Fumitaka Hirose

Simvastatin Inhibits Leukocyte Accumulation and Vascular Permeability in the Retinas of Rats with Streptozotocin-Induced Diabetes

Leukocytes play important roles in the pathogenesis of diabetic retinopathy. Recently, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors have been reported to exert various effects in addition to their lipid-lowering ability. We investigated the effects of simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, on leukocyte-induced diabetic changes in retinas. Diabetes was induced in Long-Evans rats with streptozotocin, and simvastatin administration was begun immediately after the induction of diabetes. Two weeks of treatment with simvastatin suppressed significantly the number of leukocytes adhering to retinal vessel endothelium and the number of leukocytes accumulated in the retinal tissue by 72.9% and 41.0%, respectively (P < 0.01). The expression of intercellular adhesion molecule-1 (ICAM-1) and the CD18 (the common beta-chain of ICAM-1 ligands) were both suppressed with simvastatin. The amount of vascular endothelial growth factor in the retina was attenuated in the simvastatin-treated group. To evaluate the effects of simvastatin on leukocyte-induced endothelial cell damage, vascular permeability in the retina was measured with fluorescein-labeled dextran. Treatment with simvastatin markedly reduced retinal permeability (P = 0.014). This suggests that simvastatin attenuates leukocyte-endothelial cell interactions and subsequent blood-retinal barrier breakdown via suppression of vascular endothelial growth factor-induced ICAM-1 expression in the diabetic retina. Simvastatin may thus be useful in the prevention of diabetic retinopathy.

Three-dimensional imaging of macular inner structures in glaucoma by using spectral-domain optical coherence tomography.

PURPOSE To profile macular thickness changes in glaucoma by using three-dimensional spectral-domain optical coherence tomography (3D-SD-OCT). METHODS The study included 30 eyes with suspected glaucoma and preperimetric glaucoma (SGPPG) and 35 healthy eyes. The macular thickness, including those of the total retina, nerve fiber layer (NFL), and combined inner retinal layers (IRLs)-NFL+ganglion cell layer (GCL)+inner plexiform layer (IPL)-was measured by 3D-SD-OCT raster scans in a 6 mm(2) region. The average and sectoral thicknesses were calculated on an Early Treatment of Diabetic Retinopathy Study (ETDRS) chart and a ETDRS chart with a 45° rotation (glaucoma sector chart, GSC). RESULTS The mean IRL thickness was significantly less in the SGPPG eyes than in the healthy eyes, but the mean total retinal and macular NFL thicknesses were not. In the SGPPG eyes, the IRLs were thinner in the outer macula than in the inner macula, in the inferior hemisphere than in the superior hemisphere, and in the temporal hemisphere than in the nasal hemisphere. The significantly thinned sectors were nearly identical on the GSC but only slightly overlapped on the ETDRS chart. The IRLs in the inferior temporal outer sector (GSC) had the greatest area under the receiver operating characteristic curve, which was significantly greater than those for the IRLs over the entire macula, inferior hemiretinal region, and inferior outer hemicircular region (macular subfields), and that for the circumpapillary NFL in the inferior sectors (P = 0.001-0.036). CONCLUSIONS Macular IRL thickness measured by using 3D-SD-OCT is useful for profiling macular atrophy in SGPPG.

In Vivo Evaluation of Retinal Injury After Transient Ischemia in Hypertensive Rats

Abstract—A number of studies have suggested that hypertension affects the pathogenesis of inflammatory reactions in various organs. The objective of this study was to evaluate the effects of hypertension on leukocyte–endothelial interactions after transient retinal ischemia. Transient retinal ischemia was induced for 60 minutes in spontaneously hypertensive rats (SHR) and in age-matched normotensive Wistar-Kyoto rats (WKY). At 4, 12, 24, 48, and 72 hours after reperfusion, flat-mount retinas were prepared to evaluate the density of leukocytes that had been accumulated in the retina. Intercellular adhesion molecule-1 (ICAM-1) mRNA expression was studied by semiquantitative polymerase chain reaction and ICAM-1 protein levels were studied by enzyme-linked immunosorbent assay. At 14 days after reperfusion, the retinal damage and the effect of superoxide dismutase on the damage were evaluated histologically. In SHR, the number of accumulated leukocytes peaked at 48 hours after reperfusion, and it was upregulated to 5.2-fold, as compared with that of WKY (P <0.001). ICAM-1 mRNA expression and ICAM-1 protein levels were increased significantly in the ischemia-reperfused retina in SHR compared with WKY (P <0.05). Histological examination demonstrated marked increase in the retinal ischemia/reperfusion damage in SHR (P <0.01) and a significant amelioration of the damage by treatment with superoxide dismutase in SHR (P <0.05). Oxidative stress may thus be an important mechanism for the deterioration seen in ischemia/reperfusion injury in the SHR retina.

Hemispherical Focal Macular Photopic Negative Response and Macular Inner Retinal Thickness in Open-Angle Glaucoma

PURPOSE To investigate in open-angle glaucoma the focal macular photopic negative response and spectral-domain optical coherence tomography measurements of retinal thicknesses in the superior and inferior macula. DESIGN Comparative case series. METHODS We studied 63 eyes of 63 patients with open-angle glaucoma and 41 normal eyes of 41 volunteers. Photopic negative responses were recorded using a spotlight (diameter of 15 degrees of circle), projected onto the whole or superior or inferior macula. Ganglion cell complex (nerve fiber + ganglion cell + inner plexiform layers) thickness was measured using spectral-domain optical coherence tomography. RESULTS Mean photopic negative response amplitude over the entire macula was significantly (P < .001) decreased compared with that of controls in eyes with early (n = 24; to 62.1%), moderate (n = 21; to 57.2%), and severe (n = 18; to 49.3%) open-angle glaucoma, but not significantly different among eyes with various stages of glaucoma. Mean ganglion cell complex thickness was significantly (P < .001) decreased compared with that of controls in eyes with early (86.0%), moderate (78.3%), and severe (71.2%) glaucoma, and thinning correlated positively with glaucoma severity (P < .001). Mean photopic negative response amplitude correlated significantly (P < .001) with ganglion cell complex thickness over the whole, superior, and inferior macular areas (r = 0.57 to 0.74). In 16 eyes without visual field defect in the inferior hemifield, mean photopic negative response amplitude was 56.5% of normal (P < .001), and mean ganglion cell complex thickness in the superior macula was 92.1% of normal (P = .004). CONCLUSIONS Focal macular photopic negative response amplitude correlates with ganglion cell complex thickness, but decreases more abruptly in early glaucoma compared with ganglion cell complex thickness.

In vivo evaluation of ocular inflammatory responses in experimental diabetes

Aims: Diabetic patients may have abnormal inflammatory reactions to foreign or endogenous stimuli. This study was designed to evaluate inflammatory reactions in the diabetic eye through retinal leucocyte dynamics in the inflamed eyes of diabetic rats. Methods: Three weeks after diabetes induction in Long-Evans rats, endotoxin induced uveitis was produced by footpad injection of lipopolysaccharide (LPS). After LPS injection, leucocyte behaviour was evaluated in vivo by acridine orange digital fluorography. Results: The number of rolling leucocytes increased in a biphasic manner at 12 hours and 48 hours. The number of leucocytes accumulating in the retina reached a peak at 72 hours. The maximal numbers of rolling and accumulating leucocytes in the diabetic retina decreased by 56.3% (p<0.01) and 46.7% (p<0.0001), respectively, compared with the non-diabetic retina. The levels of mRNA expression of adhesion molecules in the retina, which were upregulated after LPS injection, were also lower in diabetic rats than in non-diabetic rats. Conclusion: This study is the first to show that endotoxin induced inflammation is disturbed in the diabetic eye, based on evidence that the leucocyte-endothelial cell interactions stimulated by LPS were suppressed in the diabetic retina. These findings support the theory that ocular inflammatory reactions are impaired in diabetic patients.

Influence of anterior segment biometric parameters on the anterior chamber angle width in eyes with angle closure.

Purpose:To predict angle narrowing in eyes with angle closure in a Japanese population using anterior segment optical coherence tomography (AS-OCT) quantitative parameters. Patients and Methods:AS-OCT was used to examine 118 eyes of 118 patients with angle closure and 40 eyes of 40 patients with open angle under dark conditions. After measuring the angle opening distance 500 (AOD500), anterior chamber depth, iris thickness (IT), iris convexity (IC), pupil diameter, anterior chamber width, and crystalline lens rise, multivariate regression analyses were performed for the AOD500 in each group. Results:With the exception of IT, significant differences were observed between the AS-OCT parameters for the angle closure and open-angle groups. Anterior chamber depth, IT, and IC were the explanatory variables associated with AOD500 for each group (P⩽0.001). A significant negative association was found between IT and IC only in the angle-closure group (P<0.001). Conclusions:This study quantitatively confirmed that shallow anterior chamber depth was a major mechanism of angle narrowing, and that both IT and IC had a strong impact on angle narrowing. Moreover, the negative association found between IT and IC in only the angle closure group indicated the existence of the stretch force placed on the iris by relative pupillary block.

Lack of inducible nitric oxide synthases attenuates leukocyte-endothelial cell interactions in retinal microcirculation

Aim: To investigate the effect of inducible nitric oxide synthases (iNOS) on inflammatory reactions during endotoxin-induced uveitis (EIU) in mice by studying leukocyte–endothelial cell interactions. Methods: EIU was produced in immunosuppressed iNOS−/− mice and C57BL/6 (normal) mice by footpad injection of lipopolysaccharide. Leukocytes were labelled with acridine orange. Leukocyte rolling in the retinal microcirculation was evaluated in vivo with acridine orange digital fluorography. The number of migrated leukocytes was counted in flat-mounted retina. Results: Both leukocyte rolling and migration peaked at 48 h after lipopolysaccharide injection. The maximal numbers of rolling leukocytes in the immunosuppressed iNOS−/− mouse retina decreased by 98.2% (p<0.001) compared with that in the normal mouse retina at 48 h after lipopolysaccharide injection. In addition, the maximal numbers of migrated leukocytes in the immunosuppressed iNOS−/− mouse retina decreased by 74.0% (p<0.001) compared with that in the normal mouse retina at 24 h after lipopolysaccharide injection. Furthermore, the diameters of major retinal veins of the immunosuppressed iNOS−/− group were smaller at both 24 and 48 h after lipopolysaccharide injection than were those of the normal group (p<0.001, respectively). Conclusions: A lack of iNOS suppresses leukocyte–endothelial cell interactions in the retinas of mice with EIU. This suggests that iNOS may play a role in the management of patients with uveitis and other inflammatory conditions.

Alteration of Leukocyte–Endothelial Cell Interaction During Aging in Retinal Microcirculation of Hypertensive Rats

PurposeHypertension, one of the more common chronic diseases affecting the elderly, has been reported to influence leukocyte–endothelial cell interaction. The leukocyte-mediated inflammatory process contributes to age-related changes in vessels. This study was designed to evaluate age-related changes in leukocyte–endothelial cell interaction in the hypertensive rat retina.MethodsMale spontaneous hypertensive rats (SHR; 1.5, 3, 6, 12, and 20 months of age) and age-matched Wistar-Kyoto rats (WKY) were used. The number of accumulated leukocytes was counted in sections of flat-mounted retinal tissue. The expression of intercellular adhesion molecule-1 (ICAM-1) and CD18 (the common β-chain of ICAM-1 ligands) was evaluated. Retinal thickness was evaluated histologically.ResultsThe number of accumulated leukocytes and the expression of ICAM-1 and CD18 increased in the aged retina. The number of leukocytes that accumulated and the expression of CD 18 were significantly higher in the SHR group than in the WKY group (P < 0.01). In addition, retinal thickness decreased with age.ConclusionLeukocyte–endothelial cell interaction increased in the aged retina and these changes were more severe in SHR retina than in WKY retina. This increased interaction was first observed at 3 months, a relatively young age.

Experimental proliferative vitreoretinopathy in rabbits by delivery of bioactive proteins with gelatin microspheres

Graphical abstract Figure. No caption available. &NA; Proliferative vitreoretinopathy (PVR) is a challenging pathological condition, often causing failure of retinal detachment surgery. The purpose of this study was to evaluate the feasibility of a delivery system of bioactive proteins using anionic and cationic gelatin microspheres and to establish a new PVR model in rabbits by intraocular sustained delivery of basic fibroblast growth factor (bFGF) and interferon‐beta (IFN&bgr;). Anionic and cationic gelatin microspheres were prepared and immersed in bFGF and IFN&bgr; solution, respectively, to yield a polyion complex between gelatin matrix and a bioactive protein. The bFGF‐impregnated microspheres were injected into the subretinal space in rabbit eyes. At week 2, the IFN&bgr;‐impregnated microspheres also were injected into the same space. Control eyes received gelatin microspheres without bFGF or IFNß, or both. The eyes then were observed for 8 weeks by ophthalmoscopy, fundus photography, and fluorescein angiography. The eyes also were evaluated histologically. In the group with both bFGF and IFN&bgr;, the number of eyes with more severe PVR increased over time. Histologic examination showed retinal folds. In contrast, no proliferative changes were seen in any control groups. Subretinal implantation of bFGF and IFN&bgr;‐impregnated gelatin microspheres induced reproducible PVR in rabbit eyes. This study guaranteed delivery of bioactive proteins with gelatin microspheres.

Comparison of Mydriatic Provocative and Dark Room Prone Provocative Tests for Anterior Chamber Angle Configuration.

Purpose:To investigate the relationship between angle configuration and diagnostic provocation tests such as the mydriatic provocative test (MPT) and the dark room prone provocative test (DRPPT). Materials and Methods:Seventy eyes of 70 consecutive patients with primary angle closure suspect, primary angle closure, or primary angle closure glaucoma were included. The anterior chamber depth, angle opening distance 500, trabecular-iris space area 500, and iris thickness (IT) were quantitatively determined by anterior segment optical coherence tomography, and the MPT and DRPPT were used to investigate intraocular pressure variations. Results:Seven eyes were positive and 3 eyes were suspected positive, using the MPT, whereas 10 eyes were positive and 7 eyes were suspected positive using the DRPPT. The anterior chamber depth and angle opening distance 500 of the positive and suspected positive groups (positive group), using the MPT, were significantly less than those of the negative group (P=0.013, P=0.013, respectively). IT of the positive group, using the MPT, was significantly greater than the negative group, using the same test (P=0.003). The trabecular-iris space area 500 of the positive group was significantly less than the negative group, using both the MPT (P<0.001) and the DRPPT (P=0.004). Conclusions:Eyes from the positive group, using the MPT, contained a shallower anterior chamber, narrower angle, and greater IT than those from the negative group. These results suggested that the MPT results better correlated with the anterior chamber angle configuration in eyes with primary angle closure, than the results using the DRPPT.