Assessment of transient changes in corneal hydration using confocal Raman spectroscopy

Purpose. The degree of corneal hydration has been linked to excimer laser corneal ablation rates. Enhanced precision with excimer laser refractive surgery may result from a better understanding of the transient changes in corneal hydration. To better understand the dynamic nature of corneal hydration, bovine corneas were investigated under different surface treatments. Methods. Confocal micro-Raman spectroscopy was used to quantify corneal hydration. Water and acetone solutions were used to establish a quantitative response of the relative OH/CH Raman bands, which are consistent with the water and collagen protein bands in cornea, respectively. Intact bovine corneas were manually debrided (designated MD group) or lamellar flaps were created to expose stromal tissue (designated lamellar keratectomy or LK group). Raman spectra were recorded every 30 seconds for 6 minutes while the prepared cornea surfaces were exposed to quiescent air or to a forced nitrogen gas flow across the surface. Results. The OH and CH Raman bands yielded a linear response while the percentage of acetone was varied from 0% to 100%. For the bovine cornea under forced flow drying. the OH/CH Raman band ratio was found to decrease by 41% from the initial value for both the MD and LK treatment Groups. These decreases were significantly more (p 0.0051 and 0.054, respectively) than the 26% decrease in the OH/CH band ratio measured for the control corneas. In quiescent air. the control and MD groups exhibited a 7% and 6% decrease in the OH/CH ratio. respectively, while the LK treatment group revealed a 19% decrease in the OH/CH ratio. Conclusions. The bovine eye experiments demonstrate that significant changes in corneal hydration are realized under different drying conditions and treatment methodologies. This study elucidates the nature of transient changes in corneal hydration in a bovine model and suggests the need for further study of the role of such variations in surgical outcome for excimer laser corneal refractive procedures.