Dynamics and molecular biology of optic nerve injury response and regeneration in a novel tadpole transection model

Abstract

Purpose : Amphibians possess the ability to protect their neurons and promote axon regeneration following even complete transection of the optic nerve. However, a major obstacle to working with small animal surgical models such as tadpoles is the reproducibility of the procedures on each specimen, which yield highly variable datasets, are time-consuming and limit the power of conclusive results. To improve upon this, we developed a surgical model in Xenopus laevis (X. laevis) tadpoles using a laser microdissection platform that allows for consistent optic nerve transections at developmental stage 40-46 and the study of nerve regrowth dynamics. Our preliminary data demonstrate an early and significant difference between mammalian and X. laevis redox related biochemical dynamics in response to optic nerve injury.

Methods : Using Xla.Tg(tubb2b:mapt-GFP)^Amaya transgenic tadpoles and laser micro-optics, we visualize and reproducibly transect the optic nerve. Survival rates with this model are substantially higher than with manual surgical methods. We employ microscopy and functional vision behavioral testing to characterize temporal nerve regeneration dynamics. We modulate regrowth with microinjection of pharmacological agents to test whether the timing of redox homeostasis reestablishment correlates with optic nerve healing.

Results : Following laser transection injury, we observed degeneration of axons distal to the injury site within several hours after the injury, followed by axonal bundle sprouting from the proximal cut end in multiple, uncoordinated directions from the injury site. We have named these ‘sentinel’ axons and have shown that some of these regrowing axons eventually reach the visual targets in the brain after which this tract becomes reinforced. After 8-10 days, optic nerve regeneration is complete and virtually indistinguishable from the naïve (uninjured) optic nerve morphology, and mitochondrial migration through the optic nerve occurs.

Conclusions : We improve upon previous methods to study optic nerve regeneration in tadpoles in a reproducible and high-throughput way and explore a potential mechanism of “buffering injury” in regenerative-competent species. It is our hope that this model and our research into oxidative stress handling might one day lead to the development of new targets and therapies aimed at protecting the optic nerve and lead to better recovery outcomes.

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