An electron microscopic and nuclear magnetic resonance spectroscopic evaluation of rabbit corneal epithelial wound...

Phosphorus-31 (31P) nuclear magnetic resonance (NMR) spectroscopy is a powerful method that can be used to measure levels of phosphorus metabolites in living tissue. To determine whether NMR measurements adequately describe and quantify the wound-healing process in corneal epithelium, 31P NMR spectroscopic and electron microscopic results were correlated. Dutch belted rabbit corneal epithelium was removed with a limbus-to-limbus scrape wound and sampled from 0-30 days postwounding. Four corneas were pooled for each NMR determination of corneal adenosine triphosphate (ATP). A minimum of three animals from each time point were processed for transmission and scanning electron microscopy (EM). Similar to previous studies, migrating cells completely closed the defect by 4 days postwounding. The epithelium continued to increase in thickness and hemidesmosome density was similar qualitatively to that in control animals by 10 days postwounding. The 31P NMR ATP values showed an increase toward control values after an initial 70-75% decrease, returned to within 90-95% of nonwounded values by 10 days, and returned to control values by 30 days. After these experimental values were obtained, the predictive value of the model was tested using platelet-derived growth factor (PDGF)-treated corneas. The PDGF-treated corneas showed NMR and EM results similar to day-10 untreated corneas by 5 days after wounding. These results demonstrated the feasibility of using 31P NMR spectroscopic measurements of ATP levels as a nondestructive technique for quantifying corneal epithelial wound healing.