Large-scale in vivo femtosecond laser neurosurgery screen reveals small-molecule enhancer of regeneration

Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 10/2010; 107(43):18342-7. DOI: 10.1073/pnas.1005372107
Source: PubMed


Discovery of molecular mechanisms and chemical compounds that enhance neuronal regeneration can lead to development of therapeutics to combat nervous system injuries and neurodegenerative diseases. By combining high-throughput microfluidics and femtosecond laser microsurgery, we demonstrate for the first time large-scale in vivo screens for identification of compounds that affect neurite regeneration. We performed thousands of microsurgeries at single-axon precision in the nematode Caenorhabditis elegans at a rate of 20 seconds per animal. Following surgeries, we exposed the animals to a hand-curated library of approximately one hundred small molecules and identified chemicals that significantly alter neurite regeneration. In particular, we found that the PKC kinase inhibitor staurosporine strongly modulates regeneration in a concentration- and neuronal type-specific manner. Two structurally unrelated PKC inhibitors produce similar effects. We further show that regeneration is significantly enhanced by the PKC activator prostratin.

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Available from: Stephen J Haggarty, Sep 30, 2015
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    • "Thus, it is a challenging task to monitor the gas-evoked neuronal activities in an immobilized worm for sensory biology investigations. As an alternative, microfluidic technologies have shown great potential for manipulating this small freely moving animal and providing a precisely controlled dynamic environment for high spatiotemporal resolution imaging [9] [10] [11] [12] [13]. Various microfluidic systems have been developed for greatly facilitating the study of the nematode's sensorial ability in detecting a wide range of water-soluble chemical cues [14] [15] [16] [17] [18] [19]. "
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    ABSTRACT: It is a challenging task to realize gaseous stimulations on Caenorhabibditis elegans for in vivo neuronal analysis of chemo-sensing, due to the difficulties in gas control and worm manipulation. Here, we demonstrated an integrated microfluidic system that could accurately deliver the gaseous stimuli to the worms’ noses and track the in vivo neuronal activities simultaneously. The microfluidic chip consisted of a comb-shaped micro-valve for worm immobilization and a T-junction structure for precise gas delivery. Neuronal responses of C. elegans to polar and non-polar gases were both investigated. The microfluidic device clearly demonstrated that oxygen with increasing levels of 0-10% and 0-21% induced URX neuronal responses. BAG neuronal activities were inhibited by carbon dioxide, showing the symptom of anesthesia. The vapor of polar odorant 1-octonal evoked significant calcium transients in ASH neurons in the wild-type animals but weak signals in tph-1 and Ce-grk-2 mutants. These results indicated that the developed device could be useful to identify various odor-evoked neuronal activities.
    Sensors and Actuators B Chemical 03/2015; 209:109-115. DOI:10.1016/j.snb.2014.11.081 · 4.10 Impact Factor
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    • "The use of microfluidic systems has emerged as a powerful approach to enable immobilization for high-resolution imaging, direct in vivo manipulation, and high-throughput screens of a variety of organisms including Drosophila melanogaster embryos and larvae1819202122, Caenorhabditis elegans21232425262728293031, and zebrafish (Danio rerio) embryos and larvae32333435. Immobilization of freshwater planarians, however, presents several unique challenges: Planarians are about an order of magnitude larger than C. elegans, have a squishy, readily deformed body, vary in size depending on the species, feeding state, last asexual reproduction event, and other factors36, and are significantly more photophobic than C. elegans or D. melanogaster larvae. "
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    ABSTRACT: Planarians are an important model organism for regeneration and stem cell research. A complete understanding of stem cell and regeneration dynamics in these animals requires time-lapse imaging in vivo, which has been difficult to achieve due to a lack of tissue-specific markers and the strong negative phototaxis of planarians. We have developed the Planarian Immobilization Chip (PIC) for rapid, stable immobilization of planarians for in vivo imaging without injury or biochemical alteration. The chip is easy and inexpensive to fabricate, and worms can be mounted for and removed after imaging within minutes. We show that the PIC enables significantly higher-stability immobilization than can be achieved with standard techniques, allowing for imaging of planarians at sub-cellular resolution in vivo using brightfield and fluorescence microscopy. We validate the performance of the PIC by performing time-lapse imaging of planarian wound closure and sequential imaging over days of head regeneration. We further show that the device can be used to immobilize Hydra, another photophobic regenerative model organism. The simple fabrication, low cost, ease of use, and enhanced specimen stability of the PIC should enable its broad application to in vivo studies of stem cell and regeneration dynamics in planarians and Hydra.
    Scientific Reports 09/2014; 4:6388. DOI:10.1038/srep06388 · 5.58 Impact Factor
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    • "In addition, the ability of high-throughput cellular analysis increases the accuracy of experiments, and reduces the cost and experimental times. Despite extensive research on animal cells within microfluidic environments, including high throughput sorting, manipulation, phenotyping studies, and external stimulation, their application for plant cell studies has not been accomplished yet [2]. In this context, microfluidic platforms has recently gained attention for studying pollen tube as a model for tip growing plant cells. "
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    ABSTRACT: Pollen tube, the fastest tip growing plant cell, plays essential role in life cycle of flowering plants. It is extremely sensitive to external cues and this makes it as a suitable cellular model for characterizing the cell response to the influence of various signals involved in cellular growth metabolism. For in-vitro study of pollen tube growth, it is essential to provide an environment the mimics the internal microenvironment of pollen tube in flower. In this context, microfluidic platforms take advantage of miniaturization for handling small volume of liquids, providing a closed environment for in-vitro single cell analysis, and characterization of cell response to external cues. These platforms have shown their ability for high-throughput cellular analysis with increased accuracy of experiments, and reduced cost and experimental times. Here, we review the recent applications of microfluidic devices for investigating several aspects of biology of pollen tube elongation.
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