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).     

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.     

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)     

Pharmacokinetic interpretation of vitreous fluorophotometry

Vitreous fluorophotometry is producing an increasing amount of clinical and experimental data. In order to interpret these data and obtain quantitative values for the permeability of the blood ocular barriers, there is a need to understand better the basic phenomena governing the transport of fluorescein. We present here a refined mathematical model that we use to interpret a large body of clinical data yielding values for the inward (6.9 x 10(-6) cm/min) and outward (210 x 10(-6) cm/min) posterior permeability coefficients, the effective diffusion coefficient in the vitreous (8 x 10(-4) cm2/min), and the plasma fluorescein decay constants (1.17, 0.34, and 0.044 per hour). Moreover, we utilize the model to make predictions related to kinetic vitreous fluorophotometry and to the reliability of the procedure to calculate the permeability coefficients.     

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.     

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.     

Fluid movement across the blood-retinal barrier: a review of studies by vitreous fluorophotometry

Vitreous fluorophotometry was used to study the fluid movement across the blood-retinal barrier. The effect of plasma osmolality and intraocular pressure on the movement of the fluorescent tracer confirmed the interaction between the tracers and the fluid flow across the blood-retinal barrier. The measurement of outward and inward permeabilities to carboxyfluorescein revealed the existence of a posteriorly directed fluid flow in the vitreous cavity of cynomolgus monkey eyes with retinal detachment. Fluid movement across the blood-retinal barrier was shown to be inhibited by furosemide and enhanced by acetazolamide. Furthermore, vitreous fluorophotometry proved to be useful in estimating the in vivo fluid dynamics in the vitreous and across the human retina.     

Permeability of the blood-retinal barrier to carboxyfluorescein in eyes with rhegmatogenous retinal detachment

Outward and inward permeability of carboxyfluorescein across the blood-retinal barrier were measured fluorophotometrically in seven cynomolgus monkey eyes with experimental rhegmatogenous retinal detachment. Probenecid was used to inhibit outward transport of carboxyfluorescein. The outward permeability was 1.98 +/- 0.31 microliter/min in eyes with retinal detachment and 0.84 +/- 0.15 microliter/min in control eyes with vitrectomy alone (P less than 0.01). The inward permeability, determined separately following intravenous injection, was significantly lower than the outward permeability: 0.14 +/- 0.02 microliter/min for eyes with retinal detachment and 0.04 +/- 0.01 microliter/min for control eyes. Since the outward permeability minus the inward permeability in the presence of probenecid represents that fraction of tracer moving due to fluid flow, it may be concluded that outward flow of fluid across the blood-retinal barrier is a substantial contributor to carboxyfluorescein loss from the vitreous cavity following intravitreal injection.     

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.     

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).     

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.     

Fluid dynamics in eyes with rhegmatogenous retinal detachments

Inward and outward permeabilities to sodium fluorescein at the blood-retinal barrier were measured by kinetic vitreous fluorophotometry in ten eyes with rhegmatogenous retinal detachments. Fellow eyes were used as controls. Inward permeability of eyes with detachments was significantly larger than that of controls (P less than .005), suggesting damage to the blood-retinal barrier in eyes with detachments. Outward permeability of eyes with detachment and retinal holes was slightly less than that of controls, but the difference was not statistically significant. However, outward permeability of eyes with detachments and with retinal tears was significantly larger than that of controls (P less than .05). This increased outward permeability may be attributed to the increased fluid flow posteriorly through the break across the retinal pigment epithelium.     

Vitreous fluorophotometry in patients with Best's macular dystrophy

Ten patients with Best's macular dystrophy were examined with vitreous fluorophotometry and results were compared with a normal population. Seventeen of the 20 affected eyes demonstrated an intact blood-retinal barrier with normal inward permeability of fluorescein dye. Despite diffuse functional impairment of the retinal pigment epithelium (RPE), determined by electro-oculography, as well as accumulation of a lipofuscin or lipofuscin-like substance within virtually all RPE cells, the blood-retinal barrier function of these cells remained intact as determined by clinical fluorophotometry. Similar findings have previously been noted in another hereditary retinal disorder (fundus flavimaculatus) in which a lipofuscin-like substance also accumulates diffusely within RPE cells.     

The effect of pH on the transfer of fluorescein across the blood-retinal barrier

The blood-retinal barrier (BRB) might be governed by the same permeability principles as the blood-brain barrier (BBB). For a weak acid like fluorescein, BRB permeability would be proportional to its pH-dependent lipid solubility, according to the pH partition hypothesis. A range of metabolic acidosis was produced in 20 rats by the oral administration of NH4Cl; six additional rats received normal saline. Four hours later, vitreous fluorophotometry, venous fluorescein values, and arterial pH were measured. Significant linear relationships were found between vitreous fluorophotometry readings and blood hydrogen ion concentrations (p less than 0.025) and plasma fluorescein concentrations (p less than 0.05). According to the linear relationship, changing the pH from 7.4 to 7.3 or 6.9 would result in an increase in vitreous fluorophotometry reading of 8.5 or 72%, respectively. Since the pH partition hypothesis predicts values of 52 or 640% our results suggest that the BRB conforms less to the hypothesis than does the BBB. Furthermore, although pH changes of a magnitude able to influence vitreous fluorophotometry readings substantially may occur under experimental conditions in animals, they are unlikely to occur in ambulatory human patients.     

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.     

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.     

Vitreous fluorophotometry: mathematical analysis of the effect of peripheral leakage on axial scans

Current instrumentation in vitreous fluorophotometry allows the determination of a fluorescein concentration profile along the optical axis of the eye. Based on an assumption of a uniform blood-retinal barrier permeability, several methods have been used for the determination of the permeability from an axial scan. The assumption of a uniform permeability is not realistic and it has been unknown to what extent the calculated common permeability reflects the local permeability in different areas of the retina. Using a mathematical model for a nonuniform permeability, we have investigated the effect of localized leakage on the axial concentrations and thereby on the calculated common permeability under the assumption of free diffusion in the vitreous body. It turns out that leakage outside the large temporal vessels has to be extremely strong to have a noticeable impact on 60 min axial scans. A 100-fold increase of the permeability in the region more than 30 degrees (central angle) from the optical center of the eye leads to just a 2-fold increase of the apparent common permeability. Thus, axial vitreous fluorophotometry almost exclusively measures the condition of the retina in the vicinity of the optical center.     

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.     

Oscillatory potentials and permeability of the blood-retinal barrier in noninsulin-dependent diabetic patients without retinopathy

Electroretinography and vitreous fluorophotometry were performed in 36 eyes of 36 noninsulin-dependent diabetic patients and in 32 eyes of 32 healthy control subjects between the ages of 30 and 59 years. Fluorescein fundus angiograms showed no abnormalities in either group. Peak implicit time of the first deflection of the oscillatory potential, interpeak interval between the first and second deflections, and the sum of the amplitudes of the upward deflections were analyzed. Inward permeability of the blood-retinal barrier was calculated by vitreous fluorophotometry and computer simulation. The peak implicit time of the first deflection and the interpeak interval between the first and second deflections of the oscillatory potential were significantly longer in diabetic patients than in control subjects (P less than 0.01). No significant difference in inward permeability of the blood-retinal barrier existed between the two groups. These results indicate that a selective delay in the peak implicit time of the oscillatory potential (neurosensory retinal abnormality) may be present in noninsulin-dependent diabetic patients, representing retinal functional changes before changes in blood-retinal barrier permeability are apparent.     

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.