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.     

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.     

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.     

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.     

Binding of fluorescein and carboxyfluorescein by human serum proteins: significance of kinetic and equilibrium parameters of association in ocular fluorometric studies

The binding of fluorescein and 5- and 6-carboxyfluorescein by human serum proteins was measured at 37 and 4 degrees C by equilibrium dialysis. The equilibrium association constants (KA) for fluorescein were 3.7 X 10(3) and 7.1 X 10(3)M-1, and for carboxyfluorescein were 3.5 X 10(3) and 7.5 X 10(3)M-1 at 37 and 4 degrees C equilibrium dialysis data, was 3.9 X 10(3) binding sites in human serum, determined from the 37 degrees C equilibrium dialysis data, was 3.9 X 10(-3)M for fluorescein and 3.3 X 10(-3)M for carboxyfluorescein. Utilizing these binding parameters it was calculated that a maximum of 93.5% of the total fluorescein and 92.0% of the carboxyfluorescein would be bound by undiluted human serum proteins at 37 degrees C. Experimental binding data obtained after prolonged equilibrium dialysis (four days) at low total fluorochrome concentrations (1.5 X 10(-4)M or less) indicated that 93.1 +/- 1.0 (S.D.)% of the fluorescein and 90.1 +/- 0.7% of the carboxyfluorescein were bound at 37 degrees C by undiluted human serum proteins. Stopped-flow kinetic spectrophotometric studies of the changes in absorptivity at 487-488 and 510 nm that occurred when the fluorochromes were bound by human serum proteins, indicated that the fluorescein and carboxyfluorescein binding reactions were 99% complete within 0.65 and 1.72 sec. These had second-order association rate constants at 25 degrees C of 3.0 X 10(3) and 1.5 X 10(3)M-1 sec-1, respectively. These findings offer a basis for calculation of bound and free fluorescein and carboxyfluorescein in vivo in human subjects.     

Permeability of the isolated dog retinal pigment epithelium to carboxyfluorescein

Outward (retina to choroid) and inward (choroid to retina) permeabilities of carboxyfluorescein and fluorescein in the isolated dog retinal pigment epithelium (RPE)-choroid were determined. Outward permeability was 9 and 47 times larger than inward permeability for carboxyfluorescein and fluorescein, respectively. The outward permeability of carboxyfluorescein was seven times lower than that of fluorescein, whereas there was no statistical difference between the inward permeabilities. Carboxyfluorescein is thus distinguished from fluorescein by its low affinity to the outwardly directed organic anion transport system. 10(-4) M probenecid caused greater than 98% inhibition of the outward transport of 6 X 10(-5) M carboxyfluorescein and 6 X 10(-6) M fluorescein.     

A simple method for determination of corneal epithelial permeability in humans

A simple method for the determination of human corneal epithelial permeability to fluorescein is presented. The method consists of applying a 1% sodium fluorescein solution to the cornea for 8 minutes by means of an eye bath, rinsing the eye, and measuring corneal fluorescence by fluorophotometry. The permeability value is calculated from the corneal fluorescein concentration immediately after the bathing period. Mean permeability value determined in 86 eyes of 46 volunteers aged 15 to 67 years (mean 30.9 years) was 0.038 nm/s +/- 0.017 SD. No significant correlation with age was found (corr. coeff. = 0.17, P = 0.28). The reproducibility was within 10%.     

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).     

Cold-induced corneal edema in patients with trigeminal nerve dysfunction

Two previous cases of cold-induced corneal edema have been reported in patients with corneal anesthesia secondary to a trigeminal nerve disorder. We studied six patients with complete unilateral corneal anesthesia after trigeminal ablation. Subjects' eyes were exposed to 4 C air from a fan for one hour. We measured corneal thickness, corneal surface temperature, and endothelial permeability to fluorescein. During cold exposure, two of the six study eyes exhibited reversible corneal swelling (11% and 26% over baseline value). All anesthetic corneas were consistently colder (13.8 +/- 0.7 C) than the contralateral corneas (21.0 +/- 1.7 C, P = .001). Baseline endothelial permeability and aqueous humor flow rates were similar in both the study and control groups. After cold exposure, the study eyes had a significant transient increase in permeability compared to the controls (7.5 +/- 2.4 x 10(-4) cm/min vs 2.9 +/- 1.4 x 10(-4) cm/min, P = .007). Baseline endothelial photomicrographs also showed increased pleomorphism (fewer hexagonal cells) in the anesthetic corneas. These data suggest that sensory denervation of the eye influences ocular temperature regulation and corneal endothelial cell morphologic characteristics. Some anesthetic corneas are prone to cold-induced edema, which may result from excessive cooling.     

Estimation of corneal endothelial pump function in long-term contact lens wearers

PURPOSE: To study the effects of long-term contact lens wear on morphologic and physiologic properties of corneal endothelial cells. METHODS: The endothelial permeability to fluorescein and the rate of corneal deswelling from hypoxia-induced edema were measured in 20 long-term (mean, 17+/-9 years; range, 5-33 years) contact lens wearers and 20 age-matched control subjects. From these data, the relative endothelial pump rate in each subject was estimated, based on the pump-leak hypothesis of corneal hydration control. Corneal autofluorescence and the aqueous humor flow rate were determined by fluorescein fluorophotometry. Images of corneal endothelial cells were recorded by using specular microscopy, and morphologic indices (cell density, coefficient of variation of cell area, percentage of hexagonal cells, and skewness) were determined. RESULTS: No statistically significant differences were found between the contact lens and control groups in endothelial permeability, corneal deswelling, relative endothelial pump rate ([mean +/- SD] 1.07+/-0.33 relative pump units versus 1.01+/-0.25 relative pump units; contact lens versus control; P = 0.57), and endothelial cell density. Contact lens wearers had a significantly higher aqueous humor flow rate (3.57+/-1.03 microl/min versus 2.77+/-0.51 microl/min; P = 0.005), coefficient of variation of cell area (0.35+/-0.09 versus 0.28+/-0.04; P = 0.006), and corneal autofluorescence (3.1+/-0.6 ng/ml versus 2.3+/-0.3 ng/ml fluorescein equivalents; P < 0.001) than did non-contact lens wearers. CONCLUSIONS: Despite the known effects of long-term contact lens wear on corneal endothelial morphometry, no effect on endothelial function was found.     

Effect of oxidized glutathione on the barrier function of the corneal endothelium

Effects of glutathione on the corneal endothelium were reexamined. Four kinds of solutions were made: oxidized glutathione (GSSG) was added to a basic solution which does not contain glutathione (GSSG-0) at a concentration of 0.03 mM, 0.3 mM or 3 mM to make GSSG-0.03, GSSG-0.3 or GSSG-3, respectively. Paired rabbit corneas were perfused separately, and the endothelial permeability (Pac) to carboxyfluorescein was determined. Between the paired corneas perfused with GSSG-0 and GSSG-0 or GSSG-0 and GSSG-0.03, there was no significant difference in the Pac. A significant difference in this factor was seen between the paired corneas perfused with GSSG-0 and GSSG-0.3 or GSSG-0 and GSSG-3 (P less than 0.01). The ratio of GSSG-0 to GSSG-0.3 for Pac, 1.18 +/- 0.16, and that of GSSG-0 to GSSG-3, 1.14 +/- 0.07, were significantly greater than the left-right ratio for Pac obtained when the paired corneas were perfused with GSSG-0, 1.01 +/- 0.10 (mean +/- SD, n = 8) (P less than 0.025). The corneal swelling rate (micron/hr) was 7.9 +/- 4.9 for the corneas perfused with GSSG-0 and 8.4 +/- 5.4 (mean +/- SD, n = 6) for those perfused with GSSG-0.3; difference was not significant. Addition of GSSG at a concentration of 0.3 mM or more to the irrigating solution was further beneficial to the corneal endothelial barrier function and a solution containing GSSG may be safer for patients with vulnerable corneas.     

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.     

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.     

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.     

Effects of contact lenses on corneal endothelium - a morphological and functional study

PURPOSE: To study the corneal endothelial morphological changes and endothelial barrier function in contact lens wearers. METHODS: Specular microscopy and anterior segment fluorophotometry were performed on 116 controls (group 1) and 76 daily wear soft contact lens wearers. Group 2 patients (n = 34) had been wearing contact lenses for less than 5 years and group 3 (n = 42) for more than 5 years. The relationship of corneal thickness, endothelial cell density, hexagonal cell percentage, coefficient of variation in cell area, corneal autofluorescence and corneal endothelial permeability to the contact lens wear duration was studied. RESULTS: The average corneal thickness of contact lens wearers did not differ significantly from controls (0.533 +/- 0.031, 0.538 +/- 0.044, 0.532 +/- 0.031 mm for groups 1, 2 and 3, respectively; p = 0.89). However, there was a significant tendency toward corneal thinning with a longer history of contact lens wear (r = -0.31, p = 0.002 in groups 2 and 3). The percentage of hexagonal cells decreased with both increasing hours of contact lens wear per day (r = -0.36, p < 0.001) and the duration of contact lens wear in years (r = -0.33, p < 0.001), but there was no significant change in endothelial cell density in contact lens wearers. The coefficient of variation in cell size correlated more closely with increased hours of contact lens wear per day (r = 0.35, p = 0.002) than with the duration of contact lens wear in years (r = 0.12; p = 0.31). In the functional study, corneal autofluorescence increased in contact lens wearers (5.13 +/- 0.71 ng/ml in group 1, 6.45 +/- 2.03 ng/ml in group 2 and 7.21 +/- 1.51 ng/ml in group 3, respectively, p < 0.001) and the mean endothelial permeability decreased in contact lens wearers (3.89 +/- 0.95 x10(-4)/cm in group 1, 2.71 +/- 0.73 x 10(-4)/cm in group 2 and 2.95 +/- 0.91 x 10(-4)/cm in group 3, respectively, p = 0.003). CONCLUSIONS: Daily wear soft contact lenses caused morphological changes in the corneal endothelium. With an increasing span of contact lens wear, there was a significantly increased variation in cell size, a decreased hexagonal cell percentage, an evident intercellular dark area and rosette formation. The corneal autofluorescence increased and the overall endothelial permeability decreased as a consequence of contact lens wear. Contact lens wear also caused corneal thinning, and the cornea became thinner with increasing duration of contact lens wear.     

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.     

Corneal epithelial barrier function in diabetic patients

PURPOSE: To evaluate the corneal epithelial barrier function in diabetic patients. METHODS: In 29 eyes of 29 diabetic patients and 55 eyes of 55 nondiabetic controls, corneal epithelial permeability to fluorescein was measured using an anterior fluorophotometer. The average fluorescein concentration in the central cornea was compared between diabetic patients and controls. Multiple regression analysis was used to assess the factors that affect corneal epithelial barrier function in diabetic patients. RESULTS: The average fluorescein concentrations in diabetic patients and nondiabetic controls were 44.1 +/- 25.3 ng/mL and 29.9 +/- 19.8 ng/mL (mean +/- SD), respectively (P = 0.0057, unpaired t test). An explanatory variable relevant to the impaired corneal epithelial barrier function was the serum hemoglobin A1c (HbA1c) concentration (standardized partial regression coefficient = 0.466, P = 0.0163). CONCLUSIONS: The corneal epithelial barrier function is impaired in diabetic patients. Diabetic patients with higher serum HbA1c levels are more predisposed to impaired barrier function in the corneal epithelium.     

Effect of long-term contact lens wear on corneal endothelial cell morphology and function

Anterior segment fluorophotometry (topical) and central endothelial cell photography were performed on 40 long-term (2-23 years) contact lens wearers (four groups of ten each: hard, soft, gas permeable, and gas permeable plus prior lens usage) and 40 non-contact lens wearers of similar ages. Morphologically, the endothelial cells of contact lens wearers showed greater variability in size and shape compared to controls. The mean endothelial cell size in contact lens wearers (307 +/- 35 micron2) was smaller than that of controls (329 +/- 38 micron2, P less than 0.01). There was an increase in the coefficient of variation of cell size of the contact lens group (0.35 +/- 0.06 versus 0.25 +/- 0.04 for controls, P less than 0.0001). The endothelial cell mosaic contained a smaller percentage of hexagonal cells in contact lens wearers (66 +/- 8) compared to controls (71 +/- 7, P less than 0.01). There was a compensatory increase in five-sided cells. Functionally, there was no difference in corneal clarity, central corneal thickness or endothelial permeability to fluorescein (3.78 +/- 0.57 X 10(-4) cm/min versus 3.85 +/- 0.55 X 10(-4) cm/min for controls) between the two groups. Aqueous humor flow was increased 7% in contact lens wearers. We found no correlation between oxygen transmissibility, estimated underlying oxygen tension, or duration of wear of the contact lenses and any morphologic or functional variable. We also found no differences between the four groups of contact lens wearers except that the gas permeable lens wearers had more hexagonal and less pentagonal cells. Long-term contact lens wear induces morphologic changes in the corneal endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of tight junction properties relevant to fluid transport across rabbit corneal endothelium

Paracellular junctions could play an important role in corneal endothelial fluid transport. In this study we explored the effects of different reagents on the tight junctional barrier by assessing the translayer specific electrical resistance (TER) across rabbit corneal endothelial preparations and cultured rabbit corneal endothelial cells' (CRCEC) monolayers, the paracellular permeability (Papp) for fluorescein isothiocyanate (FITC) dextrans across CRCEC, and fluid transport across de-epithelialized rabbit corneal endothelial preparations. Palmitoyl carnitine (PC), poly-L-lysine (PLL), adenosine triphosphate (ATP), and dibutyryl adenosine 3',5'-cyclic monophosphate (dB-cAMP) were used to modulate corneal endothelial fluid transport and tight junctions (TJs). After seeding, the TER across CRCEC reached maximal values (29.2+/-1.0 Omega cm2) only after the 10th day. PC (0.1 mM) caused decreases both in TER (by 40%) and fluid transport (swelling rate: 18.5+/-0.3 microm/h), and an increase in Papp. PLL resulted in increased TER rose and Papp but decreased fluid transport (swelling rate: 10+/-0.3 microm/h). dB-cAMP (0.1 mM) and ATP (0.1 mM) decreased TER by 16% and 6%, increased Papp slightly, and stimulated fluid transport; the rates of de-swelling (in microm/h) were -5.4+/-0.3 and -12.1+/-0.4, respectively. PC might cause the junctions to open up unspecifically and thus increase passive leak. PLL is a known junctional charge modifier that may be adding steric hindrance to the tight junctions. The results with dB-cAMP and ATP are consistent with fluid transport via the paracellular route.     

A method for the in vitro determination of feline corneal endothelial permeability

A mounting block for in vitro perfusion of the cat cornea is described. Using this apparatus and the techniques of Araie, the permeability (Pac) of the normal cat corneal endothelium to carboxyfluorescein was determined to be 2.5 +/- 0.2 x 10(-4) cm/min. To assess the sensitivity of this technique in determining changes in Pac associated with alterations in endothelial, morphology, three cats underwent 2 successive unilateral, central, 10 mm diameter circular areas of endothelial debridement 6 weeks apart. Six weeks following the second wounding all 3 animals underwent morphometric analysis and Pac determination. A trend toward an elevation in Pac with extreme reductions in cell density was observed.