Non-invasive in-vivo assessment of corneal dehydration in the rabbit using confocal Raman spectroscopy

Purpose. Maintaining adequate hydration of the cornea is essential for visual acuity but heretofore accurate measurements of corneal hydration in-vivo have been unavailable. Using a confocal Raman spectroscopy (Conforam) system we studied hydrational changes of the in-vivo rabbit cornea after instillation of a dehydrating agent. Methods. The Conforam is a fiber based, diffraction limited probe with a long working distance for free mechanical movement and a CCD array allowing rapid scanning (< 10s) and low laser power (< 25 mW). The 514.5 nm line of a CW Argon-ion laser was used as excitation light. Three NZW rabbits were investigated. Axial cornea scans in the higher spectral range (2634-3820 cm-1) were taken for the whole corneal thickness. Scans before and after instillation of the dehydrating agent Muro-128 (5% NaCl Ophthalmic Ointment) were compared in the same eye, using the OH- and CH-peaks to calculate a OH/CH ratio as a measure for corneal hydration. Statistical analysis was performed using the Student's t-test. Results. The average OH/CH ratio was 60.5 (SD=6.8) in non-treated eyes and 52.4 (SD=2.9) in NaCl treated eyes. There was a statistical significant difference (p<0.05) between the treated and non-treated cornea. A high enough spectral S/N ratio was obtained during these experiments. However, the NaCl ointment obscured the CH-peak in the higher spectral region at superficial cornea scans. Conclusion. With our Confocal Raman Spectroscopy probe it is possible to assess the relative decrease in corneal hydration of healthy living rabbits after a dehydrating agent is applied. 5% NaCl decreases corneal hydration dramatically.