A microfluidic neuronal platform for neuron axotomy and controlled regenerative studies

Abstract

Understanding the basic mechanisms of neural regeneration after injury is a pre-requisite for developing appropriate treatments. Traditional approaches to model axonal lesions, such as high intensity power laser ablation or sharp metal scratching, are complex to implement, have low throughputs, and generate cuts that are difficult to modulate. We present here a novel reproducible microfluidic approach to model in vitro mechanical lesion of tens to hundreds of axons simultaneously in a controlled manner. The dimensions of the induced axonal injury and its distance from the neuronal cell body are precisely controlled while preserving both the proximal and distal portions of axons. We have observed that distal axons undergo Wallerian-like anterograde degeneration after axotomy; in contrast, proximal portions of the axons remain un-degenerated, possessing the potential to re-grow. More importantly, surpassing the previous axotomy methods performed in Petridishes in which local microenvironments cannot be tailored, our platform holds the capability to implement fine-tuned treatments to lesioned axon stumps in a local, controlled manner. Specifically, molecules such as chondroitin sulphate proteoglycans and its degrading enzyme chondroitinase ABC, hydrogels, and supporting cells have been shown to be deliverable to the lesioned site of injured axons. In addition, this system also permits double interventions at the level of the lesioned axons and the perikaryon. This proves the potential of our model by demonstrating how axonal regrowth can be evaluated under circumstances that are better mimics of biological problems. We believe that this novel mechanical microfluidic axotomy approach is easy to perform, yields high throughput axon lesions, is physiologically relevant, and offers a simplified platform for screening of potential new neurological drugs.

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

“…In the current experiments, co-cultured MN and C2C12-derived myotubes showed their typical morphologies, and with high cell viabilities. Corroborating previous studies by our laboratory (i.e., Tong et al (2015) [45]), axons reached the axotomy channel a few days after culture, crossing towards the C2C12 myotube chamber in 5-7 days. Indeed, after 7-10 days in co-culture with ventral horns of spinal cord slides, distal MN axons reached the myotubes, establishing neuromuscular junctions (NMJs) labelled with BTX-Alexa Fluor-595 to identify acetylcholine receptor (AChR) clusters on myotubes (Figure 3).…”

“…After the axotomy, we proceeded to optically stimulate the ChR2-positive spinal cord explants as above (Figure 1a, see also Methods for details). Although the behavior of single identified axons after axotomy was impossible to determine due to the large number of MN axons crossing the microgrooves, we were able to detect an increased regrowth of previously lesioned axons only after the MN optical stimulation by using Calcein TM (Supplementary Figure S4, see also Tong et al (2015) [34] for technical details). Indeed, after optical stimulation (see Material and Methods) we were able, using phase contrast microscopy, to quantify the regrowth of these axons in parallel devices.…”

“…Poly(dimethylsiloxane) (PDMS) microfluidic devices were designed in CAD software and chrome masks were prepared at IBEC Microfabspace. The lab-on-chip microfluidic device design was modified and adapted for our purposes from previously published devices [34,35]. The axotomy microfluidic device ( Figure 1) consisted of 4 circular reservoirs of 7-8 mm of Ø interconnected by two cell/explant seeding chambers (150 (high) × 1,500 (wide) µm).…”

“…The device consisted of two main chambers interconnected with 100 microchannels (10 × 10 µm cross-section and 900 µm length). In addition, a transversal axotomy-designed channel was added in the middle of the microchannels (see [34] for details of a similar device). In our experiments, we generated and differentiated ChR2-expressing C2C12 myoblasts into functional myotubes in one chamber before seeding the embryonic spinal cord explants in the other chamber of the device with the appropriate culture media added in the reservoirs (Figure 1a,b).…”

“…In Supplementary Video S7, two examples of the axotomy of ChR2-eYGP fluorescence axons crossing several microchannels are presented. In addition, Figure 4a-c show representative brightfield microscopic images of the perpendicular axotomy channel before (A), during (B), and after (C) the vacuum-assisted axotomy via aspiration using a common laboratory micropipette [34]. After the axotomy, we proceeded to optically stimulate the ChR2-positive spinal cord explants as above (Figure 1a, see also Methods for details).…”