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Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis

BACKGROUND: The efficient regenerative abilities at larvae stages followed by a non-regenerative response after metamorphosis in froglets makes Xenopus an ideal model organism to understand the cellular responses leading to spinal cord regeneration. METHODS: We compared the cellular response to spin...

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Autores principales: Edwards-Faret, Gabriela, González-Pinto, Karina, Cebrián-Silla, Arantxa, Peñailillo, Johany, García-Verdugo, José Manuel, Larraín, Juan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852093/
https://www.ncbi.nlm.nih.gov/pubmed/33526076
http://dx.doi.org/10.1186/s13064-021-00152-2
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author Edwards-Faret, Gabriela
González-Pinto, Karina
Cebrián-Silla, Arantxa
Peñailillo, Johany
García-Verdugo, José Manuel
Larraín, Juan
author_facet Edwards-Faret, Gabriela
González-Pinto, Karina
Cebrián-Silla, Arantxa
Peñailillo, Johany
García-Verdugo, José Manuel
Larraín, Juan
author_sort Edwards-Faret, Gabriela
collection PubMed
description BACKGROUND: The efficient regenerative abilities at larvae stages followed by a non-regenerative response after metamorphosis in froglets makes Xenopus an ideal model organism to understand the cellular responses leading to spinal cord regeneration. METHODS: We compared the cellular response to spinal cord injury between the regenerative and non-regenerative stages of Xenopus laevis. For this analysis, we used electron microscopy, immunofluorescence and histological staining of the extracellular matrix. We generated two transgenic lines: i) the reporter line with the zebrafish GFAP regulatory regions driving the expression of EGFP, and ii) a cell specific inducible ablation line with the same GFAP regulatory regions. In addition, we used FACS to isolate EGFP(+) cells for RNAseq analysis. RESULTS: In regenerative stage animals, spinal cord regeneration triggers a rapid sealing of the injured stumps, followed by proliferation of cells lining the central canal, and formation of rosette-like structures in the ablation gap. In addition, the central canal is filled by cells with similar morphology to the cells lining the central canal, neurons, axons, and even synaptic structures. Regeneration is almost completed after 20 days post injury. In non-regenerative stage animals, mostly damaged tissue was observed, without clear closure of the stumps. The ablation gap was filled with fibroblast-like cells, and deposition of extracellular matrix components. No reconstruction of the spinal cord was observed even after 40 days post injury. Cellular markers analysis confirmed these histological differences, a transient increase of vimentin, fibronectin and collagen was detected in regenerative stages, contrary to a sustained accumulation of most of these markers, including chondroitin sulfate proteoglycans in the NR-stage. The zebrafish GFAP transgenic line was validated, and we have demonstrated that is a very reliable and new tool to study the role of neural stem progenitor cells (NSPCs). RNASeq of GFAP::EGFP cells has allowed us to clearly demonstrate that indeed these cells are NSPCs. On the contrary, the GFAP::EGFP transgene is mainly expressed in astrocytes in non-regenerative stages. During regenerative stages, spinal cord injury activates proliferation of NSPCs, and we found that are mainly differentiated into neurons and glial cells. Specific ablation of these cells abolished proper regeneration, confirming that NSPCs cells are necessary for functional regeneration of the spinal cord. CONCLUSIONS: The cellular response to spinal cord injury in regenerative and non-regenerative stages is profoundly different between both stages. A key hallmark of the regenerative response is the activation of NSPCs, which massively proliferate, and are differentiated into neurons to reconstruct the spinal cord. Also very notably, no glial scar formation is observed in regenerative stages, but a transient, glial scar-like structure is formed in non-regenerative stage animals. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13064-021-00152-2.
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spelling pubmed-78520932021-02-03 Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis Edwards-Faret, Gabriela González-Pinto, Karina Cebrián-Silla, Arantxa Peñailillo, Johany García-Verdugo, José Manuel Larraín, Juan Neural Dev Research Article BACKGROUND: The efficient regenerative abilities at larvae stages followed by a non-regenerative response after metamorphosis in froglets makes Xenopus an ideal model organism to understand the cellular responses leading to spinal cord regeneration. METHODS: We compared the cellular response to spinal cord injury between the regenerative and non-regenerative stages of Xenopus laevis. For this analysis, we used electron microscopy, immunofluorescence and histological staining of the extracellular matrix. We generated two transgenic lines: i) the reporter line with the zebrafish GFAP regulatory regions driving the expression of EGFP, and ii) a cell specific inducible ablation line with the same GFAP regulatory regions. In addition, we used FACS to isolate EGFP(+) cells for RNAseq analysis. RESULTS: In regenerative stage animals, spinal cord regeneration triggers a rapid sealing of the injured stumps, followed by proliferation of cells lining the central canal, and formation of rosette-like structures in the ablation gap. In addition, the central canal is filled by cells with similar morphology to the cells lining the central canal, neurons, axons, and even synaptic structures. Regeneration is almost completed after 20 days post injury. In non-regenerative stage animals, mostly damaged tissue was observed, without clear closure of the stumps. The ablation gap was filled with fibroblast-like cells, and deposition of extracellular matrix components. No reconstruction of the spinal cord was observed even after 40 days post injury. Cellular markers analysis confirmed these histological differences, a transient increase of vimentin, fibronectin and collagen was detected in regenerative stages, contrary to a sustained accumulation of most of these markers, including chondroitin sulfate proteoglycans in the NR-stage. The zebrafish GFAP transgenic line was validated, and we have demonstrated that is a very reliable and new tool to study the role of neural stem progenitor cells (NSPCs). RNASeq of GFAP::EGFP cells has allowed us to clearly demonstrate that indeed these cells are NSPCs. On the contrary, the GFAP::EGFP transgene is mainly expressed in astrocytes in non-regenerative stages. During regenerative stages, spinal cord injury activates proliferation of NSPCs, and we found that are mainly differentiated into neurons and glial cells. Specific ablation of these cells abolished proper regeneration, confirming that NSPCs cells are necessary for functional regeneration of the spinal cord. CONCLUSIONS: The cellular response to spinal cord injury in regenerative and non-regenerative stages is profoundly different between both stages. A key hallmark of the regenerative response is the activation of NSPCs, which massively proliferate, and are differentiated into neurons to reconstruct the spinal cord. Also very notably, no glial scar formation is observed in regenerative stages, but a transient, glial scar-like structure is formed in non-regenerative stage animals. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13064-021-00152-2. BioMed Central 2021-02-02 /pmc/articles/PMC7852093/ /pubmed/33526076 http://dx.doi.org/10.1186/s13064-021-00152-2 Text en © The Author(s) 2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research Article
Edwards-Faret, Gabriela
González-Pinto, Karina
Cebrián-Silla, Arantxa
Peñailillo, Johany
García-Verdugo, José Manuel
Larraín, Juan
Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis
title Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis
title_full Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis
title_fullStr Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis
title_full_unstemmed Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis
title_short Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis
title_sort cellular response to spinal cord injury in regenerative and non-regenerative stages in xenopus laevis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852093/
https://www.ncbi.nlm.nih.gov/pubmed/33526076
http://dx.doi.org/10.1186/s13064-021-00152-2
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