In vivo evaluation of leukocyte dynamics in the retinal and choroidal circulation

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.     

Effect of topical nipradilol on retinal microvascular leukocyte adhesion in diabetic rats

BACKGROUND: Retinal leukostasis plays an important role in the pathogenesis of diabetic retinopathy. Objectives: We studied the effects of nipradilol, a topical antiglaucoma alphabeta-blocker and nitric oxide donor, on the retinal vascular leukocyte adhesion of rats with diabetes. METHODS: Diabetes was induced in seven Brown-Norway rats by one intravenous injection (65 mg/kg) of streptozotocin and confirmed by blood glucose levels >350 mg/dl 48 h after the injection. Nipradilol solution was instilled in the right eye and nipradilol-free base solution in the left eye for 3 weeks, after which the retinal microcirculation was evaluated by acridine orange leukocyte digital fluorography using laser scanning ophthalmoscopy. Leukocytes trapped in the retina were counted around the optic disc in a 5-disc-diameter area and compared between the right and the left eye. RESULTS: The mean retinal leukostasis count in the nipradilol-treated eyes (19 +/- 15 cells) was significantly lower than in the untreated eyes (49 +/- 19 cells; p < 0.0008). The diameter of the retinal artery in the eyes treated with nipradilol significantly increased (111 +/- 13.5%) compared with untreated eyes (p < 0.03). CONCLUSIONS: Topical nipradilol significantly reduced retinal leukostasis in the retinal microcirculation in diabetic rats and may be a prophylactic agent for early diabetic retinopathy through its nitric oxide donor effects on the microcirculation.     

Evaluation of fluorescein-labeled autologous leukocytes for examination of retinal circulation in humans

PURPOSE: Increased leukocyte-endothelium interaction have been suggested as a phenomenon contributing to capillary occlusion and/or rupture of the blood-retina barrier during human retinal vascular diseases. This study was performed to evaluate if fluorescein-labeled autologous leukocytes (FLALs) can be used for examination of leukocyte transit in the human retina. METHODS: The preparation consisted of human dextran-separated leukocytes mixed with fluorescein. After reinjection in normal subjects and in one diabetic patient, a confocal scanning laser ophthalmoscope was used to visualize them in the retinal circulation. The changes between FLALs and control leukocytes in the expression of leukocytes adhesion molecules CD11b and CD62L were evaluated by flow cytometry. RESULTS: The circulating FLALs were clearly visible in retinal vessels. The mean (+/- SD) capillaries velocity was 1.43 (+/- 1.3) mm/s in the macula and 1.82 (+/- 1.4) mm/s in the peripapillary area. No leukostasis was detected in the normal subjects, while it was detected in te diabetic patient. Flow cytometry revealed an increase in CD11b and a decrease in CD62L expression of leukocytes after labeling, suggesting that compared to normal leukocytes FLALs are more susceptible to interact with vascular endothelium. CONCLUSIONS: The use of FLAL is presently the only technique applicable in humans for study of leukocyte transit in the retina. Their preparation is technically simple and unexpensive. Precise measurement of the velocity of leukocytes in small vessels can be obtained. Despite evidence of a certain degree of leukocyte activation after the labeling procedure, no leukostasis was detected in vivo in normal subjects. Potential applications for this technique may include the detection of leukostasis in the human retina during severe forms of diabetes and retinal phlebitis.     

VEGF increases retinal vascular ICAM-1 expression in vivo.

PURPOSE: Intraocular injections of vascular endothelial growth factor (VEGF), a peptide implicated in the pathogenesis of diabetic retinopathy, can induce retinal ischemia. Diabetic retinal ischemia may be caused, in part, by the adhesion of leukocytes to the retinal vasculature. In this study, the ability of VEGF to increase the expression of intercellular adhesion molecule-1 (ICAM-1) and other adhesion molecules in capillary endothelium and the retinal vasculature was examined. METHODS: The expression of ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin on human brain capillary endothelial cell monolayers exposed to VEGF was quantitated by immunoassay. The effect of VEGF on retinal vascular ICAM-1 expression was determined in ICAM-1 immunofluorescence studies of retinal flat-mounts and in RNase protection assays. RESULTS: VEGF increased capillary endothelial cell ICAM-1 levels in a dose- and time-dependent manner (6-24 hours, plateau after 6 hours; EC50, 25 ng/ml). VEGF failed to alter E-selectin, P-selectin, or VCAM-1 levels under the conditions tested. Intravitreal injections of pathophysiologically relevant concentrations of VEGF increased ICAM-1 protein and mRNA levels in the retinal vasculature. CONCLUSIONS: VEGF increases retinal vascular ICAM-1 expression. VEGF-induced increases in ICAM-1 may promote retinal leukostasis in diabetic eyes.     

Peripheral Vessel Leakage (PVL): A New Angiographic Finding in Diabetic Retinopathy Identified with Ultra Wide-Field Fluorescein Angiography

Purpose: To characterize the relationship between peripheral vessel leakage and other angiographic features of diabetic retinopathy.

Design: Retrospective, consecutive case series.

Methods: Consecutive ultra wide-field angiographs obtained at a single institution for diabetic retinopathy were graded for angiographic characteristics including macular edema, retinal neovascularization, retinal vascular perfusion abnormalities, and retinal vascular staining and leakage.

Results: Angiographic characteristics of 264 eyes of 143 patients were evaluated. Findings included focal and diffuse angiographic macular edema (150/264, 57%), neovascularization (107/264, 41%), late peripheral vascular leakage (PVL) (107/264, 41%), and peripheral non-perfusion (142/264, 54%). Amongst all subjects untreated peripheral non-perfusion was associated with anterior neovascularization (78% vs. 48%, p?=?0.0001, Fisher exact test) and posterior neovascularization (78% vs. 43%, p?<?0.0001), but not with macular edema (p?=?0.71). PVL was associated with peripheral non-perfusion (78% vs. 38%, p?<?0.0001) and posterior neovascularization (53% vs. 35%, p?=?0.01), but not with macular edema (p?=?0.449). However, focal macular edema was strongly associated with PVL (33% vs. 13%, p?=?0.008) in eyes without peripheral non-perfusion. Amongst untreated eyes with non-proliferative retinopathy and macular edema, there was a trend for association between macular edema and peripheral non-perfusion (p?=?0.065).

Conclusion: Untreated peripheral non-perfusion and late peripheral vascular leakage detected using ultra wide-field FA are associated with neovascularization in diabetic retinopathy. PVL may be associated with focal diabetic macular leakage in this cohort.     

糖尿病视网膜病变合并白血病的诊疗进展

白血病是加重糖尿病视网膜病变（DR）的危险因素。DR合并白血病的患者常首诊于眼科,其眼底除了会出现视网膜静脉纡曲扩张、微动脉瘤和视网膜出血、渗出等典型DR表现,还会合并罗斯斑等白血病视网膜病变的表现。其在微血管异常轻微的病变早期就可能出现大量视网膜血管无灌注区及新生血管,同时玻璃体积血、纤维血管增生膜及牵引性视网膜脱离...     

VEGF164 is proinflammatory in the diabetic retina

PURPOSE: The objectives of this study were to characterize the differential potency of two major VEGF isoforms, VEGF(120) and VEGF(164), for inducing leukocyte stasis (leukostasis) within the retinal vasculature and blood-retinal barrier (BRB) breakdown and to determine whether endogenous VEGF(164) mediates retinal leukostasis and BRB breakdown in early and established diabetes. METHODS: Retinal leukostasis and BRB breakdown were simultaneously quantified by combining concanavalin A lectin (ConA) perfusion labeling with a fluorophotometric dextran leakage assay. CD45 immunohistochemistry was performed to confirm that ConA-stained cells within the vasculature were leukocytes. Retinal leukostasis and BRB breakdown were compared in nondiabetic rats receiving intravitreous injections of VEGF(120) or VEGF(164). Retinal intercellular adhesion molecule (ICAM)-1 and VEGF protein levels were studied by Western blot and ELISA, respectively. An anti-VEGF(164(165)) aptamer (EYE001) was administered by intravitreous injection to 2-week and 3-month diabetic rats, and the effect on retinal leukostasis and BRB breakdown was quantified. RESULTS: Compared with VEGF(120), VEGF(164) more potently increased retinal ICAM-1 levels (2.2-fold), leukostasis (1.9-fold), and BRB breakdown (2.1-fold, P < 0.01 for all), despite negligible differences in vitreoretinal VEGF levels at the time of evaluation (P > 0.05). Retinal leukostasis and leakage increased with the duration of diabetes (P < 0.01) and correlated closely (P < 0.01, r = 0.889). The isoform-specific blockade of endogenous VEGF(164) with EYE001 resulted in a significant suppression of retinal leukostasis and BRB breakdown in both early (72.4% and 82.6%, respectively) and established (48.5% and 55.0%, respectively) diabetes (P < 0.01). CONCLUSIONS: On an equimolar basis, VEGF(164) is at least twice as potent as VEGF(120) at inducing ICAM-1-mediated retinal leukostasis and BRB breakdown in vivo. The inhibition of diabetic retinal leukostasis and BRB breakdown with EYE001 in early and established diabetes indicates that VEGF(164) is an important isoform in the pathogenesis of early diabetic retinopathy.     

Retinal Microvascular Patency in the Diabetic Rat

To study whether the patency to erythrocytes in retinal microvessels of diabetic rats is reduced or blocked before the vessels lose their patency to plasma flow. Methods: We used recognized techniques to induce diabetic and galactose related microvascular retinal lesions in rats: (1) alloxan induction (2) streptozotocin induction (3) galactose-containing diet. The rats were followed up to 17 months. We used our vascular trichrome technique to observe the effects of the ongoing diabetes on the retinal microcirculation. Results: A focal leakage of a plasma-borne fluorescent dye was noted around the junction of the deep retinal capillaries and the ascending venules to the superficial retinal circulation in the streptozotocin and alloxan diabetic rats by the 14th month, and, by the 16th month, retinal capillary non-perfusion and retinal vascular malformations were present. The affected vessels showed patency to microspheres (0.2 μm in diameter) but no perfusion of erythrocytes. No such changes were seen in the galactose-fed rats. Conclusions: (1) The location between the deep retinal capillary net and the ascending venules may be the site of early vascular leakage in the diabetic rat model, (2) the erythrocytes’ passage in the affected retinal microcirculation was blocked before the development of complete blockage to plasma in diabetic rats. The logical assumption that during the development stage of retinal capillary occlusion there may be a transient stage of microvascular insufficiency was examined. The lathyrogen, imino-diproprionitrile (IDPN), had previously been effective for creating a fast-developing model of retinal vasculopathy. Using that model, we demonstrated a stage in which the retinal microvasculature was blocked to erythrocytes but not to plasma [1]. However, we questioned the applicability of our findings to more slowly developing microvasculopathies, such as diabetic retinopathy. We designed the current study to examine the presence of such stage in slowly developing microvasculopathy. Animal models that are known to induce “diabetic retinopathy-like” changes used [2–4]. The diabetic animals were followed for a period of 17 months. Starting at the 12th month, a few animals of each group were killed and the retains were examined with our trichrome method [1] for relative capillary patency to erythrocytes and plasma, for functionality of endothelial cells, and for disturbances in the blood–retinal barrier. The results of this study support the hypothesis that retinal microvascular insufficiency does exist as a temporary stage that precedes the development of complete capillary blockage in long-term developing rat models of diabetic retinopathy.     

Effects of peroxisome proliferator-activated receptor gamma and its ligand on blood-retinal barrier in a streptozotocin-induced diabetic model

PURPOSE: To clarify whether endogenous peroxisome proliferator-activated receptor gamma (PPARgamma) and its ligand, rosiglitazone, affect retinal leukostasis and the associated vascular leakage using an experimental diabetic model. METHODS: Diabetes was induced in heterozygous PPARgamma+/- mice and Brown Norway rats with an intraperitoneal streptozotocin (STZ) injection. Retinal leukostasis and leakage, quantified by concanavalin A (Con A) lectin perfusion labeling combined with a fluorophotometric dextran leakage assay, were investigated at 120 days in diabetic PPARgamma+/- and wild-type mice and at 21 days in diabetic rats receiving rosiglitazone or the vehicle. The retinal protein expression levels of vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-alpha, and the intercellular adhesion molecule (ICAM)-1 were investigated by means of the ELISA assay. RESULTS: In the diabetic PPARgamma+/- mice, retinal leukostasis and leakage were greater than in the diabetic wild-type mice. In addition retinal leukostasis and leakage were suppressed by treatment with rosiglitazone in experimental diabetic rats. ELISA analysis revealed that the upregulated ICAM-1 expression in the diabetic rat retina was reduced by rosiglitazone treatment. CONCLUSIONS: An endogenous pathway involving PPARgamma provides protection against retinal leukostasis and retinal leakage in diabetes and treatment with PPARgamma specific ligands inhibits retinal leukostasis and retinal leakage in diabetic rats.     

Integrin-mediated neutrophil adhesion and retinal leukostasis in diabetes

PURPOSE: A critical early event in the pathogenesis of diabetic retinopathy is leukocyte adhesion to the diabetic retinal vasculature. The process is mediated, in part, by intercellular adhesion molecule-1 (ICAM-1) and results in blood-retinal barrier breakdown and capillary nonperfusion. This study evaluated the expression and function of the corresponding ICAM-1-binding leukocyte beta2-integrins in experimental diabetes. METHODS: Diabetes was induced in Long Evans rats with streptozotocin. The expression of the surface integrin subunits CD11a, CD11b, and CD18 on rat neutrophils isolated from peripheral blood was quantitated with flow cytometry. In vitro neutrophil adhesion was studied using quantitative endothelial cell-neutrophil adhesion assays. The adhesive role of the integrin subunits CD11a, CD11b, and CD18 was tested using specific neutralizing monoclonal antibodies. CD18 bioactivity was blocked in vivo with anti-CD18 F(ab')2 fragments, and the effect on retinal leukocyte adhesion was quantitated with acridine orange leukocyte fluorography. RESULTS: Neutrophil CD11a, CD11b, and CD18 surface integrin levels were 62% (n = 5, P = 0.006), 54% (n = 5, P = 0.045), and 38% (n = 5, P = 0.009) greater in diabetic versus nondiabetic animals, respectively. Seventy-five percent more neutrophils from diabetic versus nondiabetic animals adhered to rat endothelial cell monolayers (n = 6, P = 0.02). Pretreatment of leukocytes with either anti-CD11b or anti-CD18 antibodies lowered the proportion of adherent diabetic neutrophils by 41% (n = 6, P = 0.01 for each treatment), whereas anti-CD11a antibodies had no significant effect (n = 6, P = 0.5). In vivo, systemic administration of anti-CD18 F(ab')2 fragments decreased diabetic retinal leukostasis by 62% (n = 5, P = 0.001). CONCLUSIONS: Neutrophils from diabetic animals exhibit higher levels of surface integrin expression and integrin-mediated adhesion. In vivo, CD18 blockade significantly decreases leukostasis in the diabetic retinal microvasculature. Integrin adhesion molecules may serve as therapeutic targets for the treatment and/or prevention of early diabetic retinopathy.     

Peroxynitrite decomposition catalyst, FP15, and poly(ADP-ribose) polymerase inhibitor, PJ34, inhibit leukocyte entrapment in the retinal microcirculation of diabetic rats

PURPOSE: Oxidative and nitrosative stress and activation of poly(ADP ribose) polymerase (PARP) play a role in the pathogenesis of diabetic complications. We evaluated the effectiveness of the peroxynitrite decomposition catalyst, FP15, and the PARP inhibitor, PJ34, in the treatment of leukocyte entrapment in the retinal microcirculation of diabetic rats. METHODS: Diabetes was induced in rats by intraperitoneal injection of 60 mg/kg of streptozotocin. Rats were divided into four groups: controls; untreated diabetes; diabetes treated with FP15 (10 mg/kg oral gavage twice daily) and diabetes treated with PJ34 (10 mg/kg oral gavage twice daily). All experiments were performed 4 weeks after initiation of treatment. Leukocyte entrapment in the retinal microcirculation was quantitatively evaluated in vivo with acridine orange digital fluorography. RESULTS: The density of leukocytes trapped in the retinal microcirculation 30 minutes after dye injection was significantly greater in untreated diabetes (32.1 +/- 4.7 cells/mm2) than in controls (11.3 +/- 4.5 cells/mm2) (p < 0.05). Compared with untreated diabetes, the density of trapped leukocytes significantly decreased in diabetes treated with FP15 (14.5 +/- 5.1 cells/mm2) (p < 0.0001) and diabetes treated with PJ34 (24.1 +/- 4.2 cells/mm2) (p < 0.05). CONCLUSIONS: Treatment with FP15 and PJ34 decreased enhanced leukocyte entrapment in the retinal microcirculation during the early diabetic period. The current study suggests a role for peroxynitrite production and for PARP activation in the pathogenesis of retinal microvascular leukostasis in early diabetes.     

PKC-beta inhibitor (LY333531) attenuates leukocyte entrapment in retinal microcirculation of diabetic rats

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.     

白细胞淤滞对糖尿病视网膜病变作用的研究

目的:观察糖尿病小鼠视网膜血管内白细胞淤滞和细胞间黏附因子-1(ICAM-1)在视网膜的表达.方法:选取C57型小鼠45只,随机分为2组:糖尿病组22只、正常组23只.10wk后取视网膜,荧光显微镜计数小鼠全视网膜微血管内淤滞的白细胞数目及其含有ICAM-1表达的荧光小球数目,免疫蛋白印迹法检测视网膜血管内皮生长因子(VEGF)和ICAM-1的表达.结果:10wk后,糖尿病小鼠视网膜血管内白细胞数目及含有ICAM-1表达的荧光小球数目明显高于正常对照组(P<0.01),其视网膜内VEGF和ICAM-1明显增加,有统计学意义(P<0.05).结论:视网膜血管内白细胞淤滞与糖尿病视网膜病变(DR)早期的视网膜毛细血管无灌注有关.     

First microangiographic abnormalities in childhood diabetes — types of lesions

An analysis by fluorescein angiography of the first signs of retinopathy in 161 diabetic children showed that microaneurysm-like spot dilatations, microaneurysms, focal capillary dilatations, leakage, generalized capillary dilatations, retinal hemorrhages, areas of capillary non-perfusion and capillary remodelling, in decreasing order of frequency, were features of the onset of retinopathy. However, microaneurysm-like spot dilatations and both focal and generalized capillary dilatations were considered to be too subjective for use in further quantitative analysis. Retinopathy was not found in children < 12 years of age and was detected only after at least 3 years of diabetes. The mean duration of diabetes before the occurrence of the first lesions in 118 affected eyes was 8.2 years. The mean age at which lesions occurred was 16.4 years. Although capillary non-perfusion was rarely an initial lesion, occurring after a longer duration of diabetes and at a later age than the other lesions, no significant difference could be found between the various types of lesions for either the patient's age at their onset or the duration of diabetes. The type of initial lesion was also unrelated to sex, age at the onset of diabetes, or metabolic control.Deceased     

Captopril inhibits capillary degeneration in the early stages of diabetic retinopathy

PURPOSE: This study was conducted to examine the effect of angiotensin-converting enzyme (ACE) inhibitors on the development of early stages of diabetic retinopathy. METHODS: Rats were made diabetic by injection of streptozotocin and treated with the ACE inhibitor captopril and the AT1 antagonist losartan. The extent of capillary degeneration and leukostasis in the retina were determined. RESULTS: Acellular capillaries and pericyte ghosts in the retina of diabetic animals were increased by approximately two-fold after 8 months of diabetes compared with the nondiabetic control, and captopril completely inhibited this capillary degeneration. Captopril and losartan also inhibited hyperglycemia-induced leukostasis at 6 weeks and 1 week in the retinal vasculature, respectively. In cultured retinal endothelial cells, angiotensin II-induced VCAM-1 expression was inhibited by losartan. CONCLUSIONS: Inhibition of the renin-angiotensin system can block retinal capillary degeneration and inflammation in the early stages of diabetic retinopathy.     

Comparison of venous filling times and SLO findings at each quadrant region in diabetic retinopathy

Our purpose is to evaluate the correlation of retinal venous filling time (VFT), which reflects the peripheral retinal microcirculation in diabetic retinopathy, with fluorescein angiography (FAG) findings such as microaneurysm (MA), non-perfusion area (NP), neovascularization (NV) and relative retinal venous diameter (RRVD), and further, to evaluate the correlation between arteriovenous passage time (AVPT) and these factors. Partial F-test (multiple regression analysis) was performed on patients (32 eyes of diabetic retinopathy) who underwent video FAG, using scanning laser ophthalmoscope. In the four quadrants, VFT was compared with each MA, NP, and NV at each vascular arch at a distance of one disc diameter distant from the optic disc. VFT was significantly correlated to RRVD, NP, and NV at each quadrant (r > 0.7). However, AVPT was not significantly correlated to any of the factors studied. Delayed VFT was associated with the hemodynamic change of the peripheral retinal microcirculation at each quadrant in diabetic retinopathy. VFT can be used as an index to indicate the peripheral microcirculation of the retina.     

细胞因子与糖尿病视网膜病变的研究进展

糖尿病视网膜病变（DR）是一种发生进行性视力损害的糖尿病（DM）并发症,其特征是毛细血管闭锁、微循环障碍和局部缺血性视网膜新生血管形成,其确切的发病机制尚不清楚。最近有研究认为DR可能与视网膜毛细血管炎症反应有关。由于细胞因子能引起炎症反应和黏附分子表达,因此细胞因子增加单核细胞和内皮细胞黏附的过程被认为是DR发生发展过程中的关键事件。就血管内皮生长因子（VEGF）、转化生长因子-β（TGF-β）、碱性成纤维细胞生长因子（bFGF）等多种细胞因子在DR中的作用进行综述。     

An endothelin type A receptor antagonist reverses upregulated VEGF and ICAM-1 levels in streptozotocin-induced diabetic rat retina

Diabetic retinopathy, a cause of blindness, is often associated with the upregulation of vascular endothelial growth factor (VEGF) in the retina. Recently, leukocyte adhesion (leukostasis) is claimed for the occlusion of retinal capillary vascularity, which ultimately assists in the progression of diabetic retinopathy. In addition, intercellular adhesion molecule-1 (ICAM-1), a representative factor for leukostasis, is increased in diabetic retina. Endothelin (ET)-1, a potent vasoconstrictor peptide, is closely linked to the pathogenesis of diabetic retinopathy. Different therapeutic interventions concerning VEGF have already been proposed to prevent diabetic retinopathy. However, no study has yet reported concerning the effects of ET-1 receptor antagonist on the upregulated VEGF and ICAM-1 in morphologically intact diabetic retina. The current study investigated the effect of ET(A) receptor antagonist (TA-0201; 1 mg kg(-1) day(-1)) on the expressions of VEGF and ICAM-1 in rat diabetic retina. Diabetes was induced by intraperitoneal injection of streptozotocin (70 mg/kg) in Sprague-Dawley rats, whereas control rats (Cont) received only citrate buffer. After 1 week, the streptozotocin-administered rats were randomly divided into two groups: ET(A) receptor antagonist-treated group (DM+TA-0201) and saline-treated group (DM+vehicle). After the treatment for 4 weeks, the retina was removed from the eyeball. In DM+vehicle group, the VEGF expression of retina was significantly increased (33.5 pg/mg) in comparison with that in the Cont group (25.1 pg/mg), and the upregulation of VEGF was reversed in DM+TA-0201 group (26.9 pg/mg), a phenomenon consistent with the change in VEGF mRNA levels. The expression of retinal ICAM-1 was increased in DM+vehicle group (55.1 pg/mg) compared with Cont group (43.8 pg/mg), and ET antagonism completely blocked this increase (43.8 pg/mg). Moreover, an increased leukostasis by 3.3-fold in DM+vehicle retina was returned to the control level by ET antagonism. In the current study, there was no obvious retinal morphological alteration from both the hematoxylin and eosin staining and the FITC-dextran angiography. Thus, ET(A) receptor antagonist might be useful in preventing the progression of diabetic retinopathy, as evidenced by suppressing the increase in VEGF and ICAM-1 levels as well as leukostasis in morphologically intact diabetic retina.     

Inhibition of diabetic leukostasis and blood-retinal barrier breakdown with a soluble form of a receptor for advanced glycation end products

PURPOSE: The interaction of advanced glycation end products (AGEs) with their receptors is hypothesized to be involved in the development of diabetic retinopathy. In the present study, the role of an AGE receptor, RAGE, was investigated in the development of diabetic retinopathy in vivo. METHODS: C57/BJ6 and RAGE-transgenic mice that carried human RAGE genetic DNA under the control of the murine flk-1 promoter were made diabetic with streptozocin. Three months after the onset of diabetes, the soluble form of RAGE (sRAGE) or mouse serum albumin was injected intraperitoneally at 100 mug/d for 14 consecutive days. After the final injection, blood-retinal barrier breakdown, retinal leukostasis, expression of VEGF and ICAM-1, and expression of RAGE in the retina were investigated. RESULTS: Blood-retinal barrier breakdown and increased leukostasis were associated with the experimental diabetes in the C57/BJ6 mice. These changes were significantly augmented in RAGE-transgenic mice. The blood-retinal barrier breakdown and leukostasis in the diabetic C57/BJ6 and RAGE-transgenic mice were accompanied by increased expression of VEGF and ICAM-1 in the retina. The systemic administration of sRAGE significantly inhibited blood-retinal barrier breakdown, leukostasis, and expression of ICAM-1 in the retina in both the diabetic C57/BJ6 and RAGE-transgenic mice. The expression of RAGE was slightly increased in the retinal vessels in diabetic or RAGE-transgenic mice. Furthermore, a strong induction of RAGE was observed in the retinal vessels of diabetic RAGE-transgenic mice. CONCLUSIONS: This study further demonstrates the role of the AGEs and RAGE axis in blood-retinal barrier breakdown and the retinal leukostasis, which are characteristic clinical symptoms of diabetic retinopathy. Furthermore, these data demonstrate that blocking AGE bioactivity may be effective for the treatment of diabetic retinopathy.     

Role of angiotensin II in retinal leukostasis in the diabetic rat

To study if the endogenous renin-angiotensin system affects diabetic retinal leukostasis, rats with streptozotocin-induced diabetes were treated with an ACE inhibitor (ramipril), an angiotensin II AT(1) receptor antagonist (losartan) and the Ca channel blocker, (nifedipine). In the diabetic rats, these drug treatments reduced systolic blood pressure by approximately 16 mmHg but did not change blood glucose. After 2 weeks, the rats were examined for retinal leukostasis in vivo with a scanning laser ophthalmoscope (SLO). Retinal leukostasis, which was defined as no movement of arrested leukocytes over 2 min, was markedly higher in diabetic rats than normal controls (P<0.01). Leukostasis was significantly decreased by ramipril and losartan (P<0.01 vs. untreated diabetic rats) but was still higher than normal. Retinal leukostasis after nifedipine treatment was not significantly different than in untreated diabetic rats. The same trend was observed when leukostasis was analyzed on retinal flat mounts with concanavalin A and CD45 immunofluorescence; ramipril and losartan treatment, however, decreased leukostasis to values no different than controls. Retinal leukostasis was lowered by nifedipine (P<0.05, untreated diabetes vs. nifedipine-treated) but was still higher than in normal, ramipril-, or losartan-treated rats. Assays of gene expression of retinal intercellular adhesion molecule (ICAM-1) by semi-quantitative RT-PCR indicated that ICAM-1 mRNA was increased in diabetic rats but was decreased markedly by treatment with losartan or ramipril, and modestly by nifedipine. In summary, suppressing the activity of the endogenous renin-angiotensin system markedly decreases, perhaps even normalizes, the retinal leukostasis that accompanies type I diabetes in rats. These effects seem to be partly independent of blood pressure and to be associated with a decrease in ICAM-1 gene expression. Angiotensin II may, thus, mediate retinal leukostasis in early diabetes.