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

Georg Jocher,Rüdiger Schweigreiter,Martin Offterdinger,Sidney H Mannschatz

doi:10.3389/fncel.2018.00166

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.

Highlights

Neurons are the most complex cells of the human body
From the two trigeminal ganglia (TG) per animal we obtain at most 10,000 neurons. Such a yield is poor when related to the number of neurons required to load an microfluidic chamber (MFC), the standard reference of which is the MFC device manufactured by Xona Microfluidics and described by Taylor et al (2005)
The number of neurons required per MFC varies and presumably depends on the experimenter’s experience with handling microfluidic devices, but based on the existing literature approximately 500,000 dorsal root ganglia (DRG) neurons are required per MFC (Tsantoulas et al, 2013; Jia et al, 2016, 2018)

Summary

Introduction

Neurons are the most complex cells of the human body. The cell soma extends processes that can measure more than 1 meter in length. Performing a series of experiments with such small-population neurons in MFC devices is impractical, if not unfeasible To overcome this bottleneck, we have established a simple MFC protocol that allows for a dramatic reduction in the number of required neurons to only 10,000, which equals one adult mouse per microfluidic chamber when working with TG neurons, and one adult mouse for a number of chambers when dealing with DRG neurons. We have established a simple MFC protocol that allows for a dramatic reduction in the number of required neurons to only 10,000, which equals one adult mouse per microfluidic chamber when working with TG neurons, and one adult mouse for a number of chambers when dealing with DRG neurons This advanced microfluidic protocol, along with a novel linear Sholl analysis tool, markedly reduces the consumption of lab animals, and facilitates microfluidic experiments with small-population neurons at an unprecedented speed and efficiency. The results obtained show remarkably little experimental variability and reveal specific differences in the potency of various growth factor combinations to switch neurons into an axonal growth state

Methods

Results

Conclusion