Makoto Shirasawa

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.).

Retinal morphologic changes and concentrations of cytokines in eyes with diabetic macular edema.

Purpose: To determine the relationship between the retinal morphologic changes and concentrations of intravitreal cytokines in eyes with diabetic macular edema. Methods: A retrospective comparative study was performed. The preoperative optical coherence tomography images were evaluated to determine the presence of serous retinal detachments (SRDs), retinal cystic changes, and retinal swelling. The concentrations of vascular endothelial growth factor, interleukin (IL)-6, and IL-8 in vitreous samples collected during vitrectomy were determined. The correlations between optical coherence tomography parameters, other clinical factors, and the concentration of cytokines were calculated. Results: Fifty-two eyes (52 patients) were investigated. An SRD was found in 19 of the 52 eyes (36.5%). Multivariate regression analysis showed that IL-6 was the only factor significantly associated with the presence of an SRD (P = 0.001; odds ratio, 1.268; 95% confidence interval, 1.105–1.452). The other morphologic changes, such as retinal cystic changes and retinal swellings, were not significantly associated with the concentrations of intravitreal cytokines. Conclusion: The significant association of SRD with intravitreal IL-6 indicates that inflammation may play an important role in the development of SRD in diabetic macular edema.

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.

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.

Penetration of bevacizumab and ranibizumab through retinal pigment epithelial layer in vitro.

Purpose: To determine the permeability of bevacizumab and ranibizumab through highly-polarized retinal pigment epithelial (RPE) cells in vitro. Methods: Highly-polarized RPE cells were cultured in the upper chamber of a Transwell culture system. Bevacizumab or ranibizumab was added to the upper chamber. After 3 hours, the concentrations of bevacizumab or ranibizumab were determined in the upper and lower chambers. The cytotoxicities of the two anti-vascular endothelial growth factor agents were determined histologically. The effects of inhibiting endocytosis by pharmacologic inhibitors were also evaluated. Results: The concentration of ranibizumab was higher than that of bevacizumab in the lower chamber (P < 0.05). Vascular endothelial growth factor was found mainly in the lower chamber under normal conditions. However, the concentration of vascular endothelial growth factor in the lower chamber was significantly less when ranibizumab was added to the upper chamber than when bevacizumab was added. Histology showed no obvious changes in bevacizumab-exposed or ranibizumab-exposed RPE cells. Pretreatment with protein kinase C inhibitor staurosporine had significant negative effects on the permeability of bevacizumab and ranibizumab (P < 0.05). Conclusion: Ranibizumab is more permeable than bevacizumab through the highly-polarized RPE layer at clinically equivalent concentrations, and their permeability was partially protein kinase C–dependent. Ranibizumab might be more therapeutically effective than bevacizumab on choroidal neovascularization beneath the RPE layer.

Different Effects of Thrombin on VEGF Secretion, Proliferation, and Permeability in Polarized and Non-polarized Retinal Pigment Epithelial Cells

Abstract We investigated the effect of thrombin on the secretion of vascular endothelial growth factor (VEGF), on cellular proliferation, and on the integrity of the barrier function of polarized retinal pigment epithelial (RPE) cells. In addition, we compared the responses of polarized to that of non-polarized RPE cells. Porcine polarized RPE cells were established using Transwell membranes. The polarization of the RPE cells was determined by their high transepithelial electrical resistance (TER > 200 Ω cm2) and by their differential secretion of VEGF (basal direction >apical direction by 2.5×). RPE cells were incubated with thrombin (5–20 U/ml) for 24 h. The concentration of VEGF in the culture medium was measured by enzyme-linked immunosorbent assay, and the TER was measured. Cellular proliferation was assessed by Ki-67 immunostaining. The area of laser-induced choroidal naovascularization (CNV) was measured in rat eyes and compare to that of controls with or without thrombin. Our results showed that thrombin significantly increased VEGF secretion both in polarized and non-polarized RPE cells in a dose-dependent way. Thrombin did not significantly affect the TER or the expression of tight-junctional proteins in polarized RPE cells, but decreased it in non-polarized RPE cells by inducing intercellular gaps. Ki-67-positive cells were observed in non-polarized RPE cells but not in polarized RPE cells as controls. After thrombin exposure, the number of Ki-67-positive cells increased significantly in non-polarized RPE cells but not in polarized RPE cells. The area of CNV was larger in thrombin-injected eye than control eyes. Although thrombin increased VEGF secretion regardless of cell polarity, its effects on proliferation and barrier integrity were dependent upon cell polarity. Cell polarization is an important factor for determining the response of RPE cells to thrombin, and the different responsive patterns to thrombin upon cell polarity might explain the complicated pathology of such diseases as age-related macular degeneration.