Ocular surface complex carbohydrates are modified with aging

Picric acid-paraformaldehyde-glutaraldehyde (PA-P-G) was used to stabilize chemically ocular surface-associated mucus in mice of various ages. Transmission electron microscopy was then used to examine those specimens stained with cationic ferritin (CF), dialysed iron and Alcian Blue. Collectively, all of these stains are markers for anionic sulfate or carboxyl groups. With each of them, positive labeling of the ocular surface was observed for all ages examined, even when mucus cannot be morphologically demonstrated. Except for dialysed iron, staining was weak in the youngest animals and heaviest in young adult and aged mice. The ocular surface was negative for high iron diamine (HID) in pups and older mice through 1 year of age. Scant positive staining for HID was seen at the ocular surface in 14-month-old mice indicating that mucus became slightly sulfated with aging. Treatment of eyes with neuraminidase prior to fixation reduced the number of CF binding sites in all ages of mice examined, confirming that many of the carboxyl groups at the ocular surface are associated with sialic acid residues. Comparison of percentage reduction in CF labeling following neuraminidase treatment of the eyes of 5- and 10-postnatal day mice with all other ages of mice suggested that fewer removable carboxyl groups at the ocular surface are associated with sialic acid residues in pups. The ocular surface of all eyes also stained positively at the TEM level when a periodic acid-thiocarbohydrazide-silver protein (PA-T-SP) staining sequence was used. Collectively, these data are of significance with respect to further characterization of the ocular surface, particularly with regard to development-associated changes and their possible role in defence of the eye surface.