Abstract
Purpose :
Although in vivo confocal microscopy (IVCM) is routinely used in clinics, it is challenging to precisely monitor cornea pathophysiological changes, partially due to the complexity of cornea nerve architecture. Double lacrimal gland removal (DLGR) is commonly employed to establish animal dry eye disease, with a substantial change in cornea nerve morphology and inflammatory cell infiltration. Here we describe a new method for monitoring corneal stroma change using the Heidelberg Retina Tomography III (HRT III) in a murine dry eye model.
Methods :
Sprague-Dawley rats (8-9 weeks, male) underwent DLGR to remove the infraorbital and extraorbital lacrimal glands. Male rats were chosen in this study due to a lesser hormonal cycle influence. Up to three stroma nerves per eye were selected to image and re-imaged 2 weeks and 6 weeks post-surgery. Image data were acquired as volume acquisition. The sequential images with selected stromal nerve were manually adjusted and stacked into a 3-D nerve reconstruction by Image J. To validate the result, whole rat cornea was harvested post-mortem and nerve fibers were stained with TUBBIII.
Results :
Cornea wholemounts image indicated that the stroma nerve acquired by IVCM matched the nerve architecture map. Due to the curvature of the rat cornea, three IVCM images were located within an area of 30% of the whole cornea (Fig 1A, B). In the rat cornea with dry eye disease, we observed various dendritic cells infiltration along nerve trunks. 3-D reconstruction of the corneal nerve architecture showed the differential spatial distribution of those dendritic cells (Fig 1C, D, triangles). Longitudinal follow-up of the stroma nerve after DLGR indicated that nerve reflection increase (tan arrows), nerve sprouting (white arrows). There was more dendritic cell infiltration (triangles) 6 weeks after the double lacrimal gland compared to 2 weeks post-surgery (Fig2A, B). The 3-D reconstruction of stromal nerve architecture revealed different nerve and cellular changes in the cornea stroma between the anterior side (00) and posterior side (1800).
Conclusions :
Here we demonstrated the precise mapping and 3-D reconstruction of stroma nerve architecture in a DLGR dry eye model, revealing that nerve and cellular changes exacerbation after model establishment. This work also suggested that this methodology held potential for development into a clinical diagnostic modality.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.