Abstract
Abstract: :
Purpose: To analyze and quantify the concentrations of sulfated keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) disaccharides in human LASIK corneal wounds. Methods: Human corneas with prior LASIK surgery stored in Optisol–GS (4°C, mean 5 days) were obtained from eye banks. The specimens were fixed in 70% ethanol for 24 hours, dehydrated through a graded series of alcohol, embedded in paraffin, and sectioned. Single sections were de–waxed in xylene. The LASIK scar was microdissected using the LCM with laser spots set at a diameter of 7.5µm. The samples collected contained either 1, 10, 25, 50, 100, or 200 spots/samples. They were then digested with 50µl droplets of keratanase II or chondroitinase ABC, and analyzed by ESI–MS/MS as described by Zhang et al., Analytical Chemistry 2004 (in press). Normal corneas served as controls. Results: Sulfated KS and CS/DS disaccharides were detectable in the samples containing ≥ 50 and 200 spots/sample, respectively. The average (±SD) concentration of mono–sulfated and di–sulfated KS dissacharide was 0.0018 ± 0.0002 and 0.0034 ± 0.0002 pmol/µm3. The total average concentration of sulfated CS/DS dissacharides was 0.00036 ± 0.00004 pmol/µm3 (54.6% Δdi–4S and 45.4% Δdi–6S). Controls averaged 0.0023 ± 0.0003 pmol/µm3 of mono–sulfated KS, 0.0032 ± 0.0002 pmol/µm3 of di–sulfated KS, and 0.00058 ± 0.00003 pmol/µm3 of total sulfated CS/DS dissacharides (85.0% Δdi–4S and 15.0% Δdi–6S). Conclusions: GAGs can now be analyzed and quantitatively compared using small microdissected samples (≤ 8,835 µm2) from single ethanol–fixed slide sections. These sections show that LASIK corneal stromal wounds have a 21% reduction in mono–sulfated KS, similar concentrations of di–sulfated KS, and a 38% reduction in CS/DS compared to controls. The clinical significance of these results requires further study.
Keywords: cornea: basic science • refractive surgery: LASIK • proteoglycans/glycosaminoglycans