June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Imaging murine corneal nerves in vivo using genetically-encoded calcium indicators
Author Affiliations & Notes
  • Matthew McPheeters
    Biomedical Engineering, Case Western Reserve University Case School of Engineering, Cleveland, Ohio, United States
  • Brecken Blackburn
    Biomedical Engineering, Case Western Reserve University Case School of Engineering, Cleveland, Ohio, United States
  • William J. Dupps
    Ophthalmology, Cleveland Clinic, Cleveland, Ohio, United States
  • Andrew M Rollins
    Biomedical Engineering, Case Western Reserve University Case School of Engineering, Cleveland, Ohio, United States
  • Michael W Jenkins
    Biomedical Engineering, Case Western Reserve University Case School of Engineering, Cleveland, Ohio, United States
  • Footnotes
    Commercial Relationships   Matthew McPheeters, None; Brecken Blackburn, None; William Dupps, None; Andrew Rollins, None; Michael Jenkins, None
  • Footnotes
    Support  NIH R21-EY031525
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 373. doi:
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    • Get Citation

      Matthew McPheeters, Brecken Blackburn, William J. Dupps, Andrew M Rollins, Michael W Jenkins; Imaging murine corneal nerves in vivo using genetically-encoded calcium indicators. Invest. Ophthalmol. Vis. Sci. 2021;62(8):373.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Numerous diseases detrimentally affect corneal nerves. Corneal nerve dysfunction has been linked to dry eye syndrome onset, neurotrophic keratopathy, and other diseases. The morphology and histology of corneal nerves in health and disease has been extensively studied. However, studies of corneal nerve function have been limited (e.g., electrical recordings of ciliary nerves or calcium reporter dyes in ex vivo corneas). Here, we present a non-contact method for functional imaging of corneal nerves. This tool has great potential to improve understanding of diseases of corneal nerves and the development of therapies for diminished neural function.

Methods : We made a cre-lox murine line expressing a genetically encoded calcium indicator, GCaMP6f, against the Nestin promoter for expression in the cornea nerves. We built a custom confocal imaging system with an air objective to image the corneal nerves while excluding autofluorescence from the lens. We designed various stereotactic and imaging apparati to minimize motion of the mouse cornea relative to our imaging system (see Figure 1).

Results : Figure 1 shows our cornea imaging setup and Figure 2 shows a demonstration of in vivo corneal functional neuro-imaging. Using this technique, we were able to stably image the murine corneal nerves over long periods (e.g., 10 minutes). We have also demonstrated longitudinal imaging of the same field of nerves through multiple successive experiments.

Conclusions : Our developed tools and methods can enable study of the relationship between corneal nerve function and diseases of the ocular surface. The use of an in vivo system potentially allows for the study of factors that could slow the development of neuropathies and help develop new clinical interventions and therapies, which is a major goal of this work.

This is a 2021 ARVO Annual Meeting abstract.

 

Figure 1. Image of experimental setup showing mouse, objective, stereotaxis and isoflurane nose cone.

Figure 1. Image of experimental setup showing mouse, objective, stereotaxis and isoflurane nose cone.

 

Figure 2. Non-contact functional imaging of the murine corneal nerves in vivo. (a) An imaged region and (b) calcium traces of basal activity in a live mouse using the CaImAn pipeline. Automatically identified ROIs are shown in various colors in (a). The traces show both the change in fluorescence over the baseline fluorescence (dF/F) in colors corresponding to the ROIs of the same color in the upper panel, as well as spike detection on the baseline in gray.

Figure 2. Non-contact functional imaging of the murine corneal nerves in vivo. (a) An imaged region and (b) calcium traces of basal activity in a live mouse using the CaImAn pipeline. Automatically identified ROIs are shown in various colors in (a). The traces show both the change in fluorescence over the baseline fluorescence (dF/F) in colors corresponding to the ROIs of the same color in the upper panel, as well as spike detection on the baseline in gray.

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