Abstract
Retinal ganglion cells (RGCs), the sole output cells of the retina, are a heterogeneous population of neurons that project axons to visual targets in the brain. Like most central nervous system (CNS) neurons, RGCs are considered incapable of mounting long distance axon regeneration. Using immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO) in transgenic mice, we tracked the entire paths of individual RGC axons and show that adult RGCs are highly capable of spontaneous long-distance regeneration, even without any treatment. Our results show that the Thy1-H-YFP mouse sparsely labels RGCs, consisting predominantly of regeneration-competent alpha type-RGCs (αRGCs). Following optic nerve crush, many of the YFP-labeled RGC axons extend considerable distances proximal to the injury site with only a few penetrating through the lesion. This tortuous axon growth proximal to the lesion site is even more striking with intravitreal ciliary neurotrophic factor (CNTF) treatment. We further demonstrate that despite traveling more than 5 mm (i.e. a distance equal to the length of mouse optic nerve), many of these circuitous axons are confined to the injury area and fail to reach the brain. Our results re-evaluate the view that RGCs are naturally incapable of re-extending long axons, and shift the focus from promoting axon elongation, to understanding factors that prevent direct growth of axons through the lesion and the injured nerve.
Significance Statement Retinal ganglion cells (RGCs) are viewed as being incapable of mounting lengthy axon regeneration. Using whole tissue immunolabeling, we establish a technique to visualize and trace the entire paths of small populations of genetically labeled RGC axons as they regenerate. Following optic nerve injury, few axons grow beyond the lesion, but we find these axons branch and form loops proximal to the lesion. A regeneration inducing treatment further exacerbates branching and tortuous growth, while only modestly increasing the number of RGC axons that successfully grow beyond the lesion. Our study demonstrates extensive and circuitous RGC axon elongation both in pre- and post-lesion regions, highlighting the need to better understand the factors that inhibit direct axon growth in the optic nerve.
Footnotes
Authors report no conflict of interest.
This work was supported by grants from NEI 1R01EY022961-01 (KKP), NEI 1R21EY026668-01 (KKP), NEI 1U01EY027257-01 (KKP and VPL), NEI 1F30EY025527-01 (ERB), Walter G Ross Foundation (VPL), Ziegler Foundation (KKP), Glaucoma Research Foundation (KKP) and Pew Charitable Trust (KKP), The Miami Project to Cure Paralysis, and the Buoniconti Fund (KKP, VPL, and PT). The authors thank Pingping Jia at the University of Miami Viral Vector Core, and Melissa Carballosa-Gautam at the Miami Project.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
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