Variations in amino acid neurotransmitters in the rat ventral spinal cord after hindlimb unloading.

We have measured by HPLC the neurotransmitter content in L(4) and L(5) spinal segmental levels in CONT rats, after 7 (HU7) and after 14 days (HU14) of hindlimb unloading. These segments are known to contain the hindlimb muscle motoneurons. The main result is the increase of two neuroexcitators (glutamate and aspartate) and two neuroinhibitors (glycine and GABA) at the L(5) spinal segmental level in HU7 group. Our data indicated that the neurotransmitter changes are restricted to spinal segmental level containing motoneurons from muscles which are strongly modified by HU condition.

Physiologically adaptive changes of the L5 afferent neurogram and of the rat soleus EMG activity during 14 days of hindlimb unloading and recovery.

The hindlimb unloading rat model (HU, Morey's model) is usually used to mimic and study neuromuscular changes that develop during spaceflights. This Earth-based model of microgravity induces a muscular atrophy of the slow postural muscle of hindlimbs, such as the soleus, a loss of strength, modifications of contraction kinetics, changes in histochemical and electrophoretical profiles and modifications of the tonic EMG activity. It has been suggested in the literature that some of these neuromuscular effects were due to a reduction of afferent feedback during HU. However, no direct data have confirmed this hypothesis. The aim of this study was to clearly establish if changes of the L5 afferent neurogram are closely related to the soleus EMG activity during and after 14 days of HU. Immediately after HU, the EMG activity of the soleus muscle disappeared and was associated with a decrease in the afferent neurogram. The soleus electromyographic and afferent activities remained lower than the pre-suspension levels until the sixth day of HU and were recovered between the sixth and the ninth day. On the twelfth and fourteenth days, they were increased beyond the pre-suspension levels. During the first recovery day, these activities were significantly higher than those on the fourteenth HU day and returned to the pre-suspension levels between the third and sixth recovery days. To conclude, our study directly demonstrates that the HU conditions cannot be considered as a functional deafferentation, as suggested in the literature, but only as a reduction of afferent information at the beginning of the HU period.

Dual effect of deafferentation on contractile characteristics and sarcoplasmic reticulum properties in rat soleus fibers.

The neural message is known to play a key role in muscle development and function. We analyzed the specific role of the afferent message on the functional regulation of two subcellular muscle components involved in the contractile mechanism: the contractile proteins and the sarcoplasmic reticulum (SR). Rats were submitted to bilateral deafferentation (DEAF group) by section of the dorsal roots L(3) to L(5) after laminectomy. Experiments were carried out in single skinned fibers of the soleus muscle. The maximal force developed by the contractile proteins was increased in the DEAF group compared with control, despite a decrease in muscle mass by 17%. The tension-pCa relationship was shifted toward lower calcium (Ca(2+)) concentrations. Different functional properties of the SR of DEAF soleus were examined by using caffeine-induced contractions. The caffeine sensitivity of the Ca(2+) release was decreased after deafferentation and ryanodine receptor 1 isoform was expressed at a lower level. The rate of Ca(2+) uptake was only slightly increased. The results underlined the dual effect of the afferent input on the functional regulation of both contractile proteins and SR.

Changes in rat soleus muscle phenotype consecutive to a growth in hypergravity followed by normogravity.

It has been demonstrated that a long-term stay in hypergravity (HG: 2G) modified the phenotype and the contractile properties of rat soleus muscle. The ability of this muscle to contract was drastically reduced, which is a sign of anticipated aging. Consequently, our aim was to determine whether rats conceived, born, and reared in hypergravity showed adaptative capacities in normogravity (NG: 1G). This study was performed on rats divided into two series: the first was reared in HG until 100 days and was submitted to normogravity until 115 to 220 postnatal days (HG-NG rats); the second was made up of age paired groups reared in normogravity (NG rats). The contractile, morphological, and phenotypical properties of soleus muscle were studied. Our results showed that the NG rats were characterized by coexpressions of slow and fast myosin, respectively, 76.5 and 23.5% at 115 days. During their postnatal maturation, the fast isoform was gradually replaced by slow myosin. At 220 days, the relative proportions were respectively 91.05% and 8.95%. From 115 to 220 days, the HG-NG rats expressed 100% of slow myosin isoform and they presented a slower contractile behavior compared with their age-matched groups; at 115 days, the whole muscle contraction time was increased by 35%, and by 15%, at 220 days. Our study underlined the importance of gravity in the muscular development and suggested the existence of critical periods in muscle phenotype installation.

Enhancement of hybrid-fiber types in rat soleus muscle after clenbuterol administration during hindlimb unloading.

Our objective was to determine the effects of a clenbuterol (CB) treatment orally administered (2 mg per kg) to rats submitted to 14 days of hindlimb unloading (HU). The morphological and the contractile properties as well as the myosin heavy chain isoforms contained in each fiber type were determined in whole soleus muscles. As classically described after HU, a decrease in muscle wet weight and in body mass associated with a loss of muscular force, an evolution of the contractile parameters towards those of a fast muscle type, and the emergence of fast myosin heavy chain isoforms were observed. The CB treatment in the HU rats helped reduce the decrease in 1) muscle and body weights, 2) force and 3) the proportion of slow fibers, without preventing the emergence of fast myosin isoforms. Clenbuterol induced a complex remodelling of the muscle typing promoting the combination of both slow and fast myosin isoforms within one fiber. To conclude, our data demonstrate that CB administration partially counteracts the effects produced by HU, and they allow us to anticipate advances in the treatment of muscular atrophy.

Preliminary results and evidence of early regeneration in inferior alveolar nerve fibers.

Inferior alveolar nerve (IAN) damage can occur in trauma, cyst enucleation, sagittal split osteotomy or third molar removal, and the consequences are a loss of sensation to the mandibular teeth, gingiva and lower lip. Because of its anatomical position in a bony canal, IAN suture is rarely evoked. The aim of this study was to demonstrate the reality of IAN regeneration by using electrophysiological and histological methods after experimental section and suture of this nerve in rabbits. Nine adult female animals were used for the experiments. Six months after section and suturing using 10.0 nylon with a conventional technique, electrical stimulation of the nerve was performed to record electrophysiological activity. Each rabbit was its own reference. In each case, an action potential was recorded after microsurgical repair and definitively suppressed by section of the nerve. Morphometric analysis showed a decrease in the number of nerve fibers in the operated nerve versus the control nerve. The histological study showed an increase in nerve fibers with a cross-sectional area of 19-36 and 37-73 micro m(2) and a decrease in the smaller fibers (2-4 and 5-7 micro m(2)). This preliminary study confirms the possibility of nerve regeneration in rabbits 6 months after section and conventional suturing.

Expression of myosin heavy chain isoforms along intrafusal fibers of rat soleus muscle spindles after 14 days of hindlimb unloading.

Morphological, contractile, histochemical, and electrophoretical characteristics of slow postural muscles are altered after hindlimb unloading (HU). However, very few data on intrafusal fibers (IFs) are available. Our aim was to determine the effects of 14 days of hindlimb unloading on the morphological and immunohistochemical characteristics of IF in rat soleus muscle. Thirty-three control and 32 unloaded spindles were analyzed. The number and distribution of muscle spindles did not appear to be affected after unloading. There was no significant difference in number, cross-sectional area, and histochemical properties of IF between the two groups. However, after unloading, a significant decrease in slow type 1 MHC isoform and a slight increase in slow-tonic MHC expression were observed in the B and C regions of the bag1 fibers. The alpha-cardiac MHC expression was significantly decreased along the entire length of the bag2 fibers and in the B and C regions of the bag1 fibers. In 12 muscle spindles, the chain fibers expressed the slow type 1 and alpha-cardiac MHC isoforms over a short distance of the A region, although these isoforms are not normally expressed. The most striking finding of the study was the relative resistance of muscle spindles to perturbation induced by HU.

Contractile properties and myosin expression in rats born and reared in hypergravity.

The effects of hypergravity (HG) on soleus and plantaris muscles were studied in Long Evans rats aged 100 days, born and reared in 2-g conditions (HG group). The morphological and contractile properties and the myosin heavy chain (MHC) content were examined in whole muscles and compared with terrestrial control (Cont) age-paired rats. The growth of HG rats was slowed compared with Cont rats. A decrease in absolute muscle weight was observed. An increase in fiber cross-sectional area/muscle wet weight was demonstrated, associated with an increase in relative maximal tension. The soleus muscle changed into a slower type both in contractile parameters and in MHC content, since HG soleus contained only the MHC I isoform. The HG plantaris muscle presented a faster contractile behavior. Moreover, the diversity of hybrid fiber types expressing multiple MHC isoforms (including MHC IIB and MHC IIX isoforms) was increased in plantaris muscle after HG. Thus the HG environment appears as an important inductor of muscular plasticity both in slow and fast muscle types.

Effect of the beta(2)-agonist clenbuterol on the locomotor activity of rat submitted to a 14-day period of hypodynamia-hypokinesia.

The beta(2)-adrenergic agonist clenbuterol is known for its anabolic action on normal and atrophied muscles. The aim of this work was to evaluate if chronic clenbuterol administration could prevent alterations in the locomotor activity induced by hindlimb suspension. The effects of clenbuterol were evaluated in three studies: muscle morphological characteristics, observation of locomotor movement and electromyographic activity of soleus and gastrocnemius muscles. Rats were divided into four groups: control (CON, morphological study only), hindlimb suspended (HS), clenbuterol administered (CB, 2 mg kg(-1) per day in drinking water), and hindlimb suspended+clenbuterol administered (HSCB). The soleus muscle weight was reduced in the two suspended groups (HS and HSCB) but did not change after clenbuterol treatment. By contrast, the gastrocnemius weight was not affected by suspension but was increased by clenbuterol (CB and HSCB). Some locomotor deficits were always observed in HS rats (unstable gait, ankle hyperextension, ellipsis). Clenbuterol administration did not prevent these perturbations. Cycle duration and soleus burst duration were increased in the three groups. Soleus mean EMG (burst area/duration) was decreased in HS rats, but not in the two other groups. For the gastrocnemius, burst duration was increased in CB rats, decreased in HSCB rats and unchanged in HS ones; mean EMG did not change. In conclusion, clenbuterol cannot be used as a countermeasure to reduce the alteration in locomotor performance. Moreover, our results suggest that this alteration is specifically related to changes in neuronal properties.

Plasticity of monkey triceps muscle fibers in microgravity conditions.

We examined the changes in functional properties of triceps brachii skinned fibers from monkeys flown aboard the BION 11 satellite for 14 days and after ground-based arm immobilization. The composition of myosin heavy chain (MHC) isoforms allowed the identification of pure fibers containing type I (slow) or type IIa (fast) MHC isoforms or hybrid fibers coexpressing predominantly slow (hybrid slow; HS) or fast (hybrid fast) MHC isoforms. The ratio of HS fibers to the whole slow population was higher after flight (28%) than in the control population (7%), and the number of fast fibers was increased (up to 86% in flight vs. 12% in control). Diameters and maximal tensions of slow fibers were decreased after flight. The tension-pCa curves of slow and fast fibers were modified, with a decrease in pCa threshold and an increase in steepness. The proper effect of microgravity was distinguishable from that of immobilization, which induced less marked slow-to-fast transitions (only 59% of fast fibers) and changed the tension-pCa relationships.

Effects of cutaneous receptor stimulation on muscular atrophy developed in hindlimb unloading condition.

The aim of this study was to investigate whether stimulation of the cutaneous mechanoreceptors of the rat foot sole could partially or totally prevent the soleus muscle atrophy developed after 14 days in hindlimb unloading conditions. Final experiments were achieved under deep anesthesia using pentobarbital sodium (60 mg/kg, ip injection). Atrophy was characterized by a significant decrease in muscle wet weight, fiber size, maximal twitch and tetanic tensions, contraction kinetics, and histochemical and electrophoretical changes. Our data demonstrate that the stimulation of these mechanoreceptors partially prevents the decrease in muscle weight (53%) and cross-sectional area of the soleus muscle (36%) and in all fiber types (type I: 31%; type Ic: 40%; type IIc: 49%; and type IIa: 44%) and also prevented the reductions in strength (peak twitch tension: 31%; peak tetanic tension: 25%). However, the decrease in contraction kinetics was not counteracted. Moreover, histochemical and electrophoretical changes were partially slowed. Thus our results suggest that stimulation of the sole mechanoreceptors can be used, in part, as a countermeasure to the muscular atrophy observed after a period of hindlimb unloading.

Phenotypic changes in the composition of muscular fibres in rat soleus motor units after 14 days of hindlimb unloading.

The main goal of this study was to identify the different fibre types of the motor units (MUs) contained in the soleus muscles from control (CONT) rats and from rats submitted to 14 days of hindlimb unloading (HU). The MU types were classified according to their contractile properties and also using glycogen depletion followed by adenosine triphosphatase (ATPase) staining. In CONT rats, the soleus muscle contained two MU types identified as slow and fast types. After HU, the MU distribution showed three populations: slow, intermediate and fast. All the MUs from HU soleus were heterogeneous in terms of fibre type composition, indicating a complex remodelling of the muscle.

Effect of spaceflight on single fiber function of triceps and biceps muscles in rhesus monkeys.

Primates appeared to be a good model for investigating muscle contractile and biochemical properties, as well as EMG recordings. The purpose of our study was to examine the effects of microgravity on the contractile properties of the slow-type triceps and fast-type biceps muscles during the 14-day Bion 11 spaceflight.

Short-term plasticity in primary somatosensory cortex of the rat after hindlimb suspension.

Since the last 25 years, the cortex is considered as a dynamic entity, susceptible of changes. Various types of modifications in stimuli may lead to the plasticity of the target neurons. These include immobilisation, denervation, amputation, deafferentation... In the somatosensory system, the most important changes are a substantial reorganisation of the cortical somatotopic representation, and an enlargement of the receptive fields (RF) of cortical neurons. Hindlimb suspension (HS) is characterized by the absence of weight-bearing and a reduced motor activity. In these conditions, the cutaneous receptors located on the foot sole are deactivated. Our hypothesis is that this condition of HS can produce a reorganisation of the somatosensory cortex (SmI) and a modification in the size of the cutaneous RF.

Differential effects of a six-day immobilization on newborn rat soleus muscles at two developmental stages.

Our objective was to determine the effects of a six-day immobilization on the musculoskeletal system of the rat during postnatal development at two key periods when the states of innervation are known to be different. This work was undertaken on the soleus muscle since it is well known that postural slow muscles show marked changes after a period of disuse. Thus, the soleus muscle was immobilized in a shortened position either when the innervation was polyneuronal or monosynaptic, respectively from 6 to 12 and from 17 to 23 days. The muscle modifications were followed by ATPase staining and myosin heavy chain (MyHC) isoform identification using monoclonal antibodies and SDS-PAGE. The functional properties of skinned fibre bundles were established by calcium/strontium (Ca/Sr) activation characteristics. In control muscles the maturation was characterized by a progressive increase of adult MyHCs (I and IIA) concomitant with a decrease in both the MyHC neo and the Ca affinity. Between 6 to 12 days, immobilization of the limb induced an increase in histochemical type IIC fibres. Using antibodies we identified new fibre types, classified as a function of their MyHC isoform co-expression. We observed an increase in expression of both MyHC neo and Ca affinity. From 17 to 23 days, the immobilization induced an increase in Ca affinity and marked changes in the MyHC isoform composition: disappearance of MyHC neo and expression of the fast MyHC IIB isoform, which in normal conditions is never expressed in the soleus muscle. We conclude that an immobilization imposed during polyneuronal innervation delays the postnatal maturation of the soleus muscle, whereas when the immobilization is performed under monosynaptic innervation the muscle evolves towards a fast phenotype using a default pathway for MyHC expression.

Contractile properties and myosin heavy chain composition of newborn rat soleus muscles at different stages of postnatal development.

This study was undertaken to correlate some of the functional characteristics with the myofibrillar composition in myosin heavy chain isoforms on newborn and adult rat soleus muscles. The following postnatal ages were chosen in order to determine the role of innervation in the establishment of the mature muscle phenotype: before (postnatal day 6), when (postnatal day 12), and after (days 17 and 23) the monosynaptic innervation appeared. The steady state of definitive innervation was controlled on adult muscles (i.e. approximately 13 weeks). Muscle maturation was followed by ATPase staining and fibre diversity was observed at postnatal day 12. The functional properties of skinned bundles isolated from newborn rats were determined by Calcium/Strontium activation characteristics (Tension/pCa and pSr relationships). From postnatal days 6 to 17, the Soleus bundles exhibited Calcium/Strontium activation characteristics intermediate between slow and fast fibre populations previously described in muscles. At day 23, the Calcium/Strontium activation characteristics of the soleus were closer to those of a slow type. Moreover, we observed a decrease in Ca affinity concomitant with the installation of the monosynaptic innervation, and an increase of the slow type I during postnatal development. Finally, this work reported a greater correlation between the Calcium/Strontium activation parameters and the myosin heavy chain isoform composition at the postnatal days when the mature monosynaptic innervation pattern occurred.

Differential adaptation to weightlessness of functional and structural characteristics of rat hindlimb muscles.

NASA: Soleus, vastus intermedius, tibialis anterior, and extensor digitorum longus muscles were removed from rats following space flight onboard the SLS-2 mission and from control animals. Muscle tissues were studied for their calcium and strontium activated tension characteristics and for structural changes. Muscles were also examined for myosin composition using electrophoresis. Results indicate that changes occurred in structural and functional muscle characteristics in both slow and fast muscle fiber types. These results are detailed and discussed.^ieng

Structure-function relationship of soleus muscle fibres from the rhesus monkey.

Functional and structural properties of rhesus monkey skinned fibres were studied in order to examine the relationship between calcium/strontium (Ca/Sr) activation characteristics and protein composition. The fibres were classified according to their Ca/Sr affinity into slow (61%) and fast groups (39%). According to the myosin isoform composition, two additional hybrid types were defined. Thus, four profiles were characterized: two corresponding to slow (S) and fast (F) isoforms and two corresponding to a mixed proportion of slow and fast isoforms. They were called hybrid slow (HS) or hybrid fast (HF) based on the predominant myosin isoform. Tension/pCa parameters and maximal shortening velocities were determined. S fibres showed a higher pCa threshold and affinity as well as shallower slopes of their tension/pCa curve than did F fibres. HS and HF fibres exhibited tension/pCa curves which were positioned close to those of S and F fibres, respectively. No significant difference was observed between S and HS fibres or between F and HF fibres. Maximal shortening velocity values were higher for fibres expressing predominantly fast myosin isoforms. We suggest than when both S and F isoforms of myofibrillar proteins are expressed in a muscle fibre, the functional properties are mainly governed by the predominant isoform.