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PubMed1990

Encircling photothrombotic therapy for choroidal Greene melanoma using rose bengal.

The photosensitizing dye rose bengal in combination with an argon green laser (514.5 nm) operated at low power was evaluated in 49 rabbit eyes for treatment of experimental choroidal Greene melanoma by circumferential occlusion of the choroidal vasculature. The effects of no treatment, laser alone, and rose bengal alone were observed in 16 control eyes, all of which showed rapid tumor growth. Immediately following rose bengal injection, 3 minutes of continuous irradiation at 20.4 W/cm2 (500-microns spot, 40 mW) applied in three to four circumferential revolutions around the base of tumor nodules, without direct tumor irradiation, produced peripheral vascular occlusion and consequent tumor inhibition. Similar therapy at higher laser intensity (30.6 W/cm2) and with multiple retreatment sessions (28.0 to 30.6 W/cm2) resulted in increased tumor-inhibiting effect. Low-dose rose bengal phototherapy did not appear to directly damage ocular tissues adjacent to treatment areas; however, when multiple irradiation sessions were given within a short interval, an increased incidence of retinal detachment was observed.

Animals↗

PubMed1995

Interaction between rose bengal and different protein components.

Bindings of rose bengal to several proteins were determined by Sephadex G-75 chromatography. Their respective blocking effect against dye uptake was demonstrated in an assay using a rabbit corneal epithelial cell layer. The total binding capacity of nonmucin proteins was measured using fluorometry and Scatchard analysis. The results showed that albumin, lactoferrin, transferrin, and lysozyme could--but serum prealbumin, IgA, carboxymethyl cellulose (CMC), and Sepharose 4B-purified porcine stomach mucin (PSM) could not--bind rose bengal. Lysozyme formed precipitates with rose bengal. Sufficient concentrations of albumin, lactoferrin, transferrin, or lysozyme premixed with rose bengal could block dye uptake by cells, but IgA and serum prealbumin could not. Premixed PSM was not as effective as precoated PSM in blocking dye uptake. The dissociation constant (Kd) was 1.2 x 10(-7) M, 3.6 x 10(-7) M, 3.9 x 10(-7) M, and 1.6 x 10(-6) M for albumin, transferrin, lactoferrin, and lysozyme, respectively. Based on these values, the total maximal binding capacity of nonmucin proteins in normal 7-microliters tears was extrapolated to be 0.249 micrograms rose bengal, which is too small to explain the negative staining of rose bengal on the normal ocular surface. Rose bengal, but not fluorescein, could interact with carbohydrate-containing Sephadex, CMC, and PSM to slow down its elution via Sephadex column chromatography. Therefore, the normal negative staining to rose bengal might be caused by the blocking effect of preocular mucus tear layer, which serves as a diffusion barrier. Rose bengal remains a unique dye for detecting the protective function of the preocular mucus tear.

Animals↗

PubMed1998

GH3 cells, ionic currents and cell killing: photomodification sensitized by Rose Bengal.

Photosensitization using Rose Bengal (RB) modifies membrane ionic currents and kills cultured mouse pituitary, GH3, cells. Here we investigate the dose-response relationship for ionic current modification and for cell killing to assess a possible causal link. When exposed to 0.5 microM RB and 6.5 mW/cm2 of visible light, calcium current was blocked in 1.9 +/- 0.2 min (mean +/- SEM; 0.74 +/- 0.08 J/cm2; n = 18), a transient component of potassium current, tentatively identified as a delayed-rectifier potassium current, disappeared in 52 +/- 8 s (0.34 +/- 0.05 J/cm2; n = 10) and a steady-state component of potassium current, largely a calcium-activated potassium current, disappeared in 3.5 +/- 0.4 min (1.37 +/- 0.16 J/cm2; n = 11). Conversely, the background leak current increased in magnitude. At 5 min of illumination, the longest time studied here, it continued to increase nearly linearly, making it the only current component studied that is still changing after 5 min of light. Under the conditions used, cell killing increased to 100% in the exposure range of 4-10 min of illumination (1.6 J/cm2 to 3.9 J/cm2) when assessed using fluorescent markers, ethidium homodimer and calcein and required slightly longer exposure times when assessed using trypan blue. Thus, it is difficult to ascribe a causal role in cell killing by photosensitization to alterations of standard ion channels and known ionic currents. However, the increase in leak current has the correct dose-response characteristics to be involved.

Animals↗

PubMed1992

What is actually stained by rose bengal?

It has been believed that 1% rose bengal does not stain normal, healthy cells but rather stains degenerated or dead cells and mucous strands. In contrast to this conventional knowledge, we discovered that both commercial additive-containing and additive-free rose bengal solutions stained four different types of healthy cultured cells, including rabbit corneal epithelial cells. Rose bengal staining was rapid, dose dependent, predominantly nuclear, and detectable with the naked eye at concentrations as low as 0.05% and 0.025% for the commercial additive-containing or additive-free solutions, respectively, and with the fluorescence microscope at a concentration of 0.001%. It is surprising to discover that rose bengal is not a vital dye; after staining, cells actually lost vitality, as evidenced by instant morphologic changes, subsequent loss of cellular motility, cell detachment, and cell death. Such an intrinsic toxic effect was augmented by light exposure. The rose bengal staining of live as well as detergent-treated (Triton X-100) cells could be blocked by such tear components as mucin and albumin, suggesting that normally negative rose bengal staining is due to the protective function of the preocular tear film, ie, staining is not dictated by lack of cell vitality. These data indicate that rose bengal staining ensues whenever there is poor protection of surface epithelium by the preocular tear film; this represents a new interpretation for rose bengal stains seen in various ocular surface disorders.

Animals↗

PubMed2003

Sensitization of aluminum chloride adsorbed tin(IV) oxide nanocrystalline films with Rose Bengal.

The anionic dye Rose Bengal was found to surface chelate more strongly to SnO2 nanocrystalline films previously kept immersed in a solution of washed and dried AlCl3. Dye-sensitized photoelectrochemical cells made from such films exhibit enhanced quantum and energy conversion efficiencies. The result is explained as caused by binding of AlCl3 to SnO2 surface by elimination of Cl atoms and stronger bonding of Rose Bengal to Al, enhancing dye adsorption and suppression of back electron transfer by bridging of an Al atom between Sn and the dye molecule.

Journal Article↗

PubMed1987

The antiviral effects of rose bengal and fluorescein.

We evaluated the antiviral effects of rose bengal and fluorescein sodium. The direct antiviral activity was determined by an in vitro direct neutralization assay. The 50% inhibitory dose was 16 micrograms/mL for rose bengal and 460 micrograms/mL for fluorescein. The in vivo antiviral effects of these drugs were determined in the mouse herpetic keratitis model. Following topical application, rose bengal reduced surface virus titers (swabs) 1 million-fold, and residual ocular virus (eye homogenates) 32-fold, compared with controls. No infectious virus was recovered by swabbing after topical application of rose bengal. Fluorescein had no significant effect on virus replication. Thus, rose bengal, unlike fluorescein, has significant antiviral activity, and the diagnostic use of rose bengal prior to viral culture may preclude a positive result. Also, the use of rose bengal to grade keratitis in the study of new antiviral agents should be discouraged.

Animals↗

PubMed1991

Pharmacological studies of arrhythmias induced by rose bengal photoactivation.

Singlet oxygen and superoxide production by rose bengal photoactivation leads to rapid electrophysiological changes and arrhythmias. To investigate which intermediate is causative and to probe possible mechanisms, hearts (n = at least 6/group) were perfused aerobically for 10 min without rose bengal followed by 5 min with rose bengal before illumination for 20 min. In controls, all or most hearts exhibited ventricular premature beats, ventricular tachycardia, and complete atrioventricular block. Most antioxidants tested had no protective effect; histidine, however, significantly delayed the onset of electrocardiographic (ECG) changes. In further studies, two antiarrhythmic agents (quinidine and verapamil) had no little protective effect, whereas R56865 significantly delayed the onset of ECG changes and reduced the incidence of arrhythmias. However, spectrophotometric and laser pulse radiolysis studies showed that this apparent protective effect might have resulted from an interaction between R56865 and the rose bengal molecule, leading to a reduction in singlet oxygen production. In conclusion, the electrophysiological changes induced by rose bengal photoactivation are likely to be due to singlet oxygen; antiarrhythmic drugs appear to be unable to protect against the injury unless there is some interaction with the photoactivation process.

Animals↗

PubMed1992

Rose bengal inhibits herpes simplex virus replication in vero and human corneal epithelial cells in vitro.

Rose bengal dye is thought to highlight corneal lesions induced by herpes simplex virus type 1 (HSV-1) by virtue of its binding to dead or dying HSV-1-infected epithelial cells. However, whether rose bengal binds specifically to damaged versus normal corneal epithelial cells is unclear. To determine the binding properties of rose bengal, the authors compared binding of the dye to HSV-1-infected and uninfected cells, determined the cellular binding sites of the dye, and investigated the effects of rose bengal on HSV-1 replication in dye-treated cells in vitro. Spectrophotometric analysis revealed that uninfected and infected Vero cells bound equivalent amounts of dye at several times post inoculation, indicating that rose bengal does not preferentially bind to HSV-1-infected cells. By light microscopy, rose bengal was found to bind to the cell nuclei and perinuclear region of human corneal epithelial cells (HCEC) and Vero cells. Pretreatment of Vero and HCEC with different concentrations of rose bengal and exposure to 148 microW/cm2 of white light for 2 min reduced the ability of both cell types to support HSV-1 replication. Vero cells, in the absence of rose bengal, supported HSV-1 replication, whereas pre-treatment with 0.05% rose bengal reduced the yield of HSV-1 by 99.99% (P less than 0.000001) and 1% rose bengal completely prevented HSV replication. HCEC supported HSV-1 replication in the absence of rose bengal, but pre-treatment with 1% or 0.05% rose bengal completely prevented HSV-1 replication (P less than 0.000001).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1996

Skeletal sarcoplasmic reticulum dysfunction induced by reactive oxygen intermediates derived from photoactivated rose bengal.

We investigated the role of reactive oxygen intermediates generated from photoactivation of xanthene dye rose bengal on skeletal sarcoplasmic reticulum (SR) function, which plays a major role in the regulation of intracellular Ca++ and thereby in the generation of force. We used SR microsomes of canine masseter muscle as a model system in which to explore the effect of oxidation by determining oxalate-supported Ca++ uptake, Ca++, Mg++-adenosine triphosphatase (Ca++-ATPase) activity and Ca++ permeability of the SR vesicles. Skeletal SR vesicles exposed to rose bengal (50 nM) illuminated at 560 nm resulted in significant inhibition of Ca++ uptake velocity and Ca++-ATPase activity and in stimulation of Ca++ permeability. The observed effect afforded by illuminated rose bengal was dependent on intensity of light. Most reactive oxygen species scavengers tested had no protective effect; histidine (a powerful quenching agent for singlet oxygen), however, significantly protected the effect of illuminated rose bengal on Ca++ uptake velocity and Ca++-ATPase activity. The illumination of rose bengal also caused histidine-inhibitable loss of total sulfhydryl groups of SR. The increased Ca++ permeability elicited by illuminated rose bengal was blunted by a cocktail of histidine-catalase, but not by histidine alone. Generation of reactive oxygen species (singlet oxygen, superoxide and hydroxyl radical) from photoactivation of rose bengal was studied by electron spin resonance spectroscopy by use of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 2,2,6,6-tetramethylpiperidine (TEMP). We found that illumination of rose bengal formed a 1:2:2:1 quartet, characteristic of the hydroxyl radical-DMPO spin adduct, which was effectively blunted by hydroxyl radical scavenger, dimethyl sulfoxide, and by superoxide scavenger, superoxide dismutase. The results of electron spin resonance study also showed that singlet oxygen was produced by photoactivation of rose bengal was detected as singlet oxygen-TEMP product (TEMPO); 2,2,6,6-tetramethylpiperidine-N-oxyl). The formation of TEMPO signal was strongly inhibited by histidine. Similarly, we could detect hydrogen peroxide production from illuminated rose bengal. It is suggested that photoactivation of rose bengal generated singlet oxygen, superoxide, hydrogen peroxide and hydroxyl radical, and the data obtained from the present study indicate that singlet oxygen, rather than superoxide, hydrogen peroxide and hydroxyl radical, to be the active agent in the Ca++ transport system of SR; the observed effect of singlet oxygen may be due to sulfhydryl group oxidation. Our results are also consistent with the view that singlet oxygen does not appear to be an exclusive species that increases Ca++ permeability of SR vesicles, but the increased Ca++ permeability may be caused in part by hydrogen peroxide as well as singlet oxygen.

Animals↗

PubMed1985

[Experimental evaluation of Bengal rose labeled with 123I].

Experimental evaluation of Bengal rose labeled with short-lived 123I was performed. The best diagnostic feature in 123I-Bengal rose were shown during a scintigraphic study of the rabbit liver and biliferous tract conducted in simultaneous experiments with the use of the agent labeled with 131I and 99mTc-HIDA. These were a high quality of the image of the organs, a low radiation exposure and the absence of extrahepatic uptake of the agent, particularly under conditions of the ligament of the common bile duct. The absence of a noticeable toxic effect of 123I-Bengal rose was established in experiments on rats and mice.

Animals↗

PubMed1992

Comparison of fluorescein and rose bengal staining.

The authors have recently reported that rose bengal is not a vital dye, and stains whenever the cultured cells are not covered by such components as albumin and mucin, and such a tear substitute as carboxycellulose. In this report, using cultured cells as well as normal rabbit corneas, they characterize and correlate the staining differences between rose bengal and fluorescein with the differences in their chemical structures. Fluorescein differs from rose bengal in its lack of intrinsic toxicity, photodynamic action, and ability to be blocked by the above-mentioned substances. Fluorescein staining is increased by rapid stromal diffusion and hence can manifest whenever there is disruption of cell-cell junctions. In contrast, rose bengal staining ensues whenever there is deficiency of preocular tear film protection. These experimental data may help interpret the clinical staining properties of these two dyes and enhance the understanding of the pathogenesis of various ocular surface disorders.

Animals↗

PubMed2000

PCR assessment of HSV-1 corneal infection in animals treated with rose bengal and lissamine green B.

PURPOSE: In vivo, the ophthalmic dye rose bengal displays profound antiviral effects against herpes simplex virus (HSV)-1, thus limiting its utility in diagnosis of epithelial keratitis when used before viral culture is performed. In contrast, lissamine green B does not possess significant antiviral activity in vivo. To determine whether polymerase chain reaction (PCR) could successfully detect HSV-1 DNA in ocular samples that have been exposed to ophthalmic dyes, animal models were used to observe the presence of infectious HSV-1 and viral DNA in eyes treated with rose bengal or lissamine green B. METHODS: Animals were bilaterally infected with HSV-1 strain H129, and at daily intervals up to 16 days post infection (dpi) rose bengal or lissamine green B was instilled in the left eyes. The right eyes were not treated with dyes. Swabs of the dye-treated and untreated eyes were assayed by PCR for viral infectivity by culture and the presence of DNA specific for a fragment of the HSV-1 DNA polymerase gene. RESULTS: A statistically equivalent number of samples from lissamine green B-treated and untreated eyes were positive by both viral culture and PCR. In contrast, rose bengal significantly decreased the infectious virus present in ocular secretions. A total of 44% and 78% of the rose bengal-treated and untreated eye samples, respectively, were positive by culture from 1 through 16 dpi. PCR was more sensitive than culture for detection of HSV-1 in rose bengal-treated eyes, in that 74% of rose bengal-treated samples were positive by PCR compared with 44% that were positive by culture during the 16-day period studied. It was also noted that both rose bengal and lissamine green B treatments slightly prolonged the period during which viral DNA was detectable in ocular secretions by PCR, possibly because the singlet oxygen produced by these photoreactive dyes compromised ocular cellular, humoral, and nonspecific immune factors allowing viral DNA to persist for slightly longer periods. CONCLUSIONS: PCR can successfully detect HSV-1 DNA in ocular samples that are culture negative and contain rose bengal or lissamine green B. Visualization of ocular epithelial defects with lissamine green B does not interfere with detection of infectious virus or HSV-1 DNA.

Animals↗

PubMed1979

Rose bengal strips.

I prepared rose bengal biological stain from its powder base to a 1% concentration. It was then impregnated into filter paper and allowed to air dry. The filter paper strips were sterilized and stored for individual use.

Eye Diseases↗

PubMed1992

Rose bengal activates the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

The photooxidizing xanthene dye rose bengal (10 nM to 1 microM) stimulates rapid Ca2+ release from skeletal muscle sarcoplasmic reticulum vesicles. Following fusion of sarcoplasmic reticulum (SR) vesicles to an artificial bilayer, reconstituted Ca2+ channel activity is stimulated by nanomolar concentrations of rose bengal in the presence of a broad-spectrum light source. Rose bengal does not appear to affect K+ channels present in the SR. Following reconstitution of the sulfhydryl-activated 106-kDa Ca2+ channel protein into a bilayer, rose bengal activates the isolated protein in a light-dependent manner. Ryanodine at a concentration of 10 nM is shown to lock the 106-kDa channel protein in a subconductance state which can be reversed by subsequent addition of 500 nM rose bengal. This apparent displacement of bound ryanodine by nanomolar concentrations of rose bengal is also directly observed upon measurement of [3H]ryanodine binding to JSR vesicles. These observations indicate that photooxidation of rose bengal causes a stimulation of the Ca2+ release protein from skeletal muscle sarcoplasmic reticulum by interacting with the ryanodine binding site. Furthermore, similar effects of rose bengal on isolated SR vesicles, on single channel measurements following fusion of SR vesicles, and following incorporation of the isolated 106-kDa protein strongly implicates the 106-kDa sulfhydryl-activated Ca2+ channel protein in the Ca2+ release process.

Animals↗

PubMed1995

Patient tolerance and ocular surface staining characteristics of lissamine green versus rose bengal.

PURPOSE: To determine patient tolerance and ocular surface staining characteristics of 1% lissamine green versus 1% rose bengal solutions in patients with dry eye and in those with normal ocular surfaces by comparison of subjective sensation and objective staining scores. METHODS: Twelve patients with keratoconjunctivitis sicca and eight subjects with no ocular surface disease recorded their symptoms on a graded scale (0-5) and the duration of symptoms after instillation of one drop of 1% lissamine green and 1% rose bengal on two different occasions in different order. Lissamine green and rose bengal staining patterns were recorded on a graded scale (0-4) by one of the authors. RESULTS: For patients with keratoconjunctivitis sicca mean sensation score with lissamine green (2.42) was significantly lower (P = 0.00006) than with rose bengal (4.58). The duration of symptoms was significantly longer (P = 0.0007) after rose bengal instillation as well. For subjects with no ocular surface disease, mean sensation score with lissamine green (1.375) was significantly lower (P = 0.01) than with rose bengal (2.5). In this group, duration of symptoms also was significantly longer (P = 0.001) after rose bengal instillation. In both groups, there was no difference in objective staining scores. CONCLUSION: Lissamine green is better tolerated than rose bengal by patients and is equally as effective as rose bengal in evaluating the ocular surface in keratoconjunctivitis sicca.

Adult↗

PubMed1999

Evaluation of the effect of lissamine green and rose bengal on human corneal epithelial cells.

PURPOSE: To examine the effects of lissamine green and rose bengal on proliferating human corneal epithelial (HCE) cells in vitro. METHODS: HCE cells derived from explants of discarded corneoscleral rims were cultured by the standard technique. Experimental cells were exposed to 1, 0.5, or 0.1% of either lissamine green or rose bengal for 10 min while control cells were exposed to a phosphate buffer solution (PBS). RESULT: Cell viability was 92% greater for 1% lissamine green (p = 0.013) and 81.2% greater for 0.5% lissamine green (p = 0.006) compared to 1 and 0.5% rose bengal, respectively. The difference between the effect of 0.1% rose bengal and 0.1% lissamine green on cell viability was not statistically significant (p = 0.83). Rose bengal staining of HCE cells was immediate and readily detectable with unaided eyes at all three concentrations, whereas no observable staining of healthy HCE cells was noted with lissamine green. CONCLUSION: Rose bengal adversely affects HCE cell viability and stains normal proliferating HCE cells in contrast to lissamine green, which exhibited neither of these characteristics. Therefore, we recommend the use of lissamine green over rose bengal in evaluating ocular surface disorders.

Adolescent↗

PubMed1989

Characterization of Rose Bengal binding to sinusoidal and bile canalicular plasma membrane from rat liver.

The binding of Rose bengal, a model organic anion, to sinusoidal and bile canalicular membrane fractions isolated from rat liver was compared. The fluorescence change of Rose bengal after being bound to liver plasma membranes was utilized for measuring the binding. The dissociation constants (Kd = 0.1-0.12 microM) and the binding capacities (n = 11-15 nmol/mg protein) for Rose bengal are comparable between the two membrane fractions, although the n value for sinusoidal membrane is somewhat larger than that for bile canalicular membrane. The Rose bengal binding to both membrane fractions was inhibited by various organic anions at relatively low concentrations, i.e., the half-inhibition concentrations (IC50) for Indocyanine green, sulfobromophthalein, Bromophenol blue and 1-anilino-8-naphthalene sulfonate were 0.1, 100, 1.5-2.5 and 100 microM, respectively, while taurocholate did not inhibit the Rose bengal binding to either membrane fraction at these low concentration ranges. The type of inhibition of sulfobromophthalein and Indocyanine green for Rose bengal binding is different between the two membrane domains. That is, in sinusoidal and bile canalicular membrane fractions, these organic anions exhibit mixed-type and competitive-type inhibition, respectively. It was suggested that the fluorescence method using Rose bengal may provide a simple method for detecting the specific organic anion binding protein(s) in the liver plasma membrane.

Anilino Naphthalenesulfonates↗

PubMed1994

Photoinactivation of herpes simplex virus by rose bengal and fluorescein. In vitro and in vivo studies.

Rose bengal and fluorescein are photosensitive dyes in widespread use in the evaluation of ocular surface diseases, including herpes simplex virus (HSV) keratitis. These dyes have recently been shown to penetrate living cells, and rose bengal was previously reported to possess antiviral activity. Several experiments reported herein suggest that these dyes do possess the potential for potent antiviral activity against extracellular virus, but only in the presence of light. Rose bengal is substantially more effective in vitro than fluorescein, and the effect is greater with increasing concentration of dye and duration of light exposure. Electron microscopic evaluation of treated virus showed no structural difference from untreated virus, in spite of 4- to 5-log decreases in virus titer. Intracellular virus was found to be markedly resistant to photoinactivation. In a rabbit model of acute primary HSV keratitis, daily application of topical rose bengal followed by light exposure had no therapeutic effect, although an adverse effect on culture sensitivity testing was seen.

Animals↗