Bioluminescent Optogenetics to Enhance Recovery from Peripheral Nerve Injury Open Access
Ecanow, Anna (Spring 2022)
Abstract
Functional recovery from peripheral nerve injury (PNI) is poor. Experimental therapies increasing injured neuronal activity are effective in accelerating axon growth and improving outcomes. Bioluminescent optogenetics (BL-OG) is a novel approach to increasing injured neuron activity. BL-OG uses luminopsins – light sensing ion channels (opsins) fused with light- emitting luciferase – which generate bioluminescence when exposed to a suitable substrate, such as coelenterazine (CTZ). Injured neurons expressing an excitatory luminopsin could be activated by BL-OG to promote axon regeneration. We hypothesized that induction of BL-OG after PNI would result in an increased number of motor and sensory neurons that have regenerated, relative to controls. Sciatic nerves of mice were injected unilaterally with an adeno-associated viral vector encoding either an excitatory luminopsin (eLMO3) or a mutated form (R115A) that can generate bioluminescence but not activate neurons. After waiting two weeks for retrograde viral transport and viral transduction, injected sciatic nerves were cut and repaired, and mice were then treated with a single dose of CTZ. Four weeks later, different retrograde fluorescent tracers were injected into gastrocnemius and tibialis anterior muscles to mark motor and sensory neurons innervating them. Counts of neurons were made from histological sections of spinal cords and DRGs and included those that were only retrogradely labeled and those that were both retrogradely labeled and contained green fluorescent protein (GFP), indicating presence of the luminopsin. The number of retrogradely labeled motor and sensory neurons in mice expressing eLMO3 was significantly greater than the number in mice expressing the R115A luminopsin. However, only a small proportion of labeled cells contained GFP, indicative of luminopsin expression. Treatments using CTZ to induce BL-OG enhanced axon regeneration are promising but illuminating the exact mechanism will require further investigation.
Table of Contents
Background/Introduction ................................................................................................................ 1
Methods .......................................................................................................................................... 4
Animals & Surgeries ....................................................................................................... 4
Tissue Processing & Immunohistochemistry ....................................................................... 5
Imaging ......................................................................................................................... 6
Results ............................................................................................................................................ 7
BL-OG Enhances Regeneration of Motoneurons................................................................... 7
BL-OG Enhances Regeneration of Sensory Neurons.............................................................. 8
Discussion ..................................................................................................................................... 10
References ..................................................................................................................................... 13
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