Microfluidics of Small-Population Neurons Allows for a Precise Quantification of the Peripheral Axonal Growth State

Abstract

Neurons are morphologically the most complex cell types and are characterized by a significant degree of axonal autonomy as well as having efficient means of communication between axons and neuronal cell bodies. For studying the response to axonal injury, compartmentalized microfluidic chambers (MFCs) have become the method of choice because they allow for the selective treatment of axons, independently of the soma, in a highly controllable and reproducible manner. A major disadvantage of these devices is the relatively large number of neurons needed for seeding, which makes them impractical to use with small-population neurons, such as sensory neurons of the mouse. Here, we describe a simple approach of seeding and culturing neurons in MFCs that allows for a dramatic reduction of neurons required to 10,000 neurons per device. This technique facilitates efficient experiments with small-population neurons in compartmentalized MFCs. We used this experimental setup to determine the intrinsic axonal growth state of adult mouse sensory neurons derived from dorsal root ganglia (DRG) and even trigeminal ganglia (TG). In combination with a newly developed linear Sholl analysis tool, we have examined the axonal growth responses of DRG and TG neurons to various cocktails of neurotrophins, glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and leptin. Precise quantification of axonal outgrowth revealed specific differences in the potency of each combination to promote axonal regeneration and to switch neurons into an intrinsic axonal growth state. This novel experimental setup opens the way to practicable microfluidic analyses of neurons that have previously been largely neglected simply due to insufficient numbers, including sensory neurons, sympathetic neurons and motor neurons.

A Brief Review of In Vitro Models for Injury and Regeneration in the Peripheral Nervous System

“…Injury-induced axonal changes similar to those described above have also been observed in other compartmental models, such as the 2D- and 3D microfluidic chip systems [ 53 , 54 ]. These model systems fabricated from an inert polymer material such as poly(dimethylsiloxane) [ 55 , 56 ] comprise compartments and microgrooves of varying design, which spatially separate neuronal axons and soma [ 57 ].…”

“…Another significant advancement in studying axonal injury is the adoption of microfluidic devices. Neurons cultured within microfluidic devices have been subjected to axonal injury by simply pushing fluid into intersecting microgrooves within the device [ 54 , 59 , 62 , 90 ]. Axonal stretching using a cantilever attached to a piezoelectric drive is another innovative approach to produce stretch-induced injury changes at the single axon level ( Figure 5 ).…”

Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition

“…To quantify the difference, we acquired tile scan images of the myotube compartment and isolated the neurites ( Figure S3 ). The images were converted to a mask and analyzed with a linear Scholl analysis script similar to a previously published method ( Jocher et al., 2018 ) ( Figures S3 A and S3B). The Scholl analysis creates an intersection line every 50 μm, and the number of neurite pixels per intersection was quantified ( Figure S3 C).…”

Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition

“…To assess neurite outgrowth, total number of pixel intersections were quantified utilizing a custom-made Image J 1.52p software script. The script performs a linear Scholl analysis and automatically quantifies the amount of pixel crossings per line intersection similar to a previously published method (48). The distance between each line intersection is 50μm.…”