Detection of destruction of anionic sites in the outer blood-retinal barrier and damage caused by iron]

We studied the alteration of the anionic charge in the outer blood-retinal barrier in ocular siderosis by electron microscopy using the cationic probe, polyethyleneimine (PEI). The eyes of four days after injection of iron powder and control rabbit eyes demonstrated numerous PEI-positive sites at the basement membrane of the retinal pigment epithelium, collagen fiber of Bruch's membrane, and the basement membrane of the choriocapillaris. Seven days after injection of iron powder, no change was observed in the retina by light microscopy, but PEI positive sites at the basement membrane of the retinal pigment epithelium were decreased. At 14 days, it was seen that the outer layer of the retina disappeared and was replaced by pigment containing macrophages and Müller cells. Berlin blue reaction became positive in some of those macrophages and retinal pigment epithelia. PEI positive sites decreased. At 28 days, the retina was completely disorganized, and became thin, while the retinal pigment epithelium proliferated and became double layered. Berlin blue reaction was strongly positive in pigment-laden macrophages and retinal pigment epithelium. PEI-positive sites became scarce. As a result of these methods, anionic sites decreased, despite negative iron reaction of 7 days after injection of iron powder. It was shown that in ocular siderosis at an early stage, the barrier function of anionic sites between choroid and retina decreased before histological change.     

Early effects of sodium iodate shown by detection of the destruction of anionic sites in outer blood-retinal barrier

In order to demonstrate the early effect of sodium iodate (NaIO3) on retinal pigment epithelium (RPE), Bruch's membrane and choriocapillaries, we examined the alteration of their anionic sites using a cationic probe, polyethyleneimine (PEI). We injected 10 microliters of 3% NaIO3 solution into the vitreous of pigmented rabbits and after thirty minutes or one hour, administered 0.5% PEI intravenously. In the eyes injected with intravitreously saline solution or without injection, PEI-positive particles (diameter 15-20 nm) were located in a row on both sides of the basement membrane of the RPE and choriocapillaries and periodically at the collagen fiber of inner and outer collagenous zone of Bruch's membrane. However no particle was observed on the cell membrane of the basal infolding of the RPE. Thirty minutes after injection of NaIO3, PEI particle positive sites were reduced at the basement membrane of the RPE. One hour after injection they reduced in number but remained at the basement membrane of choriocapillaris and collagens in Bruch's membrane. We showed the early effect of NaIO3 to the RPE by examining the alterations of the anionic sites.     

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.     

Effect of argon laser photocoagulation on the permeability of the blood-retinal barrier

The breakdown and repair of the blood-retinal barrier following argon laser photocoagulation were studied in pigmented rabbits by kinetic vitreous fluorophotometry using carboxyfluorescein as a tracer. The outward carboxyfluorescein transport across the blood-retinal barrier was significantly reduced in the photocoagulated eyes (p less than 0.01). This transport activity was normalized in 14 days. Ten rabbits were intraperitoneally pretreated with 0.1 M/kg probenecid, a competitive inhibitor, which was likely to decrease active transport across the outer blood-retinal barrier. In probenecid-treated animals, argon laser photocoagulation did not affect the permeability of the blood-retinal barrier. The present study suggests that some beneficial effects of photocoagulation may be attributed to replacement of diseased retinal pigment epithelium by rapid growth of new pigment epithelial cells and reestablishment of active transport across the restored barrier.     

Disruption of blood-retinal barrier at the retinal pigment epithelium after systemic urea injection.

Electron microscopic studies were done on the structural alterations of retinal pigment epithelial cells occurring under osmotic stress utilizing rhesus monkeys and lanthanum nitrate as a tracer. Fluorescein fundus angiography revealed leaking points of fluorescein from the choroid to the retina, and various degenerative alterations of the retinal pigment epithelial cells were observed in these leaking areas. Some cells included many vacuoles in their cytoplasm, but diffusion of lanthanum from the choroid to the outer segment of visual cell was obstructed by the tight junctions between the retinal pigment epithelial cells. Other cells were severely damaged with ruptured membranes. Abundant tracer material diffused through the cytoplasm into the subretinal space. Other cells were broken down in an isolated manner and here marked extravasation of lanthanum into the subretinal space was observed.     

Breakdown of the blood-retinal barrier in a model of retinal neovascularization

Breakdown in the blood-retinal barrier occurs in retinal neovascularization in a number of diseases. To study the anatomic basis of this breakdown, we examined retinal neovascularization induced by injection of 250,000 homologous fibroblasts into the vitreous cavity of pigmented rabbits. Neovascularization is evident by electron microscopy in this model 3 days after fibroblast injection. Fluorescein angiography followed by intravenous horseradish peroxidase (HRP) injection was performed prior to enucleation on 2, 3, 5, 7, and 14 days after fibroblast injection. Fluorescein leakage from retinal vessels occurs early (at day 1) and persists as the neovascularization progresses. The leakage in the early stages is concentrated near puckers from the medullary wings. In the later stages, fluorescein leakage is most prominent in the developing tips of the new vessels. Horseradish peroxidase was not observed to leak from the lumen of new vessels. "Gaps" or separations in the endothelial cell junctions were not observed in developing vessels. The breakdown of the blood-retinal barrier in this model of retinal neovascularization is therefore selective, (ie, fluorescein leaks but not HRP) and it is not due to gaps or fenestrations between endothelial cells in developing vessels.     

次声作用对大鼠血视网膜屏障超微结构的损害

目的 观察次声作用对大鼠血视网膜屏障（blood-retinal barrier，BRB）超微结构和通透性的影响。 方法 20只大鼠按暴露时间随机分为5组，每组4只。暴露于16 Hz，130 dB的次声压力仓中，2 h/d，分别暴露0(对照组)、1、7、14、 21 d后摘除眼球，硝酸镧灌注固定法制备电镜样品,超薄切片,电镜观察。 结果 随次声暴露时间 的延长，BRB的损害加重，线粒体肿胀，内质网扩张，膜盘破坏，视网膜色素上皮(retinal pigment epithelium, RPE )细胞变形坏死，核膜扩张等都随暴露时间的延长而更加明显。镧在视网膜各层都有分布，随着暴露时间的延长，镧在各层的渗漏越来越多。 结论 次声可造成一定程度的 BRB通透性损害；随着 次声暴露时间的延长，损害更严重。 （中华眼底病杂志，2003，19：46-48）     

Localised blood-retinal barrier leakage and retinal light sensitivity in diabetic retinopathy.

Twenty patients with insulin dependent diabetes mellitus were selected on the basis of morphological signs of blood-retinal barrier leakage--namely, hard exudates seen on fundus photographs and/or localised leakage of fluorescein seen on fluorescein angiograms. Computerised perimetry was carried out in visual field areas that corresponded to the morphological lesions, and the visual field data were accurately correlated with the morphology as seen on fundus photographs and fluorescein angiograms. In addition, in seven of the patients who represented the range of leakage among the patients studied, the blood-retinal barrier leakage was quantitated by vitreous fluorophotometry. In 16 cases normal light sensitivity was found in retinal areas showing localised leakage as studied on fluorescein angiograms. In four cases with pronounced maculopathy, where scotomata occurred, there was no topographical correlation between the scotomata and barrier leakage. Furthermore hard exudates often, but not consistently, caused localised scotomata when arranged in dense conglomerates. The permeability values correlated with angiographically observed hyperfluorescence in the macular area. On the basis of the techniques employed in the present study it seems that breakdown of the blood-retinal barrier is an earlier event than disturbance of neurosensory function in the development of diabetic retinopathy. However, the findings give no evidence of a causal relationship between barrier leakage and damage to sensory cell function.     

Permeability of the blood-retinal barrier to carboxyfluorescein in eyes with rhegmatogenous retinal detachment

Outward and inward permeability of carboxyfluorescein across the blood-retinal barrier were measured fluorophotometrically in seven cynomolgus monkey eyes with experimental rhegmatogenous retinal detachment. Probenecid was used to inhibit outward transport of carboxyfluorescein. The outward permeability was 1.98 +/- 0.31 microliter/min in eyes with retinal detachment and 0.84 +/- 0.15 microliter/min in control eyes with vitrectomy alone (P less than 0.01). The inward permeability, determined separately following intravenous injection, was significantly lower than the outward permeability: 0.14 +/- 0.02 microliter/min for eyes with retinal detachment and 0.04 +/- 0.01 microliter/min for control eyes. Since the outward permeability minus the inward permeability in the presence of probenecid represents that fraction of tracer moving due to fluid flow, it may be concluded that outward flow of fluid across the blood-retinal barrier is a substantial contributor to carboxyfluorescein loss from the vitreous cavity following intravitreal injection.     

Diabetic macular oedema: the effect of photocoagulation on fluorescein transport across the blood-retinal barrier

BACKGROUND/AIM: The visual loss secondary to diabetic macular oedema can be controlled to some extent by photocoagulation, though the mechanism of action is largely unknown. The purpose of the present study was to quantitate the effect of photocoagulation on the blood-retinal barrier using fluorescein as a tracer of passive and active transport. METHODS: A prospective study of 46 eyes in 34 patients with clinically significant macular oedema (CSMO) examined by vitreous fluorometry before and 6 months after macular photocoagulation treatment. RESULTS: In 23 eyes CSMO was not present at follow up (responding eyes), in another 23 other eyes CSMO was still present (non-responding eyes). With reference to the presence or absence of CSMO at follow up, the passive transport (permeability) for responding eyes decreased after photocoagulation in contrast with an increase in non-responding eyes; the difference between the groups at follow up was significant (p=0.03). The active transport for responding eyes decreased slightly at follow up, while it increased for non-responding eyes; the difference between the groups at follow up was not significant (p=0.09). CONCLUSION: Following photocoagulation a reduction of diabetic macular oedema, defined as disappearance of CSMO, is paralleled by a decrease of the passive permeability while the hypothesis of an increase in the active transport from the retina to the blood could not be supported by this study.     

糖尿病视网膜病变光凝术后角膜知觉的改变

目的 探讨糖尿病视网膜病变患者在全视网膜光凝术后的角膜知觉改变及影响因素。方法 32例(32只眼)糖尿病视网膜病变患者应用倍频ND：YAG激光行全视网膜光凝，光凝斑大小：200～300／μm，光凝时间：0．2～0．4秒，光凝斑强度以达到中等强为标准。采用Cochet--Bonnnnet角膜知觉测量仪测量光凝前及光凝后1、2、3、4、5、6周及2个月的角膜知觉。结果 糖尿病视网膜病变患者术前的角膜知觉为2．27cm，术后1、2、3、4、5、6周及2个月的角膜知觉分别为1．42cm、1．60cm、1．58cm、1．92cm、1．71cm、2．19cm和2．23cm。与术前相比，术后1～5周的角膜知觉减退值与术前的角膜知觉的相关系数为0．82。结论 糖尿病视网膜病变行全视网膜光凝后的角膜知觉有明显减退，在光凝后6周恢复至术前水平，角膜知觉的减退值与光凝的范围及术前的角膜知觉有关。     

The blood-retinal barrier in chloroquine retinopathy

Chloroquine retinopathy can result in devastating loss of vision. To date, there are no effective and reliable methods of detecting the toxicity at an early stage when retinopathy may be reversible. Chloroquine is deposited in the retinal pigment epithelium, which forms part of the blood-retinal barrier (BRB). By vitreous fluorophotometry we have shown that there is breakdown of the BRB in chloroquine retinopathy. However, in asymptomatic patients who had received varying amounts of hydroxychloroquine (up to 1067 g), the BRB remained intact.     

The blood-retinal barrier in Berlin's edema

The pathogenesis of Berlin's edema is uncertain; but one possible factor is breakdown of the blood-retinal barrier. The authors evaluated 10 affected patients with the use of vitreous fluorophotometry and fluorescein angiography. The aqueous humor dye concentration was elevated in the affected eye, compared with the unaffected eye. The means and standard deviations of the posterior vitreous penetration ratios of the involved and uninvolved eyes were 1.36 +/- 0.66 and 1.45 +/- 0.67 X 10(-6) min-1, respectively; there was no statistically significant difference. The angiograms showed no leakage in either eye. It seems unlikely that disruption of the blood-retinal barrier could be a major pathophysiologic factor in this condition without leading to abnormalities on either test. The authors recommend using the term "commotio retinae" until there is more conclusive evidence that the blood-retinal barrier is abnormal.     

The inner blood-retinal barrier in diabetes

The inner blood-retinal barrier is highly complex, dependent on a normally functioning retinal vascular endothelium and possibly adjacent perivascular cells. There are undoubtedly many different grades of barrier failure, and in the early stages an inbuilt compensatory mechanism may prevent significant fluid accumulation and delay associated structural and functional changes. In diabetes, early barrier failure may be a function of an increase in the rate of endothelial cell death and turnover, probably exaggerated by the early and widespread loss of the supporting cells and dilatation of the small blood vessels.     

Repair of outer blood-retinal barrier after severe ocular blunt trauma in rabbits

Retinal contusion is a leading cause of visual loss in ocular blunt trauma. However, its pathogenesis remains controversial. We established a rabbit model of severe retinal contusion with energy of about 2.87 J. Typical retinal edema and sometimes subretinal hemorrhage reproducibly occurred at the posterior pole after injury. These subsided 1 week later with depigmentation in the lesion. Histopathological examination revealed severe damage of the outer layer of retina, for example, disruption of retinal pigment epithelium (RPE) and photoreceptors. Electroretinography showed a decrease in the b wave by 38–47% in amplitudes (P<0.01) during the first 3 days and then returned, although not to normal level. To investigate the damage and repair of blood-retinal barrier (BRB), 5 ml 2% lanthanum solution (La) was injected via the common carotid artery 1–2 min before enucleation. La diffused in the interphotoreceptor space through the damaged junctions of RPE 1 h-3 days after injury. La also reached the nuclei level of photoreceptors up to 14 days after injury. Although a glial scar with scattered RPE cells attached to Bruch's membrane in the severely damage area, no La diffusion was found in the retina 4 weeks after trauma. These results showed incomplete repair of outer BRB after severe blunt trauma.     

Outward permeability of the blood-retinal barrier

To characterize quantitatively the active transport mechanism of the blood-retinal barrier (BRB), we estimated the inward (Pin) and outward permeability (P_out) of the BRB in monkey eyes using vitreous fluorophotometry. P_in values for fluorescein (F) and fluorescein glucuronide (FG) were simulated by computer in separate experiments following measurements of intraocular fluorescence at 1 h following the intravenous injection of F or FG. The estimated mean P_in values for F and FG were 4.7 ± 1.6 and 5.9 ± 1.7 × 10^–6 cm/min, respectively, in ten eyes of five monkeys. At 1 month thereafter, F was injected into the right vitreous cavity and the same amount of FG was injected into the left vitreous cavity. The intraocular kinetics of fluorescence were measured at 6–24 h postinjection. As estimated by the simulation model, the mean P_out values were 7.7 ± 2.6 and 1.7 ± 0.9 × 10^–4 cm/min for F and FG, respectively. The P_out/P_in ratio was 160±78 for F and 26±9 for FG. The intraperitoneal injection of probenecid resulted in a significant decrease in the P_out value for F (P < 0.005) but had no significant effect on that for FG, suggesting that F is excreted from the retina via an active transport mechanism; as characterized by the P_out/P_in ratio, the actual magnitude of the latter is far greater than that previously reported.This research was supported in part by a 1988 Grant-in-Aid for the Encouragement of Young Scientists (63771366, to A.Y.) from the Ministry of Education, Science and Culture, Japan Offprint requests to: A. Yoshida