Abstract
Purpose :
To demonstrate the feasibility of noninvasive, in vivo transpupillary two-photon imaging for quantitative analysis of axonal mitochondria dynamics in the retinal ganglion cells (RGCs) of adult C57BL/6J mice.
Methods :
Wild-type C57BL/6J mice aged 2-4 months were used in this study. To label mitochondria in RGCs, an adeno-associated virus (AAV) vector with the PHP.eB capsid serotype was systemically delivered via the retro-orbital route. This vector was constructed with the mouse γ-Synuclein promoter—a promoter specific to RGCs—to drive the expression of mito-Dendra2, a green-to-red irreversibly photoswitchable monomeric fluorescent protein fused with the mitochondrial targeting signal of cytochrome c oxidase subunit VIII. For axonal mitochondria imaging, we employed the Ultima 2P-Plus two-photon laser scanning microscope (Bruker, MA), equipped with an Insight X3 femtosecond laser (Spectra-Physics/Newport, CA). A 10X, 0.5 NA dry objective (TL10X-2P, Thorlabs, NJ) with a long working distance of 7.77 mm was utilized, along with corneal leveling achieved by applying eye gel and a cover slip.
Results :
Four weeks after systemic administration of AAV.PHP.eB-mSncg-mito-Dendra2, uniform transgene expression was noted in the majority of RGCs within all quadrants. This expression was evident in the cell bodies, dendrites, and axons of the RGCs. Utilizing the dry objective with corneal leveling, we resolved axonal mitochondria within a planar field of focus (approximately 400 µm x 400 µm), enabling the simultaneous collection of multiple individual mitochondria. Over 3-5 minutes of time-lapse imaging, dynamic mitochondrial transport was captured. Kymograph analysis unveiled both anterograde and retrograde mitochondrial transport within a single axon. Significantly, axonal transport was detected in only one out of 63 axons from six mice, suggesting that mitochondria are largely stationary in the RGCs of healthy adult mice in vivo. Additionally, a green-to-red photoconverted mitochondrial patch remained stationary for up to four days, with the red fluorescence signal markedly diminishing and green fluorescence signal intensifying, likely indicative of mito-Dendra2 turnover.
Conclusions :
We successfully demonstrated the feasibility of visualizing individual axonal mitochondria and their stationary tendency in adult mouse RGCs using in vivo transpupillary two-photon imaging.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.