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Regeneration of neurons and fibers in the mammalian spinal cord has not …


Biology Articles » Developmental Biology » Animal Development » Regeneration of the radial nerve cord in the sea cucumber Holothuria glaberrima » Figures

Figures
- Regeneration of the radial nerve cord in the sea cucumber Holothuria glaberrima

mcith_0318-2-f1.jpg 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.

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mcith_0318-2-f2.jpg 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.

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mcith_0318-2-f3.jpg 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.

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mcith_0318-2-f4.jpg 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.

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mcith_0318-2-f5.jpg 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.

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mcith_0318-2-f6.jpg 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.

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mcith_0318-2-f7.jpg Figure 7 Cellular birth dating in the regenerating radial nerve cord. (A) Quantification of the percent of BrdU-labeled nuclei for each group of regenerating animals that were injected with BrdU at different time points and sacrificed at 62 dpi. (B) BrdU-labeled nuclei (green, arrows) among Hoechst-labeled nuclei (red) in the radial nerve cord at the injury site of an animal injected during the second week of regeneration. (C) Triple labeling showing Nurr1-expressing cell (red, arrow) with BrdU-labeled nuclei (green) in the hyponeural compartment at the injury site. Blue labeling is Hoechst dye. (D) Quantification of the percent of Nurr1 labeled neurons that were BrdU-labeled for each group at 62 dpi. The only neurons labeled with BrdU at 62 dpi were from the animals injected during the second and third weeks of regeneration (groups 2 & 3). Each point represents the mean ± S.E. of at least three animals. When compared to distal, significant differences are observed at various stages in the injured area. Similarly, significant cell division occurs in the proximal and distal area at G4. *p < .05, **p < .01. Scale bars = (A) 50 μm, (B) 25 μm.

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mcith_0318-2-f8.jpg 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.

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mcith_0318-2-f9.jpg 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.

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