S. Miyahara

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.

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.

Pars Plana Vitrectomy for Epiretinal Membrane Associated with Sarcoidosis

PURPOSE To examine retrospectively the visual outcomes in patients undergoing vitrectomy for epiretinal membranes secondary to sarcoid uveitis. METHODS Eleven consecutive patients (11 eyes) with epiretinal membrane and uveitis associated with sarcoidosis underwent pars plana vitrectomy. RESULTS Nine eyes (82%) gained two or more lines of Snellen visual acuity at 1-12 months after surgery. However, 4 of these 9 eyes lost two or more lines of Snellen visual acuity by the final visit. Overall, 5 eyes (45%) had attained at least two Snellen lines of visual acuity improvement, 5 eyes (45%) were unchanged, and 1 eye (10%) had worsened by two lines at the final visit. Nine eyes (81%) achieved visual acuity of 20/40 or better by the final visit. Slit-lamp biomicroscopy and fluorescein angiography showed that cystoid macular edema had resolved in 4 of 7 eyes postoperatively; vitritis improved in all cases. Postoperative complications included cataract formation, glaucoma, and membrane recurrence. Subsequent surgeries consisted of cataract extraction in 2 eyes and membrane peeling in 1 eye. CONCLUSIONS Pars plana vitrectomy appears to have a beneficial effect on restoring vision in eyes with epiretinal membrane and uveitis associated with sarcoidosis, but final visual acuity was limited by the development of cataract and membrane recurrence.

Argatroban Attenuates Leukocyte– and Platelet–Endothelial Cell Interactions After Transient Retinal Ischemia

Background and Purpose— Argatroban, a direct thrombin inhibitor, has been shown to reduce neural injury after transient cerebral ischemia. It has also been reported that this neuroprotective effect results from an anticoagulant function. This study was designed to evaluate quantitatively the inhibitory effects of argatroban on leukocyte– and platelet–endothelial cell interactions after transient retinal ischemia. Methods— Retinal ischemia was induced for 60 minutes in male Long-Evans rats by temporary ligation of the optic sheath (n=342). Argatroban was administered just after induction of ischemia. Leukocyte and platelet behavior in the retinal microcirculation was then evaluated in vivo with scanning laser ophthalmoscopy. The expression of P-selectin and intracellular adhesion molecule-1 (ICAM-1) was evaluated by reverse transcription–polymerase chain reaction. After 10 days of reperfusion, ischemia-induced retinal damage was evaluated histologically. Results— Treatment with argatroban suppressed leukocyte–endothelial cell interactions; the maximum numbers of rolling and accumulated leukocytes were reduced by 90.1% (P <0.05) and 58.7% (P <0.05), respectively, at 12 hours after reperfusion. Treatment with argatroban also suppressed platelet–endothelial cell interactions; the maximum numbers of rolling and adhering platelets were reduced by 91.8% (P <0.01) and 78.9% (P <0.01), respectively, at 12 hours after reperfusion. The expression of P-selectin and ICAM-1 mRNA was suppressed significantly in the argatroban-treated retinas (P <0.01). Histologic examination demonstrated the protective effect of argatroban on ischemia-induced retinal damage (P <0.01). Conclusions— Argatroban treatment suppressed leukocyte– and platelet–endothelial cell interactions after transient retinal ischemia. This inhibitory effect on postischemic blood cell–endothelial cell interactions might partially contribute to its neuroprotective effects.

Neuroprotective effect of cilostazol against retinal ischemic damage via inhibition of leukocyte-endothelial cell interactions.

Summary.  Background:  Cilostazol, a selective platelet phosphodiesterase inhibitor, has been shown to reduce neuronal injury after transient cerebral ischemia. Its neuroprotective effect is thought to result from an antiplatelet function. This study was designed to evaluate the inhibitory effects of cilostazol against retinal ischemic damage focusing on leukocyte–endothelial cell interactions.

Corneal Thickness in Eyes Following Pars Plana Lensectomy for Congenital Cataracts

PurposeTo evaluate the corneal thickness following pars plana lensectomy for congenital cataracts.MethodsThe corneal thickness was measured in 24 eyes of 24 patients with congenital cataracts who had undergone pars plana lensectomy at a mean age of 24 ± 32 (SD) months. The mean age at the time of our evaluation was 15 ± 3 years. These measurements were compared with those in 15 eyes of an age-matched group of 15 normal volunteers. The central corneal thickness and endothelium were evaluated in both groups.ResultsThe mean corneal thickness of the cataract-extracted eyes (592 ± 47 µm) was significantly greater than that of the controls (529 ± 43 µm; P ≪ 0.00l). There was no significant difference in the corneal endothelial cell count between cataract-extracted eyes (3420 ± 715/mm2) and the controls (3182 ± 358/mm2; P = 0.49). However, there were significant differences in the frequency of hexagonally shaped endothelial cells (63% ± 8.4%, cataract-extracted eyes; 70% ± 7.7%, controls; P ≪ 0.0l), and in the coefficient of variation in the endothelial cell size (33 ± 6.0, cataract-extracted eyes; 26 ± 4.8, controls; P ≪ 0.01).ConclusionThe central cornea of congenital cataract-extracted eyes was significantly thicker than that of controls. Jpn J Ophthalmol 2004;48:169–171

Identification of resident and inflammatory bone marrow derived cells in the sclera by bone marrow and haematopoietic stem cell transplantation

Aims: To characterise bone marrow derived cells in the sclera under normal and inflammatory conditions, we examined their differentiation after transplantation from two different sources, bone marrow and haematopoietic stem cells (HSC). Methods: Bone marrow and HSC from green fluorescent protein (GFP) transgenic mice were transplanted into irradiated wild-type mice. At 1 month after transplantation, mice were sacrificed and their sclera examined by histology, immunohistochemistry (CD11b, CD11c, CD45), and transmission and scanning electron microscopy. To investigate bone marrow derived cell recruitment under inflammatory conditions, experimental autoimmune uveitis (EAU) was induced in transplanted mice. Results: GFP positive cells were distributed in the entire sclera and comprised 22.4 (2.8)% (bone marrow) and 28.4 (10.9)% (HSC) of the total cells in the limbal zone and 18.1 (6.7)% (bone marrow) and 26.3 (3.4)% (HSC) in the peripapillary zone. Immunohistochemistry showed that GFP (+) CD11c (+), GFP (+) CD11b (+) cells migrated in the sclera after bone marrow and HSC transplantation. Transmission and scanning electron microscopy revealed antigen presenting cells among the scleral fibroblasts. In EAU mice, vast infiltration of GFP (+) cells developed into the sclera. Conclusion: We have provided direct and novel evidence for the migration of bone marrow and HSC cells into the sclera differentiating into macrophages and dendritic cells. Vast infiltration of bone marrow and HSC cells was found to be part of the inflammatory process in EAU.

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.

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.