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)     

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.     

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 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).     

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.     

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.     

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.     

Fluorophotometric study of juvenile insulin-dependent diabetic patients

PURPOSE: The aim of this study is to find a correlation between fluorophotometric data, diabetes duration and patient's age. MATERIALS AND METHODS: A group of 19 patients (38 eyes) aged 11-23 years with juvenile-onset, insulin-dependent diabetes, treated for 2-17 years were examined using ocular fluorophotometry. Visual acuity, anterior segment and fundus as well as IOP, fluoroangiography and opacimetry were also examined. RESULTS: Anterior Chamber (AC) and Posterior Vitreous (PV) fluorescein concentration was higher in the group with mild background retinopathy (10 eyes) than in group with normal fundi (28 eyes). CONCLUSIONS: Diabetes duration over 15 years increases the risk of the development of diabetic retinopathy and increases fluorescein leakage through the blood-retinal barrier in juvenile insulin-dependent diabetes patients.     

Fluorophotometry of the vitreous body in diabetics without retinopathy or with minimal retinopathy

Vitreous fluorophotometry using the Fluorotron Master, was performed on the two eyes of 56 diabetics patients 45,65 year-old +/- 14,91 (mean +/- S.D.), duration of diabetes was 15,14 years +/- 9,17; and on one eye of 17 normal subjects 39,18 years old +/- 11,19. According to biomicroscopic and angiographic examinations, only diabetic subjects with either no or microaneurysmal retinopathy were included. Three groups of right and three groups of left eyes were made: Groups 1: no retinopathy (28 right eyes, 27 left eyes); Groups 2: 1 to 5 microaneurysms (11 right eyes, 20 left eyes); Groups 3: greater than 5 microaneurysms (11 right eyes, 9 left eyes) For the two eyes, posterior vitreous penetration ratio increased with the degree of retinopathy. In the eyes without retinopathy (Groups 1), the penetration ration is not statistically different from the normal subjects; it was the opposite for the eyes with microaneurysmal retinopathy (Groups 2 et 3). The results indicated a breakdown of the blood-retinal barrier only when clinical diabetic retinopathy was present. The relation between fluorophotometric evaluation and the degree of retinopathy showed that vitreous fluorophotometry can be useful in the follow-up of diabetic subjects.     

Alteration of the blood-retinal barrier and vitreous in sickle cell retinopathy

Nineteen eyes with background sickle cell retinopathy, eleven from patients with SC disease, eight from patients with SS disease, and twelve eyes with proliferative sickle cell retinopathy were examined by direct and indirect ophthalmoscopy, slit-lamp, fluorescein angiography and vitreous fluorophotometry. Calculation of the alteration of the blood-retinal barrier (BRB) and estimation of the diffusion coefficients of fluorescein in the vitreous were performed by fluorophotometry.In background sickle cell retinopathy, the results show a normally functioning BRB in the posterior pole. Abnormally increased fluorescence values to the mid-vitreous (peripheral leakage) were found only in 3 of these 19 eyes, at the two-hour examination (all SC patients). Similarly, mid-vitreous fluorescence values at the two-hour examination were 4.19 ± 1.52 ng/ml in eyes of patients with SC disease, compared with 2.65 ± 0.56 ng/ml in eyes of patients with SS disease. All eyes with background sickle cell retinopathy, except one, showed values for the coefficient of diffusion of fluorescein within normal limits, indicating normal vitreous gel structure.In proliferative retinopathy, the mid-vitreous fluorophotometry readings were abnormally increased, correlating well with the extent of the peripheral angiographic changes (neovascularization). The coefficient of diffusion of fluorescein in the vitreous was generally increased in the eyes with proliferative retinopathy (15.0 ± 8.4 × 10^–4cm²/min) in comparison with a mean value of 5.4 ± 1.4 × 10^–4cm²/min in the eyes wi background sickle cell retinopathy, suggesting an alteration of the vitreous structure eyes with proliferative retinopathy.Fluorophotometry is considered a useful tool to follow patients with sickle cell retinopathy by quantitating peripheral retinal vascular leakage.     

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.     

Fluorescein transport through the blood-aqueous and blood-retinal barriers in diabetic macular edema

PURPOSE: To investigate the integrity of the blood-aqueous barrier (BAB) and the blood-retinal barrier (BRB) in diabetic patients with clinically significant macular edema (CSME). METHODS: The rate constant (Kd(F)) of the BAB and the permeability of the blood-retinal barrier for non-protein bound fluorescein were determined simultaneously by fluorometry. Results were analysed pairwise in diabetic patients (n = 25) with CSME in one eye and without CSME in the other eye. RESULTS: Kd(F) for the eyes with CSME was significantly increased compared to eyes without CSME (444. 10(-6) min( -1) and 387. 10(-6) min(-1) respectively, p = 0.01). The passive permeability of the BRB was also significantly increased in CSME (5.7 nm/sec and 3.5 nm/sec respectively, p = 0.009, n = 19). CONCLUSIONS: Both the rate constant of the BAB and the BRB permeability were significantly increased in CSME indicating that eye-specific factors are common for both barriers in diabetic patients with CSME.     

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.     

Blood-retinal barrier permeability versus diabetes duration and retinal morphology in insulin dependent diabetic patients

The blood-retinal barrier permeability to fluorescein was quantitated in 54 patients (22 females and 32 males) with insulin dependent diabetes mellitus (IDDM) of different duration. Correlation was demonstrated between permeability and diabetes duration. A normal permeability was measured in patients with up to ten years diabetes duration. A pathologically increased permeability was measured with ten to 15 years diabetes duration and during the next decade the permeability increased rapidly to 5-10 times the normal value. Onset of diabetes in the decade before and after puberty did not change the pattern. However, the pathologically increased permeability after ten years duration of the disease could not be demonstrated in diabetics with onset of the disease after the age of 30 years. The permeability of the blood-retinal barrier correlated well with changes in retinal morphology as seen by ophthalmoscopy and fluorescein angiography. However, there was an overlap in permeability between groups with different retinal appearance. A significant factor was the presence of macular edema, which also apparently indicated a preproliferative state.     

Blood-retina barrier permeability in diabetes during acute ACE-inhibition.

We assessed the acute effect of ACE-inhibition (captopril) on blood-retina barrier (BRB) permeability in 10 hypertensive insulin-dependent diabetic patients with background retinopathy in a double-masked placebo controlled cross-over study. All patients underwent ophthalmological examination, fundus photography, fluorescein angiography, vitreous fluorometry, and continuous blood pressure recording within 3 h of the drug/placebo administration. The decrease in mean arterial blood pressure, from placebo treatment 149/92 +/- 17/7 to captopril treatment 132/83 +/- 14/7 mmHg (mean +/- SD), P less than 0.01 was not accompanied by a significant decrease in BRB permeability, which was 2.51 (1.24-9.15) with placebo and 3.02 (1.25-13.93).10(-7) cm/s during captopril treatment (geometric mean and-range), NS. Our study suggests that abnormal leakage through the BRB in hypertensive insulin-dependent diabetic patients with background retinopathy is caused predominantly by structural changes in the retinal vessels whereas hydrostatic forces play a minor role.     

Retinal circulation and ocular blood barrier in diabetic retinopathy]

Macular capillary blood flow velocity, the score of fluorescein angiography in posterior region and aqueous flare intensity were investigated in 38 diabetics (41 eyes) and 6 normal subjects (6 eyes) to clarify the relationship among retinal circulation, blood-retinal barrier and blood-aqueous barrier. Macular capillary blood flow velocity was measured by means of the blue field entoptic phenomenon, the score of fluorescein angiography was graded by the extent of edema in the posterior region, aqueous flare intensity was measured with a flare-cell meter. The results showed that the reduction of macular capillary blood flow is significantly associated with an increase in fluorescein angiography score and aqueous flare intensity via diabetic retinopathy (p less than 0.01).     

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.     

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.