Wallerian-Like Degeneration of Central Neurons After Synchronized and Geometrically Registered Mass Axotomy in a Three-Compartmental Microfluidic Chip

Degeneration of central axons may occur following injury or due to various diseases and it involves complex molecular mechanisms that need to be elucidated. Existing in vitro axotomy models are difficult to perform, and they provide limited information on the localization of events along the axon. We present here a novel experimental model system, based on microfluidic isolation, which consists of three distinct compartments, interconnected by parallel microchannels allowing axon outgrowth. Neurons cultured in one compartment successfully elongated their axons to cross a short central compartment and invade the outermost compartment. This design provides an interesting model system for studying axonal degeneration and death mechanisms, with a previously impossible spatial and temporal control on specific molecular pathways. We provide a proof-of-concept of the system by reporting its application to a well-characterized experimental paradigm, axotomy-induced Wallerian degeneration in primary central neurons. Using this model, we applied localized central axotomy by a brief, isolated flux of detergent. We report that mouse embryonic cortical neurons exhibit rapid Wallerian-like distal degeneration but no somatic death following central axotomy. Distal axons show progressive degeneration leading to axonal beading and cytoskeletal fragmentation within a few hours after axotomy. Degeneration is asynchronous, reminiscent of in vivo Wallerian degeneration. Axonal cytoskeletal fragmentation is significantly delayed with nicotinamide adenine dinucleotide pretreatment, but it does not change when distal calpain or caspase activity is inhibited. These findings, consistent with previous experiments in vivo, confirm the power and biological relevance of this microfluidic architecture

Microfluidics for Neuronal Imaging

In neurobiology studies, the use of well-controllable microenvironments that can actively interact with biological samples is becoming increasingly popular. Microfluidic systems due to their precise micron-size dimensions are becoming the gold standard for manipulating small-model organisms in vivo, such as the nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster as well as for assembling and interacting with neuronal cell cultures in vitro. The reproducible microenvironment, the automation of time-consuming protocols, and the low manufacturing cost of microfluidic chips offer unique experimental capabilities and a large amount of high-quality data to the neurobiologist over traditional methods. This chapter highlights a certain aspect of microfluidic technology that facilitates the study of neuronal physiology and function through imaging