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Purpose: To evaluate the relationship between vascular endothelial growth factor (VEGF) and extracellular superoxide dismutase (EC-SOD) in vitreous body and serum in patients with proliferative diabetic retinopathy (PDR), and investigate the role of EC-SOD in PDR by evaluating its angiostatic effect, using an in vitro angiogenesis model. To investigate the role of EC-SOD in PDR by evaluating its angiostatic effect, using an in vitro angiogenesis model., Methods: EC-SOD and VEGF concentrations in vitreous and serum samples from PDR and macular hole (MH) were measured by ELISA. The effects of EC-SOD on VEGF-induced proliferation, migration, and tube formation were evaluated using human umbilical vein endothelial cells (HUVECs). Moreover, the effects of EC-SOD on VEGF-induced proliferation and migration were evaluated in HUVECs and primary normal human retinal microvascular endothelial cells., Results: Intravitreal concentrations of EC-SOD were significantly higher (p<0.01) in PDR (58.0+/-23.8 ng/ml, mean+/-SD) than in MH (29.3+/-6.6 ng/ml). Intravitreal concentrations of VEGF were dramatically higher (p<0.01) in PDR (798.2+/-882.7 pg/ml) than in MH (17.7+/-15.5 pg/ml). The serum concentrations of EC-SOD and VEGF did not differ between the two patient groups. The vitreous concentrations of VEGF correlated with those of EC-SOD in all patients (rs=0.61, p<0.001). In HUVECs, EC-SOD at 100 ng/ml significantly suppressed VEGF-induced proliferation and tube formation, but not VEGF-induced migration., Conclusions: EC-SOD was increased together with VEGF in the vitreous body from PDR patients, suggesting that EC-SOD may play a pivotal role in the pathogenesis of angiogenesis.

Purpose: To assess the effects of combined therapy with latanoprost and beta blockers on optic nerve head (ONH) blood flow in normal-tension glaucoma (NTG) patients., Methods: Intraocular pressure (IOP), ONH blood flow (laser speckle flowgraphy) and blood pressure were measured in 15 eyes of 15 NTG patients (41-76 years old) before treatment or after a 1-month washout period. Similar measurements were performed at 2 months after the commencement of treatment with latanoprost and at 3 months after the start of combined therapy of latanoprost with 0.5% timolol or 2% carteolol in a crossover study using the envelope method. Measurement was carried out 2-3 hr after the morning application of eyedrops., Results: Latanoprost decreased IOP with no significant change in ONH blood flow. Concomitant use of timolol or carteolol further decreased IOP with no significant difference between these two drugs. Only the combined therapy of latanoprost with carteolol significantly (p < 0.01) increased ONH blood flow by approximately 10%, compared to initial levels. There was no significant change in mean blood pressure, ocular perfusion pressure or pulse rate as a result of these therapies., Conclusion: Topical latanoprost-carteolol combined therapy increased ONH blood flow in NTG patients, unlike latanoprost-timolol therapy. Because ocular perfusion pressure was unchanged, direct vasodilative effects were suspected as the mechanism.

Purpose: To determine the effects of mitomycin C (MMC) on the expression of chymase and mast cells in the conjunctival scar after trabeculectomy., Methods: Ten eyes of five monkeys were used. Three eyes underwent trabeculectomy with MMC (MMC-treated), four eyes had trabeculectomy without MMC (placebo-treated), and three eyes served as control eyes. Intraocular pressure was measured before and three weeks after surgery. The scores of the degree of conjunctival adhesion were evaluated. Immunohistochemistry was used to analyze the densities of proliferative cell nuclear antigen-positive cells, chymase-positive cells, and mast cells. The ratio of collagen fiber areas to conjunctival and scleral lesions was analyzed by Mallory-Azan staining., Results: After trabeculectomy, the intraocular pressure reduction of MMC-treated eyes was significantly different from placebo-treated and control eyes (p=0.032, 0.035). The adhesion score of MMC-treated eyes was also significantly lower than that of placebo-treated eyes (p=0.034). Densities of proliferative cell nuclear antigen-positive cells, chymase-positive cells, and areas of collagen fiber in conjunctival and scleral lesions were significantly decreased in MMC-treated eyes, compared with placebo-treated eyes (p=0.034, 0.034, 0.049, respectively). There was a tendency for the density of mast cells to be suppressed in MMC-treated eyes (p=0.157)., Conclusions: Chymase might be involved in one of the mechanisms by which MMC suppresses scar formation after trabeculectomy.

Objective: To determine whether subconjunctival injection of unoprostone isopropyl alters changes in the topography and blood flow of the optic disc induced by endothelin 1 (ET-1) in rabbits., Methods: From April 1, 2005, to April 28, 2006, we injected ET-1 (20 pmol) intravitreally into rabbits twice per week for 4 weeks. The observation period was 8 weeks. The first group received an intravitreal injection of ET-1 followed by a subconjunctival injection of unoprostone (0.12%, 50 microL). The second group received the same amount of ET-1 followed by a subconjunctival injection of the vehicle of unoprostone. The third group received the intravitreal vehicle of ET-1. The blood flow and topography of the optic nerve head (ONH) were measured by laser speckle flowgraphy and confocal scanning ophthalmoscopy, respectively. The number of cells in the retinal ganglion cell layer and inner nuclear layer was determined histologically., Results: We found that ET-1 decreased the ONH blood flow, decreased the cells in the ganglion cell layer and inner nuclear layer, enlarged the cup area of the ONH, and reduced the rim area of the ONH. When unoprostone was given with ET-1, no such changes occurred., Conclusion: Unoprostone can suppress the effects of ET-1 on the circulation and topography of the ONH., Clinical Relevance: Unoprostone could be a candidate for treating eyes with ischemic ONH.

In the present study, the effects of long-term angiotensin (Ang) II antagonism on the development of cardiac and endothelial disorders were examined in Spontaneously Diabetic Torii (SDT) rats. Blood glucose concentration started to increase markedly in the untreated SDT rats from 20 weeks of age, while the blood glucose concentrations of candesartan cilexetil-treated SDT rats were significantly lower until 30 weeks of age. Cardiac function deteriorated in SDT rats and was accompanied by severe cardiac fibrosis, cardiac hypertrophy, and microstructural pathologic change in cardiomyocytes. Cardiac function was very well preserved in the age-matched Sprague Dawley (SD) rats, but cardiac fibrosis developed with aging. Candesartan cilexetil treatment improved cardiac structural remodeling and cardiac function in SDT rats. Surprisingly, the degree of cardiac fibrosis in candesartan cilexetil-treated SDT rats was less than that of SD rats. Immunohistological staining confirmed that in addition to collagen deposition, fibroblasts and myofibroblasts were the main cellular components in the cardiac fibrotic areas. The diabetic hearts showed positive staining for ACE, Ang II, and AT(1) receptors. SDT rats also showed decreased endothelial function, which was improved with candesartan cilexetil treatment. These findings indicate that Ang II is involved in the development of cardiac dysfunction by accelerating cardiac remodeling and cardiomyocyte damage in the presence of hyperglycemia. On the other hand, although the mechanisms responsible for the cardiac fibrosis that occurs under normal conditions may differ greatly from those responsible for cardiac fibrosis with hyperglycemia, Ang II seems to play an important role in both.

EAU in mice is a model of human posterior uveitis. EAU is a Th1-dependent disease that has been assumed to target the neural retina and related tissues; however, in situ effector cells and the target have not yet been clearly demonstrated. In the present study, we induced EAU in B10R mice by immunizing them with human interphotoreceptor retinoid-binding protein peptide 161-180. Histological examinations revealed that EAU occurred approximately 11 days after the immunization and reached a peak on day 14. Retinae from normal or EAU mice were treated with proteases to obtain mono-dispersed cells. The mono-dispersed cells thus obtained were separated into three to four fractions by discontinuous Percoll density-gradient (e.g. PBS/40/60) centrifugation. In normal mice, 94% of the total cells were recovered in two fractions (i.e. PBS/40 and pellet); and these fractions mainly contained inner and outer segments and cell bodies of photoreceptor cells and RPE cells, respectively. In EAU mice, additional cells (i.e. inflammatory cells) were obtained at the 40/60 interface. Electron microscopic examination showed that tissue damage during EAU was initiated by non-phagocytic destruction of inner segments by Mac-1(+) mononuclear cells on day 11, followed by phagocytic activity of macrophages against outer segments and RPE cells on day 14. In vitro culturing of normal retinal cells with EAU infiltrates suggested the involvement of TNF-alpha and NO in the tissue damage. These results indicate that EAU was initiated by non-phagocytic destruction of inner segments of photoreceptor cells by Mac-1(+) mononuclear cells.

Spontaneously diabetic Torii (SDT) rats are an inbred strain of rats with a non-obese type 2 diabetes mellitus that were isolated from an outbred colony of Sprague-Dawley (SD) rats. Electroretinograms (ERGs) were recorded from SDT and SD (controls) rats at 10- and 44-weeks-of-age to determine their retinal function. The amplitudes and implicit times of the ERGs of the right and left eyes were not significantly different indicating that the intra-individual variation was small. Both amplitudes and implicit times of the ERGs in the SDT rats were not significantly different from those of SD rats at 10-weeks-of-age. At 44-weeks-of-age, however, the a- and b-waves and the oscillatory potentials were significantly reduced with prolonged implicit times in the SDT rats compared to SD rats. These depressed ERGs may reflect vascular and neuronal damage throughout the retina as are seen in the advanced stages of human diabetic retinopathy. Thus, the SDT rat can be used to study the retinal physiology of diabetic retinopathy.

Purpose: To determine the relationship between the changes in optic nerve head (ONH) circulation and the level of plasma endothelin-1 (ET-1) during the glucose tolerance test (GTT)., Methods: Twenty-six healthy volunteers with normal GTT and 15 patients with mild hyperglycemia and abnormal GTT were studied. The ONH circulation [square blur rate (SBR) value], blood pressure, intraocular pressure (IOP), blood glucose, blood insulin and plasma ET-1 were determined before and every hour up to 3 h after an oral intake of 75 g of glucose., Results: The SBR increased in the normal glucose tolerance group at all times during the GTT, but it decreased significantly in the abnormal glucose tolerance group (P < 0.05). Before the GTT, the plasma ET-1 level was not significantly different in the two groups; however, the level increased 1 h after the oral GTT in the abnormal glucose tolerance group (P < 0.05). No significant changes were observed in mean blood pressure or IOP., Conclusions: ONH circulation increased after glucose intake in the normal glucose tolerance group and remained high even after the blood glucose level had returned to its baseline. The decrease in ONH circulation in the abnormal glucose tolerance group was attributed partly to the increased ET-1.

The effects of an angiotensin II receptor blocker (ARB) on the accumulation of one of advanced glycation end products (AGEs), pentosidine, expression of vascular endothelial growth factor (VEGF) and retinal function were investigated in Spontaneously Diabetic Torii (SDT) rats. Candesartan, an ARB, was administered to SDT rats from 10 to 44 weeks of age and the results compared with untreated SDT rats and SD rats. Electroretinograms (ERGs) were recorded to evaluate retinal function. At 44 weeks of age, pentosidine was quantified in the vitreous, lens and plasma using high-performance liquid chromatography (HPLC). Real-time reverse transcription-PCR (RT-PCR) analysis was also performed in order to measure VEGF mRNA expression in the retina. Histological changes were examined and immunohistochemistry for pentosidine performed on the retina and retinal microvasculature. In untreated SDT rats, the amplitudes of a- and b-waves, oscillatory potentials were reduced significantly at 44 weeks of age compared with the 10-week levels, whereas they remained unchanged in SDT rats treated with candesartan. The concentration of pentosidine in the vitreous and lens did not change in treated SDT rats but increased in untreated SDT rats. Retinal VEGF mRNA expression was inhibited in treated SDT rats. Histologically, proliferative tissue was detected around the optic disc, with pentosidine being detected only in untreated SDT rats. Our findings indicate the ARB may inhibit the development of diabetic retinopathy by reducing the accumulation of pentosidine, one of AGEs and expression of VEGF in the retina.

Objective: To test the effect of activation of P2X7 receptors on retinal blood velocity in diabetic rabbits., Methods: Immunohistochemical analysis was performed on healthy and diabetic rabbit eyes using P2X7 receptor antibodies. Diabetes was induced using alloxan. Retinal blood velocity was measured with the laser speckle circulation analyzer. Visual function was assessed using electroretinography., Results: Cells in inner retinal layers were positive for anti-P2X7 receptor antibodies in healthy rabbits. The distribution of positive cells extended to outer layers and some small vessels stained in diabetic rabbits. When assayed 24 hours after an intravitreal injection of 150 nmol of benzoylbenzoyl adenosine triphosphate (BzATP), a P2X7 agonist, the retinal blood velocity in healthy rabbits was reduced by approximately 30%; this reduction continued for at least 4 weeks. Only in diabetic rabbits did an injection of 50 nmol of BzATP reduce retinal blood velocity by approximately 30% and the amplitudes of electroretinography a waves, b waves, and oscillatory potentials for at least 4 weeks., Conclusions: Soon after the onset of alloxan-induced diabetes, retinal blood velocity and function become more vulnerable to reduction initiated through P2X7 receptors., Clinical Relevance: Our findings support the hypothesis that the retinal circulation disorder accelerated by activation of P2X7 receptors may be involved in the early changes of diabetic retinopathy.

Background: We report a case of normal-tension glaucoma(NTG) with impaired eye movements in a young patient., Case: A 22-year-old man with enlarged optic disc cuppings and visual field defects was suspected to have NTG, and was found, upon examination, to have impaired eye movements. Intraocular pressure recordings, including diurnal variation, were under 21 mmHg. Gonioscopic findings were normal. Magnetic resonance image(MRI) showed no particular findings. The patient was diagnosed with NTG. Using the 123I-iodoamphetamine single photon emission computed tomography (123I-IMP SPECT), decreased cerebral blood flow was detected in the patient's occipital lobe and an electro-oculogram showed impaired eye movements during smooth pursuit. The patient's visual field defects have increased over the last two years, but, no accompanying change in his eye movements has been observed., Conclusion: The patient in this case has NTG with impaired eye movements and decreased cerebral blood flow suggesting that some disorder of the central nervous system may be one of the causative mechanisms of NTG.

Purpose: To evaluate regional cerebral blood flow (rCBF) in normal-tension glaucoma patients (NTGs) compared with normal volunteers (controls) in a pilot study., Design: Observational cohort study., Methods: The Iodine-123-IMP single photon emission computed tomography (SPECT) scans were acquired in 31 NTGs and 18 age-matched controls. The SPECT images in NTGs were classified and the quantitative rCBF data were compared with controls., Results: Seven of 31 NTGs (22.6%) exhibited an Alzheimer's disease (AD)-like perfusion pattern although none of them were clinically diagnosed with AD. The visual field defect of NTGs with an AD-like pattern progressed more rapidly than NTGs with a normal pattern (P=.132). The rCBF in left parietal regions of NTGs with senile sclerotic or focal ischemic disk was significantly lower than controls (P<.05)., Conclusions: A number of NTGs exhibited an AD-like perfusion pattern, suggesting that some of NTGs and AD patients might have a common pathologic mechanism.

Purpose: To compare transforming growth factor (TGF) beta(2) levels in subretinal fluid of rhegmatogenous retinal detachment with or without subretinal strand formation., Methods: We assessed total and mature TGF-beta(2) levels in subretinal fluid obtained from 24 eyes with rhegmatogenous retinal detachment using an enzyme-linked immunosorbent assay. Group I comprised 18 specimens from eyes without subretinal strands, while group II comprised 6 specimens from eyes with subretinal strands., Results: Total and mature TGF-beta(2) levels were higher in group II than in group I (p=0.01 and p=0.07, respectively)., Conclusion: The concentrations of total and mature TGF-beta(2) were higher in cases of rhegmatogenous retinal detachment associated with subretinal strand formation compared to those without subretinal strand formation., (Copyright (c) 2005 S. Karger AG, Basel.)

The purpose if this study was to elucidate how extracellular ATP causes cell death in the retinal microvasculature. Although ATP appears to serve as a vasoactive signal acting via P2X(7) and P2Y(4) purinoceptors, this nucleotide can kill microvascular cells of the retina. Because P2X(7) receptor activation causes transmembrane pores to form and microvascular cells to die, we initially surmised that pore formation accounted for ATP's lethality. To test this hypothesis, we isolated pericyte-containing microvessels from rat retinas, assessed cell viability using Trypan blue dye exclusion, detected pores by determining the uptake of the fluorescent dye YO-PRO-1, measured intracellular Ca(2+) with the use of fura-2, and monitored ionic currents via perforated patch pipettes. As predicted, ATP-induced cell death required P2X(7) receptor activation. However, we found that pore formation was minimal because ATP's activation of P2Y(4) receptors prevented P2X(7) pores from forming. Rather than opening lethal pores, ATP kills via a mechanism involving voltage-dependent Ca(2+) channels (VDCC). Our experiments suggest that when high concentrations of ATP caused nearly all microvascular P2X(7) receptor channels to open, the resulting profound depolarization opened VDCC. Consistent with lethal Ca(2+) influx via VDCC, ATP-induced cell death was markedly diminished by the VDCC blocker nifedipine or a nitric oxide (NO) donor that inhibited microvascular VDCC. We propose that purinergic vasotoxicity is normally prevented in the retina by NO-mediated inhibition of VDCC and P2Y(4)-mediated inhibition of P2X(7) pore formation. Conversely, dysfunction of these protective mechanisms may be a previously unrecognized cause of cell death within the retinal microvasculature.

Purpose: To determine if the renin-angiotensin system (RAS) is involved in diabetic retinopathy, we measured the levels of angiotensin-converting enzyme (ACE) and angiotensin II in the vitreous of patients with proliferative diabetic retinopathy (PDR). We also investigated whether the genes of the RAS factors were expressed by cultured human Müller cells., Methods: Vitreous samples were collected during vitreous surgery from patients with PDR, and from patients with idiopathic macular hole, who served as controls. The concentration of ACE was analyzed, and the level of angiotensin II was quantitatively determined by double antibody radioimmunoassay. In addition, the cDNA, prepared from the mRNA extracted from cultured human Müller cells, was used as a template for reverse transcriptase-polymerase chain reaction with primers selected for renin, angiotensinogen, ACE, and angiotensin receptor type I., Results: The mean concentration of ACE was 0.82 +/- 0.73 IU/L at 37 degrees C in the PDR group, which was significantly higher than the 0.05 +/- 0.07 IU/L at 37 degrees C in the controls. The mean concentration of angiotensin II was 8.77 +/- 4.57 pg/mL in the PDR group, which was significantly higher than the 5.1 +/- 1.7 pg/mL in the controls. mRNA for renin, angiotensinogen, ACE, and angiotensin receptor type I was detected in cultured human Müller cells., Conclusions: The RAS is locally activated in eyes with PDR, and Müller cells may play a role in this local activation.

Purpose: To investigate the changes in retinal blood flow (RBF) during a 75-g oral glucose tolerance test in the contralateral eye of patients with retinal branch vein occlusion (BRVO)., Methods: Seventeen patients with BRVO were included. None had a known history of diabetes mellitus. The blood velocity and vessel diameter of the upper temporal retinal artery in the contralateral eye were measured using laser Doppler velocimetry with an eye-tracking system before and 1, 2, and 3 h after oral intake of 75 g glucose. Blood sugar, systemic blood pressure, and intraocular pressure were monitored. Eleven healthy volunteers were examined similarly as control., Results: Nine patients (53%) with BRVO had an abnormal pattern on the glucose tolerance test. RBF significantly increased at 1, 2, and 3 h in eight patients with normal glucose tolerance. The changes in RBF primarily resulted from changes in vessel diameter; the blood velocity did not change significantly. The nine patients with impaired glucose tolerance or diabetes mellitus had a RBF pattern that was significantly lower at 2 and 3 h than those with normal oral glucose tolerance. In the control group, all 11 volunteers showed the same increases in RBF at 2 and 3 h as the BRVO patients with a normal glucose pattern., Conclusion: A relatively high number of patients with BRVO had an abnormal pattern on glucose tolerance testing. Glucose intake increased RBF in BRVO patients without diabetes and in healthy volunteers. The RBF response may differ between subjects with diabetes and those without diabetes.

Purpose: To determine the effect of caffeine on microcirculation in the human ocular fundus., Methods: The microcirculation in the ocular fundus of 10 healthy volunteers (10 eyes) was studied using a laser speckle tissue circulation analyzer. Caffeine or placebo (100 mg) was administered orally in a double-masked manner. Square blur rate (SBR), a quantitative index of blood flow velocity, was measured in a temporal site of the optic nerve head (ONH) free of surface vessels and in a middle site of the choroid-retina between the ONH and macula. Intraocular pressure (IOP), blood pressure (BP), pulse rate (PR), and central critical fusion frequency (CFF) were also measured. These parameters were measured before and for 2 hours after administration. The area under curve (AUC) of SBR was calculated for each area. Ocular perfusion pressure (OPP) was also calculated from BP and IOP., Results: The time-course of change in SBR value showed much individual difference. Caffeine decreased the AUC of SBR in the ONH (P =.0218) as well as in the choroid-retina (P =.0469) significantly. IOP, mean BP, PR, OPP, and central CFF did not change significantly., Conclusions: These results suggest that caffeine may increase blood vessel resistance and decrease blood flow in the human ONH and choroid-retina.

Purpose: To determine the role played by nitric oxide (NO) in the autoregulation of circulation in the optic nerve head (ONH)., Methods: The intraocular pressure (IOP) was increased and maintained at 50 mm Hg by the infusion of balanced saline solution (BSS) into the anterior chamber of albino rabbits. Experiments were performed with or without an intravenous injection of 10, 20, or 50 mg/kg N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor. The blood flow in the ONH was evaluated by the hydrogen clearance method, and NO metabolites (nitrite and nitrate) were measured in the ONH under the same experimental conditions in other rabbits. Visual evoked potentials (VEPs) were recorded before the IOP elevation and every 15 minutes during the 60 minutes of elevation. The effect of elevated IOP on the VEPs and the hemodynamics and NO levels in the ONH were determined. The effect of pretreatment with a NOS inhibitor on the IOP-induced changes was also investigated., Results: The implicit time of the VEP was prolonged after L-NAME in a dose-dependent manner, whereas the implicit time in the control group (saline) was less affected. Blood flow in the ONH was not reduced by an elevation of the IOP (50 mm Hg) but was significantly reduced by L-NAME (20 mg/kg). The NO metabolites, which were elevated in the ONH during IOP elevation in the control, were also depressed by L-NAME pretreatment., Conclusions: These results indicate that NO may play a role in the autoregulation of circulation in the ONH during elevated IOP. This would mean that NO provides some neuroprotection during an acute phase of ischemia in the ONH.

Purpose: To investigate the long-term effect of 0.12% isopropyl unoprostone (Rescula) on microcirculation in the human ocular fundus., Methods: A laser speckle tissue circulation analyzer was used to measure normalized blur (NB), a quantitative index of blood flow velocity, in the optic nerve head (ONH) and choroid-retina before and 4.5 hours after the instillation of a placebo into both eyes of 11 healthy volunteers. The intraocular pressure (IOP), blood pressure, and pulse rate were also recorded in this control experiment. Thereafter, a drop of unoprostone or a placebo was instilled into each eye in a double-blind manner twice a day for 21 days to form treated and untreated groups., Results: After 21 days, the NB values in the ONH and choroid-retina had increased significantly and the IOP had decreased significantly in the unoprostone-treated eyes. Ocular perfusion pressure showed no significant change., Conclusions: These results suggest that long-term application of unoprostone can increase microcirculatory blood flow in the human ocular fundus, probably due to a reduction in vascular resistance.