Optogenetic neuronal stimulation promotes axon outgrowth and myelination of motor neurons in a three‐dimensional motor neuron–Schwann cell coculture model on a microfluidic biochip

Abstract

Axonal regeneration and remyelination of peripheral motor neurons (MNs) are critical for restoring neuromuscular motor function after injury or peripheral neuropathy. We examined whether optogenetically mediated light stimulation (OMLS) could enhance the axon outgrowth and myelination of MNs using threedimensional motor neuron-Schwann cell (MN-SC) coculture on a microfluidic biochip. The biochip was designed to allow SCs to interact with the axons of MNs, while preventing direct contact between SCs and the cell bodies of MNs. Following coculture with SCs on the microfluidic biochip, MNs were transfected with a lightsensitive channelrhodopsin gene. Transfected MNs subjected to repeated light stimulation (20 Hz, 1 hr) produced significantly longer axons than nontransfected MNs. OMLS of MNs greatly increased the number of myelin basic protein (MBP)expressing SCs, promoting the initiation of myelination of MNs. Ultrastructurally, OMLS of MNs markedly enhanced the thickness of the compact myelin sheath around the MN axons such that the average thickness was closer to that of the theoretical estimates in vivo. Thus, the MN-SC coculture model on a microfluidic biochip augmented by OMLS of MNs is a feasible platform for studying the relationship of neuronal activity with regrowth and remyelination. K E Y W O R D S motor neuron (MN), myelin, optogenetically mediated light stimulation (OMLS), peripheral nerve trauma, peripheral neuropathy, Schwann cell (SC)

Neuromuscular Activity Induces Paracrine Signaling and Triggers Axonal Regrowth after Injury in Microfluidic Lab-On-Chip Devices

“…; p = 0.1157, unpaired t-test; Figure 4d). These results are in line with recently published data by Hyung et al (2019) on MN responses to optical stimulation in 3D hydrogels [49].…”

“…By taking advantage of an approach combining microfluidics, in vitro axotomy, and optogenetics, this study suggests the previously undescribed role of muscular activity through a paracrine mechanism to promote axonal regrowth of MNs after injury. Specifically, we found that optogenetic modulation of neuronal activity induces a ≈2-fold increase (although non-statistically significant) in axonal regrowth, similar to what was observed by [49]. In addition, we determined that modulating C2C12 contractility after MN axotomy by optogenetics induces an increase in the expression and release of LIF and GDNF, which in turn increases the regrowth of MN axons.…”

Neuroprotective Effect of Glial Cell-Derived Exosomes on Neurons

“…SC suspension was seeded on coverslips pre-coated with 10 μg/mL poly-Dlysine (PDL, Sigma). After 3 days, complement-mediated cytolysis was performed to remove fibroblasts from the SC culture, as previously described [18,20]. Briefly, cells were treated with DMEM containing 20 mM hydroxyethyl-piperazineethanesulfonic acid (HEPES) buffer (T and I), 10% FBS, 4 mM L-gln, and 1% P/S) for 5 min before washing with Ca2 + -and Mg2 +free Hank's balanced salt solution (HBSS, Invitrogen) containing 20 mM HEPES.…”

“…MN culturing was performed as described previously [18,20]. 12 fetuses) were harvested in Ca2 + -and Mg2 + -free HBSS containing 1% Toll-like receptor (TLR) trypsin (Worthington) for 15 min at 37°C, followed by treatment with 1% trypsin inhibitor (Sigma).…”

Optical control of ERK and AKT signaling promotes axon regeneration and functional recovery of PNS and CNS in Drosophila

“…Furthermore, using a thermonociception-based behavioral recovery assay, we found that optoRaf and optoAKT activation led to effective axon regeneration as well as functional recovery after central nervous system (CNS) injury. We note that most of the previous optogenetic control of neural repair studies were based on channelrhodopsion in C. elegans ( Sun et al, 2014 ), mouse DRG culture ( Park et al, 2015a ) or motor neuron-schwann cell co-culture ( Hyung et al, 2019 ). Another study used blue-light activatable adenylyl cyclase bPAC to stimulate neural repair in mouse refractory axons ( Xiao et al, 2015 ).…”