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


Holothuria glaberrima specimens, 10–16 cm long, were collected from the northeastern rocky shores of Puerto Rico and kept in an aquarium at 22–24°C with circulating seawater and constant oxygenation. All experiments conform to the regulations on animal research at the University of Puerto Rico.

Surgical procedures

The technique used to injure the RNC has been described elsewhere [28]. Briefly, evisceration was induced by intracoelomic injection of 0.35 M KCl (3–5 ml). One hour following evisceration, animals were anesthetized by placing them for 10 min in 0.1% 1,1,1-Trichloro-2-methyl-2-propanol hydrate (chlorobutanol) diluted in seawater. Severing of the RNC was done by pushing the dorsal bodywall through the cloaca with a blunt rod, thus exposing the coelomic surface of the bodywall. A transection-type injury was made perpendicularly to the nerve using a scalpel, after which animals were reverted to their normal orientation and returned to their aquarium. By transecting the radial nerve from the inside we could obtain a reliable injury to the RNC with minimal damage to the surrounding tissues. Control animals were taken through the same protocol, except that no transection was made.

Tissue harvesting and Histology

During the regeneration period, animals were sacrificed at different days post-injury (2, 6, 12, 20, and 28 dpi). Animals were anesthetized with chlorobutanol as before, slit open and pinned to a dissecting tray. Initially, they were directly fixed within the tray with 4% paraformaldehyde for 30 min and then the nerve-muscle complex, along with the bodywall, was excised. The tissue block was refixed overnight, washed in PBS and left in 30% sucrose until used.

Tissue blocks were embedded in OCT (Tissue Tek OCT; Miles Inc.), frozen at -35°C and longitudinally sectioned (20 μm) in a cryostat (Leica CM 1900). Sections were mounted on poly-L-lysine coated slides, left to dry for 1 hr at room temperature and stored in a dry chamber until needed. Since the RNCs are localized within the body wall, the tissue sections contained the regenerating RNC, the proximal regions that were 0.5 mm from the injury site, and the distal regions that were approximately 5 mm away.

Histological analyses

Chemical Dyes – Toluidine blue

To obtain a comprehensive view of the temporal modifications that the RNCs go through after an injury, longitudinal tissue sections were stained with toluidine blue, as described by Presnell and Schreibman [31]. In brief, sections were rinsed in PBS for 2 min, and then immersed in dye solution for 2 min. They were then rinsed with tap water, mounted in PBS-buffered glycerol and viewed under the microscope. Occasionally, staining was also done on tissue sections previously processed for immunohistochemistry (see below).

Morphometric analysis

To evaluate changes in the RNCs following injury, tissue sections stained with toluidine blue were studied under light microscopy and the shortest distance between nerve stumps of injured animals was recorded. At least ten tissue sections were measured per animal and three to five animals were used for each stage. Sections were examined and photographed using a Nikon E600 microscope. Images were collected digitally with an RT Color Spot camera (Diagnostic Inst. Inc.) with 1520 × 1080 pixel resolution, and analyzed using the MetaVue software (version 6.0; Universal Imaging, Inc.) and Image J (version 1.37;NIH, USA).


Protocols for the immunohistochemical procedures performed for this study have been described elsewhere [28]. Tissue sections were permeabilized with 0.5% Triton X-100 in PBS, blocked with goat serum (1:50), and incubated at room temp overnight with the corresponding primary antibody: α-Bromodeoxyuridine (BrdU, 1:5, Amersham), Sph2 supernatant [32], α-GFSKLYFamide (α-GFS, 1:1000, [33]), α-Galanin (1:500, [34]), α-Nurr1 (1:1000, Sigma) or RN1 (1:50,000, [35]) in a humid chamber. The following day, sections were rinsed in PBS and treated with the FITC-labeled goat-anti-mouse secondary antibody (1:50, BioSource Int.). When double labeling was performed, the Cy3-labeled goat-anti-rabbit antibody (1:2,000) was used along with the FITC labeled antibody. Sections were rinsed again in PBS and mounted in buffered glycerol. Double or triple labeling with Hoechst nuclear dye (Sigma) was done by immersing slides in 1 μM Hoescht for 15 min after the secondary antibody [28]. Sections were examined and photographed using the Nikon E600 microscope equipped with FITC, R/DII and DAPI filters. Images were recorded using the MetaVue software as mentioned previously.

Cell counts

Various cellular populations were labeled and quantified. These include (1) the overall number of cells within the RNC using Hoescht nuclear dye, (2) spherulocytes, cells that contained a large number of spherules within their cytoplasm and were labeled with monoclonal antibody Sph2, (see [32]), (3) morulas, cells that also contain vesicles or spherules and that are strongly stained with Toluidine blue (see [32]), (4) neuropeptide-containing neurons labeled with anti-GFS and (5) a neuronal population labeled with an antibody against the transcription factor NURR1. The number of cells was quantified by counting labeled cells in a 280 mm2 area of the longitudinal RNC (this is the equivalent of the microscope field of view-FOW using the 40× objective). In order to be counted, cells had to be within the nerve or in close apposition with the somas in the nerve periphery, or with the fibers that had exited the nerve stumps. Cell counts were done in two fields of view chosen at random for each corresponding area in every animal and in at least five animals per regeneration stage.

TUNEL assay

The TUNEL method was used to label apoptotic cells within the RNC. For this purpose we used the TdT-FragEl DNA Fragmentation Detection kit (Calbiochem QIA33) and followed the manufacturer specifications with only minor modifications. Tissue sections were washed for 3 min in PBS to remove the embedding medium; afterwards they were refixed in 4% formaldehyde (diluted in 1× PBS) and washed in TBS for 15 min. Tissue sections were permeabilized with 50 μl proteinase K (5 μg/ml) for 15 min and then washed in TBS. Prior to labeling apoptotic cells, tissue sections were bathed with 1× TdT Equilibration Buffer (80 μl per section) for 30 min. For the labeling reaction, 60 μl of the mixture (57 μl Labeling Reaction Mix: 3 μl TdT enzyme) were applied to each section and incubated for 1 hr at room temperature. Slides were then washed 2× for 2 min in TBS, counterstained with the nuclear dye Hoescht for 10 min, washed for 5 min in TBS, and mounted with phosphate buffered glycerol.

Proliferation Assays

Pulse-chase experiments

Incorporation of BrdU into cell nuclei was used to study the spatio-temporal distribution of proliferating cells in regenerating animals. Animals were injected intracoelomically at 2, 6, 12, 20 and 28 dpi with 100 μl of a 1 mg/ml BrdU solution (approximately 0.01 mg/g wet weight) and sacrificed 24 hrs after. Tissues were fixed and treated as described above for immunohistochemistry. Cells undergoing replication were identified with an antibody against BrdU (Amersham). Prior to the incubation with the antibody, tissue sections were bathed with 0.05 N HCl for 1 h. This was done to improve the accessibility of the antibody to the BrdU epitope, followed by an additional 15 min PBS wash. Cell division index was obtained by dividing the number of BrdU-positive cells against the total number of Hoescht-labeled nuclei within a longitudinal section of the RNC in the microscope field of view. At least two measurements of corresponding areas chosen at random from each of at least five different animals were used for each stage.

Cellular birth-dating

To determine if dividing cells were giving rise to the neurons in the regenerating RNC, we injected animals with BrdU every other day for a period of 6 days (4 BrdU injections per animal in each group). There were a total of 6 groups: 0–6, 8–14, 16–22, 24–30, 32–38, and 40–46 dpi; all animals were sacrificed at 62 dpi. The rationale behind the multiple and consecutive doses of BrdU stems from the assumption that new neurons do not differentiate immediately after the division, but that a dividing neuronal precursor will not necessarily express the neuronal markers after the last division. Multiple injections were thought to be necessary to assure that at least some cells were caught at the time of division. Sections of RNCs from the various groups were double labeled against BrdU and markers for different subpopulations (GFS, Galanin, Nurr1) to establish the date of birth of the neurons that were repopulating the new RNC tissue.

Statistical Analyses

Student's t test and ANOVA's were used for statistical analyses. P values of < 0.05 were deemed to be significant.

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