Ken Ogino

SDF-1/CXCR4 Contributes to the Activation of Tip Cells and Microglia in Retinal Angiogenesis

PURPOSE. Although stromal cell-derived factor (SDF)-1 contributes to angiogenesis, its effects on sprouting angiogenesis remain ill defined. The authors investigated how SDF-1 and its receptor, CXCR4, influence neovascular sprouting. METHODS. In vivo retinal vascular development was evaluated and ex vivo retinal angiogenesis induced by vascular endothelial growth factor (VEGF). Time-sequential observation was followed by the quantification of movements in neovascular sprouts and microglia. Real-time PCR was performed for the measurement of mRNA levels. RESULTS. Neutralizing antibodies against SDF-1 or an antagonist of CXCR4, AMD3100, reduced the radius of the vascularized area in retinal vascular development. These inhibitions disturbed the filopodial extensions in tip cells and proliferation in stalk cells, reduced the number of microglia, and decreased the mRNA levels of KDR/Flk-1, UNC5B, and PDGFB, which are abundantly expressed in tip cells. In ex vivo experiments, VEGF induced SDF-1 mRNA expression, and the inhibition of SDF-1/CXCR4 decreased the number of VEGF-induced neovascular sprouts. The authors further evaluated the kinetics of sprouts using time-lapse imaging and found that SDF-1/CXCR4 contributes to the elongation of neovascular sprouts and to the extension and retraction of leading edges. The movements of resident microglia after VEGF treatment were also reduced by SDF-1/CXCR4 inhibition. Interestingly, microglial depletion decreased VEGF-induced neovascular sprouts with the partial effects of SDF-1/CXCR4 blockade. CONCLUSIONS. SDF-1/CXCR4 promotes retinal angiogenesis by both tip cell activation and the indirect angiogenic effects of microglia.

Comprehensive Molecular Diagnosis of a Large Cohort of Japanese Retinitis Pigmentosa and Usher Syndrome Patients by Next-Generation Sequencing

PURPOSE Retinitis pigmentosa (RP), a major cause of blindness in developed countries, has multiple causative genes; its prevalence differs by ethnicity. Usher syndrome is the most common form of syndromic RP and is accompanied by hearing impairment. Although molecular diagnosis is challenging, recent technological advances such as targeted high-throughput resequencing are efficient screening tools. METHODS We performed comprehensive molecular testing in 329 Japanese RP and Usher syndrome patients by using a custom capture panel that covered the coding exons and exon/intron boundaries of all 193 known inherited eye disease genes combined with Illumina HiSequation 2500. Candidate variants were screened using systematic data analyses, and their potential pathogenicity was assessed according to the frequency of the variants in normal populations, in silico prediction tools, and compatibility with known phenotypes or inheritance patterns. RESULTS Molecular diagnoses were made in 115/317 RP patients (36.3%) and 6/12 Usher syndrome patients (50%). We identified 104 distinct mutations, including 66 novel mutations. EYS, USH2A, and RHO were common causative genes. In particular, mutations in EYS accounted for 15.0% of the autosomal recessive/simplex RP patients or 10.7% of the entire RP cohort. Among the 189 previously reported mutations detected in the current study, 55 (29.1%) were found commonly in Japanese or other public databases and were excluded from molecular diagnoses. CONCLUSIONS By screening a large cohort of patients, this study catalogued the genetic variations involved in RP and Usher syndrome in a Japanese population and highlighted the different distribution of causative genes among populations.

Characteristics of optical coherence tomographic hyperreflective foci in retinal vein occlusion.

Purpose: To evaluate the hyperreflective foci in branch retinal vein occlusion and central retinal vein occlusion depicted by spectral-domain optical coherence tomography (OCT). Methods: Consecutive series of 73 eyes of 73 patients with retinal vein occlusion (58 branch retinal vein occlusion and 15 central retinal vein occlusion) who had Spectralis OCT images obtained were retrospectively reviewed, comparing color fundus photographs and fluorescein angiography. Results: Hyperreflective foci were detected in 54 eyes (74.0%) by spectral-domain OCT, and hard exudates were detected in 17 eyes (23.3%) by color fundus photography. Hard exudates on the color photographs corresponded to the confluence of hyperreflective foci mainly around the outer plexiform layer in the unaffected areas on the spectral-domain OCT images, whereas fine hyperreflective foci were scattered in all retinal layers of the affected areas (P < 0.001). Most eyes with hyperreflective foci attached to the inner side of the external limiting membrane also had serous retinal detachment (P < 0.001). Compared with diabetic macular edema, we did not find subfoveal hard exudates in retinal vein occlusion. Conclusion: Hyperreflective foci delineated on spectral-domain OCT suggest the pathogenesis regarding the flow of extravasated blood constituents in retinal vein occlusion.

Age- and hypertension-dependent changes in retinal vessel diameter and wall thickness: an optical coherence tomography study.

PURPOSE To validate and evaluate the reliability of retinal vessel diameter measurements by optical coherence tomography (OCT). The effects of age and hypertension on vessel diameter were also examined. DESIGN Prospective, cross-sectional study. METHODS Two hundred thirty-eight eyes (238 subjects) with no ocular disease were included. Hypertension was present in 106 subjects and absent in 132 subjects. Spectralis HRA+OCT was used to scan a circular region around the optic disc. Outer and inner diameters of the 4 largest retinal arteries and veins were measured using OCT vascular wall reflections, and vessel wall thickness was calculated. RESULTS Intervisit, interexaminer, and interevaluator intraclass correlation coefficients of randomly selected vessel measurements were all greater than 0.90. Mean inner arterial and venous diameters were 87.8 ± 9.4 μm and 113.7 ± 12.5 μm, respectively. The OCT-measured mean inner arterial and venous diameters were significantly correlated to fundus photography caliber measurements (P = .005 and P = .001, respectively). Arterial and venous wall thicknesses were 17.4 ± 2.4 μm and 13.7 ± 2.1 μm, respectively, both of which were highly correlated with subject age (arterial: r = 0.612, P < .001, venous: r = 0.455, P < .001). Additionally, both mean arterial and venous wall thicknesses were significantly greater in subjects with hypertension than in age-matched subjects without hypertension (P = .020 and P = .015, respectively). CONCLUSIONS Retinal vessel diameter measurements obtained with OCT were highly reproducible and vessel wall thicknesses, calculated using outer and inner diameter measurements, were significantly thickened by both aging and systemic hypertension.

Wide-Field Fundus Autofluorescence Imaging of Retinitis Pigmentosa

PURPOSE To evaluate the clinical usefulness of wide-field fundus autofluorescence (FAF) imaging in patients with retinitis pigmentosa (RP). DESIGN Cross-sectional case series. PARTICIPANTS Seventy-five eyes of 75 patients with RP. METHODS We examined the eyes of the RP patients using the Optos 200Tx imaging system (Optos PLC, Dunfermline, United Kingdom) and identified abnormal FAF patterns such as ring hyperautofluorescence and patchy hypoautofluorescent areas. Patients with hyperautofluorescent rings or foveal hyperautofluorescence were compared with those without such findings. We determined the percentage area occupied by the FAF abnormalities within a defined region of the eye and examined the relationship between the percentage area of these abnormalities and the visual field area. Moreover, we categorized the patients into 3 different groups based on the presence of a patchy hypoautofluorescent lesion larger than 1 disc diameter: Group A consisted of those with patchy lesions smaller than 1 disc diameter, group B consisted of those with patchy lesions larger than 1 disc diameter but present in only 1 quadrant, and group C consisted of those with patchy lesions larger than 1 disc diameter and present in more than 1 quadrant. In addition, various clinical characteristics were compared among these 3 groups. MAIN OUTCOME MEASURES Predicting the visual field size and duration of the disease in RP patients based on FAF patterns. RESULTS Patients without hyperautofluorescent rings or foveal hyperautofluorescence had better visual acuity or mean deviation measured with a Humphrey perimeter. The total area of the abnormal FAF image correlated with the visual field area measured with a Goldmann perimeter (R = -0.64, P<0.001). The individuals with the large patchy hypofluorescent areas (i.e., larger than 1 disc diameter) were older than those with small patchy hypofluorescent areas (group A vs. groups B and C, P = 0.002 and P<0.001, respectively) and had experienced the symptoms for longer durations (group A vs. groups B and C, P<0.05 and P<0.001, respectively). CONCLUSIONS We can estimate the visual field in patients with RP using the objective measurements from wide-field FAF. The presence of patchy hypofluorescent lesions can be used an indicator of the duration of RP. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Choroidal thickness after intravitreal ranibizumab injections for choroidal neovascularization.

Purpose To study changes in choroidal thickness with ranibizumab treatment for choroidal neovascularization (CNV). Design Prospective case series. Methods This prospective study consisted of 60 CNV-affected eyes of 60 patients treated with intravitreal injections of ranibizumab using an on-demand protocol after an initial loading phase. The eyes studied included 20 with age-related macular degeneration (AMD), 20 with polypoidal choroidal vasculopathy (PCV), and 20 with myopic CNV. In the eyes with AMD and PCV, choroidal thickness at the fovea was measured with optical coherence tomography using enhanced depth imaging. In eyes with myopic CNV, the choroidal thickness was measured using standard optical coherence tomography without the enhanced depth imaging technique. Results With ranibizumab treatment, central retinal thickness decreased significantly (P < 0.001) and visual acuity improved significantly (P < 0.001). However, central choroidal thickness (167.2 ± 108.3 μm) showed no significant change at 1 month after the loading phase (165.2 ± 107.8 μm, P = 0.120) or at final examination (164.8 ± 107.7 μm, P = 0.115). At baseline, central retinal thickness in eyes with AMD was significantly greater that those with PCV (P = 0.005) or high myopia (P = 0.029). However, central choroidal thickness in eyes with myopic CNV was significantly thinner than in eyes with AMD (P < 0.001) or PCV (P < 0.001). In each type of disease, there was no significant change in central choroidal thickness with ranibizumab treatment. Conclusion The effect of ranibizumab on the choroidal thickness is minimal, if any.

Morphologic and Functional Changes in Retinal Vessels Associated with Branch Retinal Vein Occlusion

OBJECTIVE To study the morphologic and functional changes in retinal veins of eyes affected with branch retinal vein occlusion (BRVO) by thin sectioning with optical coherence tomography (OCT). DESIGN Prospective, observational, cross-sectional study. PARTICIPANTS Twenty-five consecutive patients (25 eyes) with acute BRVO. METHODS Major retinal veins, arteries, and arteriovenous (A/V) crossing were examined by sequential thin sectioning by Spectralis HRA+OCT (Heidelberg Engineering, Heidelberg, Germany). The retinal blood flow was mimicked in vitro and scanned with Spectralis HRA+OCT. MAIN OUTCOME MEASURES Morphologic characteristics of normal and BRVO-affected retinal vessels seen in OCT sections. RESULTS Cross-sectional OCT images revealed physiologic retinal vessels as oval configurations with 4 distinctive hyperreflectivities in a line. The vessel walls showed the innermost and outermost hyperreflectivity, and the blood flow showed internal paired hyperreflectivities with an hourglass shape. No eye with disturbed blood flow due to BRVO showed this internal hyperreflectivity pattern. In vitro, OCT sections of the blood within the glass tube without flow showed homogeneous reflectivities. Increased blood flow velocity resulted in the development of heterogeneous internal reflectivity and hourglass-shaped hyperreflectivities. In all eyes with acute BRVO, sequential sectioning with OCT visualized 3-dimensional vascular architecture and the intravascular conditions at the A/V crossing. At the affected A/V crossing, arterial overcrossing was seen in 17 eyes and venous overcrossing was seen in 8 eyes. In eyes with arterial overcrossing, the retinal vein seemed to run deep under the artery at the A/V crossing, and the venous lumen often appeared to be preserved even at the A/V crossing. In all eyes with venous overcrossing, the retinal vein appeared to be compressed and choked between the internal limiting membrane and the arterial wall at the A/V crossing. Optical coherence tomography sectioning showed intravenous thrombi in 21 eyes, and the thrombi were detected downstream of the A/V crossing in all the cases. The detection of thrombus was significantly associated with ischemic pattern in BRVO (P=0.036). CONCLUSIONS In eyes with BRVO, sequential thin sections with OCT visualized 3-dimensional retinal vasculature. The present OCT findings suggest that BRVO may occur by 2 different mechanisms, depending on the relative anatomic positions of the crossing vessels. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Outer retinal circular structures in patients with Bietti crystalline retinopathy

Background Bietti crystalline retinopathy (BCR) is a distinct retinal degenerative disease characterised by retinal degeneration with many yellow–white crystals located mainly at the posterior pole area. Using spectral domain-optical coherence tomography (SD-OCT), the structural change in retina was investigated. Methods Patients diagnosed with BCR (n=12), retinitis pigmentosa (RP, n=292) and cone dystrophy (n=16) were included in this study. The authors mainly examined fundus photographs and SD-OCT, infrared and fundus autofluorescence images of these patients. Results Crystalline deposits were detected in portions of the retinal pigment epithelium that lacked patchy degenerated lesions. SD-OCT revealed that most of the observed crystalline deposits were located adjacent to the inner side of retinal pigment epithelium layer. The change most frequently observed was circular hyper-refractive structures in the outer nuclear layer. Although the structures were considered to be previously reported “tubular formation” or “tubular degeneration”, we determined that many of these circular structures were slices of spherical structures and were typically noted in areas suspected of ongoing active degeneration. Conclusion BCR has characteristic structures in the outer nuclear layer. Although the incidence of the structure varies, it may be characteristic of retinal degeneration and can be found in many retinal degenerative diseases.

Macular Cone Abnormalities in Retinitis Pigmentosa with Preserved Central Vision Using Adaptive Optics Scanning Laser Ophthalmoscopy

Purpose To assess macular photoreceptor abnormalities in eyes with retinitis pigmentosa (RP) with preserved central vision using adaptive optics scanning laser ophthalmoscopy (AO-SLO). Methods Fourteen eyes of 14 patients with RP (best-corrected visual acuity 20/20 or better) and 12 eyes of 12 volunteers underwent a full ophthalmologic examination, fundus autofluorescence, spectral-domain optical coherence tomography (SD-OCT), and imaging with a prototype AO-SLO system. Cone density and spatial organization of the cone mosaic were assessed using AO-SLO images. Results In 3 eyes with RP and preserved central vision, cones formed a mostly regular mosaic pattern with small patchy dark areas, and in 10 eyes, the cone mosaic patterns were less regular, and large dark regions with missing cones were apparent. Only one eye with RP demonstrated a normal, regular cone mosaic pattern. In eyes with RP, cone density was significantly lower at 0.5 mm and 1.0 mm from the center of the fovea compared to normal eyes (P<0.001 and 0.021, respectively). At 0.5 mm and 1.0 mm from the center of the fovea, a decreased number of cones had 6 neighbors in eyes with RP (P = 0.002 for both). Greater decrease in cone density was related to disruption of the photoreceptor inner segment (IS) ellipsoid band on SD-OCT images (P = 0.044); however, dark regions were seen on AO-SLO even in areas of continuous IS ellipsoid on SD-OCT. Decreased cone density correlated thinner outer nuclear layer (P = 0.029) and thinner inner segment and outer segment thickness (P = 0.011) on SD-OCT. Conclusions Cone density is decreased and the regularity of the cone mosaic spatial arrangement is disrupted in eyes with RP, even when visual acuity and foveal sensitivity are good. AO-SLO imaging is a sensitive quantitative tool for detecting photoreceptor abnormalities in eyes with RP.

Macular choroidal thickness and volume in eyes with angioid streaks measured by swept source optical coherence tomography.

PURPOSE To study the mean choroidal thickness and volume of the macula in eyes with angioid streaks using swept source optical coherence tomography (OCT) in the 1050-nm wavelength range. DESIGN Prospective case series. METHODS The macular area of 39 eyes of 23 patients with angioid streaks and of 20 normal eyes of 20 matched controls (Group 1) was studied with a swept source OCT prototype system. Eyes with angioid streaks were classified into 1 of 4 groups: those without choroidal neovascularization (CNV) (Group 2); those with CNV that had no history of treatment (Group 3); those with CNV that had previously received only anti-vascular endothelial growth factor treatments (Group 4); and those with CNV that had previously received photodynamic therapy (Group 5). Using a raster scan protocol with 512 × 128 A-scans, we produced a macular choroidal thickness map (6 × 6 mm(2)). RESULTS There were no significant differences in age, axial length, or refractive error among the 5 groups. Mean choroidal thickness of the macula in Group 2 (218.9 ± 46.8 μm) was as great as that in Group 1 (218.8 ± 69.2 μm). However, the macular choroidal thickness in Group 3 (119.7 ± 49.2 μm), Group 4 (140.1 ± 64.9 μm), and Group 5 (144.0 ± 52.6 μm) was significantly less than that of Group 1 (P < .05). There were no statistical differences between Groups 3 through 5. In each group, the choroid of the nasal quadrant was significantly thinner compared to that in other quadrants (P < .05). CONCLUSIONS The choroid in eyes with angioid streaks without CNV was as thick as that in normal controls, but was significantly thinner in eyes with angioid streaks that had developed CNV.