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J Neurosci Methods
2021 Dec 01;364:109358. doi: 10.1016/j.jneumeth.2021.109358.
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Dedifferentiation of radial glia-like cells is observed in in vitro explants of holothurian radial nerve cord.
Quesada-Díaz E, Figueroa-Delgado P, García-Rosario R, Sirfa A, García-Arrarás JE.
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BACKGROUND: Among animal phyla, some of the least studied nervous systems are those of the phylum Echinodermata. Part of the problem lies in that most of their nervous components are embedded in the body wall that has calcareous skeletal components.
NEW METHOD: We have developed a novel technique for the successful isolation of the radial nerve cords (RNCs) and an in vitro system where the isolated RNCs can be cultured and are amenable to experimental manipulation. Here we use this system to isolate the RNC of the sea cucumber Holothuria glaberrima as a way to extend our studies on its regeneration capabilities.
RESULTS: The RNCs can be isolated from the surrounding tissues by collagenase treatment. The explants obtained following enzymatic dissociation can be kept in culture for up to 2 weeks. Histological and immunohistochemical studies show that the explants maintain a stable number of cells with little proliferation or apoptosis throughout the culture incubation period. The main change observed in RNCs in vitro is a progressive dedifferentiation of radial glia-like cells. This dedifferentiation corresponds to the first step in the regeneration response to injury that has been described in vivo.
COMPARISON WITH EXISTING METHODS: There are no existing methods to isolate and culture echinoderm radial nerve cord.
CONCLUSIONS: The described protocol provides a unique tool to obtain easily accessible RNC from holothurians to perform cellular, biochemical, and genomic experiments in the echinoderm nervous system without interference of adjacent tissues. The technique provides a unique opportunity to study the dedifferentiation response associated with the regeneration of the nervous system in echinoderms.
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