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

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

The regenerated RNC

Our results clearly show that the RNC of holothurians can regenerate within a month following a transection. The regenerated RNC looks amazingly similar to the non-injured RNC as judged by several histological and cellular parameters: It is well organized into ectoneural and hyponeural components, there is no scar or fibrotic tissue, the number of cells is similar to those found in uninjured tissue, neurons are localized to the nerve periphery, and spherule-containing cells that have infiltrated the injured cord have all but dissapeared. This is an amazing regeneration feat for an adult deuterostome, particularly if we consider that the RNC conforms the echinoderm CNS [24,29,30] and has been proposed to be evolutionary related to the vertebrate CNS [36-38].

Although we lack direct proof, a large amount of circumstantial evidence suggest that regeneration is not solely the joining back of the severed nerve stumps but that actually a new portion of the RNC is generated. First, from the moment it is cut, the wound forms a V-shaped structure where the nerve stumps are initially separated by a gap, and then by connective tissue. Second, most of the ongoing cellular division occurs within the regenerating RNC. Third, other tissues that surround the nerve and that also regenerate, do so by forming new tissues. This is particularly true of the surrounding muscle and connective tissues, which regenerate in parallel to the RNC and where very small differences in organization, cell density and texture distinguish the "new" tissues from the adjacent "old" [28]. Fourth, the initial and most visible regeneration event is the outgrowth of fibers from the RNC stumps. These outgrowths increase with time and eventually completely fill the space between the stumps. The latter retain their original morphology throughout regeneration, and it is the space in between that grows and becomes organized. Finally, recent work in another holothurian, Eupentacta fraudatrix, also shows regeneration of the RNC to be newly formed tissue [23].

In some ways, the formation of the new RNC can be compared to the "new" RNC segments that grow when the tip of the arm is severed in starfish or crinoids [19,20,22]. In all these cases the regenerated arm tip eventually grows to form new segments that include all the components found in the RNCs. Experimental evidence for the formation of a new RNC also comes from other closely related deuterostomes, the ascidians, that have an impressive ability to regenerate main components of their nervous system as has been shown by Bollner and colleagues [17,18] in studies where the main neural ganglion regenerates afer ablation. Similarly, lower vertebrates are able to regenerate a lost segment of the spinal cord as has been shown by nerve cord injury in amphibians [39,40] and fish [1].

Phylogenetic analysis of regeneration following transection

How does the regeneration of the holothurian RNC compares to that shown by other animals? To answer this question we will focus on those experiments where injury to the CNS is done by transection of the main nerve cord (or spinal cord). This includes studies in protostomes, such as mollusks and annelids and lower vertebrates, such as fish and urodele amphibians. It also includes developing vertebrates, since experiments have shown that spinal cord regeneration can occur at particular developmental stages of some higher vertebrates, such as chicks or marsupials, and then becomes restricted as development continues [41,42].

RNC regeneration in H. glaberrima shows strong similarities to other animal species known to be capable of axonal regeneration and/or formation of a missing part of their CNS. In most species where CNS regeneration takes place, two events can be highlighed to occur: the outgrowth of fibers from the nerve stumps and the absence of fibrotic tissue or glial scar. The growth of fibers into the lessioned area provides the first indication of the regeneration process. As has been reported by TEM in the sea cucumber E. fraudatrix [23] and by immunohistochemistry in the starfish, Asterias rubens [22], these fibers originate from the lesioned stumps and move across the lesion eventually forming a bridge between the two nerve stumps. Similarly, in urodele larvae, fibers extending from each of the cut ends grow across the gap and eventually form a bridge connecting the two nerve ends [39]. New fibers are mainly attributed to the re-growth of fibers from lesioned cells as has been shown by retrograde labeling at the time of transection [43,44]. Another common observation in regenerating animals, including holothurians, is the fact that there seems to be a directionality to the process, since fibers extending from one stump are directed toward the opposite stump and are rarely observed to be traveling toward another target, suggesting the participation of soluble factors originating from the severed stumps and/or guidance molecules in the surrounding environment.

The absence of a glial scar or fibrotic tissue in regenerating cords is another characteristic of those animals capable of regenerating their main nervous system. No scar tissue is detected or described in studies showing regeneration in urodeles, fish or echinoderms. Similarly, chick embryos that are capable of regeneration do not form scar tissue [41]. In fact, what investigators regularly show to occur during the regenerative response is an increase in cells or cell activity associated with the removal of the injury debris and of apoptotic cells.

There are also differences in the regeneration process among animals. One of these is the level or extent at which regeneration occurs. In holothurians, the regenerated RNC appears almost indistinct from the non-injured RNC. However, in other organisms where it has been studied, the regenerated cord is not fully equivalent to the normal one in spite of fibers crossing the lesion site. For example, in the regenerating chick embryo, fibers do cross the transection site and functional regeneration occurs, but the site of transection shows a slight disorganization of the gray matter [41]. Another example is the regenerating spinal cord of bullfrogs that have been transected as tadpoles, where the transection site is still visible 8 months after injury (and 6 after metamorphosis), showing a considerable constriction in size [15]. The differences can be striking, as occurs in the lizard where it has been shown that the regenerated spinal cord in the tail is considerably different from the uninjured cord and that it only contains descending fiber tracts from the old cord and scattered glial cells [45]. Other species show subtle differences, as in the case of regenerating 1 year-old goldfish, where the regenerated spinal cord appeared normal after 90 days post injury, but there were no motor horn cells in the regenerated area [46]. In H. glaberrima, the regenerated RNC has NURR 1 and GFS expressing neurons (albeit in lower numbers) and appears to be normal according to all anatomical parameters used. Nonetheless, more detailed experiments are needed to fully determine that all cell populations and fiber connections are present.

Finally, it is important to acknowledge that structural differences do exist in the regenerating nervous systems of animals. The echinoderm ganglionated nerve cord differs greatly from the regenerating nerves of some invertebrates where only fibers are found, and from the complex cellular and fiber organization of the vertebrate spinal cord. Thus, it remains unclear whether similar events occur during the regeneration of the different structures. Nonetheless, it will be by performing comparative studies, such as this one, that the commonalities and differences in nervous system regeneration among animals will be established.

Role of Cell division and apoptosis

Two related and at the same time, opposite processes are occuring within the RNC during regeneration; cell proliferation and cell death. The two show a spatial and temporal profile that seems perplexing because they seem to be occurring simultaneously; both show a peak in the nerve stumps at 6 dpi and a peak within the regenerating RNC at 12 dpi.


In vertebrates, apoptosis is associated with neurons that have been injured at the time of transection, but also with a period of secondary injury where the lesion size increases due to inflammation, immune-related events and hemorrhage [47-49]. Therefore, in our system, apoptosis within the nerve stumps seems to occur in cells whose fibers were damaged by the transection and that eventually failed to survive, undergoing apoptosis during the first week following injury. This was also observed at the TEM level in regenerating RNC of E. fraudatrix [23] and in regenerating fish CNS where apoptotic cells are seen for a long period of time following the lesion [50].

A different explanation might be involved in explaining the increase in apoptosis observed within the regenerating RNC in the second week following the transection. In this case the apoptosis event might be similar to what has been shown to occur during embryonic development in vertebrates, where about half of the neurons that are formed in the spinal cord undergo apoptosis [51]. Neuronal death in regenerating spinal cord has also been reported in the tail of the fish Sternachus [1,52]. In these fishes, younger segments of the regenerated spinal cord have a larger number of neurons than older segments, once again suggesting that the regenerating spinal cord recapitulates the known embryonic developmental process of overproducing neurons that will eventually be discarded.

Cell division

Cell division has been shown to occur in the regenerating arms of several echinoderms, mainly crinoids and starfish [20-22]. In crinoids, the nerve stump is also an active place of cell proliferation, although the dividing cells in the early stages appear to be non-neuronal [20]. In the starfish Asterias rubens, it was found that cell division in the regenerating nerve began 6–7 days following transection and increased during the next 2 weeks, gradually decreasing to control levels by day 60 post-injury [22]. Similarly, our results show cell division to occur within the ectoneural and hyponeural components of the nerve stump, but even more within the regenerating RNC. However, it is still unclear what type(s) of cells are proliferating since support cells, macrophages or some type of precursor cell could be accountable for the extent of cell division. This is a crucial issue that will be addressed below. Nonetheless, in recent studies Mashanov and colleagues [23] describe neurons and glial cells undergoing division in the regenerating RNC of E. fraudatrix.

The overall pattern of cell division within the regenerating RNC has some similarities to what has been shown to occur in the adjacent muscle and connective tissue, where cell division peaks between 6 and 12 days following injury [28]. These two weeks of extensive cell proliferation are undoubtly responsible for the finding that, at 30 dpi, animals have the same number of cells in the regenerated and in the uninjured RNCs.

Formation of new neurons has been shown to be an important process in animals where CNS regeneration is known to occur. In fish, BrdU labeling together with back-tracing techniques have shown that in the adult fish, neurons are regenerated from proliferating precursors following CNS injury [53]. In urodeles, new neurons are known to originate from proliferating ependymal cells [14]. Whether new neurons take part in the regeneration of the spinal tract observed in the chick embryo and the neonateal opposum is not known [54]. However, recent results suggest that stages where the ability to regenerate is still present, coincide with those stages where there is ongoing formation of new neurons [55]. On the other hand it is important to emphasize that some vertebrates can undergo functional regeneration with little or no contribution of neurogenesis, or that new neurons can originate from undifferentiated precursors.

In view of these findings, our results showing that neurons are present within the regenerated RNC and that some originate from dividing precursors provide for interesting comparisons to other systems. At the same time we must reconcile the finding that the densities of GFS and NURR1 neurons are lower in the regenerated RNC. One possible explanation is that the appearance of neurons in the regenerated RNC is a late event and that at 62 dpi some neuronal precursors have not fully differentiated. This would be consistent with experiments in starfish, where the first neurons observed in the regenerated segment following transection of the RNC were not observed until 28 days following injury [22]. These results might appear to contradict the observations by Mashanov and colleagues [23] in E. fraudatrix, who observed some neuronal division in the early stages of RNC regeneration, however, this might be explained when we take into account that early division was observed using TEM, while we used markers for differentiated neurons. Nonetheless, it is important to emphasize that in both E. fraudatrix and H. glaberrima neurons appear in the regenerated RNCs and that at least some of these neurons originate from dividing precursors. Similarly, in crinoid regenerating arms, some BrdU labeled neurons were recognized using electron microscopy, suggesting that in this echinoderm, neurons also originate from dividing precursors [21]. However, there are striking differences between neuronal populations since the GFS neurons were not observed to express BrdU, suggesting that they do not originate from dividing precursors. Once again this is consistent with studies in the transected starfish RNC where none of the neurons identified with the S1 neuropeptide (the starfish equivalent of GFS) showed BrdU labeling, even when enough time was allowed following the BrdU treatment for differentiaton to occur [22].

In order to fully identify the origin of neurons it will be important to determine the role of glia in holothurian RNC regeneration. Particularly in view that in E. fraudatrix Mashanov and colleagues propose that glial cells might be paving the path for neuron and fiber migration and even differentiating into neurons [23]. The role of the glial cells is not the only question that remains to be studied; several other questions remain to be answered. For example, does the observed anatomical regeneration provides for complete functional recovery? In many other model systems the extent of regeneration has been evidenced by backtracing neuronal connections and comparing the regenerated versus the uninjured connections. However, in view that little is known about the echinoderm radial nerve circuitry and nervous system physiology, it is not possible to determine at present to what extent there is morphological connectivity. Similarly, in other animals functional recovery can be documented by behavioral tests, that, at present, are not available for holothurians.

Comparison with other wound healing/regenerative processes

Transection of the nervous system, whether experimentally induced or by accidental cause rarely, if ever, exclusively affect the nervous system. Thus, it is always important to view the regeneration of the nervous component as a process that occurs parallel to the regeneration of surrounding tissues. Granted, there are nerve-specific processes, such as axonal regeneration and synaptic specificity, that only occur in the nervous system. However, it is interesting that events that occur in the surrounding muscle and connective tissue previously published by our group [28] parallel those occuring within the nerve cord itself. One of them has already been addressed above: the pattern of cell division, showing that in muscle, coelomic epithelium and connective tissue the proliferation pattern coincides with that observed in the regenerating RNC.

Another event that provides for an interesting comparison is the presence of morula or spheruloytes cells within the RNC. These cells are rarely observed in uninjured nerves, however, their numbers increase after injury. This increase in cell numbers parallels what occurs within the regenerating intestine [32] and following the transection injury in the adjacent tissues [28]. These cells have been proposed to play important roles in extracellular matrix remodeling, phagocytosis and antimicrobial activity, among others [32]. The presence of these cells within the regenerating nerve is quite intriguing and should be studied in further detail.

The extent of apoptosis observed after nerve transection can also be compared to what occurs during intestinal regeneration. We have recently completed a study of apoptosis during intestinal regeneration showing that high levels of apoptosis take place in the mesothelium 7 days following evisceration (unp. Observation). In both events, nerve regeneration and intestinal regeneration, apoptosis is rarely observed in the early days (2–5 days) following the injury or evisceration but occurs more likely during the second week (7–14 days). Moreover, apoptosis is more likely to take place within "new" tissues that have regenerated such as the newly formed intestinal blastema-like structure and the regenerated RNC, than in adjacent "old" tissues, such as the remaining nerve stumps or the intestinal mesentery.

For many investigators involved in studies of neurogenesis or neuronal regeneration, their focus has been strictly on the nervous system. However, our previous results show that the same events that occur during wound healing, also occur during intestinal regeneration [28]. And now we show that some of these events also occur during nervous system regeneration. Moreover, at the species level, the same species that can successfully regenerate their nervous systems are known to be exceptional regenerators of others tissues or organs, not just the nervous system. Thus, a major implication of our work is that the capacity to regenerate nervous system might be determined by the same processes and mechanisms in all tissues and that some animals have developed or retained these capabilities while in others they are absent. Although there are obvious exemptions to this statement, such as liver regeneration in vertebrates, the general rule remains that animals that have some capacity to regenerate their nervous system are, in general, good regenerators of many tissues or organs. If this is correct, then investigators interested in deciphering the mysteries of nervous system regeneration should also focus on regenerative or wound healing phenomena in other tissues and organs and on species where these regenerative processes are prominent.

Authors' contributions

JSMR carried out most of the experiments and helped in the analyses of the data and preparation of the manuscript. ARMS performed the birthdating experiments, analysed the data, and prepared the corresponding figures. JEGA conceived the study, participated in its design, data analyses and helped in the manuscript preparation. All authors read and approved the final manuscript.


The authors would like to thank Dr. V. Mashanov for helpful comments on the manuscript. Funded by NSF (IBN-0110692), NIH-MBRS (S06GM08102), NIH-1SC1GM084770-01), and the University of Puerto Rico. JSMR and ARMS are fellows of the NIH-MARC Program.

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