Further amputations of the tail in adult Triturus carnifex: contribution to the study on the nature of regenerated spinal cord

Abstract

Adult Urodele Amphibians, if deprived of the tail, are able to fully regenerate it. This occurs owing to a typical epimorphic phenomenon which takes place in various phases. Within this matter, in particular on the reconstruction of the caudal nervous component, literature sources refer to a great quantity of research following only one amputation of the tail. Being aware of these data we programmed to investigate the possible persistence, decrease or disappearance of the medullary regenerative power after repeated amputations of the regenerated tail. With this objective in view, we have performed on adult Triturus carnifex a series of such operations at time spaced out from one another. In previous experiments, the amputations of the tail have been before seven and then nine. In the current experiment, the same specimens have been subjected to further removals of the tail. This study has developed into three cycles going on over a period of more than ten years. Overall, our panorama rising from the integration of present results and those of former observations is in agreement with what occurs in the area which is the centre of the beginnings of medullary regeneration processes and the bibliographic information concerning the pre-blastematic and blastematic phases. In the subsequent morphogenetic and differentiative phases, however, with the recurrence of the re-establishment of the spinal cord, these events proceed more slowly (gap which reduces when the time interval starting from the operation increases) than in the spinal cords which regenerated after only one tail amputation. Furthermore, although the regenerated spinal cords, if compared to normal spinal cord, show some anomalies (regarding medullary length and diameter, distribution of the spinal nerves and ganglia), the regenerated spinal cords (as well-known uncapable to re-form the Mauthner fibres and supplied with the Rohon-Beard sensitive neurons), their nerves and ganglia reacquire the same complex structural organization as normal spinal cord (where, already known, the Rohon-Beard larval neurons lack, because they play the same role of the spinal ganglia in adult life and disappear when these ganglia first appear). Therefore, at least within numerical bounds of our tail amputations, the medullary regenerative potentialities would seem not to decrease. At the time of our starting investigations, being aware that the Authors ask questions to the morphogenesis of the regenerated spinal cord on which some aspects have not certainly been clarified, two antithetic hypotheses have been proposed. We raised the doubt that the entity of mitotic activity could alone be responsible for the quick reacquisition of a regenerated spinal cord which is superimposable to a normal one. Owing to meditation, we tended towards the hypothesis that this regeneration would be due to trans-differentiative process, which would trigger off in the tissues of the stump of the tail, induced by the impulse following the amputation. In order to obtain a complete picture of the proliferative possibilities responsible mainly, if not exclusively, for these phenomena which could support such our propension, we also programmed the current experiments on a parallel twofold approach. Therefore, we, as in past studies, have analyzed the proliferative activities in progress, through karyokineses and moreover we have attempted to unmask the possible presence of latent proliferative activities symbolized by the elements in the S phase of their vital cycle. To this end, an appropriate proliferative test has been chosen, the Proliferating Cell Nuclear Antigen (PCNA). Mitoses and signals of perspective proliferative activities, revealed by this immunocytochemical marker, are localizable in the ependyma and the periventricular grey. In the normal spinal cord there is an irrelevant karyokinetic activity coexisting with the expression of a PCNA considerably higher. Against these physiological proliferative paintings, in progress and potential, in the regenerating and regenerated spinal cords the numerical entity of the mitoses and of the cells revealing DNA synthesis has been found to be, if not negligible, modest or on the whole inadequate to sustain the regeneration events in progress and later possible ones after further amputations of the tail. Based on the evidence at present available, one could hypothesize that the impulse following the amputation of the normal tail would operate as a priority on the natural incomparable initial reserve of cyclic cells in the S phase, detected immunoreactively, which would be depositary of medullary proliferative silent potentialities, so that these cells, leaving the stand by condition in which they would be, would mobilize and passing through the M phase would set out for their differentiation. These undifferentiated cells would be, therefore, mainly responsible for the first medullary regenerative event. Such a scenario would give weight to those Authors that suggested these elements play a decisive role in the regenerative processes, Authors, that's so, have limited their observations to only one amputation of the tail. After this event, once the inizial considerable stock of undifferentiated cells has irreparably dropped, one could then suppose that the shock subsequent to each new amputation promotes in the stump of the amputated tail trans-differentiative processes which would become of primary weight for the following new medullary regenerations. This interpretation, therefore, prefigures that the shock would have a different primary target depending on whether it is connected to the first or to successive amputations of the tail. In the dispute regarding the genesis of the regenerated spinal cord in adult Urodele Amphibians, such a vision taking into consideration current data would make it possible, to a certain extent, to reconcile the two contrasting hypotheses previously advanced by Authors and put an end to the doubts expressed by us in the past at the time of previous our observations where in supporting the hypothesis regarding trans-differentiative activities, we have been hesitant in sustaining they were solely responsible for these events.