Biochemical analysis was completed without knowledge of which was the treated eye and which was the control eye. Individual scleras were thawed, blotted, cut into approximately 1-mm squares, and lyophilized overnight at 4°C. Scleras were then defatted overnight in acetone (35 vol) at 4°C, desiccated for 18 hours at room temperature (RT), and weighed, giving a dry weight. Defatted scleras (4–5 mg) were incubated with agitation in 300 μL of 0.1 M NaOH at 4°C for 16 hours to extract the GAGs. The sample was centrifuged at 15,000
g for 30 minutes, and the supernatant was collected and neutralized to pH 7.4 with 2 M acetic acid. GAGs were precipitated by the addition of 4 volumes of 100% ethanol at 4°C for 16 hours. After centrifugation at 1300
g for 30 minutes, pellets were dissolved in 400 μL of 50 mM Tris buffer, the sample was loaded on a diethylaminoethanol (DEAE) membrane (Multiscreen-DE plate; Millipore, Bedford, MA), and light vacuum was applied. Flow-through was discarded (earlier analysis revealed the absence of GAGs in flow-through samples). GAGs were retained on the DEAE membrane and were eluted with 200 μL of 0.2 M LiCl (fraction 1) followed by 200 μL of 1.5 M LiCl (fraction 2). Both fractions were then precipitated for 16 hours at 4°C with 4 vol of 100% ethanol. Fraction 1, containing hyaluronan, was resuspended in 40 μL hyase buffer (50 mM ammonium acetate, 10 mM CaCl
2, pH 6.5). Twenty microliters ABCase buffer (0.1 M Tris-HCl, 30 mM sodium acetate, pH 8.0) was added to fraction 2, which was then divided into two equal aliquots (2a and 2b) for subsequent ABCase and ACase digestions. Both aliquots were ethanol precipitated for 16 hours at 4°C, and the pelleted GAGs were resuspended in 10 μL ACase buffer (0.1 M Tris-HCl, 30 mM sodium acetate, 10 mM EDTA, pH 7.4; fraction 2a) or ABCase buffer (fraction 2b). Fraction 1 was analyzed for hyaluronan, which eluted from DEAE in 0.2 M LiCl. On digestion with group B streptococcal hyaluronidase at 37°C for 3 hours (3.75 m-units; generously provided by David Pritchard, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL), the product Δdi HA was separated by CE and quantified
(Fig. 2A) . Fraction 2a was analyzed for chondroitin 4- and 6-sulfates and chondroitin, which eluted from DEAE in 1.5 M LiCl. On digestion with chondroitinase AC (3.75 m-units; Sigma, St. Louis, MO) at 37°C for 3 hours (fraction 2a), the corresponding unsaturated disaccharide products Δdi 4S, Δdi 6S, and Δdi 0S were released, separated by CE, and quantified
(Fig. 2B) . On digestion using chondroitinase ABC (3.75 m-units, Sigma) at 37°C for 3 hours (fraction 2b), dermatan sulfate, in addition to chondroitins 4- and 6-sulfates (and chondroitin at a slow rate), was released. In this case, Δdi 4S derived from chondroitin 4-sulfate and dermatan sulfate was separated and identified and quantified by CE
(Fig. 2C) . Fraction 2b was analyzed for dermatan sulfate, which also eluted from DEAE in 1.5 M LiCl. Dermatan sulfate content was calculated as the difference between Δdi 4S released by chondroitinases ABC and AC.