Characterization of the early stages of diabetic retinopathy by vitreous fluorophotometry.

We compared insulin-dependent diabetic patients with minimal (16 eyes of 9 patients) or no retinopathy (45 eyes of 27 patients) to normal volunteers (20 eyes of 12 subjects) using a commercial vitreous fluorophotometer and different procedures for artifact correction. The influence of background autofluorescence was minimized through the use of a software program that subtracted a fluorophotometric scan obtained before administration of fluorescein from that obtained after its injection. We also compared two programs designed to minimize the contribution of the chorioretinal peak spread function to the readings in the vitreous. The fluorescein concentration in the posterior vitreous was then averaged within two different regions. We then assessed the influence of these data-processing methods on the spread of the results of the different groups. The clinical study showed that only the posterior vitreous concentration of fluorescein is relevant in the evaluation of the blood-retinal barrier. However, since there is a gradient of fluorescein concentration in the posterior vitreous, one needs a scanning device so that one can measure at a precise location in front of the retina. The posterior vitreous concentration of fluorescein was significantly increased in diabetic subjects with one or no aneurysms as compared with normals. Moreover, the eyes with minimal retinopathy, as judged by the presence of microaneurysms, had higher values than those without retinopathy. The clear differences among these three groups were not present when the midvitreous values were used.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of a noninvasive measurement of optic nervehead mechanical compliance with an invasive method.

We have postulated that abnormal mechanical support of the optic nervehead at the level of the lamina cribrosa could be the precursor of glaucomatous damage. Recent studies have shown deformations of the lamina cribrosa to be among the earliest changes in glaucoma. To evaluate the support of the nervehead, we have developed a noninvasive optical method to measure the optic nervehead compliance, namely, the displacement of the optic nervehead induced by an artificial increase in intraocular pressure. To test the validity of the method, we have compared noninvasive measurements obtained in post-mortem enucleated human eyes with those recorded using an invasive technique. Both methods had a reproducibility better than 6 microns and induced no damage capable of interfering with the results. The displacements measured by both methods were similar, thus indicating that our optical method is capable of measuring bulk motion of the optic nervehead. Our results were identical with those obtained by other authors using a third method. The data obtained also established the normal range of optic nervehead displacements induced by a range of intraocular pressure increments.

Feasibility test of a new method to measure retinal thickness noninvasively.

There are many devastating ocular diseases that are directly related to an alteration of the retinal or nerve fiber layer thickness, such as glaucoma and macular edema. To diagnose these diseases earlier and to monitor their therapy more sensitively, an accurate measurement of the tissue thickness is needed. Since no clinical method is currently available, we developed and tested a new method capable of measuring noninvasively the retinal thickness. The separation between the images of the anterior and posterior intersections is quantitated by an optoelectronic system. The theoretical performance of the method has been calculated. Tests of the method in a model eye indicated that the measurements were basically diffraction limited, their reproductivity was +/- 9 microns, and their accuracy was 5.5 microns. Tests performed in vivo indicated that two intersections between the laser and the retina are present and correspond to the anterior and posterior surfaces of the retina. These intersections can be resolved and analyzed to yield quantitative data. These encouraging results indicate that this method is feasible and could yield sensitive measurements of the retinal thickness.

Evaluation and comparison of commercial vitreous fluorophotometers.

The Metricon vitreous fluorophotometer and two models by Gamma Scientific were evaluated. The lower limit of detection of the Metricon and the 2900 Gamma Scientific were found to be satisfactory. Because of the DR2 Gamma Scientific high dark noise, an external electronic filter had to be added to achieve a lower limit of detection, which was borderline for vitreous fluorophotometry. The error of the systems was found to be acceptable except for the DR2 Gamma Scientific at low concentrations. However, it was demonstrated that the determining factor for reliability is the level of artifactual readings. It was shown that the level of artifact is acceptable for the Gamma Scientific models but high for the Metricon, in which significant error could be induced in patients with borderline fluorescein leakage. Other technical aspects of the systems are discussed.

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.

Visualization of the retinal microvasculature by targeted dye delivery.

Although fluorescein angiography has proven to be an important tool in the diagnosis and management of retinal vascular diseases, it is subject to certain limitations, namely the presence of the choroidal background, which usually precludes a detailed examination of the retinal microvasculature. Moreover, the inability to repeat the bolus reduces the chance of obtaining high-quality photographs of early phases, and does not allow for a complete binocular examination or for testing the response to induced physiologic changes. We have developed a method of targeted dye delivery that consists of encapsulating the dye in lipid vesicles, injecting them intravenously, and causing them to release their contents locally when a short heat pulse is induced in a retinal artery by a laser. This method was applied in the rhesus monkey in order to visualize the retinal microvasculature. A well-defined bolus and absence of background fluorescence permitted both following of the dye front through the vasculature and clear imaging of the capillary network over the whole posterior pole. The bolus delivery could be repeated as many as 100 times in 45 min without significant loss of contrast. The comparison of these results with conventional fluorescein angiography illustrated the advantage of the new method. The examination of the safety of the delivery system indicates that there is no major obstacle to the eventual application to humans.

Quantitative analysis of retinal hemodynamics using targeted dye delivery.

A new method designed to allow repeated mapping of retinal hemodynamics on a macro- and microcirculatory level was evaluated in the primate eye. The method, called "targeted dye delivery," consists of encapsulating a fluorescent dye in temperature-sensitive liposomes, injecting the liposomes systemically, and using a light pulse from an argon laser to release a bolus of dye in a targeted retinal vessel. The follow-up of the well-defined dye front thus generated allows calculation of the blood flow and capillary transit time. Evaluation of targeted dye delivery in a monkey indicated that centerline blood velocity and the vessel diameter can be measured with a reproducibility of 10% and 4%, respectively, in vessels that are 40 microns and larger. These measurements yielded flow values that had a reproducibility of 10% on the same day and 13% on different days. The normalization of flow rate by the vessel diameter was consistent with theoretic estimates and promises to be a circulation indicator independent of variations between individual and species. The transit time across capillary beds at different locations was found to be similar, thus indicating that the method could be used to evaluate the local viability of the microcirculation.

A potential method for local drug and dye delivery in the ocular vasculature.

We propose a new drug and dye delivery system that would allow repeated release of substances in the ocular vasculature by an externally controlled mechanism. The substances are encapsulated in heat-sensitive liposomes, which are lysed by locally applying a heat pulse produced by an argon laser. The system was tested by investigating the release of carboxyfluorescein encapsulated in the liposomes. The liposome suspension was incubated at 37 degrees or 38.5 degrees C and irradiated at different powers and pulse durations. The amount of dye released was monitored by fluorophotometry and compared with the concentration obtained when the liposomes were lysed at their transition temperature of 41 degrees C. The results showed that 85% of the encapsulated substance can be released. Moreover, a dramatic contrast was observed between the fluorescence before and after the lysis. Presently the energy density is higher than but close to the maximal permissible exposure for humans. The release mechanism with the short laser pulse appeared to be similar to that present when liposomes were heated slowly.

Studies on the technique of vitreous fluorophotometry.

We examined the sources of artifacts of vitreous fluorophotometry (VF), a technique used to assess the permeability of the blood-retinal barrier. By performing in vitro measurements in a model eye and in vivo readings in humans, we investigated the influence of various concentrations of fluorescein in the anterior chamber and in the retina on the measurements performed in the vitreous. Upper limits for the artifactual effects are set under clinical conditions, and a way to account for them is proposed. Special attention is given to depth resolution, which was investigated as a function of the region being scanned, the beam width, and the probe size. Further, preliminary results indicate that a procedure to account for light attenuation by the lens is feasible. It appears that reliable quantitative results can be obtained by VF, provided that the artifacts are acknowledged, minimized, and corrected.

Feasibility of blood flow measurement by externally controlled dye delivery.

We are developing a new method of delivering substances locally and repeatedly in the retinal vasculature under external control. This delivery system is based on encapsulating the substance in heat-sensitive lipsomes, which are injected intravenously and lysed by a heat pulse delivered by a laser. The feasibility of using this system with dyes and creating a sharp dye front was tested in vitro and in vivo. The results indicate that the background fluorescence of intact liposomes is minimal but in contrast a dramatic increase in fluorescence is achieved where the dye is released. In vivo tests indicated that only the selected vascular branch fluoresced. Moreover, a sharp dye front could be obtained repeatedly and preserved over significant distances. The presence of a sharp dye front allowed measurements, in vitro, of blood velocity which correlated well (r = 0.985, P less than 0.001) with the average blood velocity values calculated from the known flow rate.

Feasibility of targeted drug delivery to selective areas of the retina.

A new method was developed to deliver locally a bolus dose of a drug to the retinal vasculature. The targeted delivery system was based on encapsulating the drug in heat-sensitive liposomes, which are injected intravenously and lysed in the retinal vessels by a heat pulse generated by a laser. To test if substances delivered in the vessels could also penetrate into the surrounding tissue, 6-carboxyfluorescein was encapsulated in liposomes and used as a marker for drug penetration. Moderate argon laser pulses were applied to the retinal vessels of Dutch pigmented rabbits to induce breakdown of the blood-retinal barrier (BRB). A suspension of liposomes at a dose of 2 ml/kg body weight, corresponding to a carboxyfluorescein dose of 12 mg/kg, was injected into the ear vein. The dye was released from the liposomes proximal to the damaged portion of the vessel. Fundus fluorescein angiograms were recorded with a video camera and digitized for subsequent image analysis. The penetration of carboxyfluorescein into the retinal tissue was evaluated by comparing the fluorescence intensity of the area around the damaged vessel with that of an adjacent control area. The dye penetration increased with the numbers of laser applications (P less than 0.001). The leakage was localized distally to the released site and was restricted to areas with a disrupted BRB. The mass of carboxyfluorescein that penetrated gradually spread with time. Both veins and arteries could be used for the targeted delivery. These results indicated that this delivery system, which is fully controllable by laser through the pupil, can deliver drugs inside the vasculature and into the retinal tissue wherever the BRB is disrupted.

Fluorescein and fluorescein glucuronide pharmacokinetics after intravenous injection.

The permeability of the blood-retinal and blood-aqueous barriers to fluorescein (F) and the rate of aqueous flow can be estimated by measurements of F in the vitreous, aqueous, and plasma after systemic administration. F is commonly measured by fluorescence, but fluorescein glucuronide (FG), a metabolite of F, also fluoresces. To assess the influence of FG on the quantitation of F by fluorescence, we studied the pharmacokinetics of F and FG for 38 hr in the plasma of five normal subjects given 14 mg/kg of sodium fluorescein intravenously. The plasma and the plasma ultrafiltrate were measured by fluorescence and by high performance liquid chromatography. In our fluorophotometer, FG was 0.124 times as fluorescent as F. F was rapidly converted to FG, and within 10 min the concentration of unbound FG exceeded that of unbound F. The terminal half-lives of F and FG in the plasma ultrafiltrate were 23.5 and 264 min, respectively, so that FG contributed almost all of the plasma fluorescence after 4-5 hr. Because FG was less bound in the plasma than F, the ratio of the fluorescence of the plasma ultrafiltrate to that of the plasma increased with time. The greatest proportion of the total F available to penetrate into the ocular compartments occurred shortly after injection. We concluded that FG is an important contributor to the fluorescence of the plasma ultrafiltrate after intravenous injection and that accurate quantitation of physiologic parameters calculated from the plasma F requires taking this factor into account.

An interactive model eye for use with ophthalmic instruments.

A model eye was found to be a practical and versatile simulator of conditions present in the application of different surgical and diagnostic ophthalmic instruments. It realistically simulated the thermal and acoustic effects of lasers on tissues and thus could be used for teaching and practicing laser therapeutics. The geometric optics were similar to those of the human eye, and realistic conditions of scatter and fluorescence could be created.

The relation between glaucomatous damage and optic nerve head mechanical compliance.

Histologic studies seem to imply that the mechanical compliance of the optic nerve head, namely, its displacement under a given pressure, may be altered in glaucoma. We have developed a method to noninvasively measure the optic nerve head displacement. In postmortem human glaucomatous eyes, the optic nerve head compliance decreased as the visual field worsened (n = 15, r = -.34). The mean difference between the optic nerve head displacement of the two eyes in subjects with symmetric clinical findings differed significantly from the mean in subjects with asymmetry. In the latter group, the lower value was always measured in the more affected eye. No significant correlation was found between age and the optic nerve head compliance. Overall, the results indicate that, in glaucoma, there is a stiffening of the mechanical support of the optic nerve head.

In vivo evaluation of a noninvasive method to measure the retinal thickness in primates.

To diagnose certain macular diseases earlier and monitor their therapy more sensitively, we are developing a noninvasive method to measure the retinal thickness. The new instrument, which is an extension of slit-lamp biomicroscopy, was used to obtain the data, which were analyzed with an algorithm to yield thickness measurements. The measurements performed in monkeys indicated that the retinal thickness can be visualized in a region extending from the optic disc to the fovea and that quantitative results can be obtained. The retinal thickness reproducibility was 6% for the same location on the same day, 15% for the same location on different days, and 12% for the same location in different eyes. The average retinal thickness in these areas was 335 microns, indicating that the reproducibility was between 20 and 50 microns. Measurements across the foveola illustrated that retinal thicknesses as low as 80 microns could be obtained.

Vitreous fluorophotometry for clinical research. I. Description and evaluation of a new fluorophotometer.

In vitro and in vivo tests of a new commercial fluorophotometer showed that the instrument is capable of measuring fluorescein concentrations of 1 to 1,000 ng/mL accurately. The in vivo estimate of the spread functions of the lens and chorioretinal peaks yielded values of about 1% and 0.5% 3 and 6 mm, respectively, from the peak. Pigmentation did not influence the chorioretinal spread function. Reproducibility in vivo was evaluated to be 12%. Furthermore, when compared with other existing fluorophotometers, the new instrument performed superiorly.

Vitreous fluorophotometry for clinical research. II. Method of data acquisition and processing.

Several methods of data analysis permit improved evaluation of the blood-retinal barrier (BRB) by vitreous fluorophotometry. With the use of an algorithm for artifact correction, tested in vivo in situations in which large artifacts are expected, artifacts were reduced to less than the equivalent of 1 ng/mL in normal eyes 3 mm from the chorioretinal and lens peaks. A procedure developed to evaluate the lens transmittance was found to have a 12% reproducibility. In addition, the anterior and posterior sources of leakage could be separated by choosing the appropriate time of measurement after injection of the dye. Moreover, a procedure to estimate quantitatively the inward permeability of the BRB yielded a preliminary estimate of 7.2 X 10(-8) cm/s in normal subjects.

Evaluation of vitreous body integrity in retinitis pigmentosa by vitreous fluorophotometry.

We performed vitreous fluorophotometry on 23 eyes of 15 normal individuals and on 29 eyes of 15 patients with retinitis pigmentosa one hour after the systemic administration of sodium fluorescein. The concentration gradient of fluorescein in the posterior vitreous was evaluated from the results of the testing. Patients with retinitis pigmentosa showed a significantly flat gradient compared with normal subjects, implying that fluorescein is distributed more rapidly in the vitreous body of the patients. It is suggested that patients with retinitis pigmentosa have an abnormal vitreous gel structure that causes more rapid distribution of fluorescein. If verified, this would indicate that vitreous fluorophotometry could be useful to evaluate changes in the integrity of the vitreous body in vivo.

Outward transport of fluorescein from the vitreous in normal human subjects.

The permeability of the blood-vitreous barrier to fluorescein passing out of the vitreous does not necessarily equal the permeability to fluorescein passing into it. We calculated the ratio between the outward and inward permeability coefficients of the blood-vitreous barrier in eight normal men who ingested 3-g of sodium fluorescein. The calculation was based on the ratio between the serum free fluorescein and the vitreous fluorescein concentrations (as determined by fluorophotometry) when the net transport across the barrier was zero. The outward permeability to fluorescein was 31 +/- 18 times (mean +/- SD) the inward permeability. To our knowledge, this article provides the first direct evidence for a specialized transport mechanism in humans whereby fluorescein is removed from the vitreous into the blood. The malfunction of this process may be important in human diseases. Pharmacologic manipulation of this process may be possible.

Transport of fluorescein in the ocular posterior segment in retinitis pigmentosa.

The function of the blood-retinal barrier was assessed by vitreous fluorophotometry in 12 patients with various genetic types of retinitis pigmentosa and in 11 normal subjects. The measurements were corrected to minimize the effect of artifacts. We evaluated the inward penetration of fluorescein sodium across the blood-retinal barrier and determined the outward permeability coefficient for fluorescein by interpreting the data with a pharmacokinetic computer model. Patients with retinitis pigmentosa had increased inward permeability and decreased outward permeability to fluorescein in comparison with values of normal subjects.