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21 Laboratoire de Recherche sur les Biomatériaux et les Biotechnologies, Université du Littoral-Côte d'Opale, Bassin Napoléon, BP 120, 62327 Boulogne sur Mer, France2 Université du Littoral-Côte d'Opale, 220 rue F Buisson, BP699, 62228 Calais, France
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Abstract |
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(Received 5 January 2004;
accepted after revision 18 March 2004; first published online 1 April 2004)
Corresponding author D. Leterme: Laboratoire de Recherche sur les Biomatériaux et les Biotechnologies, Université du Littoral-Côte d'Opale, Bassin Napoléon, BP 120, 62327 Boulogne sur Mer, France. E-mail: damien.leterme{at}opale.univ-littoral.fr
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Introduction |
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When a nerve is crushed, axotomized motoneurones re-innervate their muscle because the regenerating axons follow their original pathway back to their muscle fibres (Kugelberg et al. 1970). This experimental technique preserves the continuity of nerve sheaths and the normal alignment of basement membranes in contrast to other surgical procedures such as nerve transection which may promote the growth of regenerating axons into inappropriate pathways (Sunderland, 1978; Brushart, 1988, 1993; Grubb et al. 1991; Kline & Hudson, 1995; Doolabh et al. 1996; Brushart et al. 1998; Rafuse & Gordon, 1998; Zhou et al. 2002). In these latter models, motor axonal regeneration and muscle re-innervation were studied using histological labelling techniques, histochemistry or electrophysiological recordings for assessment of neurotransmitter release (Carmignoto et al. 1983; Tonge, 1974). However, one important feature of regeneration and re-innervation which was often neglected is the time course of the functional recovery process. The question was recently addressed in the rat femoral model in which axons destined for skin and muscle intermingle within the nerve trunk. Re-innervation of the distal stump was observed to occur gradually (Brushart, 1993; Al-Majed et al. 2000; Brushart et al. 2002). Histological techniques of radioisotope labelling of transported proteins and of anterograde labelling of regenerating axons revealed an asynchrony of axon regeneration which was termed ‘staggered regeneration’ (Brushart et al. 2002). However, the electrophysiological counterpart of this phenomenon of progressive regeneration was not assessed.
The aim of the current experiments was to examine the axonal regeneration and the re-establishment of functional neuromuscular connections of the soleus muscle in the simple model of nerve crush. We use electrophysiological measures of the whole contractile muscle properties and of single motor units (MUs) to establish the time course of the restoration after the nerve was crushed. We find that recovery after nerve crush is characterized by a process of sequential functional re-innervation.
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Methods |
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The investigation was carried out in 39 adult male Wistar rats weighing 90–140 g and aged 5–6 weeks at the time of the initial operation. All experiments conformed to our Institutional and French guidelines on the ethical use of animals and all efforts were made to minimize both the number and suffering of the animals used in these experiments (Licence no. 62–6, Agriculture and Education Ministries). In 35 rats, surgery was performed prior to the final acute experiments. The rats were deeply anaesthetized by intraperitoneal injection of 35 mg kg–1 sodium pentobarbital and supplementary doses (about 5 mg kg–1) were given when necessary. Using sterile procedures, the right soleus muscle was exposed and its nerve gently isolated from the surrounding tissues. It was then compressed for 10 s with fine forceps at about 5 mm from its muscular insertion, so that 1 mm length of the nerve was crushed (Bridge et al. 1994). Even though axon continuity was interrupted in that space, the structural tissue was not damaged. After the operation, the skin incisions were closed with silk suture. The rats were returned to individual cages, supplied with food and water ad libitum and maintained in a temperature-controlled room (22 °C) with a 12–12 h light–dark cycle. Some preliminary crush tests were performed immediately after the operation by electrically stimulating the sciatic nerve to verify that muscular action potential and muscle contraction had been suppressed by the nerve crush. These tests were positive as no contraction was observed in the soleus muscle.
Final acute experiments were performed under anaesthesia (35 mg kg–1 sodium pentobarbital, I.P.) on 24 rats divided into four groups of six rats to test the effects of nerve crush on the contractile properties of the whole muscle at 7, 14, 28 and 56 days after surgery. The remaining 11 rats were used to measure single MU properties at 7 (n= 6) and 14 days (n= 5) after initial surgery. Four rats received no surgery (control) and were used as control for MU properties. No difference in muscle tension was found between control rats and the control hindlimb in the animals receiving surgery (experimental).
Whole muscle contractile properties
The properties of the re-innervated soleus muscles were compared to those of the contralateral intact control soleus muscle. The right and left soleus muscles were exposed and isolated from surrounding tissues to avoid damage to blood supply and the nerve trunk. The tested hindlimbs were fixed rigidly. The body temperature and the exposed portions of the limbs were regulated at 37 ± 0.5 °C with radiant heat and pools of mineral oil. The distal tendon of the soleus muscle was cut while other limb muscles were denervated by cutting their nerve supply. The distal tendon was connected via a low-compliance link to an isometric force transducer (Grass FT 03, Quincy, MA, USA). Contractions of the soleus muscle were evoked by electrical stimulation (square pulses, 0.1 ms duration) of the sciatic nerve via platinum electrodes, displayed on a thermal arraycorder (WR 9000, Graphtec, Yokohama, Japan) and on a numerical oscilloscope (Gould 630, Ballainvilliers, France) and stored on a digital tape recorder (DTR 1404, Bio Logic, Claix, France). The muscle length was adjusted to obtain the maximal twitch contraction. The nerve was stimulated every second by single pulses. The stimulus intensity was gradually increased from zero to obtain stepwise increments in twitch tension due to the successive recruitment of individual MUs until no further increment was recorded. The increments were counted and taken as an indicator of the number of MUs. This counting procedure was repeated three times and the highest number of MUs was recorded. The stimulus intensity was fixed for eliciting the maximal twitch. Twelve successive twitches were automatically averaged (0.5 Hz stimulation) to measure the maximal twitch tension (Pt), the time to peak (TTP) and the half-relaxation time (RT
). Tetanic contractions were elicited by 750 ms trains at 20, 40, 80, 120 and 200 Hz with a 2 min delay between each train and the maximal tetanic tension (P0) was measured. The soleus is a slow twitch muscle containing largely slow fibre types (80–90%) which achieve maximal force production at low rate of stimulation (30 Hz) (Close, 1967; Kugelberg et al. 1970; Kugelberg, 1973, 1976; Pullen, 1977). The high stimulation rates used here were tested to verify that the nerve injury was not followed by unexpected radical changes in MU types. The stability of the tetanic contraction was quantified by calculating the vulnerability index (VI) which was the ratio of the force developed at the 700th ms of the contraction time to P0. After crush, the re-innervated muscle could eventually show a decrease in tension during the 120 and 200 Hz stimulation. To check that the tetanic fade was not due to damage of the preparation, the same muscle was re-exposed to an 80 Hz train. The muscle developed the same force of contraction as that induced by the first 80 Hz train, indicating no deterioration. Then, a direct stimulation was applied through two silver plates flanking the soleus muscle on each side and the stimulus intensity was increased to produce the maximal twitch. Trains at 80, 120 and 200 Hz were applied to measure P0 and VI under these conditions. Finally, the fatigue resistance was evaluated by applying a 40 Hz, 330 ms train to the sciatic nerve every second for 2 min. The fatigue index (FI) was calculated as the ratio of the tension developed by the 120th contraction to the maximal tension recorded during the test (Burke et al. 1973). The soleus muscle was removed and weighed. The rats were killed by an overdose of anaesthetic at the end of the experiments.
Motor unit contractile properties
The lumbar spinal cord was exposed by laminectomy. The right ventral roots L4 and L5 were cut at the entry of the spinal cord on the crushed side. The vertebral column was rigidly secured. The spinal cord was covered by mineral oil maintained at 36.8–37.2 °C. Two Teflon-coated steel wire electrodes (Cooner wires, Chatsworth, CA, USA), without insulation at the tip, were gently inserted into the soleus muscle for recording the electrical activity of the single active MU. A reference electrode was inserted into the denervated tibialis anterior muscle. The MUs were isolated by fine dissection and splitting of L4 and L5 ventral root filaments. Recordings were performed with the same equipment described for the whole muscle recordings. The criteria for single MU activity were all-or-none mechanical twitch and all-or-none action potentials from EMG recordings. This was tested by gradual small increases and decreases of the stimulus intensity around the excitation threshold. This protocol was repeated at least three times at intervals of about 5 s.
Statistical analysis
The normality of each parameter and equal variance between groups were determined. A one-way ANOVA detected statistical differences between means for the whole muscle parameters. When the equal variance test failed, a Kruskal–Wallis analysis of variance on ranks was applied. The one-way ANOVA was followed by a pairwise comparison test (Tukey's test) for the MU properties. In some cases, the Kruskal–Wallis test was followed by a Dunn's test. Relationships between variables were analysed by linear regression and Spearman rank correlation. Differences were considered to be significant at P < 0.05.
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The whole muscle contractile properties
A typical example of recordings of twitch and tetanic contraction from a soleus muscle 7 days after crush is illustrated in Fig. 1. The twitch of the muscle was clearly reduced (Fig. 1A and F). Tetanic contractions were elicited by 500 ms trains at 20, 120 and 200 Hz and recorded from the control and the axotomized muscle (Fig. 1G–J). In the control soleus muscle, the tension remained stable at the three frequencies (Fig. 1B–D). In contrast, the tetanic contractions of the experimental muscle at 120 and 200 Hz reached a maximum amplitude during the first 200 ms stimulation and decreased in amplitude during the last 100 ms (Fig. 1H–I).
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The values of TTP and RT of the muscle 7 days after re-innervation were statistically higher than the control values (31% and 65%, respectively). They decreased progressively to reach the control value after 28 days. At day 56, the RT value was slightly but significantly higher than the control (14%). The fatigue test showed that the control soleus was, as expected, resistant to fatigue, FI ranged from 81 to 99%. Seven days after crush, FI of the experimental muscle was 11% lower than the control value and soon recovered after the 14th day.
MU contractile properties
Typical examples of EMG and tetanic contractions recorded from different MUs found in the control muscle and in the muscle at day 7 after crush are shown in Fig. 3. In the control muscle, the amplitude of the action potentials remained stable during the 750 ms stimulation. The tetanic contraction was also stable (100%). Seven days after crush, the EMG showed a decrease in amplitude of the action potentials during the first 300 ms of the tetanic stimulation. This was the typical EMG behaviour whatever the frequency of the stimulation. It was first observed at 40 Hz in 48% of the recorded MUs, for 42% at 80 Hz and at 120 Hz in the remaining 10% of MUs. During 200 Hz stimulation, four typical shapes of the MUs were observed allowing us to group the MUs into four classes (Fig. 3, Class A–D). For these MUs, an initial peak in amplitude was reached during the first 45–130 ms of stimulation. Following the peak, the twitch tension diminished gradually in amplitude for 67% of the MUs, 7% of which ended the contraction before cessation of the stimulation (Fig. 3, Classes A and B). In the remaining 33%, the first peak in amplitude was followed by a sharp decrease and an unexpected second peak was observed (Fig. 3, Classes C and D). The disruption of the tetanic contraction followed by a rebound response was systematically recorded in 33% of the cases.
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Discussion |
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We used electrophysiological recordings to quantify the rate of re-innervation both of the whole muscle and of single MUs. We found that the crushed axotomized motoneurones that re-innervate their original muscle do not reach their target simultaneously. During the first weeks after crush, a progressive axonal re-innervation characterizes the process of restoring effective neuromuscular transmission.
Other studies of nerve repair were based on different models. For example, studies of the change in nerve fibre numbers distal to a nerve repair in the sciatic nerve have also shown a progressive increase in nerve fibre counts. This result was obtained on the basis of morphometric histological evaluation and stressed the importance of timing in experimental studies (Mackinnon et al. 1991). In a nerve–nerve resuture model, the timing of muscular restoration was studied in the cat (Gordon & Stein, 1982). The success of re-innervation was also investigated in terms of numbers of re-innervated MUs and recovery of MU force in the cat medial gastrocnemius muscle but the question of the time course of the muscular restoration was not addressed (Rafuse & Gordon, 1998). Recently, the rat femoral nerve model after nerve transection was used to study the rate of axonal re-innervation of distal nerve stumps and the possibility of accelerating it by electrical stimulation (Al-Majed et al. 2000; Brushart et al. 2002). The regeneration speed was described by radioisotope labelling of transport proteins and by anterograde labelling of regenerating axons. An asynchrony of axon regeneration was observed in the non-stimulated group between 1 and 2 weeks with a progressive increase in the number of crossings. A process of staggered regeneration was thus demonstrated by these morphological findings (Brushart et al. 2002).
A similar process of regeneration is uncovered by our experimental approach providing a functional picture of the staggered regeneration. The interpretation of this event can involve many stages in the degenerative process and the restoration of the MUs which cannot all be assessed simultaneously. Our recordings of muscle tension give a description of the successive steps taken by the system to restore its normal function.
Given the site of nerve crush, the re-innervation is likely to occur by the same set of motoneurones that previously innervated the soleus muscle. The short distance between the injury site and the muscle to be re-innervated orientates the interpretation of our findings towards events that take place at regenerating axon terminals, endplates and muscle fibres. However, retrograde deficits cannot be excluded to occur centrally at the motoneurone level. Since the soleus muscle is made largely of slow MUs with axons of similar diameters (6–14 µm), the speed of axonal regeneration and restoration of neuromuscular function is expected to be similar. In contrast, some of our observations indicate a gradual recuperation from axotomized motoneurones to regenerating axons over a period of several weeks. The functional recovery is accompanied by a reduction of the twitch maximal tension, a reduction and fade in tetanic contraction of the muscle and evidence of new MU types at high frequency tetanic stimulation.
Polyneuronal innervation of muscle fibres which occurs during development (Brown et al. 1976; Thompson & Jansen, 1977; Ty & Vrbová, 1995a) takes place also after nerve injury due to axonal sprouting of intact MUs and regeneration of severed motor axons to endplates mobilized by sprouts (Ribchester, 1988). It was shown that differences in the activity of convergent synapses give advantages on the more active one (Ribchester & Taxt, 1983; Balice-Gordon & Lichtman, 1994; Ty & Vrbová, 1995b), suggesting that endogenous activity may play a role in eliminating competitive neuromuscular synapses. The role of exogenous activity was investigated in a preparation with axonotmesis of the motor nerve innervating the soleus muscle in the rabbit. The electrically stimulated soleus nerves showed a faster improvement in motor function thus proving a positive effect of electrical stimulation on the regeneration and motor recovery of nerves (Nix & Hopf, 1983). However, many observations do not fit with a decisive function for activity in synapse elimination. For example, some polyneuronal junctions persist in the presence of activity and conversely, sometimes inactive synapses have a competitive advantage over active ones (Costanzo et al. 2000). Recent studies at the level of individual identified synapses using styryl dyes, such as FM1-43 and RH414 which label terminals by staining recycling synaptic vesicles, suggest that motor endplates become occupied by regenerating motor terminals because intact axons are competitively displaced by regenerating inactive terminals (Costanzo et al. 2000). The possibility cannot be excluded that these events may be involved in our experiments in which the activity is not blocked.
The decline of MU force during tetanic contractions observed between 14 and 28 days after crush must be due to a rearrangement and a progressive stabilization of the MUs. The different classes of MUs revealed by high frequency stimulation may reflect the fact that changes in contractile properties were not homogeneous following nerve crush. The modified MUs respond by a rapid tetanic fade under high frequency stimulation which clearly differentiates them from the normal MUs in the control muscle which show no tetanic fade under the same stimulation. The facts that no instability occurs during direct stimulation of the muscle and VI is not correlated with FI suggest that one of the targets of the injury must be located in the regenerating axons which may lose their ability to carry out appropriate matching between motoneuronal activity and their capacity to transmit. Moreover the disruption of the tetanic MU response is followed by a rebound response after a delay of some 300 ms which indicates that the high frequency stimulation can still be efficient in provoking an increase in tension after some time. Possible mechanisms may involve a conduction failure in the re-innervating axons as well as some immature synapses in the regenerating neuromuscular junctions.
Further experiments which combine electrophysiological and morphological approaches are needed to describe quantitatively and simultaneously the time course of the functional re-innervation process together with the number of re-innervating axons at each step, the morphological state of endplates and of synapse reformation to fully understand the re-innervation stagger even in our simple model of nerve crush.
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Acknowledgements |
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-Dumont for discussions and comments on the manuscript, Dr C. Batini and Fabi Dell for correcting the English language and G. Richardeau for technical assistance. This work was supported by the Association Française contre les Myopathies.
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