Assessment of postnatal corneal development in the C57BL/6 mouse using spectral domain optical coherence tomography and microwave-assisted histology

The eyes of newborn mice are relatively underdeveloped and the lids remain closed for the first 2 weeks after birth. There after the eyes undergo a period of rapid growth for several weeks. Eventually the eyes reach an age at which many ocular structures stabilize for the remainder of the animal’s life, or for others, growth is significantly slowed. The central corneal thickness (CCT) is a parameter commonly reported in corneal studies. However there is a large discrepancy in values reported for adult mice as well as a lack of comprehensive values covering the time from birth through adulthood. In this study we report, for the first time, the use of spectral domain optical coherence tomography (SD-OCT) for in situ and in vivo determination of CCT from P0 to P250 for C57BL/6 mice. SD-OCT provided a reliable measure of CCT and we fit the data to an exponential rise to maximum growth curve resulting in a value of 49 μm for P0 and a maximum adult value of 106 μm. By comparison, corneas processed for conventional histology produced CCT values approximately 30–35% thicker and with greater variability. Ex vivo real-time imaging during fixation revealed swelling and gross distortion of the cornea beginning after only 10–15 min in fixative. The fixation artifacts were not observed when the cornea was processed using an optimized microwave fixation protocol. CCT values measured in corneas fixed with the microwave process compared favorably with values obtained with SD-OCT. We conclude that for corneal research, mice younger than 8 weeks of age should not be considered as adults since they are still in a rapid phase of growth up until that time. In addition we report the first use of microwave processed histological specimens for visualizing the murine cornea. Tissue processed in this manner has minimal artifacts, a CCT equivalent to that measured in vivo by SD-OCT and ultrastructural detail comparable to conventional fixation methods.

► Our results suggest that for corneal research, C57BL/6 mice younger than 8 weeks of age should not be considered as adults. ► The adult central corneal thickness for C57BL/6 mice is approximately 106 μm. ► Spectralis SD-OCT provides high resolution images of the murine anterior segment. ► Conventional histology fixation processing is prone to stromal artifacts. ► Microwave tissue processing minimizes fixation artifacts.