Hiroto Terasaki

Repeatability and Reproducibility of Subfoveal Choroidal Thickness in Normal Eyes of Japanese Using Different SD-OCT Devices

PURPOSE To compare subfoveal choroidal thickness (SCT) measurements of three different commercially available spectral-domain optical coherence tomography instruments with healthy eyes of Japanese. METHODS A prospective, cross-sectional study was performed at a single institution. SCT of the right eye of 43 normal subjects was measured using three different SD-OCTs: Heidelberg Spectralis-OCT (Spectralis), Cirrus HD-OCT (Cirrus), and Topcon 3D OCT-1000 Mark II (Topcon). Two separate measurements were performed for the same eye with a maximum by a single examiner. SCT was defined as the distance from the posterior edge of the retinal pigment epithelium to the choroid/sclera junction. After manual segmentation, measurements were made using calipers equipped on each machine by masked raters. Intraclass, interrater, and intermachine agreements were assessed. RESULTS Forty-three subjects (mean age, 30.5 years) were enrolled. Of 43 eyes, the SCT of 39 eyes (90.7%) could be measured using each machine. Intraclass correlation coefficients (95% confidence intervals) were 0.976 (0.954-9.987), 0.958 (0.919-0.978), and 0.939 (0.895-0.971) with Spectralis, Cirrus, and Topcon, respectively. Interrater correlation coefficients (95% confident interval) were 0.944 (0.893 to 0.971), 0.956 (0.831 to 0.983), and 0.924 (0.825 to 0.964) with Spectralis, Cirrus, and Topcon, respectively. The average SCT was 272.6, 272.8, and 269.2 μm with Spectralis, Cirrus, and Topcon, respectively. The intermachine correlation coefficient was significantly high among the machines (P<0.001, Spearman), 0.97 (Spectralis-Cirrus), 0.96 (Cirrus-Topcon), and 0.98 (Topcon-Cirrus). Bland-Altman plot analysis showed no typical trend among the machines. CONCLUSIONS SCT measurements obtained with three different SD-OCTs were highly correlated and could be used interchangeably. (http://upload.umin.ac.jp number, UMIN000005287.).

Choroidal Structure in Normal Eyes and After Photodynamic Therapy Determined by Binarization of Optical Coherence Tomographic Images

PURPOSE To determine changes in choroidal structure by binarization of optical coherence tomographic (OCT) images. METHODS Choroidal images were recorded by enhanced depth imaging OCT. The subfoveal choroidal images were analyzed, and the luminal and interstitial areas were converted to binary images by the Niblack method. The interrater, intrarater, and intersession agreements of the binary images were determined for healthy eyes. In eyes with age-related macular degeneration (AMD), the binary images of the choroid before photodynamic therapy (PDT) were compared to those after PDT. The untreated fellow eyes were studied as controls. RESULTS In healthy eyes, the average ratio of the luminal to choroidal area was 65.4%. The interrater agreement rate was high, with intraclass correlation coefficient (ICC) 0.985 and 0.988 for the choroid and luminal areas, respectively. The intrarater ICC was 0.996 for the choroid and 0.997 for the luminal areas. The intersession ICC was 0.993 for the choroid and 0.984 for the luminal areas. In eyes with AMD, the subfoveal choroidal area, the luminal area, and the interstitial areas were thinner 6 months after PDT (all P < 0.01, Wilcoxon signed-rank sum test). The ratio of the luminal to choroidal area was significantly decreased to 62.8% (P < 0.01, Wilcoxon signed-rank sum test). The ratio for the fellow eyes was not significantly changed. CONCLUSIONS The Niblack binarization method can be used to analyze the luminal area of choroid in an OCT image with good repeatability and reproducibility. The change in the subfoveal choroidal area after PDT is due mainly to a decrease in the luminal areas.

Comparisons of Choroidal Thickness of Normal Eyes Obtained by Two Different Spectral-Domain OCT Instruments and One Swept-Source OCT Instrument

PURPOSE We compared the subfoveal choroidal thickness (SFCT) measured on the images obtained by two spectral-domain optical coherence tomographic (SD-OCT) instruments and one swept-source OCT (SS-OCT) instrument. METHODS A cross-sectional, prospective noninterventional study was done in which SFCT was measured in the images obtained by two SD-OCT instruments; Heidelberg Spectralis-OCT (Spectralis-SD-OCT) and Topcon 3D OCT-1000 Mark II (Topcon-SD-OCT). Images also were obtained with SS-OCT Atlantis DRI OCT-1 (DRI-SS-OCT). After manual segmentation, the measurements were made using the calipers embedded in each instrument. The intrarater, interrater, and intermachine agreements were assessed. RESULTS We studied 35 subjects. The intrarater correlation coefficient (95% confidence interval) was 0.994 (0.988-0.994) for Spectralis-SD-OCT, 0.996 (0.993-0.998) for Topcon-SD-OCT, and 0.997 (0.991-0.998) for DRI-SS-OCT (P < 0.001). The interrater correlation coefficient was 0.995 (0.991-0.998) for Spectralis-SD-OCT, 0.995 (0.990-0.998) for Topcon-SD-OCT, and 0.996 (0.992-0.998) for DRI-SS-OCT (P < 0.001). The average SFCT was 273.2 μm with Spectralis-SD-OCT, 269.1 μm with the Topcon-SD-OCT, and 280.5 μm with DRI-SS-OCT. The intermachine correlation coefficient was 0.982 (0.964-0.991) for Spectralis-SD-OCT versus Topcon-SD-OCT, 0.907 (0.815-0.953) for Topcon-SD-OCT versus DRI-SS-OCT, and 0.911 (0.832-0.954) for DRI-SS-OCT versus Spectralis-SD-OCT (P < 0.001). The SFCT measured with DRI-SS-OCT was significantly thicker than that with Topcon-SD-OCT, with a mean difference of 11.41 ± 30.27 μm (P = 0.032). CONCLUSIONS In normal adult eyes, there was good reproducibility and repeatability of SFCT measurements obtained by the SD-OCT and SS-OCT instruments. However, the choroid measured with DRI-SS-OCT was thicker than that measured with both SD-OCT instruments, and, thus, the choroidal thickness should not be compared between the SD-OCT and SS-OCT instruments. (www.umin.ac.jp/ctr number, UMIN000011259.).

Dynamic Increase in Extracellular ATP Accelerates Photoreceptor Cell Apoptosis via Ligation of P2RX7 in Subretinal Hemorrhage

Photoreceptor degeneration is the most critical cause of visual impairment in age-related macular degeneration (AMD). In neovascular form of AMD, severe photoreceptor loss develops with subretinal hemorrhage due to choroidal neovascularization (CNV), growth of abnormal blood vessels from choroidal circulation. However, the detailed mechanisms of this process remain elusive. Here we demonstrate that neovascular AMD with subretinal hemorrhage accompanies a significant increase in extracellular ATP, and that extracellular ATP initiates neurodegenerative processes through specific ligation of Purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7; P2X7 receptor). Increased extracellular ATP levels were found in the vitreous samples of AMD patients with subretinal hemorrhage compared to control vitreous samples. Extravascular blood induced a massive release of ATP and photoreceptor cell apoptosis in co-culture with primary retinal cells. Photoreceptor cell apoptosis accompanied mitochondrial apoptotic pathways, namely activation of caspase-9 and translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, as well as TUNEL-detectable DNA fragmentation. These hallmarks of photoreceptor cell apoptosis were prevented by brilliant blue G (BBG), a selective P2RX7 antagonist, which is an approved adjuvant in ocular surgery. Finally, in a mouse model of subretinal hemorrhage, photoreceptor cells degenerated through BBG-inhibitable apoptosis, suggesting that ligation of P2RX7 by extracellular ATP may accelerate photoreceptor cell apoptosis in AMD with subretinal hemorrhage. Our results indicate a novel mechanism that could involve neuronal cell death not only in AMD but also in hemorrhagic disorders in the CNS and encourage the potential application of BBG as a neuroprotective therapy.

Effect of intravitreal triamcinolone acetonide or bevacizumab on choroidal thickness in eyes with diabetic macular edema.

PURPOSE We evaluated the effect of intravitreal triamcinolone acetonide (IVTA) or intravitreal bevacizumab (IVB) on subfoveal choroidal thickness (SFCT) in eyes with diabetic macular edema (DME). METHODS In this prospective, randomized, interventional comparative study, 51 DME eyes of 51 patients were randomized to receive either IVTA or IVB. The central macular thickness (CMT) and SFCT were determined by optical coherence tomography at 24 hours, 7 days, and 4, 8, and 12 weeks. The SFCT at 1500 and 3000 μm nasal or temporal to the central fovea also was measured. The values obtained before were compared to those obtained 12 weeks after the injections. RESULTS The eyes were randomly assigned to the IVTA (25 eyes) and IVB (26 eyes) groups. The SFCT was reduced significantly in the IVTA group from 24 hours to 12 weeks. The average ± SD of the SFCT expressed as the ratio to baseline thickness decreased to 94.8% ± 5.6% (P < 0.01) at 24 hours after IVTA and remained unchanged up to 12 weeks (91.8% ± 10.5%, P < 0.01, Wilcoxon signed-rank test). In the IVB group, no significant difference was found in the SFCT after IVB for 12 weeks. The CMT decreased significantly in both groups from 24 hours to 4 weeks; however, the decrease was not significant at 8 weeks or later in the IVB group. CONCLUSIONS The decrease in choroidal thickness in eyes with DME after IVTA suggests that the choroidal pathology in diabetic retinopathy might be due to steroid-sensitive factors rather than vascular endothelial growth factor. (www.umin.ac.jp/ctr number, clinical trials number UMIN000009854.).

Correlation between reflectivity of subretinal fluid in OCT images and concentration of intravitreal VEGF in eyes with diabetic macular edema.

PURPOSE The reflectivity of optical coherence tomographic (OCT) images has been used to evaluate retinal diseases. The purpose of our study was to determine whether a significant correlation exists between the reflectivity of the subretinal fluid (SRF) and the concentration of intravitreal cytokines in eyes with diabetic macular edema (DME). METHODS A retrospective comparative study was done of eyes with DME with SRF before vitrectomy. The reflectivity of the SRF was determined from the OCT images. Vitreous samples were collected during vitrectomy, and analyzed for the concentrations of VEGF, IL-6, and IL-8. To determine the factors in the SRF that could affect the reflectivity, the aqueous humor of isolated swine eyes was replaced by saline with plasma, albumin, or fibrinogen, and the reflectivity of the anterior chamber was determined by anterior segment OCT. RESULTS The average OCT reflectivity of the SRF was 3.52 arbitrary units (AU; 15 eyes; range, 0.01-20.7 AU). The average concentration of VEGF was 870.1 pg/mL, that of IL-6 was 131.7 pg/mL, and that of IL-8 was 224.1 pg/mL. The degree of OCT reflectivity was correlated significantly with the intravitreal VEGF concentration (r = 0.516, P = 0.049, Spearmans rank correlation coefficient), but not with IL-6 or IL-8. In the swine eyes, the presence of plasma, bilirubin, and fibrinogen in the anterior chamber led to significant increases in the reflectivity. CONCLUSIONS The significant correlation between the reflectivity of SRF and intravitreal VEGF indicates that OCT can be used to monitor the level of VEGF in eyes with DME.

TNF-α Decreases VEGF Secretion in Highly Polarized RPE Cells but Increases It in Non-Polarized RPE Cells Related to Crosstalk between JNK and NF-κB Pathways

Asymmetrical secretion of vascular endothelial growth factor (VEGF) by retinal pigment epithelial (RPE) cells in situ is critical for maintaining the homeostasis of the retina and choroid. VEGF is also involved in the development and progression of age-related macular degeneration (AMD). We studied the effect of tumor necrosis factor-α (TNF-α) on the secretion of VEGF in polarized and non-polarized RPE cells (P-RPE cells and N-RPE cells, respectively) in culture and in situ in rats. A subretinal injection of TNF-α caused a decrease in VEGF expression and choroidal atrophy. Porcine RPE cells were seeded on Transwell™ filters, and their maturation and polarization were confirmed by the asymmetrical VEGF secretion and trans electrical resistance. Exposure to TNF-α decreased the VEGF secretion in P-RPE cells but increased it in N-RPE cells in culture. TNF-α inactivated JNK in P-RPE cells but activated it in N-RPE cells, and TNF-α activated NF-κB in P-RPE cells but not in N-RPE cells. Inhibition of NF-κB activated JNK in both types of RPE cells indicating crosstalk between JNK and NF-κB. TNF-α induced the inhibitory effects of NF-κB on JNK in P-RPE cells because NF-κB is continuously inactivated. In N-RPE cells, however, it was not evident because NF-κB was already activated. The basic activation pattern of JNK and NF-κB and their crosstalk led to opposing responses of RPE cells to TNF-α. These results suggest that VEGF secretion under inflammatory conditions depends on cellular polarization, and the TNF-α-induced VEGF down-regulation may result in choroidal atrophy in polarized physiological RPE cells. TNF-α-induced VEGF up-regulation may cause neovascularization by non-polarized or non-physiological RPE cells.

Targeting CD9 produces stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis: A novel antiangiogenic therapy

The precise roles of tetraspanin CD9 are unclear. Here we show that CD9 plays a stimulus-independent role in angiogenesis and that inhibiting CD9 expression or function is a potential antiangiogenic therapy. Knocking down CD9 expression significantly inhibited in vitro endothelial cell migration and invasion induced by vascular endothelial growth factor (VEGF) or hepatocyte growth factor (HGF). Injecting CD9-specific small interfering RNA (siRNA-CD9) markedly inhibited HGF- or VEGF-induced subconjunctival angiogenesis in vivo. Both results revealed potent and stimulus-independent antiangiogenic effects of targeting CD9. Furthermore, intravitreous injections of siRNA-CD9 or anti-CD9 antibodies were therapeutically effective for laser-induced retinal and choroidal neovascularization in mice, a representative ocular angiogenic disease model. In terms of the mechanism, growth factor receptor and downstream signaling activation were not affected, whereas abnormal localization of integrins and membrane type-1 matrix metalloproteinase was observed during angiogenesis, by knocking down CD9 expression. Notably, knocking down CD9 expression did not induce death and mildly inhibited proliferation of quiescent endothelial cells under conditions without an angiogenic stimulus. Thus, CD9 does not directly affect growth factor-induced signal transduction, which is required in angiogenesis and normal vasculature, but is part of the angiogenesis machinery in endothelial cells during angiogenesis. In conclusion, targeting CD9 produced stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis, and appears to be an effective and safe antiangiogenic approach. These results shed light on the biological roles of CD9 and may lead to novel antiangiogenic therapies.

Resveratrol inhibits epithelial-mesenchymal transition of retinal pigment epithelium and development of proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is a serious complication of retinal detachment and ocular trauma, and its recurrence may lead to irreversible vision loss. Epithelial to mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a critical step in the pathogenesis of PVR, which is characterized by fibrotic membrane formation and traction retinal detachment. In this study, we investigated the potential impact of resveratrol (RESV) on EMT and the fibrotic process in cultured RPE cells and further examined the preventive effect of RESV on PVR development using a rabbit model of PVR. We found that RESV induces mesenchymal to epithelial transition (MET) and inhibits transforming growth factor-β2(TGF-β2)-induced EMT of RPE cells by deacetylating SMAD4. The effect of RESV on MET was dependent on sirtuin1 activation. RESV suppressed proliferation, migration and fibronectin synthesis induced by platelet-derived growth factor-BB or TGF-β2. In vivo, RESV inhibited the progression of experimental PVR in rabbit eyes. Histological findings showed that RESV reduced fibrotic membrane formation and decreased α-SMA expression in the epiretinal membranes. These results suggest the potential use of RESV as a therapeutic agent to prevent the development of PVR by targeting EMT of RPE.

Selective Gene Transfer to the Retina Using Intravitreal Ultrasound Irradiation

This paper aims to evaluate the efficacy of intravitreal ultrasound (US) irradiation for green fluorescent protein (GFP) plasmid transfer into the rabbit retina using a miniature US transducer. Intravitreal US irradiation was performed by a slight modification of the transconjunctival sutureless vitrectomy system utilizing a small probe. After vitrectomy, the US probe was inserted through a scleral incision. A mixture of GFP plasmid (50 μL) and bubble liposomes (BLs; 50 μL) was injected into the vitreous cavity, and US was generated to the retina using a SonoPore 4000. The control group was not exposed to US. After 72 h, the gene-transfer efficiency was quantified by counting the number of GFP-positive cells. The retinas that received plasmid, BL, and US showed a significant increase in the number (average ± SEM) of GFP-positive cells (32 ± 4.9; n = 7; P < 0.01 ). No GFP-positive cells were observed in the control eyes (n = 7). Intravitreal retinal US irradiation can transfer the GFP plasmid into the retina without causing any apparent damage. This procedure could be used to transfer genes and drugs directly to the retina and therefore has potential therapeutic value.