title: Wallerian-like degeneration of central neurons after synchronized and geometrically registered mass axotomy in a three-compartmental microfluidic chip.
abstract: 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.
authors.affiliation: Laboratoire de Neurobiologie des Processus Adaptatifs, UMR 7102 CNRS/UPMC, Univ. P. et M. Curie, Bat B, 6Eme Etage, Case Courrier 12, 9 Quai St. Bernard, 75252 Paris, France.
authors.forename: Devrim
authors.initials: D
authors.lastname: Kilinc
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Peyrin
authors.forename: Vanessa
authors.initials: V
authors.lastname: Soubeyre
authors.forename: Sébastien
authors.initials: S
authors.lastname: Magnifico
authors.forename: Laure
authors.initials: L
authors.lastname: Saias
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Viovy
authors.forename: Bernard
authors.initials: B
authors.lastname: Brugg
mesh_terms: D000818:Animals; D019771:Axotomy / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods*; D002490:Central Nervous System / Q000166:cytology / Q000473:pathology; D002540:Cerebral Cortex / Q000166:cytology / Q000196:embryology / Q000473:pathology*; D051379:Mice; D044085:Microfluidics / Q000379:methods*; D009474:Neurons / Q000166:cytology / Q000473:pathology*; D014855:Wallerian Degeneration / Q000473:pathology*