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Journal of Physiology (2001), 535.1, pp. 241-248
© Copyright 2001 The Physiological Society
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ABSTRACT |
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INTRODUCTION |
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The only adult vertebrates that are able to regenerate their limbs after amputation are the urodele amphibians, as first reported in 1768 by Spallanzani. The limb regeneration process is composed of three main stages: dedifferentiation, proliferation and differentiation.
Several studies have pointed out the importance of neural signals in promoting cell proliferation during limb regeneration in the newt. Indeed, the mitotic activity of blastemal cells occurring during the proliferation stage is reduced following denervation of the limb (Singer, 1952, 1978). Various mitotic factors associated with the nervous tissue such as fibroblast growth factors have been proposed to be involved in the trophic effects of nerves. Motor and sensory neurons express fibroblast growth factor-1 (FGF-1) (Elde et al. 1991) and recently Zenjari et al. (1997) have shown that nerve-blastema interactions induce FGF-1 release during limb regeneration in Pleurodeles waltlii. Peripheral nerves further express non-regulatory trophic factors required to sustain the cell cycle, such as insulin-like growth factors I and II (Hansson, 1993) and transferrin (Kiffmeyer et al. 1991), which are good candidates for mediating the neural effects on blastema growth.
However, the role of the nerve supply in limb regeneration has been revealed by suppressing the neural activity in the amputated limb (see Wallace, 1981 for review) and to date nothing is known about the effects of an increase in nerve activity on the regeneration process.
Locomotor training provides a physiological way of increasing both the sensory and the motor neural activities in the limb. Therefore, in the present study we have investigated the effects of locomotor training on muscle hindlimb regeneration in a urodele amphibian, Pleurodeles waltlii. The first proximal part of the hindlimb of the animals was amputated, the animals were trained daily in overground stepping for 8 months, and the effects of training on limb regeneration were evaluated with a combination of morphological and electrophysiological techniques. Our results reveal disrupting effects of locomotor training on limb regeneration.
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METHODS |
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Experiments were performed on 21 adult fully metamorphosed Pleurodeles waltlii taken from the breeding colony in our laboratory. Four animals possessed a dorsal supernumerary forelimb that had been previously grafted onto the back under full anaesthesia (see below). All animals were maintained individually in tanks containing tap water at 18 °C and fed with Chironomus larvae three times a week. Surgical procedures, pre- and postoperative care, and handling and housing of the animals were in accordance with protocols approved by the European Communities Council and the INSERM Animal Care Committee, and conformed to NIH guidelines.
Experimental protocol
The normal newts (n = 17) were deeply anaesthetized by immersion in a 0.1 % aqueous solution of tricaine methanesulfonate (MS 222; Sigma) and, under aseptic conditions, one hindlimb was amputated at mid-femur. Thereafter, the 17 amputee animals were initially divided into three distinct groups. In the first group (control animals; n = 7), the newts were transferred back to their tank. These animals displayed aquatic locomotor behaviours, which are characterized by limb movements of small amplitude (paddling) or no rhythmic movements at all (swimming) (see Delvolvé et al. 1997). In the second group (trained animals; n = 7) the animals were trained to walk on a wet surface 2 days after limb amputation. Training sessions were performed twice daily with an interval of 5 min resting time between each session, 5 days a week for 8 months. During each training session the animals were forced to maintain locomotion for 30 min by touching or gently squeezing the tail. At the beginning of each session, walking was faster (range: 0.04-0.053 m s-1) than at its termination (range: 0.02-0.028 m s-1). In the third group (constrained animals; n = 3), the animals were removed from their tank and placed in small boxes in which they could not locomote, and the boxes were gently rocked for 30 min. This protocol was performed twice daily over a period of 4 months. The animals did not seem to be in distress as judged from the absence of mucus secretion, escape response or aggressive behaviour.
In trained animals, the regenerated hindlimb was re-amputated under full anaesthesia (0.1 % MS 222) at mid-femur 8 months after the original amputation. The re-amputee animals were then separated into two groups: locomotor training was stopped for the animals of the first group (n = 3) while it was maintained for those of the second group (n = 2).
In the four animals with a supernumerary forelimb, both one hindlimb and the supernumerary forelimb were amputated (mid-femur and mid-humerus, respectively) following the same surgical procedure as described above for normal animals. After amputation, animals were either trained (trained supernumerary animals; n = 2) or not trained (control supernumerary animals; n = 2) to terrestrial stepping according to the procedure described above.
At the end of each experiment, animals were killed with an overdose of MS 222.
Morphology
In each group, the limb regeneration process was staged as described in Tank et al. (1976). When regenerates stopped growing and were overlapped by pigmentated skin they were removed, washed in PBS1X (phosphate-buffered saline: 130 mM NaCl, 7 mM Na2PO4, 3 mM NaH2PO4, pH 7.4) and incubated in a rapid bone decalcifier (Eurobio). Fixation was carried out in 4 % paraformaldehyde- PBS1X. Tissues were dehydrated in alcohol and immersed in toluol. Inclusions were carried out in paraffin and sectioned by use of a microtome (section thickness: 7 µm). For histological observations, sections were stained using haematoxylin and eosin/orange G.
Gel electrophoresis
The animals were anaesthetized in a 0.1 % aqueous solution of MS 222 and samples of thigh muscles were removed and quickly frozen to be subsequently stored in liquid nitrogen. In supernumerary forelimbs the muscle samples were dissected out from the arm. After muscle sampling the anaesthesia was deepened to kill the animal.
The myosin extraction procedure and the electrophoretic analysis of native myosin were performed as described previously (Chanoine et al. 1987). Briefly, the frozen muscles were cut into small pieces, washed 4 times in iced (2 °C) buffer (600 mM KCl, 40 mM NaHCO3, 10 mM Na2CO3, 1 mM MgCl2, 10 mM Na4PO7, pH 8.8) and myosin was then extracted with four volumes of iced buffer. After 90 min of gentle shaking, the tissue homogenate was centrifuged at 10 000 g and the supernatant containing myosin was diluted twice with glycerol for the preservation and study of native myosin. For the electrophoretic analysis, the running buffer was 20 mM Na4P2O7 (pH 8.5), 10 % glycerol, 0.01 % 2-mercaptoethanol, 2 mM MgCl2 and 2 mM ATP. Cylindrical gels (6 cm 
0.5 cm) contained 4 % polyacrylamide, and about 1 mg of myosin per band was loaded. Electrophoresis was carried out at 80 V for 17.5 h at -3 °C. Gels were stained with Coomassie blue R-250.
Electrophysiology
The methods used for recording the electromyographic (EMG) activities of limb muscles during stepping were described in detail elsewhere (Delvolvé et al. 1997). Briefly, under general anaesthesia (MS 222, 1 g l-1), pairs of fine Teflon-insulated stainless steel wires (70 µm in diameter) were inserted into the puboischiofemoralis internus of the intact hindlimb and the proximal part of the regenerate of the amputated hindlimb. In supernumerary animals, the biceps brachii of the supernumerary forelimb and that of either the ipsilateral or the contralateral normal forelimbs were implanted. The electrode wires were gathered together into a common cable (length around 1 m) which was sutured to the skin on the mid-dorsum. Sufficient slack was provided along implantation sites and points of attachment on the back so that the newt could move in an unimpeded fashion. After electrode implantation, the animals were placed in a tank filled with water and allowed to recover from anaesthesia for 1 h. After recovery, the newts were induced to walk on a wet, stainless steel surface by gently squeezing the tail. In some experiments (n = 6), the reflex responsiveness of the regenerated hindlimb to electrical stimulation (pulses: 1 Hz, 0.5 ms duration, < 5 mA) was tested via the inserted wires.
The voltage signals from electrodes were differentially amplified, displayed on an oscilloscope and stored with a magnetic tape recorder (Biologic DAT). The EMGs during episodes of steady locomotion were sampled (1 kHz per channel) with the use of an A/D converter (Cambridge Electronic Design 1401). The digital EMGs were full-wave rectified and smoothed by a software filter (< 100 Hz).
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RESULTS |
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Main features of the regenerated hindlimb in control and in locomotor trained animals
Morphological data
Control animals. In control animals, wound healing was completed within 8-9 days and the blastema formed and grew to achieve the late cone stage in 58-61 days. The palette stage was observed 74-76 days after hindlimb amputation and it took 55 more days to regenerate a complete hindlimb including all the five digits of the pre-amputated animals (Table 1). Our histological study further showed the reformation of all bony and muscular structures (Fig. 1A-C).
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Figure 1. Longitudinal sections of regenerates in control (A-C) and in trained animals with (D and E) and without (F and G) digits Sections were stained with eosin and haematoxylin. f, femur; fi, fibula; m, metatarsis; p, phalanges; t, tibia; ta, tarsis. | ||
Locomotor trained animals. In locomotor trained animals, wound healing needed 20-22 days to be completed and the late cone stage was achieved 176-184 days after limb amputation (Table 1). The cones were thicker and bigger than those in control animals at the same stage. During the following 60 days, the cones kept growing but without achieving the palette stage in 4 out of 5 animals. During this growth, a few digits could appear. After 234-246 days, the regenerates stopped growing and were covered with an adult pigmented skin. Trained animals exhibited a regenerated limb with none to four digits (Fig. 1D and F) while every control animal regenerated five digits (Fig. 1A). Therefore, it could be concluded that limb regeneration was both delayed and not fully achieved in locomotor trained animals.
Histological data further showed that in trained animals the femur was abnormally regenerated and remained relatively short, 4.9 % of the snout-vent length (SVL; measured from the tip of the snout to the anterior angle of the vent) vs. 8.0 % of the SVL in control animals. In contrast, the tibia and fibula regenerated totally (Fig. 1E and G). Tarsi were not correctly organized and could be absent in some trained animals. Metatarsi and phalanges were found in every regenerated digit. Muscle structures regenerated, except in the digits and interdigitations. Conjunctive tissue was thick and well developed under the epithelium and many gland structures were visible at the periphery of the proximal region (Fig. 1D-G).
Electrophysiological data
Regenerated muscles displayed rhythmical EMG bursts during stepping in both control animals and locomotor trained animals (Fig. 2A). Moreover, the EMG locomotor pattern of the regenerate limbs was well coordinated with that of the opposite hindlimb (inter-limb coordination). In regenerates, we could not identify muscles with confidence so we did not proceed further in the analysis of the EMG locomotor patterns of regenerates. On the other hand, the EMG bursts always had a smaller amplitude in regenerated than in intact hindlimbs. This is likely to have resulted from a smaller number of muscle fibres in regenerated limbs. Electrical stimulation of the regenerated hindlimb via the inserted wires induced in both control and locomotor trained animals a withdrawal of the stimulated limb. This could be associated with movements of the entire animal. Altogether the electrophysiological data showed that motor and sensory innervations were functional in regenerated hindlimbs.
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Figure 2. EMG pattern and electrophoretic pattern of myosin in normal and supernumerary forelimbs A, EMG pattern of regenerates during overground stepping in a control animal (left panel) and in a locomotor trained animal (right panel). For each channel, EMG activity was full-wave rectified and filtered. Calibrations apply to each channel. i, ipsilateral; co, contralateral; regen, regenerate; pifi, puboischiofemoralis internus. B, EMG pattern in a supernumerary forelimb during overground stepping. Conventions as in A. su, supernumerary; bic, biceps brachii. Data are from the same individual. C, electrophoretic pattern of myosin isoforms contained in muscles of contralateral forelimb (left) and supernumerary forelimb (right). Arrowheads: slow myosin isoform; thick line: intermediate myosin isoform; thin lines: fast myosin isoforms. | ||
Role of locomotor training in hindlimb regeneration
Stopping the locomotor training after 120 days did not induce further morphological changes in the regenerated hindlimb. However, in two animals, training was stopped after 30 days and, about 4 weeks later, both animals started a regeneration process with a normal timing and a complete regeneration of the limb was achieved. This suggested that the disrupting influences of locomotor training on limb regeneration could be suppressed within a short time window after limb amputation.
In order to further investigate the regenerative capacity of abnormally regenerated hindlimbs, the regenerated hindlimbs of the trained animals were re-amputated. Animals were then separated into two groups. Locomotor training was stopped for the animals of the first group, while it was maintained for those of the second group. We observed that animals of the first group completed a normal regeneration process in 3 months while hindlimb regeneration was absent in animals of the second group. Indeed, 10 months after amputation they exhibited wound healing only. These observations confirmed that locomotor training inhibited limb regeneration and that the abnormally regenerated limbs still had the capacity to regenerate normally.
In order to evaluate the influences of stress due to handling and change in locomotor medium (aquatic vs. terrestrial) on limb regeneration, some animals (constrained animals) were removed from their aquarium and placed in small boxes in which they could not locomote. Moreover, the boxes were continuously and gently rocked (two 30 min daily sessions for 4 months) in order to increase friction of the wound epithelium on the ground, which could be involved in limb regeneration (Mescher, 1976; Holder & Reynolds, 1984). Wound healing was observed 8-9 days after amputation and, thereafter, the regeneration process occurred as in controls. The hindlimb was fully regenerated with distinct digits overlaid by a pigmentated skin 118-123 days following amputation (Table 1).
In an attempt to evaluate the specificity of locomotor training, animals with a supernumerary forelimb grafted on their back were subjected to the same experimental procedure. It is worth noting that the supernumerary forelimb did not touch the ground, and hence it was not directly involved in stepping.
In a first step, we investigated whether the supernumerary forelimb muscles were rhythmically activated during stepping. Figure 2B shows, as an example, that the EMG pattern of the biceps brachii of the supernumerary forelimb during stepping consisted of bursts of activity in phase with those displayed by the biceps muscle of the ipsilateral forelimb and alternated with those displayed by the biceps muscle of the contralateral forelimb. Moreover, low intensity electrical stimulation of the supernumerary limb through electrodes inserted into the skin induced a withdrawal of the stimulated limb and/or movements of the animal. On the other hand, electrophoresis showed that the composition of myosin isoforms in muscles of the supernumerary limbs was the same as in muscles of normal forelimbs (Fig. 2C). Altogether these data strongly suggest that the motor control, the sensory innervation and the muscle fibre composition are similar in the supernumerary forelimb and in normal forelimbs.
Non-trained supernumerary animals could regenerate both their hindlimb and supernumerary forelimb (Fig. 3A and C) while the locomotor trained ones regenerated their supernumerary forelimb only (Fig. 3B and D). Hindlimb regeneration was inhibited at early stages with small and thick regeneration cones overlaid with a pigmented skin. These cones did not grow any more. These results suggest that the disrupting effects of locomotor training are localized to the hindlimb directly involved in stepping.
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Figure 3. Regeneration in supernumerary animal Regeneration of the supernumerary forelimb (arrowed) in a non-trained (A) and in a trained (B) supernumerary animal. Regeneration of the right hindlimb in a non-trained (C) and in a trained (D) supernumerary animal. | ||
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The results from the present study indicate that locomotor training alters the limb regeneration process in the adult newt. However, limb regeneration can proceed normally after stopping locomotor training either within a short time window (around 1 month) after limb amputation or after re-amputation of an abnormally regenerated limb. This suggests that locomotor training affects transiently the expression of one or several factors which play a key role in limb regeneration.
There are many experimental procedures that led to the inhibition or the disturbance of limb regeneration, such as denervation of adult limb (Singer, 1974); amputation zone closure with thick layer skin (Mescher, 1976); preventing wound healing (Goss & Holt, 1992); hypophysectomy of adult animals (Hall & Schotté, 1951); surgical appending of a piece of supernumerary limb (Bryant, 1976); and reduction of the current of injury leaving the amputation area (Borgens et al. 1979; Jenkins et al. 1996). A new protocol can now be added to this list, forced terrestrial stepping, which considerably disturbs limb regeneration.
Previous studies have shown that the limb regeneration in the newt is controlled by the nerve supply of that limb (Singer, 1974). Indeed, limb denervation performed before or early after amputation can induce a decrease of the mitotic index of blastemal cells, leading to inhibition of the regeneration process. However, when the blastema has been formed and the differentiation stage has begun, denervation does not disturb regeneration (see Mescher, 1996), although the regenerated limb is smaller than the original one (Brockes, 1984). These results point out the importance of a critical number of blastemal cells required for the initiation of differentiation and morphogenesis (Brockes, 1984). Moreover, blastemal cells derive from fibroblasts (connective tissue of dermis, muscles and nerves), muscle cells, bone cells and Schwann cells (Hay & Fischman, 1961). Brockes (1987) identified, using the 22/18 monoclonal antibody, a subpopulation of blastemal cells that are highly nerve dependent. The fact that in our experimental conditions the medial structures of the limb (e.g. tibia and fibula) completely regenerated while the proximal and distal ones did not supports the view of a positional identity of blastemal cells. Furthermore, one can assume that only a subpopulation of blastemal cells have been altered during locomotor training.
In our experimental conditions the motor and sensory nerve supplies were present and functional in the abnormally regenerated limbs. Therefore, the inhibitory influences of locomotor training on limb regeneration do not result from the absence and/or dysfunction of the nerve supply in the regenerate. The sensory and motor activities in the regenerate were instead increased by locomotor training. This increase in neural activity induced an important delay of the early stages of limb regeneration which are nerve dependent (Fekete & Brockes, 1988); the subsequent nerve-independent stages were disturbed and achieved an abnormal regenerated limb.
The promoting effects of nerves on limb regeneration have been attributed to the action of growth factors released from the peripheral axons (Wallace, 1981; Carlone & Mescher, 1985) and acting in synergy with the signals coming from the healing epidermis (Globus et al. 1980). To date, the involvement of glial growth factors (Brockes & Kitner, 1986), insulin-like growth factors I and II (Hansson, 1993), transferrin (Kiffmeyer et al. 1991) and fibroblast growth factors (FGF)-1 and -2 (Elde et al. 1991; Zenjari et al. 1997) have been investigated. The role of both FGF-1 and FGF-2 has been directly correlated to the different stages of the regeneration process. FGF-2 is expressed in nerve and in apical epithelium at early stages. It allows the expression of homeobox gene dlx3 in apical epithelium (Mullen et al. 1996). Nerves secrete FGF-1 and/or FGF-2, which help the proliferation of blastemal cells expressing FGF receptors and induce both the release of other FGFs (1, 2, 4 and 8) and the expression of dlx3 by epithelium cells with FGF receptor 2. Moreover, FGF receptor 2 has been found in cartilaginous cells, and hence FGF could also play a role in cartilage differentiation (Poulin et al. 1993). It would be interesting to investigate the effects of nerve activity on the regulatory mechanisms of these trophic factors.
In our experimental conditions, the wound epidermis could be altered because of the rhythmic contact of the wound on the table during locomotor training. This could inhibit limb regeneration (Goss & Holt, 1992). However, we observed that such injury did not disturb the overlapping of the amputation area and the formation of the cone of regeneration. Moreover, both the regeneration of the supernumerary limb submitted to friction with the rhythmically activated forelimb localized underneath and the normal regeneration in the control animals suggested that the friction of the wound epidermis played a minor role in the inhibition of regeneration evidenced in the present study. This is consistent with previous data showing that the alteration of the peripheral epithelium 1 week before amputation does not disturb the limb regeneration process (Holder & Reynolds, 1984).
Limb amputation in urodeles is associated with a natural electrical current flowing through the wounded skin ('injury current') because of the leakage of ions by the injured tissues (Borgens, 1977). It has been proposed (Jenkins et al. 1996) that the injury current leaving the amputation zone could play a role in establishing the pattern of expression of hox family genes, which are crucial for limb development and limb regeneration (Duboule, 1992; Muneoka & Sassoon, 1992; Johnson et al. 1994). Indeed, it has been shown that the reduction of the injury current can either inhibit limb regeneration or induce an abnormal regeneration process which took place with an abnormal skeletal pattern similar to that observed in the present study (Borgens et al. 1979; Jenkins et al. 1996). In our experiments, the increased level of neural activity during locomotor training might induce ion flows that might cross-talk with the natural current of injury in a similar way to the application of exogenous currents.
In summary, our present results show a nerve activity-dependent inhibition of limb regeneration in the urodele Pleurodeles waltlii. Further experiments at cellular and molecular levels are needed to identify the signals and their targets which mediate this control.
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REFERENCES |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Corresponding author
C. Chanoine: Laboratoire de Biologie du Développement et de la Différenciation Musculaire, Centre Universitaire des Saints Pères, 45 rue des Saints Pères, 75006 Paris, France.
Email: christophe.chanoine{at}biomedicale.univ-paris5.fr
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