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The fluorescein distribution in the stomach of the rat with induced gastric cancer has been studied. In all cases, the level fluorescein fluorescence in the tumor was higher than in the adjacent tissues of the stomach. Thus, the data obtained confirm the selective accumulation of fluorescein in tumors of the stomach and agree with clinical results.
PURPOSE: To derive the value of corneal epithelial barrier to fluorescein in humans from experiments in which the fluorophore in instilled in a single drop. METHOD: A commercial scanning fluorophotometer, the Fluorotron Master, was used to scan through an anterior segment. It could not resolve the tear film from the cornea, but in the early stages of measurement, the tear component predominated. After 20 minutes, the remaining fluorescein was washed out of conjunctival sac, and the amount that penetrated into the cornea was estimated. Because the resolution was not sufficient to estimate the concentrations, the total masses in the tear film and in the cornea derived from the area under the profile were used to calculate the epithelial permeability. A value for the tear film thickness had to be assumed. RESULTS: The technique led to reproducible values for epithelial permeability-a range of 2:1 in a subject. High concentrations of fluorescein were required to achieve a sufficient penetration into the cornea, and this led to error in estimating the tear film concentration of the dye. A method is presented that corrects for this effect. For 17 normal subjects, the corrected permeability was 0.15 nm/second, and the tear turnover was 0.15/minute ( +/- 0.016, standard error.) CONCLUSIONS: This technique is convenient and yields two useful physiological parameters from a single clinical procedure. The permeability values are considerably higher than those found by previous workers, and the source of the discrepancy is discussed.
The objective of this study was to identify, through kinetic analysis of individual elementary reactions, the conditions under which a simple first-order photobleaching kinetic model is sufficient for quantitative fluorescence measurements, and those under which more complex photobleaching kinetics must be considered. Three model systems of various fluorophore densities and distributions were employed to verify the kinetic analysis. The results showed that the photobleaching kinetics of free fluorescein at concentrations lower than 5 microM corresponded closely to a single exponential function and therefore involved predominantly simple unimolecular or pseudounimolecular photochemical reactions. When fluorescein was bound to polyvinyl alcohol (PVA) molecules, the photobleaching kinetics of the densely labeled PVA deviated more from a single-exponential function than sparsely labeled PVA. When fluorescein was bound to a DNA probe, the photobleaching kinetics were more complex and deviated significantly from a single-exponential function, due to one or more bimolecular processes with apparent concentration-dependent photobleaching rate constants. The practical applications of time-integrated fluorescence emission are discussed in the context of simple and complex photobleaching kinetics.
Optic disc edema (ODE) due to chronic intracranial hypertension was produced experimentally in rhesus monkeys. Serial studies of fundus changes at frequent intervals, by routine ophthalmoscopy, steroscopic color photography, and fluorescein angiography, revealed that swelling of the optic disc was the first sign of ODE. Other early signs were striation of nerve fibers on the optic disc margins and peripapillary retina, blurring of the disc margins, hyperemia of the disc and capillary dilation, hemorrhages, and other retinal vascular changes; these usually appeared in that sequence. The classically described signs of early ODE were almost always absent. A normal fluorescein fundus angiogram during the incipient stage did not rule out ODE. Stereoscopic color fundus photography was the most sensitive means of detecting early ODE. Fluorescein angiography did not show changes till edema was of a mild to moderate degree; routine ophthalmoscopy was the least reliable method.
An improved technique for high-contast, high-resolution fluorescein angiography of the optic disc has been developed that almost eliminates pseudofluorescence. Fluorescein angiography of the optic disc was performed on normal, ocular hypertensive, and glaucomatous patients. Rapid-sequence angiograms in the early arterial phases have demonstrated localized areas of hypofluorescence or filling defects of the optic disc. Two types of fluorescein filling defects were observed-absolute and relative. The number of absolute filling defects, which increased with degree of visual field loss, was greater in glaucomatous than in ocular hypertensive or normal eyes. Similarly, ocular hypertensive eyes showed a larger number of discs with filling defects than normal eyes. It is postulated that relative defects progress to absolute filling defects, which may be an indication of impending loss of visual field.
Fluorescein angiography of the optic disc was performed on normal subjects, patients with nonmyopic open-angle glaucoma dna myopic open-angle glaucoma, normal myopes, patients with optic atrophy due to chiasmal pituitary tumors, and patients with sectorial ischemic optic neuropathy due to vascular hypotension. The normal myopes, those with optic atrophy secondary to pituitary tumor, and the ischemic optic neuropathy group had optic discs similar in appearance to those with open-angle glaucoma. Absolute fluorescein filling defects occurred only in patients with open-angle glaucoma and sectorial ischemic optic neuropathy. The filling defects in open-angle glaucoma appear to be specific, indicating that the blood supply to the anterior portion of the optic nerve is involved, as in ischemic optic neuropathy. Fluorescein angiography of the optic disc may be useful to differentiate open-angle glaucoma from other entities that have similar optic discs.
Fluorescein iris angiography was performed in 42 normal blue or green human eyes to investigate the normal angiographic pattern and in vivo circulation of the iris. Brown irides were unsuitable for the angiography because the brown pigment masked fluorescence. The pattern in the iris was much more complex than in the retina when seen on fluorescein angiography, with interindividual variations in its filling, and these physiological variations may erroneously be considered pathological. Radial arteries of the iris filled rather sluggishly and the circulation in the pupillary and peripupillary region was often much slower than in the peripheral part of the iris. Various segments of the iris usually filled in an irregular order. There was no distinct venous phase, but it merged with the arteriovenous phase. The pupillary region of the iris between the collarette (which contained a fragmented and incomplete minor circle of vessels) and the pupillary margin contained a dense plexus of capillaries. The normal vessels usually showed no fluorescein leakage.
Iris fluorescein angiography was performed on 23 patients with primary iris melanocytic tumors. Four angiographic patterns were identified, the first three of which were considered to be usually indicative of a benign lesion, based on clinical duration and follow-up, biopsy specimens, and stable patterns on repeated angiography. (1) Eleven moderately to heavily pigmented placoid lesions were angiographically silent, in that they failed to display either a tumor-associated vasculature or diffuse leakage. (2) Seven generally nonpigmented lesions demonstrated a quasi-geometric filigree vascular network, approximating the caliber of the normal radial vasculature. These tumor-associated vessels fluoresced early and in synchrony with the appearance of dye in the radial vasculature from which they probably were derived, but they diffusely and confluently leaked fluorescein in the late phases of the angiogram. (3) Three brown or variably pigmented lesions manifested a mixed angiographic pattern, combining features of groups 1 and 2. (4) The final angiographic pattern was comprised of two pathologically proved mixed spindle-epithelioid cell melanomas, each of which in part or in toto showed diffuse and eventually confluent fluorescence emanating from ill-defined vascular foci. One of these two lesions additionally exhibited a large area that was perfused late by irregular leashes of vessels. The ability to distinguish benign from malignant melanocytic lesions of the iris can be greatly enhanced by iris fluorescein angiography.
Fluorescein angiography carries with it a variable incidence of nausea and vomiting. We investigated a method of prophylaxis against this side effect. One hundred patients undergoing fluorescein angiography were pretreated in a double-masked, randomized fashion with either 20 mg of intravenous metoclopramide hydrochloride or an equal volume of normal saline solution. The metoclopramide-treated group demonstrated a statistically significant decrease in the incidence of nausea and vomiting. Eleven (22%) of the control group and three (6%) of the metoclopramide-treated group had this complication. Metoclopramide is an effective drug when used prophylactically in selected patients undergoing fluorescein angiography.
Laser Doppler velocimetry provides a quantitative and noninvasive measurement of cutaneous blood flow. Piglets were used to compare laser Doppler velocimetry and fluorescein with each other and as predictors of flap viability. Twenty-five random pattern rectangular skin flaps were studied with both techniques. Eighteen of the flaps were observed for two weeks and the length of viable flap documented. A reading of 100 mV on the laser Doppler corresponded with full fluorescence after dye injection. Both fluorescein and the laser Doppler underestimated flap surviving length slightly. Laser Doppler readings taken 24 hours postoperatively predicted the length of surviving flap exactly. The laser Doppler correlated well with fluorescein dye.
The fluorescein test is widely used in clinical and experimental studies for prediction of the viability of skin flaps. A thorough search of the literature revealed that the accuracy of this test had never been studied. Therefore, this study was designed to evaluate the accuracy of the fluorescein test in predicting the viability of skin flaps. A total of 212 unipedicle porcine skin flaps were used in this study. Statistical analysis demonstrated that the surviving area of the flap was significant greater than the staining area of the flap. We suggest that caution be exercised when the fluorescein dye test is used to assess skin flap viability and that the test be used for an approximate indication and not for an accurate prediction of the surviving flap area.
The artificial lipocalin FluA with novel specificity toward fluorescein was derived via combinatorial engineering from the bilin-binding protein, BBP by exchange of 16 amino acids in the ligand pocket. Here, we describe the crystal structure of FluA at 2.0 A resolution in the space group P2(1) with two protein-ligand complexes in the asymmetric unit. In both molecules, the characteristic beta-barrel architecture with the attached alpha-helix is well preserved. In contrast, the four loops at one end of the beta-barrel that form the entrance to the binding site exhibit large conformational deviations from the wild-type protein, which can be attributed to the sidechain replacements. Specificity for the new ligand is furnished by hydrophobic packing, charged sidechain environment, and hydrogen bonds with its hydroxyl groups. Unexpectedly, fluorescein is bound in a much deeper cavity than biliverdin IX(gamma) in the natural lipocalin. Triggered by the substituted residues, unmutated sidechains at the bottom of the binding site adopt conformations that are quite different from those observed in the BBP, illustrating that not only the loop region but also the hydrophobic interior of the beta-barrel can be reshaped for molecular recognition. Particularly, Trp 129 participates in a tight stacking interaction with the xanthenolone moiety, which may explain the ultrafast electron transfer that occurs on light excitation of the bound fluorescein. These structural findings support our concept of using lipocalins as a scaffold for the engineering of so-called "anticalins" directed against prescribed targets as an alternative to recombinant antibody fragments.
The fluorescein diacetate test is a measure of both esterase enzyme activity and membrane integrity and it has been shown to be useful in assessing the viability of a variety of cells cultured in vitro, mouse embryos after freezing and thawing, and mouse embryos grown under inadequate culture conditions. In this study, the effects were examined of cyclophosphamide and sodium valproate administered respectively to pregnant inbred CBA mice 60 h after fertilization, on the viability of 84-h blastocysts. Cyclophosphamide 20 and 40 mg/kg bodyweight and sodium valproate 90 mg/kg significantly increased the number of nonviable blastocysts. Cyclophosphamide 4 mg/kg and sodium valproate 23 and 45 mg/kg did not adversely affect blastocyst viability. The intensity of embryo fluorescence correlated well with the subsequent development of the embryos in culture (r = 0.863; p less than 0.001). The test had a sensitivity of 83% and a specificity of 100%. Exposure of embryos to fluorescein diacetate under the conditions of this experiment did not adversely influence the subsequent postimplantation development of 84-h blastocysts cultured in vitro for a further 120 h. These findings suggest that the fluorescein diacetate test is a simple, rapid, and nontoxic procedure that may be useful in assessing the viability of preimplantation embryos after exposure to embryotoxic drugs and chemicals.
A protocol is described for coupling of carrier-free iodine to protein sulfhydryl groups via fluorescein maleimide. 125I is first coupled to fluorescein maleimide in the presence of chloramine T. Iodination is stopped with sodium thiosulfate, and the iodine-substituted fluorescein maleimide is reacted with free cysteines of the protein. Excess label is then removed by gel-permeation chromatography. The procedure avoids exposition of the protein to oxidative conditions and does not require purification of the labeled carrier reagent. Suitability of the method for a given protein can be evaluated spectrophotometrically without employing radioactivity. It can be applied under denaturing conditions and may be particularly useful with mutant proteins carrying engineered single cysteine residues at sites that are not functionally critical.
Mechanisms of cell death induced by toxins probably involve one or more processes such as inhibition of protein synthesis and impairment of plasma membrane integrity leading to an increase in membrane permeability. Since one of the possible actions of mastoparan, a cationic tetradecapeptide from wasp venom, is to perturb membrane phospholipids resulting in an increase in membrane permeability, we studied the effect of chemically synthesized mastoparan on lactate dehydrogenase release (LDH), ethidium bromide and fluorescein accumulation in Madin-Darby Canine Kidney (MDCK) cultured cells. Our results demonstrated that mastoparan induced cytosolic LDH release, ethidium bromide accumulation and intracellular fluorescein depletion in MDCK cells. Neomycin, a polycationic aminoglycoside, interacts with anionic polyphosphoinositides at the plasma membrane. Since both mastoparan and neomycin are cationic peptides and react with the negatively charged membrane phospholipids, we studied the interaction of these two peptides on membrane permeability. Our results demonstrated that neomycin inhibited mastoparan-induced LDH release, ethidium bromide accumulation and intracellular fluorescein depletion.
Previous reports have suggested that pregnancy does not influence the development of diabetic retinopathy. Since 1979 a prospective study using fluorescein angiography has been in progress. Preliminary results include 22 diabetic women with or without moderate background retinopathy studied until delivery. They were maintained under good metabolic control (mean HbA1: 7.68%). Five clinical and fluorescein angiography examinations were performed at prefixed intervals (before the 15th week, at the 28th and 35th weeks of pregnancy, and 6 and 15 months after delivery). The analysis of fluorescein angiography which did not induce any side effect showed that the mean number of microaneurysms increased from 42.7 before pregnancy to 56.7 at the 28th week and to 79.7 at the 35th week. Six months later the number was 62.7, and 15 months later 60.3, still higher than at the initial examination. This study suggests that pregnancy has an unfavourable influence on the background retinopathy in diabetic patients even when a good metabolic control is achieved and retinopathy is minimal.
Morphine (5 mg/kg, s.c.) caused a submaximal increase in the brain level of sodium fluorescein administered i.v. Histamine H1-antagonists, diphenhydramine and mepyramine, given either i.p. or i.c.v., had no significant influence on the effect of morphine. H2-Antagonists, cimetidine and ranitidine, administered i.c.v., but not i.p., significantly inhibited the morphine effect. alpha-Fluoromethylhistidine, a specific histidine decarboxylase inhibitor (given i.p. and i.c.v.) and antimuscarinic drugs, atropine and biperiden, but not methylatropine (given i.p.) also significantly reduced the morphine effect. Physostigimine (i.p.) significantly enhanced the effects of 0.5 and 1 mg/kg of morphine. Similar effects of histaminergic and cholinergic drugs were also observed on the buprenorphine- and DAGO-induced increase in blood-brain barrier (BBB) permeability to sodium fluorescein. None of the treatments with 6-hydroxydopamine, alpha-methyltyrosine, 5,7-dihydroxytryptamine or p-chlorophenylamine had any significant effect on the morphine-induced increase in BBB permeability. These findings suggest that the activation of brain H2-receptors by neuronal histamine and muscarinic receptors by acetylcholine is involved in the increase in BBB permeability to sodium fluorescein caused by mu opioid receptor agonists.
Iontophoresis is less apt to prove the existence of outflow channels of aqueous humour, but it is appropriate in demonstrating filtering blebs. After injecting fluorescein directly into the anterior chamber, the outflow channels are clearly marked in enucleated non-glaucomatous eyes, as well as in eyes containing a tumor, prior to enucleation. Regularly, one is able to observe sectors in which the aqueous veins are filled slowly or not filled at all. Injecting fluorescein directly in Schlemm's canal proves that in cadaver eyes without glaucoma the lumen is open all around. Sectorial filling defects of vessels leading out of Schlemm's canal are often seen. In three patients with simple glaucoma blocking of dye was noticed during passage through Schlemm's canal without reappearance. Our experiments give the impression that segmental division of Schlemm's canal is more marked in eyes with glaucoma than in normal eyes. If local occlusions within Schlemm's canal are of importance for the range of lowering of intraocular pressure, one must differentiate between the trabecular and the intrascleral part of the resistance of outflow in the operation field, in order to choose the best operating procedure. Such a differentiation seems, in principle, possible using fluorescein--primarily injected into the anterior chamber directly, and secondarily into a given part of Schlemm's canal.