Abstract
Purpose :
Scientists are equipped with tools to induce genetic mutations in mouse models to advance the understanding of disease mechanisms. We have developed a multimodality imaging system to evaluate gene expression and structural remodeling in the cornea of transgenic mice.
Methods :
An integrated system of optical coherence microscopy (OCM) and dual-channel fluorescence microscopy (DCFM) was developed to simultaneously co-register reflectance and fluorescence from the cornea and track the temporal and spatial distribution of gene expression in a triple transgenic mouse model, KeraRT; tet-O-Cre; RosamTmG, that expresses membrane-bound red tdTomato in the whole body. In this model, the red tdTomato (Kera), initially expressed in the membrane of corneal keratocytes, would progressively get deleted to enable the expression of enhanced green fluorescent proteins (EGFP) in corneal keratocytes upon doxycycline (Dox) chow treatment. This process was evaluated in three KeraRT; tet-O-Cre; RosamTmG mice sacrificed at different time points of dox chow treatment (0, 9, and 47 days)
Results :
The integrated system achieved the concurrent co-registration of reflectance and fluorescence signals at the speed of 245 kHz, a lateral resolution of 2.1 mm and axial resolution of 2.4 mm for the HROCM, 16.3 mm for the DCFM’s green channel, and 14.4 mm for the DCFM's red channel, in the cornea. Fig.1 presents the images of the cornea of the three mice. The first reporter mouse without Dox treatment (Figs. 1(a)-1(d)) showed only red signal from tdTomato with no EGFP expression as expected, except for a cellular feature that was presumed to be a Langerhans cell (Figs. 1(b) and 1(c)). The EGFP activation was effective in corneal keratocytes of the two mice treated with Dox chow for 9 days (Figs. 1(e)-1(h)) and 47 days (Figs. 1(i)-1(l))
Conclusions :
The integrated system successfully registered reflectance and fluorescence signals from deep corneal layers. This system will be used to longitudinally evaluate the timing of EGFP activation in the same reporter mouse. Future studies will elucidate the molecular mechanism underlying the signaling pathways of TGFb2 in the corneal development, homeostasis, and related pathophysiology, using quadruple transgenic mouse models of KeraRT; tet-O-Cre; RosamTmG crossed with knock-out mice of TGFb2 downstream genes.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.