Tanguy Marqueste

Functional recovery after peripheral nerve injury and implantation of a collagen guide.

Although surgery techniques improved over the years, the clinical results of peripheral nerve repair remain unsatisfactory. In the present study, we compare the results of a collagen nerve guide conduit to the standard clinical procedure of nerve autografting to promote repair of transected peripheral nerves. We assessed behavioral and functional sensori-motor recovery in a rat model of peroneal nerve transection. A 1cm segment of the peroneal nerve innervating the Tibialis anterior muscle was removed and immediately replaced by a new biodegradable nerve guide fabricated from highly purified type I+III collagens derived from porcine skin. Four groups of animals were included: control animals (C, n=12), transected animals grafted with either an autologous nerve graft (Gold Standard; GS, n=12) or a collagen tube filled with an acellular skeletal muscle matrix (Tube-Muscle; TM, n=12) or an empty collagen tube (Collagen-Tube; CT, n=12). We observed that 1) the locomotor recovery pattern, analyzed with kinetic parameters and peroneal functional index, was superior in the GS and CT groups; 2) a muscle contraction was obtained in all groups after stimulation of the proximal nerve but the mechanical muscle properties (twitch and tetanus threshold) parameters indicated a fast to slow fiber transition in all operated groups; 3) the muscular atrophy was greater in animals from TM group; 4) the metabosensitive afferent responses to electrically induced fatigue and to two chemical agents (KCl and lactic acid) was altered in GS, CT and TM groups; 5) the empty collagen tube supported motor axonal regeneration. Altogether, these data indicate that motor axonal regeneration and locomotor recovery can be obtained with the insertion of the collagen tube RevolNerv. Future studies may include engineered conduits that mimic as closely as possible the internal organization of uninjured nerve.

EMG versus oxygen uptake during cycling exercise in trained and untrained subjects

The aim of this study was to test the hypothesis that bicycle training may improve the relationship between the global SEMG energy and VO2. We already showed close adjustment of the root mean square (RMS) of the surface electromyogram (SEMG) to the oxygen uptake (VO2) during cycling exercise in untrained subjects. Because in these circumstances an altered neuromuscular transmission which could affect SEMG measurement occurred in untrained individuals only, we searched for differences in the SEMG vs. VO2 relationship between untrained subjects and well-trained cyclists. Each subject first performed an incremental exercise to determine VO2max and the ventilatory threshold, and second a constant-load threshold cycling exercise, continued until exhaustion. SEMG from both vastus lateralis muscles was continuously recorded. RMS was computed. M-Wave was periodically recorded. During incremental exercise: (1) a significant non-linear positive correlation was found between RMS increase and VO2 increase in untrained subjects, whereas the relationship was best fitted by a straight line in trained cyclists; (2) the RMS/VO2 ratio decreased progressively throughout the incremental exercise, its decline being significantly and markedly accentuated in trained cyclists; (3) in untrained subjects, significant M-wave alterations occurred at the end of the trial. These M-wave alterations could explain the non-linear RMS increase in these individuals. During constant-load exercise: (1) after an initial increase, the VO2 ratio decreased progressively to reach a plateau after 2 min of exercise, but no significant inter-group differences were noted; (2) no M-wave changes were measured in the two groups. We concluded that the global SEMG energy recorded from the vastus lateralis muscle is a good estimate of metabolic energy expenditure during incremental cycling exercise only in well-trained cyclists.

Cholecalciferol (Vitamin D3) Improves Myelination and Recovery after Nerve Injury

Previously, we demonstrated i) that ergocalciferol (vitamin D2) increases axon diameter and potentiates nerve regeneration in a rat model of transected peripheral nerve and ii) that cholecalciferol (vitamin D3) improves breathing and hyper-reflexia in a rat model of paraplegia. However, before bringing this molecule to the clinic, it was of prime importance i) to assess which form – ergocalciferol versus cholecalciferol – and which dose were the most efficient and ii) to identify the molecular pathways activated by this pleiotropic molecule. The rat left peroneal nerve was cut out on a length of 10 mm and autografted in an inverted position. Animals were treated with either cholecalciferol or ergocalciferol, at the dose of 100 or 500 IU/kg/day, or excipient (Vehicle), and compared to unlesioned rats (Control). Functional recovery of hindlimb was measured weekly, during 12 weeks, using the peroneal functional index. Ventilatory, motor and sensitive responses of the regenerated axons were recorded and histological analysis was performed. In parallel, to identify the genes regulated by vitamin D in dorsal root ganglia and/or Schwann cells, we performed an in vitro transcriptome study. We observed that cholecalciferol is more efficient than ergocalciferol and, when delivered at a high dose (500 IU/kg/day), cholecalciferol induces a significant locomotor and electrophysiological recovery. We also demonstrated that cholecalciferol increases i) the number of preserved or newly formed axons in the proximal end, ii) the mean axon diameter in the distal end, and iii) neurite myelination in both distal and proximal ends. Finally, we found a modified expression of several genes involved in axogenesis and myelination, after 24 hours of vitamin supplementation. Our study is the first to demonstrate that vitamin D acts on myelination via the activation of several myelin-associated genes. It paves the way for future randomised controlled clinical trials for peripheral nerve or spinal cord repair.

Convenient Access to Biocompatible Block Copolymers from SG1-Based Aliphatic Polyester Macro-Alkoxyamines

SG1-based poly(d,l-lactide) (PLA) or poly(epsilon-caprolactone) (PCL) macro-alkoxyamines were synthesized and further used as macroinitiators for nitroxide-mediated polymerization (NMP) of 2-hydroxyethyl (meth)acrylate (HE(M)A) to obtain the corresponding PLA- or PCL-PHE(M)A block copolymers. First, a PLA-SG1 macro-alkoxyamine was prepared by 1,2-intermolecular radical addition (IRA) of the MAMA-SG1 (BlocBuilder) alkoxyamine onto acrylate end-capped PLA previously prepared by ring-opening polymerization. The NMP of HEA monomer from the PLA-SG1 macro-alkoxyamine appeared to be well controlled in the presence of free SG1 nitroxide, contrary to that of HEMA. In the latter case, adjustable molecular weights could be obtained by varying the HEMA to macro-alkoxyamine ratio. The versatility of our approach was then further applied to the preparation of PHEMA-b-PCL-b-PHEMA copolymers from a alpha,omega-di-SG1 functionalized PCL macro-alkoxyamine previously obtained from a PCL diacrylate by IRA. Preliminary studies of neuroblast cultures on these PCL-based copolymer films showed acceptable cyto-compatibility, demonstrating their potential for nerve repair applications.

Eccentric exercise alters muscle sensory motor control through the release of inflammatory mediators

Following downhill exercise, muscle damage and local inflammatory reactions, induced by lengthening contractions, are observed and voluntary muscle activation decreases. The hypothesis that feedback carried by the group IV muscle afferents could be involved has often been raised but never measured in vivo in these conditions. In this experiment, we tested the response of the group IV muscle afferents from the lower limb to injections of KCl and lactic acid in non-exercising rats and at 1, 2, and 8 days after one running session (-13 degrees, 16 m/min). At days 1 and 2, the baseline discharge of the group IV afferents increased, but further activation by test agents was absent. After 8 days, the afferent response was equivalent to the control response. Pretreatment with betamethasone before exercise abolished the effects of downhill exercise. In non-exercising rats, arachidonic acid evoked group IV afferent discharge and suppressed their further response to another stimulus. These results demonstrate that exhaustive downhill running highly activates, for at least 2 days, the sensory feedback carried by group IV afferents through the local release of inflammatory mediators. Such an altered sensori-motor control, accompanying the post-eccentric inflammatory syndrome, could play a key role in deterioration of muscle performance and of its voluntary activation.

Effect of muscle electrostimulation on afferent activities from tibialis anterior muscle after nerve repair by self-anastomosis

Numerous previous studies were devoted to the regeneration of motoneurons toward a denervated muscle after nerve repair by self-anastomosis but, to date, few investigations have evaluated the regeneration of sensory muscle endings. In a previous electrophysiological study (Decherchi et al., 2001) we showed that the functional characteristics of tibialis anterior muscle afferents are affected after self-anastomosis of the peroneal nerve even when the neuromuscular preparation was not chronically stimulated. The present study examines the regeneration of groups I-II (mechanosensitive) and groups III-IV (metabosensitive) muscle afferents by evaluating the recovery of their response to different test agents after self-anastomosis combined or not with chronic muscle stimulation for a 10-weeks period. We compared five groups of rats: C, control; L, nerve lesion without suture; LS, nerve lesion with suture; LSE(m): nerve lesion plus chronic muscle stimulation with a monophasic rectangular current; and LSE(b): nerve lesion plus chronic stimulation with a biphasic current with modulations of pulse duration and frequency, eliciting a pattern of activity resembling that delivered by the nerve to the muscle. Compared to the control group, (1) muscle kept only its original weight in the LSE(b) group, (2) in the LS group the response curve to tendon vibration was shifted toward the highest mechanical frequencies and the response of groups III-IV afferents after fatiguing muscle stimulation lowered, (3) in the LSE(m) group, the pattern of activation of mechanoreceptors by tendon vibrations was altered as in the LS group, and the response of metabosensitive afferents to KCl injections was markedly reduced, (4) in the LSE(b) group, the response to tendon vibration was not modified and the activation of metabosensitive units by increased extracellular potassium chloride concentration was conserved. Both LSE(b) and LSE(m) conditions were ineffective to maintain the post muscle stimulation activation of metabosensitive units as well as their activation by injected lactic acid solutions. Our data indicate that chronic muscle electrostimulation partially favors the recovery of mechano- and metabosensitivity in a denervated muscle and that biphasic modulated currents seem to provide better results.

Matched adaptations of electrophysiological, physiological, and histological properties of skeletal muscles in response to chronic hypoxia

This study tried to differentiate the consequences of chronic hypoxia on the electrophysiological and physiological properties and the histological characteristics of slow and fast muscles in rats. Animals inhaled a 10% O2 concentration for a 1-month period. Then, slow [soleus (SOL)] and fast [extensor digitorum longus (EDL)] muscles were analyzed in vitro by physiological and electrophysiological measurements and histological analyses. The results were compared to those obtained in corresponding muscles of an age-matched normoxic group. After exposure to hypoxia: (1) in SOL, there was a tendency to elevated Fmax, a significant increase in twitch force and tetanic frequency and a shortening of M-wave duration, and a reduced percentage of type I fibres, whereas the proportion of type IIa fibres doubled; (2) in EDL, Fmax and tetanic frequency were lowered, the muscle became less resistant to fatigue, and the proportion of type IId/x fibres was halved. Then, after 1 month of hypoxia, in the SOL muscle, both the contractile and histological properties resemble those of a fast muscle. By contrast, the EDL became slower, despite its histology was modestly affected. Reduced muscle use in hypoxia could explain the tendency for deteriorating adaptations in EDL, and the faster properties of SOL could result from hypoxia-induced inhibition of the growth-related fast-to-slow shift in muscle fibre types.

Changes in neuromuscular function after training by functional electrical stimulation

We examined whether the neuromuscular function of rectus femoris (RF) and flexor digitorum brevis (FDB) in humans was modified after a 6‐week training period of functional electrical stimulation (FES), and whether any effects persisted at the end of a 6‐week post‐FES recovery period. In both the stimulated and contralateral nonstimulated muscles, we recorded the muscle force, surface electromyogram, and M wave, and also measured the root mean square (RMS) and the median frequency (MF) during static contraction sustained until exhaustion at 60% of maximal voluntary contraction (MVC). FES was performed with symmetric biphasic pulses, with a ramp modulation of both the stimulation frequency and pulse duration. No changes in MCV and endurance time to exhaustion occurred in nonstimulated muscles, whereas a significant MVC increase occurred immediately after FES in RF (+14 ± 5%) and FDB (+13 ± 5%), these effects persisting 6 weeks after the end of FES. In FDB, FES also elicited a significant increase in endurance time to exhaustion (+18 ± 7%). The M‐wave characteristics never varied after FES, but a marked attenuation occurred in the MF decrease and the RMS increase measured at endurance time to sustained 60% MVC, especially in FDB, which contains the higher proportion of type II fibers. These data indicate that FES improves muscle function and elicits changes in central muscle activation. The benefits of FES were greater in FDB, which is highly fatigable, and persisted for at least a 6‐week period. Muscle Nerve 28: 181–188, 2003

Selective training-induced thigh muscles hypertrophy in professional road cyclists

Muscular adaptations linked to a high volume and intensity of training have been scarcely reported. We aimed at documenting, using MRI, the cross-sectional area changes associated with a high volume and intensity of training in 11 thigh muscles of a population of professional road cyclists as compared with sport science students. We were also interested in determining, whether selective muscle hypertrophy in professional road cyclists, if any, was correlated to selective exercise-induced T2 changes during a pedaling exercise on a cycloergometer. Cross-sectional area of 11 thigh muscles was quantified in sixteen subjects (i.e. eight professional road cyclists and eight sport science students) using MRI. In addition, transverse relaxation times (T2) were measured before and just after a maximal standardized constant-load exercise in order to investigate exercise-related T2 changes in these muscles. Professional road cyclists had a significantly higher relative amount of muscle (including the whole set of thigh muscles, 90.5±3.3%) as compared to controls (81.6±7.3%). Regarding relative values expressed with respect to the total thigh muscles CSA, Vastus lateralis and Biceps femoris CSA were significantly larger in cyclists whereas CSA of the Vastus intermedius was smaller. However, this selective hypertrophy was not correlated to the exercise-induced T2-increase. We have reported, for the first time, a selective hypertrophy of Vastus lateralis and Biceps femoris in professional road cyclists confirming their involvement in pedaling task and suggesting a possible cause–effect relationship between muscle activation and hypertrophy, associated with a specific pedaling skill.

VITAMIN D3 IMPROVES RESPIRATORY ADJUSTMENT TO FATIGUE AND H-REFLEX RESPONSES IN PARAPLEGIC ADULT RATS

We previously demonstrated that vitamin D₂ (ergocalciferol) triggers axon regeneration in a rat model of peripheral nerve transection. In order to confirm the regenerative potential of this neuroactive steroid, we performed a study in which vitamin D₃ (cholecalciferol) was delivered at various doses to paralytic rats. After spinal cord compression at the T10 level, rats were given orally either vehicle or vitamin D₃ at the dose of 50 IU/kg/day or 200 IU/kg/day. Three months later, M and H-waves were recorded from rat Tibialis anterior muscle in order to quantify the maximal H-reflex (H(max)) amplitude. We also monitored the ventilatory frequency during an electrically induced muscle fatigue known to elicit the muscle metaboreflex and an increase in respiratory rate. Spinal cords were then collected, fixed and immunostained with an anti-neurofilament antibody. We show here that vitamin D-treated animals display an increased number of axons within the lesion site. In addition, rats supplemented with vitamin D₃ at the dose of 200 IU/kg/day exhibit (i) an improved breathing when hindlimb was electrically stimulated; (ii) an H-reflex depression similar to control animals and (iii) an increased number of axons within the lesion and in the distal area. Our data confirm that vitamin D is a potent molecule that can be used for improving neuromuscular adaptive mechanisms and H-reflex responses.