Neuronal substrates alter the migratory responses of nonmyelinating Schwann cells to controlled brain‐derived neurotrophic factor gradients

Abstract

Neurodegeneration and dysfunction cause mobility impairment and/or paralysis in millions of adults, worldwide. Motor deficit and recovery in adults depend upon the plasticity of the neuromuscular junction (NMJ), a tripartite, biochemical synapse that transduces electrical impulses from the brain into voluntary contraction of skeletal muscle. Nonmyelinating Schwann cells (nmSCs) of the NMJ have been increasingly recognized as active synaptic partners with motor neurons and muscle and have become recent therapeutic targets for regeneration. nmSC synaptic transmission, plasticity, and growth are strongly modulated by brain-derived neurotrophic factor (BDNF), whose regenerative abilities have been explored through emerging biomaterials and tissue-engineered systems, as well as via clinical trials. Experimental models engineered to investigate integrated NMJ response(s) to local gradients of BDNF will both advance our understanding of key modulators of synaptic activity, postinjury, and aid in the development of NMJ-targeted, regenerative therapies to restore mobility. The current study examined the ability of nmSCs to respond to microfluidically controlled BDNF signaling upon different haptotactic substrates of motor neurons (MNs) and laminin adhesion coating. Tests seeding nmSCs sequentially with MNs illustrated that sequential seeding reported a fivefold increase in levels of tropomyosin receptor kinase B expression in response to BDNF signaling and a nearly fivefold increase in migration distance along BDNF gradients. By contrast, concurrent seeding of MNs and nmSCs upon laminin adhesion coating illustrated a difference in migration distance of less than one third-fold over control. Our findings are among the first to examine migratory responses of nmSCs for regenerative strategies and highlight the potential to restabilize NMJ synaptic activity by affecting nmSC behaviors through therapeutic BDNF and seeding with MNs.

Cerebrospinal fluid replacement solutions promote neuroglia migratory behaviors and spinal explant outgrowth in microfluidic culture

“…EBS is most clinically used as a diluent of therapeutics for disorders of the meninges, which partially contain CSF and have been implicated in neuromuscular disease, neurodegenerative disorders, and some forms of cancer (Oka, Yamamoto, Nonaka, & Tomonaga, 1996; Shestakova, Healey, Zhao, Rezk, & Nakagiri, 2019). Results of our study illustrate that EBS solutions facilitate ShC survival, morphology, and proliferation similar to those measured in traditional DMEM medium, and additionally support glial chemotactic behaviors in response to the regenerative agent, brain‐derived growth factor (BDNF) (Singh, Robles, & Vazquez, 2020). Moreover, SCE cultured with EBS solutions exhibited survival rates comparable to those within conventional NB medium, and demonstrated rates of cellular outgrowth commensurate with reported in vivo measurements.…”

“…Subsequent experiments used EBS as a diluent fluid within the bridged gLL device to produce chemoattractive gradient fields of extracellular BDNF (Figure 5). Previous work from our lab and others have shown that ShC migratory responses to BDNF are important to glia‐ and NMJ‐targeted NS therapies (Singh et al., 2020). The data here illustrate that ShC responded chemotactically to BDNF stimuli generated within DMEM or EBS solutions with high similarity.…”

“…It was selected for our study, in part, because of its recent surgical applications as a therapeutic diluent for disorders of the meninges, which partially contain CSF and have been implicated in neuromuscular disease and neurodegenerative disorders (Stark et al., 2019, Garcia Molina et al., 2020). Our study used EBS as a CSF diluent of BDNF, a regenerative therapeutic often used for its ability to affect synaptic transmission, plasticity, and growth of the NMJ via ShC signaling (Pena et al., 2019, Singh et al., 2020). Clinical trials have explored BNDF delivery across the blood brain barrier (Bertram, Rauch, Chang, & Lavik, 2010) and have begun to use emerging models of CSF circulation to develop methodologies that increase local BDNF bioavailability via interstitial flow (Kaneko et al., 2018).…”

“…Boundary conditions used no slip at the inner surface walls and axial symmetry within the microchannels, while gravity effects were presumed to be negligible. An inlet concentration of C o = 100 ng/ml of BDNF was used to approach the in vivo values cited in the literature (Singh et al., 2020). Experiments used a model fluorescent molecule of comparable molecular weight (FITC‐Dextran, CAS 60842‐46‐8 [20 kDa], Sigma‐Aldrich, St. Louis, MO) to validate the concentration distributions predicted by the computational model within the gLL.…”

A Micro-Optic Stalk (μOS) System to Model the Collective Migration of Retinal Neuroblasts

“…A 20-mL volume of polydimethylsiloxane (PDMS, Dow Corning, Midland, MI) was then poured onto the final patterned wafer surface and oven cured (300 • C, 15 min) to produce an elastomer of 2-3 mm in thickness. Lastly, oxygen plasma was applied to the inner surfaces of the PDMS elastomer and a piranha-cleaned glass microscope slide using a 30-s corona treatment (Electro-technic Products Inc., Chicago, IL, USA) [49,50,58] before press fitting to create the closed µOS system, as done previously by our lab [59,60] ( Figure 3F). The μOS is manufactured using a two-step photolithography process with elastomeric micromolding.…”