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Fluorescein angiography of the iris in the management of eyes with central retinal vein occlusion.

PURPOSE: To study fluorescein angiography of the iris as a means of identifying eyes at risk for developing secondary glaucoma after central retinal vein occlusion. METHODS: Fluorescein angiography of the iris was performed with a scanning laser ophthalmoscope in 27 patients (27 eyes). Individual images were by computer, determining relative areas of fluorescein staining and leakage. RESULTS: The best predictor of secondary glaucoma was the relative area showing staining. Neither relative area of leakage nor presence of iris rubeosis, age, or initial visual acuity were effective means of predicting which eyes would develop secondary glaucoma. This occurred in six eyes; all were treated with argon laser photocoagulation. No eye was enucleated. CONCLUSION: Iris angiography can identify eyes at risk for secondary glaucoma but specificity is poor. The often recommended regime to perform laser photocoagulation in all eyes with iris neovascularisations was challenged.

Adult↗

A new method of image analysis of fluorescein angiography applied to age-related macular degeneration.

Quantitative analysis of retinal and choroidal abnormalities using current photographic techniques is complex and laborious. Digital image analysis techniques using the scanning laser ophthalmoscope overcome many of the problems with present techniques and allow reliable quantitation. A prerequisite of quantitation is accurate image acquisition and registration. The authors describe a reliable method of image analysis and apply it to the quantitation of hyperfluorescence in scanning laser fluorescein angiograms of different forms of age-related macular degeneration. Retrospective analysis of scanning laser fluorescein angiograms obtained using a standardised technique was undertaken. Eighty-six angiograms from patients with age-related macular degeneration were analysed and categorised as dry maculopathy, geographic atrophy of the retinal pigment epithelium, retinal pigment epithelial detachment (PED) or subretinal neovascularisation (SRNV). Fluorescein characteristics of both SRNV and PED showed a characteristic pattern of fluorescence. The advantages and disadvantages of the technique are discussed.

Aged↗

Fluorescein angiography-guided indocyanine green angiography for the detection of feeder vessels in subfoveal choroidal neovascularization.

AIMS: To compare the newly devised fluorescein angiography (FA) - guided indocyanine green angiography (ICGA) with conventional ICGA for detecting feeder vessels in subfoveal choroidal neovascularization (CNV). METHODS: The detection of feeder vessels was attempted in 86 AMD patients with subfoveal CNV: 44 (age 70.4+/-4.5 years) underwent conventional ICGA (control group) and 42 (age 70.9+/-4.0 years) underwent an FA-guided ICGA (FA-guided group) using a double-detector scanning laser ophthalmoscope (SLO). In the control group, indocyanine green (ICG) was injected simultaneously with fluorescein. The patients were instructed to gaze forward localizing the fovea at the centre. In the FA-guided group, fluorescein sodium was injected a few minutes prior to ICG, and the patients were instructed to fixate in the appropriate direction by referring to the ongoing FA on the monitoring screen. In both groups, a 20 degrees visual angle was used to capture good images of feeder vessels in ICGA and, in case ICGA missed the first images of the entire CNV filling, an additional injection of ICG was given in the late phase to record the choroidal filling again. The overall detection rate, single-injection detection rate, double-injection rate and examination time were analysed using Fisher's direct exact probability test or Mann-Whitney's U-test. RESULTS: There was no significant difference in the overall detection of feeder vessels between the two groups (50% in the control group and 52.3% in the FA-guided group; P=0.49 with Fisher's direct exact probability test). However, in the FA-guided group, the single injection detection rate was significantly higher (45.1 and 15.9%, respectively; P<0.001 with Fisher's direct exact probability test); significantly less double injections were required (7.1 and 50%, respectively; P=0.003 with Fisher's direct exact probability test); and significantly shorter examination times were needed (9.6+/-3.7 and 14.1+/-6.8 min, respectively; P=0.02 with Mann-Whitney's U-test). CONCLUSION: FA-guided ICGA is effective for detecting feeder vessels of subfoveal CNV, minimizing the amount of ICG injected and the examination time compared to conventional ICGA.

Aged↗

[Absolute filling defects of the optic disc in fluorescein angiograms in glaucoma--a retrospective clinical study].

BACKGROUND: Analysis of clinical importance of the size of filling defects in fluorescein angiograms in primary open-angle glaucoma (POAG), normal-tension glaucoma (NTG), ocular hypertension and subjects with physiological excavations in comparison to visual field loss, optic nerve head morphology and hemodynamics. PATIENTS AND METHODS: 75 patients (POAG, NTG, ocular hypertension) and 10 healthy subjects with physiological excavations were included in this study. In digitized video fluorescein angiograms (Scanning Laser Ophthalmoscope) the size of absolute filling defects of the optic disc was quantified in the early venous phase and expressed by percentage of the optic disc. Visual fields were obtained by conventional static perimetry (Humphrey 24-2) and graded in stages of glaucoma visual field defects (Aulhorn I-V). Optic disc excavations were evaluated as cup-to-disc-area-ratios. RESULTS: The filling defects correlated with the visual-field loss stages of Aulhorn and the visual field indices MD (mean deviation), PSD (pattern standard deviation) and CPSD (corrected pattern standard deviation). There was no correlation with the index SF (short-term fluctuation) and with systemic hemodynamics (blood pressure, perfusion pressure) or the IOP. Absolute filling defects correlated with the cup-to-disc-area-ratio in NTG. The absolute filling defects were larger in patients with glaucoma (POAG, NTG) in comparison to patients without glaucomatous visual field loss (ocular hypertension, glaucoma-like discs). No difference of filling defects was found in the glaucoma group (POAG, NTG). Patients with NTG had larger excavations and lower systolic blood pressures than patients with POAG. CONCLUSION: The size of fluorescein filling defects may be useful as a parameter for the evaluation of an ischemic lesion of the optic nerve head. Absolute filling defects may differentiate POAG from ocular hypertension and NTG from glaucoma-like discs without field defects. The results support the hypothesis that in POAG and NTG disturbances of the circulation result in similar filling defects of the optic disc and visual field loss.

Adult↗

Fluorescence endoscopy using a fluorescein-labeled monoclonal antibody against carcinoembryonic antigen in patients with colorectal carcinoma and adenoma.

BACKGROUND AND STUDY AIMS: Various methods of fluorescence excitation and detection have been developed in gastrointestinal endoscopy. This study reports an endoscopic technique using locally applied fluorescein-labeled antibodies for in-vivo detection of colorectal dysplasia and carcinoma. PATIENTS AND METHODS: Fluorescence endoscopy with a fluorescein-labeled monoclonal antibody against carcinoembryonic antigen (CEA) was carried out in 27 patients with colonic polypoid lesions. During conventional colonoscopy, the monoclonal antibody was applied directly onto the mucosal surface. After an incubation time of 10 min, specific fluorescence was visualized with a conventional endoscope whose optical range was increased via two narrow-band filters. RESULTS: Fluorescence in vivo was present in 19 out of 25 carcinomas and in three of eight adenomas. The technique failed in the presence of mucosal ulceration or bleeding. One fluorescence-positive villous adenoma showed high-grade dysplasia, and another fluorescence-positive polypoid lesion was diagnosed as carcinoma in adenoma. Normal-appearing mucosa was fluorescence-negative in all cases. Endoscopic fluorescence significantly correlated with the CEA expression of luminal epithelial cells as determined immunohistochemically (Wilcoxon-Mann-Whitney U-test, P < 0.01). In all cases without ulceration or bleeding, the specificity of fluorescence endoscopy was 100%, the sensitivity was 78.6%, and the accuracy was 89.3%. CONCLUSIONS: Fluorescence endoscopy using fluorescein-labeled monoclonal antibody against CEA was shown to be positive in most cancers and some adenomas. Further and larger studies will be needed to demonstrate the value of this technique for differential diagnosis.

Adenoma↗

Low-molecular-weight heparin and dermatan sulfate end group-labeled with tyramine and fluorescein. Biochemical and biological characterization of the fluorescent-labeled heparin derivative.

To improve the understanding of the biological functions and pharmacology of heparin and dermatan sulfate, low-molecular-weight heparin (LMWH) and low-molecular-weight dermatan sulfate (LMWDS) were labeled with tyramine (T) by covalently linking T to the terminal residue of 2,5-anhydromannose (or 2,5-anhydrotalose for dermatan sulfate). The covalent labeling was demonstrated by nuclear magnetic resonance spectroscopy. The tyramine-labeled LMWH (LMWH-T) was also labeled with fluorescein (F) by further reacting it with fluorescein isothiocyanate. The fluoresceinated LMWH-T (LMWH-T,F ) was used to analyze biological functions on blood coagulation and binding to leukocytes. The biological activities on factor Xa and thrombin inhibition remained unchanged compared with the parent compound. Flow cytometric analysis of leukocytes demonstrated binding of the modified heparin to granulocytes, monocytes, and lymphocytes, the half-live being twice as long as the antifactor Xa activity. F-labeled heparin was displaced by unlabeled heparin from all three populations of leukocytes. Binding of heparin to leukocytes may play an important role in inflammation and atherosclerosis.

Animals↗

"Plasmatic imbibition" in the rabbit flow-through venous flap, using horseradish peroxidase and fluorescein.

In the present study, the phenomenon of "plasmatic imbibition" was investigated histologically in a rabbit flow-through venous flap model, using horseradish peroxidase and fluorescein. Horseradish peroxidase introduced between the flap and the recipient bed, produced brown staining of the entire contact surface 10 min postoperatively. At 30 min, erythrocytes in the flow-through vein stained red. The flap and the erythrocytes became more intensely stained over time. When horseradish peroxidase was introduced from a central artery after a Silastic sheet had been inserted between the flap and recipient bed, the flap color became a soft brown and the erythrocytes of the flow-through vein stained red 10 min postoperatively. In the experimental groups subjected to fluorescein, introduced between the graft (or flap) and the recipient bed, fluorescence was seen 10 min postoperatively, except in the flow-through vein. At 30 and 120 min, the whole graft (or flap) showed fluorescence. Fluorescein was introduced into the femoral vein after flap elevation, or a Silastic sheet was inserted between the flap and the recipient bed. At 10 min, these groups showed a weak fluorescence only in the flow-through vein; subsequently, at 120 min, the flow-through vein and its surrounding tissue showed fluorescence, but the flap showed none. Plasmatic imbibition was proved to be acting in the flow-through vein, and it is considered as important a phenomenon in flap survival as venous flow itself.

Animals↗

[Simultaneous digital indocyanine green and fluorescein angiography].

BACKGROUND: The simultaneous recording of indocyanine green and fluorescein angiography is since more than twenty years possible. The real-time digital recording of angiograms is about since 10 years possible. Because of the poor temporal resolution due to technical limitations, both of the above methods were only seldom used routinely. Graphics workstations can digitize and store multiple video sources real-time with full video framerate. Comparing to conventional videoangiography, the quality can be improved significantly. MATERIALS AND METHODS: Simultaneous ICG and Fluorescein (SIF) angiography images of a two-channel Scanning Laser Ophthalmoscope were digitized and stored real-time at a rate of 28-50 images/sec using a graphics workstation. The digitized image sequence could be replayed as a motion picture and/or single images of the recorded sequence could be analyzed separately. RESULTS: The simultaneous digital recording of angiograms is easier, than using two video tape recorders. It was possible to enhance the quality of the stored images using standard digital image processing procedures. Morphological measurements and densitometry were done with software developed for the UNIX workstation, or with personal computers using standard software packages. CONCLUSION: Simultaneous digital ICG and fluorescein angiography has several advantages over conventional video-angiographies: the image quality is better, data retrieval is easier, it is easier to perform than conventional video angiography, image can be accessed through a computer network, it is easy to perform morphological and densitometrical measurements, etc.

Angiography↗

[Digital fluorescein angiography in follow-up of the clinical course in therapy of patients with central vein occlusion and cystoid macular edema].

BACKGROUND: Fluorescein angiography with a scanning laser ophthalmoscope allows the quantification of morphologic and dynamic changes in the retina. In two patients with central retinal vein occlusion we monitored the progression of the cystoid macular edema (CME) and changes of microcirculation with a follow up of 4 to 6 months. MATERIALS AND METHODS: Two patients with central retinal vein occlusion and CME were examined by means of digital fluorescein angiography. Arteriovenous passage times and macular capillary blood velocities in combination with quantifications of the macular angioarchitecture and the extent of the CME were evaluated. RESULTS: Hemodilution therapy improved retinal circulation, whereas macular circulation and the severity of the CME remained unchanged. With persisting CME oral acetazolamid therapy was initiated. Oral Acetazolamide led to resorption of the cystoid edema in tandem with improved macular circulation. The capillary density was altered but remained unchanged over time. CONCLUSIONS: By means of digital analysis of fluorescein angiograms a detailed monitoring and retinal and macular dynamics, macular morphology is possible. In these two cases CME improved after Acetazolamide and moreover retinal circulation returned to normal conditions.

Acetazolamide↗

[The importance of fluorescein angiography in the diagnosis of Stargardt's mascular degeneration (author's transl)].

A comparison between the fundus photographs and the fluorescein angiogram at the beginning of this disease shows the great informative value of fluorescein angiography. At first the distinct functional losses are most important; ophthalmoscopically, only slight pigment irregularities are discernible. In the fluorescein angiogram the picture of fundus flavimaculatus is already distinguishable at this stage of the disease. These findings led support to the view that Stargardt's macular degeneration and fundus flavimaculatus are identical.

Adolescent↗

[Fluorescein angiographic findings in retinal venous thromboses (author's transl)].

In addition to documenting pathologic vessel changes fluorescein angiography also makes it possible to show hemodynamic changes in retinal thrombosis. An interruption of laminary flow can be seen in the fluorescein angiogram at arteriovenous intersections. The retinal circulation time can be an aid to prognosis. If the interval does not exceed four seconds the prognosis is generally favorable but when it is considerably longer the prognosis with regard to visual acuity is generally poor and, furthermore, sequelae such as rubeosis iridis and secondary glaucoma must be expected. The therapeutic efficacy of laser coagulation can be judged more accurately by photographic and fluorescein angiographic documentation of the course.

Fluorescein Angiography↗

[Differential diagnosis of blurred optic disks by fluorescein angiography (author's transl)].

Fluorescein angiography has been underrated as an exploratory tool in the diagnosis of blurred optic disks. The ten cases presented here had blurred disks of diverse etiology; fluorescein angiography helped in the differential diagnosis and therapeutic approach. Irrigation of the disk, type, magnitude and location of dye leakage, as well as objectivation of peripapillary and retinal vascular characteristics all help to explain the pathogenic mechanisms leading to different types of optic disk edema. Although it is known tha papilledema due to increased intracranial pressure can not be differentiated from locally caused blurring of disks, there remain a number of mechanical, ischemic and inflammatory conditions that can be diagnosed by means of fluorescein angiography. Thus, optic neuritis, ischemic optic neuropathy, papillovasculitis and juxtapapillary chorioretinitis are some of the diseases in which chorioretinitis are some of the diseases in which angiography may help in differential diagnosis and lead to adequate therapeutic approaches.

Adolescent↗

Nuclear localization of pyrrole-imidazole polyamide-fluorescein conjugates in cell culture.

A series of hairpin pyrrole-imidazole polyamide-fluorescein conjugates were synthesized and assayed for cellular localization. Thirteen cell lines, representing 11 human cancers, one human transformed kidney cell line, and one murine leukemia cell line, were treated with 5 microM polyamide-fluorescein conjugates for 10-14 h, then imaged by confocal laser scanning microscopy. A conjugate containing a beta-alanine residue at the C terminus of the polyamide moiety showed no nuclear localization, whereas an analogous compound lacking the beta-alanine residue was strongly localized in the nuclei of all cell lines tested. The localization profiles of several other conjugates suggest that pyrrole-imidazole sequence and content, dye choice and position, linker composition, and molecular weight are determinants of nuclear localization. The attachment of fluorescein to the C terminus of a hairpin polyamide results in an approximately 10-fold reduction in DNA-binding affinity, with no loss of binding specificity with reference to mismatch binding sites.

Active Transport, Cell Nucleus↗

Direct visualization of receptors for thyrotropin-releasing hormone with a fluorescein-labeled analog.

Thyrotropin-releasing hormone (TRH) binds to specific receptors on GH4C1 pituitary tumor cells. A fluorescently labeled analog of TRH was synthesized by coupling pGlu-His-ProNH(CH2)6NH2 to fluorescein isothiocyanate. The fluorescein-labeled peptide (FL-TRH) stimulated prolactin synthesis and release by GH4C1 cultures and bound to TRH receptors with an apparent Kd of 400 nM. Binding of FL-TRH to unfixed, viable GH4C1 cells was followed by fluorescence microscopy. After incubation with 1.4 microM FL-TRH for 1 hr at 37 degrees C, the surface of all cells was fluorescent and patches of intense fluorescence were evident. Control cultures incubated with FL-TRH and excess TRH were not fluorescent, and a line of pituitary tumor cells which lacks TRH receptors displayed little fluorescence after incubation with FL-TRH. When GH4C1 cells were incubated with FL-TRH for 1 hr at 37 degrees C and then with excess TRH for an additional 1 hr, the fluorescence associated with the cells was diminished to control levels. The results demonstrate that the fluorescein-labeled peptide labels specific TRH receptors.

Animals↗

Production and characterization of recombinant human plasminogen(S741C-fluorescein). A novel approach to study zymogen activation without generation of active protease.

A variant of recombinant plasminogen with the plasmin active site serine (S741) replaced by cysteine was produced and labeled with fluorescein at this residue to provide the derivative Plg(S741C-fluorescein). Studies of cleavage, conformation, and fibrin-binding properties of the derivative showed it to be a good model substrate to study plasminogen activation. Both in solution and in a fully polymerized fibrin clot, cleavage of the single chain zymogen to the two-chain "plasmin" molecule was accompanied by a 50% quench of fluorescence intensity. This change allows facile, continuous monitoring of the kinetics of cleavage. Measurements of cleavage by single chain t-PA within intact, fully polymerized 3 microM fibrin yielded apparent kcat and Km values of (0.08 s-1, 0.52 microM) and (0.092 s-1, 0.098 microM) for [Glu1]- and [Lys78]Plg(S741C-fluorescein), respectively. These values are similar to those obtained by others with plasma plasminogen. The approach used here might generally be useful in simplifying the analysis of zymogen activation kinetics in cases where the product (protease) has a great influence on its own formation via positive or negative feedback loops.

Enzyme Activation↗

Photoinactivation of fluorescein isothiocyanate-modified Na,K-ATPase by 2'(3')-O-(2,4,6-trinitrophenyl)8-azidoadenosine 5'-diphosphate. Abolition of E1 and E2 partial reactions by sequential block of high and low affinity nucleotide sites.

The Na,K-ATPase activity of the sodium pump exhibits apparent multisite kinetics toward ATP, a feature that is inherent to the minimal enzyme unit, the alpha beta protomer. We have argued that this should arise from separate catalytic and noncatalytic sites on the alpha beta protomer as fluorescein isothiocyanate (FITC) blocks a high affinity ATP site on all alpha subunits and yet the modified Na, K-ATPase retains a low affinity response to nucleotides (Ward, D. G., and Cavieres, J. D. (1996) J. Biol. Chem. 271, 12317-12321). We now find that 2'(3')-O-(2,4,6-trinitrophenyl)8-azido-adenosine 5'-diphosphate (TNP-8N3-ADP), a high affinity photoactivatable analogue of ATP, can inhibit the K+-phosphatase activity of the FITC-modified enzyme during assays in dimmed light. The inhibition occurs with a Ki of 140 microM at 20 mM K+; it requires the adenine ring as 2'(3')-O-(2,4 6-trinitrophenyl) (TNP)-UDP or TNP-uridine are less potent and 2,4,6-trinitrobenzene-sulfonate is ineffective. Under irradiation with UV light, TNP-8N3-ADP inactivates the K+-phosphatase activity of the fluorescein-enzyme and also its phosphorylation by [32P]Pi. The photoinactivation process is stimulated by Na+ or Mg2+, and is inhibited by K+ or excess TNP-ADP. In the presence of 50 mM Na+ and 1 mM Mg2+, TNP-8N3-ADP photoinactivates with a K0.5 of 15 microM. Furthermore, TNP-8N3-ADP photoinactivates the FITC-modified, solubilized alpha beta protomers, even more effectively than the membrane-bound fluorescein-enzyme. These results strongly suggest that catalytic and allosteric ATP sites coexist on the alpha beta protomer of Na,K-ATPase.

Adenosine Diphosphate↗

Affinity labeling of two nucleotide sites on Na,K-ATPase using 2'(3')-O-(2,4,6-trinitrophenyl)8-azidoadenosine 5'-[alpha-32P]diphosphate (TNP-8N3-[alpha-32P]ADP) as a photoactivatable probe. Label incorporation before and after blocking the high affinity ATP site with fluorescein isothiocyanate.

ATP and its analogues act on the minimal functional unit of Na, K-ATPase, the alpha beta protomer, with high and low affinity effects. Fluorescein isothiocyanate (FITC) irreversibly blocks the high affinity, or catalytic, ATP site, and yet the surviving K+-phosphatase activity of soluble FITC-modified alphabeta protomers can be photoinactivated by 2'(3')-O-trinitrophenyl (TNP)-8N3-ADP (Ward, D. G., and Cavieres, J. D. (1998) J. Biol. Chem. 273, 14277-14284). We have now used TNP-8N3-[alpha-32P]ADP as a photoaffinity label for Na,K-ATPase. The native enzyme can be photolabeled at 5 microM TNP-8N3-[alpha-32P]ADP, and ATP or FITC treatment prevents labeling of the alpha chain. At 25 microM, however, TNP-8N3-[alpha-32P]ADP can be incorporated in the FITC-modified alpha chain, concurrently with the inactivation of the K+-phosphatase activity, to an extrapolated level of 0.5-1.2 mol of 32P-probe per mol of alpha chain. Photoinactivation and labeling are prevented by TNP-ADP, vanadate, or strophanthidin and are promoted by Na+ or Mg2+, but not K+. The cation effects suggest that the fluorescein-modified enzyme incorporates the TNP-8N3-[alpha-32P]ADP. Mg complex preferentially, and the free probe when in the E1 enzyme form and after occupation of a low-affinity Na+ site. Partial trypsinolysis reveals that the point of TNP-8N3-[alpha-32P]ADP attachment is on the C-terminal 58-kDa fragment of the FITC-modified alpha chain. The affinity labeling of the fluorescein enzyme by TNP-8N3-[alpha-32P]ADP endorses the view that two nucleotide sites can be occupied simultaneously in each alpha subunit of Na,K-ATPase.

Adenosine Triphosphate↗

Specific labeling of the bovine heart mitochondrial phosphate carrier with fluorescein 5-isothiocyanate: roles of Lys185 and putative adenine nucleotide recognition site in phosphate transport.

The amine/SH-modifying fluorescein 5-isothiocyanate (FITC) specifically labeled Lys(185) in the putative membrane-spanning region of the phosphate carrier from both the cytosolic and matrix sides of bovine heart mitochondria at 0 degrees C and pH 7.2, and the labeling inhibited the phosphate transport. Nonmodifying fluorescein derivatives having similar structural features to those of ADP and ATP (Majima, E., Yamaguchi, N., Chuman, H., Shinohara, Y., Ishida, M., Goto, S., and Terada, H. (1998) Biochemistry 37, 424-432) inhibited the specific FITC labeling and phosphate transport, but the nonfluorescein phenylisothiocyanate did not inhibit FITC labeling, suggesting that there is a region recognizing the adenine nucleotides in the phosphate carrier and that this region is closely associated with the transport activity. The phosphate transport inhibitor pyridoxal 5'-phosphate inhibited the specific FITC labeling, possibly due to competitive modification of Lys(185). In addition, FITC inhibited the ADP transport and specific labeling of the ADP/ATP carrier with the fluorescein SH reagent eosin 5-maleimide. Based on these results, we discuss the structural features of the phosphate carrier in relation to its transport activity.

Adenosine Diphosphate↗