Here we present a protocol for the husbandry of Xenopus laevis tadpoles and froglets, and procedures to study spinal cord regeneration. This includes methods to induce spinal cord injury (SCI); DNA and morpholino electroporation for genetic studies; in vivo imaging for cell analysis; a swimming test to measure functional recovery; and a convenient model for screening for new compounds that promote neural regeneration. These protocols establish X. laevis as a unique model organism for understanding spinal cord regeneration by comparing regenerative and nonregenerative stages. This protocol can be used to understand the molecular and cellular mechanisms involved in nervous system regeneration, including neural stem and progenitor cell (NSPC) proliferation and neurogenesis, extrinsic and intrinsic mechanisms involved in axon regeneration, glial response and scar formation, and trophic factors. For experienced personnel, husbandry takes 1-2 months; SCI can be achieved in 5-15 min; and swimming recovery takes 20-30 d.

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Studying the cellular composition and morphological changes of cells lining the central canal during Xenopus laevis metamorphosis could contribute to understand postnatal development and spinal cord regeneration. Here we report the analysis of central canal cells at different stages during metamorphosis using immunofluorescence for protein markers expression, transmission and scanning electron microscopy and cell proliferation assays. The central canal was regionalized according to expression of glial markers, ultrastructure, and proliferation in dorsal, lateral, and ventral domains with differences between larvae and froglets. In regenerative larvae, all cell types were uniciliated, have a radial morphology, and elongated nuclei with lax chromatin, resembling radial glial cells. Important differences in cells of nonregenerative froglets were observed, although uniciliated cells were found, the most abundant cells had multicilia and revealed extensive changes in the maturation and differentiation state. The majority of dividing cells in larvae corresponded to uniciliated cells at dorsal and lateral domains in a cervical-lumbar gradient, correlating with undifferentiated features. Neurons contacting the lumen of the central canal were detected in both stages and revealed extensive changes in the maturation and differentiation state. However, in froglets a very low proportion of cells incorporate 5-ethynyl-2'-deoxyuridine (EdU), associated with the differentiated profile and with the increase of multiciliated cells. Our work showed progressive changes in the cell types lining the central canal of Xenopus laevis spinal cord which are correlated with the regenerative capacities.

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