Overview of nerve regeneration
To assess the morphological changes that the RNC underwent during
the regeneration period, longitudinal sections of body wall were
stained with Toluidine blue and studied under light microscopy (Fig. 1). The first stage (2 dpi) was characterized by an injured RNC with fan-shaped stumps (Fig. 1B).
Remarkably, the pre-existing CT band in the stumps were practically
unaffected; if anything, it lost a bit of cohesiveness at its most
proximal end, adjacent to the injury. By 6 dpi, small bundles of fibers
had extended from both nerve stumps, giving the structure an overall
needle-like shape laying on the substratum provided by the body wall
and pointing towards the opposing stump (Fig. 1C).
At this stage, the origin of nerve bundles extending beyond the stump
seemed to be, mostly, the result of an outgrowth in the nerve EN
component, which lies closer to the body wall. Still, the lack of a
substratum in the injury gap appeared to be the main limitation for
scouting bundles. Support for this comes from observations of fiber
bundles, which in the absence of a direct intervening matrix between
the stumps, were found close to the trough of the injury gap. Nerve
re-contact occurred by 12 dpi, concurrent with the appearance of a
provisional matrix that filled the injury gap (Fig. 1D).
The regenerated RNC spanned the injury gap but remained disorganized
with empty spaces that could be seen. This was clearly observed by
using the differential interference contrast (DIC) filter in the
microscope. Although the nerve had reconnected by 12 dpi, the CT band
that normally separates the HN and EN was not present. The RNC
structure was much improved by 20 dpi (Fig. 1E).
At this stage the number and localization of nuclei, both in the HN and
EN, was similar to that of an uninjured nerve. The CT band separating
the HN and EN was already perceptible at this stage in some, but not
all animals, as traces of thin filaments along the length of the nerve.
Approximately one month after the injury (28 dpi), the regenerated
nerve had a normal architecture (Fig. 1F).
The nuclear arrangement and density of fibers was now similar to that
of a normal uninjured nerve and the CT band was already present and
compact, although in some areas it still seemed diffuse.
Regeneration of fiber subpopulations
To obtain an in-depth view of the events that occur in the
regenerating RNC, we studied the responses of different fiber
subpopulations to the injury. Using immunohistochemistry we were able
to correlate the gross appearance of the RNC, studied in light
microscopy, with the growth of particular fiber populations across the
injury site of the regenerated RNC.
This is a monoclonal antibody raised in our lab against H. glaberrima RNC tissue  that provides very specific labeling of what appears to be a large fiber population of the holothurian nervous system (Fig. 2A).
RN1 does not label the CT band that separates the HN and EN component,
thus, this band could be detected as a non-labeled distinct strip of
tissue embedded within the high density of RN1 immunoreactive fibers.
As a result, this antibody served as a two-edged marker, to measure
progress during the process of nerve regeneration, namely the tracking
of fibers as they elongate, and the appearance of the CT band as
evidence of tissue organization into HN and EN components.
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.
Most of the data collected with this antibody confirm
the observations made using the Toluidene blue dye; however, some
interesting differences were found. The fan-shaped morphology of the
nerve stumps at the injury site observed at 2 dpi was mostly the result
of a swelling (enlargement) in the EN component, while the HN component
was not severely affected (Fig. 2B).
At this stage, the HN component proximal to the injury appeared to
retract in relation to the EN. At 6 dpi, the stumps acquired a
club-shaped appearance and RN1-labeled fiber bundles could be observed
protruding toward the opposite stump (Fig. 2C).
Unfortunately, because of the high density of labeling, individual
nerve fibers inside these pioneering bundles were hard to identify.
However, the RN1 fiber bundles were always observed to be in direct
contact with the substratum of the body wall and continuous with the
injured stump. At this stage we observed the first difference in the
rate of regeneration between the EN and HN components; the EN component
provided the first scouting bundles that traverse the substratum
towards the injury site, while RN1-labeled fibers of the HN appeared to
be in a latent state. At 12 dpi, RN1 fibers from opposing nerve stumps
came together, forming the newly regenerated RNC (Fig. 2D).
At this stage, although RN1 fibers filled the area between the stumps,
the separation of the RNC into its respective components had not
occurred yet. During this stage RN1 labeled side branches could be
observed originating from the same nerve bundles that form the
connection between the stumps. These branches might be some that had
gone astray into other injured tissues or they might be precursors to
peripheral nerves. By 20 dpi the new RNC showed the first hint of
connective tissue separation between the HN and EN components, albeit,
rudimentary and irregular at times (Fig. 2E).
The EN component was now structurally comparable to that of an
uninjured RNC, as its wandering bundles of fibers were no longer
present or at least apparent. Additionally, the regenerated RNC had a
more cylindrical shape in accordance with the rest of the nerve;
nonetheless, the staining intensity was now less than in uninjured
areas. In spite of all these improvements in the EN component, the HN
in most animals was still underdeveloped compared to uninjured
counterparts in the same section. The new RNC was restored by 28 dpi
with an HN and an EN component clearly separated from each other by the
CT band between them (Fig. 2F).
is a polyclonal antibody made in our lab against the neuropeptide
GFSKLYF-amide that recognizes a subpopulation of neuronal fibers and
somas in both HN and EN components of the RNC of H. glaberrima .
At 2 dpi, immunolabeling with GFS clearly showed the fan-shaped
morphology of the cut RNC and the fraying of the nerve fibers close to
the injury (Fig. 3A).
The reduction in fiber density close to the injury was accompanied by
an increase in labeling intensity close to the nerve stumps, which
could be attributed to the appearance of α-GFS-labeled
varicosities along the fibers. At 6 dpi, GFS-immunoreactive fibers had
sprouted from the nerve stumps and the overall structure was clearly
pointing towards the opposite stump (Fig. 1B).
It is important to note that that the previously observed varicosities
were also present in these migrating fibers. Similar to what was
observed for RN1, fibers expressing the GFS neuropeptide also
reconnected by 12 dpi (Fig. 3C).
The labeling with GFS showed that immunoreactive fibers in the HN
followed those of the EN; instead of using the body wall as a
substrate. Even when the path taken by some fibers might be twisted and
indirect, the overall axis of the fibers inside the regenerated EN was
parallel to the long axis of the RNC. By 20 dpi both components were
present in the regenerated RNC. No major differences were observed a
month after transection (28 dpi) between regenerated RNC segment and
its uninjured counterpart (Fig. 3D).
In the RNC this antibody labels a relatively small population of fibers .
At 2 dpi, galanin immunoreactive fibers underwent a change in their
morphology forming round varicosities with very intense labeling. This
change was more conspicuous closer to the injury site, where the nerve
fibers have became frayed; they were present in the HN and EN component
of the RNC. At 6 dpi, varicosities were also found in the needle-shaped
bundle of fibers exiting the nerve stump (Fig. 4A).
Once the RNC reconnected at 12 dpi, the area of the injury could be
easily identified by the presence of these large varicosities. At 20
dpi, galanin-positive fibers were present in both components of the
RNC, although with a higher density in the EN (Fig. 4B).
Galanin-positive fibers passing between these two components were also
apparent at multiple points along the regenerated RNC, as is usual in
an uninjured nerve. At 28 dpi, the large varicosities had disappeared
and α-galanin produced a labeling that was virtually undistinguishable from that of a normal RNC.
In summary, all neuronal markers used showed that the
injury gap that separates the two RNC stumps is slowly filled by a
growth of fibers that originate from the stump ends in the first two
weeks of regeneration (Fig. 4C). This process is complete by 20 dpi and a RNC with high similarity to the uninjured RNC is in place by 28 dpi.
RNCs are ganglionated nerves where the neuronal
soma and supporting cells can be mainly found in the periphery. To
study the possibility that cell division is occurring in the RNC as a
result of the injury, animals were injected with the thymidine analog,
BrdU, and then checked for its incorporation into the nuclei.
Initially, at 2 dpi few cells within the proximal stumps, both in the
HN and EC bands, of the injured nerves were undergoing cell division;
no cell division was seen distal to the injury site (Fig. 5).
At 6 dpi, the highest numbers of dividing cells were seen in the
proximal region, with about 6% of cells (6.81 ± 0.42 in HN and 5.36 ±
0.4 in EN) undergoing cell division. Cell division distal to the injury
site never exceeded 2%. By 12 dpi, cell division in the proximal stumps
remained comparatively similar (6.54 ± 0.23 in HN and 383 ± 0.21 in EN)
to the previous stage. However, an impressive surge in cell division
occurred in the regenerating RNC that is forming across the injury
site, where close to 20% of the cells (22.2 ± 1.06 in HN and 14.9 ±
1.15 in EN) were undergoing cell division. Subsequently, at 20 dpi, a
sharp drop in cell division was observed in the newly formed RNC.
Proximal to the injured nerve, cell division did not exceed 2%. At the
last stage studied (28 dpi), cell division was mainly restricted to the
HN of the regenerated RNC, where cell division was 3.28 ± 0.7% in HN
and 1.26 ± 0.3 in EN, everywhere else, cell division was less than 1%.
Cell regeneration and birth dating
The next step
was to determine whether the neurons in the regenerated RNC were
originating from dividing precursors. For this we used two neuronal
markers that label independent neuronal populations in the RNC,
anti-GFS  and anti-Nurr1 (unp. obs.). These markers labeled neuronal cell bodies in both the EN and HN components of the RNC (Fig. 6A &6B).
Initially, experiments were made by injecting animals with BrdU at
different time periods and sacrificing animals at 30 dpi. However,
these animals showed few if any GFS or Nurr1 neurons in their
regenerated RNC. Thus, we decided to extend the experiment until day 62
dpi. For this, animals were separated into 6 groups and each group
received BrdU injections during a particular 6-day period. All animals
were sacrificed at 62 dpi. The rationale for this experiment was that
only those cells that stop proliferation close to the last BrdU
injections would retain the BrdU label until 62 dpi. Cells that kept
proliferating would dilute out the BrdU and not be labeled. Thus, we
would expect that dividing neuroblasts would stop dividing and then
differentiate, so that most of the labeled cells that had stopped
dividing within the RNC would be neurons.
Sections of the regenerated RNC were obtained and the
cells in an area of equivalent length of RNC were determined on the
microscope field of view (40×) were counted at 62 dpi. Results showed
that the total number of cells (as measured by counting Hoescht-labeled
nuclei) within the regenerated RNC did not differ from that in the
proximal and distal RNC, suggesting that cell regeneration had occurred
after 2 months (Fig. 6C).
At this time, neurons expressing GFS or Nurr1 were also found within
the regenerated RNC, albeit, in smaller numbers when compared to those
within the proximal and distal RNC (Fig. 6D &6E)
Birthdating studies showed a peak during the second and third week
of regeneration (groups 2 & 3, 8–22 dpi) when close to 3% of the
cells (group 2 – 3.16 ± 0.54%, group 3 – 2.81 ± 0.59%) stopped
dividing, and were still BrdU labeled at 62 dpi (Fig. 7A &7B).
This would suggest that it is during this time period that most neurons
have stopped dividing and started differentiation. To further verify
that some of the dividing cells were neurons, we used double labeling
with the neuronal markers GFS and NURR1. The results were somewhat
unexpected. None of the cells that expressed GFS were double labeled
with BrdU in any of the injected groups, even when over 300 GFS neurons
were observed. In contrast, some cells labeled with Nurr1 were also
labeled with BrdU (Fig. 7C).
These cells were present only in the HN component of animals injected
with BrdU during 8–22 dpi (which corresponds to the peak in cells that
stopped proliferation). However, the number of neurons is rather small
as only 2.5% of all NURR1 immunoreactive cells present in these groups
were BrdU labeled (Fig. 7D).
To determine the cellular events that occurred during RNC
regeneration following a transection-type injury, we decided to examine
if programmed cell death was occurring (Fig. 8).
As soon as 2 dpi, apoptotic cells were present in the HN component of
the proximal stumps. The highest number of apoptotic cells in the
proximal stumps occurred at 6 dpi, in both the HN and EC bands,
although the magnitude of apoptotic cells was higher in the HN band. By
12 dpi and on to 28 dpi, few if any apoptotic cells were observed in
the nerve proximal to the injury site. In contrast, at 12 dpi, a large
wave of apoptosis occurred in the regenerated RNC that had just formed
over the injury gap. The number of apoptotic cells in the regenerating
RNC dropped in the two subsequent stages, until at 28 dpi, no apoptosis
was observed in the EN and only in 1% of cells of the HN band.
Two types of spherule-containing cells have been described in our
system: morulas, which are highly basophilic and stain dark purple with
toluidine blue, and spherulocytes, which are recognized by a monoclonal
antibody prepared against sea cucumber tissues [28,32]. Only about 5% of spherule-containing cells are recognized by both markers.
Few (less than1 per visual field), if any, morula cells were found within the normal RNCs (see Fig. 1).
(As mentioned earlier, fields of view, FOV, were obtained with the 40×
objective and encompassed an equivalent area in all animals). Changes
in their number within the RNC were already seen at 2 dpi, when they
invaded the nerve stumps. They achieved their highest numbers at this
stage, when the average number of cells per field of view within the
proximal stumps was 6.9 + 0.2 cells per FOV (Fig. 9A, and see Fig. 1).
Most cells were closely apposed to the CT band that separates the HN
and EN components; this is the same location where they are normally
found in an uninjured nerve. However, as a response to the injury,
morulas were also found interspersed within the neuropile, more
commonly where the nerve stumps were engorged, or fan-shaped. By 6 dpi,
the number of cells in the proximal stump remained the same (6.4 + 0.24
cells per FOV) as in the previous stage. However, some of these cells
were also associated with the RNC cell bodies, in both the HN and EN,
adjacent to the coelomic side and also with the regrowing fibers that
originated from the nerve stumps. At 12 dpi the number of morulas in
the proximal RNC decreased to levels just slightly higher (1.6 + 0.1
cells per FOV) than the basal level and remained at this level until 28
dpi. The changes in the morula population only occurred proximally to
the injury site since distal to the injury site the cell numbers were
not significantly different from uninjured nerves at any stage studied.
The number of cells was much higher in the new RNC bridging the injury
site at 12 dpi, on average there were 7.12 ± 0.35 cells per FOV. At 20
dpi, the average number of cells in the regenerated RNC declined to
2.61 ± 0.25 cells per FOV; similar to the number of cells found at 28
dpi (2.89 ± 0.23 cells per FOV)(see Fig. 1).
The number of spherulocytes within the RNCs also underwent changes during regeneration (Fig. 9D).
These cells were not normally found in uninjured nerves, however, as
soon as 2 dpi they were found within the proximal stumps (1.8 ± 0.27
cells per FOV); and to a lesser extent distal to the injury (1.1 ± 0.
07 cell per FOV). A consecutive increase was seen in the RNC stump by 6
dpi, when on average 3.23 ± 0.34 cells per FOV were observed in the
RNC. By 12 dpi, the number of spherulocytes proximal to the regenerated
RNC decreased, but still remained significantly higher than the basal
level (see Fig. 9B).
This was accompanied by a relatively high infiltration of these cells
into the new RNC that had on average 4.34 ± 0.51 cells per FOV. The
number of cells seen in the regenerated RNC by 20 dpi decreased to 2.24
± 0.25 cells per FOV; the range of cells in proximal and distal parts
relative to the injury site was 1.59 ± 0.17 to 1.34 ± 0.04 per FOV.
Distally, the spherulocyte population remained unchanged from 3 dpi
until 20 dpi at less than 1.4 cells per FOV. At 28 dpi, spherulocytes
could only be detected in the regenerated portion of the RNC (see Fig. 9C),
and although their levels were lower than those of previous stages,
they were still significantly higher than in uninjured nerves.