Fast, volumetric calcium imaging of murine corneal nerves

Abstract

Purpose : The corneal nerves sense the state of the ocular surface, regulate ocular surface homeostasis and change in form and function in disease states. Corneal nerve dysfunction is a key cause/result of ocular surface disorders such as dry eye syndrome, neurotrophic keratopathy, and others. Understanding how the function of the corneal nerves changes in these circumstances is important both to understanding disease progression and assessing therapies. Here, we build on previous work and demonstrate volumetric functional imaging in the murine cornea over a large field of view using a custom spinning-disk confocal imaging system.

Methods : We imaged a cre-lox murine line expressing GCaMP6f against the Nestin promoter using a custom-built spinning disk confocal imaging system with an air objective. Isoflurane anesthesia, a custom support, an objective piezo, and image registration were all used to reduce motion artifacts. By imaging multiple depths a wider field of view could be assembled even when imaging off center.

Results : The murine stereotaxis and anesthesia system is shown in Fig. 1 with the imaging system objective at an oblique angle to better position the optic axis normal to the central corneal. Fig. 2 demonstrates volumetric functional imaging of cornea nerves over ~8 minutes. Fig. 2a shows an intensity projection of a 40 µm thick volume and Fig. 2b shows calcium traces in time for representative ROIs in the same color in Fig. 2a.

Conclusions : The significant expansion of the field of view and enhanced imaging speed enables examination of a broad area of the murine cornea. This improvement greatly facilitates research on nerve function in the context of injuries or in response to stimuli affecting the entire corneal surface. We expect approximately an order of magnitude improvement in imaging speed with this system compared to our current results once we have optimized data throughput. This in vivo approach will enable the study of both functional and structural changes in murine corneal nerves over time within disease models, such as evaporative dry eye disease and diabetes mellitus.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Figure 1: Mice are imaged in custom stereotaxis under isoflurane anesthesia. The stereotaxis and imaging objective have a wide range of motion to allow for precise positioning.

 

Figure 2. An example volume projection is shown with calcium traces for colored ROIs. Scale bar is 100µm.