Quantitative estimation of HRP-labeled sensory and motor neurons during nerve-dependent and nerve-independent periods of urodele limb regeneration.

The relationship between urodele regeneration and possible regeneration in mammalian prospects is hard to evidence, but the idea of possible regeneration of neural elements in people is an area of potential clinical importance that is under investigation. One of the great challenges of the future is to understand enough about the basic biology of animal regeneration and to use it for the betterment of the mankind. It is well established that the initial stages of urodele limb regeneration depend on the presence of intact nerve fibres connected to their cell bodies. The nerve fibres severed at the limb amputation level, regrow and invade the blastema, providing blastema cells with indispensable factors. These factors are elaborated within the neuron perikarya and transported via their axons to the blastema. Numerous studies have been so far performed and have elucidated the quantitative relationships between nerve fibres and limb regeneration. However, there are no reports dealing with the individual nerve cells at work. The aim of the present investigation was to analyse the quantitative participation and qualitative distinction of nerve cells innervating regenerating parts of the urodele limb and their possible interrelationship with the nerve-dependent and nerve-independent periods of regeneration. The cells under study are housed in the dorsal ganglia (sensory neurons) and in the ventral aspect of the spinal cord grey matter (motor neurons). As a means of visualizing the direct implication of these neurons during various regeneration periods, the enzyme horseradish peroxidase was chosen. A total of 34 animals were used, 21 experimental and 13 controls, in order to study labeled nerve cell fluctuations. The results are summarized as follows: (a) The first nerve cells incorporating HRP within 5 days post amputation are found in the dorsal ganglia. Motor neurons in the grey matter are labeled within 7 days. (b) The number of labeled perikarya increases during the nerve-dependent regeneration period (0-21 dpa). The percentage of implicated sensory neurons exceeds that found in the control series. (c) During the next, nerve-independent period, the number of participating labeled neurons decreases gradually. Such fluctuations in the number of labeled neurons might represent the metabolic status of these cells in their effort to provide the blastema cells with the factors needed at the appropriate time. The current findings support previous observations that the periods of dependence and independence of urodele limb regeneration from the integrated control of brachial nerves reflect changes in the metabolism of individual sensory and motor neurons.

Volumen und Proteinsynthese der Kerne des Stratum Basale in der Stumpfepidermis während des Wundverschlusses nach Amputation der Vorderextremität bei Triturus vulgaris: (limb regeneration/wound closure/autoradiography/protein synthesis/automatic image analysis).

After injection of tritiated phenylalanine, microautoradiographs of longitudinal sections of the stumps of the forelegs of Triturus vulgairs 1 hour, 2, 3 and 9 hours after amputation and also of the opposite limb (reference) were prepared for quantitative measurements. The epidermal tissue (stratum basale) was examined with regard to proximo-distal changes of nuclear volume and protein synthesis. In the stratum basale of the stump the values for the parameters examined were similar to those of the reference limbs 1h and 2h after amputation, but 3h and 9h after amputation areas of increased nuclear volume were observed as well as an increased rate of protein synthesis per unit of nuclear volume. Protein synthesis in the epidermis of the stump was extremely low 1h after amputation. Protein synthesis in the reference epidermis was also reduced at this time, but then increased although the nuclear volume remained constant.

[Autoradiographic investigations on protein metabolism during limb regeneration in urodeles]. / Autoradiographische Untersuchungen über den Eiweißstoffwechsel bei der Extremitätenregeneration der Urodelen.

Protein synthesis during limb regeneration has been studied by investigating the incorporation of thioamino acids-S-35 and leucine-H-3 in young postmetamorphicTriturus vulgaris andTr. alpestris. Amputation causes in all stump tissues considerable alterations of the amino acid incorporation rates. In epidermis, musculature and nerve sheaths incorporation is nearly doubled during the first hours after amputation. The increase of incorporation in proximo-distal direction is gradual.The protein synthesis of the epidermal cells decreases during wound closure when the cells are losing the connection with the stump epidermis. The reduced amino acid incorporation occurs with cytological alterations comparable to the typical aging processes of normal epidermal cells. During blastema multiplication and differentiation the protein synthesis of the epidermis reaches a maximum at about three times the rate of the proximal epidermis. Near the end of the differentiation phase this high rate of synthesis is reduced to 1.5 times of that of the normal epidermis.In the stump musculature a maximum of synthesis at the beginning of sarcolysis is shifted proximally and corresponds with the loss of the striation. In sarcoplasmic fragments without nuclei the progression in lysis is correlated to the breakdown of protein synthesis. In contrast sarcoplasmic fragments with nuclei intensify their protein synthesis. At the begin of blastema formation and multiplication their rate of synthesis reaches 10-15 times the amount of that of uninfluenced musculature. After amputation the incorporation rate of the sheath cells in the distal part of the nerves is doubled during the first days and increases furtheron. It reaches five times the rate of the proximal sheath cells.During blastema formation and multiplication the amino acid incorporation rate of the mesodermal blastema cells migrating distally increases steadily. Their highest incorporation rate has been found immediately before redifferentiation takes place. It is more than 30 times the rate of uninfluenced muscles in the proximal part of the stump and three times the rate of the proximal epidermis. The periosteum of the bone stump seems to react later than epidermis, muscles and nerves. Normally no extended lysis takes place. During the blastema formation stage the amino acid incorporation of the periosteum cells increases to five times the rate of that of the uninfluenced periosteum cells. At the begin of the redifferentiation of the regenerate it corresponds nearly to the rate of the mesodermal blastema cells. Together with the remaining fibrocytes in the stump they represent the blastematas to restitute the humerus and the connective tissue derivatives in the stylopodium. As soon as the specific determination of mesodermal blastema cells becomes visible protein synthesis is reduced. During histological differentiation synthesis increases again. The incorporation rates during histogenesis of the different tissues in the zeugopodium and autopodium are egual to the rates in corresponding tissues of the restituting stylopodium. The area of the presumptive dermis in the limb regenerate is characterized by cells with less protein synthesis than that of the mesodermal blastema cells.The investigations demonstrate that in each regeneration state protein synthesis in the different tissues and cell types occur with characteristic maxima, which often overlap eachother.