In vivo evaluation of Retinal Ganglion Cells (RGCs) in mice following Optic Nerve Crush (ONC) using Fluorescence SLO and Temporal Speckle Averaging Optical Coherence Tomography (TSA-OCT).

Abstract

Purpose : To present the results of longitudinal in vivo visualization and quantification of Retinal Ganglion Cells (RGCs) and Retinal Nerve Fiber Layer (RNFL) bundles in mice following Optic Nerve Crush (ONC).

Methods : We used our custom-built mouse retinal Scanning Laser Ophthalmoscopy / Optical Coherence Tomography (SLO/OCT) system to acquire serial OCT volumes (with corresponding SLO intensity and fluorescence data) to allow longitudinal studies in mice following ONC. To validate our ability to monitor in vivo individual Retinal Ganglion Cells (RGC) and Retinal Nerve Fiber Layer (RNFL) bundles by TSA-OCT, two mouse lines with fluorescently labeled RGC based on RGCs transcription factor (Brn3b-mCherry and Isl2-GFP) were imaged at baseline (before ONC) and at 1, 3, 7, 14 and 28 days post-ONC. After 28 days post-ONC, immunohistochemistry was performed on flat-mounted retinas to validate in vivo and ex vivo images of RGCs.

Results : TSA-OCT enhanced the image quality of OCT images and enabled RGC and RNFL bundle visualization (see Figure). Use of mice with fluorescently labeled RGCs allowed mapping of RGC position using a fluorescent SLO system, providing a direct comparison with RGC soma visualized by non-invasive TSA-OCT. Additionally, fluorescent labeling of the RGC helped with further validation of TSA-OCT imaging by histology, providing a one-to-one mapping between in vivo and ex vivo images of RGC.

Conclusions : In these studies, we used a novel TSA-OCT to allow visualization of RGC and RNFL bundles in mice in vivo following ONC. Our ability to visualize and follow RGCs and RNFL bundles in vivo using a non-invasive imaging method allows a reduction in the number of animals needed in future studies. These results provide a foundation for efficient in vivo monitoring of cellular morphology in animal models of glaucoma during disease progression and therapeutic intervention.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Figure 1. Longitudinal in vivo imaging of mouse retina before and after ONC using UC Davis OCT/SLO system. Changes in the intrinsic fluorescence of RGC following ONC are clearly visible on SLO images (top row). TSA-OCT (middle and bottom row) greatly enhanced the image quality of RNFL and highlighted changes in optic nerve head morphology.