Subjective vs. objective analysis of the corneal endothelial cells in the rabbit cornea by scanning electron microscopy - a comparison of two different methods of corneal fixation.
OBJECTIVE
To illustrate a method of quantitative scanning electron microscopy (SEM) for the evaluation of the corneal endothelia of small animals by comparing two commonly used fixation methods.


METHODS
Female New Zealand white rabbits, aged 10-12 weeks.


METHODS
The corneas were either dissected from the eye and placed in fixative (2% glutaraldehyde in 80 mm cacodylate, pH 7.4) or the whole eyeball immersed in fixative solution and the cornea dissected later. Lower (x 200) and high magnification (x 1,000-5,000) images were inspected for overall appearance. Magnification images (x 500) were used to measure the areas of 100 cells, and cell density (CD) calculated from the average area.


RESULTS
Both fixation protocols yielded an intact endothelial surface, but the dissect- then-fix protocol resulted in more creasing and distortion artifacts that were avoided with the whole-eyeball fixation. Overall, the CD values were higher if the dissect-then-fix method was used, and the uniformity of the cell mosaic was less. The median CD values for the central, mid-peripheral and peripheral regions following dissect-then-fix protocols were 7,693, 7,353, and 7,071 cells/mm(2) (average eight corneas at each location). If the whole-globe fixation was used, the median CD values were 6,098, 5,747, and 4,785 cells/mm(2).


CONCLUSIONS
Cell density values in SEM can be very different according to the fixation method used. A distinct regional difference in CD was evident, which was more pronounced if the cornea was fixed prior to being dissected from the eye.
Department of Vision Sciences, Glasgow-Caledonian University, Glasgow G4 OBA, Scotland. m.doughty@gcal.ac.uk
Michael J
MJ
Doughty
D000818:Animals; D002452:Cell Count / Q000662:veterinary; D003315:Cornea / Q000166:cytology / Q000648:ultrastructure; D004728:Endothelium, Corneal / Q000166:cytology* / Q000648:ultrastructure*; D005260:Female; D005404:Fixatives / Q000494:pharmacology; D008855:Microscopy, Electron, Scanning / Q000379:methods / Q000662:veterinary*; D011817:Rabbits; D016707:Tissue Fixation / Q000379:methods / Q000662:veterinary*
D005404:Fixatives