HSCs改善糖尿病小鼠血-视网膜屏障功能的研究

目的探讨自体造血干细胞动员是否可使成体造血干细胞（HSCs）环境诱导分化，修复受损的视网膜微血管内皮细胞，使血-视网膜屏障得到功能性重建。方法糖尿病小鼠和正常小鼠各分为干细胞动员组和生理盐水对照组。流式细胞术以CD34-／low和scal^＋为标记鉴定外周血HSCs。小鼠视网膜病理切片行苏木精-伊红染色观察和VEGF免疫组织化学分析，伊文思蓝定量检测血-视网膜屏障的破坏程度。结果自体干细胞动员使糖尿病小鼠视网膜的VEGF表达量明显下降。糖尿病小鼠经干细胞动员后清蛋白的渗出量明显下降（P=0．033）。结论自体HSCs动员可以通过外周血干细胞倍增使血-视网膜屏障得到一定程度的功能性修复。     

Effect of pyridoxine on the blood-retina barrier in experimentally induced hyperglycemia in rats.

We studied the effects on the blood-retina barrier of pyridoxal S phosphate, the biologically active form of vitamin B6 (pyridoxine), in 23 male Long-Evans rats in which hyperglycemia had been induced by means of intraperitoneal injection of streptozocin. Twelve rats were treated with pyridoxine, and 11 rats served as the control group. Vitreous fluorophotometry was used to quantitatively evaluate the effect of pyridoxine. The procedure was done before and 2 weeks after the induction of hyperglycemia, readings being taken 5 and 60 minutes after injection of the dye. In both groups the mean vitreous fluorescein levels after hyperglycemia was induced were significantly higher than those before hyperglycemia was induced (p less than 0.05). The mean levels before hyperglycemia was induced were not significantly different between the two groups. The mean level 60 minutes after injection of the dye after hyperglycemia was induced was lower in the treated group than in the untreated group, although not significantly so. The incidence of lens opacities at 5 weeks was higher in the untreated group than in the treated group (p less than 0.01). We conclude that administration of pyridoxine to hyperglycemic rats for 5 weeks has no significant effect on the permeability of the blood-retina barrier.     

光损伤对大鼠血-视网膜屏障功能的影响

目的：探讨强光对大鼠血－视网膜屏障功能的影响。方法：大鼠随机分为光照组及对照组,光照组大鼠经散瞳后进行10000lx强光照射（12h光照,12h避光,连续1～14d）,对照组只接受自然光线照射。分别于强光照射后第1、3、7、14 d 摘除相应的光照组和对照组大鼠双侧眼球;并用HE染色观察视网膜各层结构变化,用电镜观察视网膜超微结构变化,用伊凡思蓝（Evans blue,EB）灌注后激光共聚焦显微镜下微循环成像及分光光度法定量检测视网膜微循环通透性变化,来评估血－视网膜屏障变化。结果：大鼠在强光照射1d后就出现视网膜光感受器细胞变性、外节膜盘脱落、外核层厚度变薄等超微结构改变,并随着强光照射持续而逐渐加重,3 d后出现光感受器细胞凋亡,至14 d时外核层厚度已明显变薄、细胞数也明显减少。大鼠在强光照射1 d后视网膜血管就出现EB染料渗漏,至14 d时EB染料渗漏最明显。结论：强光照射可导致大鼠视网膜外核层光感受器细胞变性、凋亡,外核层厚度变薄、细胞数减少,血－视网膜屏障结构、功能破坏。     

Effect of calcium dobesilate on the blood-retinal barrier in early diabetic retinopathy

Summary The effect of calcium dobesilate on the alteration of the blood-retinal barrier was studied in 41 adult-onset, non-insulin dependent diabetic patients with minimal or no retinopathy, randomly assigned to receive either oral calcium dobesilate (1000 mg twice daily) or a placebo for 12 months. The posterior vitreous value and the penetration ratio, determined by vitreous fluorophotometry, reflected stabilisation of blood-retinal barrier permeability in the calcium dobesilate patients and deterioration of blood retinal barrier in those given placebo. During the relatively short period of the study, one year, no significant change in microaneurysm and capillary closure gradings was observed. No side effects were associated with calcium dobesilate.     

Blood-retina barrier permeability is independent of trace substance lipid solubility in retinitis pigmentosa and in the healthy eye

Differential ocular spectrofluorometry was used toassess the passive permeability of the blood-retinabarrier in healthy subjects and in patients withretinitis pigmentosa by determination of the rate ofinward leakage of fluorescein and fluoresceinglucuronide after intravenous injection offluorescein.In five healthy subjects we found permeabilities of1.3 (0.6–2.8) nm/s [log-mean (range)] for fluoresceinand 1.3 (0.6–3.1) nm/s for fluorescein glucuronide.Six patients with retinitis pigmentosa all had amarkedly increased blood-retina barrier leakage, withinward permeabilities of 8.2 (3.4–25) nm/s forfluorescein and 8.2 (5.6–27) nm/s for fluoresceinglucuronide.Since no detectable difference was found between thepermeabilities of the two tracers the passive permeability of the blood-retina barrier appears to beindependent of the 18-fold difference in lipidsolubility between the two tracers, both in retinitispigmentosa and in healthy subjects. Presumably, thestructural substrate for leakage of small hydrophilicmolecules through the blood-retina barrier is awater-filled pore, since diffusion through lipidcellular membranes would favor fluorescein over itsmore water soluble glucuronide.     

Alteration of the blood-retina barriers in cases of viral retinitis

Summary This paper presents three cases of serologically documented viral retinitis, and the great value of fluorescein angiography in outlining the structural abnormalities and the site of the lesions.Of our series in the case of influenza retinitis, the fluoroangiographic findings showed dye leakage from retinal vessels in the posterior pole. The dye leakage did not appear completely in the sites of macular edema. This edema was similar to the clinical appearance of cotton-wool spots and was arranged in a star-like pattern.One of the cases of cytomegalovirus retinitis (Case 3), a previously healthy adult with dysfunction of the cellular immune system, seems to be a further example of an inflammation in the inner retina presenting cotton-wool spots at the early stage.Case 2, a previously healthy adult, fulfilled the criteria for Vogt-Koyanagi-Harada syndrome. The patient had serologically documented cytomegalovirus infection with dysfunction of the cellular immune system. The fluorescein angiographic examination showed alteration both in the inner and the outer blood-retina barriers as it is characteristic in cases of Harada syndrome. The cytomegalovirus infection might be assumed to play a role in the clinical picture as well as in the etiology of this disease.     

芪灯明目胶囊对高血糖大鼠血视网膜屏障影响的研究

目的:观察链脲佐菌素(STZ)诱发糖尿病大鼠在造模后血-视网膜屏障(blood-retina barrier,BRB)变化情况,并以阳性药为对照研究中药芪灯明目胶囊对STZ诱发糖尿病大鼠的视网膜血管渗漏影响。方法:采用STZ腹腔注射制作糖尿病大鼠模型,在造模后6mo内各时点(2wk;1,2,3,4,5,6mo)采用伊文思蓝灌注示踪显示血-视网膜的渗漏情况,在造模后3mo开始用中药芪灯明目胶囊(低中高剂量组分别给予125,250,500mg/kg体质量剂量的胶囊内容物灌胃),对照组用安多明胶囊(200mg/kg体质量剂量,相当于10倍成人剂量),灌胃3mo,观察药物对BRB的影响。结果:STZ糖尿病大鼠在2wk即可出现BRB的损害,并随着高血糖状态的持续而不断加重。对造模3moSTZ糖尿病模型大鼠连续灌胃中药芪灯治疗3mo,结果提示:中药芪灯对STZ糖尿病BRB有保护作用,可明显减少视网膜血管的渗漏。结论:STZ糖尿病模型大鼠在早期即可出现BRB损害,并随着高血糖的持续而加重,中药芪灯明目胶囊可减少高血糖导致的BRB损害。     

Effect of nucleotide P2Y2 receptor agonists on outward active transport of fluorescein across normal blood-retina barrier in rabbit

BACKGROUND: To evaluate the effect of nucleotide P2Y(2) receptor agonists INS542 and uridine 5'-triphosphate (UTP) on the outward active transport of fluorescein across rabbit blood-retina barrier (BRB) in vivo. METHODS: Injection (0.1 ml) of INS542 (0.1 or 1mM), phosphate buffered solution, or UTP (1 or 10mM) was made in Dutch-belted rabbits. Differential vitreous fluorophotometry (DVF) was performed 3hr later and the fluorescein (F)/fluorescein monoglucuronide (FG) ratio was then calculated. F/FG ratios are inversely proportional to outward active transport of F across BRB at the level of the retinal pigment epithelium (RPE). In another set of experiments, the effect of 0.1 ml vitreous injection of INS542 (1mM) on F/FG ratios was evaluated at different time points ranging from 0.5 to 48hr before conducting DVF. RESULTS: F/FG ratios obtained 3hr after intravitreal injection were as follows (mean+/-standard error): 0.49+/-0.14 (0.1mM INS542), 0.19+/-0.04 (1mM INS542), 0.48+/-0.09 (PBS), 0.40+/-0.08 (1mM UTP) and 0.36+/-0.05 (10mM UTP). The F/FG ratio for 1mM INS542 was significantly lower than in the other groups (P<0.05). In the time course experiments, a significant decrease in the F/FG ratios was observed between 1 and 12hr following administration of INS542 when compared with F/FG ratios obtained in the contralateral (untreated) eye. CONCLUSION: Intravitreal administration of INS542 (but not UTP) enhances outward active transport of F across RPE in intact rabbit eye, indicating that activation of P2Y(2) receptors in vivo directly stimulates RPE active transport.     

Alteration of the blood-retinal barrier and vitreous in sickle cell retinopathy

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 × 10^–4cm²/min) in comparison with a mean value of 5.4 ± 1.4 × 10^–4cm²/min in the eyes wi 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.     

Effect of argon laser photocoagulation on fluorescein transport across the blood-retinal barrier

The effect of argon laser photocoagulation on the active transport of fluorescein across the blood-retinal barrier was examined in the rabbit, before and after healing of the photocoagulation scars.The outward fluorescein transport across the blood-retinal barrier, which was assessed by vitreous fluorophotometry after intravitreal administration of sodium fluorescein, was greatly reduced in the first days after panretinal photocoagulation with the argon laser. However, this alteration of the barrier recovered progressively with time, depending on the intensity of the photocoagulation burns. When moderate panretinal laser photocoagulation was used (500 μm, 0·1 sec, 0·1 W), the transport activity was markedly altered by 6 days, but there was almost complete recovery at the end of 30 days. When severe photocoagulation was performed (500 μ, 0·1 sec and 0·25–0 · 35 W), very little recovery was observed, even 90 days afterwards.     

基质金属蛋白酶抑制剂GM6001对糖尿病大鼠血-视网膜屏障的影响

背景 研究表明,多种基质金属蛋白酶（MMPs）家族成员在糖尿病视网膜病发（DR）的发病过程中发挥作用,但是否MMPs抑制剂能改善MMPs对血-视网膜屏障（BRB）的破坏作用尚不十分清楚.目的 探讨人工合成MMPs抑制剂GM6001对糖尿病大鼠BRB的影响.方法 24只成年清洁级SD大鼠按随机数字表法随机分为对照组、糖尿病组和糖尿病＋GM6001组.采用链脲佐菌素诱导腹腔内注射法诱导大鼠糖尿病模型,对照组以相同的方法注射等体积枸橼酸盐缓冲液.造模成功后3d和14d,糖尿病＋GM6001组大鼠玻璃体腔内注射10μl（100tμmol/L）GM6001各1次,对照组和糖尿病组大鼠以相同的方法注射等体积生理盐水.注射后1个月摘取大鼠一侧眼球,采用逆转录PCR（RT-PCR）法检测大鼠视网膜中MMP-2 mRNA和MMP-9 mRNA的相对表达量;用伊文思蓝（EB）灌注大鼠右侧颈静脉,120 min后以约120 mmHg（1 mmHg=0.133 kPa）的灌注压于大鼠左心室灌注枸橼酸钠缓冲液配制的质量分数1％多聚甲醛溶液,2 min后摘取大鼠另一侧眼球,观察大鼠视网膜EB的渗漏量.结果 RT-PCR检测显示MMP-2、MMP-9和GAPDH的反应条带分别位于436、536和484 bp.3个组大鼠视网膜中MMP-2 mRNA和MMP-9 mRNA相对表达量总体差异均有统计学意义（F=20.336,P=0.000;F=8.742,P=0.002）;其中糖尿病组和糖尿病＋GM6001组大鼠视网膜中MMP-2 mRNA和MMP-9 mRNA的相对表达量明显高于对照组大鼠,差异均有统计学意义（P＜0.01）,而糖尿病＋GM6001组大鼠视网膜中MMP-2 mRNA和MMP-9 mRNA的相对表达量明显低于糖尿病组大鼠,差异均有统计学意义（P=0.01、0.02）.对照组、糖尿病组和糖尿病＋GM6001组大鼠视网膜中标准化EB质量分数分别为（12.60±3.50）、（26.52±7.14）和（17.55±2.65） ng/mg,总体差异有统计学意义（F=17.032,P＜0.01）,其中糖尿病组大鼠视网膜中标准化EB质量分数值明显高于对照组,而糖尿病＋GM6001组较糖?     

两种糖尿病大鼠模型血-视网膜屏障难EB通透性的比较

目的探讨2种糖尿病大鼠模型血-视网膜屏障对伊凡思蓝（Evans-Blue，EB）通透性的改变。方法对照组及1周、4周和16周时Sprague-Dawley（SD）和Brown-Norway（BN）糖尿病大鼠，EB45mg·kg^-1颈静脉注入，分别检测视网膜中EB含量。结果对照组及1周、4周和16周时SD和BN糖尿病大鼠视网膜标准化EB含量分别为（12．24±3．11）ng·mg^-1、（21．68±4．41）ng·mg^-1、（21．78±3．17）ng·mg^-1、（23．56±4．05）ng·mg^-1和（13．22±3．59）ng·mg^-1、（23．41±4．47）ng·mg^-1、（26．40±5．00）ng·mg^-1、（31．01±4．46）ng·mg^-1，糖尿病大鼠视网膜标准化EB含量明显高于对照组，其差异均有显著性（P〈0．05）。2种不同种系大鼠相比，BN糖尿病大鼠1周、4周和16周时视网膜标准化EB含量均显著高于SD大鼠（P〈0．05），而对照组差异无显著性（P〉0．05）。结论通过检测视网膜内EB含量，可以比较精确的发现早期糖尿病大鼠视网膜病变中血-视网膜通透性的改变。不同大鼠种系的差异性决定了其对STZ诱导的糖尿病大鼠视网膜通透性改变的不同。     

Effect of morphological abnormalities on blood retinal barrier permeability in diabetic retinopathy

The morphological base for the impaired function of the blood retinal barrier was studied in 50 eyes of 10 insulin dependent and 21 non-insulin dependent patients with various levels of diabetic retinopathy. The permeability of the blood retinal barrier (P_BRB) was determined by vitreous fluorophotometry with correction for autofluorescence, lenstransmission and non-protein bound plasma fluorescein concentration. Morphological abnormalities of diabetic retinopathy assessed by fundus photography and fluorescein angiography were individually scored on a decimal scale and related to the P_BRB by multiple regression analysis. The P_brb was not correlated to morphological abnormalities of non-proliferative retinopathy [(1) microaneurysms, (2) hard exudates, (3) soft exudates, (4) intraretinal hemorrhages, (5) fluorescein leakage, and (6) capillary closure, p > 0.3]. The P_BRB was correlated to morphological abnormalities of (pre)proliferative retinopathy [(1) intraretinal microvascular abnormalities (S_irma) and (2) new vessels (S_neo): p_brb = A – B.S_IRMA – C.S_neo with P_BRB in nm/sec, A = 1.5 ± 0.5, B = 0.9 ± 0.2 and C = 1.7 ± 0.4, R² = 0.65, p < 0.0001]. It can be concluded that the increased blood retinal barrier permeability in diabetic patients is mainly due to (pre)proliferative abnormalities and not to non-proliferative abnormalities.     

Clinical and metabolic factors associated with the blood retinal barrier permeability in insulin dependent diabetes mellitus without retinopathy

The authors assessed a study pointing out the relationship between the permeability of Blood Retinal Barrier (BRB), using vitreous fluorophotometry, and several risk clinical and laboratory data in Insulin Dependent Diabetes Mellitus (IDDM). Eighty eyes of 40 IDDM patients were evaluated. Their mean age was 14.8 ± 3.2 years. Twenty healthy volunteers served as control group (mean age 15.3 ± 3.2 years). Preexisting diabetic retinopathy was dismissed after funduscopy and fluorescein angiography. The studied risk factors were: age, duration of diabetes, HLA antigens, blood pressure, cholesterol and triglycerides levels, glycosylated hemoglobin, insulin dose/kg body weight and fructosamine. Vitreous and lens fluorophotometry was performed in both groups (VPRt). The mean VPRt in IDDM patients was 3.56 ± 1.47 × 10–6 min^–1. In healthy subjects it was 2.53 ± 0.55 × 10–6 min^–1, establishing a significant difference (p < 0.01). We found a statistically significant correlation between VPRt and duration of diabetes, insulin dose/kg body weight and HbAlc. No correlation was found between VPRt and the rest of parameters. We conclude that vitreous fluorophotometry is a valid method to measure BRB; and the three factors mentioned above are related to the BRB permeability.Abbreviations BRB blood retinal barrier - DR diabetic retinopathy - VF vitreous fluorophotometry     

伊凡思蓝在测定血视网膜屏障 破坏中的作用

目的探讨能否应用伊凡思蓝(Evans blue, EB)作为示踪物定量诊断血视网膜屏障破坏(blood retinal barrier breakdown, BRBB)。方法 实验动物选用雄性Sprague-Dawley大鼠。首先应用EB测定由血管内皮生长因子(vascular endothelial growth factor, VEGF)引起的视网膜血管渗漏。然后用EB测 量1周糖尿病导致的BRBB。以甲酰胺提取视网膜中的EB,用分光光度计测量提取液中的EB浓度。最后根据视网膜重量标准化视网膜内的EB含量。结果VEGF 处理眼视网膜内的EB含量显著高于未处理眼视网膜内的EB含量(P<0.0001)。早期糖尿病大鼠视网膜内的EB含量亦显著高于正常对照组视网膜内的EB含量(P<0.05)。结论EB是一种可以定量测定BRBB的敏感示踪物。(中华眼底病杂志,2001,17:221-223)     

Effect of cyclospasmol on early diabetic retinopathy

A randomized, double-blind, placebo controlled study to investigate the long-term effect of Cyclospasmol^R (cyclandelate) on the abnormal permeability of the blood-retinal barrier was performed in 26 patients with insulin-dependent diabetes mellitus for at least 1 year and minimal retinopathy. Cyclospasmol 400 mg or placebo capsules were taken 4 times daily for 12 months by equal numbers in both groups.Each patient underwent a routine ophthalmoscopic examination, retinal fluorescein angiography and quantitative vitreous fluorophotometry to assess the permeability of the blood-retinal barrier just before the trial and following 6 and 12 months of therapy. Laboratory tests for determining blood and urine glucose levels and blood HbA₁-levels were also carried out at these assessments.Statistically significant changes in diabetic control, in HbA₁-levels or in the frequency of retinal microaneurysms could not be shown in either treatment group during the trial, nor were there any significant differences in these parameters between the two groups. Analysis of fluorophotometric data on fluorescein penetration into the left posterior vitreous demonstrated significant reductions in this parameter during the trial compared to the pretreatment level in Cyclospasmol treated diabetics. These changes in the pretreatment level after 6 and 12 months also differed significantly between the two groups.However, this statistically significant beneficial reduction in fluorescein penetration into the left posterior vitreous did not occur in the right eye in the Cyclospasmol group.In placebo treated patients a consistently deleterious trend for this parameter was observed for both eyes during the one year study. The consistently beneficial trend in the right eye following Cyclospasmol and the consistently deleterious trend in both eyes following placebo with regard to the abnormal permeability of the blood-retinal barrier of diabetics with minimal retinopathy could probably be explained by the small number of patients in both groups. No side-effects were reported.     

巴曲酶对糖尿病大鼠血视网膜屏障和视网膜血管内皮生长因子表达的影响

目的 研究巴曲酶对糖尿病大鼠血视网膜屏障以及视网膜血管内皮生长因子（VEGF）表达的影响。 方法 将60只大鼠用链尿佐菌素腹腔注射制成糖尿病大鼠模型，分成糖尿病组（n＝20）、40mg/kg巴曲酶注射组（n＝20）和20 mg/kg巴曲酶注射组（n＝20）。另25只正常大鼠为正常对照组。7d 后处死全部大鼠，通过伊凡思蓝法观察各组大鼠血视网膜屏障情况，酶连免疫吸附法分析视网膜总蛋白中的VEGF含量，比较各组结果。 结果 正常对照组大鼠视网膜内渗漏的伊凡思蓝含量明显低于另外3个糖尿病大鼠组（P<0.01）,不同剂量巴曲酶治疗组之间伊凡思蓝含量无明显差异（P>0.05），巴曲酶治疗的2组大鼠伊凡思蓝含量均比糖尿病组大鼠低（P<0.05）。正常对照组大鼠、不同剂量巴曲酶注射的2组大鼠视网膜内VEGF含量明显低于糖尿病大鼠（P<0.01）；正常对照组视网膜内VEGF含量与20 mg/kg巴曲酶注射组比较无明显差异（P＝0.06）；40mg/kg 巴曲酶注射组视网膜内VEGF含量比正常对照组低(P＝0.01)；不同剂量的巴曲酶治疗组之间VEGF含量无明显差异（P＝0.78）。 结论 巴曲酶治疗减轻了糖尿病大鼠血视网膜屏障功能的损伤，降低了VEGF的表达，提示巴曲酶对糖尿病大鼠血视网膜屏障功能有一定的保护作用。 (中华眼底病杂志, 2006, 22: 16-19)     

The effect of flurbiprofen 0.03% eye drops on the blood aqueous barrier in extracapsular cataract extraction with IOL implantation

Preoperative and postoperative anterior chamber fluorophotometry were performed after intravenous administration of fluorescein sodium in patients undergoing extracapsular extraction and posterior chamber lens implantation. The topical application of aqueous flurbiprofen sodium 0.03% solution before and after surgery significantly decreased the surgery mediated disturbance of the blood aqueous barrier as compared to vehicle application (placebo controlled). In contrast to similar studies, cortical steroids were not given topically or systemically to either group of patients during the study. The present fluorophotometric results correlate well with slitlamp biomicroscopy of postoperative inflammation. The data of this study indicate that flurbiprofen ophthalmic solution is effective in protecting the blood aqueous barrier (BAB) in human eyes during cataract surgery and IOL implantation.