Morphologic correlations with fluorophotometric data from monkey eyes with anterior uveitis.

Acute anterior uveitis was induced in monkeys by unilateral intravitreal injection of 1.0 ng of Escherichia coli endotoxin. Twenty-four hours later, each animal received an intravenous injection of 250 mg/kg body weight of fluoresceinated horseradish peroxidase (F-HRP), and fluorophotometric measurements were taken for 90 min. The animals were killed, and both eyes were processed for HRP demonstration. In the anterior chamber aqueous humor of normal control eyes, F-HRP concentrations were less than 0.002 mg/ml at 90 min. The F-HRP concentration was elevated consistently in the endotoxin-injected eyes; however, the magnitude of the effect varied. By fluorophotometry, inflamed eyes fell into two distinct groups. At 90 min, most had an anterior chamber F-HRP concentration of 0.014-0.06 mg/ml, although others had 0.39 mg/ml. In the latter group, an appreciably shorter latency was observed between the time of tracer injection and its detection in the anterior chamber. Aqueous humor protein concentrations, although highest in the most F-HRP-permeable eyes, followed more of a continuum in their distribution and identified less clearly the subpopulations seen by fluorophotometry. Normal eyes had no tracer leakage across either the ciliary epithelial or iris vascular endothelial barriers. All inflamed eyes had HRP leakage across the ciliary epithelium, but the subpopulation of eyes with shorter latencies and higher F-HRP concentrations by fluorophotometry also had iris vascular leakage.

The source of protein in the aqueous humor of the normal monkey eye.

In vivo aqueous fluorophotometry, morphology, and computational modeling were combined to examine the source of protein and the pathway by which protein enters the aqueous humor of monkeys. A computational model was developed to determine the likelihood of a diffusional route for delivering plasma proteins from ciliary body capillaries via the iris to anterior chamber aqueous humor, bypassing the posterior chamber. Model predictions were compared to aqueous fluorophotometric data obtained from monkeys following a single intravenous injection of fluoresceinated horseradish peroxidase (F-HRP, 250 mg/kg body mass). Model predictions of the magnitude and time course of anterior chamber F-HRP concentration agree with the fluorophotometric measurements. For example, of anterior chamber F-HRP concentration as a percentage of initial plasma F-HRP concentration at 90 min and 180 min post-injection was predicted to be 0.02% and 0.05%, respectively, and was measured to be 0.01-0.03% and 0.03-0.06%, respectively. In addition, model predictions in the case of a constant plasma protein level also are consistent with experimental data. The steady-state anterior chamber total protein concentration as a percentage of plasma protein concentration was predicted to be 0.2% and was assayed to be 0.05-0.2%. As in our previous study of the normal rabbit eye, morphologic and tracer localization evidence combined with the good agreement between model predictions and experimental data lead to the conclusion that a significant amount of the plasma protein normally present in monkey aqueous humor originates in ciliary body capillaries and diffuses anteriorly through the iris and into the anterior chamber.

Background-protein effects on fluorophotometric data.

Fluorescent tracers are commonly used in fluorophotometric studies of ocular fluids and tissues that contain background protein. Background-protein concentrations were found to decrease or increase significantly the measure of fluorescence emitted from solutions containing sodium fluorescein, fluorescein-labeled dextran, or fluorescein-labeled horseradish peroxidase. The effect of background protein on fluorescence was expressed as a function of the specific fluorescent tracer, tracer concentration, and background-protein concentration; it can be corrected in the analysis of fluorophotometric data. Fluorophotometric studies--particularly those in which the background-protein level is expected to be abnormally high, such as postoperative and pathologic studies--may need to include either a data correction based on measured effects of background protein on tracer fluorescence or, in the case of clinical investigations, recognize at least the potential for a range of possible interpretations.

The source of proteins in the aqueous humor of the normal rabbit.

Aqueous fluorophotometric, tracer localization and modeling methods were combined to document the existence of a pathway in the normal rabbit for the diffusion of proteins from the ciliary and iridial process stromas through the iris stroma into the aqueous humor of the anterior chamber. A new custom-conjugated tracer, fluoresceinated horseradish peroxidase (F-HRP), was used. Anesthetized rabbits were injected intravenously with F-HRP (250 mg/kg). In some animals, aqueous fluorophotometric and tracer localization studies were performed on the same eyes. Anterior chamber fluorescence was detected 2-10 min post-injection and rose to concentrations of 0.01-0.05 mg/ml 60 min post-injection. Subsequent tracer localization studies of these eyes revealed that the morphologic components of the blood-aqueous barrier were intact, that is, no leakage of F-HRP from the iris vasculature or across the ciliary epithelium was observed. Separate tracer localization studies were performed to examine the time course of the route(s) by which tracer entered the anterior chamber. These studies revealed a "wave" of tracer that migrated from the ciliary and iridial process stromas, through the iris, and arrived at the anterior iris surface approximately 8 min post-injection. A pharmacokinetic model based on the diffusional pathway was developed to describe the time course of the concentration of plasma macromolecules in the ciliary body, iris and anterior chamber. Model predictions were consistent with aqueous fluorophotometric and tracer localization results. The diffusion model can account for a major fraction of protein entering the aqueous humor of normal rabbit eyes.