A transcutaneous wire interface for small mammals using an expanded PTFE patch.

The objective of this paper is to provide the reader with a method of surgical implantation of a transcutaneous wire interface for chronic instrumentation of small mammals, utilizing a non-bioreactive expanded polytetrafluoroethylene (ePTFE) material (W.L. Gore and Associates, Inc., Flagstaff, AZ). We describe the implant assembly, as well as details of the surgical implantation, which will facilitate successful data acquisition. EMG signal from the implant is of a high quality during both rest and activity of the animal and the signal quality is maintained up to at least 7 weeks post-implantation without any sign of infection or other adverse reaction. This represents an improvement in the viability of long-term physiological signal collection via a surgically implanted back plug.

Is the spring quality of muscle plastic?

During locomotion, major muscle groups are often activated cyclically. This alternate stretch-shorten pattern of activity could enable muscle to function as a spring, storing and recovering elastic recoil potential energy. Because the ability to store and recover elastic recoil energy could profoundly affect the energetics of locomotion, one might expect this to be an adaptable feature of skeletal muscle. This study tests the hypothesis that chronic eccentric (Ecc) training results in a change in the spring properties of skeletal muscle. Nine female Sprague-Dawley rats underwent chronic Ecc training for 8 wk on a motorized treadmill. The spring properties of muscle were characterized by both active and passive lengthening force productions. A single "spring constant (Deltaforce/Deltalength) from the passive length-tension curves was calculated for each muscle. Results from measurements on long heads of triceps brachii muscle indicate that the trained group produced significantly more passive lengthening force (P = 0.0001) as well as more active lengthening force (P = 0.0001) at all lengths of muscle stretch. In addition, the spring constants were significantly different between the Ecc (1.71 N/mm) and the control (1.31 N/mm) groups. A stiffer spring is capable of storing more energy per unit length stretched, which is of functional importance during locomotion.

Eccentric ergometry: increases in locomotor muscle size and strength at low training intensities.

Lengthening (eccentric) muscle contractions are characterized by several unusual properties that may result in unique skeletal muscle adaptations. In particular, high forces are produced with very little energy demand. Eccentrically trained muscles gain strength, but the specific nature of fiber size and composition is poorly known. This study assesses the structural and functional changes that occur to normal locomotor muscle after chronic eccentric ergometry at training intensities, measured as oxygen uptake, that do not influence the muscle when exercised concentrically. Male subjects trained on either eccentric or concentric cycle ergometers for 8 wk at a training intensity starting at 54% and ending at 65% of their peak heart rates. The isometric leg strength increased significantly in the eccentrically trained group by 36%, as did the cross-sectional area of the muscle fiber by 52%, but the muscle ultrastructure remained unchanged. There were no changes in either fiber size, composition, or isometric strength in the concentrically trained group. The responses of muscle to eccentric training appear to be similar to resistance training.

Fiber size and myosin phenotypes of selected rhesus lower limb muscles after a 14-day spaceflight.

Muscle biopsies were taken from the rhesus (Macaca mulatta) soleus (Sol, a slow ankle extensor), medial gastrocnemius (MG, a fast ankle extensor), tibialis anterior (TA, a fast ankle flexor), and vastus lateralis (VL, a fast knee extensor) muscles in vivarium controls (n=5) before and after either a 14-day spaceflight (Bion 11, n=2) or a 14-day ground-based flight simulation (n=3). Myosin heavy chain (MHC) composition (gel electrophoresis), fiber type distribution (immunohistochemistry), and fiber size were determined. Although there were no significant changes, each muscle showed trends towards adaptation.

Fiber size and myosin phenotypes of selected Rhesus hindlimb muscles after a 14-day spaceflight.

Open muscle biopsies were obtained from Rhesus soleus (slow ankle extensor), medial gastrocnemius (fast ankle extensor) and tibialis anterior (fast ankle flexor) muscles before and after either a 14-day spaceflight (BION 11, n=2) or ground-based flight simulation (n=3) and in time-matched controls (n=5). Fiber type distribution (immunohistochemistry), myosin heavy chain (MHC) composition (gel electrophoresis) and fiber size were determined. There was a large amount of inter-animal variability and there were no significant pre-post differences for any variable under any condition for any muscle studied. However, each muscle showed trends towards adaptation. Based on the immunohistochemical analyses, the percentage of type I fibers in the soleus was 68 and 86% in pre and 43 and 70% in post biopsies of the simulation and flight groups. The number of hybrid (containing both fast and slow MHC) fibers increased in both groups. MHC composition changed in a similar direction. Type I and hybrid fibers were 23 and 31% smaller after than before flight. In the medial gastrocnemius, type I fibers were 16, 14 and 32% smaller in post compared to pre biopsies in control, simulation and flight Rhesus. In the tibialis anterior, type I fibers were approximately 14% smaller in post- than pre-flight biopsies. As expected the soleus, a slow anti-gravity muscle, was most affected after 14 days of weightlessness. Further, slow fibers in each muscle were more responsive to microgravity than fast fibers. All changes, however, were smaller than those observed in rats after the same duration of flight. This differential effect may be related to the partial restraint of Rhesus in the chaired position compared to the free-floating position of rats in the cage and/or to differences in the contractile protein turnover rates between species.

Cyclical passive stretch influences the mechanical properties of the inactive cat soleus.

The effects of cyclical, passive manipulation (PM, 30 min day(-1), 5 days week(-1) for 6 months) mimicking the length excursions observed during stepping on the mechanical and associated biochemical properties of the inactive cat soleus muscle were determined in five cats. Inactivity was produced via spinal cord isolation (SI), i.e. complete spinal cord transections at low thoracic and high sacral levels and bilateral dorsal rhizotomy between the transection sites. Passive manipulation was administered to one leg of each SI cat. Compared with normal controls, SI resulted in approximately 70% decrease in weight, an 80% decrease in maximum tetanic tension (Po) and an approximately 100% increase in maximum rate of shortening (Vmax) and myosin adenosine triphosphatase (mATPase) activity of the soleus. The passive manipulation regime partially ameliorated these effects. When compared with the control SI soleus, the SI-PM soleus weight and maximum tetanic tension were 12 and 21% higher, respectively, and the Vmax and mATPase activity 21 and 12% (p > 0.05) lower, respectively. Thus, inactivity resulted in a smaller and faster muscle, whereas passive manipulation for only 30 min a day tended to maintain these properties closer to normal control values. The results suggest a potential therapeutic effect of short bouts of cyclical, passive manipulation on otherwise inactive skeletal muscles.

Myosin heavy chain mRNA and protein expression in single fibers of the rat soleus following reinnervation.

Myosin heavy chain (MHC) expression is regulated by many factors including neural input. To gain a better understanding of myosin transformation following reinnervation we examined both MHC protein and mRNA in single fibers of the soleus. A midthigh sciatic nerve lesion resulted in reinnervation of the soleus by motoneurons from both original and foreign motor pools. MHC expression was examined in individual fibers 8 and 16 weeks post injury in situ histochemistry and immunohistochemistry. Following a sciatic nerve lesion, the reinnervated soleus underwent a transformation from slow toward fast based on physiologic and biochemical measurements. At 8 weeks, fast MHC mRNA isoforms (IIa and IIx) were upregulated and slow mRNA was downregulated, however, the predominant protein isoform was MHC I. At both 8 and 16 weeks, many fibers expressed multiple mRNA isoforms. At 16 weeks there was limited co-expression of slow and fast MHC mRNAs, but continued co-expression of fast MHC mRNAs. Sixteen weeks following reinnervation the predominant fast mRNA and protein in the soleus was IIx MHC.

Skeletal muscle fiber cross-sectional area: effects of freezing procedures.

The cross-sectional area (CSA) of individual fibers is an important measure of skeletal muscle plasticity. To investigate the effects of different freezing procedures on CSA measurements, the CSA of type-identified fibers in the cat tibialis anterior were determined following quick-freezing the muscle at a fixed physiological length (ipsilateral, frozen at length) and compared to the fiber CSA following quick-freezing a mid-portion of the muscle where the fibers were allowed to freely shorten during tissue preparation (contralateral, frozen as a block). The mean CSA of each fiber type was significantly smaller in the muscles frozen at length vs. frozen as a block in both a deep (close to the bone) and a superficial (away from the bone) region of the muscle, except for the slow oxidative (SO) fibers in the superficial region. The percent difference in mean fiber CSA was smaller for the SO compared to the fast oxidative glycolytic and fast glycolytic fibers in both the deep (41, 47 and 56%, respectively) and superficial (20, 36 and 48%, respectively) regions. In addition, the differences in the mean CSAs of the fast fibers between the two freezing procedures were approximately 10% larger in the deep compared to the superficial region. The fiber-type differential responses may be related, at least in part, to the architectural features of the fibers. These data indicate that the freezing procedures used to prepare the muscle tissue are an important consideration when determining the CSA of individual skeletal muscle fibers and consequently the specific tension, tension per unit CSA, of the muscle units.

Further evidence of incomplete neural control of muscle properties in cat tibialis anterior motor units.

To examine the influence of a motoneuron in maintaining the phenotype of the muscle fibers it innervates, myosin heavy chain (MHC) expression, succinate dehydrogenase (SDH) activity, and cross-sectional area (CSA) of a sample of fibers belonging to a motor unit were studied in the cat tibialis anterior 6 mo after the nerve branches innervating the anterior compartment were cut and sutured near the point of entry into the muscle. The mean, range, and coefficient of variation for the SDH activity and the CSA for both motor unit and non-motor unit fibers for each MHC profile and from each control and each self-reinnervated muscle studied was obtained. Eight motor units were isolated from self-reinnervated muscles using standard ventral root filament testing techniques, tested physiologically, and compared with four motor units from control muscles. Motor units from self-reinnervated muscles could be classified into the same physiological types as those found in control tibialis anterior muscles. The muscle fibers belonging to a unit were depleted of glycogen via repetitive stimulation and identified in periodic acid-Schiff-stained frozen sections. Whereas muscle fibers in control units expressed similar MHCs, each motor unit from self-reinnervated muscles contained a mixture of fiber types. In each motor unit, however, there was a predominance of fibers with the same MHC profile. The relative differences in the mean SDH activities found among fibers of different MHC profiles within a unit after self-reinnervation and those found among fibers in control muscles were similar, i.e., fast-2 < fast-1 < or = slow MHC fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional and cellular adaptation to weightlessness in primates.

Considerable data has been collected on the response of hindlimb muscles to unloading due to both spaceflight and hindlimb suspension. One generalized response to a reduction in load is muscle fiber atrophy, although not all muscles respond the same. For example, predominantly slow extensor muscles like the Sol exhibit a large reduction in fiber size to unloading, while fast extensors like the plantaris and fast flexors like the tibialis anterior show little, if any, atrophy. Our understanding of how muscles respond to microgravity, however, has come primarily from the examination of hindlimb muscles in the unrestrained rat in space. The non-human primate spaceflight paradigm differs considerably from the rodent paradigm in that the monkeys are restrained, usually in a sitting position, while in space. Recently, we examined the effects of microgravity on muscles of the Rhesus monkey by taking biopsies of selected hindlimb muscles prior to and following spaceflights of 14 and 12 day durations (Cosmos 2044 and 2229). Our results revealed that the monkey's response to microgravity differs from that of the rat. The apparent differences in the atrophic response of the hindlimb muscles of the monkey and rat to spaceflight may be attributed to 1) a species difference, 2) a difference in the manner in which the animals were maintained during the flight (i.e., chair restraint or "free-floating"), and/or 3) an ability of the monkeys to counteract the effects of spaceflight with resistive exercise.

Adaptations in myosin heavy chain profile in chronically unloaded muscles.

In this review, myosin heavy chain (MHC) adaptations in response to several models of decreased neuromuscular activity (i.e. electrical activation and loading of a muscle) are evaluated. In each of these "reduced-activity" models it is important to: a) quantify the changes in electrical activation of the muscle as a result of the intervention; b) quantify the forces generated by the muscle; and c) determine whether the neuromuscular junction remains normal. Most of the models, including spaceflight, hindlimb suspension, spinal cord isolation, spinal cord transection, denervation, and limb immobilization in a shortened position, result in increases in the percentage of fast MHCs (or fast MHC mRNA) in normally slow rat muscles. It also can be inferred from histochemical data that increases in fast MHCs occur with TTX application and bed rest. The only "reduced-activity" model to consistently increase slow muscle myosin mRNA, and slow fibers is limb immobilization in a stretched position; however, this model results in at least a temporary increase in tension. It appears that the most common feature of these models that might induce MHC adaptations is the modification in loading rather than a change in the neuromuscular activity.

Level of independence of motor unit properties from neuromuscular activity.

Neuromuscular activity was eliminated in the tibialis anterior muscle of adult cats for 6 months by spinal isolation (SI), i.e., complete spinal cord transections at T-12-13 and at L-7-S-1, plus bilateral dorsal rhizotomy between the two transection sites. One motor unit from each muscle was isolated using ventral root teasing procedures and physiologically tested. The fibers belonging to each motor unit were visualized in PAS-stained sections by the loss of glycogen following prolonged repetitive stimulation. Qualitatively, the normal enzymatic interrelationships among fibers identified by myosin heavy chain composition were unchanged by SI. Generally, each motor unit from SI cats were of a single myosin immunohistochemical type. The same physiological motor unit types that typify control muscles were found in SI cats. In SI compared to control cats, there was approximately a 10% increase in the number of muscle fibers expressing fast myosin. Mean fiber activity levels of ATPase and SDH for a given fiber type (based on MHC antibody reactions) decreased by approximately 10% and 25%, whereas GPD activity increased approximately 35%. It is concluded that differential levels or patterns of activity are not essential to maintain the range of histochemical and physiological motor unit types found in the tibialis anterior of normal adult cats.

Interactive effects of growth hormone and exercise on muscle mass in suspended rats.

Measures to attenuate muscle atrophy in rats in response to stimulated microgravity [hindlimb suspension (HS)] have been only partially successful. In the present study, hypophysectomized rats were in HS for 7 days, and the effects of recombinant human growth hormone (GH), exercise (Ex), or GH+Ex on the weights, protein concentrations, and fiber cross-sectional areas (CSAs) of hindlimb muscles were determined. The weights of four extensor muscles, i.e., the soleus (Sol), medial (MG) and lateral (LG) gastrocnemius, and plantaris (Plt), and one adductor, i.e., the adductor longus (AL), were decreased by 10-22% after HS. Fiber CSAs were decreased by 34% in the Sol and by 17% in the MG after HS. In contrast, two flexors, i.e., the tibialis anterior (TA) and extensor digitorum longus (EDL), did not atrophy. In HS rats, GH treatment alone maintained the weights of the fast extensors (MG, LG, Plt) and flexors (TA, EDL) at or above those of control rats. This effect was not observed in the slow extensor (Sol) or AL. Exercise had no significant effect on the weight of any muscle in HS rats. A combination of GH and Ex treatments yielded a significant increase in the weights of the fast extensors and in the CSA of both fast and slow fibers of the MG and significantly increased Sol weight and CSA of the slow fibers of the Sol. The AL was not responsive to either GH or Ex treatments. Protein concentrations of the Sol and MG were higher only in the Sol of Ex and GH + Ex rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of incomplete neural control of motor unit properties in cat tibialis anterior after self-reinnervation.

1. The mechanical, morphological and biochemical properties of single motor units from the anterior compartment of the tibialis anterior muscle in adult cats were studied six months after the nerve branches to that compartment were cut and resutured in close proximity to the muscle. 2. In these self-reinnervated muscles, the maximum tetanic tensions were lower in slow than fast units, a relationship similar to that observed among motor units from control adult muscles. The maximum tetanic tensions produced by the fast units were larger than those produced by the same motor unit types in control muscles. Direct counts of muscle fibres belonging to a motor unit showed that factors controlling the number of muscle fibres innervated by a motoneurone type persist during the reinnervation process in that fast motoneurones reinnervated more muscle fibres than slow motoneurones. Thus, the number of muscle fibres reinnervated by a motoneurone principally accounted for the difference in the maximum tension outputs among motor unit types, a relationship similar to that observed in control tibialis anterior muscles. 3. Monoclonal antibodies for specific myosin heavy chains were used to differentiate fibre types. By this criterion, motor units from control muscles were found to contain a homogeneous fibre type composition. In contrast, a heterogeneous, yet markedly biased, fibre type composition was observed in each unit analysed from self-reinnervated muscles. 4. Although not all of the muscle fibres of a motor unit developed the same type-associated parameters after reinnervation, the relationships among myosin heavy chain profile, succinate dehydrogenase activity and the fibre size were similar in fibres of control and self-reinnervated muscles. 5. The processes which dictate both motor unit size and the matching between motoneurone and muscle fibre type during the reinnervation process must be interdependent and result from a hierarchy of decisions which reflects their relative importance. The mechanisms responsible for these two processes may be a combination of: (1) selective innervation which may or may not incorporate a pruning process if multiple synaptic connections are initially formed and/or (2) conversion of enough fibres of a motor unit to form a predominant type.

Cloning and in situ hybridization of type 2A and 2B rat skeletal muscle myosin tail region: implications for filament assembly.

Changes in fast myosin expression play a critical role in skeletal muscle adaptation. Two fast myosin isoforms, type 2A and type 2B, are commonly expressed by fast muscle fibers but their sequences have not been determined to allow mRNA expression studies. A complete set of rat skeletal muscle myosins was amplified by PCR of cDNAs derived from skeletal muscle mRNA, cloned in a TA cloning vector, and sequenced. Specificity was demonstrated by in situ hybridization against skeletal muscle and myosin protein identification using monoclonal antibodies. Two novel sequences were cloned: A type 2A myosin which consisted of a 642 bp segment from the 3' end and a type 2B myosin which consisted of a 624 bp segment also from the 3' end. This region encodes that portion of the myosin molecule implicated in the control of filament assembly. The two fast myosins showed 88% homology in the open reading frame and 95% homology at the amino acid level. Based on this homology, it is unlikely that selective myosin filament assembly occurs during muscle fiber type transformation between type 2A and 2B.

Fibre size and type adaptations to spinal isolation and cyclical passive stretch in cat hindlimb.

Impulse activity is known to have a strong influence in determining the characteristics that distinguish skeletal muscle fibres into types. The control of muscle proteins by the neural systems that innervate the muscles, however, is not complete (Edgerton et al. 1985, 1990). The purpose of the present study, therefore, was to determine the effects of inactivity for 6 months on the size and fibre type composition of selected cat hindlimb muscles. Inactivity was produced by isolating the lumbar region of the spinal cord, i.e. transecting the cord at T12-T13 and again at L7-.S1 and then performing a bilateral dorsal rhizotomy between the transection sites (SI). In each SI cat, one hindlimb was passively manipulated for 30 min per day through a range of motion at the ankle mimicking a step cycle. SI resulted in an atrophic response in most muscles, with predominantly slow extensors showing the largest effect. In general, the predominant fibre type, which also had the largest mean size, in each muscle atrophied the most. The mean fibre size of all fibre types were similar after SI, suggesting that there may be a minimal size for inactive intact fibres. In comparison with control animals, all muscles in the SI cats had a higher proportion of fast fibres. Further, the relative contribution of the slow fibres to the total cross-sectional area of the muscle was decreased following SI. Some slow fibres in each muscle, however, were resistant to change. These data demonstrate the extent to which size and myosin type of mammalian muscle fibres are independent of activation characteristics.

Mechanical and morphological properties of chronically inactive cat tibialis anterior motor units.

1. The lumbar spinal cord was functionally isolated in ten cats by cord transection at the junctions of segments T12-T13 and L7-S1 and cutting bilaterally all dorsal roots between the two transections. Two 24 h EMG recording sessions were used to verify that muscles in the lower limb were virtually electrically silent. The cats were maintained in excellent health for 6 months. 2. Six months after spinal cord isolation, an acute experiment was performed to isolate a single motor unit from the tibialis anterior of each hindlimb using ventral root splitting techniques. Each motor unit was characterized physiologically as either fast fatigable (FF, n = 11), fast fatigue resistant (FR, n = 4), fast intermediate (FI, n = 2), or slow (S, n = 1), and repetitively stimulated to deplete the motor unit of its glycogen. 3. Maximum tensions of the fast motor units were lower than mean maximum tensions of control, whereas the S motor unit remained within the range observed in controls. In general, the isometric contractile properties, as well as fatigability, were within the ranges for each of the motor unit types in control cats. The mean fibre cross-sectional areas of the fibres within the FR and FF motor units were approximately 40 and 50% smaller than control, while the mean fibre size of the fibers within the S motor unit was similar to control. 4. Innervation ratios and specific tensions for all experimental motor units were within the ranges of those reported for tibialis anterior motor units in control cats. Thus, it appears that the decrease in maximum tension of the fast motor units was primarily related to a reduction in fibre size. 5. The spatial distribution of the fibres within fast motor units of a spinally isolated cat, as measured by interfibre distances of the motor unit fibres, was similar to that reported for control tibialis anterior motor units. 6. These data suggest that factors independent of activity play a prominent, if not dominant, role in maintaining the complement of motor unit types typical of adult cat muscles. In addition, normal innervation patterns appear to be maintained in the absence of activity.

EMG patterns of rat ankle extensors and flexors during treadmill locomotion and swimming.

Intramuscular electromyography (EMG) was used to determine and compare the recruitment patterns of the rat soleus (Sol), tibialis anterior (TA), and a deep and a superficial portion of the medial gastrocnemius (MG) during treadmill locomotion at various speeds and inclines and during swimming. Raw EMG signals for 10-20 step or stroke cycles were rectified, averaged, and processed to determine cycle period (EMG onset of one cycle to EMG onset of the next cycle), EMG burst duration, and integrated area of the rectified burst (IEMG). Mean EMG per burst was calculated as IEMG/burst duration. IEMG/min was calculated as IEMG times the number of bursts (cycles) per minute. Cycle period and burst duration of the extensors decreased hyperbolically, while the TA burst duration was unchanged, with increased treadmill speed. With increased treadmill speed, IEMG was decreased in the Sol and unchanged in the MG and TA, whereas IEMG/min decreased in the Sol and increased in the MG and TA. An elevation in treadmill incline resulted in an increase in the activation levels of the MG but not in the Sol or TA. These data indicate that the additional power required at increased speeds and/or inclines of treadmill locomotion is derived from the recruitment of the fast extensors, e.g., the MG. The mean cycle period during swimming was similar to that observed during the fastest treadmill locomotion. EMG burst durations and amplitudes, however, were higher in the TA, relatively similar in the MG, and lower in the Sol during swimming than treadmill locomotion.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of 7 days of hindlimb suspension and intermittent weight support on rat muscle mechanical properties.

Unloading the rat hindlimb results in a decrease in mass, especially in those muscles that normally have a load-bearing function. The present study was designed to evaluate the effect of intermittent periods of weight support in ameliorating this atrophic response. Adult male Sprague-Dawley rats were assigned to either a control (CON), a hindlimb suspended (HS), or a hindlimb suspended plus intermittent weight support (HS-WS) group. HS-WS rats were walked slowly on a treadmill at 0.2 m/s and a 19% incline for 10 min, every 6 h. After 7 d, the in situ mechanical properties of the soleus (Sol) and medial gastrocnemius (MG) were studied. Body weights of HS and HS-WS rats were 9 and 13% lower than CON. The SOl weight relative to body weight was 21 and 9% lower in HS and HS-WS than CON. Maximum tetanic tension relative to muscle mass was significantly lower in HS than CON, whereas HS-WS had values similar to CON. The MG weight relative to body weight was significantly lower in both suspended groups. The maximum tetanic tension relative to muscle weight was significantly elevated in HS-WS compared to CON, suggesting that weight support may have preferentially maintained the contractile protein component of the muscle. Contraction times were 25% faster (p less than 0.05) in the Sol and unchanged in teh MG of HS rats. For each muscle, the fatigue properties were similar in all groups. These data indicate that a low-force, short-duration exercise regime results in a significant functional recovery in the "slow" Sol, whereas the "fast" MG is less affected.(ABSTRACT TRUNCATED AT 250 WORDS)