Canine ocular surface epithelia secrete hyaluronan

Purpose. We have previously shown the presence of hyaluronic acid (HA) in tears. We now investigate the site of HA secretion onto the ocular surface. Methods. a) Corneal and conjunctival tissue were cultured in DMEM for 24h in chambers enabling uncontaminated sampling of medium from the apical aspect of the tissue. Fragments of lacrimal gland were also maintained in vitro for 24h. HA concentrations in culture media were assessed in a plate assay with biotinylated HA-binding protein (bHABP). b) Cornea, conjunctiva and lacrimal gland were fixed in formol saline or 95% ethanol, and wax embedded. HA was located using bHABP. Results. Corneal and conjunctival epithelia secreted detectable levels of HA. When related to epithelial surface area, conjunctiva produced approximately twice as much HA as cornea. High levels of HA were also found in lacrimal gland culture medium. Fixation methods had a profound influence on the histological visualisation of HA. Staining of cell membranes was demonstrated most consistently in conjunctiva, and most obviously in basal epithelial cells. We could not demonstrate staining of lacrimal and corneal epithelia reliably. Conclusions. Ocular surface epithelia secrete HA in vitro, with greater secretion by the conjunctiva. Histological evidence indicates HA associated with conjunctival epithelial cell membranes. Our failure to demonstrate epithelial HA in sections of cornea or lacrimal gland may be due to the presence of low molecular weight (MW) HA. This may be labile during histological processing. HA detected in lacrimal gland cultures, may be derived from fibroblasts. Comparison of the MW of ocular/lacrimal epithelial vs. interstitial HA will assist in investigating this possibility.