Optical Stimulation and Electrophysiological Analysis of Regenerating Peripheral Axons

Abstract

Although axons in the peripheral nervous system can regenerate, functional recovery after nerve injuries is poor. Activity-based therapies, such as exercise and electrical stimulation, enhance the regeneration of cut peripheral axons. Despite their effectiveness, clinical application of these experimental techniques has been limited. At least part of the basis for this translational barrier has been a lack of information as to the precise mechanism of activity-based therapies on peripheral axon regeneration. To evaluate the requirements for neuron-type specific activation to promote regeneration using these therapies, in the current protocol, we employed optogenetics. Utilizing the advantages of transgenic mouse lines we targeted opsin expression to different neuron types. Using fiber optics we activated those neurons with high temporal specificity as a model of activity-based intervention after nerve injury and to measure functional recovery achieved after such a treatment.

Neuronal androgen receptor is required for activity dependent enhancement of peripheral nerve regeneration

“…We performed in vivo conditional deletion of the DNA‐binding dependent domain of the androgen receptor from subsets of motoneurons and DRG neurons. Brief neuronal activity in the form of 1 hr of electrical stimulation promoted axon elongation in wild‐type neurons from male and female mice (Figure 1), which agrees with many in vivo studies from our lab and others demonstrating the regeneration enhancing effect of brief neuronal activity (Al‐Majed et al., 2000; Gordon et al., 2010; Jaiswal et al., 2018; Ward et al., 2018; Ward & English, 2019; Zuo et al., 2020). When this same treatment was applied in mice with selective deletion of the DNA‐binding dependent domain of the androgen receptor, the axon elongation enhancing effect of electrical stimulation was prevented in males (but not females).…”

“…Mice of each genotype (SLICK‐AR fl/y , SLICK‐AR fl/fl , or SLICK) and sex were randomly assigned to one of two treatments: transected/repaired and untreated or transected/repaired and treated with electrical stimulation for 1 hr. For electrical stimulation, a small bipolar cuff electrode was constructed from an incised piece of silastic tubing with two sets of fine stainless steel wires sewn into the inner diameter of tubing (Ward & English, 2019). At the time of surgery, nerves were supramaximally stimulated continuously for one hour with 0.1‐ms duration constant voltage pulses delivered through a stimulus isolation unit at 20 Hz.…”

Compliant peripheral nerve interfaces

“…Therefore, by shining light onto the whole nerve, it is possible to interact only with opsin-expressing fibers, which respond exclusively to a preselected wavelength in the visible range. Aside from neuromodulation purposes, optogenetic techniques are also being applied to promote nerve regrowth from specific cell populations, which can allow a better understanding of axonal growth mechanisms [140,165]. In the implementation of optogenetics, undesirable side effects, both at the cellular and neural circuit level, can arise due to: the presence of the lightsensitive proteins, the consequence of photothermal effects, and the activation or silencing of specific cell populations [166].…”

Disruption of Core Stress Granule Protein Aggregates Promotes CNS Axon Regeneration

“…Two unipolar needle electrodes (Neuroline monopolar, 28 Ga; Ambu/AS, Columbia, MD) were placed on either side of the sciatic nerve, separated from each other by approximately 1 mm. Lead wires from the needles were connected to an optically isolated constant voltage stimulator under computer control ( 69 ).…”