Abstract
Purpose: :
Lumican deficiency leads to abnormal collagen fibril aggregation and loss of corneal transparency, especially within the posterior stroma after eyelid opening. The purpose of this study was to determine if NLO imaging of SHG signals could be used to detect developmental differences in the corneal collagen organization between wild type (WT) and Lum -/- mice.
Methods: :
WT and Lum -/- mouse corneas were obtained at postnatal (PN) days 4, 8, 10, 12, 16 and 20. Eyes were fixed in 4% paraformaldehyde and then imaged using the 40× oil-immersion objective lens (NA=1.3) of Axiovert 200 microscope (Zeiss, Jena, Germany). NLO SHG signals were generated using a femtosecond laser tuned to 800nm wavelength (Chameleon, Coherent Inc, Santa Clara, CA) and collected by the 388-409nm band-pass filter. Images were recorded as 8 bit, 512 × 512 and were analyzed by the LSM image browser software (Zeiss, Jena, Germany).
Results: :
As can be seen in Fig. 1, images of the posterior stroma using NLO imaging of the forward scattered (cyan) and backward scattered (magenta) SHG signal showed details of collagen fibril organization of the WT (a) and Lum -/- (b) mouse at PN8. In the WT mouse, collagen fibers were organized into broad bundles that showed interweaving characteristic of stromal collagen lamellae. By comparison, images from the Lum-/- mouse cornea showed a general breakdown in the lamellar organization, with smaller collagen fiber bundles and irregular collagen fiber signals, particularly in the forward scattered images. These differences were noted at all postnatal ages from day 4 to day 20.
Conclusions: :
These findings indicate that NLO imaging of SHG signals can be used to evaluate the postnatal development of corneal stromal collagen organization and to detect changes that lead to the loss of corneal transparency. The data also indicate that lumican deficiency results in abnormal collagen fibril organization very early in development.
Keywords: cornea: stroma and keratocytes • microscopy: confocal/tunneling • transgenics/knock-outs