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1% lymphazurin vs 10% fluorescein for sentinel node mapping in colorectal tumors.

HYPOTHESIS: Ten percent fluorescein may be successfully used as an alternative to 1% Lymphazurin (1% isosulfan blue; US Surgical Corp, North Haven, Conn) in sentinel lymph node (SLN) mapping for the accurate staging of colorectal tumors. DESIGN: Review of prospectively gathered data. SETTING: University-affiliated regional medical center. PATIENTS: Sentinel lymph node mapping was performed in 120 consecutive patients with colorectal malignancies. INTERVENTIONS: The first 1 to 4 blue nodes detected within 5 minutes were designated as Lymphazurin-detected SLNs. The first 1 to 4 fluorescent nodes seen under the Wood light were designated as fluorescein-detected SLNs. Multilevel serial sections for hematoxylin-eosin and immunohistochemistry studies for cytokeratin were performed on all SLNs. MAIN OUTCOME MEASURES: Successful mapping, accuracy, skip metastasis, adverse reactions, occult micrometastases detection, and cost. RESULTS: Mapping was successful using Lymphazurin in 99% of the patients vs 97% of the patients using fluorescein (P =.89). The accuracy of predicting nodal metastases with each tracer was 95.8% vs 93.1%, respectively (P =.82). The skip metastases rate was 4.2% for Lymphazurin vs 6.9% for fluorescein (P =.37). The 5 patients in whom nodal disease was only identified as occult micrometastasis in the SLNs had a total of 5 SLNs, all of which were identified by both tracers. No adverse reactions occurred. The cost for Lymphazurin was $99.00, while the cost for fluorescein was $2.10. CONCLUSIONS: With the exception of cost, there were no statistically significant differences between the 2 dyes. While easy availability and lower cost remain distinct advantages of fluorescein, Lymphazurin remains the gold standard. In patients with known hypersensitivity to Lymphazurin and when availability and cost are an issue, fluorescein may be used effectively for SLN mapping in colorectal tumors.

Adult↗

Identification of unexpected modifications of fluorescein-labeled oligodeoxynucleotides by nuclease P1 digestion and mass spectrometric techniques.

Fluorescein-labeled oligodeoxynucleotides (ODNs) from automated synthesis commonly produce multiple peaks in high performance liquid chromatography (HPLC) chromatograms. We found that these peaks are due to chemical modifications of the ODNs instead of the common perception of isomers. To identify the modifications, a model ODN, fluorescein-T(25), was synthesized and five compounds were isolated. Nuclease P1 (NP1) digestion was employed to cleave these compounds into nucleotides and fluorescein-nucleotides in order that the modifications be determined by mass spectrometry (MS). Analyses of NP1 digestion products containing fluorescein by MS revealed the expected product F1-T (M) and four unexpected compounds with MWs at M-1, M-17, M-16 and M + 16, respectively. Collision-induced dissociation (CID) spectra of these digestion products indicate that all modifications occur on the thiourea linkage [-NH-C( = S)-NH-] to the fluorescein moiety and the adjacent phosphate group, and the modifications were determined. The modifications were also confirmed by accurate mass measurement with Fourier transform mass spectrometry (FT-MS), by the synthesis of a reference compound, and by a mechanistic study using model compounds. These results demonstrate the power of the mass spectrometric techniques by determining the structures of two pairs of ODNs with MW difference of 1 Da. The results also suggest that fluorescein phosphoramidite with a thiourea linkage is not appropriate for the automated synthesis of fluorescein-labeled ODNs of high purity.

Chromatography, High Pressure Liquid↗

Enhancer effect of fluorescein on the luminol-H2O2-horseradish peroxidase chemiluminescence: energy transfer process.

The chemiluminescence of the luminol-H2O2-horseradish peroxidase system is increased by fluorescein. Fluorescein produces an enhancement of the luminol chemiluminescence similar to that of phenolphthalein, by an energy transfer process from luminol to fluorescein. The maximum intensity and the total chemiluminescence emission (between 380 and 580 nm) of luminol with fluorescein was more than three times greater than without fluorescein; however, the emission duration was shorter. The emission spectra in the presence of fluorescein had two maxima (425 and 535 nm) and the enhancement was dependent on pH and fluorescein concentration. A mechanism is proposed to explain these effects.

Energy Transfer↗

Highly sensitive and simple fluorescence staining of proteins in sodium dodecyl sulfate-polyacrylamide-based gels by using hydrophobic tail-mediated enhancement of fluorescein luminescence.

Fluorescein has an extremely low luminescence intensity in acidic aqueous media. However, when it was bound to proteins, subsequent increase of luminescence intensity took place. Furthermore, when a hydrophobic tail, such as aliphatic hydrocarbons, was introduced to fluorescein, more dramatic increase of luminescence intensity was observed upon binding to proteins. In the present study, by utilizing this luminescence enhancement, three hydrophobic fluorescein dyes (5-dodecanoyl amino fluorescein, 5-hexadecanoyl amino fluorescein, and 5-octadecanoyl amino fluorescein) were examined as noncovalent fluorescent stains of protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Effective incorporation of the dyes to proteins in gels was accomplished either simply by adding dyes at the protein fixation step, or by treating gels with a staining solution after the fixation. The sensitivity of this staining method using the fluorescein derivatives was approximately 1 ng/band for most proteins. For some cases, protein bands containing as low as 0.1 ng were successfully visualized. In addition, the detection sensitivity showed much less protein-to-protein variation than silver staining. This new staining method was also successfully applied to two-dimensional electrophoresis of rat brain proteins. Its overall sensitivity was comparable to that of silver staining.

Electrophoresis, Gel, Two-Dimensional↗

Relationship between skin fluorescence and blood flow in normal and in chronically ischemic subjects dosed with fluorescein.

To elucidate parameters diagnostic of chronic ischemia, the fluorescence of skin on the foot, leg, arm, and forehead of six chronically ischemic patients and six normal subjects injected with fluorescein was measured serially using a surface-measurement fluorometer (dermofluorometer). Simultaneously collected plasma samples were assayed spectrofluorometrically for unmetabolized fluorescein. The time courses of plasma fluorescein content and dermofluorometer readings were jointly analyzed by combining a standard pharmacokinetic model, a model predicting skin site from plasma concentrations of fluorescein, and a model predicting the dermofluorometer response to those skin concentrations. Fluorescein plasma clearance (0.22 +/- 0.06 versus 0.46 +/- 0.20 L/h/kg) in ischemic patients was only half, and half-life was double (2.4 +/- 1.0 versus 1.3 +/- 0.3 h) those in normal subjects, with volume of distribution (Vdss = 0.46 L/kg) being similar. Despite the ischemia diagnosis for all patients involving claudication of the lower extremities, patients could be distinguished statistically from normal subjects on the basis of fluorescence readings taken on the arm, but not those using the foot or leg. The rate constant describing flux of fluorescein from the arm skin site in patients was only half that in normal subjects, and the peak reading on the arm occurred at 42 +/- 14 min after fluorescein injection in patients, but at only 15 +/- 6 min in normal subjects. Lack of discrimination between subject groups via leg and foot readings may be due to several physiologic and/or experimental factors, including the need to take skin surface readings much earlier than previously recognized.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

The mucosal blood flow in pelvic pouches in man. A methodologic study of fluorescein flowmetry.

Fluorescein flowmetry implies the measurement of capillary blood flow, expressed as an index between the maximum fluorescence after the first circulatory passage of sodium fluorescein (NaF) and the rise time, defined as the time interval between ten and 90 percent of the maximum fluorescence. A mathematic model based on fluorescein flowmetry was deduced to distinguish a mucosal and muscular blood flow in an intact (unopened) intestine during surgery in man. The hypothesis was that if, at a certain point in time, there is a fixed relationship between the seromuscular fluorescence and the mucosal maximum fluorescence, obtained during the first circulatory passage of NaF, and if the rise times were equal, then a mucosal blood flow could be calculated based on the seromuscular fluorescence. The model was tested in intestinal anastomoses on 16 patients. A fixed relationship between the numeric value of the mucosal maximum fluorescence and the seromuscular fluorescence was found. After five minutes, the ratio was 1:1 and the correlation coefficient at its highest (0.97). It was also found that the rise times were practically identical (r = 0.92). The validity of the model was then tested by comparing it with fluorescein flowmetry, and the correlation coefficient was 0.85. The model was therefore accepted and named indirect mucosal fluorescein flowmetry. Indirect mucosal fluorescein flowmetry was applied to measure blood flow in pelvic pouches in 14 patients, and fluorescein flowmetry in the ileoanal anastomoses in eight patients. The mucosal blood flow in the reservoir, compared with the normal intestine, was reduced to 58 percent if the ileocolic artery or distal branches of the mesenteric artery were ligated, and to 88 percent if the vessels were left intact (P less than 0.05). In the ileoanal anastomosis the mucosal blood flow was reduced to 23 percent compared with the normal intestine (P less than 0.01). The results suggest that stretching and compressing the mesentery might be critical for circulation in the ileoanal anastomoses.

Capillaries↗

Ocular fluorescein kinetics before and after vitrectomy on swine.

PURPOSE: To study the quantitative effects of vitrectomy on fluorescein transport kinetics across the ocular barriers. METHODS: Thirty-six domestic swine were used in this study. Twenty anesthetized swine were given a standardized fluorescein intravenous injection immediately after unilateral vitrectomy. This was followed by one single central sample aspiration from the vitreous and the anterior chamber of both eyes in individual animals at increasing intervals up to 24 h after the injection. Fluorescein concentrations in the samples were determined by high-pressure liquid chromatography (HPLC). Eight swine underwent unilateral vitrectomy followed by anterior chamber and vitreous fluorophotometry on both eyes 1 month later. The fluorescein concentrations determined using this method were followed for 24 h. Similar examinations were performed in a control group of eight swine that did not undergo vitrectomy. Anterior chamber, vitreous, and plasma fluorescein concentration/time courses were analyzed kinetically by iterative nonlinear regression analysis. RESULTS: The barrier surrounding the anterior chamber of the eye was immediately impaired after vitrectomy, as evidenced by an increased area under the fluorescein concentration versus time curve, but the transport kinetics were restored within 1 month after surgery. The blood-retinal barrier was, however, persistently altered following vitrectomy. Transport rate and extent of drug penetration into the vitreous were increased, while drug elimination from the vitreous remained unchanged. CONCLUSION: Vitrectomy led to persistent kinetic fluorescein transport changes in the blood-retinal barrier resulting in faster and increased drug penetration to the vitreous, whereas similar alterations in the anterior chamber barrier transport were only transitory.

Animals↗

Effect of acetate on transport of organic acid (fluorescein) in renal proximal tubules of frog.

The effect of acetate on active fluorescein transport in intact proximal tubules of surviving frog kidney was studied. When the kidneys were incubated in a 120 mM Na+ medium, 10 mM acetate stimulated fluorescein uptake in the tubules. The stimulation was more pronounced if the kidneys had been previously preincubated for 3 h in the substrate-free solution. Lowering of the Na+ concentration in the bathing medium to 10 mM resulted in the disappearance of the acetate effect. Preincubation of the kidneys with acetate at 2-4 degrees C gave rise to stimulation of the fluorescein transport only in the 120 mM Na+ acetate-free medium. The acetate effect on the fluorescein uptake was partially prevented by ouabain. The stimulation of the uptake by acetate in the 120 mM Na+ medium correlated with an increase in the extent of reduction of pyridine nucleotides in the tubules. The pyridine nucleotides were reduced more markedly after incubation of the kidneys in the 10 mM Na+ medium, when acetate had no effect on the fluorescein transport. In both the 120 MM and the 10 mM Na+ media, the cold preincubation of the kidneys with 2.5 mM ADP or 2.5 mM ATP resulted in only slight stimulation of the fluorescein uptake. But in both media the uptake was significantly enhanced after cold preincubation of the kidneys with 2 mM NADH. After the cold preincubation with ADP, stimulation of the fluorescein transport by acetate was observed in the case of the 10 mM Na+ medium also. The absence of any stimulatory effect of acetate on the organic acid transport in the 10 mM Na+ medium is explained as the result of the transformation of mitochondria in the tubular cells into the inactive state 4 due to a decrease in the intracellular ADP level. Reducing equivalents are supposed to take part in energization and/or regulation of transport processes in plasma membranes of the renal proximal tubules.

Acetates↗

Safety of fluorescein angiography during pregnancy.

We sent 424 retina specialists questionnaires on fluorescein angiography performed on pregnant women; 399 specialists responded. Of these, 309 (77%) had never performed fluorescein angiography on a pregnant woman. Ninety specialists (23%) had performed at least one fluorescein angiogram on a pregnant woman; detailed information was obtained on 105 patients. Authors of previous reports that included fluorescein angiography during pregnancy provided information about an additional 11 patients. Substantiated side effects were nausea or vomiting in seven patients. Anomalies at birth, an undescended testicle and syndactyly, were reported in two children. There was one stillbirth with pathologic findings classic for toxemia, and one fetal death occurred several months after fluorescein angiography. One therapeutic abortion was performed for complications in toxemia. One spontaneous abortion occurred three days after fluorescein angiography in a patient who was four weeks pregnant. Eight children born to toxemic mothers had low birth weights. We conclude that fluorescein angiography does not cause a high rate of birth anomalies or complications during pregnancy.

Abnormalities, Drug-Induced↗

Nerve fiber layer and optic disc fluorescein defects in glaucoma and ocular hypertension.

Photographs of the optic discs and fluorescein angiograms of 31 patients with open-angle glaucoma and 43 patients with ocular hypertension were evaluated for nerve fiber layer (NFL) defects and absolute fluorescein filling defects. All of the glaucomatous eyes showed both defects. Of the 43 ocular hypertensive eyes, in which both NFL and absolute fluorescein filling defects were evaluated, 9% had only NFL defects, 19% had only fluorescein filling defects, 14% had both defects, and 58% had neither defect. The percent area of fluorescein defect in the optic disc increased with severity of NFL defect in glaucoma and ocular hypertension. This study confirms the relationship of fluorescein filling defects and NFL defects to glaucomatous abnormalities and thus the association between vascular damage to the optic nerve and axon loss in glaucoma. The earliest objective evidence of glaucomatous damage can be detected with a combination of NFL evaluation and optic disc fluorescein angiography.

Aged↗

Raman and FTIR spectroscopies of fluorescein in solutions.

Raman and Fourier transform-infra red (FT-IR) spectroscopies of fluorescein in aqueous solutions have been investigated in the pH range from 9.1 to 5.4. At pH 9.1 fluorescein is in the dianion form. At pH 5.4, fluorescein is a mixture of monoanion (approximately 85%), dianion and neutral forms (together approximately 15%). The fluorescence quantum yield drops from 0.93 for the dianion form to 0.37 for the monoanion form. The Raman and FT-IR studies focused on the frequency range from 1000 to 1800 cm(-1) which contains the skeletal vibrational modes of the xanthene moiety of fluorescein. At pH 9.1, the spectroscopic feature of fluorescein dianion are consistent with a picture of an electron delocalized among the xanthene moiety and two identical oxygens attached to opposite ends of the xanthene moiety, forming a very symmetric structure. The characteristic of fluorescein dianion is the presence of the phenoxide-like stretch at 1310 cm(-1). At pH 5.4, fluorescein monoanion has lost the symmetric structure characteristic of the dianion. The spectra of the monoanion have distinctive contributions from the phenolic bend at 1184 cm(-1). The assignments of the vibrational bands shown in Raman and FT-IR spectra are given based on both literature and the ab initio calculations at the Hartree-Fock level with HF/6-31 + +G* basis set. Excellent correlation is found between the experimental and calculated spectra.

Contrast Media↗

Time resolution of fluorescence changes observed in titrations of fluorescein 5'-isothiocyanate-modified Na,K-ATPase with monovalent cations.

Equilibrium fluorometric titrations of fluorescein 5'-isothiocyanate-modified Na,K-ATPase with cations have usually been interpreted by assuming that an enhancement reports the conformational change from E2 to E1. We report time resolution of the fluorescence change into three phases when fluorophore-modified enzyme is mixed with the chloride salt of either sodium or choline in a stopped-flow instrument. The first phase is an increase in fluorescence within the dead time of the instrument that is also observed when fluorescein 5'-isothiocyanate (FITC) reacted with lysine is substituted for fluorescein-labeled enzyme. The other two phases occur on millisecond and second time scales. Three phases are also observed when fluorophore-modified enzyme preincubated in KCl is mixed with NaCl, but in this case the slowest phase is absent when choline chloride replaces NaCl. The two faster effects in phases one and two can be eliminated either by controlling the ionic strength or by anti-fluorescein antibody. Labeling the enzyme with fluorescein 5'-isothiocyanate in the presence of its substrate, adenosine 5'-triphosphate, practically eliminates the slowest effect. These results demonstrate that fluorescein reports three events that occur on three different time scales. The fastest phase reports the ionic strength jump of unbound fluorophore. The intermediate phase reports the ionic strength jump of fluorescein at "antibody-accessible" sites [Abbott, A. J., Amler, E., & Ball, W. J., Jr. (1991) Biochemistry 30, 1692-1701]. Only the slowest phase reports the enzyme conformational change implicated in transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Fluorescein angiography versus ERG for predicting the prognosis in central retinal vein occlusion.

PURPOSE: It is not easy to predict which patients with a central retinal vein occlusion will develop rubeosis and which will not. We have compared two methods for doing so, fluorescein angiography and full-field electroretinogram (ERG). Our aim was to improve our possibilities for predicting rubeosis in patients with central vein occlusion. METHODS: 32 patients with a central retinal vein occlusion with a duration of less than 14 days were included in the study. Fluorescein angiography and ERG were performed in all patients. The fluorescein angiograms were studied by two independent examiners in a masked mode. The patients were then followed for at least one year. RESULTS: Development of rubeosis in patients with central retinal vein occlusion could be predicted by fluorescein angiography in 82% of the patients and with ERG in 94% of the patients. The non-ischemic central retinal vein occlusions were identified in 62% by fluorescein angiography and in 100% with ERG. Fluorescein angiography misjudged 9 patients 28%, whereas ERG only misjudged 1 patient, 3%. CONCLUSION: ERG seems to be a better method for predicting the prognosis in central retinal vein occlusion than fluorescein angiography.

Adult↗

Fluorescein dermofluorometry for the assessment of diabetic microvascular disease.

BACKGROUND/AIMS: Fluorescein dermofluorometry can be used to relate the uptake of fluorescein in the skin to blood flow. We have characterized the uptake of the dye by a wash-in time constant that is inversely proportional to the local blood flow. The purpose of this study was to explore the use of dermofluorometry in the assessment of patients with diabetic microvascular disease. METHODS: Fluorescein dermofluorometry was performed in four groups of patients: non-diabetic control patients, diabetic control patients, diabetic patients with chronic foot ulcers, and diabetic patients with acute foot ulcers. The outcomes of the patients with foot ulcers were documented 4-14 months after participation. Following an intravenous injection of sodium fluorescein, the change in the fluorescein signal with time was continuously measured at the plantar surface of the foot. Both the initial slope of the signal and the wash-in time constant were calculated in each subject. RESULTS: Significant differences in the wash-in time constant were found between diabetic and non-diabetic subjects and between diabetic subjects with and without foot ulcers. Of the eight patients with foot ulcers, two of them did not display an early wash-out in the dermofluorometer signal and later both required amputations. CONCLUSION: The fluorescein wash-in time constant demonstrated better correlation with the presence of diabetic microvascular disease than did the initial slope of the signal. Differences in the wash-in time constants of non-diabetic and diabetic subjects support the hemodynamic hypothesis for the development of microvascular disease. The indication of early wash-out of the fluorescein signal may also be useful in the prediction of ulcer healing.

Diabetic Angiopathies↗

[Automatic measurement of dye filling of simultaneous digital ICG- and fluorescein angiography sequences].

BACKGROUND: The ICG filling is supposed to be faster than Fluorescein filling. Interestingly the filling characteristics of these dyes were never correlated directly using precise quantitative methods. Since ICG and Fluorescein are injected as a mixture, the simultaneous 2-channel angiography provides a suitable method to correlate the filling characteristics of the dyes. MATERIAL AND METHODS: The simultaneous ICG and Fluorescein angiograms were recorded with a Rodenstock Scanning Laser Ophthalmoscope. The angiographic images were digitized real-time with a graphic workstation. Filling characteristics of the two dyes was calculated after off-line eye tracking in different regions of interests (ROIs) on the central retina. RESULTS: The Fluorescein filling was faster than the ICG filling in 56.5% of our patients. In 26% of our patients was a mixed filling detectable. Depending on the position of the ROIs the Fluorescein or ICG filling was faster. In only 17.5% of our cases was the ICG filling faster than the Fluorescein filling. CONCLUSION: Our results show that Fluorescein filling in more than 50% of the cases is faster than ICG filling and only a minority of the patients has a faster ICG filling. According to our experience the filling pattern of the two dyes is individual, there is no rule of thumb for the filling.

Angiography, Digital Subtraction↗

Reversible photobleaching of fluorescein conjugates in air-saturated viscous solutions: singlet and triplet state quenching by tryptophan.

Fluorescence recovery after photobleaching (FRAP) measurements on air-saturated aqueous solutions of fluorescein made viscous with glycerol or sucrose revealed a rapid component of fluorescence recovery with exponential time constants of 30-120 microseconds at viscosities of 15-300 cP. The rapid recovery process was not related to fluorophore translational diffusion and was insensitive to fluorophore concentration and the additive used to increase solution viscosity. At constant viscosity, the rate of reversible photobleaching recovery increased 2.5-fold in an O2- vs N2-saturated solution. The relative efficiency of reversible-to-irreversible photobleaching decreased with increasing photobleaching time and/or beam intensity. Reversible photobleaching was also detected for conjugates of fluorescein with dextrans and proteins in viscous media. In screening triplet state quenchers that might influence the reversible recovery, it was found that tryptophan enhanced the rate of reversible photobleaching recovery (two-fold increase at 8 mM) and quenched the fluorescein singlet state (Stern-Volmer constant, 12 M-1). Analysis of fluorescein lifetimes and photobleaching parameters for a series of fluorescein-labeled proteins with different numbers of tryptophans were also carried out. The results provide evidence for an oxygen-dependent, reversible photobleaching mechanism for the fluorescein chromophore involving triplet state relaxation. The identification of reversible fluorescein photobleaching has important implications for FRAP measurements of rapid solute diffusion in biological systems.

Fluoresceins↗

The Saccharomyces cerevisiae weak-acid-inducible ABC transporter Pdr12 transports fluorescein and preservative anions from the cytosol by an energy-dependent mechanism.

Growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid results in the induction of the ATP-binding cassette (ABC) transporter Pdr12 in the plasma membrane (P. Piper, Y. Mahe, S. Thompson, R. Pandjaitan, C. Holyoak, R. Egner, M. Muhlbauer, P. Coote, and K. Kuchler, EMBO J. 17:4257-4265, 1998). Pdr12 appears to mediate resistance to water-soluble, monocarboxylic acids with chain lengths of from C(1) to C(7). Exposure to acids with aliphatic chain lengths greater than C(7) resulted in no observable sensitivity of Deltapdr12 mutant cells compared to the parent. Parent and Deltapdr12 mutant cells were grown in the presence of sorbic acid and subsequently loaded with fluorescein. Upon addition of an energy source in the form of glucose, parent cells immediately effluxed fluorescein from the cytosol into the surrounding medium. In contrast, under the same conditions, cells of the Deltapdr12 mutant were unable to efflux any of the dye. When both parent and Deltapdr12 mutant cells were grown without sorbic acid and subsequently loaded with fluorescein, upon the addition of glucose no efflux of fluorescein was detected from either strain. Thus, we have shown that Pdr12 catalyzes the energy-dependent extrusion of fluorescein from the cytosol. Lineweaver-Burk analysis revealed that sorbic and benzoic acids competitively inhibited ATP-dependent fluorescein efflux. Thus, these data provide strong evidence that sorbate and benzoate anions compete with fluorescein for a putative monocarboxylate binding site on the Pdr12 transporter.

ATP-Binding Cassette Transporters↗

Acute effect of the carbon dioxide laser on the epicerebral microcirculation. Experimental study by fluorescein angiography.

The effect of laser radiation on the central nervous system has been studied in cases of clinical and experimental tumors. However, no report yet exists on the effects of laser radiation on the cerebral microcirculation in vivo. Cerebral fluorescein angiography permits observations of small vessels that are not possible by conventional angiography. In this study, disturbance in the epicerebral microcirculation after carbon dioxide laser radiation was localized. On fluorescein angiograms, a circular zone of nonfilling of fluorescein dye around the site of impact, 1 to 1.5 mm in diameter, was seen throughout from the arterial to later venous phase. Around the nonfilling area, thrombus formation in small vessles and extravasation of the dye were demonstrated. Such extravasation of the fluorescein remained after the dye had faded from the venules and veins. Microscopically, coagulation necrosis was observed to coincide with the area of nonfillin of fluorescein dye in the fluorescein angiograms. In areas surrounding this, edema, dilatation or rupture of the capillaries, and thrombus formation in the arterioles were observed. Such areas coincided with those of extravasation of the fluorescein dye.

Animals↗