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).     

Estimation of the permeability of the blood-retinal barrier in normal individuals

The fluorescein kinetics in the vitreous was simulated with a computer to consider several factors such as permeability of the blood-retinal barrier, outward active transport, plasma fluorescein dynamics, diffusion of fluorescein in the vitreous, and fluorescein leakage from the blood-aqueous barrier. Kinetic vitreous fluorophotometry was performed in normal individuals to estimate the inward and outward permeability of the blood-retinal barrier based on the theory of the simulation model. The results of the simulation studies suggest that the fluorescein concentration in the posterior vitreous after intravenous administration is dependent mainly on the inward permeability and on the plasma concentration and that the outward permeability has little influence on the fluorescein kinetics at the early phase. In the pharmacokinetic analysis of the results of kinetic vitreous fluorophotometry, we obtained average values of 1.8 X 10(-5) cm/min and 5.6 X 10(-4) cm/min for the inward permeability and outward permeability coefficients, respectively. The diffusion coefficient of fluorescein in the vitreous was estimated at 7.9 X 10(-4) cm2/min on the average. The outward permeability of the blood-retinal barrier is approximately 31 times the inward permeability. This suggests that a facilitated process that transports fluorescein outward from the vitreous cavity exists in the blood-retinal barrier of human eyes.     

Measurement of blood-retinal barrier permeability: a reproducibility study in normal eyes

Fluorescein penetration into the posterior vitreous depends on plasma-free fluorescein concentration and blood-retinal barrier (BRB) permeability. The reproducibility of two methods of deriving BRB permeability was studied in 19 normal eyes of 14 subjects using vitreous fluorophotometry on two separate occasions. Plasma-free fluorescence was measured at intervals over 1 hr and posterior vitreous fluorescence was measured before (background scan), within 6 min (bolus) and at 60 min (measurement) after intravenous fluorescein (14 mg X kg-1). A computer algorithm subtracted background fluorescence from the measurement scan which was then corrected for signal spread by using a "spread" function derived from the bolus scan. BRB permeability coefficient and vitreous diffusion coefficients were derived by fitting a mathematical model to the plasma and corrected vitreous fluorescence data. A permeability index was also calculated by dividing the area under the vitreous fluorescence by the area under the plasma fluorescence curve. There were no significant differences in the results between right and left eyes. Mean +/- SD values on first and second occasions for all eyes were permeability coefficient: (1.91 +/- 0.94) and (2.08 +/- 0.95) X 10(-7) cm X s-1; diffusion coefficient: (1.33 +/- 0.68) and (1.19 +/- 0.54) X 10(-5) cm2 X s-1; and permeability index: (2.05 +/- 1.03) and (2.11 +/- 1.02) X 10(-7) cm X s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

The blood-retinal barrier permeability in essential hypertension

In a series of 8 patients with recently discovered moderate essential hypertension the blood-retinal barrier permeability to fluorescein was determined by aid of quantitative vitreous fluorophotometry before and after normalization of the systemic blood pressure. The permeability before medical treatment was 1.69 X 10(-7) cm/sec (+/- SEM = 0.14) and after medical treatment and normalization of the blood pressure 1.27 X 10(-7) cm/sec (+/- SEM = 0.12), corresponding to the permeability in a normal population. Thus the blood-retinal barrier permeability is pathologically increased even in moderate degrees of essential hypertension, but this increase in permeability is reversible.     

Breakdown of the blood-retinal barrier in multiple sclerosis measured by vitreous fluorophotometry

Periphlebitis retinae in multiple sclerosis appears as transitory cellular infiltrations around veins in an otherwise normal retina. Similar cellular infiltrations have been found around veins in the central nervous system. In the present study the blood-retinal barrier has been investigated by vitreous fluorophotometry. Eight multiple sclerosis patients with actual periphlebitis retinae and 9 patients with previous but not active periphlebitis retinae were included in this study. Abnormal leakage of fluorescein was manifest in the group of multiple sclerosis patients with periphlebitis retinae. Permeability (1.8 +/- 0.2 X 10(-7) cm/sec; mean +/- SEM) but not in the control group as a whole permeability (1.3 +/- 0.1 X 10(-7) cm/sec; mean +/- SEM) compared to 17 normals (permeability 1.1 +/- 0.005). It is thus concluded that breakdown of the blood-retinal barrier may be transitory when connected with periphlebitis retinae in multiple sclerosis.     

Blood-retinal barrier permeability to carboxyfluorescein and fluorescein in monkeys

Lipid solubility is a major determinant of permeability across the blood-brain barrier, to which the blood-retinal barrier (BRB) has many similarities. Carboxyfluorescein is a dye with about 1/1000 the lipid solubility of fluorescein, but their molecular sizes and spectral characteristics are similar. We studied the importance of lipid solubility in BRB permeability by comparing the BRB permeabilities to these two dyes. Dye in the vitreous and plasma of four monkeys was measured by fluorophotometry. The estimated inward permeability coefficients (Pin) were 11 +/- 7.4 X 10(-6) cm/min (mean and SD) for carboxyfluorescein and 21 +/- 5.9 X 10(-6) cm/min for fluorescein. The ratio of the means was 1/1.9, far from the expected 1/1000. This finding suggests that the BRB does not function as a continuous lipid membrane and that other factors are more important determinants of permeability for these dyes than lipid solubility.     

Calculation of the permeability of the blood-retinal barrier to fluorescein

A method is presented, for calculation of the permeability of the blood-retinal barrier to fluorescein which is based upon stimultaneous determination of the free fluorescein concentration in plasma and the fluorescein concentration profile in the vitreous body. By aid of a simplified mathematical model of the eye the blood-retinal barrier permeability is calculated automatically on a computer from corresponding values of the fluorescein concentration in plasma and in the vitreous body. The present method eliminates some of the factors of uncertainty, which have been present in earlier applied fluorophotometric methods, thus contributing to increasing the exactness of the fluorophotometric method for the estimation of the permeability of the blood-retinal barrier to fluorescein. Apart from the permeability of the barrier, the diffusion coefficient for fluorescein in the vitreous body is also estimated by the present method.     

Blood aqueous barrier permeability versus age by fluorophotometry

Values of the diffusion coefficient into the anterior chamber and the blood aqueous barrier permeability as a function of age were determined by fluorophotometry in 58 healthy volunteers. The diffusion coefficient was calculated from aqueous fluorescein concentration and the time integral of non-protein bound fluorescein concentration in plasma. Blood-aqueous barrier permeability was calculated using diffusion coefficient values, the area of fluorescein inflow into the anterior chamber and anterior chamber volume. Values for diffusion coefficient as well as permeability were found to be independent of age between 13 y and 72 y (lin. corr. coeff. 0.2, p = 0.11) mean values were 4.7 .10(-4) min-1 +/- 1.5. 10(-4) SD and 15.4 nm/s +/- 4.8 SD, respectively. The difference between permeability values calculated from fluorophotometric scans at 30, 55 and 65 mins. after fluorescein injection was less than 5% and the 7 months reproducibility was within 15%. There was no significant correlation between simultaneously measured values of blood-retinal and blood-aqueous barrier permeability (lin. corr. coeff. 0.13, p = 0.4).     

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.     

Inward permeability of the blood-retinal barrier by fluorophotometry

A method for assessing the inward permeability of the blood-retinal barrier by fluorophotometry is presented. The permeability value is calculated with the fluorophotometer computer from fluorophotometric scan values in vitreous and non-protein-bound fluorescein concentration values in plasma. No diffusion coefficient of fluorescein in vitreous was required in the calculations. Corrections were performed for lens transmission, corneal transmission, and the spatial resolution of the apparatus. The method was applied to 58 healthy volunteers, aged 13-72 yr. An insignificant average increase of permeability values was found from 4.8 nm/s at 10 yr, up to 6.1 nm/s at 70 yr (P = 0.14; standard deviation: 1.8 nm/s). Permeability values showed an average increase of 10% between 30 min and 60 min after injection (P less than 0.001).     

Transport of fluorescein in the rabbit eye after treatment with sodium iodate

The outward active transport and the inward permeability of the blood-retinal barrier were studied in the rabbit eye after i.v. administration of sodium iodate. The active transport was evaluated from the half-time of disappearance of the vitreous fluorescein following intravitreal administration, and the inward permeability was evaluated from the vitreous concentration of fluorescein monoglucuronide after i.v. administration. The half-time of the vitreous fluorescein was 3.5 +/- 0.3 (mean +/- S.D.) hr, and 3.9 +/- 0.2 hr before and within 6 hr after iodate administration, respectively. After 24 hr, the half-time was 11.7 +/- 1.7 hr, similar to that of fluorescein monoglucuronide, 12.0 +/- 2.7 hr. The vitreous and the anterior chamber concentration of fluorescein monoglucuronide was measured at 1 hr after the i.v. dye injection. The vitreous concentration in the rabbits given iodate 3 hr before the dye injection was significantly greater than in the normal eyes, while the anterior chamber concentration was not different. Since fluorescein is rapidly metabolized to fluorescein monoglucuronide, differences in parameters determined using systemic fluorescein under two treatments or in disease states may be the result of alteration of the dynamics of fluorescein, fluorescein monoglucuronide, or both.     

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.     

Blood-ocular barrier permeability in monkeys.

The permeability of the blood-ocular barrier was investigated in five monkeys using vitreous fluorophotometry (VFP). Inward permeability (Pin) of the blood-retinal barrier was calculated by a computer simulation method. Kinetic VFP was performed after intravitreal injection of fluorescein (F) or fluorescein monoglucuronide (FG). The estimated mean value of Pin (x10(-6) cm/min) was 4.8 (SD 1.2). The mean rates of loss (per hour) of F from the anterior chamber (Ka) and the vitreous (Kv) were 0.11 (SD 0.01) and 0.13 (SD 0.03), respectively, which were approximately three and four times greater than those of FG (0.04 (SD 0.01) and 0.03 (SD 0.01), respectively). Probenecid administered intraperitoneally decreased both the Ka and the Kv of F significantly but had no effect on the Ka or the Kv of FG, suggesting that F was excreted from the eye with the aid of the active transport mechanism. The results of comparative studies of the rates of loss of F from the anterior chamber (Ka) and from the vitreous (Kv) suggested that active transport was more predominant in the blood-retinal barrier than in the blood-aqueous barrier.     

Movement of fluorescein and fluorescein glucuronide across the isolated rabbit iris-ciliary body

Movement of fluorescein and fluorescein glucuronide, a fluorescent metabolite of fluorescein, across the isolated iris-ciliary body of the albino rabbit was determined under short-circuit conditions using a modified Ussing's chamber. The permeabilities of this tissue to these dyes were calculated. The outward permeability (from the aqueous to the stromal side) of the iris-ciliary body preparation averaged 6.63 +/- 0.86 for fluorescein and 1.51 +/- 0.47 X 10(-6) cm/sec for fluorescein glucuronide, and the inward permeability (from the stromal to the aqueous side) was 1.68 +/- 0.41 for fluorescein and 1.37 +/- 0.77 X 10(-6) cm/sec for fluorescein glucuronide, respectively. Application of probenecid or ouabain decreased the outward permeability of fluorescein, but it had no significant effect on the fluorescein glucuronide movement. Application of 10(-5) M 2,4-dinitrophenol showed no significant effect on the fluorescein or fluorescein glucuronide movement, but application of 5 X 10(-4) M 2,4-dinitrophenol decreased the outward fluorescein transfer, which was also markedly suppressed by incubation at 0 degrees C. It is possible that an active transport mechanism is involved in the outward fluorescein movement across the iris-ciliary body, while the inward movement of fluorescein and also the fluorescein glucuronide movement across this tissue is mainly by passive diffusion.     

Photometric oculometry: A slit-lamp technique for determination of intraocular distances

The optics of slit-lamp fluorophotometry were analyzed. This analysis forms the basis of the formulation of a slit-lamp technique called photometric oculometry, which makes it possible to measure intraocular distances.A comparison between ultrasonography and photometric oculometry in the determination of the ocular axial length was carried out. Measurements in 11 eyes showed statistical agreement, with a mean of 22.90 mm, SD ±1.815 (ultrasound), and a mean of 23.17 mm, SD ±1.667 (photometry).The principle involved in photometric oculometry is of special importance for the calculation of the blood-retinal barrier permeability to fluorescein by vitreous fluorophotometry, since the method allows the volume of the vitreous body to be estimated directly from the slit-lamp fluorophotometer recording.     

Vitreous fluorophotometry in aphakic or pseudophakic eyes with persistent cystoid macular edema

By vitreous fluorophotometry, the degree of disruption of the blood-vitreous barrier was studied in 16 aphakic or pseudophakic eyes with persistent cystoid macular edema (CME) and 11 aphakic or pseudophakic eyes without CME; postoperative periods ranged from 7 to 22 months in both groups. The rate of fluorescein penetration into the vitreous was determined within 30 minutes after intravenous injection. It represented the permeability of the blood-vitreous barrier and averaged 10.28 +/- 4.52 (SD) X 10(-6) min-1 in eyes with CME and 3.05 +/- 1.21 X 10(-6) min-1 in eyes without CME; the difference between the two groups was statistically significant (P less than 0.002, Student t-test). The peak fluorescein concentration in the mid-vitreous (Cv) was determined and the concentration of free fluorescein in the blood serum (Cs) at the corresponding time was also estimated: the Cs/Cv ratio represented the balance between the inward and outward transport of fluorescein across the blood-vitreous barrier. The Cs/Cv ratio was 7.91 +/- 2.94 in eyes with CME and 12.91 +/- 3.68 in eyes without CME: the difference was statistically significant (P less than 0.001). In 6 eyes with the Cs/Cv ration of 5.0-9.2 the condition of CME deteriorated or remained unchanged during the follow-up of 4-8 months, but in 4 eyes with the ratio of 9-14.2 CME showed an improvement during the same period. In 3 eyes with vitreous tug syndrome, anterior vitrectomy improved the ratio from an average of 4.8 to 17.4. It was concluded that a functional disturbance of the blood-vitreous barrier underlies the development of persistent CME.     

Carboxyfluorescein. A dye for evaluating the corneal endothelial barrier function in vivo

The relationship of the cornea-aqueous distribution ratio (r ca) and concentration in vitro was established for fluorescein and carboxyfluorescein. The value of rca for fluorescein was found to fall from 3.20 +/- 0.25 (mean +/- S.D., n V 6) to 1.78 as the concentration of the free fluorescein in the bathing medium rose from 5.8 X 10(-8) to 5.9 X 10(-5) g ml-1. For carboxyfluorescein, it remained unchanged over the same concentration range, and the average for total determinations was 1.29 +/- 0.16 (n = 20). The value of rca for carboxyfluorescein determined in vivo was 1.62 +/- 0.23 (mean +/- S.D., n = 6) and the corneal endothelial permeability to carboxyfluorescein in normal rabbits was 3.31 +/- 0.66 X 10(-4) cm min-1 (n = 11), which was 35% lower than that for fluorescein. Because of its lower endothelial permeability and a value of rca which is unchanged over a wide range of concentration, carboxyfluorescein may be better suited for the in vivo evaluation of the barrier function of the corneal endothelium than fluorescein.     

Analysis of transport of fluorescein across the isolated retinal pigment epithelium-choroid using an ussing type chamber

Retinal pigment epithelium (RPE)-choroid preparations from albino rabbits were sealed in an Ussing type chamber under stabilized conditions for 3 hours. The transepithelial potential was 1.2 +/- 0.08 mV and the transepithelial resistance was 175.2 +/- 9.1 omega.cm2 (mean +/- SE, n = 16). The transport of fluorescein across the isolated rabbit RPE-choroid was studied under short circuit condition and outward (vitreous----choroid) and inward (choroid----vitreous) permeability to fluorescein were determined. The outward permeability was 1.63 +/- 0.20 x 10(-5) cm/sec and inward permeability was 0.44 +/- 0.13 x 10(-5) cm/sec (mean +/- SE, n = 8). The former was 4 times greater than the latter (p less than 0.01). The outward permeability was decreased to 1.02 +/- 0.08 x 10(-5) cm/sec (n = 7), 0.75 +/- 0.11 x 10(-5) cm/sec (n = 5), 0.67 +/- 0.11 x 10(-5) cm/sec (n = 6) by 10(-6) M of ouabain, 10(-5) M of 2,4-dinitrophenol and 10(-4) M of probenecid, respectively. Low temperatures (0.5-1.0 degree C) markedly decreased the outward permeability to 0.05 +/- 0.04 x 10(-5) cm/sec (n = 4, mean +/- SE). These results suggest that active transport plays a role in the outward movement of fluorescein across the rabbit RPE-choroid.     

Fluorescein and fluorescein glucuronide in the vitreous body of diabetic patients

Fluorescein (F) and fluorescein glucuronide (FG) were determined in the vitreous of four diabetic patients by a double-filter slit-lamp fluorophtometric technique. Determinations were performed 60–80 min after i.v. injection of fluorescein. F and FG were also determined in plasma ultrafiltrate 5, 15, 30, 60 and 120 min after injection by high-pressure liquid chromatography. The concentration of FG in the vitreous was 3 times that of F. After correction for plasma concentrations of FG higher than those of F, the penetration index of FG through the blood-retinal barrier was found to be twice the penetration index of F. This is not what would be expected if passive transport alone were involved. Accordingly, it is suggested that active transport mechanisms contribute to the movement of F and FG across the blood-retinal barrier.Presented at the XVth Meeting of the Club Jules Gonin, Copenhagen, 10–15 August 1986