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Impact of brief exposure to balanced salts solution or cetylpyridinium chloride on the surface appearance of the rabbit corneal epithelium—a scanning electron microscopy study

Michael J Doughty
Current Eye Research 2003, 26 (6): 335-46

PURPOSE: To examine the surface of pre-corneal mucous layer by scanning electron microscopy (SEM) with the view to assessing whether it was an amorphous, fibrous or porous structure.

METHODS: Healthy female albino rabbits (2 kg) were euthanised at 15.00 h and the corneal surface immediately fixed with buffered glutaraldehyde fixative pH 7.2 to 7.4 at 35 degrees C, carefully rinsed with balanced salts solution (BSS) and then fixed, or treated with the same fixative containing 0.05 to 0.25% w/v cetylpyridinium chloride (CPC). The corneas were critical point dried and gold-palladium coated, prior to examination at 1000x or 15000x magnification.

RESULTS: Buffered glutaraldehyde fixation resulted in an SEM image of polygonal cells, with very contrasting electron reflexes (light, medium and dark) and distinct cell surface microplicae and large crater-like features (average diameter 2.8 +/- 0.9 microm). Along the cell-cell borders was a distinct line (referred to as "caulking"), perhaps composed of mucous. Rinsing of the corneal surface with BSS just before fixation reduced the cell contrast, the surface was then covered with very short strands or small clumps of presumed mucus, the cell-cell border features were changed, but the crater-like features were largely unchanged (average diameter 2.5 +/- 0.7 microm). Fixation in the presence of 0.05% or 0.1% CPC resulted in the appearance of very short fine strands and occasional coarse mucous strands or even small plaques of mucous on the cell surfaces. The microplicae and craters were still often evident. Fixation with 0.2 or 0.25% CPC however yielded a surface with a uniform grey reflex that included numerous ultramicroscopic debris particles (average diameter of 72 nm) and was punctuated by numerous pores. No microplicae or craters were evident. The average pore diameter was 70 nm, their density averaged 59/5 microm(2), but a radial distribution analysis indicated no substantial pattern.

CONCLUSIONS: The pre-corneal mucous layer can be dispersed, by careful rinsing with a divalent cation containing solution, into small fibrils or clumps, but the cell surface craters are unaffected. After precipitation with a low concentrations of a polycation, some coarse fibrils are also formed. The use of high concentrations of a polycation in the fixative transforms the more superficial aspects of the pre-corneal mucous layer into a pore-like gel. The crater-like features therefore do not appear to be mucous, but the pore-like structure may be indicative of the organisation of the mucous layer on the surface of the living eye.

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