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Sangly P Srinivas, Wanachat Chaiyasan, Pattravee Niamprem, Katelyn Keefer, Waree Tiyaboonchai, Uday B Kompella; Penetration of Polar Sulforhodamine B into the Cornea. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4138.
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© ARVO (1962-2015); The Authors (2016-present)
Several fluorescent dyes are employed as tracers to investigate barrier function of the corneal epithelia and as drug analogs to assess pharmacokinetics of topical drugs. Unlike fluorescein and its derivatives, the fluorescence of Sulforhodamine B (SRB) is pH independent and therefore can serve as a better tracer in the presence of changes in pH. This study has examined the transcorneal dynamics of SRB using a custom-built confocal scanning microfluorometer (CSMF; depth resolution ~ 7 μm).
SRB (0.1%) was injected into a/c of pig eyes (n = 3). The epithelium was exposed to a dish containing the dye (n = 3). CSMF with a water-immersion objective (Zeiss 40x; 0.75 NA and wd = 1.2 mm) was employed to quantify the penetration dynamics of SRB. The output of a white LED modulated at 10 kHz was filtered through an interference filter (565 + 10 nm) and led to the excitation port of the CSMF. The SRB fluorescence ( > 585 nm) and scattered light passing through a parfocal exit slit in the eyepiece were detected by 2 photomultiplier tubes coupled to 2 lock-in amplifiers. Measurements were performed with eyeballs held underneath the objective on a motorized linear stage.
Exposure of the corneal epithelium to SRB over 3-12 hrs led to significant but variable fluorescence in the stroma (n= 3). The fluorescence distribution showed a marked discontinuity at the interface between epithelium and stroma, with gradient in the stroma itself. Injection of SRB into the anterior chamber also produced increase in fluorescence from the stroma over time with noticeable discontinuity at the interface between endothelium and anterior chamber (n = 3). Unlike a small peak of fluorescence in the epithelium, there was no notable fluorescence from the endothelial layer and stromal gradient was also much smaller. These transcorneal fluorescence profiles of SRB are very much similar to those obtained with hydrophilic carboxyfluorescein administered into the a/c.
A significant increase in the stromal fluorescence with a small fluorescence increase corresponding to the epithelium and negligible increase in fluorescence corresponding to the endothelium indicates that SRB crosses barriers of the corneal epithelia via penetration through the tight junctions. Therefore, SRB could be used as a polar tracer to assess barrier function of the ocular epithelia in situations of marked changes in pH.
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