San Miguel-Ruiz JE et al. (2009), Regeneration of the radial nerve cord in the se...

ECB-ART-41004

BMC Dev Biol 2009 Jan 06;9:3. doi: 10.1186/1471-213X-9-3.

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Regeneration of the radial nerve cord in the sea cucumber Holothuria glaberrima.

San Miguel-Ruiz JE , Maldonado-Soto AR , García-Arrarás JE .

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BACKGROUND: Regeneration of neurons and fibers in the mammalian spinal cord has not been plausible, even though extensive studies have been made to understand the restrictive factors involved. New experimental models and strategies are necessary to determine how new nerve cells are generated and how fibers regrow and connect with their targets in adult animals. Non-vertebrate deuterostomes might provide some answers to these questions. Echinoderms, with their amazing regenerative capacities could serve as model systems; however, very few studies have been done to study the regeneration of their nervous system. RESULTS: We have studied nerve cord regeneration in the echinoderm Holothuria glaberrima. These are sea cucumbers or holothurians members of the class Holothuroidea. One radial nerve cord, part of the echinoderm CNS, was completely transected using a scalpel blade. Animals were allowed to heal for up to four weeks (2, 6, 12, 20, and 28 days post-injury) before sacrificed. Tissues were sectioned in a cryostat and changes in the radial nerve cord were analyzed using classical dyes and immunohistochemistry. In addition, the temporal and spatial distribution of cell proliferation and apoptosis was assayed using BrdU incorporation and the TUNEL assay, respectively.We found that H. glaberrima can regenerate its radial nerve cord within a month following transection. The regenerated cord looks amazingly similar in overall morphology and cellular composition to the uninjured cord. The cellular events associated to radial cord regeneration include: (1) outgrowth of nerve fibers from the injured radial cord stumps, (2) intense cellular division in the cord stumps and in the regenerating radial nerve cords, (3) high levels of apoptosis in the RNC adjacent to the injury and within the regenerating cord and (4) an increase in the number of spherule-containing cells. These events are similar to those that occur in other body wall tissues during wound healing and during regeneration of the intestine. CONCLUSION: Our data indicate that holothurians are capable of rapid and complete regeneration of the main component of their CNS. Regeneration involves both the outgrowth of nerve fibers and the formation of neurons. Moreover, the cellular events employed during regeneration are similar to those involved in other regenerative processes, namely wound healing and intestinal regeneration. Thus, holothurians should be viewed as an alternative model where many of the questions regarding nervous system regeneration in deuterostomes could be answered.

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Genes referenced: LOC100887844 LOC115919910 LOC115925415 tfdp1

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	Figure 1. Uninjured and regenerating stages of the radial nerve cord (RNC) of H. glaberrima. Longitudinal tissue sections of (A) uninjured and regenerating RNC at (B) 2, (C) 6, (D) 12, (E) 20 and (F) 28 days post injury (dpi) were stained with Toluidene Blue. (A) The uninjured RNC clearly shows the large ectoneural (EN) component separated from the thinner hyponeural (HN) component by a connective tissue band. (B) At 2 dpi the area is filled with debris and the open end of the RNC is visible (arrowhead). (C) By 6 dpi, the RNC stumps have organized into club-shaped structures. (D) By 12 dpi the injury gap has been filled with nervous tissue that now forms a continuous extension that joins the stumps. (E) By 20 dpi the RNC still appears slightly disorganized and no connective tissue band is observed. (F) At 28 dpi, the RNC has recovered much of the structure and organization found within non-injured RNC including the separation between the EN and HN components by a connective tissue band. D-dermis, EN-epineural component, HN-hyponeural component, RNC-radial nerve cord. X's denote the injury site; Asterisks show the presence of tattoo ink used to label the injury site; arrows signal morula cells within the RNC. Bar = 300 μm.
	Figure 2. RN1 labeling of the regenerating radial nerve cord. Longitudinal tissue sections of (A) uninjured and regenerating radial nerve cords at (B) 2, (C) 6, (D) 12, (E) 20 and (F) 28 days post injury (dpi) were labeled with the monoclonal antibody RN1. (A) Labeling in the uninjured RNC is highly specific to the nervous components, both ectoneural (EN) and hyponeural (HN) component while the connective tissue band remains unlabeled (arrowhead). (B) At 2 dpi nerve fiber debris is visible around the injury site and the nerve stumps can be seen to be highly disorganized. (C) By 6 dpi, the RNC stumps have organized into club-shaped structures from which some nerve fibers can be observed to be extending (arrows). (D) By 12 dpi the injury gap has been filled with nervous tissue that now forms a continuous extension that joins the stumps. (E) By 20 dpi the RNC still appears slightly disorganized and the CT band is not present along the entire length of the regenerated RNC. (F) At 28 dpi, the RNC has recovered much of the structure and organization found within non-injured RNC including the connective tissue that separates the EN and HN components. EN-epineural component, HN-hyponeural component, RNC-radial nerve cord. X's denote the injury site; Bar = 300 μm.
	Figure 3. Double labeling of regenerating radial nerve cords for the fibers expressing the neuropeptide GFS and proliferating cells with BrdU. Longitudinal tissue sections of regenerating RNC at (A) 2, (B) 6, (C) 12, and (E) 28 days post injury (dpi) were labeled with anti-GFS (green) and anti-BrdU (red). Animals that had undergone a transection of their radial nerve were injected with BrdU 24 hrs prior to sacrifice. (B) GFS expressing fibers can be seen to elongate out of the nerve stump by 6 dpi (arrow). (D) Restoration of GFS-expressing fibers in the radial nerve cord is complete by 28 dpi. In terms of cell division, little cell division is observed at (A) 2 or (D) 28 dpi. Actively dividing cells were mainly observed in the periphery of the RNC where most of the nuclei are present (arrows). Some cell division is also observed among cells in the neuropile. Cell division begins around (B) 6 dpi and peaks around (C) 12 dpi in both hyponeural and ectoneural components (arrows). EN-epineural component, HN-hyponeural component, RNC-radial nerve cord. X's denote the injury site; arrows signal some of the BrdU-labeled cells within the RNC. Bar = 300 μm.
	Figure 4. Galanin-like immunoreactivity in regenerating radial nerves. Longitudinal tissue sections of regenerating radial nerve cords at (A) 6, (B) 12 and (C) 28 days post injury (dpi) were labeled with anti-galanin (green) and counterstained with Hoescht (red) to label cell nuclei. (A) Galanin immunoreactivity disappears from the injury edge of the cord (asterisk) and the proximal stump acquires a punctuated labeling at 6 dpi. (B) At 12 dpi, the immunoreactive fibers have crossed the injury gap and labeling appears more homogeneous. (C) At 28 dpi labeling is similar to that in the uninjured cord (not shown). EN-ectoneural component, HN-hyponeural component, RNC-radial nerve cord. X's denote the injury site. Bar = 300 μm.
	Figure 5. Patterns of cell proliferation in the radial nerve cord. The percentage of BrdU-labeled cells compared to Hoescht labeled nuclei in the (A) ectoneural and (B) hyponeural component of the radial nerve cord are shown. Measurements were done at the injury site and compared with sites proximal and distal to the injury. Animals were injected with BrdU 24 hrs before sacrificed. Quantification of cell division at the injury site shows a peak in cell proliferation at 12 dpi both in the hyponeural and ectoneural components. A smaller increase in cell division is also observed in the nerve stumps proximal to the injury site, but not so distal to the injury. Regenerated tissues in the injured area do not appear until 12-dpe. Each point represents the mean ± S.E. of at least five animals. When compared to distal, significant differences are observed at various stages in the proximal and injured areas p < .05, *p < .01.
	Figure 6. Neuronal labeling in the regenerating radial nerve cord. Neurons (arrows) within the regenerated radial nerves were observed 62 dpi using (A) anti-GFS and (B) anti-Nurr1 markers. (C) The total number of cells within the regenerated radial nerve is not significantly different from the number of cells at proximal or distal sites. In contrast, quantification of the percent of neurons at the three sites showed that the percentage of neurons at the injury site was observed to remain significantly lower than at the other two sites. Each point represents the mean ± S.E. of at least three animals. p < .05, *p < .01. Scale bars = 50 μm.

	Figure 8. Apoptosis during radial nerve cord regeneration. (A.) Longitudinal section of regenerating RNC at 6 dpi showing TUNEL labeling (green) in cells undergoing apoptosis (arrows) (counter-stained with Hoescht-red). Apoptosis occurs in both (B) hyponeural and (C) ectoneural components of the regenerating radial nerve cord peaking at day 6 dpi in the proximal nerve stumps and at 12 dpi in the regenerating cord that is forming at the injury site. Little or no apoptosis occurs in areas distal to the injury. Regenerated tissues in the injured area do not appear until 12-dpe. Each point represents the mean ± S.E. of five animals. When compared to distal, significant differences are observed at various stages in the proximal and injured areas p < .05, *p < .01. HN – hyponeural component, EN – ectoneural component. Bar = 25 μm.
	Figure 9. Changes in spherule-containing cells during radial nerve cord regeneration. The number of morula cells and spherulocytes increases in the regenerating radial nerve cord following transection. Morulas were detected using Toluidene blue (see Fig. 1). Quantitative analysis show that (A) the number of morulas peak between 2 and 6 dpi in the proximal nerve stumps and at 12 dpi in the regenerating cord that is forming at the injury site. Few changes are observed distal to the injury site. (B-C) Spherulocytes (green, arrows) were detected using the Sph2 antibody, a monoclonal antibody that recognizes the spherulocyte population. Longitudinal sections of animals at (B) 12 and (C) 28 dpi show the presence of spherulocytes within the nerve cord in early but not in late regenerating stages. Sections were also labeled with anti-galanin (red) and Hoeschst nuclear dye (blue). X's denote the injury site (D) Quantitative analysis show that the number of spherulocytes peaks at 6 dpi in the proximal stumps and at 12 dpi at the injury site, with little change at distal sites. Regenerated tissues in the injured area do not appear until 12-dpe. Each point represents the mean ± S.E. of five animals. When compared to distal, significant differences are observed at various stages in the proximal and injured areas p < .05, *p < .01.

References [+] :

Anderson, Regeneration of spinal neurons in inframammalian vertebrates: morphological and developmental aspects. 1983, Pubmed