Search

Aims: To detect collateral vessels using optical coherence tomography angiography (OCTA) in eyes with branch retinal vein occlusion (BRVO) and to investigate the associations with visual outcomes and macular oedema., Methods: Eyes with macular oedema secondary to BRVO that underwent OCTA at baseline and were followed up for more than 6 months were enrolled. The presence of collaterals, whether the collaterals were leaky or not, and the associations with visual outcomes and macular oedema were investigated., Results: Twenty-eight eyes of 28 patients (8 men and 20 women; mean age, 68 years) were enrolled. Collaterals were detected in 23 eyes (82%) and already existed at the initial visit. Collaterals were more frequently detected in eyes with major BRVO or ischaemic type. One-third of the collaterals were leaky and all of the leaky collaterals had microaneurysms (MAs) inside. Macular oedema in eyes with collaterals was more quickly and frequently resolved than that in eyes without collaterals, but there were no significant differences. Collateral vessel formation did not seem to impact on visual outcomes, but the mean baseline central retinal thickness (CRT) was significantly higher in eyes with collaterals, and the mean CRT reduction at 6 months after treatments was significantly greater than in eyes without collaterals., Conclusions: These results suggest that collateral vessels are formed at the acute phase in eyes with BRVO. In addition, the presence of collaterals might be associated with absorption of macular oedema, but MAs formed in collaterals sometimes can cause macular oedema., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To evaluate the location of microvascular abnormalities using wide-field fluorescein angiography (WFFA) and investigate the impact on visual outcome in eyes with branch retinal vein occlusion (BRVO)., Methods: Forty eyes of 39 patients (24 males and 15 females with an average age of 71 years) were retrospectively reviewed. One patient had BRVO bilaterally. WFFA was performed in all patients to evaluate perfusion status and detect microvascular abnormalities. The WFFA images were divided into 3 zones: zone 1, posterior pole; zone 2, mid-periphery; zone 3, far periphery, in order to document the presence of microvascular abnormalities. Scatter retinal photocoagulation (PC) was performed for retinal neovascularization (NV) and/or widespread nonperfused areas (NPAs)., Results: The incidence of microvascular abnormalities in zone 3 was significantly (p < 0.0001) less than in zones 1 and 2. The presence of larger NPAs in zone 1, but not in zone 3, was associated with the incidence of NV and vitreous hemorrhage. The presence of peripheral lesions and the application of PC did not affect the visual outcome., Conclusion: The presence of peripheral abnormalities or scatter PC for NPAs did not affect the visual outcome in eyes with BRVO., (© 2018 S. Karger AG, Basel.)

Purpose: This study evaluates the retinal hemodynamics using optical coherence tomography angiography (OCTA) before and after anti-vascular endothelial growth factor (VEGF) therapy in patients with macular edema associated with retinal vein occlusion (RVO)., Methods: Twelve patients (23 eyes; mean age, 64 years) were included (eight eyes with branch RVO, four with central RVO, and 11 unaffected fellow eyes. The best-corrected visual acuity (BCVA) and central retinal thickness (CRT) were measured before and 6 months after treatment. The foveal avascular zone (FAZ), nonperfused areas (NPAs), and flow area were evaluated with OCTA before and after treatment., Results: The BCVA and CRT improved significantly after treatment. In eyes with RVO, the baseline FAZ in the retinal deep capillary layer was larger than in fellow eyes and enlarged in the retinal superficial and deep capillary layers after therapy; NPAs decreased after therapy, especially in the retinal deep capillary layer; and the baseline flow area was smaller than in fellow eyes and improved after therapy, especially in the retinal deep capillary layer., Conclusions: Optical coherence tomography angiography can evaluate the retinal hemodynamics in patients with RVOs. Anti-VEGF therapy reduced the NPA size and improved retinal blood flow, especially in the retinal deep layer. The current results suggested that anti-VEGF therapy might improve retinal deep ischemia in patients with RVO in the retinal deep layer, which is abundant in capillaries.

Purpose: The purpose of this study was to compare the angiographic findings of polypoidal choroidal vasculopathy (PCV) detected by indocyanine green angiography (ICGA) and en face optical coherence tomography angiography (OCTA)., Methods: The study design was a retrospective chart review of 20 eyes with a clinical diagnosis of treatment-naive PCV, seen at Nagoya City University Hospital between December 2014 and January 2016. Indocyanine green angiography was performed with Heidelberg Retina Angiography 2 and OCTA was performed by using Avanti RTVue XR., Results: Twenty eyes of 20 patients (18 male, 2 female) were studied. Average age was 71.9 years. Manual segmentation was required to detect the pathologic lesions of PCV in all cases. The polypoidal lesions were detected in 20 eyes (100%) by ICGA, and in 17 eyes (85%) by OCTA. The number of polypoidal lesions detected by OCTA averaged 2.6 ± 1.9, with an average of 2.0 ± 1.1 by ICGA (P < 0.05). The branching vascular network (BVN) was detected in 14 eyes (70%) by ICGA and in 14 eyes (70%) by OCTA. All of the BVNs detected by OCTA were located between the RPE and Bruch's membrane., Conclusions: Despite the manual segmentation required, en face OCTA enabled us to analyze the angiographic features of PCV combined with longitudinal image (B-scan). En face OCTA may be useful for understanding the pathogenesis of PCV and managing PCV.

Purpose: To study the association between the distributions of microaneurysms detected by en face optical coherence tomography angiography (OCTA) and diabetic macular edema (DME)., Methods: The study design was a retrospective chart review of 27 patients (33 eyes) with DME. The eyes were scanned using OCTA (6 × 6 mm) and spectral-domain (SD) OCT macular cube. Each of the images of the capillary plexus was overlaid onto the image of the topographic map, and the densities of the microaneurysms were measured by ImageJ software. The association between the distribution of microaneurysms and macular edema was evaluated., Results: For microaneurysms in areas with and without edema, 77.3 ± 8.1% of these microaneurysms were located in the deep capillary plexuses (DCP). However, in areas of edema where the retinal thickness was more than 400 μm, 91.3 ± 9.1% of the microaneurysms were found in the DCP. This difference was statistically significant (P < 0.001). In the macular edema area, there was a significantly higher density of microaneurysms in the DCP compared to the superficial capillary plexuses (1.71/mm2 vs. 0.17/mm2, P < 0.001). There was also a significant correlation between the macular volume and the density of microaneurysms in the DCP in edema (r = 0.63, P < 0.001)., Conclusions: Our study demonstrated a high proportion of microaneurysms in the DCP, as well as a novel association between the distributions of microaneurysms detected by OCTA and DME. Results also indicated that microaneurysms located in the DCP contribute to the pathogenesis of DME.

Purpose: To develop a new method to quantify the choroidal vessel density by binarizing ultra-wide-field indocyanine green angiography (ICGA) images and determine whether values are altered in diseased eyes., Design: Reliability and validity analysis., Methods: Ultra-wide-field fluorescein angiography (FA) and ICGA images were obtained using an ultra-wide-field imaging device (Optos California ultra-wide-field imaging device; Dunfermline, Scotland, UK) in 11 eyes of 11 patients without chorioretinal diseases. The angiographic signals of the choroidal vessels were determined by subtracting those of the retinal vasculature and optic disc on the FA images from the ICGA images, binarized by Niblack's method, and the choroidal vessel density calculated. Reproducibility of the method was assessed by calculating the coefficient of variance, coefficient of repeatability, and intraclass correlation coefficient. The relationships between age, spherical equivalent refractive error (SERE), and intraocular pressure and the vasculature density were assessed. To investigate possible impacts of chorioretinal diseases on the vasculature density, 10 eyes of 7 patients with central serous chorioretinopathy (CSC) were compared with the 11 control eyes., Results: Choroidal vessels were contrasted by binarizing ICGA images. The method to quantify the choroidal vessel density showed high reproducibility. The SERE was correlated significantly (r = 0.573, P < .05) with the vasculature density. In the 11 control eyes, the vasculature density was 34.26% ± 0.77% in the entire area, 31.37% ± 0.97% in the superior portion, 36.98% ± 0.88% in the inferior portion, 37.01% ± 1.44% in the posterior portion, and 34.17% ± 0.77% in the peripheral portion. In eyes with CSC, the density was significantly (P < .05) higher: 36.46% ± 0.49%, 34.02% ± 0.97%, 38.65% ± 0.27%, 41.04% ± 0.82%, and 36.36% ± 0.51%, respectively., Conclusions: Binarization of ultra-wide-field ICGA images enabled quantification of the choroidal vessel density, which was altered in eyes with CSC. This method of measuring the choroidal vessel density may provide new insights into diagnosing and treating chorioretinal diseases., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: To evaluate the area of the foveal avascular zone (FAZ) detected by en face OCTA (AngioVue, Avanti OCT; Optovue) in healthy and diabetic eyes., Methods: Retrospective chart review of patients who underwent fundus examination including en face OCTA. Eyes with proliferative diabetic retinopathy and history of laser photocoagulation were excluded. The FAZ area in the superficial and deep plexus layers were measured and evaluated using ImageJ software., Results: The FAZ area in the superficial layer was 0.25 ± 0.06 mm² in healthy eyes (n = 19), whereas it was 0.37 ± 0.07 mm² in diabetic eyes without retinopathy (n = 24) and 0.38 ± 0.11 mm² in eyes with diabetic retinopathy (n = 20). Diabetic eyes showed statistically significant FAZ enlargement compared with healthy eyes, regardless of the presence of retinopathy (P < 0.01). The FAZ area in the deep plexus layer was also significantly larger in diabetic eyes than in healthy eyes (P < 0.01)., Conclusion: Our data suggest that diabetic eyes show retinal microcirculation impairment in the macula even before retinopathy develops. En face OCTA is a useful noninvasive screening tool for detecting early microcirculatory disturbance in patients with diabetes.

Purpose: We reviewed various methodologies for studying leukocyte-retinal endothelial interactions in vivo, and summarized the information obtained from studies employing these methods., Procedures: Fluorescence dye-enhanced scanning laser ophthalmoscopy facilitates study of leukocyte-cell interactions in the retinal microcirculation., Results: Various methods such as adaptive optics scanning laser ophthalmoscopy (AO-SLO), acridine orange digital angiography, and intravital microscopy provided evidence of the mechanisms of leukocyte recruitment in the retina and of their importance in the pathogenesis of various retinal diseases., Conclusions: Leukocyte behavior can be easily examined in the retina in vivo because the optical media is transparent. SLO substantially contributes to visualizing leukocyte-endothelial interactions in retinal vessels, although most of the methods employing SLO could only be used for animal studies. AO-SLO noninvasively demonstrates the movement of each leukocyte in the parafoveal capillaries in humans. Message: AO-SLO could be useful in investigating the leukocyte-retinal endothelial interactions in various diseases in humans., (Copyright © 2011 S. Karger AG, Basel.)

Purpose: Dysregulation of the polyol pathway has been implicated as a major cause of diabetic retinopathy. The aldose reductase inhibitor fidarestat was recently reported to prevent retinal oxidative stress and overexpression of vascular endothelial growth factor (VEGF) protein in diabetic rats. In this study, we investigated the effect of fidarestat on leukocyte-endothelial cell interactions in an in vivo experimental model for diabetic retina., Materials and Methods: Diabetes was induced in six-week-old male Long-Evans rats by intraperitoneal injection of streptozotocin (STZ) (75 mg/kg). The rats were divided into four experimental groups: non-diabetic control rats, untreated diabetic rats, and diabetic rats treated with a low (4 mg/kg/day) or high (16 mg/kg/day) oral dose of fidarestat. After four weeks of treatment, accumulated leukocytes in the retina were counted in vivo by acridine orange digital fluorography. Intercellular adhesion molecule-1 (ICAM-1) and VEGF-164 mRNA levels in the retina were analyzed using the quantitative reverse transcription-polymerase chain reaction. ICAM-1 protein expression in the retina was investigated by immunohistochemistry., Results: Fidarestat treatment significantly decreased concentrations of sorbitol and fructose in the retinas of STZ-induced diabetic rats. Leukocyte accumulation in the retinas of fidarestat-treated rats was significantly less than in the untreated diabetic group (P < 0.01). Fidarestat treatment significantly reduced the expression ICAM-1 mRNA, but not VEGF-164 mRNA, in the retina of diabetic rats. Immunohistochemical study also revealed the suppressive effect of fidarestat on expression of ICAM-1., Conclusions: Oral administration of fidarestat attenuated leukocyte accumulation in the retina of STZ induced-diabetic rats, suggesting that fidarestat may have a therapeutic role in preventing the progression of diabetic retinopathy.

Purpose: Leukocytes play a critical role in ocular diseases such as uveitis, diabetic retinopathy, and choroidal neovascularization. Intercellular adhesion molecule (ICAM)-1 is essential for the migration of leukocytes. Control of ICAM-1 expression may lead to therapies for these diseases. Small-interfering ribonucleic acids (siRNAs) are efficient specific modulators of endogenous gene expression. The authors describe the application of siRNA to suppress ICAM-1 expression on the murine neurosensory retina or retinal pigment epithelial (RPE) cells using a hydrodynamics-based transfection technique (HT) and intravitreal injection (IV) in vivo., Methods: ICAM-1-specific plasmid siRNAs designed from the murine gene sequence were transfected into the retina using HT and IV in vivo. Green fluorescent protein (GFP) expression plasmid vector is used as a transfection marker in the retinal cells. ICAM-1 expression was analyzed by enzyme-linked immunosorbent assay and flow cytometry. ICAM-1 upregulation was induced by retinal laser photocoagulation and streptozotocin (STZ)., Results: After the administration of GFP expression plasmid with HT and IV, histologic analysis showed GFP fluorescence in every layer of the murine retina. After photocoagulation, ICAM-1 expression in the neurosensory retina or RPE cells transferred with plasmid ICAM-1 siRNA was significantly decreased compared with cells that were not transfected or cells transferred with scrambled control siRNA. Plasmid siRNAs silenced ICAM-1 expression after STZ administration compared with control or naked siRNA injection., Conclusions: SiRNA expression mediated by this plasmid causes efficient and specific downregulation of ICAM-1 expression, suggesting that it can be silenced by plasmid siRNA in murine retina in vivo. This technology may lead to novel concepts to reduce retinal neovascular disease by inhibiting leukocyte infiltration.

Purpose: Macular edema is one of the serious side effects associated with panretinal photocoagulation (PRP). The inhibitory effect of triamcinolone acetonide (TA) on leukocyte-endothelial cell interactions in vivo after PRP was evaluated., Methods: Argon laser photocoagulation was performed in one half of the retinas in male Brown Norway rats. Experimental rats were injected with 2 mg TA (50-microL volume) in the posterior sub-Tenon space, and the vehicle-treated rats were injected with the same amount of saline (50 microL) immediately after PRP. Untreated rats were used as the control. Leukocyte dynamics in retinal microcirculation and retinal vessel diameters were evaluated 1 day after laser photocoagulation with the use of acridine orange digital fluorography. Retinal thickness was evaluated with optical coherence tomography., Results: The number of rolling leukocytes and accumulating leukocytes in the retina decreased by 66% in the TA-treated rats (P < 0.01) and by 24% (P < 0.05), respectively, compared with the number in the vehicle-treated rats. Retinal thickness in the vehicle-treated rats was significantly thicker than that in control rats 1 day after laser photocoagulation (P < 0.01). Retinal thickness in the TA-treated rats was significantly suppressed compared with that in the vehicle-treated rats (P < 0.05)., Conclusions: Sub-Tenon administration of TA significantly suppressed leukocyte dynamics in rat retinal microcirculation and decreased retinal edema after laser photocoagulation. The results suggest that the suppression of leukocyte-endothelial cell interactions in retinal microcirculation may be one mechanism responsible for the therapeutic effect of sub-Tenon TA on postlaser retinal edema.

Purpose: Recent studies have reported that intravitreal or posterior sub-Tenon's injection of triamcinolone acetonide (TA) is effective in the treatment of macular edema resulting from retinal microcirculatory disturbances such as diabetic retinopathy and retinal vein occlusion. The effects of periocular administration of TA on leukocyte-endothelium interactions were studied after transient retinal ischemia., Methods: Transient retinal ischemia was induced by temporary ligation of the optic nerve sheath for 60 minutes in male Long-Evans rats. After the induction of ischemia, experimental eyes received a periocular injection of TA (2 mg). In control animals, the same volume of saline was administered. Leukocyte dynamics were evaluated in the retinal microcirculation using acridine orange digital fluorography. Also, retinal thickness was studied by using optical coherence tomography and a histologic, Method: The retinal mRNA expression of P-selectin and intercellular adhesion molecule (ICAM)-1 was semiquantitatively studied with RT-PCR., Results: The leukocytes rolling along retinal vein linings increased after ischemia in the vehicle-treated rats (32.5 +/- 2.1 cells/min). No rolling leukocytes, however, were seen in the TA-treated rats. The number of accumulated leukocytes was significantly lower in the TA-treated rats (831 +/- 99 cells/mm2) than in the control (971 +/- 81 cells/mm2, P < 0.05). The treatment decreased the retinal thickness and the mRNA expression of P-selectin and ICAM-1., Conclusions: The present study demonstrated that the periocular injection of TA effectively decreased retinal thickness and inhibited leukocyte-endothelium interactions in the retina after ischemia. Downregulation of adhesion molecules of retinal vascular endothelium induced by TA may play a role in the course.

The levels of lipid hydroperoxide (LHPs) in vitreous are elevated in a variety of retinal disorders. Recently, we have shown that increased levels of LHPs in the vitreous enhanced leukocyte-endothelium interaction in the retina, which should contribute to the initial disturbance of the retinal microcirculation. Based upon the previous work, the purpose of the present study was to investigate the effect of polyethylene glycol-superoxide dismutase (PEG-SOD), one of the important enzyme antioxidants, on leukocyte-endothelial interaction in the retinal microcirculation under LHP-induced oxidative stress. Male Brown-Norway rats weighing approximately 250 g were used. LHP(18:2) was made from linoleic acid (LA) with lipoxygenase and 10 microg of LHP dissolved in 5 microl of sodium borate buffer (SBB, 0.02 m) was slowly injected into the vitreous using a 33-gauge needle. PEG-SOD (5000 units/kg) was given intravenously 5 min before LHP injection. At 2, 4, 6, 12, 24 and 48 hr after the vitreous injections, we evaluated the number of rolling leukocytes along the major retinal veins and the number of leukocytes that accumulated in the retinal microvasculature with acridine orange digital fluorography. In LHP-treated rats, leukocyte rolling along the major retinal veins was maximal at 6 hr after LHP injection. The number of rolling leukocytes in the PEG-SOD-treated rats was decreased to 5.5% of those in the LHP-treated rats at 6 hr after LHP injection (P<0.01). No rolling leukocytes were observed in either control or vehicle-treated eyes. The number of accumulated leukocytes in LHP-treated eyes started to increase at 12 hr, and peaked at 24 hr which was significantly higher than in both control and vehicle-treated eyes (P<0.01). The number of accumulated leukocytes in the PEG-SOD-treated rats was reduced by 88.0% at 24 hr (P<0.01). Intravenous injection of PEG-SOD significantly inhibited the leukocyte rolling and its accumulation under LHP-induced oxidative stress. These results suggest that PEG-SOD might attenuate various retinal microcirculatory disorders associated with LHP.

The purpose of this study was to evaluate the effects of insulin on leukocyte-endothelial cell adhesion in the retinal microcirculation in vitro and in vivo. Human retinal endothelial cells (HRECs) were cultured in medium with or without insulin, and neutrophils allowed to adhere. Adherent neutrophils were quantified by measuring myeloperoxidase activity. Surface expression of endothelial adhesion molecules were studied with the use of an enzyme immunoassay. Insulin at concentrations of 50 and 100 microU/ml significantly increased neutrophil adhesion to HRECs compared with the control cells (P < 0.01, respectively). Surface expression of intercellular adhesion molecule-1 (ICAM-1) significantly increased when HRECs were exposed to 100 microU/ml insulin, as compared with the control cells (P < 0.05). Anti-ICAM-1 antibody significantly inhibited neutrophils adhesion to HRECs (P < 0.0001). Brown-Norway rats received subcutaneous injection of 0.2 U per 100 g body weight insulin three times. Control rats received the same amount of phosphate-buffered saline. Leukocyte entrapment in the retina was evaluated using acridine orange leukocyte fluorography. The number of leukocytes trapped in the retina of insulin-treated rats was significantly elevated compared with that in the control animals (P < 0.0001). These results suggested that insulin enhances leukostasis in retinal microcirculation. Hyperinsulinemia may be a risk factor of retinal microcirculatory disturbances.

Background: Involvement of leukocytes in three major pathologic conditions in the retina, i.e., edema, ischemia, and angiogenesis, has recently been thoroughly investigated. The accumulated evidence will lead to an integrated understanding of the relationship between leukocytes and retinal disorders, which is required for clinical applications of new medical treatment., Method: Leukocyte roles in the retinal vascular pathology were assessed by reviewing the related literature., Results: Vascular endothelial growth factor(VEGF)-induced leukocyte adhesion to the retinal vessels was shown to result in blood-retinal barrier breakdown, and the leukocyte-mediated pathogenesis was also seen in a model of diabetic retinopathy. A possible mechanism by which retinal angiogenesis develops intraviteral proliferation instead of compensating the avascular retina is that VEGF-producing monocytes/macrophages infiltrate the vitreous with subsequent migration of retinal vascular endothelial cells. On the other hand, cytotoxic T lymphocyte-mediated apoptosis of endothelial cells appears to cause the suppression of pathological retinal angiogenesis and the formation of ischemic retina secondary to vascular regression., Conclusion: Leukocytes are considered to regulate retinal edema, ischemia, and angiogenesis.

Purpose: Vascular endothelial cells and leukocytes express several inflammatory adhesion receptors, such as selectins and cell adhesion molecules, after retinal ischemia. They mediate the transmigration process of leukocytes. For further understanding of the role of leukocytes after retinal ischemia-reperfusion injury, the responses of the leukocyte-endothelial cell adhesion molecules P-selectin and intercellular adhesion molecule (ICAM)-1 during retinal ischemia and reperfusion were examined., Methods: Male pigmented rats were subjected to retinal ischemia by a 1-hour ligation of the optic nerve followed by reperfusion. Gene and protein expression of P-selectin and ICAM-1 were studied at 0.5, 1, 6, 12, and 24 hours after the onset of reperfusion with semiquantitative polymerase chain reaction and Western blot analysis. Immunohistochemical methods were used to detect specific lesions expressing ICAM-1., Results: Significant upregulation of P-selectin and ICAM-1 mRNA (at 6, 12, and 24 hours of reperfusion) were observed, with the expression peaks of both P-selectin and ICAM-1 mRNA occurring at 12 hours after reperfusion. P-selectin protein gradually increased and reached a maximum 6 hours after reperfusion, whereas ICAM-1 protein increased until 24 hours after reperfusion. Immunostaining with ICAM-1 antibodies was positive in endothelial cells after ischemia-reperfusion injury., Conclusions: Retinal ischemia-reperfusion stimulates P-selectin and ICAM-1 expression. Endothelial ICAM-1 expression in retinal vessels was observed. Upregulation of P-selectin and ICAM-1, which contribute to leukocyte rolling and adhesion, were observed after retinal ischemia.

Platelet microthrombi are present in the diabetic retinal vasculature of humans and rodents; however, the mechanisms and consequences of their presence have not been defined. The current study demonstrates that platelet containing microthrombi accumulate in the retinal vasculature of the rat within 2 weeks of experimental diabetes, a timepoint at which leukocyte-mediated endothelial cell injury and death are known to occur. Platelet accumulation increased with the duration of diabetes, and crossover experiments revealed that maximal platelet accumulation required both diabetic platelets and a diabetic endothelium. Platelet accumulation also coincided with the expression of Fas and FasL in the diabetic retina. When endothelial cell apoptosis was inhibited with an anti-FasL neutralizing antibody, platelet accumulation was effectively suppressed. When platelets were depleted from the systemic circulation with an anti-platelet antibody, blood-retinal barrier breakdown worsened in the diabetic animals. These findings suggest that platelet accumulation in the diabetic retinal vasculature is secondary to endothelial cell death and serves, in part, to suppress blood-retinal barrier breakdown.

Retinal ischemia can cause vision-threatening pathological neovascularization. The mechanisms of retinal ischemia are not fully understood, however. Here we have shown that leukocytes prune the retinal vasculature during normal development and obliterate it in disease. Beginning at postnatal day 5 (P5) in the normal rat, vascular pruning began centrally and extended peripherally, leaving behind a less dense, smaller-caliber vasculature. The pruning was correlated with retinal vascular expression of intercellular adhesion molecule-1 (ICAM-1) and coincided with an outward-moving wave of adherent leukocytes composed in part of cytotoxic T lymphocytes. The leukocytes adhered to the vasculature through CD18 and remodeled it through Fas ligand (FasL)-mediated endothelial cell apoptosis. In a model of oxygen-induced ischemic retinopathy, this process was exaggerated. Leukocytes used CD18 and FasL to obliterate the retinal vasculature, leaving behind large areas of ischemic retina. In vitro, T lymphocytes isolated from oxygen-exposed neonates induced a FasL-mediated apoptosis of hyperoxygenated endothelial cells. Targeting these pathways may prove useful in the treatment of retinal ischemia, a leading cause of vision loss and blindness.

Leukocyte dynamics were evaluatyed in vivo in rat retinal microcirculation following exposure to lipid hydroperoxide (LHP) in the vitreous.Various amounts (1, 5, 10 or 100 microg) of LHP (18:2) dissolved in 5 microl of sodium borate buffer (SBB, 0.02M) were injected into the vitreous of Brown-Norway rats. As a comparative study, 10 microg of linoleic acid (LA) dissolved in 5 microl of SBB was injected in the same way. Rats that did not undergo injection were evaluated as un-treated. At 2 to 48 hr after LHP exposure, the following were examined: (1) the flux of rolling leukocytes along the major retinal veins, (2) the number of leukocytes that accumulated in the retinal microvasculature using acridine orange digital fluorography and (3) the diameter of major retinal vessels. In the LHP-treated eyes, leukocyte rolling along the major retinal veins was observed and the number increased in a dose-dependent manner ( 1 to 10 microg). The flux of rolling leukocytes peaked at 6 hr after LHP (10-100 microg) injection. No rolling leukocytes were observed in LA-treated or un-treated eyes. The number of accumulated leukocytes started to increase at 4 hr and peaked at 24 hr after LHP (10 microg) injection. This number was significantly higher than that in LA-treated and un-treated eyes. Venous dilation was seen from 4 hr after LHP (10 microg) injection and became significant at 6 and 24 hr as compared with LA-treated and un-treated eyes. The results indicate that increased LHP levels in the vitreous due to oxidative stress enhance leukocyte-enothelium interaction in the retinal microcirculation.

Otsuka Long-Evans Tokushima Fatty (OLETF) rats are a spontaneously diabetic strain with clinical features resembling those of human noninsulin-dependent diabetes mellitus. These rats show increased leukocyte entrapment in the retina. The present study was designed to investigate the immunolocalization of intercellular adhesion molecules-1 (ICAM-1) and P-selectin in the retinas of OLETF rats. Four 72-week-old male OLETF rats and 4 age- and sex-matched Long-Evans Tokushima Otsuka (LETO) control rats were used. Western blot analysis and immunohistochemical study were performed using an anti-P-selectin monoclonal antibody (mAb) and an anti-ICAM-1 mAb. Western blot analysis showed increased expression of both ICAM-1 and P-selectin in the retinas of OLETF rats. Immunohistochemically, OLETF rats expressed greater amounts of ICAM-1 and P-selectin in the retinal vascular endothelium than did LETO rats. These findings demonstrated the upregulation of ICAM-1 and P-selectin in the retina of OLETF rats. The enhanced expression of these adhesion molecules might participate in the pathogenesis of early diabetic microangiopathy in the retina., (Copyright 2002 S. Karger AG, Basel)

Purpose: Recent reports have shown that ischemic preconditioning induces strong protection against retinal damage by subsequent prolonged ischemia and that this protection is mediated by mechanisms involving the adenosine A1 receptor. This study was designed to evaluate quantitatively the effects of ischemic preconditioning on leukocyte-mediated reperfusion injury after transient retinal ischemia and to define the role of the adenosine A1 receptor in these effects., Methods: Transient retinal ischemia was induced in male rats by temporary ligation of the optic nerve. Ischemic preconditioning (5 minutes of ischemia) was induced 24 hours before 60 minutes of ischemia. The adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) was administered intramuscularly immediately after ischemic preconditioning. Leukocyte behavior in the retina after 60 minutes of ischemia was evaluated in vivo with acridine orange digital fluorography., Results: Ischemic preconditioning inhibited leukocyte rolling. The maximum number of rolling leukocytes was reduced to 3.0% at 12 hours after reperfusion (P < 0.01). Subsequent leukocyte accumulation was also decreased with ischemic preconditioning. The maximum number of accumulated leukocytes was reduced to 22.6% at 24 hours after reperfusion (P < 0.01). These inhibitory effects were suppressed by administration of DPCPX (P < 0.0001). The numbers of rolling leukocytes at 12 hours after reperfusion and accumulated leukocytes at 24 hours after reperfusion were 102.7% (NS) and 83.4% (P < 0.01), respectively, compared with the number without ischemic preconditioning., Conclusions: The present study demonstrates the inhibitory effects of ischemic preconditioning on leukocyte rolling and subsequent leukocyte accumulation during retinal ischemia-reperfusion injury. Furthermore, the adenosine A1 receptor may play an important role in these inhibitory effects.

Purpose: To evaluate leukocyte dynamics in the retinal microcirculation of hypercholesterolemic rats in vivo using acridine orange digital fluorography., Methods: 18 male pigmented rats were divided into 3 groups according to their diet; (A) control diet (0.1% cholesterol) for 8 weeks, (B) control diet for an initial 4 weeks followed by a high-cholesterol (HC) diet (5% cholesterol) for another 4 weeks and (C) HC diet for 8 weeks. Leukocyte dynamics were observed with acridine orange digital fluorography. The diameter of major retinal vessels and the number of leukocytes trapped in the retina were evaluated quantitatively., Results: Both groups B and C showed approximately 4-fold higher serum cholesterol levels than in group A. The diameters of retinal arteries and veins were not significantly different among the three groups. Also, no rolling leukocytes were observed in any of the groups. In the retinal microcirculation at 30 minutes after acridine orange injection, the density of trapped leukocytes in both groups B and C was significantly greater than in group A (23.19 +/- 2.13, 28.56 +/- 1.96 and 13.67 +/- 1.45 cells/mm(2), respectively, p < 0.01). Furthermore, group C exhibited a significantly greater number of trapped leukocytes than in group B (p < 0.01)., Conclusion: Enhanced leukocyte entrapment in the retinal microcirculation was induced in the early stage of hypercholesterolemia. Entrapment was increased as the period of hypercholesterolemia was prolonged. Leukocyte accumulation in the retina may be an early vascular dysfunction leading to arteriosclerosis in hypercholesterolemia.

Purpose: The activity of protein kinase C (PKC), preferentially beta isoform of PKC, has been shown to be elevated in the diabetic retina. Recently, LY333531, a specific inhibitor of PKC-beta, has been reported to improve the decrease of retinal blood flow in early diabetes. Increased leukocyte entrapment has been suggested to be involved in blood flow disturbances in the early diabetic retina. This study was designed quantitatively to evaluate leukocyte entrapment in the retinal microcirculation of diabetic rats and the effect of LY333531 on leukocyte entrapment., Methods: Diabetes was induced in male Long-Evans rats by intraperitoneal injection of streptozotocin (60 mg/kg). LY333531 (0.1, 1.0, or 10.0 mg/kg/d) was administered orally during a 4-week diabetic period. Leukocyte entrapment in the retinal microcirculation was quantitatively evaluated in vivo with acridine orange digital fluorography., Results: The number of leukocytes trapped in the retinal microcirculation of diabetic rats (mean +/- SEM; 14.3 +/- 1.3 cells/mm2) was significantly increased, compared with nondiabetic control rats (7.5 +/- 0.3 cells/mm2; P < 0.0001). Oral administration of LY333531 significantly decreased the number of leukocytes trapped in the retinal microcirculation of diabetic rats (10.9 +/- 0.6, 11.3 +/- 0.7, and 10.4 +/- 0.4 cells/mm2 with LY333531 0.1, 1.0, and 10.0 mg/kg/d, respectively; P < 0.05)., Conclusions: Treatment with LY333531 attenuated the increase of leukocyte entrapment in the retinal microcirculation during the period of early diabetes. This effect may contribute to the improvement of abnormal retinal blood flow in early diabetes with LY333531. LY333531 might have a therapeutic efficacy in preventing microcirculatory flow disturbances by trapped leukocytes in the early diabetic retina.

Platelets and leukocytes are thought to play a leading role in the pathogenesis of many inflammatory conditions. To recruit flowing blood cells to the inflammatory region, it would be necessary for them to interact with vascular endothelial cells. Recently, many reports have indicated the resistance of spontaneous hypertensive rats (SHR) to endotoxic sepsis. Their resistance might be derived from suppressed interaction between these blood cells and endothelial cells. Therefore, SHR and age-matched Wistar-Kyoto rats (WKY) were induced with endotoxic sepsis by intravenous injection of lipopolysaccharide (LPS). At 4, 12, 24, and 48 hours after induction, leukocyte-endothelial interactions in the retina were evaluated in vivo with acridine orange digital fluorography. Fluorescently labeled platelets were also injected to investigate platelet-endothelial interactions in the retina in endotoxic sepsis. Leukocyte rolling in SHR after LPS injection was significantly suppressed; the maximum number of rolling leukocytes was reduced by 80.1% at 12 hours after LPS injection in SHR compared with WKY. Subsequent leukocyte infiltration into the vitreous cavity was significantly inhibited in SHR. Furthermore, platelet-endothelial interactions in the retina were also suppressed in SHR treated with LPS. The maximum numbers of rolling and adherent platelets were reduced by 59.5% and 62.6%, respectively, in SHR compared with WKY. In both strains, leukocyte- and platelet-endothelial interactions were substantially inhibited by the blocking of P-selectin. These suppressed interactions could contribute to the reduction of leukocyte- and platelet-mediated tissue injury in endotoxic sepsis in SHR, resulting in their resistance to endotoxemia.

Two prominent vascular endothelial growth factor (VEGF)-induced retinal effects are vascular permeability and capillary nonperfusion. The mechanisms by which these effects occur are not completely known. Using a rat model, we show that intravitreous injections of VEGF precipitate an extensive retinal leukocyte stasis (leukostasis) that coincides with enhanced vascular permeability and capillary nonperfusion. The leukostasis is accompanied by the up-regulation of intercellular adhesion molecule-1 expression in the retina. The inhibition of intercellular adhesion molecule-1 bioactivity with a neutralizing antibody prevents the permeability and leukostasis increases by 79% and 54%, respectively. These data are the first to demonstrate that a nonendothelial cell type contributes to VEGF-induced vascular permeability. Additionally, they identify a potential mechanism for VEGF-induced retinal capillary nonperfusion.

Purpose: The purpose of this study is to confirm the reliability of scanning laser Doppler flowmetry in the rat retina and optic nervehead, and the validity of measuring changes of retinal blood flow in rats while breathing 100% oxygen., Methods: We used a commercially available scanning laser Doppler flowmeter. To ascertain reliability, five consecutive and separate perfusion measurements of 12 eyes of 12 anesthetized pigmented rats were performed. To evaluate the validity of the system, repeated measurements were taken in anesthetized rats breathing room air or 100% oxygen. This series of measurements was repeated three times., Results: The reliability coefficients of volume, flow, and velocity in the optic nervehead and the retina ranged from 0.80 to 0.83 and 0. 77 to 0.82, respectively. After the first exposure to oxygen, the measured values of volume, flow, and velocity were reduced by an average of 20.9-24.0%, 21.2-28.2%, and 19.5-24.5%, respectively. After the values returned to the basal condition, the second and third exposures to oxygen yielded measured values that were reduced by the same amounts as at the first exposure., Conclusions: Scanning laser Doppler flowmetry provided relatively good reliability in measurements of blood flow in the rat retina and optic nervehead. This study has indicated the possibility of applying this system to the rat retina.

We have developed a new method to visualize leukocytes and evaluate their kinetics in the chorioretinal microcirculation of the living eyes. Nuclear staining dyes and a scanning laser ophthalmoscope were used to image leukocytes in the fundus. Acridine orange was used to visualize leukocytes in the retinal microcirculation. For imaging leukocytes in the choroid, indocyanine green was injected intravenously. Dynamics of leukocytes in the capillaries of the retina and choroid were quantitatively estimated in monkeys and rats. This method also allowed evaluation of leukocyte-endothelial interactions, such as rolling or firm adhesion, in vivo. Acridine orange leukocyte fluography was used to study leukocyte dynamics in the following experimentally induced microcirculatory disturbances of the retina: 1) interferon-associated retinopathy, 2) ischemia-reperfusion injury of the retina, and 3) experimental diabetes mellitus. 1) Interferon-associated retinopathy Systemic administration of interferon alpha enhanced leukocyte-endothelial interactions in the retina, which resulted in leukocyte rolling and entrapment in the retinal capillary beds. Leukocyte accumulation was also detected in the lung. The entrapment or accumulation of leukocytes in the microcirculation was inhibited by simultaneous administration of corticosteroids or other agents. These results suggested that leukocytes play a major role in the development of adverse effects of interferon, such as retinopathy or interstitial pneumonia. 2) Ischemia-reperfusion injury of the retina During reperfusion period after transient (60 min) retinal ischemia by optic nerve ligation, the rolling of leukocytes in the retinal veins was prominent and numerous leukocytes were trapped in the retinal capillaries. The number of rolling leukocytes was at a maximum 12 hours after reperfusion. Leukocyte entrapment peaked at 24 hours after reperfusion. By blocking adhesion molecules on the vascular endothelium, these leukocyte-endothelial interactions were effectively inhibited. Postischemic retinal atrophy was also inhibited by blocking adhesion molecules. These results suggested that leukocytes may be major players in the pathophysiology of ischemia reperfusion injury of the retina. 3) Experimental diabetes mellitus Leukocyte dynamics in the retina were studied in streptozotocin-induced diabetes and spontaneous diabetes (OLETF rats). In both diabetic models, leukocyte entrapment in the retinal capillaries was increased even in the early stages of diabetes. Fluorescein angiography revealed that trapped leukocytes disturbed the regional capillary blood flow in the downstream. Enhanced expression of adhesion molecules was observed in the capillary endothelium of the retina in the diabetic rats. Leukocyte entrapment in the retinal capillaries might cause microvascular occlusions and dysfunction, in turn causing diabetic retinopathy.

Purpose: This study was designed to develop a new method to evaluate the dynamics of platelets in the retinal microcirculation in vivo and to investigate quantitatively the platelet-endothelial interactions in rat retina with the use of this system., Methods: Isolated platelet samples were labeled with carboxyfluorescein diacetate succinimidyl ester. After intravenous administration, platelet behavior in the retinal microcirculation was evaluated with a scanning laser ophthalmoscope. The images were recorded on S-VHS videotape and analyzed with a computer-assisted image analysis system. The platelet- endothelial interactions in the retinal microcirculation were also investigated with the use of lipopolysaccharide-stimulated endothelium or platelets activated with thrombin., Results: Fluorescent platelets were recognized as distinct dots in the retinal microcirculation and could be traced frame by frame. The velocity of platelets in the retinal arteries, capillaries, and veins was 26.1+/-6.4, 1.6+/-0.4, and 19.9+/-8.2 mm/sec, respectively. In control rats, even the activated platelets showed minimal interaction with retinal endothelial cells. In contrast, stimulated retinal endothelium showed active platelet- endothelial interactions; many platelets were observed rolling and adhering along the major retinal veins. The interactions between platelets and stimulated endothelial cells were substantially inhibited with the injection of P-selectin monoclonal antibody., Conclusions: The present study demonstrated a new method to visualize platelet behavior in the retinal microcirculation in vivo. This method will allow quantitative evaluation of platelet dynamics and platelet- endothelial interactions in retinal pathologic conditions.

Diabetic retinopathy is a leading cause of adult vision loss and blindness. Much of the retinal damage that characterizes the disease results from retinal vascular leakage and nonperfusion. This study shows that diabetic retinal vascular leakage and nonperfusion are temporally and spatially associated with retinal leukocyte stasis (leukostasis) in the rat model of streptozotocin-induced diabetes. Retinal leukostasis increases within days of developing diabetes and correlates with the increased expression of retinal intercellular adhesion molecule-1 (ICAM-1). ICAM-1 blockade with a mAb prevents diabetic retinal leukostasis and vascular leakage by 48.5% and 85.6%, respectively. These data identify the causal role of leukocytes in the pathogenesis of diabetic retinopathy and establish the potential utility of ICAM-1 inhibition as a therapeutic strategy for the prevention of diabetic retinopathy.

Purpose: To evaluate the effects of irradiation on leukocyte dynamics in the rat retinal microcirculation., Methods: Thirty-five Brown-Norway rats received a dose of 10 Gy irradiation in one fraction. Leukocyte dynamics were studied with acridine orange digital fluorography, in which a nuclear fluorescent dye of acridine orange is used and examined by scanning laser ophthalmoscope. This technique allowed visualization of fluorescent leukocytes in vivo. The leukocyte dynamics were evaluated at 0, 4, 7, 14, 30, and 60 days after the irradiation., Results: Mean leukocyte velocity in the retinal capillaries decreased immediately. It partially recovered on day 4 but then gradually decreased up to the 2-month mark. Low-dose irradiation induced entrapment of leukocytes in the retinal microcirculation. The number of entrapped leukocytes gradually increased with time. The major retinal vessels significantly constricted immediately after irradiation. The diameter was reduced by 76% in arteries and 75% in veins, 2 months after irradiation., Conclusions: Entrapped leukocytes may be activated and exacerbate vascular injury and microinfarction and thus may participate in the pathogenesis of radiation retinopathy.

Purpose: To evaluate quantitatively the effects of blocking of adhesion molecules (P-selectin or intercellular adhesion molecule-1 [ICAM-1]) on leukocyte dynamics in the retinal microcirculation in vivo during ischemia-reperfusion injury and the therapeutic efficacy of the blocking of adhesion molecules on retinal ischemia-reperfusion injury., Methods: Retinal ischemia was induced for 60 minutes in anesthetized pigmented rats by temporary ligation of the optic nerve. P-selectin or ICAM-1 monoclonal antibody (mAb) was administered at 5 minutes before reperfusion. At 4, 12, and 24 hours after onset of reperfusion, leukocyte behavior in the retinal microcirculation was evaluated in vivo with acridine orange digital fluorography. After 7 or 14 days of reperfusion, retinal damage was evaluated histologically., Results: P-selectin mAb significantly inhibited leukocyte rolling along the major retinal veins after reperfusion. Subsequently, the number of accumulated leukocytes decreased in the P-selectin-inhibited rats. ICAM-1 mAb also inhibited leukocyte accumulation during the reperfusion period in a more substantial manner than P-selectin mAb. Histologic examination demonstrated the protective effect of the blocking of P-selectin or ICAM-1. In accordance with a reduction in leukocyte accumulation, the protective effect of mAb on retinal ischemia-reperfusion injury was more substantial in ICAM-1-inhibited rats., Conclusions: The present study demonstrates the inhibitory effect of P-selectin and ICAM-1 mAb on leukocyte accumulation and subsequent tissue injury during retinal ischemia-reperfusion injury.

Purpose: Nitric oxide is a reactive species that could be protective or destructive to the retina depending on the stage of the evolving ischemic process. This study was conducted to obtain a better understanding of the roles of constitutive nitric oxide synthase (cNOS) during reperfusion after ischemia in rat retina., Methods: Retinal ischemia was induced for 60 minutes in Sprague-Dawley rats by ligating the optic nerve. Gene expression for endothelial and neuronal nitric oxide synthases (eNOS and nNOS) was studied by reverse transcription-polymerase chain reaction (RT-PCR). To inhibit cNOS, NG-nitro-L-arginine (L-NNA) was injected intraperitoneally four times (every 6 hours) beginning 2 hours after reperfusion, for a total dose of 80 mg/kg. Retinal damage was assessed by the rate of a- and b-wave recovery on electroretinograms and by the thickness of the retinal layers. Retinal circulation and vessel diameter were evaluated by the dye-dilution technique., Results: After ischemia ended, eNOS mRNA initially decreased until 6 hours, then increased to a peak at 12 hours, and decreased progressively beyond 24 hours until the final measurement at 96 hours of reperfusion. nNOS mRNA decreased to nearly undetectable levels during the same measurement periods. L-NNA treatment enhanced reduction of a- and b-wave amplitudes and increased thinning of the inner retina in postischemic eyes. Retinal mean circulation time was markedly prolonged in L-NNA-treated postischemic eyes. Arterial mean transit times were 2.1-fold and 4.5-fold longer in L-NNA-treated postischemic eyes than in L-NNA-treated nonischemic eyes and in D-NNA-treated postischemic eyes, respectively., Conclusions: This study shows that postischemic inhibition of NOS worsens retinal damage after ischemia-reperfusion and alters postischemic retinal circulation. Nitric oxide may play an important role in protecting the retina from ischemic injury, possibly by preventing postischemic hypoperfusion.

Purpose: Leukocytes have been reported to be less deformable and more activated in diabetes. It has also been suggested that they cause microvascular occlusions that may cause diabetic microangiopathy. This study was designed to evaluate in vivo leukocyte dynamics in the retinal microcirculation of diabetic rats., Methods: Streptozotocin (STZ)-induced diabetic rats 4 weeks after diabetes induction and spontaneously diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats with 6 weeks' duration of diabetes were used in this study. Leukocyte dynamics were observed with acridine orange digital fluorography, using a nuclear fluorescent dye of acridine orange and high-resolution images from a scanning laser ophthalmoscope., Results: There was no significant difference in capillary leukocyte velocity between the STZ-induced diabetic rats (1.27 +/- 0.12 mm/sec, mean +/- SD) and nondiabetic control subjects (1.38 +/- 0.07 mm/sec) or between OLETF rats (1.31 +/- 0.17 mm/sec) and the nondiabetic controls, Long-Evans Tokushima Otsuka (LETO) rats (1.29 +/- 0.11 mm/sec). In contrast, the density of leukocytes trapped in the retinal microcirculation was significantly elevated in the STZ-induced diabetic (2.5-fold; P < 0.01) and the OLETF rats (2-fold; P < 0.01) compared with leukocyte density in the control subjects., Conclusions: Pharmacologically induced and spontaneously diabetic rats showed increased leukocyte entrapment in the living retina in the early stages of diabetes. In light of the damaging potential of leukocytes, accumulation of leukocytes in diabetic retinas from the preretinopathy stage could cause microvascular occlusions and dysfunction, in turn causing diabetic retinopathy.

Background and Purpose: Tacrolimus, an immunosuppressant agent, has been shown to reduce tissue injury and leukocyte accumulation after transient ischemia. This study was designed to evaluate quantitatively the inhibitory effects of tacrolimus on leukocyte rolling and on subsequent leukocyte accumulation in vivo after transient retinal ischemia and the protective effects of tacrolimus on ischemia-induced neural damage., Methods: Retinal ischemia was induced for 60 minutes in anesthetized pigmented rats by temporary ligation of the optic sheath. Tacrolimus was administered at 10 minutes after ischemic induction. At 4, 12, 24, and 48 hours after reperfusion, leukocyte behavior in the retinal microcirculation was evaluated in vivo with acridine orange digital fluorography. After 7 days of reperfusion, ischemia-induced retinal damage was evaluated histologically., Results: Treatment with tacrolimus suppressed leukocyte rolling; the maximum number of rolling leukocytes was reduced by 60.1% at 12 hours after reperfusion (P<0.05). In tacrolimus-treated rats, the velocity of rolling leukocytes was significantly faster than in vehicle-treated rats (P<0.01). The subsequent leukocyte accumulation was reduced by 61.6% at 24 hours after reperfusion (P<0.01). Histological examination demonstrated the protective effect of tacrolimus on ischemia-induced retinal damage, which was more substantial in the inner retina (P<0.01)., Conclusions: The present study demonstrated the inhibitory effect of tacrolimus on leukocyte rolling and on subsequent leukocyte accumulation and the therapeutic potency to neural injury after transient retinal ischemia.

Purpose: To evaluate quantitatively leukocyte dynamics in vivo in the rat retinal microcirculation during ischemia reperfusion injury with the use of acridine orange digital fluorography., Methods: Retinal ischemia was induced in anesthetized pigmented rats by a temporary ligation of the optic nerve. After 60 minutes of ischemia, leukocyte behavior in the retinal microcirculation was evaluated, with acridine orange digital fluorography--consisting of a scanning laser ophthalmoscope and the fluorescent nuclear dye, acridine orange--during reperfusion at 1, 2, 4, 6, 12, 24, 48, 96, and 168 hours. The obtained images were recorded on videotape and analyzed with a computer-assisted image analysis system., Results: Rolling leukocytes along the major retinal veins were observed in treated rats during the reperfusion period; no rolling leukocytes were observed in the control rats. The number of rolling leukocytes gradually increased and peaked at 102 +/- 40 cells/minute 12 hours after reperfusion; few rolling leukocytes were observed at 96 hours. The velocity of rolling leukocytes at 12 hours (19.1 +/- 3.5 microns/second; P < 0.05) was significantly lower than that at the other three times. No rolling leukocytes were observed along the arterial walls throughout the experiments. The number of accumulated leukocytes increased as time elapsed, peaked at 931 +/- 187 cells/mm2 24 hours after reperfusion, and decreased thereafter., Conclusions: Leukocyte dynamics in the retinal microcirculation can be quantitatively evaluated during ischemia reperfusion injury.

Purpose: To perform acridine orange digital fluorography on rats after scatter photocoagulation to investigate alterations of retinal microcirculation at the capillary level, the authors used leukocyte dynamics as a parameter., Methods: Twenty-five pigmented rats (Long-Evans) were studied. Argon laser photocoagulation, extending 6 disc diameters from the optic disc, was delivered to one half of the retina, and the other half was untreated. The total number of burns was 200 +/- 10. Leukocyte hemodynamics in retinal microcirculation were evaluated 4, 7, 14, and 28 days after photocoagulation by acridine orange digital fluorography. The fundus image was obtained by using an argon laser in a scanning laser ophthalmoscope and was recorded on magnetic tapes at a video rate. The images were analyzed by a personal computer-based image analysis system., Results: Leukocyte velocities in the retinal capillaries were significantly decreased immediately after photocoagulation. In the laser-treated area, mean capillary leukocyte velocities were 0.73, 0.92, 1, and 1.3 mm/second on days 4, 7, 14, and 28, respectively (velocity in normal control animals 1.4 mm/second). In addition, leukocyte hemodynamics were compromised in the untreated retina: Mean capillary leukocyte velocities were 0.88, 1.1, 1.2, and 1.3 mm/second on days 4, 7, 14, and 28, respectively. Twenty-eight days after photocoagulation, the velocities recovered to normal values in the treated and the untreated areas of the retina., Conclusions: Retinal capillary hemodynamics were impaired after scatter photocoagulation, and the hemodynamics in the untreated retina were also affected. Photocoagulation to the retina may influence capillary hemodynamics by diffusible chemical substances and by direct tissue injury.

Purpose: Interferon (IFN) alpha has been suggested as a possible treatment for choroidal neovascularization. However, the pathogenesis of retinal complications after IFN therapy still is unknown. Previously, we have shown that IFN alpha induced leukocyte entrapment in retinal microcirculation. The current study was designed to determine if leukocyte entrapment can be reduced by the agents that modulate leukocyte-endothelial adherence., Methods: Interferon alpha was administered intravenously in rats. Simultaneously, prednisolone (PSL), platelet-activating factor receptor antagonist (CV-6209), or superoxide dismutase (SOD) was given to the rats. Leukocyte dynamics were observed with acridine orange (AO) digital fluorography, which uses a nuclear fluorescent dye of AO and scanning laser ophthalmoscopy. The number of trapped leukocytes in each group was assessed with a personal computer-based image analysis., Results: Interferon alpha induced leukocyte entrapment in retinal microcirculation and increased leukocyte adherence to the vessel walls. The simultaneous administration of PSL, CV-6209, or SOD inhibited leukocyte adherence to the venous walls and significantly reduced the number of trapped leukocytes., Conclusions: Acridine orange digital fluorography was helpful to quantitate leukocyte-endothelial interactions in retinal microcirculation. The results suggested that increased leukocyte adherence after IFN alpha administration was reduced significantly by PSL, CV-6209, or SOD. These agents may be useful to prevent IFN-induced microcirculatory disturbances.

Purpose: The interaction between leukocytes and vascular endothelial cells plays an important role in various inflammatory disorders. This study evaluated leukocyte behavior in the retina during endotoxin-induced uveitis (EIU) in vivo., Methods: EIU was induced in female Lewis rats by footpad injection of lipopolysaccharide (LPS). The time-course changes of retinal leukocyte behavior were followed at 1.5, 3, 4.5, 6, 12, 24, 48, 72, and 120 hours after LPS treatment using acridine orange digital fluorography, consisting of high-resolution images from a scanning laser ophthalmoscope and a fluorescent nuclear dye of acridine orange., Results: Major retinal vessels were significantly dilated (P < 0.05) at 4.5 hours after LPS injection. The vasodilation, marked in veins, became maximum at 24 hours and subsided at 72 hours. Leukocytes were observed rolling along the walls of major veins at 4.5 hours. The number of rolling leukocytes gradually increased and reached a peak level of 33.8 +/- 3.4 cells/minute per major vein at 12 hours. Leukocyte rolling was still observed at 72 hours. No rolling of leukocytes was observed along the arterial walls throughout any experiments. The velocities of rolling leukocytes were determined at 6, 12, 24, and 48 hours. The leukocyte rolling velocity at 6 hours was significantly slower (33.3 +/- 2.8 microns/second, P < 0.05) than at the other three times (average, 46.6 microns/second). Cellular infiltration into the vitreous cavity was detected at 24 hours and reached its maximum at 48 hours., Conclusions: This study demonstrates that it is possible to evaluate EIU by investigating retinal leukocyte behavior and that vasodilation of major retinal vessels and leukocyte-endothelial interactions precede inflammatory cell emigration into the vitreous. This method may be useful to quantify the severity of inflammation in EIU.

Purpose: Recent studies have demonstrated that leukocytes play an important role in microcirculatory flow disturbances. A few methods are available to investigate leukocyte dynamics in retinal microcirculation. The authors explored leukocyte dynamics in the retina of rats with acridine orange digital fluorography., Methods: Acridine orange digital fluorography produces high-resolution images from a scanning laser ophthalmoscope with the use of a fluorescent nuclear dye of acridine orange, which has been used for staining nucleic acids of cells in histochemical and cytochemical studies. The images were recorded on S-VHS tapes and were investigated with a personal computer-based image analysis system., Results: Fluorescent leukocytes in the retinal microcirculation were visualized. Highly magnified images could be obtained because of the high dioptric power of the rat eye compared with the primate eye. It was possible to observe leukocyte deformation in narrow capillaries and nuclei of vascular endothelium. At arteriolar bifurcations, leukocytes moved preferentially into the branch with the higher flow rate. "Preferential channels" were identified in which predominantly leukocytes were delivered; the channels were characterized by high flow velocity and a straight, short capillary route. The average leukocyte velocities of the arteries, veins, and capillaries are 29.5 +/- 7.3, 17.4 +/- 5.3, and 1.4 +/- 0.4 mm/second, respectively., Conclusions: This study demonstrates that microcirculatory dynamics of leukocytes can be visualized and analyzed quantitatively in rats in vivo with acridine orange fluorography. This method may be a promising tool to reveal how leukocytes contribute to retinal flow disturbances under various pathologic conditions.

Background: Interferon alfa has been suggested as a possible treatment for choroidal neovascularization. However, retinal complications following interferon therapy have been reported., Objective: To evaluate the effects of interferon alfa on leukocyte dynamics in the rat retinal microcirculation., Methods: Interferon alfa of different doses was intravenously administered in rats. Leukocyte dynamics were observed with acridine orange digital fluorography, which uses a nuclear fluorescent dye of acridine orange and scanning laser ophthalmoscopy. This technique allows visualization of leukocyte movements in the retinal microcirculation in vivo., Results: After interferon alfa was administered, leukocytes adhered to vascular walls and became trapped in the retinal microcirculation. Leukocyte trapping was dose-dependent., Conclusions: Interferon alfa increased leukocyte adherence to vascular endothelium and subsequent leukocyte trapping in the retinal capillaries. Interferon alfa may activate leukocytes, and activated leukocytes may be involved in the pathogenesis of microinfarction associated with interferon-induced retinopathy.

Purpose: To evaluate retinal microcirculation in the spontaneous diabetic GK (Goto-Kakizaki) rat over an extended time., Methods: The dye-dilution technique with scanning laser ophthalmoscope-based fluorescein angiography was used to evaluate retinal circulation in GK rats with diabetes of 1, 3, and 5 months' duration and in age-matched controls. Scanning laser ophthalmoscope fluorescein angiograms were recorded after a 10-microliter bolus of 10% sodium fluorescein was injected into the tail vein, followed by a flush of 0.1 ml saline. Retinal mean circulation times (MCTs), vessel diameters, and retinal segmental blood flows (SBFs) were determined using computer-assisted image analysis on a frame-by-frame basis., Results: The MCTs were significantly prolonged (P< 0.01) in the GK rat groups (2.60 +/- 0.31, 2.74 +/- 0.28, and 2.84 +/- 0.38 seconds at 1, 3, and 5 months' duration of diabetes, respectively) compared to the age-matched controls (1.94 +/- 0.20, 1.99 +/- 0.12, and 1.91 +/- 0.22 seconds, respectively). No significant differences were observed in the retinal arterial and venous diameters between groups at each time period. The SBFs were significantly reduced (P< 0.03) in the GK rat groups (12.0 +/- 1.5, 12.1 +/- 2.0, and 11.8 +/- 2.5 x 10(2) micrometer squared/second at 1, 3, and 5 months' duration of diabetes, respectively) compared to the controls (16.0 +/- 2.2, 16.7 +/- 1.8, and 17.2 +/- 2.5 x 10(2) micrometer squared/second, respectively). In either group, no significant changes with growth were observed in MCT, vessel diameters, or SBF, although the MCTs in the GK rat group tended to lengthen, and arterial and venous diameters in the GK rat group tended to increase with duration of diabetes. Goto-Kakizaki rats did not exhibit dense cataracts, the retinal circulation could be observed, and morphologic changes of diabetic retinopathy did not develop throughout the experimental period., Conclusions: A significant prolongation in MCT and a significant reduction in SBF appeared in GK rats at an early stage in diabetes. This tendency continued until 5 months' duration of diabetes. These results suggest that retinal circulatory abnormalities are found before observable retinopathy development in GK rats and that there may be some mechanism causing a reduction in SBF without changing major retinal vessel diameters at an early stage in non-insulin-dependent diabetes mellitus (NIDDM). In addition, this study demonstrates that the GK rat will be a useful model of non-insulin-dependent diabetes mellitus to evaluate retinal circulation over an extended time.

We investigated a new method for in vivo evaluation of the retinal microcirculation in rats using a cell-permeant fluorescent dye, acridine orange (AO), which stains cell nuclei and cytoplasm, and a scanning laser ophthalmoscope (SLO). AO, which binds and interacts with DNA and RNA, and thus stains cell nuclei and cytoplasm, was administered intravenously to rats. Fluorescein angiography was performed after administration of the AO, and fundus images were recorded on S-VHS videotape by means of an SLO. Argon laser was used as an exciter of the dye. The retinal vessels were stained with the dye, rendering the retinal microvasculature clearly visible. Cell nuclei and vessel walls were observed as greater fluorescence and lesser fluorescence, respectively. Leukocytes were also observed as highly fluorescent dots moving through the vessels. The results suggest that SLO visualization of AO uptake by cells may be a useful procedure for the evaluation of retinal microcirculation in vivo in rats.

Purpose: Leukocyte rheology may play a key role in microcirculation because leukocytes have unique properties, such as large cell volume, high cytoplasmic rigidity, and low deformability. However, only a few methods are available to study the dynamic behavior of leukocytes in retinal microcirculation. The authors developed a new method to analyze directly movements of leukocytes in the retinal vessels of primates., Methods: Acridine orange, which has been used as a nuclear stain in histochemistry and cytochemistry, was injected intravenously into cynomolgus monkeys for a vital staining of leukocytes. The fundus image was generated with the argon blue laser and a scanning laser ophthalmoscope. The images were recorded on a magnetic tape and evaluated with a personal computer-based image analysis system., Results: Each leukocyte was recognized as a single fluorescent dot moving in the retinal vessels. It was possible to analyze the spatial and temporal dynamics of individual leukocytes in the capillaries. Some leukocytes passed through the capillaries, plugging transiently under the physiological condition. Leukocytes that stayed in the same position for a few minutes may have stuck to the endothelium as a result of leukocyte-endothelial interactions. In the postcapillary vessels, leukocytes tended to be displaced from the center stream toward the vessel walls. The mean flow velocity of leukocytes in the perifoveal capillary was 0.92 +/- 0.32 mm/sec., Conclusions: This study clearly demonstrated that rheologic behaviors of leukocytes in the retinal microcirculation can be studied through the vital staining with acridine orange in vivo. The authors' results suggest a potential role of leukocytes in retinal vascular flow disturbances. This study may open the door to the investigation of leukocyte hemodynamics in the retinal microcirculation in vivo.

Local laser targeted delivery of a platelet aggregating agent to occlude retinal and choroidal vessels was evaluated in rabbits and rats. Liposomes containing adenosine diphosphate (ADP) were administered intravenously and an argon laser was used to lyse the liposomes in main retinal arteries. Control vessels were treated with the same energy of laser without administering ADP. Fluorescein angiography performed 2 weeks later showed that all the control vessels were perfused. Ninety percent of the ADP-treated arteries showed complete or partial occlusion. Successful occlusion increased with the laser energy and decreased with increasing vessel diameter. Histopathology showed that occlusion was achieved in retinal as well as choroidal vessels. The inner retina remained relatively unaffected at the treatment site but the outer retina was thermally damaged. These preliminary results suggest that targeted delivery of a platelet aggregating agent holds promise for occluding vessels in the fundus.

Nineteen eyes with background sickle cell retinopathy, eleven from patients with SC disease, eight from patients with SS disease, and twelve eyes with proliferative sickle cell retinopathy were examined by direct and indirect ophthalmoscopy, slit-lamp, fluorescein angiography and vitreous fluorophotometry. Calculation of the alteration of the blood-retinal barrier (BRB) and estimation of the diffusion coefficients of fluorescein in the vitreous were performed by fluorophotometry. In background sickle cell retinopathy, the results show a normally functioning BRB in the posterior pole. Abnormally increased fluorescence values to the mid-vitreous (peripheral leakage) were found only in 3 of these 19 eyes, at the two-hour examination (all SC patients). Similarly, mid-vitreous fluorescence values at the two-hour examination were 4.19 +/- 1.52 ng/ml in eyes of patients with SC disease, compared with 2.65 +/- 0.56 ng/ml in eyes of patients with SS disease. All eyes with background sickle cell retinopathy, except one, showed values for the coefficient of diffusion of fluorescein within normal limits, indicating normal vitreous gel structure. In proliferative retinopathy, the mid-vitreous fluorophotometry readings were abnormally increased, correlating well with the extent of the peripheral angiographic changes (neovascularization). The coefficient of diffusion of fluorescein in the vitreous was generally increased in the eyes with proliferative retinopathy (15.0 +/- 8.4 X 10(-4) cm2/min) in comparison with a mean value of 5.4 +/- 1.4 X 10(-4) cm2/min in the eyes with background sickle cell retinopathy, suggesting an alteration of the vitreous structure eyes with proliferative retinopathy. Fluorophotometry is considered a useful tool to follow patients with sickle cell retinopathy by quantitating peripheral retinal vascular leakage.