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.     

Human corneal endothelial permeability to fluorescein and fluorescein glucuronide

The corneal endothelial permeability coefficient (Pac) for fluorescein and fluorescein glucuronide was determined in ten normal young volunteers. After oral administration of fluorescein, the apparent concentrations of both dyes in the corneal stroma and the anterior chamber were measured by differential fluorometry. The apparent dye levels calculated directly from the in vivo fluorometric measurements were converted to the true ones, based on the result of a normalization experiment performed in rabbit eyes. The value of Pac averaged 5.44 +/- 1.77 X 10(-4) cm/min for fluorescein and 3.77 +/- 1.10 X 10(-4) cm/min for fluorescein glucuronide (mean +/- SD, N = 20); the former was significantly greater than the latter (paired t-test, P less than 0.001). The aqueous-cornea distribution ratio was 0.50 +/- 0.14 for fluorescein and 0.66 +/- 0.16 for fluorescein glucuronide; the latter was significantly greater than the former (paired t-test, P less than 0.001). It was suggested that the previously reported values of Pac for fluorescein in the human eye were underestimates.     

Transport of fluorescein monoglucuronide out of the vitreous

The transport of fluorescein monoglucuronide, a fluorescent metabolite of fluorescein, from the vitreous was studied. Ten microliter of 1 mM fluorescein monoglucuronide or fluorescein solution was injected into the rabbit vitreous body, and the vitreous concentration was measured every 2 hr. The rate of loss from the vitreous was 0.066 +/- 0.012 (SD)/hour for fluorescein monoglucuronide and 0.22 +/- 0.029/hour for fluorescein. Systemically administered probenecid caused a statistically significant reduction in the loss from the vitreous of each of them.     

Decreased endothelial permeability in transplanted corneas

We measured the endothelial permeability to fluorescein of 30 clear corneal transplants from one to six years after keratoplasty. The mean permeability of 1.51 +/- 0.82 X 10(-4) cm/min was significantly lower than that of normal corneas (P less than .00001). We detected a statistically significant negative correlation between permeability and mean endothelial cell size (P less than .03), indicating that corneas with larger endothelial cells tended to be less permeable to fluorescein. We calculated a relative endothelial pump rate for each cornea; the pump rate was also decreased and negatively correlated with mean endothelial cell size (P less than .0004).     

Quantitative control of the function of the corneal endothelium by fluorophotometry in the anterior eye segment

Whereas specular microscopy yields mostly qualitative information, fluorophotometry furnishes quantitative data of endothelial cell function. Determination of fluorescein permeability of the endothelial cell layer reflects the function of the endothelial barrier. Following topical application of fluorescein, the time-dependent change in the fluorescein concentration in corneal stroma and aqueous yields the transfer coefficient (Kc) of the corneal endothelium. With the Fluorotron Master, Kc was 3.77 +/- 0.57 X 10(-3)/min. for normal eyes; patients with cornea guttata or Fuchs's dystrophy had a significantly higher transfer coefficient (Kc = 7.9 +/- 2.88 X 10(-3)/min.). Normal Kc values were found 6-63 months (average 33.5 months) after phacoemulsification with implantation of a posterior chamber lens.     

Quantitative vitreous fluorophotometry applying a mathematical model of the eye

A slit-lamp fluorophotometric method is presented that permits calculation of a blood-retinal barrier permeability to fluorescein (P) and a diffusion coefficient for fluorescein in the vitreous body (D). The calculations are performed by relating the time course of the free--not protein bound--fluorescein concentration in the bloodstream with the fluorescein concentration profile in the vitreous body. The combination is performed automatically on a computer by applying a simplified mathematical model of the eye. P refers to the area of the barrier of the model eye. In a group of six normal persons, the mean P was (1.1 +/- 0.4) X 10(-7) cm/sec (mean +/- SD), while in six diabetic patients with background retinopathy and macular edema the mean P was (7.1 +/- 3.8 ) X 10(-7) cm/sec. The mean D was (7.4 +/- 3.4) X 10(-6) cm2/sec in the normal group and (9.6 +/- 2.0) X 10(-6) cm2/sec in diabetic patients, corresponding as a first approximation to free diffusion in water. Model calculations show that knowing the fluorescein concentration in the bloodstream is considerably significant for the calculation of the permeability, contributing factors up to 50%. For the low-permeation situation, subtraction of the preinjection scan contributes a factor of 50% for both permeability and diffusion coefficient. The exact placement in the vitreous body of the concentration profile, by applying a formalism that transforms slit-lamp movement to intraocular distance, contributes a factor of 20% on the diffusion coefficient. The permeability obtained with the model can be calculated as the ratio between area of vitreous and plasma fluorescein concentration curves within 20%. Active transport of fluorescein across the blood-retinal barrier in the direction of vitreous to blood does not seem to be significant within the first 2 hr after fluorescein injection.     

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 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.     

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.     

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.     

A reevaluation of corneal endothelial permeability to fluorescein

The permeability of the corneal endothelium and its aqueous-cornea distribution ratio were reevaluated in the rabbit eye. Both parameters were determined in an individual eye by applying the dye first by iontophoresis and then by intravitreal injection, which allows the influence of fluorescein glucuronide on the fluorophotometric measurements to be excluded. The corneal endothelial permeability coefficient was 5.13 +/- 1.64 X 10(-4) cm min-1, and the aqueous-cornea distribution ratio was 0.25 +/- 0.06 (mean +/- S.D., n = 11) on the average, and the former was considerably greater than the previous results, while the latter was considerably smaller.     

Acute effects of topical phenylephrine on aqueous humor dynamics and corneal endothelial permeability in man

Acute effects of topical phenylephrine, an alpha-1 adrenergic agonist, on the aqueous humor dynamics and corneal endothelial permeability were studied by means of the oral fluorescein method in 11 normal young volunteers. Twenty microliters of phenylephrine HCL (10%) was instilled in one eye and the placebo in the other eye in a double masked manner. The instillations were carried out 0.5 hour before, and 2, 4 and 6 hours after the fluorescein ingestion. Fluorophotometric measurements were carried out in the central cornea, anterior chamber and plasma ultrafiltrate, and the aqueous-cornea transfer coefficient in reference to the corneal volume (kc.ac), the transfer coefficient in the anterior chamber by diffusion (kd.pa) and by flow (kfa) were calculated in each eye. The thickness of the cornea (CT), intraocular pressure (IOP) and anterior chamber volume (Va) were also measured. No significant difference was found in the CT, kc.ac, corneal endothelial permeability (kc.ac x the thickness of the stroma), Va, IOP and kfa between the experimental and control eyes, while the kd.pa was significantly smaller in the experimental eyes (paired t-test, P less than 0.01). The iris permeability factor (kd.pa x Va) decreased significantly to 0.74 +/- 0.26 (Mean +/- SD) of the control (P less than 0.01). The aqueous flow rate (kfa x Va) averaged 0.95 +/- 0.21 of the control, and the difference from unity was not significant (P greater than 0.1).     

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.     

Paracellular permeability of corneal and conjunctival epithelia

The paracellular permeability of normal rabbit cornea and conjunctiva was studied in vivo and in vitro. After intravenous administration, horseradish peroxidase was found to percolate to the intercellular space of conjunctival epithelia and was restricted by the tight junctions of the superficial epithelium. Only minimal tracer was present in the limbus and cornea. The difference between corneal and conjunctival paracellular pathways was further compared in vitro by tissue perfusion studies using various tracers from subepithelial space to apical surface. The intact full-thickness cornea was permeable to mannitol (MW 182) but not to inulin or dextran. The conjunctiva was permeable to mannitol, inulin and FITC-dextran (MW 20,000). The quantitative permeability to 3H-mannitol (X10(-8) cm/sec) of adult rabbit cornea was 0.12 +/- 0.02, which is about 55-fold and 50-fold lower than that of conjunctiva (6.78 +/- 0.21) and peritoneum (6.12 +/- 0.63), respectively. Removal of the corneal epithelium increased the permeability 40-fold; however, removal of the endothelium had little effect on the solute permeation. When both corneal epithelium and endothelium were debrided, the bare stroma became edematous and the permeability increased 70-fold. The permeability of 1-week-old rabbit cornea was 1.32 +/- 0.18, which decreased to 0.46 +/- 0.06 in 2-week-old rabbits, and became similar to the adult level at 4 weeks of age. When Tenon's capsule was included in the perfusion, the conjunctival permeability decreased 2.5-fold. With the apposition of bare corneal stroma to the conjunctiva and Tenon's capsule, the permeability decreased further (4-fold).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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%.     

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.     

Variations in human corneal endothelial cell morphology and permeability to fluorescein with age

Fluorophotometry with topically applied fluorescein and endothelial cell photography were performed on 80 normal subjects (age 5-79 yr). Variations in endothelial cell morphology and function, flow of aqueous humor, and intraocular pressure were recorded. The mean endothelial cell size was 332.3 +/- 46.3 micron 2. A 28% increase in endothelial cell size was measured over the eight decades (r = 0.53, P less than 0.001). The coefficient of variation of cell size also increased with age (r = 0.41, P less than 0.001). The percentage of hexagonal endothelial cells decreased by 14% (r = -0.48, P less than 0.001), while the percentage of pentagonal and heptagonal cells increased by 50% (r = 0.44, P less than 0.001) and 40% (r = 0.33, P less than 0.002), respectively, with age. The mean endothelial permeability to fluorescein was 4.03 +/- 0.63 x 10(-4) cm min-1. A 23% increase in endothelial permeability with age was observed (r = 0.44, P less than 0.001). No change in central corneal thickness or endothelial pump rate was found. Flow of aqueous humor remained stable with age, despite a 25% increase in intraocular pressure (r = 0.50, P less than 0.001). Polarization of fluorescence of fluorescein in the corneal stroma decreased with age (r = -0.46, P less than 0.001). We conclude that with age the human corneal endothelium becomes morphologically less regular and may become more permeable to fluorescein.     

Effect of acetazolamide on outward permeability of blood-retina barrier using differential vitreous flyorophotometry.

PURPOSE: To measure fluorescein (F) and fluorescein monoglucuronide (FG) concentrations in the vitreous and evaluate the effect of acetazolamide (AZM) on the outward permeability of the blood-retina barrier (BRB) using differential vitreous fluorophotometry (DVF). METHODS: DVF was performed 180 minutes after intravenous injection of AZM (5 mg/kg) and 50 mg of sodium fluorescein in six rabbits (AZM group). DVF also was performed in six rabbits injected intravenously with only 50 mg of sodium fluorescein (control group). The F/FG ratio was calculated based on the concentrations of F and FG obtained by DVF. DVF also was performed 180 minutes after 50 mg of intravenous injection of sodium fluorescein in five rabbits given probenecid (150 mg/kg) intraperitoneally (probenecid group). RESULTS: The average F/FG ratio was 0.36 +/- 0.17 (range, 0.22-0.66) in the AZM group, which was significantly smaller than the control value of 0.74 +/- 0.22 (range, 0.50-1.60). The average F/FG ratio at 180 minutes after injection was 1.51 +/- 0.46 (range, 0.94-2.00) in the probenecid group, which was significant higher (p < 0.05) than that of the AZM or control group. CONCLUSIONS: This study showed that the F/FG ratio might be a good indicator of the estimated outward permeability of the BRB using DVF and that AZM may accelerate the outward active transport function of the BRB.