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Samuel D. Hanlon, Evelyn S. Brown, Margaret M. Gondo, Alan R. Burns; Microwave Processing of Murine Corneas for Light and Electron Microscopy. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2536.
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Interlamellar separations are frequently seen in corneal histological specimens presumably as a result of fixation. These separations, as well as additional swelling or shrinkage, result in a large variability in reported central corneal thickness (CCT). Previously we reported the use of spectral domain optical coherence tomography (SD-OCT) to obtain in vivo CCT values in C57BL/6 mice. The purpose of the current study was to establish a method of histological fixation that produces CCT values similar to in vivo SD-OCT measurements and with minimal stromal artifacts.
Whole eyes were removed from euthanized C57BL/6 mice (n=39, age ≥50 days), placed in 2.5% glutaraldehyde and processed with low wattage microwave radiation in a vacuum using a Pelco Biomicrowave. For comparison, mouse eyes (n=27, age ≥50 days) were also fixed using a conventional protocol (room temperature buffered glutaraldehdye fixation for 2h). All corneas were sectioned transversely and imaged by light and electron microscopy. ImageJ was used for all measurements of captured images.
Transverse sections prepared from microwave fixed corneas exhibited excellent histological preservation with little or no evidence of interlamellar separation. CCT values for these corneas showed remarkable agreement with in vivo measurements obtained previously by SD-OCT (101.2+ 11.1 µm and 103.4 + 7.9 µm, respectively). In contrast, corneas fixed using the conventional protocol exhibited much greater CCT values (130.9+27.8 µm) and frequent posterior interlamellar separations. Ultrastructurally, the collagen interfibrillar spacing was significantly greater and more variable in conventionally fixed corneas compared to corneas processed in the microwave (71.8+18.5 nm and 54.7+9.6 nm, respectively).
The microwave fixation protocol produced CCT values similar to in vivo measurements and with minimal separation between stromal lamellae. The larger CCT measurements from the conventionally fixed corneas, in the absence of interlamellar separations, were determined to be due to increased stromal thickness, attributed to increased collagen interfibrillar spacing. Interfibrillar spacing in the microwave fixed corneas is very similar to that reported with X-ray diffraction suggesting microwave fixation preserves the native collagen structure to a much better degree than can be obtained by conventional methods.
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