Non-invasive assessment of ocular pharmacokinetics using Confocal Raman Spectroscopy.

A Laser Scanning Confocal Raman Spectroscopy (LSCRS) system was applied for the non-invasive quantification of the transport of a drug through the rabbit cornea in vivo. Employing LSCRS, the changes in the amplitude of a drug-specific Raman signal were assessed over time in the tearfilm and corneal epithelium of the living rabbit eye (n = 6), after topical application of 25 microL Trusopt 2%. This allowed for quantification of pharmacokinetic variables. The effect of the drug on corneal hydration was also monitored. LSCRS demonstrated adequate sensitivity and reproducibility, for continuous real-time monitoring of the Trusopt concentration. Each concentration-time curve had a bi-phasic trend; the rapid initial phase (t<8 min.) corresponds to the nonproductive losses of Trusopt from the tears (k10 = 0.24+/-0.04 min(-1), and the slower later phase (t>20 min.) is the result of transfer of the drug from the corneal epithelium to the stroma (k23 = 0.0047+/-0.0004 min(-1). Drug absorption into the corneal epithelium occurred at a rate of k12 = 0.034+/-0.006 min(-1). Trusopt caused an acute dehydrating effect, with a maximum decrease in corneal hydration of approximately 15% at approximately 60 min. following application of the drug. LSCRS has the specificity, sensitivity, reproducibility and spatial resolution for employment as a potentially valuable tool for the study of ocular pharmacokinetics.

Noninvasive assessment of the hydration gradient across the cornea using confocal Raman spectroscopy.

PURPOSE: The feasibility of Raman spectroscopy for the noninvasive assessment of axial corneal hydration was investigated. METHODS: A scanning confocal Raman spectroscopy system, with an axial resolution of 50 microns, was used to assess noninvasively the water (OH-bond) to protein (CH-bond) ratio as a measure of the hydration in collagen-based phantom media and rabbit corneas. RESULTS: Raman spectra with high signal-to-noise ratios were obtained under in vitro and in vivo conditions within a range of corneal hydration (H = 0.0-8.3 mg water/mg dry wt). The Raman intensity ratio OH/CH showed a strong correlation with the hydration of the phantom medium (R2 > 0.99) and the rabbit corneas (R2 > 0.95). A degree of reproducibility was seen in measurements performed at a specific depth within the cornea (SD = 1.2%-2.7%). Quantitatively, the spatially resolved corneal water content, as assessed with our method, showed an increasing gradient from the anterior to the posterior region, with a difference of approximately 0.9. Significant qualitative differences in the axial hydration gradient were observed between the in vitro and in vivo situation, caused by the presence of an intact tear-film in vivo. Characterization of the axial corneal hydration using Raman spectroscopy provided a reliable estimation of total corneal hydration compared with conventional measurements using pachymetry and lyophilization. CONCLUSIONS: The proposed noninvasive confocal Raman spectroscopic technique has the potential to assess the axial corneal water gradient with a degree of sensitivity and reproducibility.