Bioluminescent Optogenetics for Studying Sympathetic Axonal Regeneration

Abstract Body : Introduction: Peripheral nerve injuries affect >500,000 people globally each year and can lead to permanent loss of motor, sensory, and autonomic functions with potential for lifelong disabilities. After surgical nerve reconstruction, spontaneous nerve regeneration is slow and inadequate. With no approved non-surgical interventions, discovering therapies that enhance regeneration is critical for functional recovery. Brief neuronal activation can enhance motor and sensory axon growth; however, the regenerative ability of postganglionic sympathetic axons in response to neuronal activation is unknown.

Objective: Test the effects of neuronal activation on the regeneration of sympathetic axons after severe nerve injury.

Methods: Bioluminescent optogenetics (BL-OG) enables spatiotemporal selective activation of targeted neurons with intrinsic biological light production via genetically encoded luminopsins. To validate our BL-OG-engineered mouse (ThCre:eLMO3), I measured light emission from the lumbar sympathetic ganglia after coelenterazine (CTZ) application. Additionally, local field potentials (LFPs) were measured in the sympathetic ganglia in response to an external light source (465-495 nm, blue light) or CTZ. To study sympathetic regeneration 2 weeks post-injury, I performed immunohistochemistry on sciatic nerve sections to visualize regenerating sympathetic axons. I quantified the number of regenerated sympathetic neurons via fluorescent retrograde tracing. ThCre:eLMO3 mice are compared to littermate controls, which do not simultaneously express both genetic constructs.

Results: Intrinsic light emission from the sympathetic ganglia of ThCre:eLMO3 exhibits a 23.8x increase after CTZ administration, eLMO3 control vs ThCre:eLMO3 (p=0.0006, Welch’s ANOVA + Tamhane’s T2 post hoc, n=4 blank, n=4 eLMO3, n=4 ThCre:eLMO3), which induces a 2.8x change in lumbar sympathetic neuron LFPs (p=0.0218, 2-tailed t-test, n=2 eLMO3, n=3 ThCre:eLMO3). External blue light induces a 24.9x change in LFPs (p=0.0022, two-tailed t-test, n=4 eLMO3, n=4 ThCre:eLMO3). Sympathetic axonal regeneration was not enhanced with optical neuronal activation (ns between treatment groups, 2-way RM ANOVA, n=4 ThCre, n=5 ThCre:eLMO3). Bioluminescent activation of sympathetic neurons also decreased the number of sympathetic neurons whose axons had reached 5mm of growth from the injury site (p=0.0013, Mann-Whitney test, n=8 ThCre, n=8 ThCre:eLMO3).

Conclusions: Lumbar sympathetic ganglia are reactive to external blue light and emit sufficient intrinsic light to elicit neuronal excitation. Thus, BL-OG is a viable paradigm to study the effects of sympathetic activation on sympathetic axonal regeneration. Using this novel optogenetic tool, I showed that neuronal activation inhibits sympathetic regeneration.

Significance: Neuronal activity-based therapies have shown success in enhancing motor and sensory regeneration, but my data indicates that activation of sympathetic neurons impairs their regeneration. This means that electrical stimulation, a form of neuronal activation which is currently in clinical trials, may be detrimental to the functional recovery of sympathetic neurons. I identified targets that underlie sympathetic regenerative failure with RNAseq. I will test these targets to elucidate the mechanism of regenerative failure and lay the framework to discover a clinically-relevant treatment to enhance the regeneration of all axon types.