Dichlorotriazinyl Aminofluorescein Research Articles

Detection of Dibutyl Phthalate in Surface Water by Fluorescence Polarization Immunoassay.

Dibutyl phthalate (DBP) is widely used as a plasticizer in the production of polymeric materials to give them flexibility, strength and extensibility. However, due to its negative impact on human health, in particular reproductive functions and fetal development, the content of DBP must be controlled in food and the environment. The present study aims to develop a sensitive, fast and simple fluorescence polarization immunoassay (FPIA) using monoclonal antibodies derived against DBP (MAb-DBP) for its detection in open waters. New conjugates of DBP with various fluorescein derivatives were obtained and characterized: 5-aminomethylfluorescein (AMF) and dichlorotriazinylaminofluorescein (DTAF). The advantages of using the DBP-AMF conjugate in the FPIA method are shown, the kinetics of binding of this chemical with antibodies are studied, the analysis is optimized, and the concentration of monoclonal antibodies is selected for sensitivity analysis-16 nM. The calibration dependence of the fluorescence polarization signal for the detection of DBP was obtained. The observed IC50 (DBP concentration at which a 50% decrease in the fluorescence polarization signal occurs, 40 ng/mL) and the limit of detection (LOD, 7.5 ng/mL) values were improved by a factor of 45 over the previously described FPIA using polyclonal antibodies. This technique was tested by the recovery method, and the high percentage of DBP discovery in water ranged from 85 to 110%. Using the developed method, real water samples from Lake Onega were tested, and a good correlation was shown between the results of the determination of DBP by the FPIA method and GC-MS. Thus, the FPIA method developed in this work can be used to determine DBP in open-water reservoirs.

DTAF dye concentrations commonly used to measure microscale deformations in biological tissues alter tissue mechanics.

Identification of the deformation mechanisms and specific components underlying the mechanical function of biological tissues requires mechanical testing at multiple levels within the tissue hierarchical structure. Dichlorotriazinylaminofluorescein (DTAF) is a fluorescent dye that is used to visualize microscale deformations of the extracellular matrix in soft collagenous tissues. However, the DTAF concentrations commonly employed in previous multiscale experiments (≥2000 µg/ml) may alter tissue mechanics. The objective of this study was to determine whether DTAF affects tendon fascicle mechanics and if a concentration threshold exists below which any observed effects are negligible. This information is valuable for guiding the continued use of this fluorescent dye in future experiments and for interpreting the results of previous work. Incremental strain testing demonstrated that high DTAF concentrations (≥100 µg/ml) increase the quasi-static modulus and yield strength of rat tail tendon fascicles while reducing their viscoelastic behavior. Subsequent multiscale testing and modeling suggests that these effects are due to a stiffening of the collagen fibrils and strengthening of the interfibrillar matrix. Despite these changes in tissue behavior, the fundamental deformation mechanisms underlying fascicle mechanics appear to remain intact, which suggests that conclusions from previous multiscale investigations of strain transfer are still valid. The effects of lower DTAF concentrations (≤10 µg/ml) on tendon mechanics were substantially smaller and potentially negligible; nevertheless, no concentration was found that did not at least slightly alter the tissue behavior. Therefore, future studies should either reduce DTAF concentrations as much as possible or use other dyes/techniques for measuring microscale deformations.

Localization of EPS components secreted by freshwater diatoms using differential staining with fluorophore-conjugated lectins and other fluorochromes

Diatoms produce extracellular polymeric substances (EPS), which mainly consist of carbohydrates and may form different morphological structures. We studied the localization and structure of EPS secreted by 17 diatom species that were isolated from epilithic biofilms from the littoral zone of Lake Constance (Germany). We used six different lectins labelled with FITC (fluorescein isothiocynate) and DAPI (diamidino phenylindole) to localize and visualize the structure of secreted EPS and/or cell-wall-associated EPS, while DTAF (dichlorotriazinyl aminofluorescein) was used to label cell-wall-associated EPS only. The diatoms were categorized according to the structure of the secreted EPS i.e. pads, capsules, tubes or stalks. Among eight pad-producing diatoms, three Fragilaria species showed variable lectin binding indicating the presence of different carbohydrate components. Other pad-forming diatoms like Synedra, Diatoma, Asterionella, and Melosira generally showed binding to at least two different lectins. On the other hand, we did not observe any lectin binding to the capsules of Staurosira and one Achnanthes isolate. We further detected differences in the carbohydrate composition of tube-like EPS structures in two Cymbella species and were able to demonstrate developmental stages of tube formation. The stalk secreting species Gomphonema and Cymbella showed labelling by only one lectin, while EPS of Caloneis alpestris were stained by all lectins and fluorochromes used indicating a highly complex composition.

Stroma remodelling during healing of corneal surface irregularities induced by PTK

To study the histopathology of the remodelling process in the stroma after excimer-laser-induced corneal irregular injuries. Seven New Zealand white rabbits received in one eye a transepithelial plano photoablation (60 microm) and an additional plano ablation (25 microm). On the denuded stroma, an electron microscopy specimen grid was placed and another 25 microm ablation was applied to produce surface irregularities. Dichlorotriazinyl aminofluorescein (DTAF) was then applied for 45 seconds. Another seven right eyes of seven rabbits were ablated the same way but without using the grid, resulting in a plano ablation. All the rabbits were killed at weekly intervals after treatment. The harvested corneas from both eyes were further processed for haematoxylin-eosin staining and were also stained with monoclonal antibodies directed against Ki-67 antigen and alpha-smooth muscle actin (alpha-SMA). All specimens were examined under light and fluorescence microscope. The corneal wounds were covered by epithelium during the first week. The 25 microm x 25 microm x 25 microm stromal irregularities were clearly discernible up to 3 weeks after treatment, during which time they melted and disappeared. A homogeneous zone was formed in which stroma cells laid down an initially disorganized stroma. This was sharply visible under a fluorescence microscope as a dark area between the dichlorotriazinyl aminofluorescein (DTAF) fluorescent stroma and autofluorescent epithelium. Very little response was seen in the plano-ablated wound microscopically and in terms of positive stained cells. As the irregularities are flattened and the homogenous zone becomes repopulated with keratocytes forming extracellular matrix material (ECM), the cornea regains its previous architecture in both groups. The irregular wound surface promotes wound-healing reactivity, a process that allows the cornea to compensate for the irregularities and heal to a functional state.

Fluorescently labeled collagen binding proteins allow specific visualization of collagen in tissues and live cell culture

Visualization of the formation and orientation of collagen fibers in tissue engineering experiments is crucial for understanding the factors that determine the mechanical properties of tissues. In this study, collagen-specific fluorescent probes were developed using a new approach that takes advantage of the inherent specificity of collagen binding protein domains present in bacterial adhesion proteins (CNA35) and integrins (GST-α 1I). Both collagen binding domains were obtained as fusion proteins from an Escherichia coli expression system and fluorescently labeled using either amine-reactive succinimide (CNA35) or cysteine-reactive maleimide (GST-α 1I) dyes. Solid-phase binding assays showed that both protein-based probes are much more specific than dichlorotriazinyl aminofluorescein (DTAF), a fluorescent dye that is currently used to track collagen formation in tissue engineering experiments. The CNA35 probe showed a higher affinity for human collagen type I than did the GST-α 1I probe (apparent K d values of 0.5 and 50 μM, respectively) and showed very little cross-reactivity with noncollagenous extracellular matrix proteins. The CNA35 probe was also superior to both GST-α 1I and DTAF in visualizing the formation of collagen fibers around live human venous saphena cells. Immunohistological experiments on rat tissue showed colocalization of the CNA35 probe with collagen type I and type III antibodies. The fluorescent probes described here have important advantages over existing methods for visualization of collagen, in particular for monitoring the formation of collagen in live tissue cultures over prolonged time periods.

Mitomycin C alters corneal stromal wound healing and corneal haze in rabbits after argon-fluoride excimer laser photorefractive keratectomy.

To investigate the effects of mitomycin C (MMC) on rabbit cornea wound healing after photorefractive keratectomy (PRK). Rabbit corneas were stained with dichlorotriazinyl aminofluorescein immediately after PRK. MMC was applied to the right eye and phosphate-buffered salt solution (PBS) to the left. Corneal epithelial wound healing rate and corneal haze were examined. Ultrasound pachymetry was performed. Stromal collagen regeneration was evaluated by fluorescent microscopy. We used terminal deoxyribonucleotidyl transferase-mediated D-uridine 5'-triphosphated-digoxigenin nick-end labeling (TUNEL) assay and transmission electron microscopy (TEM) to evaluate keratocyte apoptosis. In eyes treated with MMC, there was no delay to the healing rate of corneal epithelial wound, and less haze 4 weeks after PRK. Ultrasound pachymetry showed thinner corneal thickness in MMC-treated eyes at week 4. Corneal stromal thickness regression was less in MMC-treated eyes observed by fluorescent microscope at week 4. Keratocyte apoptosis was noted in both MMC- and PBS-treated eyes by TUNEL assay and TEM observation. This study discovered the phenomenon that MMC prolongs keratocyte apoptosis. Applying MMC after PRK is an effective method to decrease haze formation and corneal stromal thickness regression in rabbit corneas. The effect may be related to MMC prolonging keratocyte apoptosis.

A Transmembrane Tight Junction Protein Selectively Expressed on Endothelial Cells and Platelets

Searching for cell surface proteins expressed at interendothelial cell contacts, we have raised monoclonal antibodies against intact mouse endothelial cells. We obtained two monoclonal antibodies, 1G8 and 4C10, that stain endothelial cell contacts and recognize a protein of 55 kDa. Purification and identification by mass spectrometry of this protein revealed that it contains two extracellular Ig domains, reminiscent of the JAM family, but a much longer 120-amino acid cytoplasmic domain. The antigen is exclusively expressed on endothelial cells of various organs as was analyzed by immunohistochemistry. Immunogold labeling of ultrathin sections of brain as well as skeletal muscle revealed that the antigen strictly colocalizes in capillaries with the tight junction markers occludin, claudin-5, and ZO-1. Upon transfection into MDCK cells, the antigen was restricted to the most apical tip of the lateral cell surface, where it colocalized with ZO-1 but not with beta-catenin. In contrast to JAM-1, however, the 1G8 antigen does not associate with the PDZ domain proteins ZO-1, AF-6, or ASIP/PAR-3, despite the presence of a PDZ-binding motif. The 1G8 antigen was not detected on peripheral blood mouse leukocytes, whereas similar to JAM-1 it was strongly expressed on platelets and megakaryocytes. The 1G8 antigen supports homophilic interactions on transfected Chinese hamster ovary cells. Based on the similarity to the JAM molecules, it is plausible that the 1G8 antigen might be involved in interendothelial cell adhesion.

Effect of Collagenase Inhibitors on Corneal Haze after PRK

The aim of the study was to determine whether collagenase inhibitors reduce corneal haze after photorefractive keratectomy (PRK). Inhibition of the initial removal phase of healing may limit the subsequent repair and replacement phases responsible for haze and regression. Thirty rabbits received −6.00D 5mm right PRK. They were randomized to five treatment groups: G. cysteine, G. ethylene diamine tetra-acetic acid (EDTA), G. ascorbate, Oc. tetracycline or no drops. Dichlorotriazinyl aminofluorescein (DTAF) was applied to the wound immediately after surgery in two rabbits of each group, to delineate newly-synthesized from original tissue. Corneal haze was assessed by a video-linked frame grabber with computerized grey scale analysis. Corneas were taken for histology at 1 or 3 months post-operatively. Corneal haze was not significantly different between the treatment groups and controls. The severity of the histological changes varied between individuals. Within the ablation zone the epithelium was on average 10% thicker (3–4μ m) than outside, and in some rabbits there were irregularities of the epithelial–stromal junction. The new subepithelial tissue had a mean depth of 7.8μ m, and the superficial stroma was disorganized to a mean depth of 49μ m. No particular treatment demonstrated significant benefits over controls; but of the treatments used, cysteine tended to produce the best results. Eyes treated with EDTA fared worst in most respects. The collagenase inhibitors used did not improve the outcome of PRK in rabbits. It remains to be determined whether firstly, the new more potent agents would have an effect, and secondly, whether collagenase inhibitors are of benefit in humans.

Glutamic acid decarboxylase-like immunoreactive axon terminals in synaptic contact with mesencephalic trigeminal nucleus neurons in the rat

The purpose of the present study was to obtain reliable evidence for the presence of γ-aminobutyric acid-ergic (GABAergic) synapses upon mesencephalic trigeminal nucleus (MTN) neurons in the rat. For confocal laser-scanning microscopy, phosphate-activated glutaminase-like immunoreactivity (-IR) of MTN neurons was visualized with red fluorescence of Texas Red, while glutamic acid decarboxylase (GAD)-IR of GABA axons was visualized with green fluorescence of dichlorotriazinyl aminofluorescein. Many GAD-axon terminals were in close apposition to the cell bodies of MTN neurons. For electron microscopy, MTN neurons were labeled with wheat germ agglutinin-horseradish peroxidase injected into the masseter nerve, while axon terminals were labeled with GAD-IR. GAD-axon terminals were in symmetric synaptic contact with the cell bodies of MTN neurons. Primary proprioceptive neurons in the orofacial regions might be regulated post-synaptically by GABA neurons.

Leakage responses to L-NAME differ with the fluorescent dye used to label albumin.

Nitric oxide synthase (NOS) inhibitors have been reported to increase as well as to decrease microvascular transport of macromolecules in a variety of models. This study was performed to determine whether the influence of NOS inhibition on albumin leakage was dependent on the fluorescent dyes used to label albumin. Albumin leakage was assessed in rat mesenteric venules during control conditions and after exposure to the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME). Albumin was labeled with any one of four dyes: FITC, sulforhodamine 101 [Texas Red (TR)], dichlorotriazinyl aminofluorescein (DTAF), or Oregon Green 514 (OG). Superfusion with L-NAME (10(-4) M) was accompanied by an increase in leakage of FITC-labeled albumin (n = 12) but not of albumin labeled with DTAF (n = 10), TR (n = 10), or OG (n = 4). In vessels perfused with both FITC- and TR-labeled albumin (n = 12), superfusion with L-NAME increased leakage of FITC- but not TR-labeled albumin. In conclusion, albumin leakage responses to L-NAME differ among various fluorescent dyes. Therefore, caution is advised in comparison of albumin leakage results that utilize different fluorescent dyes.

Evaluation of pentoxifylline in the prevention of haze after photorefractive keratectomy in the rabbit.

Pentoxifylline (PTX) is a methylxanthine derivative that, besides its hemorrheologic properties, possesses multiple physiologic effects at the cellular level. It has been used in keloid prevention due to its ability to inhibit the secretion of collagen and glycosaminoglycans from activated fibroblasts. Ten New Zealand White (NZW) rabbits underwent a -7.00 diopters, 6.0 mm diameter photorefractive keratectomy after laser ablation of the epithelium with a VISX 20/20 excimer laser. The bare stroma was stained with fresh 0.5% dichlorotriazinyl aminofluorescein (DTAF). The procedure was performed on both eyes, 4 days apart. One eye received 1% Pentoxifylline qid and the other balanced salt solution qid as a control for 4 weeks, starting the same day of surgery. Two masked observers graded the amount of haze at 2, 4, 6, and 8 weeks after surgery using slit-lamp biomicroscopy. Three rabbits were sacrificed at 2 and 4 weeks followed by two rabbits at 6 and 8 weeks. The area between the DTAF-stained collagen to the base of the epithelium was measured using a digital image analyzer. There was no statistically significant difference in the amount of haze either by slit-lamp microscopy or by histological analysis between the pentoxifylline-treated eyes and the controls at any time interval (Student's t-test: 0.16 to 0.92) Pentoxifylline did not seem to affect haze formation in a PRK rabbit model. As no signs of toxicity were observed, further studies might examine higher concentrations or dose frequencies.

Dichlorotriazinyl aminofluorescein inhibits human keratocyte proliferation in vitro.

Dichlorotriazinyl aminofluorescein (DTAF) has been used to stain corneal stromal collagen as part of in vivo experimentation. Toxicity of this drug, if present, might alter the observed wound healing. To determine if this drug has any deleterious effect on keratocytes, we evaluated it in vitro. Human keratocytes in 96 well plates were exposed to different concentrations of DTAF (10e-7, 10e-6, 10e-5, 10e-4, 10e-3, 10e-2, and 10e-1 mg/ml of media). Exposure times of 1 and 24 hours at each concentration of DTAF were evaluated. The cell number was measured 1 and 3 days after exposure to the drug using a coulter-counter and a hemocytometer. The proliferation of keratocytes after 24 hours of exposure to the drug was inhibited in a dose dependent manner by DTAF, but 1 hour exposure of keratocytes to the drug did not inhibit keratocyte proliferation. These results suggest that DTAF has inhibitory effects on human keratocyte proliferation after 24 hours of exposure, while exposure limited to 1 hour does not induce such a change.

The effect of dichlorotriazinyl aminofluorescein on human keratocytes in vitro.

Dichlorotriazinyl aminofluorescein (DTAF) has been used to stain corneal stromal collagen as part of in vivo animal experiments for many years. Toxicity of this drug, if present, might alter the observed wound healing. To determine if this drug has any deleterious effect on keratocytes, we evaluated it in vitro. Human keratocytes prepared in 24-well plates were exposed to varying concentrations of DTAF (10(-4), 10(-3), 10(-2), 1, 10, 10(2) micrograms/ml). Exposure times of 1 hour and 24 hours at each concentration of DTAF were evaluated. The cell number was measured 1 and 3 days after initiation of exposure to DTAF using a Coulter counter. Keratocyte proliferation was not affected by 1-hour exposure to DTAF, but keratocyte proliferation measured 3 days after initiation of exposure to DTAF for 24 hours was inhibited in a dose-dependent manner (p = 0.02) and was significantly inhibited at concentrations of 10 and 100 micrograms/ml (p < 0.05). Fluorescent microscopy showed binding of DTAF to keratocytes. We have demonstrated that prolonged exposure to DTAF inhibits proliferation of cultured keratocytes. These results suggest that DTAF-induced cytotoxicity may alter net production of collagen in the corneal stroma in animal models.

Practical and quantitative aspects in the analysis of FITC and DTAF amino acid derivatives by capillary electrophoresis and LIF detection

Practical aspects related to the preparation of the fluorescein isothiocyanate (FITC) and dichlorotriazinylaminofluorescein (DTAF) derivatives of amino acids for purposes of quantitative analysis are examined and factors affecting quantification are discussed. It is shown that the labelling reaction for both reagents can be speeded up by operating at 40°C. The difficulties with derivatizing amino acids at detectable concentrations are highlighted. In spite of the high sensitivity of CE-LIF, detection sensitivity in real applications is limited by factors external to the analytical process, such as the label chemistry.

Fine specificity analysis indicates that the primary and secondary fluorescein-specific cytotoxic T cell receptor repertoires are indistinguishable.

Previous data from this laboratory have shown that fluorescein-specific cytotoxic T cells derived from naive mice are capable of distinguishing between different isomeric forms of the fluorescein hapten, as well as between the iodoacetamido-, isothiocyanate- and dichlorotriazinyl amino-fluorescein derivatives. In this report, we show that T cells derived from previously immunized animals, while demonstrating a stronger and more cross-reactive response overall, display identical fine specificity patterns to those of primary T cells for a panel of fluorescein homologues. We interpret this finding to indicate that the antigen receptor repertoires utilized by primary and secondary cytotoxic T cells are indistinguishable and that the enhanced response of memory as compared with naive CTL results from factors such as a higher density of cell surface receptors or receptor-associated molecules and/or a higher frequency of antigen-responsive T cells.

A Prototype Erodible Mask Delivery System for the Excimer Laser

The authors developed an erodible mask delivery system for the argon-fluoride 193-nm excimer laser, which offers the possibility of correcting hyperopia and astigmatism as well as myopia. Masks were made of polymethylmethacrylate on a quartz window, with intended corrections for myopia and hyperopia of 2.5 and 5 diopters (D). Ablations using the mask and control ablations using an expanding diaphragm were performed in 30 eyes of 15 pigmented rabbits with an Excimed UV200 laser (Summit Technology, Inc, Waltham, MA). The rabbits were followed for 134 days with regular biomicroscopy and retinoscopic examination by two observers. Ablations with the mask to correct myopia were successful and produced stable corrections, although the higher-power mask produced undercorrections. Hyperopic masks produced paradoxic myopic corrections, possibly due to the lack of a transition zone at the edge of the mask. Corneas ablated with the mask had less sub-epithelial haze than those ablated with the diaphragm at all examinations. Results of histopathologic examination showed epithelial hyperplasia over the ablation zone in all eyes. Dichlorotriazinyl aminofluorescein collagen staining showed subepithelial new collagen in all eyes, but there was no relation between the depth of ablation at any point on the cornea and the amount of new collagen deposited there. Myopic ablations are feasible with the erodible mask, although additional calibration is needed. Hyperopic ablations were unsuccessful with the current design. Corneas ablated with the mask may be clearer than corneas ablated with the diaphragm, possibly due to a smoother ablated surface. Regression of effect after laser ablation in the rabbit model is likely due more to epithelial hyperplasia than to stromal remodeling.

Quantitative Analysis of Wound Healing after Cylindrical and Spherical Excimer Laser Ablations

Superficial corneal ablation using the 193 nm excimer laser for the correction of myopia and astigmatism produces histologic changes in the corneal profile that can be quantitatively analyzed. Using the excimer laser, spherical and cylindrical corneal ablations were performed on rabbit eyes at depths of 2, 4, 6, and 8 diopters. After 12 weeks, the corneas were examined histologically using a dichlorotriazinyl aminofluorescein stain for collagen, and the results were quantified using a digital video image analysis system. Epithelial thickness and thickness of new collagen were positively correlated with the depth of ablation after spherical ablations. With astigmatic ablations, epithelial thickness and new collagen production were correlated with ablation depth in one axis only. These results suggest that during cylindrical corneal ablations with the excimer laser, radially asymmetrical stromal ablations produce quantitatively asymmetrical wound healing.

Temporal variations in the fine specificity of IgM anti‐fluorescyl antibodies

This study compares the fine specificities of the primary and secondary fluorescein (FITC)-specific immunoglobulin M (IgM) repertoires in BALB/c mouse serum and monoclonal antibodies (MoAb) and has found reproducible, immunization-dependent differences. FITC and four of its homologues; iodoacetamido fluorescein (IAF), dichlorotriazinyl aminofluorescein (DTAF), substituted rhodamine isothiocyanate (XRITC) and tetramethyl rhodamine isothiocyanate (TRITC), each conjugated to bovine serum albumin (BSA), were used to determine reactivity patterns of serum IgM from mice immunized once or twice with FITC-haemocyanin (FITC-Hy). Reactivity patterns were also obtained for 20 IgM MoAb, eight of which were produced by fusions of SP2/0 myeloma cells with splenocytes from mice immunized once (primary) and 12 from mice immunized twice (secondary) with FITC-Hy. Each MoAb exhibited a unique fine specificity pattern, evidence of extensive heterogeneity in the FITC-specific repertoire. Reactivities of IgM MoAb with certain homologues were found to be more characteristic of either the primary or secondary response. Polyclonal serum IgM also showed reproducible immunization-dependent variations in fine specificity. Such a pattern could result from idiotypic suppression of primary antibodies, from the expansion of subsets of IgM memory cells utilizing novel genes and/or from somatic mutation absent in primary IgM antibodies.

Normal band 3-cytoskeletal interactions are maintained on tanktreading erythrocytes

Normal nonnucleated erythrocytes subjected to continuous hydrodynamic shear exhibit membrane deformation or "tanktreading," a process important for reduction of the bulk viscosity of circulating blood. To characterize the effect of this unique process on the erythrocyte membrane we have measured the lateral diffusion of band 3 during tanktreading. Band 3 is normally constrained through interactions with the spectrin-actin cytoskeleton, therefore, any significant disruption of these interactions would result in alterations in band 3 dynamics. Band 3 of human erythrocytes was labeled with dichlorotriazinyl amino fluorescein. After laser photobleaching of an equatorial stripe, fluorescence images were recorded from cells in the presence or absence of shear. The amplitude of induced nonuniformity in the surface distribution of fluorescence was calculated directly from images of unsheared cells. In shear the bleached line rotated with the tanktreading motion of the cells. The surface integral of fluorescence oscillated with this motion. For this case, the amplitude of photobleaching-induced nonuniformity was defined as the amplitude at the fundamental frequency of fast Fourier transforms in time of the oscillations. Shear stress-induced membrane flow did not interrupt the linkage of band 3 with the erythrocyte cytoskeleton. Diffusion coefficient and mobile fraction (1.5 +/- 0.5 x 10(-10) cm2/s and 54 +/- 11%, respectively) were unaffected by shear. The rate of fluorescence recovery of cells in shear was also similar at the centers and at the edges, where in-plane shear forces are maximal.

Corneal healing following laser refractive keratectomy.

A 193-nm excimer laser system was used to ablate 4.5-mm optically contoured zones in the corneal stroma of rabbits to achieve optical flattening of 2, 4, 8, and 16 diopters. We studied the effect of edge profile and wound depth on reepithelialization and stromal remodeling using dichlorotriazinyl aminofluorescein, a vital dye that covalently binds to the stromal bed and delineates the boundaries of new collagen synthesis. All the corneas reepithelialized; no subsequent recurrent erosions occurred. All seven corneas that received an ablation of less than 50 microns were clear centrally at 8 weeks. At an ablation depth of approximately 100 microns, opacification and scarring were observed biomicroscopically and histopathologically in two specimens. No evidence of new collagen formation or epithelial hyperplasia was found in any of the seven corneas that remained clear. Stromal remodeling was observed in the two corneas that exhibited scarring.