Axonal regeneration of cultured mouse hippocampal neurons studied by an optical nano-surgery system.

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  • 04 April 2012
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During development, the axons of neurons in the mammalian  central nervous system lose their ability to regenerate after injury. In order to study the regeneration process, we developed a  system  integrating an optical tweezers and a laser dissector to manipulate the sample. A sub-nanosecond pulsed UVA laser was used to inflict a partial damage to the axon of mouse hippocampal neurons at early days in vitro. Partial axonal transections were  performed in a highly controlled and reproducible way without  affecting the regeneration process. Force spectroscopy  measurements, during and after the ablation of the axon, were  performed by optical tweezers with a bead attached to the  neuronal membrane. Thus, the release of tension in the neurite could be analyzed in order to quantify the inflicted damage. After dissection, we monitored the viscoelastic properties of the axonal  membrane, the cytoskeleton reorganization, and the dynamics of  the newly formed growth cones during regeneration. In order to  follow cytoskeleton dynamics in a long time window by tracking a bead attached to the neuron, we developed a real-time control of  the microscope stage position with sub-illisecond and nanometer  resolution. Axonal regeneration was documented by long-term  (24-48 hours) bright-field live maging using an optical microscope equipped with a custom-built cell culture incubator.