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Staining Characteristics and Antiviral Activity of Sulforhodamine B and Lissamine Green B James Chodosh,* Richard D. Dix,* R. Clark Howell* William G. Stroop,\ and Scheffer C. G. Tseng* Purpose. Fluorescein and rose bengal are dyes used routinely in the examination of the ocular surface. As part of an ongoing search for a superior ophthalmic dye with optimal specificity and sensitivity and a lack of interference with subsequent viral cultures, and as part of studies that use chemical dyes to understand better the pathophysiology of ocular surface disorders, the staining characteristics and antiviral activity of sulforhodamine B and lissamine green B were investigated. Methods. Staining of rabbit corneal epithelial cell cultures by sulforhodamine B and lissamine green B was compared to that of fluorescein and rose bengal. Diffusion of each dye through a collagen gel was measured. Uptake of lissamine green B by herpes simplex virus type 1 (HSVl)-infected Vero cell cultures was compared at several times postinfection. The effect of sulforhodamine B and lissamine green B on HSV-1 plaque formation in Vero cells was determined. The cellular toxicity of sulforhodamine B and lissamine green B in vitro was examined by a quantitative 14C-amino acid uptake assay and by a qualitative cell viability assay. Finally, the effect of sulforhodamine B and lissamine green B on viral replication was compared in vivo with that of rose bengal in a rabbit model of herpetic epithelial keratitis. Results. Rose bengal vividly stained cell monolayers of explant cultures of rabbit corneal epithelium. By light microscopy, sulforhodamine B and lissamine green B, like fluorescein, did not stain the epithelial cells, but did stain the corneal explant stroma. Pretreatment of epithelial cells with 0.25% trypsin for 5 minutes failed to induce dye uptake; however, pretreatment with 0.5% Triton X-100 for 5 minutes resulted in nuclear staining by lissamine green B, but not sulforhodamine B. When added to a collagen gel, the relative diffusion rate was fluorescein > lissamine green B > sulforhodamine B > rose bengal. By spectrophotometric analysis, HSV-1infected and uninfected Vero cells bound equivalent amounts of lissamine green B until late in infection, when infected cells took up more dye (P < 0.001). A direct neutralization assay showed that 0.06% lissamine green B or 0.5% sulforhodamine B reduced HSV-1 plaque formation in Vero cells by greater than 50%, when present at the time of viral adsorption. By a quantitative 14C-amino acid uptake assay, lissamine green B was toxic to Vero cells in a dose-dependent manner, whereas sulforhodamine B was relatively nontoxic at the concentrations tested. By a cell viability assay, however, neither dye showed significant cellular toxicity. In a rabbit model of herpetic epithelial keratitis, rose bengal significantly reduced viral replication and recovery, whereas sulforhodamine B and lissamine green B had no effect. Conclusions. Neither sulforhodamine B nor lissamine green B stain healthy, normal cells. Lissamine green B stains membrane-damaged epithelial cells, but sulforhodamine B does not. Both sulforhodamine B and lissamine green B stain corneal stroma. Lissamine green B inhibits HSV-1 plaque formation at low concentrations of dye in vitro, which correlates with suppression of cellular metabolism as demonstrated by a 14C-amino acid uptake assay, but does not From the *Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami School of Medicine, Miami, Florida, and the -\Cullen Eye Institute, Department of Ophthalmology, and Division of Molecular Virology, Baylor College of Medicine, and Ophthalmology Research Laboratory, Houston Veterans Affairs Medical Center, Houston Texas. Presented in part at the Association for Research in Vision and Ophthalmology Annual Meeting, Sarasota, Florida, May 2-7, 1993. Supported by National Institutes of Health grarits NINCDS 1-PO-NS25569 (RDD), DC 01706 (WGS), NEIEY06819 (SCGT), and EY02I80 (Bascom Palmer 1046 Eye Institute); and the Department of Veterans' Affairs (WGS). WGS holds a Jules and Doris Stein Research to Prevent Blindness Professorship. JC was supported by a fellowship from the Heed Ophthalmic Foundation for 1992-1993. Submitted for publication June 8, 1993; revised September 22, 1993; accepted October 7, 1993. Proprietary interest category: N. Reprint requests: Scheffer C. G. Tseng, Bascom Palmer Eye Institute, 900 N.W. 17 Street, Miami, FL 33136. Investigative Ophthalmology & Visual Science, March 1994, Vol. 35, No. 3 Copyright © Association for Research in Vision and Ophthalmology Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933180/ on 05/14/2017 1047 Sulforhodamine B and Lissamine Green B affect cell viability. Neither sulforhodamine B nor lissamine green B inhibit viral replication or recovery in vivo. Invest Ophthalmol Vis Sci. 1994; 35:1046-1058. £ luorescein and its halide derivative, rose bengal, are invaluable in the evaluation of ocular surface diseases. Research has focused on the staining properties and specificity of these two dyes,1'2 and on the antiviral nature of rose bengal.3 Studies have shown that staining of the cornea by fluorescein occurs when surface epithelial cell-to-cell junctions are disrupted.1 In contrast, rose bengal stains all healthy, normal cells in vitro, but does not stain the normal, healthy corneal or conjunctival epithelium in vivo, because the normal preocular tear film blocks access to the cells.12 Although fluorescein and rose bengal are the two ophthalmic dyes used routinely in clinical practice in the United States today, their clinical superiority to other available dyes remains unproven. For example, because of the intrinsic antiviral effect of rose bengal, its application in herpetic epithelial keratitis before viral culture has been discouraged.3'4 One dye thought to be similar in its staining properties to rose bengal is lissamine green B.5 It is not known, however, whether lissamine green B shares the antiviral capacity of rose bengal. Another dye, sulforhodamine B, has been reported to be superior to fluorescein for the visualization of both preocular tear film and conjunctival epithelial lesions.6 Although sulforhodamine B, a member of the aminoxanthene dye family, structurally resembles fluorescein and rose bengal, which are hydroxyxanthene dyes, the staining properties of sulforhodamine B have not been determined. As part of an ongoing search for a superior ophthalmic dye with optimal specificity and sensitivity and lack of antiviral activity, and as part of studies using chemical dyes to understand better the pathophysiology of ocular surface disorders, we investigated the staining characteristics and antiviral activity of both sulforhodamine B and lissamine green B. As a first step in the evaluation of sulforhodamine B and lissamine green B, we compared their staining with that of fluorescein and rose bengal in rabbit corneal epithelial (RCE) cell explant cultures. Because lissamine green B has been reported to stain herpetic epithelial keratitis,5 we compared the binding to herpes simplex virus type 1 (HSV-l)-infected and uninfected Vero cells in vitro. We then investigated the effect of sulforhodamine B and lissamine green B on HSV-1 plaque formation in vitro. We examined the cellular toxicity of each dye by a quantitative 14C-amino acid uptake assay and by a qualitative cell viability assay. Finally, we compared the antiviral effect of sulforhodamine B, lissamine green B, and rose bengal in a rabbit model of herpetic epithelial keratitis. MATERIALS AND METHODS Cell Cultures Primary RCE cell cultures were obtained, as previously reported,2 using an explant culture of an approximately 1 X 1 X 1 mm3 piece of endothelium-free but epithelium-containing rabbit cornea. Explants were cultured in 100-mm2 chambers (Nunc, Inc., Naperville, IL), containing Dulbecco's modified Eagle's medium (DMEM/F-12), 2.5 mg/ml amphotericin B, 5% fetal bovine serum (GIBCO, Inc., Grand Island, NY), 0.5% dimethyl sulfoxide, 2 ng/ml epidermal growth factor, 33 ng/ml cholera toxin, 1 mg/ml insulin, and 50 mg/ml gentamicin sulfate (Sigma Chemical, Inc., St. Louis, MO). After 9 to 12 days of culture, epithelial cells had migrated from the explant and formed a confluent cell layer with areas of stratification. Vero cells, a continuous line of African green monkey kidney cells, were cultured in 25-cm2 flasks containing DMEM with 10% fetal bovine serum, 2.5 mg/ml amphotericin B, and 50 mg/ml gentamicin. All cultures were maintained at 37°C in 5% CO2 and 95% humidity. All animals used in this study were handled in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" and the ARVO Resolution on the Use of Animals in Research. Studies adhered to the tenets of the Declaration of Helsinki. Virus The HI 29 strain of HSV-1, used throughout these experiments, was originally isolated from a fatal case of herpes encephalitis by Klassen and colleagues. Dix et al have shown that HI29 is virulent in mice after peripheral infection.7'8 Using thymidine kinase assays, we have shown HI29 to be thymidine kinase positive (data not shown). In a rabbit model of acute and immunosuppression-induced reactivated infection, HI 29 causes severe epithelial keratitis and focal temporal lobe necrotizing encephalitis.9"11 Comparison of Dye Uptake by Rabbit Corneal Epithelial Cells Sulforhodamine B, lissamine green B (Sigma), rose bengal (Aldrich, Milwaukee, WI), and fluorescein (Alcon, Fort Worth, TX) were diluted with calcium-free, phosphate-buffered saline (PBS). RCE cell explant cultures were washed once with PBS. Serial dilutions of each dye were applied to each culture well for 5 minutes, followed by three washes with PBS. The cul- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933180/ on 05/14/2017 1048 Investigative Ophthalmology & Visual Science, March 1994, Vol. 35, No. 3 ture chamber slides were examined grossly and by light microscopy, and photographed. Because sulforhodamine B and lissamine green B failed to stain RCE cell monolayers (see Results), additional monolayers were pretreated for 5 minutes with 0.25% trypsin in PBS with 0.02% ethylenediamine tetraacetic acid (Sigma), followed by the addition of fetal bovine serum to stop the enzymatic action of the trypsin, or with 0.5% Triton X-100 for 5 minutes before dye application.12 Diffusion of Dyes in Collagen Gel As shown in Results, sulforhodamine B and lissamine green B appeared to stain rabbit corneal explant stroma, and thus the relative ability of each dye to diffuse through a collagen gel was tested.1 Rat tail tendons were obtained and sterilized in 70% ethanol at 4°C for 24 hours. Collagen was extracted for 48 hours at 4°C in 0.1% acetic acid solution (150 ml/g tendon), then filtered through sterile gauze, and centrifuged at 16,000g for 1 hour at 4°C. To form the gel, the collagen supernatant was mixed simultaneously with 0.34 M NaOH and XI0 concentrated DMEM to a final ratio of 20:1:2 (v/v). The mixture was dispensed in test tubes and incubated at 37°C for 1 hour to allow it to become gelatinous. One milliliter of dye was added to each tube and allowed to sit for 30 minutes before three washes of the surface of the gel with PBS. Photographs of the gel-filled tubes were taken at several times postapplication of dye. Spectrophotometric Analysis of Lissamine Green B Binding Flask cultures (25 cm2) of Vero cells were washed once with PBS, and then infected with HSV-1 at an approximate multiplicity of infection of ten plaque-forming units (PFU)/cell. Control cell cultures were mock-infected with PBS. After an adsorption period of 1 hour, cultures were washed with PBS, and incubated. At 6 and 12 hours postinfection, triplicate cultures of infected and uninfected cells were stained with 1% lissamine green B, rinsed three times with PBS, carefully scraped from the flasks, and resuspended in 5 ml of PBS. Each sample's cells were counted by hemocytometry to ensure equal numbers of cells in all flasks. The cells were then centrifuged at 500g for 5 minutes, lysed in 2 ml of 50% ethanol in PBS, vortexed, and centrifuged again. The absorbance at 630 nm was measured on 1 ml of the supernatant from the lysed cells. For the 24 hours postinfection samples, cells from the infected cultures were gently knocked loose from the flasks, centrifuged, resuspended in PBS, counted, centrifuged, and stained with 1% lissamine green B for 5 minutes. Uninfected control cell cultures were trypsinized, centrifuged, resuspended in PBS, counted, centrifuged, and stained with 1% lissamine green B. Both infected and uninfected sample were thrice washed gently with PBS followed by centrifugation, before lysis in 50% ethanol in PBS, centrifugation, and quantitative spectrophotometry. The data were analyzed by a two-factor analysis of variance in which infection was one variable and day of infection was a blocking variable. HSV-1 Plaque Reduction Vero cells were cultured in six-well plates to confluence. Serial dilutions of sulforhodamine B or lissamine green B in PBS were mixed 1:1 with 100 PFU of HSV-1 in PBS and allowed to adsorb onto the cells for 1 hour. Each culture was then washed three times with PBS, overlayed with methylcellulose, and incubated for 3 days, at which time plaques were counted. Cellular Toxicity Because, as shown in Results, both lissamine green B and sulforhodamine B reduced HSV-1 plaque formation, we wished to determine whether the apparent antiviral effect was the result of cellular toxicity of the dyes. Uptake of 14C-amino acids into acid-precipitable protein was used to assess the effect of lissamine green B and sulforhodamine B on Vero cell metabolism, as described previously.13 Individual monolayers of Vero cells were treated in duplicate with serial dilutions of each dye for 1 hour, washed four times with PBS, and then incubated for 23 hours in DMEM containing one-tenth the normal concentration of amino acids, and supplemented with 3 to 4 mCi/ml of 14C-amino acid mixture (Amersham, Inc., Arlington Heights, IL). Cells were harvested by scraping, then pelleted, and washed twice in PBS. The final cell pellet was resuspended in 0.2 ml of PBS, frozen at -70°C, and then thawed. Next, 0.01 ml of each sample was spotted in duplicate on GF/C Whatman filter paper, dried for 15 minutes at 150°C, and washed twice at 4°C with trichloroacetic acid for a total of 30 minutes. This was followed by three 10-minute washes at 4°C with 95% ethanol. After drying at 150°C, 2.5-cm squares of filter paper containing acid-unsoluble material were placed in vials containing 7.0 ml of scintillation fluid. Radioactivity was counted on a Beckman (Columbia, MD) LS5801 scintillation counter. Staining Characteristics and Antiviral Activity in an Animal Model of Herpetic Epithelial Keratitis Four- to 5-pound, specific pathogen-free, New Zealand White rabbits were obtained from Myrtle's Rabbitry (Thompson Station, TN). Nine rabbits were bilaterally inoculated with the HI29 strain of HSV-1 by placing 2 X 106 PFU of virus in 100 ml onto the corneas, and gently massaging the eyelids. These nine Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933180/ on 05/14/2017 1049 Sulforhodamine B and Lissamine Green B rabbits were divided into three groups. Beginning at 1 day postinfection (DPI), the right eyes of the rabbits in each group of three rabbits received 50 ml of 1% lissamine green B, 1% sulforhodamine B, or 1% rose bengal dye, respectively. Treatments were given daily through 11 DPI, except for the 6th DPI. To check for obvious toxicity of daily dye treatment, the right eyes of an additional three rabbits, nonvirus infected, were treated with the dyes in parallel (one rabbit per dye). The left eyes of these uninfected rabbits served as untouched controls. After instillation of the dyes into the rabbits' right eyes on DPI 1 to 4 and 7 to 11, the left and right eyes of the virus-infected animals were swabbed with sterile cotton applicators. Each swab was separately placed into 1 ml of tissue culture media, which was subsequently titered for viral infectivity on Vero cells by a tissue culture infectious dose assay. External eye photography was performed on each rabbit in this study at 5, 7, 9, and 11 DPI, immediately after the swabbing procedure. RESULTS Lissamine Green B, but Not Sulforhodamine B, Stains Membrane-Damaged Cells Because of evidence that rose bengal stains normal, healthy cells in vitro, whereas fluorescein staining of normal cells is minimal and recordable only with a fluorescence microscope, we first sought to characterize the propensity of sulforhodamine B and lissamine green B to stain healthy cells in vitro. Serial dilutions of each dye were added to RCE explant cultures (Fig. 1). Although rose bengal vividly stained the cell monolayers at concentrations greater than or equal to 5 X 10~4 M, sulforhodamine B and lissamine green B, like fluorescein, did not stain the epithelial cells by gross inspection or light microscopy at any concentration tested. Because others have suggested that both sulforhodamine B and lissamine green B are taken up by damaged cells,56 RCE cell monolayers were artificially damaged to induce staining (Fig. 2). Monolayers were treated before dye application with trypsin to break cell-matrix and cell-to-cell adhesions (Fig. 2B), or with the detergent, Triton X-100, to damage cell membranes (Fig. 2C). Neither treatment induced cellular uptake of sulforhodamine B (data not shown). Treatment with trypsin failed to facilitate subsequent uptake of lissamine green B (Fig. 2B), but Triton X-100 pretreatment resulted in visible cellular uptake (Fig. 2C). The dye appeared to bind predominantly to the epithelial cell nuclei. Because rose bengal staining can be blocked by mucin,1 we wished to determine if lissamine green B CO 8 ffl <D C 1 # E CO o 3 "5 FIGURE i. Staining of RCE cell explant cultures with serial dilutions of rose bengal, lissamine green B, sulforhodamine B, or fluorescein. Concentrations of dye are as follows: 1 = 10"a M, 2 = 5 X 1(T3 M, 3 = 10"3 M, 4 = 5 X 1(T4 M, 5 = 1(T4 M, 6 = 5 X 1CT5 M, 7 = 10"5, 8 = phosphate buffered saline control. Note that only rose bengal visibly stains the cell monolayer. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933180/ on 05/14/2017 1050 Investigative Ophthalmology & Visual Science, March 1994, Vol. 35, No. 3 FIGURE 2. Alteration of RCE cell monolayers to facilitate lissamine green B uptake. (A) Untreated monolayer after exposure to lissamine green B. (B) Monolayer pretreated with 0.25% trypsin, followed by lissamine green B. (C) Monolayer pretreated with 0.5% Triton X-100, followed by lissamine green B; or with (D) 0.5% Triton X-100, followed by coating with porcine stomach mucin, and then lissamine green B. Lissamine green B uptake occurred only in Triton X-100-pretreated cells, and was not blocked by coating the cells with mucin before dye application. Note that Triton X-100 pretreatment before lissamine green B results in nuclear staining of epithelial cells (C,D). The left lower corner of each micrograph is the area from adjacent to the explant and appears defocused. uptake by membrane-damaged cells could be blocked in a similar fashion. Triton X-100-damaged cells were coated with 1% porcine stomach mucin before application of lissamine green B. As shown in Figure 2D, mucin failed to block lissamine green B uptake by membrane-damaged cells. The treatment of cells with porcine stomach mucin also failed to affect subsequent attempts to stain them with sulforhodamine B (data not shown). Neither sulforhodamine B nor lissamine green B bound to porcine stomach mucin when mixed together with the mucin and passed through a G-75 gel filtration column (data not shown). Lissamine Green B and Sulforhodamine B Diffusion in Collagen Gels Is Intermediate Between Rose Bengal and Fluorescein Although sulforhodamine B and lissamine green B, like fluorescein, did not stain healthy RCE cells, all three dyes stained the corneal explant stroma. In contrast, rose bengal did not appreciably stain the stroma (Fig. 1). To test the relative diffusion of each dye through a collagenous stroma, rat tail collagen was extracted and used to form a collagen gel. Each dye was exposed to the surface of a gel-filled tube for 30 minutes before removal of the excess dye by washing with PBS. Photographs of the gel-filled tubes, taken at several times after dye application, revealed a relative diffusion rate of fluorescein > lissamine green B > sulforhodamine B > rose bengal (Fig. 3). Lissamine Green B Binds to HSV-1-Infected Vero Cells Late in the Infectious Cycle Because Norn5 observed that lissamine green B stains dendritic herpetic epithelial keratitis in a manner similar to rose bengal, and because it has been shown that lissamine green B binds preferentially to the measlesinfected conjunctival epithelium in measles keratoconjunctivitis,14 we wished to examine if lissamine green B uptake was enhanced in virus-infected epithelium. Flask cultures of Vero cells were infected with HSV-1 or mock-infected with PBS (Fig. 4A). By spectrophotometric analysis, HSV-1-infected and uninfected Vero cells bound equivalent amounts of lissamine green B at 6 and 12 hours postinfection (Fig. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933180/ on 05/14/2017 Sulforhodamine B and Lissamine Green B CQ (0 O> Ire a) o CO •o CQ c Si 5 4) o) o 1051 4B). However, because we had already shown that cell membrane damage was necessary to induce lissamine green B uptake by RCE cells (Fig. 2C), we postulated that uptake of lissamine green B by HSV-1-infected Vero cells might occur maximally at a time late in the viral replicative cycle, when virus-induced cell membrane alterations are most prominent.15 We tested the uptake of lissamine green B at 24 hours postinfecdon, just before cell lysis (Fig. 4A), and found that infected cells took up more dye than uninfected cells (P < 0.001; Fig. 4B). Lissamine Green B and Sulforhodamine B Inhibit HSV-1 Plaque Formation In Vitro Rose bengal possesses an intrinsic antiviral capacity, and may reduce the predictive value of a negative ocular surface culture for HSV-1, if the dye is applied just before culture.34 This fact limits the clinical usefulness of rose bengal in ambiguous cases of epithelial keratitis where a culture is desirable. Therefore, we tested lissamine green B, a dye thought to be specific for epithelial keratitis,5 for its antiviral activity by a direct neutralization assay.4 We found that 0.06% lissamine green B, when present at the time of viral adsorption, reduced HSV-1 plaque formation in Vero cells by greater than 50% (Fig. 5A). In contrast, at least 0.5% sulforhodamine B was necessary to show any significant decrease in plaque formation (Fig. 5B). FIGURE 3. Diffusion of dyes in collagen gel. The times shown reflect the number of minutes after application of dye to the gel. Arrowhead indicates the level of {fluorescein) dye within the gel. The relative diffusion rate isfluorescein> lissamine green B > sulforhodamine B > rose bengal. PBS = phosphate-buffered saline as a control. Lissamine Green B Has Intrinsic Cellular Toxicity In our plaque reduction experiment, Vero cells were incubated with a dye—virus mixture for 1 hour. Hence, it was of interest to determine whether the reduction in HSV plaque formation was due to cellular toxicity of the dyes. We therefore explored the effect of each dye on the incorporation of 14C-amino acids into acidprecipitable protein in Vero cells as a measure of dye effect on cell metabolism. Cells were treated with various concentrations of lissamine green B or sulforhodamine B for 1 hour, and then labeled for 23 hours with 14C-amino acids. By measuring incorporation of 14 C-amino acids into cellular acid-precipitable protein, we showed that lissamine green B was toxic to Vero cells in a dose-dependent manner (Fig. 6A), whereas sulforhodamine B was relatively nontoxic at all concentrations tested (Fig, 6B). These results indicate that the mild plaque reduction effect of sulforhodamine B was not due to cellular toxicity by the dye, but does not exclude this possibility for lissamine green B. We also performed a qualitative cell viability assay that has been used previously to demonstrate the toxicity of rose bengal.2 In this assay, RCE cells in culture were exposed to sulforhodamine B or lissamine green B for only 5 minutes before washing the cells with PBS; this Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933180/ on 05/14/2017 Investigative Ophthalmology & Visual Science, March 1994, Vol. 35, No. 3 B Hours Post Infection FIGURE 4. (A) Phase contrast appearance of Vero cells at 0, 6, 12, and 24 hours after infection with HSV-1. (B) Spectrophotometric measurement of binding of lissamine green B to infected versus uninfected Vero cells at 6, 12, and 24 hours after infection with HSV-1. Binding is expressed as the percent of binding in (infected cells/uninfected cells) X 100. By quantitative spectrophotometry, a significant difference in dye uptake was found between infected and uninfected cultures only at 24 hours postinfection, when infected cells bound more dye (P < 0.001). Error bars = standard error of the mean. more closely correlates with the human patient, in which tears dilute the dye immediately after dye application. By this assay, we found no effect on cell viability for either dye, even up to the 1% concentration (data not shown). Lissamine Green B and Sulforhodamine B Do Not Inhibit HSV-1 Replication In Vivo The staining characteristics and antiviral properties of lissamine green B and sulforhodamine B on HSV-1-infected corneas were studied and compared to rose bengal in rabbits experimentally infected with the HI 29 strain of HSV-1. Strain HI 29 typically produces large epithelial defects as well as dendritic keratitis in New Zealand White rabbits10 (Stroop, personal observations). For this experiment, both eyes of each rabbit were infected, but the dyes were applied only to the right eyes, leaving the left eyes to serve as virus-infected, non-dye-treated controls for the virus titration studies. Three animals per group were treated daily from 1 to 5 and 7 to 10 DPI by instillation of 50 ml of 1% solutions of each dye. Swabs from both the dyetreated right and non-dye-treated left eyes were titered separately for viral infectivity. To check for evidence of toxicity, the right eye of one uninfected rabbit per dye was treated in parallel. Despite its apparent antiviral properties in vitro, lissamine green B did not appreciably affect the replication of HSV-1 in rabbit corneas in vivo (Fig. 7A). No statistical difference in the titers recovered from the three dye-treated right eyes or the three non—dyetreated left eyes were found at any DPI. The dyetreated and non-dye-treated eyes all ceased shedding measurable amounts of virus by 10 DPI (Fig. 7A). Virus-induced, large, geographic corneal epithelial defects were stained easily by lissamine green B (Fig. 8), suggesting that this dye stained the exposed stroma. Lissamine green B also stained punctate and dendritic lesions that often were seen at the periphery of the large geographic lesions (Fig. 8). The geographic lesions became larger from 5 to 9 DPI, but had shrunk considerably by 11 DPI. Corneal healing appeared to correlate with the rapid decrease in viral titers observed in these animals from 9 to 11 DPI (Fig. 7A). The uninfected control animal that was treated with lissamine green B showed no obvious signs of ocular surface toxicity through 11 DPI. Like lissamine green B, sulforhodamine B exhibited no antiviral effect in vivo (Fig. 7B). Interestingly, between 7 and 8 DPI, there appeared to be a resurgence in viral replication in the corneas of the dyetreated eyes. This increase in titer, however, was not statistically significant, and was due to the reappear- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933180/ on 05/14/2017 Sulforhodamine B and Lissamine Green B 1053 bengal was profoundly antiviral, it is not surprising that the corneal lesions stained by this dye were less conspicuous than those seen in lissamine green Btreated eyes. No toxicity was observed in the eye of the uninfected control rabbit treated with rose bengal. 120- 0.000 0.01S 0.030 0.060 0.125 0.250 Lissamine Green B (%) O 80 o O 60 0.060 B 0 0.125 0.0039 Sulforhodamine B (%) 0 . 0 1 5 0.062 0.25 Lissamine Green B (%) FIGURE 5. HSV-1 plaque reduction caused by (A) lissamine green B and (B) sulforhodamine B. Note that 0.06% lissamine green B, or 0.5% sulforhodamine B, when present at the time of viral adsorption, reduced HSV-1 plaque formation in Vero cells by greater than 50%. Error bars indicate the standard errors of the means of numbers of plaques counted in triplicate cultures at each concentration of dye. ance of virus in the right eye secretions of one of the three rabbits studied. By analogy to other studies, the reappearance of virus in this single eye may have been due to the length of time required for HSV-1 to spread to the ganglion (2 to 3 days), replicate within it (1 to 2 days), and return to the eye (2 to 3 days).9"11 Sulforhodamine B did not stain the eyes of infected rabbits as well as lissamine green B (data not shown). No signs of toxicity were observed in the eye of the uninfected control animal treated with sulforhodamine B. As expected from our previous studies with rose bengal,3 this dye exhibited significant antiviral effect in vivo (Fig. 7C). Virus was recovered from the three rose bengal-treated eyes only on DPI 2 and 3; at all other DPI, virus could not be recovered from the swabbing medium. At 5 DPI, rose bengal stained the edges, but not the centers, of the large virus-induced corneal epithelial defects. This suggests that rose bengal stained the epithelium at the edge of the lesion, but did not stain the exposed corneal stroma. At 7 and 9 DPI, rose bengal staining revealed only very small, punctate and dendritic epithelial defects. Because rose o o B 0.0039 0.015 0.062 0.25 Sulforhodamine B (%) 6. Cellular toxicity of (A) lissamine green B and (B) sulforhodamine B as measured by l4C-amino acid incorporation into cellular acid-precipitable protein in Vero cells. Whereas lissamine green B was toxic to cells in a dose-dependent fashion, sulforhodamine B toxicity was relatively minor at clinically relevant concentrations. Error bars indicate standard errors of the means of acid-precipitable counts from triplicate cultures for each concentration of each dye. FIGURE Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933180/ on 05/14/2017 Investigative Ophthalmology 8c Visual Science, March 1994, Vol. 35, No. 3 1054 DISCUSSION 10* 10* 10' 10' 10 10 : 1 I s r 0/\r 5 r/ - 4 r1 3 1 1 1 1 1 1 1 1 : Ai r 6 1 ' ^ ^ ^ * < L T I I I 1 r 2 r 1 r 0 T ? ] I 0 Q LJ 1 1 1 | 10' 7 HIM 10 I I 1 2 3 4 5 I I I I I 6 7 8 9 I I I I I I 10 11 I \ V 10' O Z 10' CD GO 10' 10 10' 10' a: LJ a: LJ 10 101 I I I I I I I I 1 2 3 4 5 6 7 8 9 I I I 10 11 10 1 2 3 4 5 6 7 8 9 10 11 DAYS POST INFECTION ' In the evaluation of ocular surface disease, fluorescein is applied to improve visualization of corneal and conjunctival epithelial defects, to stain preocular tear film in evaluation of tear breakup time,16 and to evaluate tear clearance.17 Sulforhodamine B has been reported to be superior to fluorescein for the visualization of both preocular tear film and conjunctival epithelial lesions.6 In contrast, rose bengal is applied to visualize better the lesions of corneal epithelial keratitis, to assess the interpalpebral ocular surface in keratoconjunctivitis sicca, and to delineate the extent of intraepithelial dysplasia or squamous cell carcinoma at the corneal limbus.18 Lissamine green B has been touted as more sensitive than rose bengal for the screening of xerophthalmia,19"22 but this was limited by a lack of specificity for early xerosis.23'24 Lissamine green B also has been used to highlight the lesions of herpetic epithelial keratitis,5 to quantify the ocular surface changes in keratoconjunctivitis sicca,25"28 to assess the function of meibomian gland orifices,29 to test the effects of air currents on corneal epithelium,30 and to assess injury to corneal epithelium during cataract surgery.31 We have summarized the staining characteristics, antiviral activity, and cellular toxicity of these four dyes in Table 1, by combining our present studies with those published previously.1"3 Our recent work1 is consistent with the long-held hypothesis that staining of the ocular surface by fluorescein occurs when surface epithelial cell-to-cell junctions are disrupted. Fluorescein passage between epithelial cells or into corneal stroma occurs when the surface epithelium is absent, damaged, or not fully differentiated, and hence without tight junctions. Our in vitro data suggest that sulforhodamine B may act in much the same way as fluorescein. We could not induce visible staining of cells by sulforhodamine B in vitro, but did observe staining of corneal stroma. In vivo, sulforhodamine B stained HSV-1 -induced corneal lesions poorly. Our in FIGURE 7. Recovery of HSV-1, strain HI29 from the ocular secretions of New Zealand White rabbits treated with and without (A) lissamine green B, (B) sulforhodamine B, and (C) rose bengal. Animals were bilaterally infected at 0 DPI, and 50 ml of 1% solutions of the dyes were added to the right eyes of three rabbits per dye on DPI 1 to 5 and 7 to 10. The left eyes were not treated with dye. The figures show the titer of virus recovered from swabs of the dye-treated right (•) and non-dye-treated left eyes (O) (n = 3 per time point). Although lissamine green B and sulforhodamine B had no effect on viral recovery, daily application of rose bengal significantly reduced viral titers. The error bars indicate the standard error of the mean. Downward arrows indicate that the titer was below the lower limit of the assay (< 10 tissue culture infectious dose/ml). Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933180/ on 05/14/2017 Sulforhodamine B and Lissamine Green B 1055 FIGURE 8. An HSV-1 strain H129-infected rabbit eye stained with lissamine green B at 7 DPI. Note the large geographic epithelial lesions in the upper left quadrant of the cornea and the dendritic lesions in the lower right quadrant. Lissamine green B stained the epithelial edges of each ulcer, and also stained the deepithelialized cornea stroma. TABLE l. vitro results are in accordance with the work of Araie and Maurice,32 who showed that epithelial permeability by sulforhodamine B is similar to that of fluorescein, and that both dyes enter subepithelial stroma primarily by passing between epithelial cells rather than directly through them. In the same study, the authors also showed that fluorescein diffused through corneal stroma more quickly than did sulforhodamine B. This is also in agreement with our results, which showed that sulforhodamine B diffused into collagen gel at an intermediate rate between that of fluorescein (maximal) and rose bengal (minimal), and stained the corneal stroma of HSV-1-induced corneal epithelial defects more distinctly than rose bengal, but not nearly as well as lissamine green B. Lissamine gTeen B diffused into collagen gel at a rate faster than sulforhodamine B, but less than fluorescein, and stained corneal stroma well both in vitro and in vivo. In contrast to the concept put forth by Norn,5 that rose bengal stains only dead and devitalized ocular surface epithelium, our previous data1"3 suggest that rose bengal stains both healthy and "devitalized" cells in vitro. Therefore, if a vital dye is defined as any dye that can distinguish between normal or healthy and abnormal or unhealthy cells, then fluorescein may be a vital dye, but rose bengal is not. Rose bengal does not Staining Characteristics and Antiviral Activity of Ophthalmic Dyes Experimental data Stains healthy cells* Stains dead or degenerated cells Staining blocked by mucin Intrinsic toxicity Photo toxic ity Relative speed of diffusion through collagenous stroma Clinical extrapolation Staining promoted by Antiviral activity Inhibits HSV-1 plaque formation in vitro Inhibits HSV-1 replication in vivo Rose Bengal Lissamine Green B Sulforhodamine Fluorescein No No Yes Yes No Yes No No Yes No NAJ No Yes Yes Yes§ No ND No ND Fastest Slowest Fast Flow Disruption of cell-cell junctions Insufficient protection by preocular tear film Cell death or degeneration; and disruption of cell-cell junctions Disruption of cell-cell junctions No Yes Yes Weakly No Yes No ND = noi done. * Staining is defined by clinical means of detection, i.e., by the naked eye or cobalt blue filtered microscopy, f Possibly "Yes" if appropriate excitation/barrier filter is used for observations/' % Not applicable since no staining was observed without mucin precoating. § Metabolic suppression as shown by 14C-amino acid uptake, but no effect on cell viability. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933180/ on 05/14/2017 B Nof 1056 Investigative Ophthalmology 8c Visual Science, March 1994, Vol. 35, No. 3 discriminate between normal and abnormal cells in vitro, yet often acts like a vital dye in vivo, because of a complex and dynamic interaction between the surface epithelial cells and the preocular tear film. For instance, we have postulated previously that rose bengal highlights the edge of a herpetic dendritic or geographic ulceration, because the swollen, infected epithelial cells at the ulcer's edge are unable to bind or produce a component of the preocular tear film that normally would block penetration by rose bengal.3 We noted that the critical tear film component that blocks rose bengal staining of the normal ocular surface may be a corneal epithelial-cell derived mucin.33 Based on these findings, one might speculate that rose bengal staining in keratoconjunctivitis sicca could result from squamous metaplasia,34 which would produce a deficiency of this protective mucin layer. In the setting of limbal dysplasia or frank carcinoma,18 rose bengal staining could be explained by the same mechanism. This explanation, however, cannot apply to staining of the ocular surface by lissamine green B. As shown in this report, the dye is not blocked by mucin, but does stain those cells with detergent-induced membrane damage. Therefore, lissamine green B is a vital dye. Our report is not the first to demonstrate that lissamine green is a vital dye. Goldacre and Sylven35 reported the selective capacity of lissamine green to stain necrotic tumor cells. Holmberg36 showed that healthy cells were impermeable to lissamine green B, but cells damaged by freezing took up the dye in a dose—response fashion where repeated freeze—thaw cycles resulted in a greater percentage of dye-positive cells. Jans and Hassard37 demonstrated that lissamine green B was an effective "supravital" stain for determination of corneal endothelial cell viability, and suggested it might be useful in the pretransplantation assessment of donor corneal tissue. More recently, the dye has been used as a marker for in vitro cell-killing effects of antitumor drugs.38 We noticed that lissamine green B uptake in membrane-damaged cells appeared maximal in the nucleus. Rose bengal also binds to the cell nucleus,2'3 but we found that although rose bengal binding to cells was not reversible by repeated washes of the cells, the binding of lissamine green B to membrane-damaged cells was at least partially reversible by repeated washings (data not shown). Lissamine green has been documented previously to bind to serum proteins, in vitro,39 and we postulate that, like rose bengal,40 lissamine green B might bind to nuclear histones, although more reversibly than rose bengal. We also found that lissamine green B stains cells in the late stages of cytopathic viral infection in vitro, and highlights the epithelial edge of HSV dendritic and geographic corneal ulceration in vivo. Our data are consistent with previous reports that lissamine green B stains viral-infected epithelium of HSV epithelial keratitis,5 and that the dye binds preferentially to measlesinfected conjunctival epithelium in measles keratoconjunctivitis.14 In the latter study, however, measlesinfected Vero cells did not stain with lissamine green. Possibly, the dye was applied to the measles-infected Vero cells too early in the infectious cycle to show staining. We found, in HSV-1-infected Vero cells, that significant lissamine green B uptake occurred only late in infection, when virus-induced cell membrane alterations are prominent.15 Prior studies have demonstrated that rose bengal possesses antiviral activity, and that rose bengal application before viral culture could reduce the predictive value of a negative culture for HSV.3'4 The current study, in which the daily application of rose bengal to the ocular surface of HSV-1-infected rabbits reduced the intensity and duration of infection, confirms the antiviral capacity of rose bengal (Fig. 7C). Because the antiviral activity of sulforhodamine B and lissamine green B was unknown, we tested each dye for antiviral effect both in vitro and in vivo. Using a direct neutralization assay,4 lissamine green B, but not sulforhodamine B, caused a significant reduction in HSV plaque formation at very low doses in vitro. In vivo, however, neither lissamine green B nor sulforhodamine B significantly reduced viral replication. Possible explanations for the apparent antiviral effect of lissamine green B in vitro include direct inactivation of the virus via chemical or photoinactivation, prevention of viral adsorption and penetration into the cell by dye bound to the virion membrane or virus receptor site on the surface of the cell, or direct dye-induced cellular toxicity whereby the cells are unable to support viral replication. Our in vivo data suggest that direct inactivation of the virus or prevention of viral adsorption are unlikely mechanisms for the plaque reduction found in vitro. By a quantitative assay of cellular metabolism,13 in which cells were exposed to dye for 1 hour so as to parallel the plaque reduction assay, we found that cellular toxicity of lissamine green B and sulforhodamine B roughly paralleled HSV-1 plaque reduction. In contrast, by a cell viability assay,2 in which cells were exposed to lissamine green B or sulforhodamine B for only 5 minutes before washing the cells with PBS, we found no effect on viability. The 5-minute exposure to dye more closely mimics the situation in vivo, in which tears begin to dilute and clear the dye immediately after instillation, and any effect of dye on cellular metabolism is minimized. The mechanism of HSV-1 plaque reduction by lissamine green B in vitro may be a result of a prolonged exposure of the cells to the dye. Yet, we hesitate to interpret our data as proof of a cause-and-effect relationship between cell toxicity and plaque reduction. Feenstra and Tseng2 have shown previously that rose bengal is toxic to cells by the viabil- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933180/ on 05/14/2017 Sulforhodamine B and Lissamine Green B ity assay. Chodosh and Stroop have shown that rose bengal-pretreated human corneal epithelial cells, although unable to support HSV-1 replication, did support the replication of adenovirus types 5 and 8 in vitro (unpublished observations). Together, these data suggest that rose bengal, although toxic to cells, mediates the suppression of subsequent herpes viral replication, not by cellular toxicity, but rather by a direct inactivation of the virus. In summary, we have demonstrated that, in contrast to rose bengal, neither sulforhodamine B nor lissamine green B stain healthy, normal cells in vitro. Lissamine green B, but not sulforhodamine B, selectively stains damaged epithelial cells, and is therefore a vital dye. Like fluorescein, both sulforhodamine B and lissamine green B stain corneal and collagenous stroma, and could be used to demonstrate corneal and conjunctival epithelial defects. The capacity of lissamine green B to stain membrane-damaged epithelial cells, combined with its ability to stain denuded corneal stroma, could make the dye a useful alternative to rose bengal and fluorescein in the diagnosis of ocular surface diseases. Although rose bengal remains a unique dye to detect the protective status of the preocular tear film,12 it should be reemphasized 3 that the predictive value of a negative ocular surface culture for herpes simplex may be significantly lowered by the application of rose bengal before culture. For demonstration of the morphology of corneal epithelial keratitis in the human or animal research subject with HSV epithelial keratitis, lissamine green B would be preferable to rose bengal, except at the end point of the study. As we continue to search for the ideal vital dye, other properties unique to sulforhodamine B and lissamine green B are still under investigation, and may help us in the future to understand better the pathophysiology of ocular surface diseases. 1057 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Key Words herpes simplex virus type 1, lissamine green dye, sulforhodamine B, vital dye 16. Acknowledgments 17. The authors thank William Feuer for his help with statistical analyses, and Careene Banks for technical assistance. 18. References 1. Feenstra RPG, Tseng SCG. Comparison of fluorescein and rose bengal staining. Ophthalmology. 1992;99:6O5-617. 2. Feenstra RPG, Tseng SCG. What is actually stained by rose bengal? Arch Ophthalmol. 1992; 110:984-993. 3. Chodosh J, Banks MC, Stroop WG. Rose bengal inhibits herpes simplex virus replication in Vero and human corneal epithelial cells in vitro. Invest Ophthalmol VisSci. 1992; 33:2520-2527. 4. 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Wyon NM, Wyon DP. Measurement of acute response to draught in the eye. Ada Ophthalmol. 1987; 65:385392. 31. Norn MS. Preoperative protection of cornea and conjunctiva. Ada Ophthalmol. 1981; 59:587-594. 32. Araie M, Maurice D. The rate of diffusion of fluoro- 33. 34. 35. 36. 37. 38. 39. 40. phores through the corneal epithelium and stroma. ExpEyeRes. 1987;44:73-87. Mui MM, Tseng SCG. Characterization of monoclonal antibodies against mucosal epithelium membrane-associated mucin-like protein (MEM). Invest Ophthalmol VisSci. 1992; 33(suppl): 1176. Pflugfelder SC, Huang AJW, Feuer W, et al. Conjunctival cytologic features of primary Sjogren's syndrome. Ophthalmology. 1990; 97:985-991. Goldacre RJ, Sylven B. A rapid method for studying tumour blood supply using systemic dyes. Nature. 1959;184:63-64. Holmberg B. On the permeability to lissamine green and other dyes in the course of cell injury and cell death. Exp Cell Res. 1961; 22:406- 414. Jans RG, Hassard DTR. Lissamine green: a supravital stain for determination of corneal endothelial viability. Can J Ophthalmol. 1967;2:297-302. Kopf-Maier P, Wagner W, Kopf H. In vitro cell growth inhibition by metallocene dichlorides. Cancer Chemother Pharmacol. 1981;5:237-241. Brackenridge CJ. Factors affecting the uptake of lissamine green by serum proteins. / Clin Pathol. 1960;13:149-155. Zhang SH, Tseng SCG. Interactions between rose bengal (RB) and tear components. Invest Ophthalmol VisSci. 1992;33(suppl):1286. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933180/ on 05/14/2017