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I. Schmack, D.G. Dawson, Y. Zhang, H.E. Grossniklaus, G.W. Conrad, H.F. Edelhauser; Analysis and Quantification of Sulfated Glycosaminoglycan (GAG) Dissacharides in Human LASIK Corneal Wounds Using Single Sections Microdissected by Laser Capture Microdissection (LCM) and Evaluated by Electronspray Ionization Tandem Mass Spectrometry (ESI–MS/MS) . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2132.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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