLOGY INC) 47 ( 12 ) 5545 - 5552 2006.12

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. To investigate the retinal neural damage that occurs during inflammation and the therapeutic effects of the angiotensin II type 1 receptor (AT1R) blocker, telmisartan, using a model of endotoxin- induced uveitis (EIU).
METHODS. The localization of AT1R and AT2R was shown by immunohistochemistry. EIU was induced by intraperitoneal injection of lipopolysaccharide (LPS). Animals were treated with telmisartan for 2 days and were evaluated 24 hours later. Expression levels of angiotensin II, STAT3 activation induced by inflammatory cytokines, and retinal proteins essential for neural activities (e. g., synaptophysin, rhodopsin) were analyzed by immunoblot. An AT2R antagonist was administered to evaluate the contribution of AT2R signaling in this therapy. Dark-adapted full-field electroretinography ( ERG) was also performed.
RESULTS. AT1R and AT2R were expressed in presynaptic terminals in most of the retinal neurons. AT1R was also expressed in Muller glial cells. During inflammation, angiotensin II expression was elevated, STAT3 was activated, and synaptophysin and rhodopsin expression were reduced. The expression of glial fibrillary acidic protein (GFAP), downstream of STAT3 activation, was induced in Muller glial cells. However, treatment with telmisartan successfully avoided all these changes. An AT2R antagonist lowered synaptophysin expression despite the treatment. STAT3 activity was negatively correlated with rhodopsin expression. Furthermore, ERG responses, which were mostly prevented by telmisartan, were disturbed during inflammation.
CONCLUSIONS. Retinal protein expression and visual function are both disturbed by inflammation. Treatment with the AT1R blocker telmisartan efficiently prevented these signs of retinal neural damage through the reduction of local angiotensin II expression, the blockade of AT1R, and the relative upregulation of AT2R function.

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Pars plana vitrectomy with internal limiting membrane removal for macular hole associated with proliferative diabetic retinopathy.

Kurihara T, Noda K, Ishida S, Inoue M†

Graefes Arch Clin Exp Ophthalmol. (SPRINGER) 243 ( 7 ) 724 - 726 2005.01

Lead author, Accepted, ISSN 0721-832X

Background: We describe the outcome of vitreous surgery in three eyes of three patients with macular hole associated with proliferative diabetic retinopathy (PDR) in the absence of fibrovascular proliferation, a combination of conditions where efficacy is incompletely known. Methods: The patients, all male were 62, 65, and 66 years old. Panretinal photocoagulation had been performed preoperatively in all, and one eye had undergone vitreous surgery. No fibrovascular tissue causing macular traction was observed in any case. Fluorescein angiography and optical coherence tomography (OCT) demonstrated persistent diabetic macular edema surrounding the hole. Affected eyes were treated with vitrectomy including internal limiting membrane (ILM) peeling; 20% sulfur hexafluoride gas (SF6) was introduced for tamponade. Results: Anatomical closure of the macular hole as well as resolution of macular edema was achieved in all cases, and vision improved considerably by more than two Snellen lines. Conclusions: Vitreous surgery with ILM peeling was effective for macular hole associated with PDR, attaining not only macular hole closure but also resolution of persistent diabetic macular edema.

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A case of retinal detachment associated with acquired retinoschisis and macular pucker

Toshihide Kurihara, Hajime Shinoda, Tadahiko Eshita, Shizuaki Kitamura, Kei Shinoda, Makoto Inoue, Masaro Ogata

Japanese Review of Clinical Ophthalmology 98 ( 3 ) 205-208 - 208 2004.03

Research paper (scientific journal), Joint Work

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