Group III and IV muscle afferents: role on central motor drive and clinical implications.

The present review is focused on neural mechanisms responsible of group III and IV muscle afferent actions on central motor drive during physical exercise in both healthy and pathological populations. It seems that these mechanisms contribute to improve muscle performance by regulating the peripheral fatigue development and by avoiding excessive muscle impairments. Therefore, a great deal of attention is paid to their influences on motor unit activation during fatiguing exercise both in human and animal models. Recent evidence indicated that these afferents from a given active muscle could contribute to regulate the motor activity of the homonymous as well as surrounding skeletal muscles by acting at both spinal and supraspinal levels. In addition, given that the recovery of the sensory feedback plays a key role in the improvement of motor function following numerous neuromuscular traumas, the role of these afferents in preclinical and clinical situations is also explored in animal and human models. It is supposed that studying the motor and autonomic functions of group III and IV afferents might help healthcare professionals in the future to find appropriate treatments and rehabilitation programs.

Recovery pattern of motor reflex after a single bout of neuromuscular electrical stimulation session.

We aimed at determining the recovery pattern of neural properties of soleus muscle after a single bout of neuromuscular electrical stimulation (NMES) session. Thirteen subjects performed an NMES exercise (75 Hz, 40 contractions, 6.25 s per contraction). Maximal voluntary contraction (MVC), H-reflex at rest and during voluntary contraction fixed at 60% of MVC (respectively, H(max) and H(sup) ) and volitional (V) wave were measured before and during the recovery period following this exercise [i.e., immediately after, 2 h (H2), 2 days (D2) and 7 days (D7)]. MVC exhibited an immediate and a delayed declines at 2 days (respectively, -29.8±4.6%, P<0.001; -13.0±3.4%, P<0.05). Likewise, V/M(sup) was decreased immediately and 2 days after NMES session (respectively, -43.3±11.6%, P<0.05; 35.3±6.6%, P<0.05). The delayed decrements in MVC and V-wave occurred concomitantly with muscle soreness peak (P<0.001). It could be concluded that motor command alterations after an NMES resistance session contributed to the immediate and also to the delayed decreases in MVC without affecting resting and active H-reflex excitability. These results suggested that spinal circuitry function of larger motoneurons was inhibited by NMES (as indicated by the depressed V-wave responses) contrary to the smaller one (indicated by the unchanged H-reflex responses).

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.

[Pulmonary function testing of 156 children exposed to automobile pollution in the municipality of Cotonou]. / Evaluation Du DEM(25) Chez 156 Enfants Exposes A La Pollution Automobile Dans La Municipalite De Cotonou.

AIMS: To evaluate the risk of disruption of distal airways in children exposed to pollutants automobiles. MATERIAL AND METHODS: Study included 156 children selected assigned in groups "Most Exposed" and "Less Exposed" separated respectively 15 meters and 150 meters of road traffic. Children in both groups were subjected to lung function tests before and after an exercise test that was to perform a series of flexion / extension of the knees to the fatigue. FEV and MEF(25) were parameters selected. RESULTS: Change in FEV post exercise of each group is below 5%. The group "Less Exposed" presented a mean value of MEF(25) before exercise similar to that recorded after exercise. Within the group "Most Exposed", the mean value of MEF(25) post exercise is significantly lower than that observed at rest of 8.65%. CONCLUSION: The fact of living permanently near the traffic, poses serious risks of disruption of the distal airways.

Chronic electrostimulation after nerve repair by self-anastomosis: effects on the size, the mechanical, histochemical and biochemical muscle properties.

This study tests the effects of chronic electrostimulation on denervated/reinnervated skeletal muscle in producing an optimal restoration of size and mechanical and histochemical properties. We compared tibialis anterior muscles in four groups of rats: in unoperated control (C) and 10 weeks following nerve lesion with suture (LS) in the absence of electrostimulation and in the presence of muscle stimulation with either a monophasic rectangular current (LSEm) or a biphasic modulated current (LSEb). The main results were (1) muscle atrophy was reduced in LSEm (-26%) while it was absent in LSEb groups (-8%); (2) the peak twitch amplitude decreased in LS and LSEm but not in LSEb groups, whereas the contraction time was shorter; (3) muscle reinnervation was associated with the emergence of type IIC fibers and proportions of types I, IIA and IIB fibers recovered in the superficial portion of LSEb muscles; (4) the ratio of oxidative to glycolytic activities decreased in the three groups with nerve injury and repair; however, this decrease was more accentuated in LSEm groups. We conclude that muscle electrostimulation following denervation and reinnervation tends to restore size and functional and histochemical properties during reinnervation better than is seen in unstimulated muscle.

[Dudley Joy Morton's foot syndrome]. / Le pied de Dudley Joy Morton.

Morton's foot syndrome is a hereditary syndrome characterized by a short first metatarsal bone, posterior displacement of the sesamoids, and hypertrophy of the second metatarsal, causing excessive weight to be borne by the second metatarsal head. This condition results in callus formation under the second metatarsal. Pain and tenderness are usually felt at the base of the first two metatarsal bones and at the head of the second. Pain (metatarsalgia) may be disabling. Conservative treatment consists of placing a flexible pad under the first metatarsal and toe to increase the range of motion and weight-bearing along the first metatarsophalangeal joint and the hallux. Surgical treatment consists of removing a small portion of bone from one or two joints to bring the toe down to the desired length. Lengthening short toes by placing a silicon implant into one of the joints is also possible.

[Peripheral nerve repair: 30 centuries of scientific research]. / La réparation nerveuse périphérique: 30 siècles de recherche.

INTRODUCTION: Nerve injury compromises sensory and motor functions. Techniques of peripheral nerve repair are based on our knowledge regarding regeneration. Microsurgical techniques introduced in the late 1950s and widely developed for the past 20 years have improved repairs. However, functional recovery following a peripheral mixed nerve injury is still incomplete. STATE OF ART: Good motor and sensory function after nerve injury depends on the reinnervation of the motor end plates and sensory receptors. Nerve regeneration does not begin if the cell body has not survived the initial injury or if it is unable to initiate regeneration. The regenerated axons must reach and reinnervate the appropriate target end-organs in a timely fashion. Recovery of motor function requires a critical number of motor axons reinnervating the muscle fibers. Sensory recovery is possible if the delay in reinnervation is short. Many additional factors influence the success of nerve repair or reconstruction. The timing of the repair, the level of injury, the extent of the zone of injury, the technical skill of the surgeon, and the method of repair and reconstruction contribute to the functional outcome after nerve injury. CONCLUSION: This review presents the recent advances in understanding of neural regeneration and their application to the management of primary repairs and nerve gaps.

[Metabolic stability and physiological adaptation of muscle under conditions of exercise]. / Métabosensibilité musculaire et adaptations physiologiques au cours de l'exercice.

The first role played by group III (thin myelinated) and group IV (unmyelinated) afferent fibres from skeletal muscle is to transmit nociceptive information from muscle to the central nervous system. The second role of these free endings located in the interstitium of the muscle is to induce cardiovascular and respiratory adjustments during muscular exercise. These respiratory and circulatory responses during muscular exercise may be reflexly induced via muscular afferents. Indeed, static contraction of hindlimb muscles in anaesthetised mammals has been shown to reflexly increase the ventilatory function, the myocardial contractility and heart rate. The mechanical muscle deformation and the accumulation of metabolites in its intersitium are the cause of raised activity in small nerve fibres which in turn induces the physiological responses. It is also admitted that the central locomotor areas on the medullary and spinal neuronal pools control ventilatory and cardiovascular function during exercise. This mechanism is called "central command". Furthermore, adjustments of the locomotor activity during exercise is mediated by the thinly myelinated and unmyelinated fibres with endings in the working muscle. These fibres, also called "metaboreceptor" may be responsible of the coupling between the ventilation and the locomotion. Thickly myelinated muscle afferents (i.e. group I and II) appear to play little role in causing the reflex autonomic responses to contraction.

[Bioenergetic characteristics of the diaphragm after spinal cord injury]. / Caractéristiques bioénergétiques du diaphragme après une lésion spinale.

INTRODUCTION: C2 spinal cord hemisection interrupts descending bulbospinal respiratory axons coursing through the cervical spinal cord. One consequence of this type of spinal cord injury is the partial deafferentation of the ipsilateral phrenic nucleus, which renders the hemidiaphragm paralysed. The biochemical properties of the intact or denervated locomotor skeletal muscles have been studied extensively, whereas the biochemical properties of the diaphragm after a cervical spinal cord injury received relatively little attention. This seems unfortunate, given that the diaphragm is the most important mammalian respiratory muscle and is the only skeletal muscle considered essential for survival. Therefore, the purpose of this work was to analyse the bioenergetic characteristics of the diaphragm following a cervical spinal cord injury. METHODS: Segments of the crural diaphragm from rat were homogenised to determine the activities of a glycolytic enzyme, lactate dehydrogenase (LDH), and a Krebs cycle enzyme, citrate synthase (CS). RESULTS: Data show a significant decrease of the CS activity on the ipsilateral hemidiaphragm to the cervical hemisection. No change in the LDH is observed between the animal without or with a cervical spinal cord injury. CONCLUSIONS: These data suggested that the CS activity could be dependent of the nervous influx from the central respiratory rhythm generator and that LDH activity could be maintained by a peripheral respiratory activity or by the cross phrenic phenomenon activation. Biochemical data should be considered in subjects presenting respiratory deficiency induced by a cervical spinal cord injury. Furthermore, the diaphragmatic muscle metabolic activity could be used to evaluate the functional respiratory recovery observed spontaneously or experimentally after using repair strategies of the spinal cord.

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.

Regeneration of acutely and chronically injured descending respiratory pathways within post-traumatic nerve grafts.

Central respiratory neurons, which are acutely axotomized by peripheral nerve grafts implanted at the level of the descending respiratory pathways within the C2 spinal cord, can regenerate their axons within the grafts and still transmit normal physiological messages [Decherchi et al., 1996. Exp. Neurol. 137, 1-14]. The present work investigated the extent to which mature central neurons, acutely or chronically axotomized by a spinal lesion, still maintain the potential to regenerate an axon following post-traumatic nerve grafting within supra-lesional spinal structures and remained functional. This study is an extension of earlier work employing the more chronic lesions, that investigated whether respiratory neurons chronically axotomized by a spinal cord injury can retain the ability to regenerate their axonal process within a post-traumatic peripheral nerve graft. Here implantation was performed into the supra-lesional ventrolateral part of the ipsilateral C2 spinal cord (at the level of the descending respiratory pathways) previously hemisected at the C3 level. In the present study, these post-traumatic peripheral nerve grafts were performed either acutely (group I, n=15, 2.5 h post-injury: acute conditions) or chronically (group II, n=17, 3 weeks; group III, n=6, 3 months: chronic conditions) after the injury.Electrophysiological recording of teased filaments (n=2362) within the post-traumatic peripheral nerve grafts revealed the presence of regenerated nerve fibers with spontaneous unitary impulse traffic (graft units, n=954) in all animals. These graft units were respiratory (n=247) and non-respiratory (n=707). Respiratory discharges originated from central respiratory neurons which remained functional with preserved afferent connections. Except for the group III, post-traumatic C2 peripheral nerve grafts of the groups I and II contained a significantly higher occurrence rate (13.2+/-2% and 11.6+/-1.9%) of respiratory units than C2 spinal peripheral nerve grafts (5.9+/-1.6%) realized without previous CNS injury. The main conclusion of our study is that for a prolonged period of 3 weeks following a spinal cord injury, central respiratory neurons have the potential to remain functional and to regenerate their axonal process within post-traumatic peripheral nerve grafts inserted rostrally to the spinal damage. This indicates that supra-lesional post-traumatic nerve grafts may constitute an efficient delayed strategy for inducing axonal regrowth of chronically axotomized adult central neurons. This suggests that surgical intervention which is not always possible immediately after a spinal cord injury may be satisfactorily carried out after an appropriate delay.

Effects of chronic hypoxemia on the afferent nerve activities from skeletal muscle.

An acute reduction of the oxygen supply to contracting muscles not only affects their metabolism but also modifies their sensorimotor control through changes in afferent discharge of the group I and group III-IV nerve fibers, the latter playing a pivotal role in the protective mechanisms against muscle fatigue. The effects of chronic hypoxemia on the muscle sensitivity are totally unknown. In the present study, group I fibers (mechanosensory afferents) and group III-IV fibers (mechanosensory and chemosensory afferents) from the anterior tibial muscle were recorded in normoxemic and chronic hypoxemic rats. Hypoxemic rats breathed for 45 d a gas mixture containing 9.5 to 10% O(2) in N(2). The data were compared with those obtained in normoxemic animals of the same age. To activate the different muscle afferents, we used different test agents, including electrically induced fatigue (EIF), KCl, lactic acid injections, as well as tendon vibrations. The conduction velocity of all nerve fibers was significantly (p < 0.01) higher in hypoxemic rats than in the normoxemic group. Chronic hypoxemia significantly depressed the response of the group III-IV muscle afferents to KCl injections and even abolished their response to lactic acid and EIF. However, the response to tendon vibrations of the group I afferents was similar in hypoxemic and normoxemic rats. These results suggest that chronic hypoxemia markedly alters the chemosensitivity of the group III-IV muscle afferents, which may explain the higher fatigability of hypoxemic subjects.

Changes in afferent activities from tibialis anterior muscle after nerve repair by self-anastomosis.

In order to study sensory nerve plasticity after nerve injury and repair, recordings were made from afferent axons innervating the tibialis anterior muscle in rats under several different experimental conditions. In two groups of rats, reinnervation of the denervated tibialis anterior was examined 2.5 months (group A) and 7 months (group B) after section, along with self-anastomosis of the common peroneal nerve. The other rats (group C) were examined 2.5 months after the nerve was cut and ligatured to its stumps to avoid axonal regeneration. No evoked potentials and no activation in response to any test agent were found in group C rats. We found a significant increase in the proportion of group I-II fibers and a significant decrease in group IV fibers in the group B rats when compared with group A (P < 0.05 and P < 0.01) and control animals (P < 0.01 and P < 0.01). A higher conduction velocity was measured in group IV fibers in group B rats when compared with group A (P < 0.01) and the controls (P < 0.01). The proportion of afferent units showing an optimal discharge in response to tendon vibration at 70 Hz (range 0-100 Hz) was higher in groups A and B (72.2 and 80%, respectively) than in the controls (36.8%). The response of muscle afferents to KCl (1-20 mM) and lactic acid (0.5-3 mM) concentrations was markedly depressed in group A rats (P < 0.05), whereas it was restored and even accentuated in group B animals when compared with the controls (P < 0.05). Electrically induced fatigue (3 min, 10 Hz) significantly activated (P < 0.05) muscle afferents only in controls. The present study indicates that after self-anastomosis of a cut hindlimb muscle nerve, sensory innervation was markedly modified in the direction of enhanced mechanosensitivity to high-frequency tendon vibration and depressed metabosensitivity.

Regrowth of acute and chronic injured spinal pathways within supra-lesional post-traumatic nerve grafts.

The present work investigates the extent to which mature central neurons acutely or chronically axotomized by a spinal lesion still maintained the potential to regenerate an axon following post-traumatic nerve grafting within supra-lesional spinal structures. In adult rats, a C3 cervical hemisection (injury) was made and an autologous segment of the peroneal nerve was implanted 2mm rostrally into the ventrolateral part of the ipsilateral C2 spinal cord. Nerve graft implantations were carried out acutely at the time of injury (group I, acute conditions) or chronically, three weeks post-injury (group II, chronic conditions). Central neurons axotomized by the spinal lesion were labeled by True Blue injected at the lesion site at the time of trauma. Central neurons regenerating axons within the nerve grafts were labeled with either horseradish peroxidase (only in group I, n=4) or Nuclear Yellow (group I, n=3 and group II, n=6) applied two to four months post-grafting to the distal cut end of the nerve grafts. Neurons with dual staining (True Blue/Nuclear Yellow) represented central regenerating neurons which were previously axotomized by the spinal lesion and which had retained the capacity for axonal regeneration for a delayed period after injury. In group I (acute injury conditions), all types of labeled cells were found to be scattered with a clear bimodal distribution within the spinal cord and the brainstem. No labeled cells were found within the motor cortex. There was no statistically significant difference between horseradish peroxidase and all cells containing Nuclear Yellow (Nuclear Yellow and True Blue/Nuclear Yellow). In group II (chronic injury conditions), Nuclear Yellow- and True Blue/Nuclear Yellow-labeled cells had a similar dual distribution to that of group I, but were found to be significantly less represented (P=0.019). These differences are discussed in terms of capacity for cell survival and axonal regrowth after acute and chronic injury. The main conclusion is based on the evidence of dual staining of central neurons in both groups, which demonstrates that brainstem and spinal neurons involved in acute and chronic axotomy after spinal C3 lesion can survive the trauma and still maintain the capacity to regenerate lesioned axons within nerve grafts inserted rostrally (C2 spinal cord) to the primary site of injury. Although exhibited to a lesser extent in chronic than in acute conditions, this capacity was found to occur for as long as three weeks post-injury. These results indicate that supra-lesional post-traumatic nerve grafts may constitute an efficient delayed strategy for inducing axonal regrowth of chronically axotomized adult central neurons. We suggest that surgical intervention, which is not always possible immediately after a spinal cord injury, may be satisfactorily carried out after an appropriate delay.

Mechanisms of fatigue-induced activation of group IV muscle afferents: the roles played by lactic acid and inflammatory mediators.

We already showed that group IV muscle afferents are activated during electrically-induced fatigue. The purpose of this study is to identify the mechanisms of stimulation of these muscle afferents by electrically-induced fatigue at a high (100 Hz; high frequency fatigue, HFF) or a low rate (10 Hz; low frequency fatigue, LFF) of stimulation. In 23 paralyzed and anaesthetized rabbits, group IV afferent activity from the tibialis anterior muscle was recorded before and after 3-min HFF or 5-min LFF runs eliciting the same force failure. Plasma lactic acid concentration (LA) was also measured in leg venous blood. We tested the effects of dichloroacetate (DCA), which reduces lactic acid production, and of acetylsalicylic acid (ASA), a blocker of cyclooxygenase, on fatigue-induced activation of group IV muscle afferents after HFF and LFF trials. Pretreatment by ASA or DCA did not modify HFF-induced activation of muscle afferents. On the other hand, LFF-induced response was markedly depressed by each pharmacological agent (-44% after ASA and -75% after DCA). We verified that DCA markedly lowered LA production after LFF. The present results show that the activation of group IV muscle afferents by LFF or HFF results from different mechanisms and also demonstrate the major role played by lactic acid production and, to a lesser extent, the release of inflammatory mediators in LFF-induced activation of group IV muscle afferents.

Modifications of afferent activities from Tibialis anterior muscle in rat by tendon vibrations, increase of interstitial potassium or lactate concentration and electrically-induced fatigue.

Although previous experiments with a partially similar objective have been described in dogs, cats and rabbits, the purpose of this study was to identify and characterize mechanosensitive and chemosensitive muscle afferents in the anaesthetized rat since it is a widely used laboratory animal. The peroneal nerve innervating the tibialis anterior muscle was studied. Measurement of conduction velocities from compound action nerve potentials evoked by peripheral nerve stimulation allowed identification of group I-II (10.79+/-1.02 m/s), group III (2.96+/-0.58 m/s) and group IV (0.46+/-0.07 m/s) afferent fibers. Computation of the different compound potential areas showed that afferents I and II arising from spindles and tendon organs represented 9.65+/-2.2%, whereas afferents III and IV arising from free nerve endings in muscle represented 90.35+/-2.2% (III, 46.66+/-2.71% and IV, 43.69+/-2.52%). Action potentials were recorded from teased nerve filaments. Mechanical tendon vibrations (10 to 90 Hz) were used to activate mechanoreceptors. Peak increase in afferent discharge (fimpulses) was measured at 50 Hz (n = 12/19 units) or 70 Hz (n = 7/19 units). Intra-arterial bolus injections of different concentrations of potassium chloride (KCl: 1 to 20 mM) or lactic acid (LA: 0.5 to 3 mM) elicited marked activation of III and IV afferents (n = 124). Enhancement of fimpulses was not proportional to the increase in [KCl] or [LA]. Activation of afferents plateaued when [KCl] was equal or greater than 5 mM while fimpulses peaked, then decreased, when [LA] was 1 mM. Muscle fatigue induced by direct electrical muscle stimulation (EIF) markedly activated group III-IV (n = 17/18) afferents (176.9+/-29.7% of control) which persisted for the 3 minutes of recovery from fatigue. Maximal fimpulses increases in response to LA (+67%) and KCl (+46.9%) injections and to EIF (+76.9%) were similar. This procedure for characterizing the functional properties of sensory nerve endings in a skeletal muscle may be used to assess further changes in sensory muscle paths in experimental rodent pathophysiological systems.

CNS axonal regeneration with peripheral nerve grafts cryopreserved by vitrification: cytological and functional aspects.

To test cool-warm protocols for storing peripheral nerves, 4-cm-long-nerve segments were removed from the hindleg of adult rats and cryopreserved using a vitrification solution (or cryoprotective mixture) containing a mixture of polyalcohols (2,3-butanediol, 1,2-propanediol, polyethylene glycol, and Belzer U.W. medium). Schwann cell viability and morphology were studied with regard to the effect of (i) cryoprotective mixture concentration (100, 50, and 30% diluted in human serum albumin at 4%), (ii) duration of exposure (10, 15, or 30 min in a single step) of nerves to the cryoprotective mixture, (iii) cooling rate (F1/F2, F3, and F4: 3, 12, and 231 degrees C/min, respectively), and (iv) type of replacement of cryoprotectant (T1, one step; or T2, perfusion) after warming. Nerves exposed 10 min to cryoprotective mixture 50% (2,3-butanediol, 1.926 mol.liter-1; 1,2-propanediol, 3.063 mol.liter-1; polyethylene glycol, 0.084 mol.liter-1; and Belzer U.W., 22.4 mosm-1) and cooled-warmed with the F2/F3/F4-T2 protocols contained live and correctly cryopreserved Schwann cells. The capacity of these cryopreserved nerve segments (n = 6) to be subsequently repopulated by regenerating axons from central neurons was compared to that of fresh nerves when used as peripheral nerve autografts implanted within the spinal cord at the level of the descending respiratory pathways. All cryopreserved nerve grafts were successfully reinnervated by regenerated central axons. Unitary spontaneous action potentials propagated along these axons were assessed by recording the discharge of tested nervous filaments (T) from the grafts in artificially ventilated and paralyzed animals. Out of 535 T, 32 (6 +/- 1.2%) presented spontaneous unitary activity with respiratory (R, n = 2) and nonrespiratory (NR, n = 30) pattern of discharge. The T mean number, the occurrence rate referenced to the total number of T (R/T, NR/T, and R + NR/T) and the mean number of spontaneous units (R, NR, R + NR) were compared to those of fresh spinal peripheral nerve grafts. Except for T, cryopreserved peripheral nerve grafts contained statistically significantly (P < 0.05) less spontaneous R and NR unitary activity, which represented, respectively, 6.2 +/- 6.2 and 26.8 +/- 5.7% of that found in the control group. These data indicate that nerves cryopreserved with the protocols described above contain viable Schwann cells which constitute a suitable support to induce regeneration of central fibers. The effectiveness of nerve cryopreservation by vitrification is discussed with regard to Schwann cell viability following cool-warm protocols and to subsequent reinnervation of the cryopreserved peripheral nerve grafts.

Dual staining assessment of Schwann cell viability within whole peripheral nerves using calcein-AM and ethidium homodimer.

A membrane permeant nucleic acid stain, ethidium homodimer was used in combination with calcein-AM to document the viability of Schwann cells (SCs) in whole nerves after cold storage assays. Segments of peripheral nerves were, (i) kept intact in buffer (viability controls), (ii) thawed after a cryopreservation, according to a protocol which has been previously shown to maintain the integrity of most nerve components [Ruwe and Trumble, J. Reconstr. Microsurg., 1990, 6: 239-244; Gauthier et al., In 3rd International Symposium on Axonal Regrowth in the Mammalian Spinal Cord and Peripheral Nerve, Deauville, France, 1995, p. 24, abstract], (iii) killed by chemical injury, or (iv) by successive freezing-thawing. Teased preparations of nerve fibers were prepared from the various types of nerve segments and incubated with calcein-AM and ethidium homodimer, which stain, respectively, living and dead cells. In control or cryopreserved nerves, staining with calcein-AM resulted in bright green fluorescence in the cytoplasm of SCs, with no red fluorescence of ethidium homodimer. In contrast, in killed nerve preparations, intense ethidium red fluorescence was observed in SC nuclei, with negligible green calcein cytoplasmic fluorescence. Thus, the combination of calcein-AM/ethidium homodimer appeared as an effective tool for assessing the viability of SCs and determine the quality of cold stored nerve preparations used in graft repair procedures. In addition, the generated fluorescence enabled clear visualization of myelinated fibers by confocal imaging.

[Regrowth of central respiratory pathways in neural graft. From research tool on the axonal regeneration to a strategy of post-traumatic reparation]. / Repousse des voies respiratoires centrales dans des greffes nerveuses. De l'outil de recherche sur la régénération axonale à une stratégie de réparation post-traumatique.

This review focuses on the regrowth of respiratory pathways after nerve grafting within the central nervous system of the adult rat. After a general presentation of the background and of the grafting procedure, we summarize our nerve grafting results of while it is now well established that severed axons of adult central neurons can regenerate within segments of peripheral nerve partially implanted within the brain or spinal cord, the functional properties of the regenerating neurons remain generally unknown. With a view to assessing the extent to which the functional capacities of central neurons can be maintained after axonal regeneration, we have carried out experiments on central respiratory neurons which are a good example of a highly organized neuronal network with characteristic patterns of spontaneous discharge. We have shown that axonal regrowth of central respiratory neurons was successfully induced in blind-ended medullary and spinal autografts implanted respectively within the respiratory centers of the medulla oblongata and within the cervical spinal cord at the level of descending respiratory pathways. The grafts consisted of true "supplementary nerve" in which normal afferent and efferent respiratory pathways were confirmed by recording respiratory unitary discharges from teased fibers within the grafts. The efferent discharges reflected the activity of central respiratory neurons that had regenerated axons within the grafts: these neurons manifested spontaneous activity and normal responsiveness to respiratory stimuli that resemble those of normal respiratory cells. In order to evaluate the possibility of experimental nerve banking, the feasibility of using short-term and long-term stored nerves as potential spinal nerve grafts was established using in vitro pre-degenerated nerve and cryopreserved nerve grafts after assessment of Schwann cell viability. The extent of respiratory reinnervation of the different grafts (medullary, spinal and stored nerve grafts) was compared. The discussion focuses on the main data and the strategy for future nerve grafting is evoked: functional characteristics of regenerating respiratory axons, extent of graft reinnervation, functional schwann cell survey within stored/grafted nerve and post-traumatic grafting.

In vitro pre-degenerated nerve autografts support CNS axonal regeneration.

In the present study we compared, in adult rats, the axonal regeneration of central respiratory neurons within autologous fresh (f-; grafted immediately after removal) and pre-degenerated (pd-; grafted after being stored during 3 days in saline at +8 degrees C) peripheral nerve grafts (PNGs) implanted within the C2 cervical spinal cord. The proximal end of the left peroneal nerve was implanted in the site of projection of medullary respiratory neurons (ventro-lateral quadrant) and the distal part of each nerve graft was left unconnected (blind-ended graft). PNGs were examined 2 to 4 months after grafting. Central neurons regenerating axons within the PNGs were studied by recording spontaneous unit activity from small strands teased from the grafts. In control f-PNGs (n = 9), 248 filaments had spontaneous activities, 58 of these were respiratory-related, i.e. had discharge patterns identical to those of normal respiratory (inspiratory and expiratory) neurons. The presence of regenerated nerve fibers with spontaneous unitary impulse traffic (n = 216) was found in all pd-PNGs (n = 5). Thirty-four had respiratory patterns identical to those found within f-PNGs and corresponded to efferent activity. No statistically significant differences in axonal regrowth were found between f- and pd-PNGs. In conclusion, f- and pd-PNGs were equally capable of promoting axonal regeneration of central neurons. The neural components (Schwann cells and others) required for axonal regeneration of adult central neurons are still effective following 3 days of in vitro peripheral nerve degeneration without special storage conditions (oxygenation, medium inducing ATP synthesis). These results have clinical implications for nerve graft surgery when time is required for typing the tissues of both donor and recipient (post-mortem allografts) or transportation of graft material.