Microscopic quantification of fluorescein distribution in retinas of normal and diabetic rats

To identify more precisely the site and the nature of the abnormality of the Blood-Retinal Barrier (B.R.B.) in diabetes, quantitative fluorescence microscopy was used to measure time-dependent changes of fluorescence in ocular tissues of normal and diabetic rats, after intravenous fluorescein injection. Fluorescein distribution across the retinal layers was studied in control and streptozotocin diabetic rats at 5, 30 and 60 minutes after dye injection. Fluorescein intensities of choriocapillaris and retina were compared with plasma fluorescein levels. The results show a B.R.B. dysfunction in diabetic rats arising from abnormal leakage of fluorescein into the retina. After 60 minutes there was a greater fluorescence intensity localized to the inner retinal layers, consistent with a probable inhibition or saturation of active transport mechanisms for dye removal through the retinal vessels.     

Expression and distribution of tissue transglutaminase in normal and injured rat cornea

PURPOSE: To determine the expression and distribution of tissue transglutaminase (TG(C)) and extracellular matrix (ECM) proteins in rat cornea during epithelial wound healing. METHODS: Corneal epithelial defects were created in rat corneas, and TG(C) expression was examined by Northern blot analysis, in situ hybridization, and immunohistochemical staining after the injury. The presence of fibrinogen, laminin-1, nidogen/entactin, and type collagen was also determined immunohistochemically. RESULTS: TG(C) was expressed in normal corneas. During the early wound healing process, TG(C) mRNA expression was up-regulated and TG(C) immunoreactivity was predominantly expressed in the migrating epithelial cells. ECM proteins were also expressed in a similar pattern as TG(C). CONCLUSIONS: The sites and time course of TG(C) expression indicate that TG(C) probably plays a role in maintaining the homeostasis of the cornea and in promoting epithelial wound healing. The simultaneous expression of TG(C) and ECM proteins suggests that the ECM proteins probably operate in concert with TG(C) in corneal wound healing.     

Centripetal movement of fluorescein dextrans in the cornea: relevance to arcus

The centripetal movement of fluorescein and fluorescein-labelled dextrans (4 to 150 kD) from sclera or cut edge of the cornea was determined in isolated rabbit corneas at 4 and 24 h. Corneas were divided into 5.5 mm diameter central core, inner 5.5 to 8 mm donut, 8 to 12 mm peripheral donut and, where applicable, scleral rim. For all molecules greater than sodium fluorescein (376 D) tracer concentrations in the 5.5 mm core and the 5.5 to 8 mm donut were equal. Without sclera rim, the more central portions of the cornea (5.5 mm core and 5.5 to 8 mm donut) had tracer concentrations equal to those of corneas-with-sclera for all tracers greater than 10 kD. The tracer concentrations in the central cornea were the same in the presence or absence of sclera. The data indicate a physiological barrier to the lateral diffusion of molecules greater than 10 kD between the peripheral and more central cornea.     

Calibration of measurements in vivo of fluorescein in the cornea

Quenching of fluorescence of fluorescein is not observed with broad field fluorophotometers. Fluorophotometric equipment which measures the fluorescence in a tiny spot has, however, been reported to underestimate the molarity of fluorescein in the rabbit corneal stroma by as much as a factor of two. In this experiment, quenching was measured in the rabbit cornea with two scanning fluorophotometers. The quenching was measured by four different techniques: (1) by elution of fluorescein, (2) by elution of albumin, (3) by polarization of fluorescence, and (4) by spectrofluorophotometry. It was estimated by all four methods that quenching in the living rabbit cornea with these instruments is approximately 20%. Taken together, the four experiments suggest that the quenching of fluorescence of fluorescein can be explained entirely on the basis of the interaction of fluorescein and albumin in the stroma.     

The diffusion of fluorescein in the stroma of rabbit cornea

The diffusion rate and the distribution ratio of fluorescein in the stroma was studied in rabbit cornea. A strip of corneal stroma was mounted in a chamber and fluorescein in a phosphate buffer solution was circulated across the end of the strip for 24hr. The change in fluorescence intensity was measured along the strip and the diffusion coefficient was calculated using Fick's diffusion equation. The mean coefficient of diffusion was 1·21±0·24 (±s.d.) × 10^?6 cm²/sec at 19 °C. The ratio of fluorescence between the stroma and the solution was 1·34 to 1·33 for the concentrations ranging from 0·1 to 10 g/ml.     

Anterior segment fluorescein angiography in inflammatory diseases of the cornea.

To study the vascular changes in inflammatory diseases of the cornea 22 patients with various corneal inflammations were examined by means of anterior segment fluorescein angiography. Simple avascular central and marginal corneal ulcers stained with fluorescein in the late phase of angiography. An inflamed limbus and an early microscopic pannus adjacent to the ulcer were seeen in simple corneal ulcers. Progressive pannus with pronounced fluorescein leakage was observed in chronic corneal ulcer, disciform keratitis, Mooren's ulcer, and complicated acute keratoconus. In sclerokeratouveitis and in gutter associated with rheumatoid arthritis the corneal vessels showed less leakage. The iris vessels showed fluorescein leakage as a sign of irritative iritis during the active stage of simple and chronic corneal ulcers, in disciform keratitis, Mooren's ulcer, and in graft rejection. It is concluded that anterior segment fluorescein angiography gives valuable information of the vascular architecture, flow and leakage in inflammatory diseases of the cornea.     

Epithelial dendritic cell distribution in normal and inflamed human cornea: in vivo confocal microscopy study

PURPOSE: To evaluate dendritic cell (DCs) density, distribution, and morphology in central corneal and limbal epithelium in normal subjects and patients with immune-mediated corneal inflammation using in vivo confocal microscopy (IVCM). DESIGN: Comparative case-controlled, observational confocal microscopy study. METHODS: A total of 135 eyes of 135 patients were investigated. Group 1 (normal eyes) included 45 eyes of 45 healthy volunteers, group 2 photorefractive keratectomy (PRK-treated eyes) included 45 myopic eyes of 45 patients treated with PRK, and group 3 (inflamed eyes) comprised 45 eyes of 45 patients affected by immune-mediated corneal inflammation. The central cornea and limbus were examined for epithelial dendritic-shaped cells using laser scanning IVCM. DCs density was calculated using image analysis software. RESULTS: Cells with a branching dendritic morphology were visualized in the basal epithelial layer, basal lamina, and subbasal nerve plexus, in the central cornea, and in the basal layer and basal membrane of the limbal epithelium. The limbal epithelium demonstrated DCs in 93.3%, 89%, and 97.7% of eyes in group 1, 2, and 3, respectively (P = ns). Central epithelial DCs were observed in 20.0% and 13.3% of eyes in group 1 and 2 (P = ns), while in 93.3% of eyes in group 3 (P < .001). DCs were found to be significantly higher at the limbus compared with central cornea in each group (P < .001). Cell densities observed in group 3 were significantly greater than groups 1 and 2, at both locations (P < .05). CONCLUSIONS: Laser scanning IVCM is a useful method for evaluating epithelial DCs distribution at the limbus and central cornea in both healthy and diseased eyes.     

The distribution of gentamicin in the rabbit cornea following iontophoresis to the central cornea.

The purpose of this study was to evaluate the penetration of gentamicin into the central, midperipheral and peripheral cornea of rabbits following iontophoresis to the central 3 mm of the cornea. Four groups (groups 1-4) of five rabbits (one eye per rabbit) underwent corneal iontophoresis using gentamicin dissolved in agar. Low (1 mg/ml) and high (10 mg/ml) concentrations of gentamicin in agar were used for one or ten minutes. Two control groups (groups 5 and 6) of five eyes each underwent mock iontophoresis with low and high concentrations of agar-gentamicin mixture. Following sacrifice of the rabbits, the central, midperipheral and peripheral parts of each cornea were excised. Gentamicin concentration was determined in each part of every cornea. High concentrations of gentamicin (951.6 +/- 369.4 microg/ml to 26.6 +/- 41.34 microg/ml) were obtained in the central parts of all the iontophoresis-treated corneas. In each group, except group 6, central corneas had higher concentrations of gentamicin compared to midperipheral corneas (p = 0.038 to p = 0.021), and midperipheral corneas had higher levels than peripheral corneas (p = 0.038 to p = 0.021). Following iontophoresis, gentamicin is found in all portions of the corneas; however, the highest concentration of the drug remains in the central cornea.     

Aqueous outflow pathway imaging in the rabbit with fluorescein and indocyanine green

PURPOSE: The purpose of this study was to visualise the rabbit aqueous outflow pathway using a numeric imaging system with ICG and fluorescein injection in the anterior chamber. MATERIAL AND METHODS: We performed a simultaneous injection of Indocyanin Green (ICG) and Fluorescein into the anterior chamber of rabbit eyes. Using a digital camera, we took several sequenced pictures to visualize the distribution of the dyes within the outflow pathway. We observed the dynamics of the outflow over time. RESULTS: In the early phases, the shape of the outflow canal around the limbus was clearly seen. Several collecting veins close to the recti muscles were also identified. There was only slight fluorescein leakage during the early phases, allowing adequate visualization of the morphology of the outflow system. In the late phases, the sclera was stained with the fluorescein, and no details were thus visible. The ICG dye allowed better recognition of the fine details of the outflow structure. CONCLUSION: This method was relatively simple, safe and precise, and allowed us to visualise the details of the outflow pathways in the rabbit eyes. These results could be of great value in further evaluating the outcome of filtering surgeries in animal models.     

Modeling glucose distribution in the cornea

The central cornea obtains its glucose by diffusion through the cornea from the aqueous humor to the epithelium. The diffusion of glucose in the cornea is analogous to the flow of current in an electrical resistance network. The cellular consumption of glucose can be compared to shunting a portion of the charge to electrical ground. An electrical analog model of the cornea was developed to predict the availability of glucose to the epithelium and the distribution of glucose in the stroma. The glucose constant concentration lines in the normal stroma are parallel to the corneal surface and have decreasing values from 880 to 580 micrograms/ml. The effects on epithelial glucose concentration by implanting an intracorneal lens (ICL) of varying diameter, depth, permeability and thickness can be modeled. Glucose permeability through the intracorneal lens has the most significant effect on glucose availability. The ICL profile i.e. power, can also be an important fact in determining glucose availability. A minus power design requires a thin central lens zone with a thick peripheral zone. The design results in relatively more glucose flux through the optical zone of the lens and thus improves central epithelial glucose availability.     

正常眼角膜Q值检测分析

目的:研究正常眼角膜Q值及其分布特点。方法:采用Allegrettowavetopolayzer角膜地形图仪检测20例39眼正常眼角膜地形图,计算角膜Q值,并作统计学分析。结果:正常眼39眼角膜平均Q值为-0.28±0.09;水平方向Q值为-0.29±0.09,垂直方向为-0.28±0.12,水平和垂直方向Q值差异无显著性意义(t=-0.482,P=0.631);右眼Q值为-0.28±0.08,左眼为-0.27±0.02,双眼Q值差异无显著性意义(t=-0.512,P=0.612)。结论:正常眼角膜多呈中央屈力高而周边屈力低的长椭圆体,其正常眼角膜Q值基本呈正态分布。     

The blood-retinal barrier permeability to fluorescein in normal subjects and in juvenile diabetics without retinopathy

The blood-retinal barrier permeability to fluorescein was determined in 20 eyes from 17 normal volunteers (mean age 31 years) and in 20 eyes from 19 juvenile diabetics without apparent retinopathy (mean age 35 years - mean duration of diabetes 6 years). The permeability was in normal subjects (1.1 +/- 0.4) X 10(-7) cm/sec (mean +/- 2 X SD) and in juvenile diabetics (1.1 +/- 0.7) X 10(-7) cm/sec (mean +/- 2 X SD). Thus a break-down of the blood-retinal barrier cannot be demonstrated as a very early and general phenomenon in the early course of the diabetic disease. The fluorescein diffusion coefficient in the vitreous body was determined and juvenile diabetics without apparent retinopathy showed a diffusion coefficient of (0.80 +/- 0.25) X 10(-5) cm2/sec (mean +/- 2 X SD), which was the same as in normals where the diffusion coefficient was (0.69 +/- 0.46) X 10(-5) cm2/sec (mean +/- 2 X SD).     

Lymphocytes and Langerhans cells in the normal human cornea

The distribution of B- and T-lymphocytes, of OKIa positive cells, and of HLA-ABC antigens in the normal cornea was investigated using monoclonal antibodies. The lymphocytes and Langerhans cells are present mainly in the well-vascularized limbic region but also occur albeit in small number in the center of the cornea. HLA-ABC antigens are strongly expressed on the epithelial cells of the cornea and the limbus.     

Movement of fluorescein monoglucuronide in the rabbit cornea. Diffusion in the stroma and endothelial permeability

The movement of fluorescein monoglucuronide, a fluorescent metabolite of fluorescein, was studied in the rabbit cornea in vitro and in vivo. A stromal strip was exposed to fluorescein monoglucuronide, and the diffusion rate and the distribution in the stroma were measured every hr for 24 hr. The diffusion coefficient was 0.94 +/- 0.11 (+/- S.D.) X 10(-6) cm2/sec, and the saline/stroma distribution ratio was in a range of 0.67 to 0.69. The concentration of fluorescein monoglucuronide in the anterior chamber and the cornea was measured every hr for 8 hr following intravenous administration. The endothelial permeability was 4.7 +/- 1.0 X 10(-4) cm/min, and the aqueous/cornea distribution ratio was 0.56 +/- 0.05. It appears that the corneal endothelial permeability in the living eye determined hitherto from systemic administration of fluorescein is most likely the permeability to fluorescein monoglucuronide.     

正常角膜伴近视散光眼角膜各形态参数分布特征及与全眼屈光参数的关系

目的 探讨正常角膜伴近视散光眼角膜各形态参数分布特征及与全眼屈光参数的关系.方法 收集102例(204眼)近视患者,分别应用非接触眼压计、检影仪、A超、角膜超声测厚仪、Pentacam三维眼前节分析仪进行眼压、屈光度、眼轴长度、角膜厚度、角膜前后表面形态参数等测量,应用SPSS 13.0统计学软件进行数据分析.结果 正常角膜伴近视散光眼角膜中央厚度(537.06±25.45)μm,角膜中央前表面高度(2.35±1.39)μm,中央后表面高度(3.60±2.73) μm,角膜Q值(8 mm范围)-0.30±0.10,角膜最薄点距角膜顶点距离(0.59±0.19)mm,角膜最薄点位置多位于角膜顶点颞下方,占91.67％.角膜最薄点至周边厚度增长变化指数为1.04±0.13.角膜中央前后表面高度、角膜Q值、角膜最薄点至周边厚度增长变化指数与等效球镜、眼压、眼轴长度无明显相关性(均为P＞0.05).结论 正常角膜伴近视散光眼角膜形态各项参数分布值各有特点,与全眼屈光参数无明显相关性.     

The permeability of the corneal endothelium to fluorescein in the normal human eye

Endothelial permeability to fluorescein was measured as part of fluorophotometric studies of aqueous humor flow in 112 normal subjects whose ages span six decades. The permeability was 2.4 +/- 0.2 X 10(-4) cm/min (mean +/- SD). Females had a slightly higher permeability than males. No correlation between age and permeability was noted. The precision of this method of measurement was estimated to be +/- 22%.