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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Mammals are unable to regenerate its spinal cord after a lesion, meanwhile, anuran amphibians are capable of spinal cord regeneration only as larvae, and during metamorphosis, this capability is lost. Sox2/3+ cells present in the spinal cord of regenerative larvae are required for spinal cord regeneration. Here we evaluate the effect of the transplantation of spinal cord cells from regenerative larvae into the resected spinal cord of non-regenerative stages (NR-stage). Donor cells were able to survive up to 60 days after transplantation in the injury zone. During the first 3-weeks, transplanted cells organize in neural tube-like structures formed by Sox2/3+ cells. This was not observed when donor cells come from non-regenerative froglets. Mature neurons expressing NeuN and Neurofilament-H were detected in the grafted tissue 4 weeks after transplantation concomitantly with the appearance of axons derived from the donor cells growing into the host spinal cord, suggesting that Sox2/3+ cells behave as neural stem progenitor cells. We also found that cells from regenerative animals provide a permissive environment that promotes growth and regeneration of axons coming from the host. These results suggest that Sox2/3 cells present in the spinal cord of regenerative stage (R-stage) larvae are most probably neural stem progenitor cells that are able to survive, proliferate, self-organize and differentiate into neurons in the environment of the non-regenerative host. In addition, we have established an experimental paradigm to study the biology of neural stem progenitor cells in spinal cord regeneration.

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Mammals are not capable of regenerating their central nervous system (CNS); anamniotes, however, can regenerate in response to injury. The mechanisms that explain the different regenerative capabilities include: (i) extrinsic mechanisms that consider the cellular environment and extracellular matrix composition, (ii) intrinsic factors implicating the presence or absence of genetic programs that promote axon regeneration, and (iii) the presence or absence of neural stem and progenitors cells (NSPCs) that allow neurogenesis. Xenopus laevis is able to regenerate its CNS during larval stages (i.e., the regenerative stage [R-stage]). However, concomitant with metamorphosis this capacity decreases and is lost completely in juvenile froglets (i.e., nonregenerative stages [NR-stages]). The loss of the regenerative ability correlates with a reduction in the percentage of Sox2 + cells, which are putative NSPCs. This protocol shows the effect of transplantation of spinal cord cells from R-stage Xenopus larvae into NR-stage froglets. Using this procedure, it is possible to study axon regeneration and stem cell biology in vivo. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. ReagentsBovine serum albumin (BSA; 0.5 mg/mL in L-15 Medium) Ethyl 3-aminobenzoate methanesulfonate salt (0.02% [w/v] in 0.1× MBS [MS-222; Sigma-Aldrich A5040]) The solution can be stored for 1 mo at 4˚C. Fibrinogen (100 mg/mL in phosphate-buffered saline [PBS]; Sigma-Aldrich F6755) Final medium L-15 Medium (Sigma-Aldrich L4386) 2 Correspondence: jlarrain@bio.puc.cl From the Xenopus collection, edited by Hazel L. Sive.

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